ENGINEERED OXALATE DECARBOXYLASES

REFERENCE TO A SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith as file name CX7-220US2_ST26.xml, with a creation date of Feb. 10, 2023 and a file size of 3.11 megabytes, is part of the specification and is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to engineered oxalate decarboxylase (ODC) polypeptides and compositions thereof, polynucleotides encoding the engineered oxalate decarboxylase polypeptides, and methods of using the engineered oxalate decarboxylase polypeptides and compositions thereof for therapeutic and industrial purposes.

BACKGROUND

Oxalate is found in many foods, such as leafy greens, and is also generated in the body as a metabolic product from the digestion of amino acids and ascorbic acid. In healthy individuals, oxalate is excreted in the urine (urinary oxalate: UOx), about half of which is derived from diet and the rest of which is generated as a metabolic product (Holmes et al., Kidney Int, 2001, 59(1):270-6). UOx can be variable in healthy individuals, but a level above 40 mg/day is considered hyperoxaluria.

Hyperoxaluria is generally divided into two categories. Primary hyperoxaluria is a rare genetic condition caused by mutations in alanine:glyoxylate aminotransferase (AGT) or glycolate oxidase (GO). Secondary hyperoxaluria, sometimes referred to as enteric hyperoxaluria, is a condition in which excess oxalate is absorbed in the gastrointestinal tract, which may arise from a high oxalate diet, fat malabsorption, changes in flora of intestinal oxalate-degrading microorganisms, or genetic variations of intestinal oxalate transporters (Robijn et al., Kidney Int′l, 2011, 80(11):1146-1158). Numerous conditions that may lead to secondary hyperoxaluria include Crohn’s disease (Hueppelshaeuser et al., Pediatr Nephrol., 2012, 27(7):1103-9), bariatric surgery (Lieske et al., Semin Nephrol., 2008, 28(2):163-73.; Agrawal et al., Surg Obes Relat Dis., 2014, 10(1):88-94), short bowel syndrome (Emmett et al., Am J Kidney Dis., 2003, 41(1):230-7), cystic fibrosis (Chidekel et al., Yale J Biol Med., 1996, 69(4):317-321), and celiac disease (Ciacci et al., J Urol., 2008, 180(3):974-9).

A major effect of hyperoxaluria is urinary calcium oxalate (CaOx) formation. In normal individuals, calcium binds dietary oxalate in the colon to form CaOx, which is excreted in stools. In hyperoxaluria, dietary calcium is depleted by binding to non-absorbed fatty acids, and excess oxalate remains soluble, allowing it to passively diffuse across the colonic lumen and into circulation (Nazzal et al., Nephrol Dial Transplant., 2016, 31(3):375-82). Accumulation of circulating oxalate in the kidneys results in high levels of excretion in the urine and buildup of CaOx stones in the kidney (nephrolithiasis). Furthermore, renal calcification (nephrocalcinosis) often results and contributes to the development of chronic kidney disease (CKD) and end-stage renal disease (ESRD). Many patients with hyperoxaluria have recurrent kidney stones and experience severe kidney damage over time, which can lead to CKD, ESRD, and death. UOx levels appear to be directly correlated with the risk for increased frequency of kidney stones and CKD (Curhan et al., Kidney Int., 2008, 73(4):489-96).

Treatments for hyperoxaluria include increasing fluid intake to increase urine excretion and thereby dilute UOx and treatment with alkali citrate, which forms a complex with calcium, thereby reducing precipitation of CaOx. While patients with primary hyperoxaluria may not benefit significantly from dietary oxalate restriction, patients with secondary hyperoxaluria are advised to avoid foods high in oxalate content and/or consume a diet high in calcium or calcium supplements. Although increasing dietary calcium appears to have an impact on increasing oxalate precipitation in the colon, high-dose calcium supplementation may increase CaOx urine supersaturation if high urine output is not maintained. A different treatment approach uses bile acid sequestration agents such as organic marine hydrocolloids (OMH) and cholestyramine to reduce the adsorption of oxalate. However, the efficacy of these molecules is not clear, and there are potential issues with long-term use due to worsening of steatorrhea and malabsorption of folic acid and fat-soluble vitamins (Harper et al., Postgrad Med., 1991, 67(785):219-222).

Therapeutics in development based on recombinant technology includes an engineered bacterium (SYNB8802) that degrades oxalate in the gastrointestinal tract (Puurunen et al., J Urology, 2021, 206(Supplement 3):e378-e378), a recombinant oxalate decarboxylase from B. subtilis formulated as crystals to increase its stability and activity (Reloxaliase®; Pfau et al., Nephrology Dialysis Transplantation, 2021, 36(5):945-948), and an oxalate decarboxylase from Synechococcus elongatus that upon oral delivery degrades oxalate in the gastrointestinal tract (Quintero et al., Kidney360, 2020, 1:1284-1290). While the current enzyme-based therapies under development appear to reduce plasma oxalate in healthy subjects or patients with severe enteric hyperoxaluria, desirable are alternative robust therapeutics for treating hyperoxaluria.

SUMMARY

The present disclosure provides engineered oxalate decarboxylase (ODC) polypeptides, biologically active fragments and compositions thereof, as well as polynucleotides encoding the engineered oxalate decarboxylase (ODC) polypeptides. In some embodiments, the oxalate decarboxylase polypeptides of the disclosure are engineered to have an improved property, including, among others, enhanced catalytic activity, reduced sensitivity to proteases, and/or increased tolerance to and activity at low pH environments. The present disclosure further provides methods for using the engineered oxalate decarboxylase polypeptides and compositions thereof for treating hyperoxaluria and other disorders characterized by elevated levels of oxalate.

In one aspect, the present disclosure provides an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-1622, or to a reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 4, 5, 6, 7, 10, 11, 13, 14, 16, 17, 18, 19, 22, 26, 31, 33, 35, 37, 40, 43, 44, 46, 52, 54, 60, 61, 62, 63, 76, 79, 80, 82, 83, 85, 94, 96, 97, 103, 104, 106, 110, 117, 121, 123, 124, 125, 126, 128, 141, 149, 153, 155, 156, 160, 162, 164, 166, 169, 173, 174, 176, 180, 182, 183, 186, 187, 188, 189, 190, 193, 195, 196, 197, 199, 200, 205, 206, 208, 210, 212, 216, 219, 226, 227, 232, 233, 234, 240, 242, 243, 263, 265, 266, 267, 269, 270, 273, 274, 277, 284, 297, 301, 303, 304, 314, 316, 318, 331, 335, 339, 342, 343, 346, 347, 350, 351, 356, or 359, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 13, 14, 16, 17, 26, 31, 37, 60, 79, 83, 96, 162, 174, 195, 196, 210, 226, 277, 301, or 318, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 31, 210, or 318, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 346, 124, 359, 174, 173, 123, 196, 304, 301, 347, 11, 284, 210, 169, 216, 195, 339, 4, 6, 180, 80, 243, 182, 7, 226, 156, 183, 227, 219, 62, 343, 16/26, 16/26/242, 16/26/183/232, 155/206/242, 16/26/339, 16/26/155/206/339, 26, 26/206/339, 31/82/210, 31/82, 31/356, 31/97/226, 31/240/270, 31/82/226, 31/240, 31, 31/210/318, or 31/210, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13, 212, 124/196/210/226, 5, 16/26/124/155/195/210/284, 196/226, 16/155/195/210/226, 16/195/210/226, 16/155/174/196, 16/195/226, 318, 16/195/196/210, 16/26/124/155/174/196/210, 195/210, 26/155/174/210, 316, 60, 16/155/174, 16/124/174/196, 16/226, 16/26/174/196/226, 17, 331, 124/195, 16/174/196, 188, 195/196/210, 174/196/210, 16/26/155/174, 16/155/195/196/226, 195/226/284, 16, 240, 16/26/124/155/195/196/226, 183/232/339/343, 183/206, 63, 173/347, 16/26/174/196, 16/284, 46, 16/124/195/196, 274, 174/196, 155/174/196, 174/196/226, 16/124/155/174/195, 16/26/155/174/196, 26/174/196/210/284, 16/195/196/284, 174, 16/124/174/195/210/226/284, 16/174/195/284, 26/174/196/210/226/284, 174/196/226/284, 124/174/196, 26/174/195/210/226, 162, 16/124/195/196/284, 206/343, 16/195/196, 16/26/174/195/196/210/226, 155/195/196/226, 18, 335, 124/155/174/195/226, 26/155/174/195/226, 195/196, 153, 155/174/195, 174/195/210/284, 174/195, 37, 124/174/195/226/284, 176, 16/155/174/195/196/226/284, 10, 169/173, 155, 33, 173/183/343/347, 104/265, or 233, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/196, 17/212/331, 17/26/196/212/226, 17/26/196/212/226/331, 13/17/26/196/331, 17/60/196/226, 17/46/212, 26/60/196/226/331, 17/46/196/212/274/331, 212, 17/196/226, 13/17/26/212/331, 13/17/212/226/331, 196/212/331, 13/17/196/331, 13/17/26/212/226/331, 17/60/196/212/226/331, 13/17/212, 13/17/26/331, 13/17, 13/17/26/196, 46/196/212/226, 17/60/196, 17/196/331, 13/17/46/226/331, 17/196/226/331, 17/46/196/212/331, 17/46/212/331, 17/196, 13/17/212/226, 17/212, 17/26/212, 13/17/226/331, 17/196/212/331, 17/196/212/226/331, 13/17/26/226/331, 46/60/196/226/331, 17/46/196/331, 13/17/46/196, 196/212, 13/17/26, 17/83/263, 17/83/173/227, 17/227/301, 17/83/227/263, 17/83/125, 17/83/173, 17/263/301, 17/125, 17/83/301, 17/173, 17/83/227, 17/227, 17, 17/125/227, 17/83, 128, 76, 141, 110, 79, 117, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/79, 13/14/17/60/79, 13/17/79/83, 13/17/79/301, 13/14/17/60/79/212, 17/60/197, 13/60/212, 13/14/17/60/79/83/301, 13/17/60/79, 17/60/301, 14/17/79, 17/60/83, 17/79, 13/17/60/79/83/301, 342, 52/190, 94/190, 190/342/351, 13/79, 13/96, 273, 126, 96, 79, 269, 40, 44, 267, 266, 160, 277, 149, 22, 234, 54, 297, 106, or 13/43, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 37, 125, 162, 94, 195, 85, 103, 232, 189, 186, 155, 193, 342, 196, 63, 33, 351, 314, 187, 303, 164, 153, 346, 183, 19, 123, 350, 205,284, 199, 343, 200, 208, 169, 190, or 121, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 14/26/60/318, 14/26/60/94/162/212, 60/162/226, 60, 14/125/162/212/318, 60/125/226, 14/125/162/226, 26/60/162/226/318, 14/26/94/162/212/226, 14/60/162, 14/60/162/226, 14/94/162/318, 14/94/162, 14/60/226, 14/60/94/212, 14/60/162/212, 14/162, 14/94/212/318, 14/60, 14/26/162, 14, 14/318, 14/162/226/318, 14/125/212, 14/125/226/318, 14/26/60/162, 14/125/162/212/226, 14/94/125/162/318, 14/125/226, 277, 166, 233, 83, 5, 342, 96, 284, or 26, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 5/26/83/342, 5/26/277, 5/96/277, 5/83/277/342, 26/83/277, 5/26/83/277, 5/277/342, 5/26/166/277, 83/277/342, 83/96/277/342, 83/96/277, 26/83/277/342, 5/26/83/277/342, 5/83/277, 26/277, 83/277, 5/35/277, 5/26/83/96/277, 5/26/96/277, 5/277, 5/166/277, 26/83/96/277/342, 277, 26/277/342, 5, or 5/83/96/277/342, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 26, 31, 96, 60, 318, 210, 277, 37, 16, 195, 79, 17, 13, 83, 162, or 174, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 174/318, 26/96, 60/318, 31/318, 162/174/318, 174/277/318, 26/195/318, 60/162/195, 60/162/318, 174/195/277, 16/17/174, 60/195/277, 37/277/318, 16/162/195/277, 26/79/83/162, 26/31/60/318, 16/174/195/277, 13/37/83/162, 16/17/60/195, 13/17/37/83, 31/37/162/174/318, 13/16/31/83/162, 16/60/174/195/318, 17/60/96/162/195/277, 13/26/60/83/162/174, 17/31/60/162/174/277, 16/17/26/83/96/277, 17/26/37/174/277/318, 13/16/31/60/162/174, 13/17/31/83/277/318, 13/16/17/31/83/162/318, 13/16/60/83/162/195/318, 13/16/17/31/37/83/96/162, 13/17/26/37/60/83/162/210/318, 13/16/26/31/83/96/162/174/318, 13/16/17/31/60/83/96/162/174/195/318, 13/16/26/37/60/83/162/174/195/210/318, 13/16/37/79/83/96/162/174/195/210/277, 13/17/26/31/37/60/79/83/96/162/174/210, 13/16/17/26/37/79/83/96/174/195/210/318, 13/26/31/37/60/83/96/162/174/195/210/277/318, 13/16/17/31/37/60/79/83/96/162/174/195/210/318, 13/16/17/31/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/37/60/79/96/162/174/195/210/277/318, 13/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/96/162/174/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/318, 13/16/17/26/31/37/60/79/96/162/174/210/277/318, 13/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/26/31/37/60/79/83/96/162/174/195/210/277/318, 16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, or 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set of an engineered oxalate decarboxylase polypeptide set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to SEQ ID NO: 2 or 4.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, or a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 616-1622.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-1622, or to the reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or to the reference sequence corresponding to SEQ ID NO: 172, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13, 212, 124/196/210/226, 5, 16/26/124/155/195/210/284, 196/226, 16/155/195/210/226, 16/195/210/226, 16/155/174/196, 16/195/226, 318, 16/195/196/210, 16/26/124/155/174/196/210, 195/210, 26/155/174/210, 316, 60, 16/155/174, 16/124/174/196, 16/226, 16/26/174/196/226, 17, 331, 124/195, 16/174/196, 188, 195/196/210, 174/196/210, 16/26/155/174, 16/155/195/196/226, 195/226/284, 16, 240, 16/26/124/155/195/196/226, 183/232/339/343, 183/206, 63, 173/347, 16/26/174/196, 16/284, 46, 16/124/195/196, 274, 174/196, 155/174/196, 174/196/226, 16/124/155/174/195, 16/26/155/174/196, 26/174/196/210/284, 16/195/196/284, 174, 16/124/174/195/210/226/284, 16/174/195/284, 26/174/196/210/226/284, 174/196/226/284, 124/174/196, 26/174/195/210/226, 162, 16/124/195/196/284, 206/343, 16/195/196, 16/26/174/195/196/210/226, 155/195/196/226, 18, 335, 124/155/174/195/226, 26/155/174/195/226, 195/196, 153, 155/174/195, 174/195/210/284, 174/195, 37, 124/174/195/226/284, 176, 16/155/174/195/196/226/284, 10, 169/173, 155, 33, 173/183/343/347, 104/265, or 233, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or to the reference sequence corresponding to SEQ ID NO: 320, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/196, 17/212/331, 17/26/196/212/226, 17/26/196/212/226/331, 13/17/26/196/331, 17/60/196/226, 17/46/212, 26/60/196/226/331, 17/46/196/212/274/331, 212, 17/196/226, 13/17/26/212/331, 13/17/212/226/331, 196/212/331, 13/17/196/331, 13/17/26/212/226/331, 17/60/196/212/226/331, 13/17/212, 13/17/26/331, 13/17, 13/17/26/196, 46/196/212/226, 17/60/196, 17/196/331, 13/17/46/226/331, 17/196/226/331, 17/46/196/212/331, 17/46/212/331, 17/196, 13/17/212/226, 17/212, 17/26/212, 13/17/226/331, 17/196/212/331, 17/196/212/226/331, 13/17/26/226/331, 46/60/196/226/331, 17/46/196/331, 13/17/46/196, 196/212, 13/17/26, 17/83/263, 17/83/173/227, 17/227/301, 17/83/227/263, 17/83/125, 17/83/173, 17/263/301, 17/125, 17/83/301, 17/173, 17/83/227, 17/227, 17, 17/125/227, 17/83, 128, 76, 141, 110, 79, 117, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to sequence of residues 1-359 of SEQ ID NO: 396, or to the reference sequence corresponding to SEQ ID NO::396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO::396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/79, 13/14/17/60/79, 13/17/79/83, 13/17/79/301, 13/14/17/60/79/212, 17/60/197, 13/60/212, 13/14/17/60/79/83/301, 13/17/60/79, 17/60/301, 14/17/79, 17/60/83, 17/79, 13/17/60/79/83/301, 342, 52/190, 94/190, 190/342/351, 13/79, 13/96, 273, 126, 96, 79, 269, 40, 44, 267, 266, 160, 277, 149, 22, 234, 54, 297, 106, or 13/43, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 670, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 37, 125, 162, 94, 195, 85, 103, 232, 189, 186, 155, 193, 342, 196, 63, 33, 351, 314, 187, 303, 164, 153, 346, 183, 19, 123, 350, 205, 284, 199, 343, 200, 208, 169, 190, or 121, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 14/26/60/318, 14/26/60/94/162/212, 60/162/226, 60, 14/125/162/212/318, 60/125/226, 14/125/162/226, 26/60/162/226/318, 14/26/94/162/212/226, 14/60/162, 14/60/162/226, 14/94/162/318, 14/94/162, 14/60/226, 14/60/94/212, 14/60/162/212, 14/162, 14/94/212/318, 14/60, 14/26/162, 14, 14/318, 14/162/226/318, 14/125/212, 14/125/226/318, 14/26/60/162, 14/125/162/212/226, 14/94/125/162/318, 14/125/226, 277, 166, 233, 83, 5, 342, 96, 284, or 26, wherein the amino acid positions are relative to the reference sequence corresponding SEQ ID NO: 616.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 5/26/83/342, 5/26/277, 5/96/277, 5/83/277/342, 26/83/277, 5/26/83/277, 5/277/342, 5/26/166/277, 83/277/342, 83/96/277/342, 83/96/277, 26/83/277/342, 5/26/83/277/342, 5/83/277, 26/277, 83/277, 5/35/277, 5/26/83/96/277, 5/26/96/277, 5/277, 5/166/277, 26/83/96/277/342, 277, 26/277/342, 5, or 5/83/96/277/342, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 750.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 174/318, 26/96, 60/318, 31/318, 162/174/318, 174/277/318, 26/195/318, 60/162/195, 60/162/318, 174/195/277, 16/17/174, 60/195/277, 37/277/318, 16/162/195/277, 26/79/83/162, 26/31/60/318, 16/174/195/277, 13/37/83/162, 16/17/60/195, 13/17/37/83, 31/37/162/174/318, 13/16/31/83/162, 16/60/174/195/318, 17/60/96/162/195/277, 13/26/60/83/162/174, 17/31/60/162/174/277, 16/17/26/83/96/277, 17/26/37/174/277/318, 13/16/31/60/162/174, 13/17/31/83/277/318, 13/16/17/3⅛3/162/318, 13/16/60/83/162/195/318, 13/16/17/31/37/83/96/162, 13/17/26/37/60/83/162/210/318, 13/16/26/31/83/96/162/174/318, 13/16/17/31/60/83/96/162/174/195/318, 13/16/26/37/60/83/162/174/195/210/318, 13/16/37/79/83/96/162/174/195/210/277, 13/17/26/31/37/60/79/83/96/162/174/210, 13/16/17/26/37/79/83/96/174/195/210/318, 13/26/31/37/60/83/96/162/174/195/210/277/318, 13/16/17/31/37/60/79/83/96/162/174/195/210/318, 13/16/17/31/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/37/60/79/96/162/174/195/210/277/318, 13/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/96/162/174/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/318, 13/16/17/26/31/37/60/79/96/162/174/210/277/318, 13/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/26/31/37/60/79/83/96/162/174/195/210/277/318, 16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, or 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set of an oxalate decarboxylase polypeptide set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence of an engineered oxalate decarboxylase polypeptide set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence comprising SEQ ID NO: 4; an amino acid sequence comprising residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614; an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614; or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 616-1622, optionally wherein the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises residues 1-359 of SEQ ID NO: 172, 320, or 396, or comprises SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, optionally wherein the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

In some embodiments of the foregoing, the amino acid sequence of the engineered oxalate decarboxylase polypeptide includes 1, 2, 3, 4, or 5 substitutions.

In some embodiments, the engineered oxalate decarboxylase polypeptide of the present disclosure has oxalate decarboxylase activity and exhibits one or more improved enzyme properties compared to a reference oxalate decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or an amino acid sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396. In some embodiments, the improved property of the engineered oxalate decarboxylase polypeptide is selected from (a) increased activity on oxalate, (b) increased thermal stability, (c) increased stability at acidic pH, (d) increased activity at acidic pH, (e) increased activity at neutral pH, (f) increased expression, (g) increased solubility, and (h) increased resistance to proteolysis, or any combination of (a), (b), (c), (d), (e), (f), (g) and (h), compared to a reference oxalate decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or an amino acid sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396. Exemplary improved propertied are provided in the Examples and Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2,

In some embodiments, the reference oxalate decarboxylase polypeptide for comparison of the improved property is the oxalate decarboxylase polypeptide having an amino acid sequence corresponding to SEQ ID NO: 2 or 4. In some embodiments the reference oxalate decarboxylase polypeptide is the wild-type oxalate decarboxylase of Gemmata sp. SH-PL17.

In some embodiments, an engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference amino acid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or to a reference amino acid sequence corresponding to residues 1-424 of SEQ ID NO: 6, wherein the amino acid sequence comprises one or more substitutions in its amino acid sequence.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid comprising residues 1-424 of SEQ ID NO: 6. In some embodiments, the oxalate decarboxylase polypeptides are provided as compositions, particularly pharmaceutical compositions.

In some embodiments, the engineered oxalate decarboxylase polypeptide described herein is purified or is a purified preparation.

In another aspect, the present disclosure provides compositions comprising at least one engineered oxalate decarboxylase polypeptide provided herein. In some embodiments, the composition is a pharmaceutical composition comprising at least one engineered oxalate decarboxylase of the present disclosure, and a pharmaceutically acceptable excipient or carrier.

In a further aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide described herein.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 3, a reference polynucleotide sequence corresponding to nucleotide residues 1-1077 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613, a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 615-1621, wherein the recombinant polynucleotide encodes an engineered oxalate decarboxylase polypeptide.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 33, 171, 319, 395, 615, 669, 749, or 905, or to a reference polynucleotide sequence corresponding to nucleotide residues 1-1077 of SEQ ID NO: 171, 319, or 395, wherein the recombinant polynucleotide encodes an engineered oxalate decarboxylase polypeptide.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence which is codon-optimized. In some embodiments, the polynucleotide sequence is codon optimized for expression in a bacterial cell, fungal cell, or mammalian cell.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 1 or 3; a polynucleotide sequence comprising nucleotide residues 1-1077 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 615-1621.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 3, 171, 319, 395, 615, 669, 749, or 905, or comprising nucleotide residues 1-1077 of SEQ ID NO: 171, 319, or 395.

In another aspect, the present disclosure also provides an expression vector comprising a recombinant polynucleotide encoding an engineered oxalate decarboxylase polypeptide. In some embodiments, the expression vectors further comprise at least one control sequence operably linked to the recombinant polynucleotide. In some embodiments, the control sequence comprises a promoter. In some further embodiments, the promoter is a heterologous promoter.

In a further aspect, the present disclosure also provides host cells transformed with at least one recombinant polynucleotide or an expression vector described herein. In some embodiments, the host cell comprises a recombinant polynucleotide described herein. In some embodiments, the host cell comprises an expression vector described herein. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

In another aspect, the present disclosure also provides a method of producing an engineered oxalate decarboxylase polypeptide described herein, the method comprising culturing a host cell comprising a recombinant polynucleotide encoding an engineered oxalate decarboxylase polypeptide under suitable culture conditions such that the encoded engineered oxalate decarboxylase polypeptide is produced. In some embodiments, the method further comprises recovering the engineered oxalate decarboxylase polypeptide from the culture and/or host cells. In some embodiments, the methods further comprise a step of purifying the expressed engineered oxalate decarboxylase polypeptide.

In another aspect, the present disclosure provides a composition comprising an engineered oxalated decarboxylase described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an engineered oxalate decarboxylase, and a pharmaceutically acceptable carrier or excipient.

In another aspect, the engineered oxalate decarboxylase is applied to various therapeutic and industrial uses. In some embodiments, an engineered oxalate decarboxylase is used for reducing levels of oxalate in a subject. In some embodiments, a method for reducing levels of oxalate in a subject comprises administering an effective amount of an engineered oxalate decarboxylase described herein to a subject in need thereof to reduce levels of oxalate in the subject. In some embodiments, the method is used to reduce levels of oxalate in urine and/or plasma of the subject.

In some embodiments, the engineered oxalate decarboxylase is used for treating hyperoxaluria and/or preventing the symptoms of hyperoxaluria in a subject, the method comprising administering to a subject with hyperoxaluria an effective amount of an engineered oxalate decarboxylase or a composition thereof described herein. In some embodiments, the subject is treated to ameliorate one or more symptoms of hyperoxaluria. In some embodiments, the levels of oxalate in the urine and/or plasma is reduced. In some embodiments, the subject can eat a diet that is less restricted in oxalate content than diets required by subjects who are afflicted with hyperoxaluria or have pathogenically elevated levels of oxalate. In some embodiments, the subject is an infant, child, young adult, or adult.

In another aspect, the present disclosure provides use of the engineered oxalate decarboxylase, or a pharmaceutical composition thereof, for the treatment of a disorder or condition associated with elevated levels of oxalate. In some embodiments, the use of the engineered oxalate decarboxylase is for the preparation of a medicament for treating a disease or condition associated with elevated levels of oxalate. In some embodiments, the use is in the treatment of hyperoxaluria.

In another aspect, the engineered oxalate decarboxylase can be useful in industrial applications, such as for reducing formation of calcium oxalate deposits/precipitates. Deposits or precipitates of calcium oxalate are problematic in pulp and paper industries, including removal of oxalate from industrial wastewater.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of percent change in Uox values for individual animals in three treatment groups orally administered different doses of an engineered oxalate decarboxylase (SEQ ID NO: 906). Bars represent the % change in UOx from HOD to HOD + Treatment (HOD: high oxalate diet). Dotted line at -20% (y-axis) identifies the minimum threshold reduction value used to evaluate efficacy of treatment.

FIG. 2 shows a graph of change in UOx (mg/24 hr) by treatment group. The white bars represent groups receiving HOD, and patterned bars represent groups receiving HOD + Tx. Treatment groups received the engineered oxalate decarboxylase (SEQ ID NO: 906) dosed at 5,139 U, 10,122 U, or 41,029 U. Statistical significance between HOD and HOD + Tx was determined using a One-way ANOVA in GraphPad Prism 9.

DETAILED DESCRIPTION

The present disclosure provides engineered oxalate decarboxylase (ODC) polypeptides and compositions thereof; polynucleotides encoding the engineered oxalate decarboxylase polypeptides; and uses of the engineered oxalate decarboxylase polypeptide for therapeutic and industrial purposes. In some embodiments, the oxalate decarboxylase polypeptides are engineered to have improved properties, including, among others, improved catalytic activity, reduced sensitivity to proteolysis, and/or increased tolerance to and stability at low pH environments.

Abbreviations and Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It is also to be understood that the invention herein is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole.

As used herein, the singular “a”, “an,” and “the” include the plural references, unless the context clearly indicates otherwise.

Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

The term “about” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

It is to be further understood that where description of embodiments use the term “comprising” and its cognates, the embodiments can also be described using language “consisting essentially of” or “consisting of.”

As used herein, the term “at least one” or “at least a” is not intended to limit the invention to any particular number of items. It is intended to encompass one, two, three, four, five, six, seven, eight, nine, ten, or more items, as desired.

“EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

“ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

“Oxalate decarboxylase polypeptide” or “oxalate decarboxylase” or “ODC” refers to a member of the enzyme class EC 4.1.1.2 or an enzyme that catalyzes the conversion of oxalate to formate and CO₂. In some embodiments, oxalate decarboxylase may additionally refer to enzymes that catalyzes the conversion of oxalate to hydrogen peroxide and CO₂.

“Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

“Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about α-carbon (Cα). For example, whereas “Ala” designates alanine without specifying the configuration about the α carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the α-carbon and lower-case letters designate amino acids in the D-configuration about the α-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When amino acid sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

“Polynucleotide” or “nucleic acid” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for the genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2′-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2′-deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5′ to 3′ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid.

“Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.

“Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

“Coding sequence” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

“Percent (%) sequence identity” is used herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted (e.g., by the local homology algorithm of Smith and Waterman; Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA., 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (See e.g., Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., Nucleic Acids Res., 1977, 25:3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (See, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or amino acid sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, the phrase “reference sequence based on SEQ ID NO: 2 having a valine at the residue corresponding to X60” refers to a reference sequence in which the corresponding residue at position X60 in SEQ ID NO: 2 (e.g., a threonine), has been changed to valine.

“Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

“Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered ODC, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

“Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded amino acid sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original amino acid sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original amino acid sequence. Any number of mutations may be present in a given sequence. In some embodiments, the “substitution” comprises the deletion of an amino acid and can be denoted by “-” symbol.

“Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of an amino acid sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X60 as compared to SEQ ID NO: 2” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 60 of SEQ ID NO: 2. Thus, if the reference polypeptide of SEQ ID NO: 2 has a threonine at position 60, then a “residue difference at position X60 as compared to SEQ ID NO: 2” refers to an amino acid substitution of any residue other than threonine at the position of the polypeptide corresponding to position 60 of SEQ ID NO: 2. In some instances herein, the specific amino acid residue difference, e.g., substitution, at a position is indicated as “XnY” where “Xn” specifies the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances, the original amino acid is not indicated and the amino acid difference indicated as nY (e.g., 60V). In some embodiments, the phrase “an amino acid residue nY” denotes the presence of the amino acid residue in the engineered polypeptide, which may or may not be a substitution in context of a reference sequence.

In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “/” (e.g., X60Y/X60W/X60R or X60Y/W/R or 60Y/W/R). The present disclosure includes engineered amino acid sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions.

“Amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within an amino acid sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the engineered polypeptides listed in any of the Tables in the Examples. In these substitution sets, the individual substitutions are separated by a semicolon (“;”; e.g., A16S;A26E or abbreviated 16S;26E) or slash (“/”; e.g., A16S/A26E, or abbreviated 16S/26E).

“Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions include the substitution of A, L, V, or I with other aliphatic residues (e.g., A, L, V, I) or other non-polar residues (e.g., A, L, V, I, G, M); substitution of G or M with other non-polar residues (e.g., A, L, V, I, G, M); substitution of D or E with other acidic residues (e.g., D, E); substitution of K or R with other basic residues (e.g., K, R); substitution of N, Q, S, or T with other polar residues (e.g., N, Q, S, T); substitution of H, Y, W, or F with other aromatic residues (e.g., H, Y, W, F); or substitution of C or P with other non-polar residues (e.g., C, P).

“Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and/or retaining the improved properties of an engineered oxalate decarboxylase enzyme. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

“Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full length engineered ODC of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant ODC polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant ODC polypeptides provided herein are isolated polypeptides.

“Substantially pure” or “purified” polypeptide or protein refers to a composition in which the polypeptide or protein species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure ODC polypeptide composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant ODC polypeptides are substantially pure polypeptide compositions.

“Improved enzyme property” refers to an engineered ODC polypeptide that exhibits an improvement in any enzyme property as compared to a reference ODC polypeptide, such as a wild-type ODC polypeptide (e.g., wild-type ODC corresponding to SEQ ID NO: 2) or another engineered ODC polypeptide. Improved properties include but are not limited to such properties as increased enzymatic activity, increased specific activity, increased protein production, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased substrate specificity and/or affinity, increased resistance to substrate and/or end-product inhibition, increased chemical stability, improved solvent stability, increased stability to acidic pH, increased resistance to proteases, reduced aggregation, increased solubility, and reduced immunogenicity (i.e., reduced capability of inducing and/or eliciting an immune response).

“Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered ODC polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) and/or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of ODC) as compared to the reference ODC enzyme (e.g., wild-type ODC and/or another engineered ODC). Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmax or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25- fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring ODC or another engineered ODC from which the ODC polypeptides were derived. In some specific embodiments, the engineered ODC enzyme exhibits improved enzymatic activity in the range of 1.5 to 10 fold, 1.5 to 25 fold, 1.5 to 50 fold, 1.5 to 100 fold or greater, than that of the reference ODC enzyme.

ODC enzyme activity can be measured by any standard assay known in the art (e.g., by monitoring depletion of reactants or formation of products). Exemplary methods of measuring ODC activity are provided in the Examples, such as Example 3. In some embodiments, comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells, in order to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

“Increased storage stability” means that an engineered ODC polypeptide according to the invention will retain more activity compared to a reference ODC in a standard assay (e.g., as described in the Examples) after it has been produced in its storage form (e.g., solution, lyophilization, or spray-drying), and stored for a period of time ranging from a few days to multiple months at a defined temperature or temperatures (e.g., -20° C., 0° C., 4° C., 25° C., 30° C., 37° C., 45° C., 55° C., etc.).

“Conversion” refers to the enzymatic conversion (or biotransformation) of substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of an ODC polypeptide can be expressed as “percent conversion” of the substrate to the product in a specific period of time.

“Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5× Denhart’s solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. “High stringency hybridization” refers generally to conditions that are about 10° C. or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018 M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5× Denhart’s solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65° C. and washing in 0.1x SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism’s genome. In some embodiments, the polynucleotides encoding the ODC enzymes are codon optimized for optimal production from the host organism selected for expression.

“Control sequence” refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. In some embodiments, the control sequences are provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

“Operably linked” or “operatively linked” refers to a functional relationship between two or more nucleic acid segments. By way of illustration, the term refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and where relevant, the encoded polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

“Substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the ODC polypeptide. “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the ODC polypeptide on the substrate.

“Culturing” or “cultured” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

“Vector” as used herein refers to a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the DNA sequence. In some embodiments, an “expression vector” has a promoter sequence operably linked to the DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

“Expression” as used herein includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

“Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or amino acid sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

“Host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., a polynucleotide sequences encoding at least one ODC variant). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

“Subject” as used herein encompasses mammals such as humans, non-human primates, livestock (e.g., cows, pigs, camels, etc.), companion animals (e.g., cats, dogs, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, and lagamorphs). It is intended that the term encompass females as well as males. A subject who is a “patient” means any subject that is being assessed for, treated for, or is experiencing disease.

“Infant” refers to a child in the period of the first month after birth to approximately one (1) year of age. As used herein, the term “newborn” refers to child in the period from birth to the 28th day of life. The term “premature infant” refers to an infant born after the twentieth completed week of gestation, yet before full term, generally weighing ~500 to ~2499 grams at birth.

“Child” refers to a person who has not attained the legal age for consent to treatment or research procedures. In some embodiments, the term refers to a person between the time of birth and adolescence.

“Adult” refers to a person who has attained legal age for the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term “young adult” refers to a person less than 18 years of age, but who has reached sexual maturity.

“Composition” and “formulation” encompass products comprising at least one engineered ODC of the present disclosure, intended for any suitable use (e.g., pharmaceutical compositions, dietary/nutritional supplements, feed, etc.).

“Therapeutic” as used herein refers to an agent administered to a subject who shows signs or symptoms of pathology having beneficial or desirable medical effects.

“Pharmaceutical composition” as used herein refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of an engineered ODC polypeptide encompassed by the invention and an acceptable carrier.

“Carrier” when used in reference to a pharmaceutical composition means any of the standard pharmaceutical carrier, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

“Excipient” refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API; e.g., the engineered ODC polypeptides of the present invention). Excipients are typically included for formulation and/or administration purposes.

“Pharmaceutically acceptable” means a material that can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.

“Effective amount” refers to an amount sufficient to produce the desired result. The effective amount can be determined by one of general skill in the art by methods known in the art and the guidance provided herein.

“Therapeutically effective amount” when used in reference to symptoms of disease/condition refers to the amount and/or concentration of a compound (e.g., engineered ODC polypeptides) that ameliorates, attenuates, or eliminates one or more symptom of a disease/condition or prevents or delays the onset of symptom(s) (e.g., hyperoxaluria). In some embodiments, the term is use in reference to the amount of a composition that elicits the biological (e.g., medical) response by a tissue, system, or animal subject that is sought by the researcher, physician, veterinarian, or other clinician. “Therapeutically effective amount” when used in reference to a disease/condition refers to the amount and/or concentration of a composition that ameliorates, attenuates, or eliminates the disease/condition.

“Treating” or “treatment” of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As such, the terms “treating,” “treat” and “treatment” encompass preventative (e.g., prophylactic), as well as palliative treatment. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition can be made by those skilled in the art.

Engineered Oxalate Decarboxylase Polypeptides

In one aspect, the present disclosure provides engineered oxalate decarboxylase (ODC) polypeptides for conversion/degradation of oxalate. In some embodiments, the engineered oxalate decarboxylase polypeptides are variants of the wild-type oxalate decarboxylase of Gemmata sp. SH-PL17. In some embodiments, in the present disclosure when a particular engineered oxalate decarboxylase variant (i.e., an engineered ODC polypeptide) is referred to by reference to amino acid differences in particular amino acids residues in the sequence of a wild-type oxalate decarboxylase or reference oxalate decarboxylase, it is to be understood that variants of another oxalate decarboxylase modified in the equivalent position(s) (as determined from the optional amino acid sequence alignment between the respective amino acid sequences) are to be encompassed herein.

Furthermore, the present disclosure describes exemplary engineered ODC polypeptides having oxalate decarboxylase activity. The Examples and the exemplary engineered oxalate decarboxylase presented in the Tables (i.e., Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2) provide sequence structural information correlating specific amino acid sequence features with the functional activity (e.g., improved property) of the engineered oxalate decarboxylase polypeptides. This structure-function correlation information is provided in the form of specific amino acid residue differences relative to the reference engineered oxalate decarboxylase polypeptide of SEQ ID NO: 2 or 4, as well as associated experimentally determined activity data for the exemplary engineered ODC polypeptides.

In some embodiments, an engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-1622, or to the reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 4C/R/S, 5C/S, 6A/S/W, 7G, 10A/Q/R, 11R, 13A/C/D/E/F/G/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 14L, 16C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 17A/C/D/E/F/G/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 18G/Q/R, 19C, 22R, 26C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 31A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 33R, 35V, 37A/C/D/E/F/G/H/I/L/M/N/P/Q/R/S/T/V/W/Y, 40S, 43C, 44S, 46R, 52T, 54L, 60A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/V/W/Y, 61M, 62G, 63A/S, 76S, 79C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 80L, 82I, 83A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/V/W/Y, 85E/G/R, 94N/T, 96C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 97T, 103Q/S/V, 104Q, 106C, 110V, 117A, 121W, 123G/Q/S, 124A/C/G/Q/R/T, 125E/F/S, 126T, 128A, 141Q, 149T, 153H/Q/S, 155L/P/R/V, 156E, 160T, 162A/C/D/E/F/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 164R/V, 166R, 169A/G/L, 173Q/V, 174A/C/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 176L, 180E, 182R, 183C/E/I/K, 186N, 187L/R/S, 188A, 189R, 190G/Q, 193A/G, 195A/C/D/E/F/G/H/I/K/L/M/N/P/Q/S/T/V/W/Y, 196F/M/R/S/V, 197E, 199G/V, 200N, 205A, 206M, 208G, 210A/C/D/E/F/G/H/I/K/L/M/N/Q/R/S/T/V/W/Y, 212A/F/G/L/S/V, 216S/W, 219V, 226S, 227E/S, 232T, 233D/H/R, 234L, 240E, 242D, 243V, 263S, 265C, 266V, 267R, 269S, 270L, 273A, 274Q/S, 277A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W, 284A/R/S, 297Y, 301I/Q/T, 303T, 304D/G, 314S, 316K/V, 318A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 331I/L/P/V, 335R, 339G, 342A/E/P/R, 343R/W, 346G/L/Q/W, 347F/R, 350R, 351A/E/H, 356S, or 359L/W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution P4C/R/S, T5C/S, F6A/S/W, M7G, V10A/Q/R, P11R, H13A/C/D/E/F/G/I/K/L/M/N/P/Q/R/S/T/V/W/Y, V14L, A16C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, H17A/C/D/E/F/G/I/K/L/M/N/P/Q/R/S/T/V/W/Y, K18G/Q/R,, D19C, T22R, A26C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, E31A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, K33R, I35V, K37A/C/D/E/F/G/H/I/L/M/N/P/Q/R/S/T/V/W/Y, G40S, A43C, T44S, V46R, K52T, I54L, T60A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/V/W/Y, L61M, E62G, P63A/S, A76S, A79C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, F80L/I, L82I, T83A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/V/W/Y, H85E/G/R, Q94N/T, A96C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, A97T, G103Q/S/V, P104Q, D106C, F110V, M117A, L121W, D123G/Q/S, K124A/C/G/Q/R/T, P125E/F/S, A126T, F128A, G141Q, I149T, P153H/Q/S, A155L/P/R/V, L156E, N160T, G162A/C/D/E/F/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, P164R/V, S166R, D169A/G/L, K173Q/V, D174A/C/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, V176L, R180E, A182R, V183C/E/I/K, A186N, T187L/R/S, P188A, A189R, A190G/Q, Q193A/G, R195A/C/D/E/F/G/H/I/K/L/M/N/P/Q/S/T/V/W/Y, K196F/M/R/S/V, L197E, P199G/V, L200N, E205A, L206M, E208G, P210A/C/D/E/F/G/H/I/K/L/M/N/Q/R/S/T/V/W/Y, H212A/F/G/L/S/V, K216S/W, R219V, T226S, R227E/S, K232T, T233D/H/R, V234L, D240E, E242D, P243V, D263S, N265C, I266V, S267R, T269S, M270L, S273A, H274Q/S, Y277A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W, K284A/R/S, S297Y, V301I/Q/T, D303T, R304D/G, T314S, N316K/V, E318A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, A331I/L/P/V, A335R, S339G, T342A/E/P/R, S343R/W, E346G/L/Q/W, K347F/R, K350R, Q351A/E/H, A356S, or K359L/W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 13A/C/D/E/F/G/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 14L, 16C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 17A/C/D/E/F/G/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 26C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 31A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 37A/C/D/E/F/G/H/I/L/M/N/P/Q/R/S/T/V/W/Y, 60A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/V/W/Y, 79C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 83A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/V/W/Y, 96C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 162A/C/D/E/F/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 174A/C/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 195A/C/D/E/F/G/H/I/K/L/M/N/P/Q/S/T/V/W/Y, 196F/M/R/S/V, 210A/C/D/E/F/G/H/I/K/L/M/N/Q/R/S/T/V/W/Y, 226S, 277A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W, 301I/Q/T, or 318A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 13P, 14L, 16S, 17H/G, 26E, 31K, 37G, 60V, 79G, 83W, 96L, 162K, 174K, 195L, 196V, 210A/E, 226S, 277V, 301I, or 318Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 31K, 210A/E, 318Q, or 31K/210A/318Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises as least a substitution or substitution set at amino acid position(s) 16, 26, 174, 195, 196, 210, 226, or 16/26/174/195/196/210/226, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 16S, 26E, 174K, 195L, 196V, 210E, 226S, or 16S/26E/174K/195L/196V/210E/226S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P, 17H, 196K, or 13P/17H/196K, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 17G, 60V, 301I, or 17G/60V/301I, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 37, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 37G, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 14, 60, 162, 226, or 14/60/162/226, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 14L, 60V, 162K, 226T, or 14L/60V/162K/226T, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 83W, 96L, 277V, or 83W/96L/277V, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 346, 124, 359, 174, 173, 123, 196, 304, 301, 347, 11, 284, 210, 169, 216, 195, 339, 4, 6, 180, 80, 243, 182, 7, 226, 156, 183, 227, 219, 62, 343, 16/26, 16/26/242, 16/26/183/232, 155/206/242, 16/26/339, 16/26/155/206/339, 26, 26/206/339, 31/82/210, 31/82, 31/356, 31/97/226, 31/240/270, 31/82/226, 31/240, 31, 31/210/318, or 31/210, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 346G, 346L, 124G, 124T, 359L, 174K, 173V, 123Q, 196F, 304D, 301Q, 347F, 359W, 124Q, 124A, 11R, 284R, 210L, 169L, 304G, 216W, 347R, 196V, 284A, 195L, 339G, 4S, 196R, 6A, 124C, 195Y, 180E, 80L, 243V, 182R, 210E, 7G, 226S, 174A, 196M, 4C, 156E, 183E, 227S, 219V, 62G, 174G, 169G, 124R, 210V, 343R, 216S, 210R, 6W, 4R, 6S, 16S/26E, 16S/26E/242D, 16S/26E/183I/232T, 155P/206M/242D, 16S/26E/339G, 16S/26E/155P/206M/339G, 26E, 26E/206M/339G, 31K/82I/210A, 31K/82I, 31K/356S, 31K/97T/226S, 31K/240E/270L, 31K/82I/226S, 31K/240E, 31K, 31K/210A/318Q, or 31K/210A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set E346G, E346L, K124G, K124T, K359L, D174K, K173V, D123Q, K196F, R304D, V301Q, K347F, K359W, K124Q, K124A, P11R, K284R, P210L, D169L, R304G, K216W, K347R, K196V, K284A, R195L, S339G, P4S, K196R, F6A, K124C, R195Y, R180E, F80L, P243V, A182R, P210E, M7G, T226S, D174A, K196M, P4C, L156E, V183E, R227S, R219V, E62G, D174G, D169G, K124R, P210V, S343R, K216S, P210R, F6W, P4R, F6S, A16S/A26E, A16S/A26E/E242D, A16S/A26E/V183I/K232T, A155P/L206M/E242D, A16S/A26E/S339G, A16S/A26E/A155P/L206M/S339G, A26E, A26E/L206M/S339G, E31K/L82I/P210A, E31K/L82I, E31K/A356S, E31K/A97T/T226S, E31K/D240E/M270L, E31K/L82I/T226S, E31K/D240E, E31K, E31K/P210A/E318Q, or E31K/P210A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13, 212, 124/196/210/226, 5, 16/26/124/155/195/210/284, 196/226, 16/155/195/210/226, 16/195/210/226, 16/155/174/196, 16/195/226, 318, 16/195/196/210, 16/26/124/155/174/196/210, 195/210, 26/155/174/210, 316, 60, 16/155/174, 16/124/174/196, 16/226, 16/26/174/196/226, 17, 331, 124/195, 16/174/196, 188, 195/196/210, 174/196/210, 16/26/155/174, 16/155/195/196/226, 195/226/284, 16, 240, 16/26/124/155/195/196/226, 183/232/339/343, 183/206, 63, 173/347, 16/26/174/196, 16/284, 46, 16/124/195/196, 274, 174/196, 155/174/196, 174/196/226, 16/124/155/174/195, 16/26/155/174/196, 26/174/196/210/284, 16/195/196/284, 174, 16/124/174/195/210/226/284, 16/174/195/284, 26/174/196/210/226/284, 174/196/226/284, 124/174/196, 26/174/195/210/226, 162, 16/124/195/196/284, 206/343, 16/195/196, 16/26/174/195/196/210/226, 155/195/196/226, 18, 335, 124/155/174/195/226, 26/155/174/195/226, 195/196, 153, 155/174/195, 174/195/210/284, 174/195, 37, 124/174/195/226/284, 176, 16/155/174/195/196/226/284, 10, 169/173, 155, 33, 173/183/343/347, 104/265, or 233, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P, 212S, 124A/196V/210E/226S, 5S, 13E, 16S/26E/124A/155P/195L/210E/284A, 196V/226S, 16S/155P/195L/210E/226S, 16S/195L/210E/226S, 16S/155P/174K/196V, 16S/195L/226S, 13L, 318E, 16S/195L/196V/210E, 16S/26E/124A/155P/174K/196V/210E, 195L/210E, 26E/155P/174K/210E, 316V, 60V, 16S/155P/174K, 16S/124A/174K/196V, 16S/226S, 16S/26E/174K/196V/226S, 17H, 331V, 124A/195L, 16S/174K/196V, 17A, 188A, 195L/196V/210E, 174K/196V/210E, 5C, 16S/26E/155P/174K, 16S/155P/195L/196V/226S, 195L/226S/284A, 17N, 16L, 240E, 16S/26E/124A/155P/195L/196V/226S, 331P, 1831/232T/339G/343R, 183I/206M, 63A, 173V/347R, 16S/26E/174K/196V, 16S/284A, 60R, 46R, 16S/124A/195L/196V, 274S, 331I, 174K/196V, 155P/174K/196V, 174K/196V/226S, 331L, 16S/124A/155P/174K/195L, 16S/26E/155P/174K/196V, 26E/174K/196V/210E/284A, 212A, 16S/195L/196V/284A, 174K, 16S/124A/174K/195L/210E/226S/284A, 16S/174K/195L/284A, 26E/174K/196V/210E/226S/284A, 212L, 174K/196V/226S/284A, 124A/174K/196V, 26E/174K/195L/210E/226S, 162A, 16S/124A/195L/196V/284A, 206M/343R, 16S/195L/196V, 16S/26E/174K/195L/196V/210E/226S, 155P/195L/196V/226S, 18Q, 335R, 124A/155P/174K/195L/226S, 17C, 26E/155P/174K/195L/226S, 195L/196V, 162T, 153H, 316K, 155P/174K/195L, 153Q, 174K/195L/210E/284A, 174K/195L, 153S, 212F, 37C, 212V, 124A/174K/195L/226S/284A, 176L, 16S/155P/174K/195L/196V/226S/284A, 10Q, 169G/173V, 155L, 18G, 60L, 10A, 18R, 33R, 212G, 10R, 13R, 173V/1831/343R/347R, 16R, 104Q/265C, 233R, or 233D, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/196, 17/212/331, 17/26/196/212/226, 17/26/196/212/226/331, 13/17/26/196/331, 17/60/196/226, 17/46/212, 26/60/196/226/331, 17/46/196/212/274/331, 212, 17/196/226, 13/17/26/212/331, 13/17/212/226/331, 196/212/331, 13/17/196/331, 13/17/26/212/226/331, 17/60/196/212/226/331, 13/17/212, 13/17/26/331, 13/17, 13/17/26/196, 46/196/212/226, 17/60/196, 17/196/331, 13/17/46/226/331, 17/196/226/331, 17/46/196/212/331, 17/46/212/331, 17/196, 13/17/212/226, 17/212, 17/26/212, 13/17/226/331, 17/196/212/331, 17/196/212/226/331, 13/17/26/226/331, 46/60/196/226/331, 17/46/196/331, 13/17/46/196, 196/212, 13/17/26, 17/83/263, 17/83/173/227, 17/227/301, 17/83/227/263, 17/83/125, 17/83/173, 17/263/301, 17/125, 17/83/301, 17/173, 17/83/227, 17/227, 17, 17/125/227, 17/83, 128, 76, 141, 110, 79, 117, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P/17H/196K, 17H/212S/331V, 17H/26A/196K/212S/226T, 17H/26A/196K/212A/226T/331V, 13P/17H/26A/196K/331V, 17H/60R/196K/226T, 17H/46R/212S, 26A/60V/196K/226T/331V, 17H/46R/196K/212A/274Q/331V, 212S, 17H/196K/226T, 13P/17H/26A/212A/331V, 13P/17H/212S/226T/331V, 196K/212S/331V, 13P/17H/196K/331V, 13P/17H/26A/212A/226T/331V, 17H/60R/196K/212A/226T/331V, 13P/17H/212S, 13P/17H/26A/331V, 13P/17H, 13P/17H/26A/196K, 46R/196K/212S/226T, 17H/60R/196K, 17H/196K/331V, 13P/17H/46R/226T/331V, 17H/196K/226T/331V, 17H/60V/196K/226T, 17H/46R/196K/212S/331V, 13P/17H/212A, 17H/46R/212S/331V, 17H/196K, 13P/17H/212S/226T, 17H/212S, 17H/26A/212S, 13P/17H/226T/A331V, D17H/V196K/212S/331V, 17H/196K/212S/226T/331V, 13P/17H/26A/226T/331V, 46R/60V/196K/226T/331V, 17H/46R/196K/331V, 17H/212A/331V, 13P/17H/46R/196K, 196K/212S, 13P/17H/26A, 17G/83E/263S, 17G/83E/173Q/227E, 17G/227E/301I, 17G/83E/227E/263S, 17G/83E/125E, 17G/83E/173Q, 17G/263S/301I, 17G/125E, 17G/83E/301I, 17G/173Q, 17G/83E/227E, 17G/227E, 17G, 17G/125E/227E, 17G/83E, 17G/263S/301T, 128A, 76S, 141Q, 110V, 79S, 117A, 79G, or 61M, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/79, 13/17/79, 13/14/17/60/79, 13/17/79/83, 13/17/79/301, 13/14/17/60/79/212, 17/60/197, 13/60/212, 13/14/17/60/79/83/301, 13/17/60/79, 17/60/301, 14/17/79, 17/60/83, 17/79, 13/17/60/79/83/301, 342, 52/190, 94/190, 190/342/351, 13/79, 13/96, 273, 126, 96, 79, 269, 40, 44, 267, 266, 160, 277, 149, 22, 234, 54, 297, 106, or 13/43, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13H/17G/79G, 13H/14V/17G/60V/79G, 13H/17G/79G/83E, 13H/17G/79G/301I, 13H/14V/17G/60V/79G/212S, 17G/60V/197E, 13H/60V/212S, 13H/14V/17G/60V/79G/83E/301I, 13H/17G/60V/79G, 17G/60V/301I, 14V/17G/79G, 17G/60V/83E, 17G/79G, 13H/17G/60V/79G/83E/301I, 342P, 52T/190Q, 94N/190Q, 190Q/342P/351E, 13H/79G, 13H/96L, 273A, 126T, 96K, 79S, 269S, 40S, 96L, 44S, 96R, 79G, 267R, 266V, 160T, 277T, 149T, 22R, 234L, 54L, 297Y, 277A, 106C, or 13H/43C, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 37, 125, 162, 94, 195, 85, 103, 232, 189, 186, 155, 193, 342, 196, 63, 33, 351, 314, 187, 303, 164, 153, 346, 183, 19, 123, 350, 205, 284, 199, 343, 200, 208, 169, 190, or 121, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 37G, 125F, 162K, 94T, 195W, 85R, 103S, 232T, 189R, 186N, 155R, 193A, 342A, 196V, 63S, 33R, 351H, 193G, 125S, 314S, 187R, 303T, 103V, 103Q, 155V, 164R, 153S, 37R, 346Q, 346G, 351A, 183K, 19C, 187L, 123S, 85G, 187S, 183C, 123G, 350R, 85E, 205A, 284R, 199V, 346W, 343W, 164V, 200N, 342E, 208G, 169A, 190G, 284A, 199G, 121W, or 196S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 14/26/60/318, 14/26/60/94/162/212, 60/162/226, 60, 14/125/162/212/318, 60/125/226, 14/125/162/226, 26/60/162/226/318, 14/26/94/162/212/226, 14/60/162, 14/60/162/226, 14/94/162/318, 14/94/162, 14/60/226, 14/60/94/212, 14/60/162/212, 14/162, 14/94/212/318, 14/60, 14/26/162, 14, 14/318, 14/162/226/318, 14/125/212, 14/125/226/318, 14/26/60/162, 14/125/162/212/226, 14/94/125/162/318, 14/125/226, 277, 166, 233, 83, 5, 342, 96, 284, or 26, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 14L/26A/60V/318E, 14L/26A/60V/94T/162K/212S, 60V/162K/226T, 60V, 14L/125F/162K/212S/318E, 60V/125F/226T, 14L/125F/162K/226T, 26A/60V/162K/226T/318E, 14L/26A/94T/162K/212S/226T, 14L/60V/162K, 14L/60V/162K/226T, 14L/94T/162K/318E, 14L/94T/162K, 14L/60V/226T, 14L/60V/94T/212S, 14L/60V/162K/212S, 14L/162K, 14L/94T/212S/318E, 14L/60V, 14L/26A/162K, 14L, 14L/318E, 14L/162K/226T/318E, 14L/125F/212S, 14L/125F/226T/318E, 14L/26A/60V/162K, 14L/125F/162K/212S/226T, 14L/94T/125F/162K/318E, 14L/125F/226T, 277R, 277C, 277T, 166R, 233H, 83R, 60M, 277I, 277M, 5S, 342R, 277V, 60R, 96R, 284S, 26R, 26N, or 83W, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 5/26/83/342, 5/26/277, 5/96/277, 5/83/277/342, 26/83/277, 5/26/83/277, 5/277/342, 5/26/166/277, 83/277/342, 83/96/277/342, 83/96/277, 26/83/277/342, 5/26/83/277/342, 5/83/277, 26/277, 83/277, 5/35/277, 5/26/83/96/277, 5/26/96/277, 5/277, 5/166/277, 26/83/96/277/342, 277, 26/277/342, 5, or 5/83/96/277/342, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least substitution or substitution set 5S/26A/83W/342R, 5S/26A/277T, 5S/96L/277T, 5S/83W/277T/342R, 26A/83W/277T, 5S/26A/83W/277V, 5S/277T/342R, 5S/83W/277V/342R, 5S/26A/166R/277T, 83W/277T/342R, 83R/96L/277V/342R, 83W/96L/277T, 26A/83W/277V/342R, 5S/26A/83W/277V/342R, 5S/83R/277T/342R, 5S/26A/83W/277T/342R, 5S/83R/277V, 26A/277T, 83W/277T, 5S/35V/277T, 5S/26A/83R/96R/277V, 5S/26A/96L/277T, 5S/277T, 5S/26A/83R/277T, 5S/83R/277T, 26A/83R/277V, 5S/26A/277V, 5S/166R/277T, 26A/83W/277V, 26A/83W/96L/277V/342R, 277T, 26A/277T/342R, 83W/277V/342R, 83R/277V, 5S, 26A/277V, 83R/277T, 5S/83W/96R/277T/342R, 5S/277V, 277V, 5S/277V/342R, 83W/96L/277V, or 83W/277V, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 26, 31, 96, 60, 318, 210, 277, 37, 16, 195, 79, 17, 13, 83, 162, or 174, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 26L, 31W, 96K, 60R, 318D, 26G, 210R, 31R, 277F, 37M, 96P, 16V, 60A, 26R, 31G, 195G, 318R, 26V, 79M, 16M, 210A, 96R, 37E, 16G, 17W, 13G, 79E, 277T, 60Q, 31L, 96G, 96T, 60G, 16W, 13T, 79R, 26Y, 26S, 195R, 17R, 13V, 277E, 37L, 210G, 60L, 13L, 83Y, 37W, 277G, 83R, 60Y, 210S, 318S, 17I, 37S, 79L, 16R, 195S, 318G, 26C, 13R, 195Y, 60E, 162V, 16C, 96V, 83M, 31M, 174V, 277L, 26M, 318M, 210N, 26A, 277R, 96F, 174G, 16Q, 277A, 13Q, 79F, 16L, 195N, 79V, 17P, 60P, 37P, 96W, 162T, 195M, 318W, 37R, 16T, 210M, 162Y, 16P, 83E, 277M, 174L, 17V, 17E, 17L, 96M, 318V, 174R, 13S, 174T, 13Y, 195A, 277W, 60M, 17A, 318A, 174H, 83S, 277I, 13E, 17K, 26K, 96A, 174D, 210V, 31S, 195C, 79K, 60W, 174S, 13I, 60H, 31E, 96E, 83K, 31Q, 16K, 174M, 37V, 79D, 318T, 195F, 26T, 174Q, 83L, 96I, 13C, 13A, 96Q, 13H, 83T, 31Y, 13W, 277P, 195T, 60S, 26Q, 210P, 174N, 83A, 162E, 37F, 37Q, 31T, 174A, 83F, 60T, 96S, 83V, 210I, 16D, 37C, 83G, 13N, 31V, 79S, 96Y, 17F, 17M, 16A, 83P, 318L, 79Q, 17S, 26W, 195D, 195W, 277S, 174E, 16E, 162F, 79Y, 79A, 17C, 17Y, 195Q, 26P, 195I, 60F, 26I, 17D, 318K, 60N, 16I, 318H, 37A, 195V, 210Y, 195E, 26H, 13D, 31P, 210W, 318P, 83C, 174P, 210T, 17T, 79T, 96H, 79W, 162L, 318E, 31A, 210C, 17N, 195K, 318Y, 17H, 13M, 60D, 277K, 16N, 79C, 13K, 60K, 37N, 60I, 162D, 162I, 16Y, 162S, 96C, 210K, 174I, 210L, 26D, 26N, 210D, 13F, 318N, 277Y, 174W, 31H, 31D, 79N, 37K, 31I, 37H, 318C, 31F, 79P, 195P, 318I, 162A, 277Q, 26F, 277C, 79H, 31C, 79I, 37I, 17Q, 31N, 277N, 37D, 277H, 277D, 96D, 174C, 162P, 318F, 83N, 60C, 83I, 162M, 210H, 195H, 174Y, 37Y, 210Q, 37T, 96N, 83H, 16H, 174F, 83D, 83Q, 162H, 162W, 162N, 16F, 162Q, 162G, 162R, 210F, or 162C, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 174/318, 26/96, 60/318, 31/318, 162/174/318, 174/277/318, 26/195/318, 60/162/195, 60/162/318, 174/195/277, 16/17/174, 60/195/277, 37/277/318, 16/162/195/277, 26/79/83/162, 26/31/60/318, 16/174/195/277, 13/37/83/162, 16/17/60/195, 13/17/37/83, 31/37/162/174/318, 13/16/31/83/162, 16/60/174/195/318, 17/60/96/162/195/277, 13/26/60/83/162/174, 17/31/60/162/174/277, 16/17/26/83/96/277, 17/26/37/174/277/318, 13/16/31/60/162/174, 13/17/31/83/277/318, 13/16/17/31/83/162/318, 13/16/60/83/162/195/318, 13/16/17/31/37/83/96/162, 13/17/26/37/60/83/162/210/318, 13/16/26/31/83/96/162/174/318, 13/16/17/31/60/83/96/162/174/195/318, 13/16/26/37/60/83/162/174/195/210/318, 13/16/37/79/83/96/162/174/195/210/277, 13/17/26/31/37/60/79/83/96/162/174/210, 13/16/17/26/37/79/83/96/174/195/210/318, 13/26/31/37/60/83/96/162/174/195/210/277/318, 13/16/17/31/37/60/79/83/96/162/174/195/210/318, 13/16/17/31/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/37/60/79/96/162/174/195/210/277/318, 13/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/96/162/174/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/318, 13/16/17/26/31/37/60/79/96/162/174/210/277/318, 13/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/26/31/37/60/79/83/96/162/174/195/210/277/318, 16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, or 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 174D/318E, 26A/96A, 60T/318E, 31E/318E, 162G/174D/318E, 174D/277Y/318E, 26A/195R/318E, 60T/162G/195R, 60T/162G/318E, 174D/195R/277Y, 16A/17D/174D, 60T/195R/277Y, 37K/277Y/318E, 16A/162G/195R/277Y, 26A/79A/83T/162G, 26A/31E/60T/318E, 16A/174D/195R/277Y, 13H/37K/83T/162G, 16A/17D/60T/195R, 13H/17D/37K/83T, 31E/37K/162G/174D/318E, 13H/16A/31E/83T/162G, 16A/60T/174D/195R/318E, 17D/60T/96A/162G/195R/277Y, 13H/26A/60T/83T/162G/174D, 17D/31E/60T/162G/174D/277Y, 16A/17D/26A/83T/96A/277Y, 17D/26A/37K/174D/277Y/318E, 13H/16A/31E/60T/162G/174D, 13H/17D/31E/83T/277Y/318E, 13H/16A/17D/31E/83T/162G/318E, 13H/16A/60T/83T/162G/195R/318E, 13H/16A/17D/31E/37K/83T/96A/162G, 13H/17D/26A/37K/60T/83T/162G/210P/318E, 13H/16A/26A/37K/83T/162G/174D/195R/318E, 13H/16A/26A/31E/83T/96A/162G/174D/318E, 13H/16A/17D/31E/60T/83T/96A/162G/174D/195R/318E, 13H/16A/26A/37K/60T/83T/162G/174D/195R/210P/318E, 13H/16A/37K/79A/83T/96A/162G/174D/195R/210P/277Y, 13H/17D/26A/31E/37K/60T/79A/83T/96A/162G/174D/210P, 13H/16A/17D/26A/37K/79A/83T/96A/174D/195R/210D/318E, 13H/26A/31E/37K/60T/83T/96A/162G/174D/195R/210P/277Y/318E, 13H/16A/17D/31E/37K/60T/79A/83T/96A/162G/174D/195R/210P/318E, 13H/16A/17D/31E/60T/79A/83T/96A/162G/174D/195R/210P/277Y/318E, 13H/16A/17D/26A/37K/60T/79A/96A/162G/174D/195R/210P/277Y/318E, 13H/26A/31E/37K/60T/79A/83T/96A/162G/174D/195R/210P/277Y/318E, 13H/16A/17D/26A/31E/37K/60T/96A/162G/174D/195R/210P/277Y/318E, 13H/16A/17D/26A/31E/37K/79A/83T/96A/162G/174D/195R/210P/318E, 13H/16A/17D/26A/31E/37K/60T/79A/96A/162G/174D/210P/277Y/318E, 13H/17D/26A/31E/37K/60T/79A/83T/96A/162G/174D/195R/210P/277Y/318E, 13H/16A/26A/31E/37K/60T/79A/83T/96A/162G/174D/195R/210P/277Y/318E, 16A/17D/26A/31E/37K/60T/79A/83T/96A/162G/174D/195R/210P/277Y/318E, or 13H/16A/17D/26A/31E/37K/60T/79A/83T/96A/162G/174D/195R/210P/277H/318E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/31/210/318, 31/210/212/318, 31/124/196/210/226/318, 5/31/210/318, 16/26/31/124/155/195/210/284/318, 31/196/210/226/318, 16/31/155/195/210/226/318, 16/31/195/210/226/318, 16/31/155/174/196/210/318, 31/210, 16/31/195/196/210/318, 16/26/31/124/155/174/196/210/318, 31/195/210/318, 26/31/155/174/210/318, 31/210/316/318, 31/60/210/318, 16/31/155/174/210/318, 16/31/124/174/196/210/318, 16/31/210/226/318, 16/26/31/174/196/210/226/318, 17/31/210/318, 31/210/318/331, 31/124/195/210/318, 16/31/174/196/210/318, 31/188/210/318, 31/195/196/210/318, 31/174/196/210/318, 16/26/31/155/174/210/318, 16/31/155/195/196/210/226/318, 31/195/210/226/284/318, 16/31/210/318, 31/210/240/318, 16/26/31/124/155/195/196/210/226/318, 31/183/210/232/318/339/343, 31/183/206/210/318, 31/63/210/318, 31/173/210/318/347, 16/26/31/174/196/210/318, 16/31/210/284/318, 31/46/210/318, 16/31/124/195/196/210/318, 31/210/274/318, 31/155/174/196/210/318, 31/174/196/210/226/318, 16/31/124/155/174/195/210/318, 16/26/31/155/174/196/210/318, 26/31/174/196/210/284/318, 16/31/195/196/210/284/318, 31/174/210/318, 16/31/124/174/195/210/226/284/318, 16/31/174/195/210/284/318, 26/31/174/196/210/226/284/318, 31/174/196/210/226/284/318, 31/124/174/196/210/318, 26/31/174/195/210/226/318, 31/162/210/318, 16/31/124/195/196/210/284/318, 31/206/210/318/343, 16/26/31/174/195/196/210/226/318, 31/155/195/196/210/226/318, 18/31/210/318, 31/210/318/335, 31/124/155/174/195/210/226/318, 26/31/155/174/195/210/226/318, 31/153/210/318, 31/155/174/195/210/318, 31/174/195/210/284/318, 31/174/195/210/318, 31/37/210/318, 31/124/174/195/210/226/284/318, 31/176/210/318, 16/31/155/174/195/196/210/226/284/318, 10/31/210/318, 31/169/173/210/318, 31/155/210/318, 31/33/210/318, 31/173/183/210/318/343/347, 31/104/210/265/318, or 31/210/233/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises a substitution or substitution set 13P/31K/210A/318Q, 31K/210A/212S/318Q, 31K/124A/196V/210E/226S/318Q, 5S/31K/210A/318Q, 13E/31K/210A/318Q, 16S/26E/31K/124A/155P/195L/210E/284A/318Q, 31K/196V/210A/226S/318Q, 16S/31K/155P/195L/210E/226S/318Q, 16S/31K/195L/210E/226S/318Q, 16S/31K/155P/174K/196V/210A/318Q, 16S/31K/195L/210A/226S/318Q, 13L/31K/210A/318Q, 31K/210A, 16S/31K/195L/196V/210E/318Q, 16S/26E/31K/124A/155P/174K/196V/210E/318Q, 31K/195L/210E/318Q, 26E/31K/155P/174K/210E/318Q, 31K/210A/316V/318Q, 31K/60V/210A/318Q, 16S/31K/155P/174K/210A/318Q, 16S/31K/124A/174K/196V/210A/318Q, 16S/31K/210A/226S/318Q, 16S/26E/31K/174K/196V/210A/226S/318Q, 17H/31K/210A/318Q, 31K/210A/318Q/331V, 31K/124A/195L/210A/318Q, 16S/31K/174K/196V/210A/318Q, 17A/31K/210A/318Q, 31K/188A/210A/318Q, 31K/195L/196V/210E/318Q, 31K/174K/196V/210E/318Q, 5C/31K/210A/318Q, 16S/26E/31K/155P/174K/210A/318Q, 16S/31K/155P/195L/196V/210A/226S/318Q, 31K/195L/210A/226S/284A/318Q, 17N/31K/210A/318Q, 16L/31K/210A/318Q, 31K/210A/240E/318Q, 16S/26E/31K/124A/155P/195L/196V/210A/226S/318Q, 31K/210A/318Q/331P, 31K/183I/210A/232T/318Q/339G/343R, 31K/183I/206M/210A/318Q, 31K/63A/210A/318Q, 31K/173V/210A/318Q/347R, 16S/26E/31K/174K/196V/210A/318Q, 16S/31K/210A/284A/318Q, 31K/60R/210A/318Q, 31K/46R/210A/318Q, 16S/31K/124A/195L/196V/210A/318Q, 31K/210A/274S/318Q, 31K/210A/318Q/331I, 31K/174K/196V/210A/318Q, 31K/155P/174K/196V/210A/318Q, 31K/174K/196V/210A/226S/318Q, 31K/210A/318Q/331L, 16S/31K/124A/155P/174K/195L/210A/318Q, 16S/26E/31K/155P/174K/196V/210A/318Q, 26E/31K/174K/196V/210E/284A/318Q, 31K/210A/212A/318Q, 16S/31K/195L/196V/210A/284A/318Q, 31K/174K/210A/318Q, 16S/31K/124A/174K/195L/210E/226S/284A/318Q, 16S/31K/174K/195L/210A/284A/318Q, 26E/31K/174K/196V/210E/226S/284A/318Q, 31K/210A/212L/318Q, 31K/174K/196V/210A/226S/284A/318Q, 31K/124A/174K/196V/210A/318Q, 26E/31K/174K/195L/210E/226S/318Q, 31K/162A/210A/318Q, 16S/31K/124A/195L/196V/210A/284A/318Q, 31K/206M/210A/318Q/343R, 16S/31K/195L/196V/210A/318Q, 16S/26E/31K/174K/195L/196V/210E/2265/318Q, 31K/155P/195L/196V/210A/226S/318Q, 18Q/31K/210A/318Q, 31K/210A/318Q/335R, 31K/124A/155P/174K/195L/210A/226S/318Q, 17C/31K/210A/318Q, 26E/31K/155P/174K/195L/210A/226S/318Q, 31K/195L/196V/210A/318Q, 31K/162T/210A/318Q, 31K/153H/210A/318Q, 31K/210A/316K/318Q, 31K/155P/174K/195L/210A/318Q, 31K/153Q/210A/318Q, 31K/174K/195L/210E/284A/318Q, 31K/174K/195L/210A/318Q, 31K/153S/210A/318Q, 31K/210A/212F/318Q, 31K/37C/210A/318Q, 31K/210A/212V/318Q, 31K/124A/174K/195L/210A/226S/284A/318Q, 31K/176L/210A/318Q, 16S/31K/155P/174K/195L/196V/210A/226S/284A/318Q, 10Q/31K/210A/318Q, 31K/169G/173V/210A/318Q, 31K/155L/210A/318Q, 18G/31K/210A/318Q, 31K/60L/210A/318Q, 10A/31K/210A/318Q, 18R/31K/210A/318Q, 31K/33R/210A/318Q, 31K/210A/212G/318Q, 10R/31K/210A/318Q, 13R/31K/210A/318Q, 31K/173V/183I/210A/318Q/343R/347R, 16R/31K/210A/318Q, 31K/104Q/210A/265C/318Q, 31K/210A/233R/318Q, or 31K/210A/233D/318Q, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/16/17/26/31/174/195/210/226/318, 16/17/26/31/174/195/196/210/212/226/318/331, 16/17/31/174/195/210/212/318, 16/17/31/174/195/210/212/318/331, 13/16/17/31/174/195/210/226/318/331, 16/17/26/31/60/174/195/210/318, 16/17/26/31/46/174/195/196/210/212/226/318, 16/31/60/174/195/210/318/331, 16/17/26/31/46/174/195/210/212/226/274/318/331, 16/26/31/174/195/196/210/212/226/318, 16/17/26/31/174/195/210/318, 13/16/17/31/174/195/196/210/212/226/318/331, 13/16/17/26/31/174/195/196/210/212/318/331, 16/26/31/174/195/210/212/226/318/331, 13/16/17/26/31/174/195/210/226/318/331, 13/16/17/31/174/195/196/210/212/318/331, 16/17/26/31/60/174/195/210/212/318/331, 13/16/17/26/31/174/195/196/210/212/226/318, 13/16/17/31/174/195/196/210/226/318/331, 13/16/17/26/31/174/195/196/210/226/318, 13/16/17/31/174/195/210/226/318, 16/26/31/46/174/195/210/212/318, 16/17/26/31/60/174/195/210/226/318, 16/17/26/31/174/195/210/226/318/331, 13/16/17/26/31/46/174/195/196/210/318/331, 16/17/26/31/174/195/210/318/331, 16/17/26/31/46/174/195/210/212/226/318/331, 16/17/26/31/46/174/195/196/210/212/226/318/331, 16/17/26/31/174/195/210/226/318, 13/16/17/26/31/174/195/196/210/212/318, 16/17/26/31/174/195/196/210/212/226/318, 16/17/31/174/195/196/210/212/226/318, 13/16/17/26/31/174/195/196/210/318/331, 16/17/26/31/174/195/210/212/226/318/331, 16/17/26/31/174/195/210/212/318/331, 13/16/17/31/174/195/196/210/318/331, 16/26/31/46/60/174/195/210/318/331, 16/17/26/31/46/174/195/210/226/318/331, 13/16/17/26/31/46/174/195/210/226/318, 16/26/31/174/195/210/212/226/318, 13/16/17/31/174/195/196/210/226/318, 16/17/26/31/83/174/195/196/210/226/263/318, 16/17/26/31/83/173/174/195/196/210/226/227/318, 16/17/26/31/174/195/196/210/226/227/301/318, 16/17/26/31/83/174/195/196/210/226/227/263/318, 16/17/26/31/83/125/174/195/196/210/226/318, 16/17/26/31/83/173/174/195/196/210/226/318, 16/17/26/31/174/195/196/210/226/263/301/318, 16/17/26/31/125/174/195/196/210/226/318, 16/17/26/31/83/174/195/196/210/226/301/318, 16/17/26/31/173/174/195/196/210/226/318, 16/17/26/31/83/174/195/196/210/226/227/318, 16/17/26/31/174/195/196/210/226/227/318, 16/17/26/31/174/195/196/210/226/318, 16/17/26/31/125/174/195/196/210/226/227/318, 16/17/26/31/83/174/195/196/210/226/318, 16/26/31/128/174/195/196/210/226/318, 16/26/31/76/174/195/196/210/226/318, 16/26/31/141/174/195/196/210/226/318, 16/26/31/110/174/195/196/210/226/318, 16/26/31/79/174/195/196/210/226/318, 16/26/31/117/174/195/196/210/226/318, or 16/26/31/61/174/195/196/210/226/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P/16S/17H/26E/31K/174K/195L/210E/226S/318Q, 16S/17H/26E/31K/174K/195L/196V/210E/212S/226S/318Q/331V, 16S/17H/31K/174K/195L/210E/212S/318Q, 16S/17H/31K/174K/195L/210E/212A/318Q/331V, 13P/16S/17H/31K/174K/195L/210E/226S/318Q/331V, 16S/17H/26E/31K/60R/174K/195L/210E/318Q, 16S/17H/26E/31K/46R/174K/195L/196V/210E/212S/226S/318Q, 16S/31K/60V/174K/195L/210E/318Q/331V, 16S/17H/26E/31K/46R/174K/195L/210E/212A/226S/274Q/318Q/331V, 16S/26E/31K/174K/195L/196V/210E/212S/226S/318Q, 16S/17H/26E/31K/174K/195L/210E/318Q, 13P/16S/17H/31K/174K/195L/196V/210E/212A/226S/318Q/331V, 13P/16S/17H/26E/31K/174K/195L/196V/210E/212S/318Q/331V, 16S/26E/31K/174K/195L/210E/212S/226S/318Q/331V, 13P/16S/17H/26E/31K/174K/195L/210E/226S/318Q/331V, 13P/16S/17H/31K/174K/195L/196V/210E/212A/318Q/331V, 16S/17H/26E/31K/60R/174K/195L/210E/212A/318Q/331V, 13P/16S/17H/26E/31K/174K/195L/196V/210E/212S/226S/318Q, 13P/16S/17H/31K/174K/195L/196V/210E/226S/318Q/331V, 13P/16S/17H/26E/31K/174K/195L/196V/210E/226S/318Q, 13P/16S/17H/31K/174K/195L/210E/226S/318Q, 16S/26E/31K/46R/174K/195L/210E/212S/318Q, 16S/17H/26E/31K/60R/174K/195L/210E/226S/318Q, 16S/17H/26E/31K/174K/195L/210E/226S/318Q/331V, 13P/16S/17H/26E/31K/46R/174K/195L/196V/210E/318Q/331V, 16S/17H/26E/31K/174K/195L/210E/318Q/331V, 16S/17H/26E/31K/60V/174K/195L/210E/318Q, 16S/17H/26E/31K/46R/174K/195L/210E/212S/226S/318Q/331V, 13P/16S/17H/26E/31K/174K/195L/196V/210E/212A/226S/318Q, 16S/17H/26E/31K/46R/174K/195L/196V/210E/212S/226S/318Q/331V, 16S/17H/26E/31K/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/196V/210E/212S/318Q, 16S/17H/26E/31K/174K/195L/196V/210E/212S/226S/318Q, 16S/17H/31K/174K/195L/196V/210E/212S/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/196V/210E/318Q/331V, 16S/17H/26E/31K/174K/195L/210E/212S/226S/318Q/331V, 16S/17H/26E/31K/174K/195L/210E/212S/318Q/331V, 13P/16S/17H/31K/174K/195L/196V/210E/318Q/331V, 16S/26E/31K/46R/60V/174K/195L/210E/318Q/331V, 16S/17H/26E/31K/46R/174K/195L/210E/226S/318Q/331V, 16S/17H/26E/31K/174K/195L/196V/210E/212A/226S/318Q/331V, 13P/16S/17H/26E/31K/46R/174K/195L/210E/226S/318Q, 16S/26E/31K/174K/195L/210E/212S/226S/318Q, 13P/16S/17H/31K/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/83E/174K/195L/196V/210E/226S/263S/318Q, 16S/17G/26E/31K/83E/173Q/174K/195L/196V/210E/226S/227E/318Q, 16S/17G/26E/31K/174K/195L/196V/210E/226S/227E/3011/318Q, 16S/17G/26E/31K/83E/174K/195L/196V/210E/226S/227E/263S/318Q, 16S/17G/26E/31K/83E/125E/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/83E/173Q/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/174K/195L/196V/210E/226S/263S/301I/318Q, 16S/17G/26E/31K/125E/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/83E/174K/195L/196V/210E/226S/301I/318Q, 16S/17G/26E/31K/173Q/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/83E/174K/195L/196V/210E/226S/227E/318Q, 16S/17G/26E/31K/174K/195L/196V/210E/226S/227E/318Q, 16S/17G/26E/31K/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/125E/174K/195L/196V/210E/226S/227E/318Q, 16S/17G/26E/31K/83E/174K/195L/196V/210E/226S/318Q, 16S/17G/26E/31K/174K/195L/196V/210E/226S/263S/301T/318Q, 16S/26E/31K/128A/174K/195L/196V/210E/226S/318Q, 16S/26E/31K/76S/174K/195L/196V/210E/226S/318Q, 16S/26E/31K/141Q/174K/195L/196V/210E/226S/318Q, 16S/26E/31K/110V/174K/195L/196V/210E/226S/318Q, 16S/26E/31K/79S/174K/195L/196V/210E/226S/318Q, 16S/26E/31K/117A/174K/195L/196V/210E/226S/318Q, 16S/26E/31K/79G/174K/195L/196V/210E/2265/318Q, or 16S/26E/31K/61M/174K/195L/196V/210E/226S/318Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q, 14V/16S/17G/26E/31K/60V/79G/174K/195L/210E/226S/318Q, 16S/17G/26E/31K/79G/83E/174K/195L/210E/226S/318Q, 16S/17G/26E/31K/79G/174K/195L/210E/226S/3011/318Q, 14V/16S/17G/26E/31K/60V/79G/174K/195L/210E/212S/226S/318Q, 13P/16S/17G/26E/31K/60V/174K/195L/197E/210E/226S/318Q, 16S/17H/26E/31K/60V/174K/195L/210E/212S/226S/318Q, 14V/16S/17G/26E/31K/60V/79G/83E/174K/195L/210E/226S/301I/318Q, 16S/17G/26E/31K/60V/79G/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/60V/174K/195L/210E/226S/301I/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/60V/83E/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q, 16S/17G/26E/31K/60V/79G/83E/174K/195L/210E/226S/301I/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/318Q/342P, 13P/16S/17H/26E/31K/52T/174K/190Q/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/94N/174K/190Q/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/190Q/195L/210E/226S/318Q/342P/351E, 16S/17H/26E/31K/79G/174K/195L/210E/226S/318Q, 16S/17H/26E/31K/96L/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/273A/318Q, 13P/16S/17H/26E/31K/126T/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/96K/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/79S/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/269S/318Q, 13P/16S/17H/26E/31K/40S/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/96L/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/44S/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/96R/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/79G/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/267R/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/266V/318Q, 13P/16S/17H/26E/31K/160T/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/277T/318Q, 13P/16S/17H/26E/31K/149T/174K/195L/210E/226S/318Q, 13P/16S/17H/22R/26E/31K/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/234L/318Q, 13P/16S/17H/26E/31K/54L/174K/195L/210E/226S/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/297Y/318Q, 13P/16S/17H/26E/31K/174K/195L/210E/226S/277A/318Q, 13P/16S/17H/26E/31K/106C/174K/195L/210E/226S/318Q, or 16S/17H/26E/31K/43C/174K/195L/210E/226S/318Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/125F/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/162K/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/94T/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195W/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/85R/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/103S/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/232T/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/189R/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/186N/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/155R/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/193A/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/342A, 13P/14V/16S/17G/26E/31K/79G/174K/195L/196V/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/63S/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/33R/79G/174K/195L/210E/226S/318Q), 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/351H, 13P/14V/16S/17G/26E/31K/79G/174K/193G/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/125S/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/314S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/187R/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/303T/318Q, 13P/14V/16S/17G/26E/31K/79G/103V/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/103Q/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/155V/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/164R/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/153S/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37R/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/346Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/346G, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/351A, 13P/14V/16S/17G/26E/31K/79G/174K/183K/195L/210E/226S/318Q, 13P/14V/16S/17G/19C/26E/31K/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/187L/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/123S/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/85G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/187S/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/183C/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/123G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/350R, 13P/14V/16S/17G/26E/31K/79G/85E/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/205A/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/284R/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/199V/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/346W, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/343W, 13P/14V/16S/17G/26E/31K/79G/164V/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/200N/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/318Q/342E, 13P/14V/16S/17G/26E/31K/79G/174K/195L/208G/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/169A/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/190G/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/210E/226S/284A/318Q, 13P/14V/16S/17G/26E/31K/79G/174K/195L/199G/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/79G/121W/174K/195L/210E/226S/318Q, or 13P/14V/16S/17G/26E/31K/79G/174K/195L/196S/210E/226S/318Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/16/17/31/37/60/79/174/195/210/226, 13/16/17/31/37/60/79/94/162/174/195/210/212/226/318, 13/14/16/17/26/31/37/60/79/162/174/195/210/318, 13/14/16/17/26/31/37/60/79/174/195/210/226/318, 13/16/17/26/31/37/79/125/162/174/195/210/212/226, 13/14/16/17/26/37/60/79/125/174/195/210/318, 13/16/17/26/3/1/7/79/125/162/174/195/210/318, 13/14/16/17/31/37/60/79/162/174/195/210, 13/16/17/31/37/79/94/162/174/195/210/212/318, 13/16/17/26/31/37/60/79/162/174/195/210/226/318, 13/16/17/26/31/37/60/79/162/174/195/210/318, 13/16/17/26/31/37/79/94/162/174/195/210/226, 13/16/17/26/31/37/79/94/162/174/195/210/226/318, 13/16/17/26/31/37/60/79/174/195/210/318, 13/16/17/26/31/37/60/79/94/174/195/210/212/226/318, 13/16/17/26/31/37/60/79/162/174/195/210/212/226/318, 13/16/17/26/31/37/79/162/174/195/210/226/318, 13/16/17/26/31/37/79/94/174/195/210/212/226, 13/16/17/26/31/37/60/79/174/195/210/226/318, 13/16/17/31/37/79/162/174/195/210/226/318, 13/16/17/26/31/37/79/174/195/210/226/318, 13/16/17/26/31/37/79/174/195/210/226, 13/16/17/26/31/37/79/162/174/195/210, 13/16/17/26/31/37/79/125/174/195/210/212/226/318, 13/16/17/26/31/37/79/125/174/195/210, 13/16/17/31/37/60/79/162/174/195/210/226/318, 13/16/17/26/31/37/79/125/162/174/195/210/212/318, 13/16/17/26/31/37/79/94/125/162/174/195/210/226, 13/16/17/26/31/37/79/125/174/195/210/318, 13/14/16/17/26/31/37/79/174/195/210/226/277/318, 13/14/16/17/26/31/37/79/166/174/195/210/226/318, 13/14/16/17/26/31/37/79/174/195/210/226/233/318, 13/14/16/17/26/31/37/79/83/174/195/210/226/318, 5/13/14/16/17/26/31/37/79/174/195/210/226/318, 13/14/16/17/26/31/37/79/174/195/210/226/318/342, 13/14/16/17/26/31/37/79/96/174/195/210/226/318, 13/14/16/17/26/31/37/79/174/195/210/226/284/318, or 13/14/16/17/26/31/37/79/174/195/210/226/318, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P/16S/17G/31K/37G/60V/79G/174K/195L/210E/226S, 13P/16S/17G/31K/37G/60V/79G/94T/162K/174K/195L/210E/212S/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/318Q, 13P/14V/16S/17G/26E/31K/37G/60V/79G/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/125F/162K/174K/195L/210E/212S/226S, 13P/14V/16S/17G/26E/31K/37G/60V/79G/125F/174K/195L/210E/318Q, 13P/16S/17G/26E/31K/37G/79G/125F/162K/174K/195L/210E/318Q, 13P/14V/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E, 13P/16S/17G/31K/37G/79G/94T/162K/174K/195L/210E/212S/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/318Q, 13P/16S/17G/26E/31K/37G/79G/94T/162K/174K/195L/210E/226S, 13P/16S/17G/26E/31K/37G/79G/94T/162K/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/174K/195L/210E/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/94T/174K/195L/210E/212S/226S/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/212S/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/162K/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/94T/174K/195L/210E/212S/226S, 13P/16S/17G/26E/31K/37G/60V/79G/174K/195L/210E/226S/318Q, 13P/16S/17G/31K/37G/79G/162K/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S, 13P/16S/17G/26E/31K/37G/79G/162K/174K/195L/210E, 13P/16S/17G/26E/31K/37G/79G/125F/174K/195L/210E/212S/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/125F/174K/195L/210E, 13P/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E/226S/318Q, 13P/16S/17G/26E/31K/37G/79G/125F/162K/174K/195L/210E/212S/318Q, 13P/16S/17G/26E/31K/37G/79G/94T/125F/162K/174K/195L/210E/226S, 13P/16S/17G/26E/31K/37G/79G/125F/174K/1951/210E/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/277R/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/277C/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/277T/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/166R/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/233H/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/83R/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/60M/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/277I/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/277M/318Q, 5S/13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/318Q/342R, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/2265/277V/318Q, 13P/14V/16S/17G/26E/31K/37G/60R/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/96R/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26E/31K/37G/79G/174K/195L/210E/226S/284S/318Q, 13P/14V/16S/17G/26R/31K/37G/79G/174K/195L/210E/226S/318Q, 13P/14V/16S/17G/26N/31K/37G/79G/174K/195L/210E/226S/318Q, or 13P/14V/16S/17G/26E/31K/37G/79G/83W/174K/195L/210E/2265/318Q, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 5/13/16/17/31/37/60/79/83/162/174/195/210/318/342, 5/13/16/17/31/37/60/79/162/174/195/210/277/318, 5/13/16/17/26/31/37/60/79/96/162/174/195/210/277/318, 5/13/16/17/26/31/37/60/79/83/162/174/195/210/277/318/342, 13/16/17/31/37/60/79/83/162/174/195/210/277/318, 5/13/16/17/31/37/60/79/83/162/174/195/210/277/318, 5/13/16/17/26/31/37/60/79/162/174/195/210/277/318/342, 5/13/16/17/31/37/60/79/162/166/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/162/174/195/210/277/318/342, 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318/342, 13/16/17/31/37/60/79/83/162/174/195/210/277/318/342, 5/13/16/17/31/37/60/79/83/162/174/195/210/277/318/342, 5/13/16/17/26/31/37/60/79/83/162/174/195/210/277/318, 13/16/17/31/37/60/79/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/162/174/195/210/277/318, 5/13/16/17/26/31/35/37/60/79/162/174/195/210/277/318, 5/13/16/17/31/37/60/79/83/96/162/174/195/210/277/318, 5/13/16/17/31/37/60/79/96/162/174/195/210/277/318, 5/13/16/17/26/31/37/60/79/162/174/195/210/277/318, 5/13/16/17/26/31/37/60/79/162/166/174/195/210/277/318, 13/16/17/31/37/60/79/83/96/162/174/195/210/277/318/342, 13/16/17/26/31/37/60/79/162/174/195/210/277/318, 13/16/17/31/37/60/79/162/174/195/210/277/318/342, 5/13/16/17/26/31/37/60/79/162/174/195/210/318, 5/13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318/342, or 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase comprises at least a substitution or substitution set 5S/13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/318Q/342R, 5S/13P/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/96L/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/83W/162K/174K/195L/210E/277T/318Q/342R, 13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/277T/318Q/342R, 5S/13P/16S/17G/26E/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q/342R, 5S/13P/16S/17G/31K/37G/60V/79G/162K/166R/174K/195L/210E/277T/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/162K/174K/195L/210E/277T/318Q/342R, 13P/16S/17G/26E/31K/37G/60V/79G/83R/96L/162K/174K/195L/210E/277V/318Q/342R, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277T/318Q, 13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q/342R, 5S/13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q/342R, 5S/13P/16S/17G/26E/31K/37G/60V/79G/83R/162K/174K/195L/210E/277T/318Q/342R, 5S/13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/277T/318Q/342R, 5S/13P/16S/17G/26E/31K/37G/60V/79G/83R/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E/277T/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/26E/31K/35V/37G/60V/79G/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/31K/37G/60V/79G/83R/96R/162K/174K/195L/210E/277V/318Q, 5S/13P/16S/17G/31K/37G/60V/79G/96L/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/31K/37G/60V/79G/83R/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/83R/162K/174K/195L/210E/277T/318Q, 13P/16S/17G/31K/37G/60V/79G/83R/162K/174K/195L/210E/277V/318Q, 5S/13P/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E/277V/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/162K/166R/174K/195L/210E/277T/318Q, 13P/16S/17G/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q/342R, 13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/277T/318Q, 13P/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E/277T/318Q/342R, 13P/16S/17G/26E/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q/342R, 13P/16S/17G/26E/31K/37G/60V/79G/83R/162K/174K/195L/210E/277V/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/318Q, 13P/16S/17G/31K/37G/60V/79G/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83R/162K/174K/195L/210E/277T/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/83W/96R/162K/174K/195L/210E/277T/318Q/342R, 5S/13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/277V/318Q, 5S/13P/16S/17G/26E/31K/37G/60V/79G/162K/174K/195L/210E/277V/318Q/342R, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, or 13P/16S/17G/26E/31K/37G/60V/79G/83W/162K/174K/195L/210E/277V/318Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/96/162/174/210/277/318, 13/16/17/3⅓7/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/96/162/195/210/277/318, 13/16/17/26/37/60/79/83/96/162/174/195/210/277/318, 16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/96/162/174/195/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/277/318, 13/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/83/96/162/174/195/210/277/318, 13/16/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277, 13/16/17/26/31/37/60/79/83/96/162/174/195/210/318, 13/16/17/26/31/60/79/83/96/162/174/195/210/277/318, or 13/16/17/26/31/37/60/79/83/96/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26L/31K/37G/60V/T9G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31W/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96K/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60R/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318D, 13P/16S/17G/26G/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210R/277V/318Q, 13P/16S/17G/26E/31R/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277F/318Q, 13P/16S/17G/26E/31K/37M/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96P/162K/174K/195L/210E/277V/318Q, 13P/16V/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60A/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26R/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31G/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195G/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318R, 13P/16S/17G/26V/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79M/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16M/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210A/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96R/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37E/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16G/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17W/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13G/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79E/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277T/318Q, 13P/16S/17G/26E/31K/37G/60Q/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31L/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96G/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96T/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60G/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16W/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13T/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79R/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26Y/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26S/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/210E/277V/318Q, 13P/16S/17R/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13V/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210F/277F/318Q, 13P/16S/17G/26E/31K/37L/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210G/277V/318Q, 13P/16S/17G/26E/31K/37G/60L/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13L/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83Y/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37W/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277G/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83R/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60Y/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210S/277V/318Q, 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13P/16S/17G/26E/31K/37G/60V/79G/83W/96S/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83V/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210I/277V/318Q, 13P/16D/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37C/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83G/96L/162K/174K/195L/210E/277V/318Q, 13N/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31V/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79S/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96Y/162K/174K/195L/210E/277V/318Q, 13P/16S/17F/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17M/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83P/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318L, 13P/16S/17G/26E/31K/37G/60V/79Q/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17S/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26W/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195D/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195W/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277S/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174E/195L/210E/277V/318Q, 13P/16E/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162F/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79Y/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17C/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17Y/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195Q/210E/277V/318Q, 13P/16S/17G/26P/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195I/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60F/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26I/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318K, 13P/16S/17G/26E/31K/37G/60N/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16I/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318H, 13P/16S/17G/26E/31K/37A/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195V/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210Y/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195E/210E/277V/318Q, 13P/16S/17G/26H/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13D/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31P/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210W/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318P, 13P/16S/17G/26E/31K/37G/60V/79G/83C/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174P/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210T/277V/318Q, 13P/16S/17T/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79T/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96H/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79W/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162L/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V, 13P/16S/17G/26E/31A/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210C/277V/318Q, 13P/16S/17N/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195K/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Y, 13P/16S/17H/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13M/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60D/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277K/318Q, 13P/16N/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79C/83W/96L/162K/174K/195L/210E/277V/318Q, 13K/165/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60K/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37N/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60I/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162D/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162I/174K/195L/210E/277V/318Q, 13P/16Y/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162S/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96C/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210K/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174I/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210L/277V/318Q, 13P/16S/17G/26D/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26N/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210D/277V/318Q, 13F/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318N, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174W/195L/210E/277V/318Q, 13P/16S/17G/26E/31H/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31D/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79N/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31I/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37H/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318C, 13P/16S/17G/26E/31F/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79P/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195P/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318I, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162A/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277Q/318Q, 13P/16S/17G/26F/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277C/318Q, 13P/16S/17G/26E/31K/37G/60V/79H/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31C/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79I/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37I/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17Q/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31N/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277N/318Q, 13P/16S/17G/26E/31K/37D/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277H/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277D/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96D/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174C/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162P/174K/195L/210F/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318F, 13P/16S/17G/26E/31K/37G/60V/79G/83N/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60C/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83I/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162M/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210H/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195H/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174Y/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37Y/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210Q/277V/318Q, 13P/16S/17G/26E/31K/37T/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96N/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83H/96L/162K/174K/195L/210E/277V/318Q, 13P/16H/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174F/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83D/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83Q/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162H/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162W/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162N/174K/195L/210E/277V/318Q, 13P/16F/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162Q/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162R/174K/195L/210E/277V/318Q, 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162K/174K/195L/210F/277V/318Q, or 13P/16S/17G/26E/31K/37G/60V/79G/83W/96L/162C/174K/195L/210E/277V/318Q, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/16/17/26/31/37/60/79/83/96/162/195/210/277, 13/16/17/31/37/60/79/83/162/174/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/277, 13/16/17/26/37/60/79/83/96/162/174/195/210/277, 13/16/17/26/31/37/60/79/83/96/195/210/277, 13/16/17/26/31/37/60/79/83/96/162/195/210, 13/16/17/31/37/60/79/83/96/162/174/210/277, 13/16/17/26/31/37/79/83/96/174/210/277/318, 13/16/17/26/31/37/79/83/96/174/195/210/277, 13/16/17/26/31/37/60/79/83/96/162/210/318, 13/26/31/37/60/79/83/96/162/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/210/318, 13/16/17/26/31/60/79/83/96/162/174/195/210, 13/17/26/31/37/60/79/83/96/174/210/318, 13/16/17/31/37/60/96/174/195/210/277/318, 13/16/17/37/79/83/96/162/174/195/210/277, 13/17/26/31/37/60/79/83/96/162/210/318, 16/17/26/31/60/79/96/174/195/210/277/318, 13/26/31/37/79/83/96/162/174/210/277/318, 16/26/31/60/79/96/162/174/195/210/277/318, 13/16/17/26/60/79/83/96/195/210/277, 17/26/37/60/79/96/174/195/210/277/318, 13/17/26/31/37/79/83/96/162/210/277, 13/16/26/31/37/79/83/174/210/318, 16/17/31/37/79/96/195/210/277/318, 13/16/26/37/79/83/96/195/210/318, 13/31/37/60/79/162/174/195/210/318, 13/16/31/60/79/83/96/162/195/210, 17/26/37/79/83/96/195/210/277/318, 16/26/37/60/79/96/162/174/195/210, 26/37/60/79/96/174/195/210/277, 17/26/31/37/79/96/174/210/277, 26/60/79/174/195/210/277/318, 16/31/79/96/174/195/277, 17/31/60/79/96/210/277, 17/37/60/79/195/210/277, 26/37/79/210/277, 17/31/79/96/277, 17/26/31/60/318, 16/195/277/318, 31/60/162/210/277, 16/17/79, 26/277, 26/37, 31/83, 16/17, 79/83, 60/277, 83/195, 16, 17, 13, or 277, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set of an engineered oxalate decarboxylase polypeptide set forth in any of Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to SEQ ID NO: 2 or 4.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, or a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 616-1622.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 770%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, it includes the proviso that the engineered oxalate decarboxylase polypeptide does not include the amino acid sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-1622, or to a reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 4, 5, 6, 7, 10, 11, 13, 14, 16, 17, 18, 19, 22, 26, 31, 33, 35, 37, 40, 43, 44, 46, 52, 54, 60, 61, 62, 63, 76, 79, 80, 82, 83, 85, 94, 96, 97, 103, 104, 106, 110, 117, 121, 123, 124, 125, 126, 128, 141, 149, 153, 155, 156, 160, 162, 164, 166, 169, 173, 174, 176, 180, 182, 183, 186, 187, 188, 189, 190, 193, 195, 196, 197, 199, 200, 205, 206, 208, 210, 212, 216, 219, 226, 227, 232, 233, 234, 240, 242, 243, 263, 265, 266, 267, 269, 270, 273, 274, 277, 284, 297, 301, 303, 304, 314, 316, 318, 331, 335, 339, 342, 343, 346, 347, 350, 351, 356, or 359, or combinations thereof, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or amino acid residue 4C/R/S, 5C/S, 6A/S/W, 7G, 10A/Q/R, 11R, 13A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 14L/V, 16A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 17A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 18G/Q/R, 19C, 22R, 26A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 31A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 33R, 35V, 37A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 40S, 43C, 44S, 46R, 52T, 54L, 60A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 61M, 62G, 63A/S, 76S, 79A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 80L, 82I, 83A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 85E/G/R, 94N/T, 96A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 97T, 103Q/S/V, 104Q, 106C, 110V, 117A, 121W, 123G/Q/S, 124A/C/G/Q/R/T, 125E/F/S, 126T, 128A, 141Q, 149T, 153H/Q/S, 155L/P/R/V, 156E, 160T, 162A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 164R/V, 166R, 169A/G/L, 173Q/V, 174A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 176L, 180E, 182R, 183C/E/I/K, 186N, 187L/R/S, 188A, 189R, 190G/Q, 193A/G, 195A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 196F/K/M/R/S/V, 197E, 199G/V, 200N, 205A, 206M, 208G, 210A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 212A/F/G/L/S/V, 216S/W, 219V, 226S/T, 227E/S, 232T, 233D/H/R, 234L, 240E, 242D, 243V, 263S, 265C, 266V, 267R, 269S, 270L, 273A, 274Q/S, 277A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 284A/R/S, 297Y, 3011/Q/T, 303T, 304D/G, 314S, 316K/V, 318A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 331I/L/P/V, 335R, 339G, 342A/E/P/R, 343R/W, 346G/L/Q/W, 347F/R, 350R, 351A/E/H, 356S, or 359L/W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or amino acid residue 13A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 14L/V, 16A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 17A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 26A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 31A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 37A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 60A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 79A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 83A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 96A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 162A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 174A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 195A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 196F/K/M/R/S/V, 210A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 226S/T, 277A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 301I/Q/T, or 318A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitutions at amino acid position 31, 210, or 318, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 31K, 210A/E, or 318Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or to the reference sequence corresponding to SEQ ID NO: 172, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13, 212, 124/196/210/226, 5, 16/26/124/155/195/210/284, 196/226, 16/155/195/210/226, 16/195/210/226, 16/155/174/196, 16/195/226, 318, 16/195/196/210, 16/26/124/155/174/196/210, 195/210, 26/155/174/210, 316, 60, 16/155/174, 16/124/174/196, 16/226, 16/26/174/196/226, 17, 331, 124/195, 16/174/196, 188, 195/196/210, 174/196/210, 16/26/155/174, 16/155/195/196/226, 195/226/284, 16, 240, 16/26/124/155/195/196/226, 183/232/339/343, 183/206, 63, 173/347, 16/26/174/196, 16/284, 46, 16/124/195/196, 274, 174/196, 155/174/196, 174/196/226, 16/124/155/174/195, 16/26/155/174/196, 26/174/196/210/284, 16/195/196/284, 174, 16/124/174/195/210/226/284, 16/174/195/284, 26/174/196/210/226/284, 174/196/226/284, 124/174/196, 26/174/195/210/226, 162, 16/124/195/196/284, 206/343, 16/195/196, 16/26/174/195/196/210/226, 155/195/196/226, 18, 335, 124/155/174/195/226, 26/155/174/195/226, 195/196, 153, 155/174/195, 174/195/210/284, 174/195, 37, 124/174/195/226/284, 176, 16/155/174/195/196/226/284, 10, 169/173, 155, 33, 173/183/343/347, 104/265, or 233, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set H13P, H212S, K124A/K196V/A210E/T226S, T5S, H13E, A16S/A26E/K124A/A155P/R195L/A210E/K284A, K196V/T226S, A16S/A155P/R195L/A210E/T226S, A16S/R195L/A210E/T226S, A16S/A155P/D174K/K196V, A16S/R195L/T226S, H13L, Q318E, A16S/R195L/K196V/A210E, A16S/A26E/K124A/A155P/D174K/K196V/A210E, R195L/A210E, A26E/A155P/D174K/A210E, N316V, T60V, A16S/A155P/D174K, A16S/K124A/D174K/K196V, A16S/T226S, A16S/A26E/D174K/K196V/T226S, D17H, A331V, K124A/R195L, A16S/D174K/K196V, D17A, P188A, R195L/K196V/A210E, D174K/K196V/A210E, T5C, A16S/A26E/A155P/D174K, A16S/A155P/R195L/K196V/T226S, R195L/T226S/K284A, D17N, A16L, D240E, A16S/A26E/K124A/A155P/R195L/K196V/T226S, A331P, V183I/K232T/S339G/S343R, V183I/L206M, P63A, K173V/K347R, A16S/A26E/D174K/K196V, A16S/K284A, T60R, V46R, A16S/K124A/R195L/K196V, H274S, A331I, D174K/K196V, A155P/D174K/K196V, D174K/K196V/T226S, A331L, A16S/K124A/A155P/D174K/R195L, A16S/A26E/A155P/D174K/K196V, A26E/D174K/K196V/A210E/K284A, H212A, A16S/R195L/K196V/K284A, D174K, A16S/K124A/D174K/R195L/A210E/T226S/K284A, A16S/D174K/R195L/K284A, A26E/D174K/K196V/A210E/T226S/K284A, H212L, D174K/K196V/T226S/K284A, K124A/D174K/K196V, A26E/D174K/R195L/A210E/T226S, G162A, A16S/K124A/R195L/K196V/K284A, L206M/S343R, A16S/R195L/K196V, A16S/A26E/D174K/R195L/K196V/A210E/T226S, A155P/R195L/K196V/T226S, K18Q, A335R, K124A/A155P/D174K/R195L/T226S, D17C, A26E/A155P/D174K/R195L/T226S, R195L/K196V, G162T, P153H, N316K, A155P/D174K/R195L, P153Q, D174K/R195L/A210E/K284A, D174K/R195L, P153S, H212F, K37C, H212V, K124A/D174K/R195L/T226S/K284A, V176L, A16S/A155P/D174K/R195L/K196V/T226S/K284A, V10Q, D169G/K173V, A155L, K18G, T60L, V10A, K18R, K33R, H212G, V10R, H13R, K173V/V183I/S343R/K347R, A16R, P104Q/N265C, T233R, or T233D, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or to the reference sequence corresponding to SEQ ID NO: 320, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/196, 17/212/331, 17/26/196/212/226, 17/26/196/212/226/331, 13/17/26/196/331, 17/60/196/226, 17/46/212, 26/60/196/226/331, 17/46/196/212/274/331, 212, 17/196/226, 13/17/26/212/331, 13/17/212/226/331, 196/212/331, 13/17/196/331, 13/17/26/212/226/331, 17/60/196/212/226/331, 13/17/212, 13/17/26/331, 13/17, 13/17/26/196, 46/196/212/226, 17/60/196, 17/196/331, 13/17/46/226/331, 17/196/226/331, 17/46/196/212/331, 17/46/212/331, 17/196, 13/17/212/226, 17/212, 17/26/212, 13/17/226/331, 17/196/212/331, 17/196/212/226/331, 13/17/26/226/331, 46/60/196/226/331, 17/46/196/331, 13/17/46/196, 196/212, 13/17/26, 17/83/263, 17/83/173/227, 17/227/301, 17/83/227/263, 17/83/125, 17/83/173, 17/263/301, 17/125, 17/83/301, 17/173, 17/83/227, 17/227, 17, 17/125/227, 17/83, 128, 76, 141, 110, 79, 117, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set H13P/D17H/V196K, D17H/H212S/A331V, D17H/E26A/V196K/H212S/S226T, D17H/E26A/V196K/H212A/S226T/A331V, H13P/D17H/E26A/V196K/A331V, D17H/T60R/V196K/S226T, D17H/V46R/H212S, E26A/T60V/V196K/S226T/A331V, D17H/V46R/V196K/H212A/H274Q/A331V, H212S, D17H/V196K/S226T, H13P/D17H/E26A/H212A/A331V, H13P/D17H/H212S/S226T/A331V, V196K/H212S/A331V, H13P/D17H/V196K/A331V, H13P/D17H/E26A/H212A/S226T/A331V, D17H/T60R/V196K/H212A/S226T/A331V, H13P/D17H/H212S, H13P/D17H/E26A/A331V, H13P/D17H, H13P/D17H/E26A/V196K, V46R/V196K/H212S/S226T, D17H/T60R/V196K, D17H/V196K/A331V, H13P/D17H/V46R/S226T/A331V, D17H/V196K/S226T/A331V, D17H/T60V/V196K/S226T, D17H/V46R/V196K/H212S/A331V, H13P/D17H/H212A, D17H/V46R/H212S/A331V, D17H/V196K, H13P/D17H/H212S/S226T, D17H/H212S, D17H/E26A/H212S, H13P/D17H/S226T/A331V, D17H/V196K/H212S/A331V, D17H/V196K/H212S/S226T/A331V, H13P/D17H/E26A/S226T/A331V, V46R/T60V/V196K/S226T/A331V, D17H/V46R/V196K/A331V, D17H/H212A/A331V, H13P/D17H/V46R/V196K, V196K/H212S, H13P/D17H/E26A, D17G/T83E/D263S, D17G/T83E/K173Q/R227E, D17G/R227E/V301I, D17G/T83E/R227E/D263S, D17G/T83E/P125E, D17G/T83E/K173Q, D17G/D263S/V301I, D17G/P125E, D17G/T83E/V301I, D17G/K173Q, D17G/T83E/R227E, D17G/R227E, D17G, D17G/P125E/R227E, D17G/T83E, D17G/D263S/V301T, F128A, A76S, G141Q, F110V, A79S, M117A, A79G, or L61M, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or to the reference sequence corresponding to SEQ ID NO:396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 13/17/79, 13/14/17/60/79, 13/17/79/83, 13/17/79/301, 13/14/17/60/79/212, 17/60/197, 13/60/212, 13/14/17/60/79/83/301, 13/17/60/79, 17/60/301, 14/17/79, 17/60/83, 17/79, 13/17/60/79/83/301, 342, 52/190, 94/190, 190/342/351, 13/79, 13/96, 273, 126, 96, 79, 269, 40, 44, 267, 266, 160, 277, 149, 22, 234, 54, 297, 106, or 13/43, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set P13H/H17G/A79G, P13H/L14V/H17G/T60V/A79G, P13H/H17G/A79G/T83E, P13H/H17G/A79G/V301I, P13H/L14V/H17G/T60V/A79G/H212S, H17G/T60V/L197E, P13H/T60V/H212S, P13H/L14V/H17G/T60V/A79G/T83E/V301I, P13H/H17G/T60V/A79G, H17G/T60V/V301I, L14V/H17G/A79G, H17G/T60V/T83E, H17G/A79G, P13H/H17G/T60V/A79G/T83E/V301I, T342P, K52T/A190Q, Q94N/A190Q, A190Q/T342P/Q351E, P13H/A79G, P13H/A96L, S273A, A126T, A96K, A79S, T269S, G40S, A96L, T44S, A96R, A79G, S267R, I266V, N160T, Y277T, I149T, T22R, V234L, I54L, S297Y, Y277A, D106C, or P13H/A43C, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 670, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 670, or the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at amino acid position 37, 125, 162, 94, 195, 85, 103, 232, 189, 186, 155, 193, 342, 196, 63, 33, 351, 314, 187, 303, 164, 153, 346, 183, 19, 123, 350, 205, 284, 199, 343, 200, 208, 169, 190, or 121, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding SEQ ID NO: 670.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution 37G, 125F, 162K, 94T, 195W, 85R, 103S, 232T, 189R, 186N, 155R, 193A, 342A, 196V, 63S, 33R, 351H, 193G, 125S, 314S, 187R, 303T, 103V, 103Q, 155V, 164R, 153S, 37R, 346Q, 346G, 351A, 183K, 19C, 187L, 123S, 85G, 187S, 183C, 123G, 350R, 85E, 205A, 284R, 199V, 346W, 343W, 164V, 200N, 342E, 208G, 169A, 190G, 284A, 199G, 121W, or 196S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises a substitution K37G, P125F, G162K, Q94T, L195W, H85R, G103S, K232T, A189R, A186N, A155R, Q193A, T342A, K196V, P63S, K33R, Q351H, Q193G, P125S, T314S, T187R, D303T, G103V, G103Q, A155V, P164R, P153S, K37R, E346Q, E346G, Q351A, V183K, D19C, T187L, D123S, H85G, T187S, V183C, D123G, K350R, H85E, E205A, K284R, P199V, E346W, S343W, P164V, L200N, T342E, E208G, D169A, A190G, K284A, P199G, L121W, or K196S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 14/26/60/318, 14/26/60/94/162/212, 60/162/226, 60, 14/125/162/212/318, 60/125/226, 14/125/162/226, 26/60/162/226/318, 14/26/94/162/212/226, 14/60/162, 14/60/162/226, 14/94/162/318, 14/94/162, 14/60/226, 14/60/94/212, 14/60/162/212, 14/162, 14/94/212/318, 14/60, 14/26/162, 14, 14/318, 14/162/226/318, 14/125/212, 14/125/226/318, 14/26/60/162, 14/125/162/212/226, 14/94/125/162/318, 14/125/226, 277, 166, 233, 83, 5, 342, 96, 284, or 26, wherein the amino acid positions are relative to the reference sequence corresponding SEQ ID NO: 616.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set V14L/E26A/T60V/Q318E, V14L/E26A/T60V/Q94T/G162K/H212S, T60V/G162K/S226T, T60V, V14L/P125F/G162K/H212S/Q318E, T60V/P125F/S226T, V14L/P125F/G162K/S226T, E26A/T60V/G162K/S226T/Q318E, V14L/E26A/Q94T/G162K/H212S/S226T, V14L/T60V/G162K, V14L/T60V/G162K/S226T, V14L/Q94T/G162K/Q318E, V14L/Q94T/G162K, V14L/T60V/S226T, V14L/T60V/Q94T/H212S, V14L/T60V/G162K/H212S, V14L/G162K, V14L/Q94T/H212S/Q318E, V14L/T60V, V14L/E26A/G162K, V14L, V14L/Q318E, V14L/G162K/S226T/Q318E, V14L/P125F/H212S, V14L/P125F/S226T/Q318E, V14L/E26A/T60V/G162K, V14L/P125F/G162K/H212S/S226T, V14L/Q94T/P125F/G162K/Q318E, V14L/P125F/S226T, Y277R, Y277C, Y277T, S166R, T233H, T83R, T60M, Y277I, Y277M, T5S, T342R, Y277V, T60R, A96R, K284S, E26R, E26N, or T83W, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 616.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 5/26/83/342, 5/26/277, 5/96/277, 5/83/277/342, 26/83/277, 5/26/83/277, 5/277/342, 5/26/166/277, 83/277/342, 83/96/277/342, 83/96/277, 26/83/277/342, 5/26/83/277/342, 5/83/277, 26/277, 83/277, 5/35/277, 5/26/83/96/277, 5/26/96/277, 5/277, 5/166/277, 26/83/96/277/342, 277, 26/277/342, 5, or 5/83/96/277/342, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 750.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set 5S/26A/83W/342R, 5S/26A/277T, 5S/96L/277T, 5S/83W/277T/342R, 26A/83W/277T, 5S/26A/83W/277V, 5S/277T/342R, 5S/83W/277V/342R, 5S/26A/166R/277T, 83W/277T/342R, 83R/96L/277V/342R, 83W/96L/277T, 26A/83W/277V/342R, 5S/26A/83W/277V/342R, 5S/83R/277T/342R, 5S/26A/83W/277T/342R, 5S/83R/277V, 26A/277T, 83W/277T, 5S/35V/277T, 5S/26A/83R/96R/277V, 5S/26A/96L/277T, 5S/277T, 5S/26A/83R/277T, 5S/83R/277T, 26A/83R/277V, 5S/26A/277V, 5S/166R/277T, 26A/83W/277V, 26A/83W/96L/277V/342R, 277T, 26A/277T/342R, 83W/277V/342R, 83R/277V, 5S, 26A/277V, 83R/277T, 5S/83W/96R/277T/342R, 5S/277V, 277V, 5S/277V/342R, 83W/96L/277V, or 83W/277V, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 750.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set T5S/E26A/T83W/T342R, T5S/E26A/Y277T, T5S/A96L/Y277T, T5S/T83W/Y277T/T342R, E26A/T83W/Y277T, T5S/E26A/T83W/Y277V, T5S/Y277T/T342R, T5S/T83W/Y277V/T342R, T5S/E26A/S166R/Y277T, T83W/Y277T/T342R, T83R/A96L/Y277V/T342R, T83W/A96L/Y277T, E26A/T83W/Y277V/T342R, T5S/E26A/T83W/Y277V/T342R, T5S/T83R/Y277T/T342R, T5S/E26A/T83W/Y277T/T342R, T5S/T83R/Y277V, E26A/Y277T, T83W/Y277T, T5S/I35V/Y277T, T5S/E26A/T83R/A96R/Y277V, T5S/E26A/A96L/Y277T, T5S/Y277T, T5S/E26A/T83R/Y277T, T5S/T83R/Y277T, E26A/T83R/Y277V, T5S/E26A/Y277V, T5S/S166R/Y277T, E26A/T83W/Y277V, E26A/T83W/A96L/Y277V/T342R, Y277T, E26A/Y277T/T342R, T83W/Y277V/T342R, T83R/Y277V, T5S, E26A/Y277V, T83R/Y277T, T5S/T83W/A96R/Y277T/T342R, T5S/Y277V, Y277V, T5S/Y277V/T342R, T83W/A96L/Y277V, or T83W/Y277V, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 750.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution E26L, K31W, L96K, V60R, Q318D, E26G, E210R, K31R, V277F, G37M, L96P, S16V, V60A, E26R, K31G, L195G, Q318R, E26V, G79M, S16M, E210A, L96R, G37E, S16G, G17W, P13G, G79E, V277T, V60Q, K31L, L96G, L96T, V60G, S16W, P13T, G79R, E26Y, E26S, L195R, G17R, P13V, V277E, G37L, E210G, V60L, P13L, W83Y, G37W, V277G, W83R, V60Y, E210S, Q318S, G17I, G37S, G79L, S16R, L195S, Q318G, E26C, P13R, L195Y, V60E, K162V, S16C, L96V, W83M, K31M, K174V, V277L, E26M, Q318M, E210N, E26A, V277R, L96F, K174G, S16Q, V277A, P13Q, G79F, S16L, L195N, G79V, G17P, V60P, G37P, L96W, K162T, L195M, Q318W, G37R, S16T, E210M, K162Y, S16P, W83E, V277M, K174L, G17V, G17E, G17L, L96M, Q318V, K174R, P13S, K174T, P13Y, L195A, V277W, V60M, G17A, Q318A, K174H, W83S, V277I, P13E, G17K, E26K, L96A, K174D, E210V, K31S, L195C, G79K, V60W, K174S, P13I, V60H, K31E, L96E, W83K, K31Q, S16K, K174M, G37V, G79D, Q318T, L195F, E26T, K174Q, W83L, L96I, P13C, P13A, L96Q, P13H, W83T, K31Y, P13W, V277P, L195T, V60S, E26Q, E210P, K174N, W83A, K162E, G37F, G37Q, K31T, K174A, W83F, V60T, L96S, W83V, E210I, S16D, G37C, W83G, P13N, K31V, G79S, L96Y, G17F, G17M, S16A, W83P, Q318L, G79Q, G17S, E26W, L195D, L195W, V277S, K174E, S16E, K162F, G79Y, G79A, G17C, G17Y, L195Q, E26P, L195I, V60F, E26I, G17D, Q318K, V60N, S16I, Q318H, G37A, L195V, E210Y, L195E, E26H, P13D, K31P, E210W, Q318P, W83C, K174P, E210T, G17T, G79T, L96H, G79W, K162L, Q318E, K31A, E210C, G17N, L195K, Q318Y, G17H, P13M, V60D, V277K, S16N, G79C, P13K, V60K, G37N, V60I, K162D, K162I, S16Y, K162S, L96C, E210K, K174I, E210L, E26D, E26N, E210D, P13F, Q318N, V277Y, K174W, K31H, K31D, G79N, G37K, K31I, G37H, Q318C, K31F, G79P, L195P, Q318I, K162A, V277Q, E26F, V277C, G79H, K31C, G79I, G37I, G17Q, K31N, V277N, G37D, V277H, V277D, L96D, K174C, K162P, Q318F, W83N, V60C, W83I, K162M, E210H, L195H, K174Y, G37Y, E210Q, G37T, L96N, W83H, S16H, K174F, W83D, W83Q, K162H, K162W, K162N, S16F, K162Q, K162G, K162R, E210F, or K162C, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) 174/318, 26/96, 60/318, 31/318, 162/174/318, 174/277/318, 26/195/318, 60/162/195, 60/162/318, 174/195/277, 16/17/174, 60/195/277, 37/277/318, 16/162/195/277, 26/79/83/162, 26/31/60/318, 16/174/195/277, 13/37/83/162, 16/17/60/195, 13/17/37/83, 31/37/162/174/318, 13/16/31/83/162, 16/60/174/195/318, 17/60/96/162/195/277, 13/26/60/83/162/174, 17/31/60/162/174/277, 16/17/26/83/96/277, 17/26/37/174/277/318, 13/16/31/60/162/174, 13/17/31/83/277/318, 13/16/17/31/83/162/318, 13/16/60/83/162/195/318, 13/16/17/31/37/83/96/162, 13/17/26/37/60/83/162/210/318, 13/16/26/31/83/96/162/174/318, 13/16/17/31/60/83/96/162/174/195/318, 13/16/26/37/60/83/162/174/195/210/318, 13/16/37/79/83/96/162/174/195/210/277, 13/17/26/31/37/60/79/83/96/162/174/210, 13/16/17/26/37/79/83/96/174/195/210/318, 13/26/31/37/60/83/96/162/174/195/210/277/318, 13/16/17/31/37/60/79/83/96/162/174/195/210/318, 13/16/17/31/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/37/60/79/96/162/174/195/210/277/318, 13/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/31/37/60/96/162/174/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/318, 13/16/17/26/31/37/60/79/96/162/174/210/277/318, 13/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/26/31/37/60/79/83/96/162/174/195/210/277/318, 16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, or 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set K174D/Q318E, E26A/L96A, V60T/Q318E, K31E/Q318E, K162G/K174D/Q318E, K174D/V277Y/Q318E, E26A/L195R/Q318E, V60T/K162G/L195R, V60T/K162G/Q318E, K174D/L195R/V277Y, S16A/G17D/K174D, V60T/L195R/V277Y, G37K/V277Y/Q318E, S16A/K162G/L195R/V277Y, E26A/G79A/W83T/K162G, E26A/K31E/V60T/Q318E, S16A/K174D/L195R/V277Y, P13H/G37K/W83T/K162G, S16A/G17D/V60T/L195R, P13H/G17D/G37K/W83T, K31E/G37K/K162G/K174D/Q318E, P13H/S16A/K31E/W83T/K162G, S16A/V60T/K174D/L195R/Q318E, G17D/V60T/L96A/K162G/L195R/V277Y, P13H/E26A/V60T/W83T/K162G/K174D, G17D/K31E/V60T/K162G/K174D/V277Y, S16A/G17D/E26A/W83T/L96A/V277Y, G17D/E26A/G37K/K174D/V277Y/Q318E, P13H/S16A/K31E/V60T/K162G/K174D, P13H/G17D/K31E/W83T/V277Y/Q318E, P13H/S16A/G17D/K31E/W83T/K162G/Q318E, P13H/S16A/V60T/W83T/K162G/L195R/Q318E, P13H/S16A/G17D/K31E/G37K/W83T/L96A/K162G, P13H/G17D/E26A/G37K/V60T/W83T/K162G/E210P/Q318E, P13H/S16A/E26A/G37K/W83T/K162G/K174D/L195R/Q318E, P13H/S16A/E26A/K31E/W83T/L96A/K162G/K174D/Q318E, P13H/S16A/G17D/K31E/V60T/W83T/L96A/K162G/K174D/L195R/Q318E, P13H/S16A/E26A/G37K/V60T/W83T/K162G/K174D/L195R/E210P/Q318E, P13H/S16A/G37K/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y, P13H/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/E210P, P13H/S16A/G17D/E26A/G37K/G79A/W83T/L96A/K174D/L195R/E210D/Q318E, P13H/E26A/K31E/G37K/V60T/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E, P13H/S16A/G17D/K31E/G37K/V60T/G7T9A/W83T/L96A/K162G/K174D/L195R/E210P/Q318E, P13H/S16A/G17D/K31E/V60T/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E, P13H/S16A/G17D/E26A/G37K/V60T/G79A/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E, P13H/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E, P13H/S16A/G17D/E26A/K31E/G37K/V60T/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E, P13H/S16A/G17D/E26A/K31E/G37K/G79A/W83T/L96A/K162G/K174D/L195R/E210P/Q318E, P13H/S16A/G17D/E26A/K31E/G37K/V60T/G79A/L96A/K162G/K174D/E210P/V277Y/Q318E, P13H/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q3 18E, P13H/S16A/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q3 18E, S16A/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q3 18E, or P13H/S16A/G17D/E26A/K31E/G37K/V60T/G79AW83T/L96A/K162G/K174D/L195R/E210P/V27 7H/Q318E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution at an amino acid position set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution set forth in any of Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises the amino acid sequence of an engineered oxalate decarboxylase polypeptide set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, or corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, optionally wherein the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, insertions and/or deletions. In some embodiments, optionally the amino acid sequence of the engineered oxalate decarboxylase polypeptide includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, and/or deletions.

In some embodiments of the foregoing, optionally the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, or 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 28-174. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 176-394. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 396-530. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 532-614. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 616-728. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 730-822. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 824-908. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 910-1516. In some embodiments, the amino acid sequence of the engineered oxalate decarboxylase polypeptide comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 1518-1622.

In some embodiments, the engineered oxalate decarboxylase has oxalate decarboxylase activity. In some embodiments, the engineered oxalate decarboxylase has oxalate decarboxylase activity and exhibits one or more improved enzyme properties compared to a reference oxalate decarboxylase having a sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the engineered oxalate decarboxylase polypeptide has increased enzyme activity compared to the reference oxalate decarboxylase. In some embodiments, the engineered oxalate decarboxylase polypeptide has increased activity at about pH 6 or less, about pH 5.5 or less, about pH 4.5 or less, about pH 3 or less, about pH 2.6 or less, or about pH 2.4 or less, to about pH 2 compared to the reference oxalate decarboxylase.

In some embodiments, the engineered oxalate decarboxylase polypeptide has increased stability at about pH 6 or less, about pH 5.5 or less, about pH 4.5 or less, about pH 3 or less, about pH 2.6 or less, or about pH 2.4 or less, to about pH 2 compared to the reference oxalate decarboxylase polypeptide.

In some embodiments, the engineered oxalate decarboxylase polypeptide has increased thermostability compared to the reference oxalate decarboxylase.

In some embodiments, the engineered oxalate decarboxylase polypeptide has increased resistance to proteolysis at pH of about 2.2 to 3.2 relative to the reference oxalate decarboxylase polypeptide. In some embodiments, the engineered oxalate decarboxylase polypeptide described herein exhibits increased resistance to proteolysis at pH of about 2.2.

In some embodiments, the engineered oxalate decarboxylase polypeptide described herein exhibits increased resistance to proteolysis by digestive enzymes, such as pepsin or chymotrypsin, particularly pepsin, including at the pHs described in the foregoing, compared to the reference oxalate decarboxylase.

In some embodiments, the improved enzyme property of the engineered oxalate decarboxylase polypeptide is selected from (a) increased activity on oxalate, (b) increased thermal stability, (c) increased stability at acidic pH, (d) increased activity at acidic pH, (e) increased activity at neutral pH, (f) increased expression, (g) increased solubility, and (h) increased resistance to proteolysis, or any combination of (a), (b), (c), (d), (e), (f), (g) and (h), compared to a reference oxalate decarboxylase having a sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906.

In some embodiments, the improved property of the engineered oxalate decarboxylase polypeptide is relative to the reference oxalate decarboxylase polypeptide corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference amino acid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or to a reference amino acid sequence corresponding to residues 1-424 of SEQ ID NO: 6, wherein the amino acid sequence comprises one or more substitutions in its amino acid sequence.

In some embodiments, the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid comprising residues 1-424 of SEQ ID NO: 6. In some embodiments, the foregoing engineered oxalate decarboxylase are provided as compositions, particularly pharmaceutical compositions, as further described herein.

In some embodiments, the present disclosure provides fusion proteins comprising the engineered oxalate decarboxylase polypeptides described herein. In some embodiments, the engineered oxalate decarboxylase polypeptide can be fused to a variety of amino acid sequences, such as polypeptide tags that can be used for detection and/or purification. In some embodiments, the fusion protein of the engineered oxalate decarboxylase polypeptides comprises a glycine-histidine or histidine-tag (His-tag), such as the exemplary engineered oxalate decarboxylase polypeptides with the His-tag sequences disclosed herein. In some embodiments, the fusion protein of the engineered oxalate decarboxylase polypeptides comprises an epitope tag, such as c-myc or hemagglutinin (HA). In some embodiments, the fusion is to the amino (N-) terminus of an engineered oxalate decarboxylase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of an engineered oxalate decarboxylase polypeptide. In some embodiments, when an engineered oxalate decarboxylase polypeptide amino acid sequence herein is presented as a fusion protein, it is also to be understood that the present disclosure also encompasses the engineered oxalate decarboxylase polypeptide without the fusion polypeptide.

It is also known in the art that protein synthesis is initiated with N-terminal methionine in eukaryotes or formylmethionine in prokaryotes and mitochondria. In eukaryotes, the initiating methionine is removed by cleavage of an N-terminal signal peptide present in secreted proteins or by the action of a methionine amino peptidase (MAP). In prokaryotes, the formylmethionine can be removed by formylmethionine deformylase and the resulting methionine removed by a methionine amino peptidase. Accordingly, it is to be understood that for each and every embodiment of an engineered oxalate decarboxylase polypeptide described herein that contains an N-terminal initiating methionine or formylmethionine, the present disclosure also provides engineered oxalate decarboxylase polypeptide lacking the initiating methionine or formylmethionine. By way of example and not limitation, when the present disclosure provides an engineered oxalate decarboxylase polypeptide as an amino acid sequence comprising residues 1-359 in reference to a specified sequence, where amino acid position 1 is an initiating methionine or formylmethionine, in some embodiments, the present disclosure also provides an engineered oxalate decarboxylase polypeptide comprising residues 2 to 359 in reference to the specified sequence, which lacks the initiating methionine or formylmethionine. Similarly, when the present disclosure provides an engineered oxalate decarboxylase polypeptide comprising a sequence corresponding to a specific SEQ ID NO., in some embodiments, the present disclosure also provides an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence corresponding to the specific SEQ ID NO. lacking the N-terminal initiating methionine or formylmethionine at amino acid position 1.

In some embodiments, the present disclosure provides functional or biologically active fragments of the engineered oxalate decarboxylase polypeptides described herein. In some embodiments, functional or biologically active fragments comprise at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the engineered oxalate decarboxylase polypeptide from which it was derived (i.e., the parent engineered oxalate decarboxylase). In some embodiments, functional or biologically active fragments comprise at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the parent sequence of the engineered oxalate decarboxylase polypeptide. In some embodiments the functional or biologically active fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, or less than 50 amino acids.

In some embodiments, the functional or biologically fragments comprise at least about 95%, 96%, 97%, 98%, or 99% of the activity of the engineered oxalate decarboxylase polypeptide from which it was derived (i.e., the parent engineered oxalate decarboxylase).

In some embodiments, the functional or biologically active fragment of the engineered oxalate decarboxylase polypeptide described herein includes at least a mutation or mutation set in the amino acid sequence of the engineered oxalate decarboxylase polypeptide described herein. Accordingly, in some embodiments, the functional or biologically active fragments of the engineered oxalate decarboxylase polypeptide displays the enhanced or improved property associated with the mutation or mutation set in the parent engineered oxalate decarboxylase polypeptide. In some embodiments, the functional or biologically active fragments are characterized by an improved property relative to the reference engineered oxalate polypeptide having a sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906. In some embodiments, the functional or biologically active fragments exhibits one or more improved enzyme property of increased activity on oxalate, increased thermal stability, increased stability at acidic pH, increased activity at acidic pH, and increased resistance to proteolysis, relative to the reference oxalate decarboxylase polypeptide having a sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906. In some embodiments, the improved property of the functional or biologically active fragment is relative to the reference engineered oxalate carboxylase of SEQ ID NO: 2 or 4.

Polynucleotides Encoding Engineered Polypeptides. Expression Vectors and Host Cells

In another aspect, the present disclosure provides recombinant polynucleotides encoding the engineered oxalate decarboxylase polypeptides described herein. In some embodiments, the recombinant polynucleotide is operatively linked to one or more heterologous regulatory sequences that control gene expression to create a polynucleotide, e.g., an expression vector, capable of expressing the polypeptide. In some embodiments, expression constructs containing at least one heterologous polynucleotide encoding the engineered oxalate decarboxylase polypeptide(s) is introduced into appropriate host cells to express the encoded engineered oxalate decarboxylase polypeptide(s).

As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode an engineered oxalate decarboxylase polypeptide. Thus, the present invention provides methods and compositions for the production of each and every possible variation of recombinant polynucleotides that encode the engineered oxalate decarboxylase polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and/or 5-2).

In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria, preferred codons used in fungi are typically used for expression in fungi, and preferred codons used in mammals are used for expression in mammals and mammalian cells. In some embodiments, codon optimized polynucleotides encoding the engineered oxalate decarboxylase polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

As discussed above, it is to be understood that the present disclosure provides recombinant polynucleotides encoding each and every one of the oxalate decarboxylase polypeptides described above.

Accordingly, by way of example and not limitation, in some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-1622, or to a reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence of SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 4, 5, 6, 7, 10, 11, 13, 14, 16, 17, 18, 19, 22, 26, 31, 33, 35, 37, 40, 43, 44, 46, 52, 54, 60, 61, 62, 63, 76, 79, 80, 82, 83, 85, 94, 96, 97, 103, 104, 106, 110, 117, 121, 123, 124, 125, 126, 128, 141, 149, 153, 155, 156, 160, 162, 164, 166, 169, 173, 174, 176, 180, 182, 183, 186, 187, 188, 189, 190, 193, 195, 196, 197, 199, 200, 205, 206, 208, 210, 212, 216, 219, 226, 227, 232, 233, 234, 240, 242, 243, 263, 265, 266, 267, 269, 270, 273, 274, 277, 284, 297, 301, 303, 304, 314, 316, 318, 331, 335, 339, 342, 343, 346, 347, 350, 351, 356, or 359, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 16, 17, 26, 31, 37, 60, 79, 83, 96, 162, 174, 195, 196, 210, 226, 277, 301, or 318, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising as least a substitution or substitution set at amino acid position(s) 31, 210, 318, or 31/210/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising as least a substitution or substitution set at amino acid position(s) 16, 26, 174, 195, 196, 210, 226, or 16/26/174/195/196/210/226, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 13, 17, 196, or 13/17/196, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 17, 60, 301, or 17/60/301, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at position 37, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 14, 60, 162, 226, or 14/60/162/226, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 83, 96, 277, or 83/96/277, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 346, 124, 359, 174, 173, 123, 196, 304, 301, 347, 11, 284, 210, 169, 216, 195, 339, 4, 6, 180, 80, 243, 182, 7, 226, 156, 183, 227, 219, 62, 343, 16/26, 16/26/242, 16/26/183/232, 155/206/242, 16/26/339, 16/26/155/206/339, 26, 26/206/339, 31/82/210, 31/82, 31/356, 31/97/226, 31/240/270, 31/82/226, 31/240, 31, 31/210/318, or 31/210, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at an amino acid position set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered oxalate decarboxylase polypeptide set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to SEQ ID NO: 2 or 4.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, or a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 616-1622.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 28-1622, or to the reference sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 4, 5, 6, 7, 10, 11, 13, 14, 16, 17, 18, 19, 22, 26, 31, 33, 35, 37, 40, 43, 44, 46, 52, 54, 60, 61, 62, 63, 76, 79, 80, 82, 83, 85, 94, 96, 97, 103, 104, 106, 110, 117, 121, 123, 124, 125, 126, 128, 141, 149, 153, 155, 156, 160, 162, 164, 166, 169, 173, 174, 176, 180, 182, 183, 186, 187, 188, 189, 190, 193, 195, 196, 197, 199, 200, 205, 206, 208, 210, 212, 216, 219, 226, 227, 232, 233, 234, 240, 242, 243, 263, 265, 266, 267, 269, 270, 273, 274, 277, 284, 297, 301, 303, 304, 314, 316, 318, 331, 335, 339, 342, 343, 346, 347, 350, 351, 356, or 359, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or amino acid residue 4C/R/S, 5C/S, 6A/S/W, 7G, 10A/Q/R, 11R, 13A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 14L/V, 16A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 17A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 18G/Q/R, 19C, 22R, 26A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 31A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 33R, 35V, 37A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 40S, 43C, 44S, 46R, 52T, 54L, 60A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 61M, 62G, 63A/S, 76S, 79A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 80L, 82I, 83A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 85E/G/R, 94N/T, 96A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 97T, 103Q/S/V, 104Q, 106C, 110V, 117A, 121W, 123G/Q/S, 124A/C/G/Q/R/T, 125E/F/S, 126T, 128A, 141Q, 149T, 153H/Q/S, 155L/P/R/V, 156E, 160T, 162A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 164R/V, 166R, 169A/G/L, 173Q/V, 174A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 176L, 180E, 182R, 183C/E/I/K, 186N, 187L/R/S, 188A, 189R, 190G/Q, 193A/G, 195A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 196F/K/M/R/S/V, 197E, 199G/V, 200N, 205A, 206M, 208G, 210A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 212A/F/G/L/S/V, 216S/W, 219V, 226S/T, 227E/S, 232T, 233D/H/R, 234L, 240E, 242D, 243V, 263S, 265C, 266V, 267R, 269S, 270L, 273A, 274Q/S, 277A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 284A/R/S, 297Y, 301I/Q/T, 303T, 304D/G, 314S, 316K/V, 318A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, 331I/L/P/V, 335R, 339G, 342A/E/P/R, 343R/W, 346G/L/Q/W, 347F/R, 350R, 351A/E/H, 356S, or 359L/W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 16, 17, 26, 31, 37, 60, 79, 83, 96, 162, 174, 195, 196, 210, 226, 277, 301, or 318, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 31, 210, or 318, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or to the reference sequence corresponding to SEQ ID NO: 172, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 13, 212, 124/196/210/226, 5, 16/26/124/155/195/210/284, 196/226, 16/155/195/210/226, 16/195/210/226, 16/155/174/196, 16/195/226, 318, 16/195/196/210, 16/26/124/155/174/196/210, 195/210, 26/155/174/210, 316, 60, 16/155/174, 16/124/174/196, 16/226, 16/26/174/196/226, 17, 331, 124/195, 16/174/196, 188, 195/196/210, 174/196/210, 16/26/155/174, 16/155/195/196/226, 195/226/284, 16, 240, 16/26/124/155/195/196/226, 183/232/339/343, 183/206, 63, 173/347, 16/26/174/196, 16/284, 46, 16/124/195/196, 274, 174/196, 155/174/196, 174/196/226, 16/124/155/174/195, 16/26/155/174/196, 26/174/196/210/284, 16/195/196/284, 174, 16/124/174/195/210/226/284, 16/174/195/284, 26/174/196/210/226/284, 174/196/226/284, 124/174/196, 26/174/195/210/226, 162, 16/124/195/196/284, 206/343, 16/195/196, 16/26/174/195/196/210/226, 155/195/196/226, 18, 335, 124/155/174/195/226, 26/155/174/195/226, 195/196, 153, 155/174/195, 174/195/210/284, 174/195, 37, 124/174/195/226/284, 176, 16/155/174/195/196/226/284, 10, 169/173, 155, 33, 173/183/343/347, or 104/265, 233, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, or relative to the reference sequence corresponding SEQ ID NO: 172.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or to the reference sequence corresponding to SEQ ID NO: 320, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 13/17/196, 17/212/331, 17/26/196/212/226, 17/26/196/212/226/331, 13/17/26/196/331, 17/60/196/226, 17/46/212, 26/60/196/226/331, 17/46/196/212/274/331, 212, 17/196/226, 13/17/26/212/331, 13/17/212/226/331, 196/212/331, 13/17/196/331, 13/17/26/212/226/331, 17/60/196/212/226/331, 13/17/212, 13/17/26/331, 13/17, 13/17/26/196, 46/196/212/226, 17/60/196, 17/196/331, 13/17/46/226/331, 17/196/226/331, 17/46/196/212/331, 17/46/212/331, 17/196, 13/17/212/226, 17/212, 17/26/212, 13/17/226/331, 17/196/212/331, 17/196/212/226/331, 13/17/26/226/331, 46/60/196/226/331, 17/46/196/331, 13/17/46/196, 196/212, 13/17/26, 17/83/263, 17/83/173/227, 17/227/301, 17/83/227/263, 17/83/125, 17/83/173, 17/263/301, 17/125, 17/83/301, 17/173, 17/83/227, 17/227, 17, 17/125/227, 17/83, 128, 76, 141, 110, 79, 117, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 320, or relative to the reference sequence corresponding to SEQ ID NO: 320.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to sequence of residues 1-359 of SEQ ID NO: 396, or to the reference sequence corresponding to SEQ ID NO: 396, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 13/17/79, 13/14/17/60/79, 13/17/79/83, 13/17/79/301, 13/14/17/60/79/212, 17/60/197, 13/60/212, 13/14/17/60/79/83/301, 13/17/60/79, 17/60/301, 14/17/79, 17/60/83, 17/79, 13/17/60/79/83/301, 342, 52/190, 94/190, 190/342/351, 13/79, 13/96, 273, 126, 96, 79, 269, 40, 44, 267, 266, 160, 277, 149, 22, 234, 54, 297, 106, or 13/43, wherein the amino acid positions are relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 670, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 37, 125, 162, 94, 195, 85, 103, 232, 189, 186, 155, 193, 342, 196, 63, 33, 351, 314, 187, 303, 164, 153, 346, 183, 19, 123, 350, 205, 284, 199, 343, 200, 208, 169, 190, or 121, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 670.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 616, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 616.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 14/26/60/318, 14/26/60/94/162/212, 60/162/226, 60, 14/125/162/212/318, 60/125/226, 14/125/162/226, 26/60/162/226/318, 14/26/94/162/212/226, 14/60/162, 14/60/162/226, 14/94/162/318, 14/94/162, 14/60/226, 14/60/94/212, 14/60/162/212, 14/162, 14/94/212/318, 14/60, 14/26/162, 14, 14/318, 14/162/226/318, 14/125/212, 14/125/226/318, 14/26/60/162, 14/125/162/212/226, 14/94/125/162/318, 14/125/226, 277, 166, 233, 83, 5, 342, 96, 284, or 26, wherein the amino acid positions are relative to the reference sequence corresponding SEQ ID NO: 616.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 750, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 750.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 5/26/83/342, 5/26/277, 5/96/277, 5/83/277/342, 26/83/277, 5/26/83/277, 5/277/342, 5/26/166/277, 83/277/342, 83/96/277/342, 83/96/277, 26/83/277/342, 5/26/83/277/342, 5/83/277, 26/277, 83/277, 5/35/277, 5/26/83/96/277, 5/26/96/277, 5/277, 5/166/277, 26/83/96/277/342, 277, 26/277/342, 5, or 5/83/96/277/342, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 750.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 906, wherein the amino acid sequence comprises one or more amino acid substitutions relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 26, 31, 96, 60, 318, 210, 277, 37, 16, 195, 79, 17, 13, 83, 162, or 174, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 174/318, 26/96, 60/318, 31/318, 162/174/318, 174/277/318, 26/195/318, 60/162/195, 60/162/318, 174/195/277, 16/17/174, 60/195/277, 37/277/318, 16/162/195/277, 26/79/83/162, 26/31/60/318, 16/174/195/277, 13/3⅞3/162, 16/17/60/195, 13/17/37/83, 31/37/162/174/318, 13/16/31/83/162, 16/60/174/195/318, 17/60/96/162/195/277, 13/26/60/83/162/174, 17/31/60/162/174/277, 16/17/26/83/96/277, 17/26/37/174/277/318, 13/16/31/60/162/174, 13/17/31/83/277/318, 13/16/17/31/83/162/318, 13/16/60/83/162/195/318, 13/16/17/31/37/83/96/162, 13/17/26/37/60/83/162/210/318, 13/16/26/31/83/96/162/174/318, 13/16/17/31/60/83/96/162/174/195/318, 13/16/26/37/60/83/162/174/195/210/318, 13/16/37/79/83/96/162/174/195/210/277, 13/17/26/31/37/60/79/83/96/162/174/210, 13/16/17/26/37/79/83/96/174/195/210/318, 13/26/31/37/60/83/96/162/174/195/210/277/318, 13/16/17/31/37/60/79/83/96/162/174/195/210/318, 13/16/17/31/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/37/60/79/96/162/174/195/210/277/318, 13/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/17/26/3⅓7/60/96/162/174/195/210/277/318, 13/16/17/26/31/37/79/83/96/162/174/195/210/318, 13/16/17/26/31/37/60/79/96/162/174/210/277/318, 13/17/26/31/37/60/79/83/96/162/174/195/210/277/318, 13/16/26/31/37/60/79/83/96/162/174/195/210/277/318, 16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, or 13/16/17/26/31/37/60/79/83/96/162/174/195/210/277/318, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 906.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at an amino acid position set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set of an oxalate decarboxylase polypeptide set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising: an amino acid sequence comprising SEQ ID NO: 4; an amino acid sequence corresponding to residues 1-359 of an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614; an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 28-614; or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 616-1622, optionally wherein the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the encoded engineered oxalate decarboxylase polypeptide optionally includes 1, 2, 3, 4, or 5 substitutions.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence comprising residues 1-359 of SEQ ID NO: 172, 320, or 396, or comprising SEQ ID NO: 172, 320, 396, 616, 670, 750, or 906, optionally wherein the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the encoded engineered oxalate decarboxylase polypeptide optionally includes 1, 2, 3, 4, or 5 substitutions.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to: a reference polynucleotide sequence corresponding to SEQ ID NO: 3; a reference polynucleotide sequence corresponding to nucleotide residues 1-1077 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 615-1621, wherein the recombinant polynucleotide encodes an engineered oxalate decarboxylase polypeptide.

In some embodiments, the recombinant polynucleotide comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 33, 171, 319, 395, 615, 669, 749, or 905, or to a reference polynucleotide sequence corresponding to nucleotide residues 1-1077 of SEQ ID NO: 171, 319, or 395, wherein the recombinant polynucleotide encodes an engineered oxalate decarboxylase polypeptide.

In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotide sequence described herein encoding an engineered oxalate decarboxylase e.g., a recombinant polynucleotide provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2, or a reverse complement thereof. In some embodiments, the reference polynucleotide sequence corresponds to SEQ ID NO: 1 or 3; a polynucleotide sequence corresponding to nucleotide residues 1-1077 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; or a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 615-1621, or a reverse complement thereof, or a polynucleotide sequence encoding any of the other engineered oxalate decarboxylase provided herein. In some embodiments, the recombinant polynucleotide encodes an engineered oxalate decarboxylase and hybridizes under highly stringent conditions to a reverse complement of a reference polynucleotide sequence corresponding to SEQ ID NO: 1 or 3; a polynucleotide sequence corresponding to nucleotide residues 1-1077 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; or a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 615-1621.

In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reverse complement of a reference polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide, wherein the engineered oxalate decarboxylase polypeptide comprises an amino acid sequence having one or more amino acid differences relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 320, 396, 616, 670, 750, or 906, or relative to the reference sequence corresponding to residues 1-359 of SEQ ID NO: 172, 320, or 396, at residue positions selected from any positions as set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2. In some embodiments, the polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 1 or 3; a polynucleotide sequence corresponding to nucleotide residues 1-1077 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 27-613; or a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 615-1621. In some additional embodiments, the polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to a polynucleotide sequence set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference amino acid sequence corresponding to SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or to a reference amino acid sequence corresponding to residues 1-424 of SEQ ID NO: 6.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered oxalate decarboxylase polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference amino acid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or to a reference amino acid sequence corresponding to residues 1-424 of SEQ ID NO: 6, wherein the amino acid sequence comprises one or more substitutions in its amino acid sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or to a reference polynucleotide sequence corresponding to nucleotide residues 1-1272 of SEQ ID NO: 5, wherein the recombinant polynucleotide encodes an oxalate decarboxylase.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or comprising nucleotide residues 1-1272 of SEQ ID NO: 5.

In some embodiments, a recombinant polynucleotide encoding any of the engineered oxalate decarboxylase polypeptides herein is manipulated in a variety of ways to facilitate expression of the engineered polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are provided as expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and/or polypeptides. In some embodiments, the recombinant expression vector comprises a polynucleotide encoding an engineered oxalate decarboxylase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, suitable promoters are selected based on the host cells selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80: 21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha- amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken β-actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or from Gallus β-actin.

In some embodiments, the control sequence is a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the engineered oxalate decarboxylase polypeptide. Any terminator which is functional in the host cell of choice finds use in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.

In some embodiments, the control sequence is a suitable leader sequence, 5′-cap modification, 5′ UTR, etc. In some embodiments, these regulatory sequence elements mediate binding to molecules involved in mRNA trafficking and translation, inhibit 5′-exonucleolytic degradation and confer resistance to de-capping. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the engineered oxalate decarboxylase polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). Suitable leaders for mammalian host cells include but are not limited to the 5′-UTR element present in orthopoxvirus mRNA.

In some embodiments, the control sequence comprises a 3′ untranslated nucleic acid region and/or polyadenylation tail nucleic acid sequence, sequences operably linked to the 3′ terminus of the protein coding nucleic acid sequence which mediate binding to proteins involved in mRNA trafficking and translation and mRNA half-life. Any polyadenylation sequence and 3′ UTR which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3′ UTR sequences for mammalian host cells include, but are not limited to, the 3′-UTRs of α- and β-globin mRNAs that harbor several sequence elements that increase the stability and translation of mRNA.

In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Bio., 1995, 15:5983-5990).

In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway). In some embodiments, the 5′ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5′ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to those from the genes for immunoglobulin gamma (IgG). In some embodiments, the signal peptide is a signal peptide of a protein expressed in human cells.

In some embodiments, the control sequence is also a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

In the embodiments herein, the recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the engineered oxalate decarboxylase polynucleotide sequence, and/or expression of the encoded engineered oxalate decarboxylase polypeptide. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.

In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

In another aspect, the present disclosure provides a host cell comprising at least one recombinant polynucleotide or an expression vector encoding at least one engineered ODC polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered ODC enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors described herein are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).

In another aspect, the present disclosure provides a method of producing the engineered oxalate decarboxylase polypeptide, where the method comprises culturing a host cell capable of expressing a polynucleotide encoding the engineered oxalate decarboxylase polypeptide under conditions suitable for expression of the encoded polypeptide. In some embodiments, the method further comprises a step of isolating or recovering the expressed engineered oxalate decarboxylase polypeptide, e.g., from the culture and/or cells. In some embodiments, the method further comprises a step of purifying the expressed oxalate decarboxylase polypeptide. Purification of the polypeptide can use methods known in the art, including, by way of example and not limitation, precipitation and/or chromatography.

Chromatographic techniques for isolation/purification of the oxalate polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ionexchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the engineered oxalate decarboxylase polypeptide. In some embodiments, the affinity chromatography can use a tag on the polypeptide, such as a his-tag. In some embodiments, the affinity chromatography can use any antibody that specifically binds an engineered oxalate decarboxylase polypeptide of interest. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized with an engineered oxalate decarboxylase polypeptide, or a fragment thereof. In some embodiments, the engineered oxalate decarboxylase polypeptide or fragment for immunization is attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group for immunizing an animal.

Appropriate culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing polynucleotides for expression of the ODC polypeptides into cells will find use in the present invention. Suitable techniques include, but are not limited to electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

The engineered oxalate decarboxylase polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered oxalate decarboxylase polypeptide to any suitable mutagenesis and/or directed evolution methods known in the art, and/or as described herein. An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (See e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

For example, mutagenesis and directed evolution methods can be readily applied to oxalate decarboxylase encoding polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods find use in the present invention and are well known in the art (See e.g., U.S. Pat. Nos, 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related US and non-US counterparts; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 370:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; WO 2009/152336; and U.S. Pat. Appln. Publ. Nos. 2011/0082055, 2014/0005057, 2014/0214391, 2014/0221216, 2015/0133307, 2015/0134315, and 2015/0050658; all of which are incorporated herein by reference).

In some embodiments, the enzyme clones obtained following mutagenesis treatment are screened by subjecting the enzyme preparations to a defined temperature, acid pH conditions, exposure to proteases, or other assay conditions, and measuring the amount of enzyme activity after such treatments. Clones containing the polynucleotide encoding an engineered oxalate decarboxylase polypeptide of interest are then isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art, such as described in the Examples.

For engineered polypeptides of known sequence, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tet. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors).

Accordingly, in some embodiments, a method for preparing the engineered oxalate decarboxylase polypeptide can comprise: (a) synthesizing a polynucleotide encoding an engineered oxalate decarboxylase polypeptide comprising an amino acid sequence corresponding to any polypeptide variant as described herein, and (b) expressing the engineered oxalate decarboxylase polypeptide encoded by the polynucleotide. In some embodiments of the method, the polynucleotide encoding the polypeptide can be designed or engineered to encode a polypeptide optionally having one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions are conservative or non-conservative substitutions.

As noted above, the expressed engineered ODC polypeptide can be evaluated for any desired improved property or combination of properties (e.g., activity, stability, protease resistance, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

Compositions of Engineered Oxalate Decarboxylase

In another aspect, the present disclosure provides compositions comprising the engineered oxalate decarboxylase polypeptides described herein for various uses, including but not limited to use as pharmaceuticals, dietary/nutritional supplements, food, feed, and fine chemical production. For example, in some embodiments, the present disclosure provides a composition comprising a food and/or feeds and at least one engineered oxalate decarboxylase polypeptide and/or at least one polynucleotide encoding at least one engineered oxalate decarboxylase polypeptide.

In some embodiments, the engineered oxalate decarboxylase polypeptide find use in any suitable edible enzyme delivery matrix. In some embodiments, the engineered oxalate decarboxylase polypeptide variants are present or formulated in an edible enzyme delivery matrix designed for rapid dispersal of the engineered oxalate decarboxylase polypeptide within the digestive tract of an animal or subject upon ingestion of the polypeptide.

In some embodiments, the engineered oxalate decarboxylase polypeptides are prepared as a pharmaceutical or dietary composition. Depending on the mode of administration, the pharmaceutical composition comprises an engineered oxalate decarboxylase polypeptide described herein, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the engineered oxalate decarboxylase polypeptide. In some embodiments, the pharmaceutical or dietary composition is in the form of a solid, semi- solid, or liquid. In some embodiments, the compositions include other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. A description of pharmaceutically acceptable excipients and carriers is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) and Remington: The Science and Practice of Pharmacy, A. Adejare ed., 23 Ed., Academic Press 2020).

In some embodiments, the engineered oxalate decarboxylase polypeptides are formulated for use in oral pharmaceutical compositions. Any suitable form for use in delivering the engineered oxalate decarboxylase polypeptides find use in the present invention, including but not limited to pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks, suspensions (including but not limited to oil-based suspensions, oil-in water emulsions, etc.), slurries, syrups, controlled release formulations, suppositories, etc. In some embodiments, the engineered oxalate decarboxylase polypeptides are provided in a format suitable for injection (i.e., in an injectable formulation).

In some embodiments, compositions comprising the engineered oxalate decarboxylase polypeptides of the present invention include one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol, and/or sorbitol), starches (e.g., corn, wheat, rice, potato, or other plant starch), cellulose (e.g., methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxy- methylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin, collagen, etc.). Additional components in oral formulations may include coloring and or sweetening agents (e.g., glucose, sucrose, and mannitol) and lubricating agents (e.g., magnesium stearate), as well as enteric coatings (e.g., methacrylate polymers, hydroxyl propyl methyl cellulose phthalate, and/or any other suitable enteric coating known in the art). In some embodiments, disintegrating or solubilizing agents are included (e.g., cross-linked polyvinyl pyrrolidone, agar, alginic acid or salts thereof, such as sodium alginate). In some embodiments, the engineered oxalate decarboxylase polypeptide are combined with various additional components, including but not limited to preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and/or emulsifiers, particularly in liquid formulations.

In some embodiments, the engineered oxalate decarboxylase polypeptide are combined with various additional components, including but not limited to preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and/or emulsifiers, particularly in liquid formulations.

In some embodiments, the engineered oxalate decarboxylase polypeptides are provided in biocompatible matrices such as sol- gels, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the engineered oxalate decarboxylase polypeptides are encapsulated. In some alternative embodiments, the engineered oxalate decarboxylase polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres, liposomes, etc.).

It is not intended that the present invention be limited to any particular delivery formulation and/or means of delivery. In some embodiments, the engineered oxalate decarboxylase polypeptides be administered by any suitable means known in the art, including but not limited to parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implants, etc.

In some embodiments, the engineered oxalate decarboxylase polypeptides are chemically modified by glycosylation, pegylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (See e.g., Ikeda, Amino Acids, 2005, 29:283-287; US Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,553,653; U.S. Pat. Appln. Publ. Nos. 2013/0039898, 2012/0177722, etc.).

In some additional embodiments, the engineered oxalate decarboxylase polypeptides are provided in formulations comprising matrix-stabilized enzyme crystals. In some embodiments, the formulation comprises a cross-linked crystalline engineered oxalate decarboxylase polypeptide and a polymer with a reactive moiety that adheres to the enzyme crystals. The present invention also provides engineered oxalate decarboxylase polypeptides in polymers.

It is to be understood that for the compositions herein, including the pharmaceutical compositions, any of the engineered oxalate decarboxylase polypeptides described herein, including an engineered oxalate carboxylase set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 5-1, and 5-2 can be used in the compositions.

In some additional embodiments, the pharmaceutical composition comprises an oxalate decarboxylase comprising an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or an amino acid comprising residues 1-424 of SEQ ID NO: 6.

Uses of the Engineered Oxalate Decarboxylases

In a further aspect, the engineered decarboxylase polypeptides are used for therapeutic, diagnostic, or industrial purposes.

In some embodiments, the engineered oxalate decarboxylase polypeptides described herein are used to reduce levels of oxalate in a subject, such as a patient afflicted with abnormally elevated or pathological levels of oxalate, such as in the urine and/or plasma. In some embodiments, a method of reducing levels of oxalate in a subject comprises administering to a subject in need thereof an effective amount of an engineered oxalate decarboxylase polypeptide described herein or a composition comprising the engineered oxalate decarboxylase, to reduce levels of oxalate in the subject. In some embodiments, an effective amount of the engineered oxalate decarboxylase is administered for reducing the levels of oxalate in urine and/or plasma of the subject.

In some embodiments, the engineered oxalate decarboxylase polypeptides are used to treat and/or prevent symptoms of hyperoxaluria. In some embodiments, a method for treating and/or preventing the symptoms of hyperoxaluria in a subject comprises administering to a subject with hyperoxaluria an effective amount of an engineered oxalate decarboxylase polypeptide described herein or a composition comprising the engineered oxalate decarboxylase. In some embodiments, the subject is a patient afflicted with primary hyperoxaluria. In some embodiments, the subject is a patient afflicted with secondary hyperoxaluria. In some embodiments, the secondary hyperoxaluria is enteric hyperoxaluria. In some embodiments, the subject is afflicted with idiopathic hyperoxaluria.

In some embodiments, an effective amount of the engineered oxalate decarboxylase is administered is a therapeutically effective amount such that the symptoms of hyperoxaluria are ameliorated. In some embodiments, an effective amount of the engineered oxalate decarboxylase is administered such that the level of oxalate in urine and/or plasma is reduced. In some embodiments, the subject treated with an engineered oxalate carboxylase is able to eat a diet that is higher in its oxalate content compared to diets required by subjects who are afflicted with hyperoxaluria.

In some embodiments of the methods above, the subject treated with an engineered oxalate decarboxylase polypeptide is an infant, child, young adult, or adult.

In some embodiments, the dosage of engineered oxalate decarboxylase polypeptide(s) administered to a patient depends upon, among others, the general condition of the patient, age, sex, weight, and the disease/condition being treated, and other factors known to those in the art. In some embodiments, the compositions are intended for single or repeat administration to a patient. Preferably, the compositions are administered repeatedly. In some embodiments, the amount of engineered oxalate decarboxylase polypeptide(s) in the composition(s) administered to a patient is sufficient to effectively treat, ameliorate and/or prevent the symptoms of the disease or condition.

In some embodiments, the engineered oxalate decarboxylase is administered at about 0.1 to about 3 g/dose. In some embodiments, the engineered oxalate decarboxylase is administered at a dose of about 0.2 to about 2.5 g, about 0.25 to about 2 g, or about 0.5 to about 1.5 g. In some embodiments, the engineered oxalate decarboxylase is administered at a dose of about 0.1, 0.2, 0.5, 1, 1.5, 2. 2.5, or 3 g.

In some embodiments, the engineered oxalated decarboxylase is administered at about 100 U/kg body weight to about 5000 U/kg body weight. In some embodiments, the engineered oxalated decarboxylase is administered at about 300 U/kg body weight to about 3000 U/kg body weight. In some embodiments, the engineered oxalated decarboxylase is administered at about, 500 U/kg to about 2000 U/kg body weight. A Unit of enzyme is provided in Example 12.

In some embodiments, the engineered oxalate decarboxylase is administered 1-5 times per day, 1-4 times per day, 1-3 times per day, or 1-2 times per day. In some embodiments, the engineered oxalate decarboxylase is administered 2-5 time per day, 2-4 times per day, or 2-4 times per day. In some embodiments, the engineered oxalate decarboxylase is administered once (1) per day, 2 times per day, 3 times per day, 4 times per day, or 5 times per day.

In some embodiments, the engineered oxalate decarboxylase is administered prior to, concurrently with, or subsequent to ingestion of food or meal. In some embodiments, the engineered oxalate decarboxylase is administered orally prior to, concurrently with, or subsequent to ingestion of food or meal. In some embodiments, the engineered oxalate decarboxylase is administered together with food or a meal.

In some embodiments, the engineered oxalate decarboxylase polypeptides are used in diagnostics, e.g., for detecting oxalate (Costello et al, J. Lab. Clin. Med., 1976, 87(5):903-908); Parkinson et al., Clin Chim Acta., 1985, 152(3):335-45). In some embodiments, a method of determining amount of oxalate in a sample comprises contacting a sample with an engineered oxalate decarboxylase described herein and determining amount of formate product. In some embodiments, the amount of formate can be determined by use of formate dehydrogenase or formyl-CoA transferase (U.S. Pat. No. 5,604,111).

In some embodiments, the engineered oxalate decarboxylase is used for industrial purposes in reducing levels of oxalate, such as in reducing formation of calcium oxalate deposits/precipitates. Deposits or precipitates of calcium oxalate are problematic in pulp and paper industries, including removal of oxalate from industrial wastewater. In some embodiments, the engineered oxalate decarboxylase is added to industrial plant filtrates containing oxalate, e.g., to prevent or reduce scaling.

The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples. These Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

EXAMPLES

In the experimental disclosure below, the following abbreviations apply where appropriate: ppm (parts per million); M (molar); mM (millimolar), uM and µM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and µg (micrograms); L and l (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and µm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); psi and PSI (pounds per square inch); °C (degrees Centigrade); RT and rt (room temperature); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); AUC (area under the curve); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); LC (liquid chromatography); MS (mass spectroscopy); LC-MS/MS (liquid chromatography with two mass spectrometers); SPE (solid phase extraction); IPTG (isopropyl β-D-1-thiogalactopyranoside); PLP (pyridoxal 5′-phosphate); BSA (bovine serum albumin); BW (body weight); FIOPC (fold improvements over positive control); FIOP (fold improvement over parent); LB (Luria broth); TB (Terrific broth).

Example 1
Oxalate Decarboxylase Gene Acquisition and Construction of Expression Vectors

Bacterial ODCs from different organisms in Table 1-1 were codon optimized for expression in E. coli, synthesized and cloned into the E. coli expression vector pCK110900 vector system (See e.g., U.S. Pat. Appln. Publn. 2006/0195947, which is hereby incorporated by reference herein). The plasmid construct was transformed into an E. coli strain derived from W3110. The E. coli strains containing the ODC genes were grown in 96-well format and assayed for ODC activity (40 mM oxalate, pH 4.0) as described below in Example 3. The activity obtained from these ODCs are shown in Table 1-1.

TABLE 1-1

Unchallenged activity at pH 4.0 for wild type oxalate decarboxylases

SEQ ID NO: (nt/aa)
Source Organism
Activity at pH 4.0 Relative to SEQ ID NO: 2

½

Gemmata sp. SH-PL17
++++

⅚

Flammulina velutipes

+++

⅞

Bacillus subtilis

+

9/10

Bacillus vallismortis

++++

11/12

Bacillus glycinifermentans

+

13/14

Bacillus atrophaeus

+

15/16

Bacillus megaterium

++

17/18

Agrobacterium tumefaciens str. C58
++++

19/20

Rhizobiales bacterium

++++

21/22

Hyphomicrobium sp. 802
+++

23/24

Shinella sp.
+

25/26

Legionella maceachernii

++

All activities are expressed as a change in absorbance at 340 nm (ΔA340). Levels of activity are defined as follows: “+” 0.15 to 0.50, “++” > 0.50, “+++” > 1.0,“++++”>1.5.

Example 2
Engineered ODC Variants Expression and Construction

Based on the results obtained from Table 1-1, the ODC from SEQ ID: 2 was subcloned with and without a C-terminal 6xHis-tag into the pJV110900 vector system (See e.g., U.S. Pat. Appln Publ. 2017/213758) to generate SEQ ID: 4 and 2 respectively. The plasmid construct was transformed into an E. coli strain derived from W3110. Directed evolution techniques generally known by those skilled in the art were used to generate libraries of gene variants from this plasmid construct (See e.g., US Pat. No. 8,383,346, WO2010/144103, and references cited herein) as well as its derivatives.

Example 3
High-Throughput (HTP) Growth of ODC Variants and Screening Conditions
High-Throughput (HTP) Growth of Escherichia Coli ODC and Variants

Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose with selection. After overnight incubation at 37° C., colonies were placed into the wells of 96-well shallow flat bottom plates (NUNC™, Thermo-Scientific) filled with 180 µl/well LB supplemented with 1% glucose and selection. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30° C., and 85% relative humidity; Kuhner).

Overnight growth samples (20 µL) were transferred into Costar 96-well deep plates filled with 380 µL of Terrific Broth supplemented with a selection compound. The plates were incubated for approximately 2 hours in a shaker (250 rpm, 30° C., and 85% relative humidity; Kuhner). The cells were then induced with 40 µL of 10 mM IPTG and 10 mM manganese chloride in sterile water and incubated overnight for 20-24 hours in a shaker (250 rpm, 30° C., and 85% relative humidity; Kuhner). The cells were pelleted (4000 rpm × 20 min), the supernatants were discarded, and the cells were frozen at -80° C. prior to analysis.

Lysis of HTP Pellets

First, 400 µL of lysis buffer (1X PBS, 1 mg/ml lysozyme, and 0.5 mg/ml polymyxin B sulfate) were added to the cell pellets. The mixture was agitated for 1.5-2 hours at room temperature, and centrifuged (4000 rpm × 15 min) prior to use of the clarified lysates in the various HTP assays described herein. Analysis of these lysates by SDS-PAGE revealed the presence of an overexpressed protein bands at an apparent MW of 40-50 kDa, consistent with the expected MW of ODC. Sometimes additional dilutions of clarified lysate were performed with PBS buffer prior to challenges and analysis.

Analysis of Clarified Lysates for ODC Activity

ODC activity was assessed using potassium oxalate as a substrate and quantifying formate produced using formate dehydrogenase (FDH). Oxalate assay solution was prepared by dissolving 4.44-44.4 mM potassium oxalate in McIlvaine buffer pH 2.0-4.0 or pH 5.5-6.5. Lysate was diluted 4-200x in 1X PBS pH 7.0-7.4. For dilutions >10x, sequential dilutions were performed, wherein the first dilution was in 1X PBS, pH 7.0-7.4 and the secondary dilutions were in water. To set up ODC activity reactions, 10 µL diluted ODC was added to 90 µL oxalate solution in a round bottom plate (Costar), except in Table 1-1, where 10uL lysate was added to 40 µL 50 mM oxalate solution. Reactions were incubated at 37° C. for 1 hour and quenched by mixing 1:1 with 1 M potassium phosphate dibasic. Formate detection assay solution was prepared by dissolving 25 mM nicotinamide adenine dinucleotide (NAD) in 125 mM potassium phosphate pH 7.5. To set up formate detection reactions, 10-25 µL quenched oxalate assay sample was added to 20 µL formate detection solution with 0-15 µL water added to bring to 45 µL total, before 5 µL 10 g/L formate dehydrogenase (FDH; Codexis) was added in a UV-Star® half area, flat bottom plate (Greiner). Reactions were incubated at 25° C. for 10 min, shaking 200 rpm, and absorbance at 340 nm was read on a Molecular Devices Spectramax plate reader. Background subtracted absorbance (Table 1-1) or absorbance relative to that of the round backbone (fold improvement over parent (FIOP)) were used for activity values.

HTP Analysis of Clarified Lysates Pretreated With Low pH and Pepsin

The activities of variants were determined after pre-incubation at low pH in the presence of pepsin to simulate the environment of the stomach. First, clarified lysate was diluted 0x -8x in PBS buffer, then mixed 1:1 with McIlvaine buffer pH 2.2-3.2 with 0.2-1.6 mg/mL pepsin from porcine gastric mucosa (Sigma) in a round bottom plate (Costar), for a final challenge pH of 2.2-3.0 and a final pepsin concentration of 0.1-0.8 mg/mL. Samples were mixed then incubated for 1-2 hours at 37° C. Samples were then analyzed for residual ODC activity as described in Examples 3.3, except that samples were taken directly into oxalate assay solution (pH 2.4-3.0) without diluting in PBS.

Example 4
Screening Results of ODC Variants Based on SEQ ID NO: 4

Library variants were generated from homologs diversity and mutagenesis at surface positions based on a structural model of SEQ ID NO: 4. These variants were screened in triplicates for ODC activity at pH 3.0 with 10 mM potassium oxalate after a 1 hour simulated gastric challenge (0.2 mg/mL pepsin, pH 3.0) and unchallenged ODC activity at pH 3.0 with 4 mM and 10 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 4 are listed in Table 4-1.

TABLE 4-1

Oxalate decarboxylase activity Relative to SEQ ID NO: 4

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 2 or 4)
FIOP Unchallenged Activity at pH 3.0, 10 mM oxalate Relative to SEQ ID NO: 4
FIOP Unchallenged Activity at pH 3.0, 4 mM oxalate Relative to SEQ ID NO: 4
FIOP Pepsin Challenge Relative to SEQ ID NO: 4

¾

+
+
+

27/28
E346G

+
+

29/30
E346L

+
++

31/32
K124G
+
+
+

33/34
K124T
+
+
+

35/36
K359L
+
+
++

37/38
D174K
+
+
++

39/40
K173V
+
+
++

41/42
D123Q

+
+

43/44
K196F
+
+
+

45/46
R304D

+

47/48
V301Q

+

49/50
K347F

+

51/52
K359W
+
+
+

53/54
K124Q
+
+
++

55/56
K124A
+
+
++

57/58
P11R

+

59/60
K284R
+
+
+

61/62
P210L
+
+
+

63/64
D169L
+
++
+

65/66
R304G
+
+
+

67/68
K216W

+

69/70
K347R
+
+
+

71/72
K196V
+
++
++

73/74
K284A
+
+
+

75/76
R195L
+
+
++

77/78
S339G
+
+
++

79/80
P4S

+
+

81/82
K196R
+
+
+

83/84
F6A

+

85/86
K124C
+
+
+

87/88
R195Y
+
+
+

89/90
R180E
+
+
+

91/92
F80L
+
+
+

93/94
P243V

+
+

95/96
A182R
+
+
+

97/98
P210E
++
+
++

99/100
M7G

+

101/102
T226S
+
++
+

103/104
D174A
+
+
+

105/106
K196M

+
+

107/108
P4C
++
++
++

109/110
L156E

+
+

111/112
V183E
+
+
+

113/114
R227S
+
+
+

115/116
R219V

+
+

117/118
E62G

+

119/120
D174G
+
+
+

121/122
D169G
+
+
+

123/124
K124R
+
+
+

125/126
P210V
+
++
++

127/128
S343R
+
+
+

129/130
K216S
+
++
++

131/132
P210R
+
+
+

133/134
F6W

+
+

135/136
P4R

+

137/138
F6S

+

139/140
A16S/A26E

+
++

141/142
A16S/A26E/E242D

+
+

143/144
A16S/A26E/V183I/K232T

+
++

145/146
A155P/L206M/E242D

+
++

147/148
A16S/A26E/S339G
+
+
++

149/150
A16S/A26E/A155P/L206M/S339G

+
++

151/152
A26E
+
+
++

153/154
A26E/L206M/S339G
+
+
++

155/156
E31K/L82I/P210A
+
+
+++

157/158
E31K/L82I
+
+
++

159/160
E31K/A356S
+
+
++

161/162
E31K/A97T/T226S

+
++

163/164
E31K/D240E/M270L
+
+
+++

165/166
E31K/L82I/T226S
+
++
+++

167/168
E31K/D240E
+
+
++

169/170
E31K
+
+
++

171/172
E31K/P210A/E318Q
+
++
+++

173/174
E31K/P210A
+
+
+++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 4. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” > 1.1, “+++” > 2

Example 5
Screening Results of ODC Variants Based on SEQ ID NO: 172

Based on the results from Table 4-1, SEQ ID NO: 172 was chosen as the backbone. Beneficial mutations identified from Table 4-1 were recombined into the backbone. The variants were assayed in triplicate for ODC activity at pH 2.6 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.1 mg/mL pepsin, pH 2.8) and unchallenged ODC activity at pH 2.6 with 4 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 172 are listed in Table 4-2.

TABLE 4-2

Oxalate decarboxylase activity Relative to SEQ ID NO: 172

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 172)
Amino Acid Differences (Relative to SEQ ID NO: 4)
FIOP Unchallenged Activity at pH 2.6 Relative to SEQ ID NO: 172
FIOP Pepsin Challenge Relative to SEQ ID NO: 172

175/176
H13P
H13P/E31K/P210A/E318Q
+++
+++

177/178
H212S
E31K/P210A/H212S/E318Q
+++
+++

179/180
K124A/K196V/A210E/ T226S
E31K/K124A/K196V/P210E/T226S/E318Q
+++
+++

181/182
T5S
T5S/E31K/P210A/E318Q
+++
++

183/184
H13E
H13E/E31K/P210A/E318Q
+++
++

185/186
A16S/A26E/K124A/A155P/R195L/A210E/K284A
A16S/A26E/E31K/K124A/A155P/R195L/P210E/K284A/E318Q
+++
+++

187/188
K196V/T226S
E31K/K196V/P210A/T226S/E318Q
+++
+

189/190
A16S/A155P/R195L/A210E/T226S
A16S/E31K/A155P/R195L/P210E/T226S/E318Q
+++
+++

191/192
A16S/R195L/A210E/T226S
A16S/E31K/R195L/P210E/T226S/E318Q
+++
+++

193/194
A16S/A155P/D174K/K196V
A16S/E31K/A155P/D174K/K196V/P210A/E318Q
+++
+++

195/196
A16S/R195L/T226S
A16S/E31K/R195L/P210A/ T226S/E318Q
+++
+++

197/198
H13L
H13L/E31K/P210A/E318Q
+++
++

199/200
Q318E
E31K/P210A
++
+++

201/202
A16S/R195L/K196V/A210E
A16S/E31K/R195L/K196V/ P210E/E318Q
++
+++

203/204
A16S/A26E/K124A/A155P/D174K/K196V/A210E
A16S/A26E/E31K/K124A/A155P/D174K/K196V/P210E/E318Q
++
+++

205/206
R195L/A210E
E31K/R195L/P210E/E318Q
++
+++

207/208
A26E/A155P/D174K/A210E
A26E/E31K/A155P/D174K/P210E/E318Q
++
+++

209/210
N316V
E31K/P210A/N316V/E318Q
++
+++

211/212
T60V
E31K/T60V/P210A/E318Q
++
+++

213/214
A16S/A155P/D174K
A16S/E31K/A155P/D174K/P210A/E318Q
++
+++

215/216
A16S/K124A/D174K/K196V
A16S/E31K/K124A/D174K/ K196V/P210A/E318Q
++
+++

217/218
A16S/T226S
A16S/E31K/P210A/T226S/E318Q
++
++

219/220
A16S/A26E/D174K/K196V/T226S
A16S/A26E/E31K/D174K/K196V/P210A/T226S/E318Q
++
+++

221/222
D17H
D17H/E31K/P210A/E318Q
++
+++

223/224
A331V
E31K/P210A/E318Q/A331V
++
+++

225/226
K124A/R195L
E31K/K124A/R195L/P210A/E318Q
++
+++

227/228
A16S/D174K/K196V
A16S/E31K/D174K/K196V/P210A/E318Q
++
+++

229/230
D17A
D17A/E31K/P210A/E318Q
++
+++

231/232
P188A
E31K/P188A/P210A/E318Q
++
++

233/234
R195L/K196V/A210E
E31K/R195L/K196V/P210E/E318Q
++
+++

235/236
D174K/K196V/A210E
E31K/D174K/K196V/P210E/E318Q
++
+++

237/238
T5C
T5C/E31K/P210A/E318Q
++
++

239/240
A16S/A26E/A155P/D174K
A16S/A26E/E31K/A155P/D174K/P210A/E318Q
++
+++

241/242
A16S/A155P/R195L/K196V/T226S
A16S/E31K/A155P/R195L/K196V/P210A/T226S/E318Q
++
+++

243/244
R195L/T226S/K284A
E31K/R195L/P210A/T226S/K284A/E318Q
++
+++

245/246
D17N
D17N/E31K/P210A/E318Q
++
++

247/248
A16L
A16L/E31K/P210A/E318Q
++
+++

249/250
D240E
E31K/P210A/D240E/E318Q
++
++

251/252
A16S/A26E/K124A/A155P/R195L/K196V/T226S
A16S/A26E/E31K/K124A/A155P/R195L/K196V/P210A/T226S/E318Q
++
++

253/254
A331P
E31K/P210A/E318Q/A331P
++
++

255/256
V183I/K232T/S339G/S343R
E31K/V183I/P210A/K232T/ E318Q/S339G/S343R
++
+

257/258
V183I/L206M
E31K/V183I/L206M/P210A/E318Q
++
++

259/260
P63A
E31K/P63A/P210A/E318Q
++
++

261/262
K173V/K347R
E31K/K173V/P210A/E318Q/K347R
++
+

263/264
A16S/A26E/D174K/K196V
A16S/A26E/E31K/D174K/K196V/P210A/E318Q
++
++

265/266
A16S/K284A
A16S/E31K/P210A/K284A/E318Q
++
+++

267/268
T60R
E31K/T60R/P210A/E318Q
++
++++

269/270
V46R
E31K/V46R/P210A/E318Q
++
+++

271/272
A16S/K124A/R195L/K196V
A16S/E31K/K124A/R195L/ K196V/P210A/E318Q
++
+++

273/274
H274S
E31K/P210A/H274S/E318Q
++
+++

275/276
A331I
E31K/P210A/E318Q/A331I
++
+++

277/278
D174K/K196V
E31K/D174K/K196V/P210A/E318Q
++
+++

279/280
A155P/D174K/K196V
E31K/A155P/D174K/K196V/P210A/E318Q
++
+++

281/282
D174K/K196V/T226S
E31K/D174K/K196V/P210A/T226S/E318Q
++
+++

283/284
A331L
E31K/P210A/E318Q/A331L
++
+++

285/286
A16S/K124A/A155P/D174K/R195L
A16S/E31K/K124A/A155P/D174K/R195L/P210A/E318Q
++
+++

287/288
A16S/A26E/A155P/D174K/K196V
A16S/A26E/E31K/A155P/D174K/K196V/P210A/E318Q
++
+++

289/290
A26E/D174K/K196V/A210E/K284A
A26E/E31K/D174K/K196V/P210E/K284A/E318Q
++
+++

291/292
H212A
E31K/P210A/H212A/E318Q
++
+++

293/294
A16S/R195L/K196V/K284A
A16S/E31K/R195L/K196V/P210A/K284A/E318Q
++
+++

295/296
D174K
E31K/D174K/P210A/E318Q
++
+++

297/298
A16S/K124A/D174K/R195L/A210E/T226S/K284A
A16S/E31K/K124A/D174K/ R195L/P210E/T226S/K284A/E318Q
++
+++

299/300
A16S/D174K/R195L/K284A
A16S/E31K/D174K/R195L/ P210A/K284A/E318Q
++
+++

301/302
A26E/D174K/K196V/A210E/T226S/K284A
A26E/E31K/D174K/K196V/P210E/T226S/K284A/E318Q
++
+++

303/304
H212L
E31K/P210A/H212L/E318Q
++
+++

305/306
D174K/K196V/T226S/ K284A
E31K/D174K/K196V/P210A/T226S/K284A/E318Q
++
+++

307/308
K124A/D174K/K196V
E31K/K124A/D174K/K196V/P210A/E318Q
++
+++

309/310
A26E/D174K/R195L/A210E/T226S
A26E/E31K/D174K/R195L/P210E/T226S/E318Q
++
+++

311/312
G162A
E31K/G162A/P210A/E318Q
++
+

313/314
A16S/K124A/R195L/K196V/K284A
A16S/E31K/K124A/R195L/K196V/P210A/K284A/E318Q
++
+++

315/316
L206M/S343R
E31K/L206M/P210A/E318Q/S343R
++
+

317/318
A16S/R195L/K196V
A16S/E31K/R195L/K196V/P210A/E318Q
++
+++

319/320
A16S/A26E/D174K/R195L/K196V/A210E/T226S
A16S/A26E/E31K/D174K/R195L/K196V/P210E/T226S/E318Q
++
+++

321/322
A155P/R195L/K196V/T226S
E31K/A155P/R195L/K196V/P210A/T226S/E318Q
+
++

323/324
K18Q
K18Q/E31K/P210A/E318Q
+
++

325/326
A335R
E31K/P210A/E318Q/A335R
+
+

327/328
K124A/A155P/D174K/R195L/T226S
E31K/K124A/A155P/D174K/R195L/P210A/T226S/E318Q
+
+++

329/330
D17C
D17C/E31K/P210A/E318Q
+
++

331/332
A26E/A155P/D174K/R195L/T226S
A26E/E31K/A155P/D174K/R195L/P210A/T226S/E318Q
+
+++

333/334
R195L/K196V
E31K/R195L/K196V/P210 A/E318Q
+
++

335/336
G162T
E31K/G162T/P21OA/E318Q
+
++

337/338
P153H
E31K/P153H/P210A/E318Q
+
++

339/340
N316K
E31K/P210A/N316K/E318Q
+
++

341/342
A155P/D174K/R195L
E31K/A155P/D174K/R195L/P210A/E318Q
+
+++

343/344
P153Q
E31K/P153Q/P210A/E318Q
+
++

345/346
D174K/R195L/A210E/ K284A
E31K/D174K/R195L/P210E/K284A/E318Q
+
+++

347/348
D174K/R195L
E31K/D174K/R195L/P210A/E318Q
+
+++

349/350
P153S
E31K/P153S/P210A/E318Q
+
++

351/352
H212F
E31K/P210A/H212F/E318Q
+
++

353/354
K37C
E31K/K37C/P210A/E318Q
+
+

355/356
H212V
E31K/P210A/H212V/E318Q
+
+++

357/358
K124A/D174K/R195L/ T226S/K284A
E31K/K124A/D174K/R195L/P210A/T226S/K284A/E318Q
+
+++

359/360
V176L
E31K/V176L/P210A/E318Q
+
+

361/362
A16S/A155P/D174K/R195L/K196V/T226S/K284A
A16S/E31K/A155P/D174K/R195L/K196V/P210A/T226S/K284A/E318Q
+
+++

363/364
V10Q
V10Q/E31K/P210A/E318Q
+
++

365/366
D169G/K173V
E31K/D169G/K173V/P210A/E318Q
+
+

367/368
A155L
E31K/A155L/P210A/E318Q
+
+

369/370
K18G
K18G/E31K/P210A/E318Q
+
+++

371/372
T60L
E31K/T60L/P210A/E318Q
+
++

373/374
V10A
V10A/E31K/P210A/E318Q
+
++

375/376
K18R
K18R/E31K/P210A/E318Q
+
++

377/378
K33R
E31K/K33R/P210A/E318Q
+
++

379/380
H212G
E31K/P210A/H212G/E318Q
+
+

381/382
V10R
V10R/E31K/P210A/E318Q
+
++

383/384
H13R
H13R/E31K/P210A/E318Q
+
+

385/386
K173V/V183I/S343R/K347R
E31K/K173V/V183I/P210A/E318Q/S343R/K347R
+
+

387/388
A16R
A16R/E31K/P210A/E318Q
+
+

389/390
P104Q/N265C
E31K/P104Q/P210A/N265C /E318Q
+
+

391/392
T233R
E31K/P210A/T233R/E318Q
+
++

393/394
T233D
E31K/P210A/T233D/E318Q
+
++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 172. Levels of increased activity are defined as follows: “+” 0.5 to 0.9, “++” > 0.9, “+++” > 1.1,“+++”> 2.

Example 6
Screening Results of ODC Variants Based on SEQ ID NO: 320

Based on the results from Table 4-2, SEQ ID NO: 320 was chosen as the backbone. Beneficial mutations identified from Table 4-2 and homologs diversity were recombined into the backbone. Additionally, to generate new diversity, variants with mutations at active site positions were also constructed on SEQ ID NO: 320. The variants were assayed in triplicate for ODC activity at pH 2.6 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.1 mg/mL pepsin, pH 2.8) and unchallenged ODC activity at pH 2.4 and 6.5 with 4 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 320 are listed in Table 4-3.

TABLE 4-3

Oxalate decarboxylase activity Relative to SEQ ID NO: 320

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 320)
Amino Acid Differences (Relative to SEQ ID NO: 2 or 4)
FIOP Unchallenged Activity at pH 2.4 Relative to SEQ ID NO: 320
FIOP Unchallenged Activity at pH 6.5 Relative to SEQ ID NO: 320
FIOP Pepsin Challenge Relative to SEQ ID NO: 320

395/396
H13P/D17H/V196K
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/E318Q
+

++

397/398
D17H/H212S/A331V
A16S/D17H/A26E/E31K/D174K/R195L/K196V/P210E/H212S/T226S/E318Q/A331V
+++

+++

399/400
D17H/E26A/V196K/H212S/S226T
A16S/D17H/E31K/D174K/R195L/P210E/H212S/E318Q
++

+++

401/402
D17H/E26A/V196K/H212A/S226T/A331V
A16S/D17H/E31K/D174K/R195L/P210E/H212A/E318Q/A331V
+

+++

403/404
H13P/D17H/E26A/V196K/A331V
H13P/A16S/D17H/E31K/D174K/R195L/P210E/T226S/E318Q/A331V
++

++

405/406
D17H/T60R/V196K/S226T
A16S/D17H/A26E/E31K/T60R/D174K/R195L/P210E/E318Q
++
+
+++

407/408
D17H/V46R/H212S
A16S/D17H/A26E/E31K/V46R/D174K/R19SL/K196V/P210E/H212S/T226S/E318Q
+++

+

409/410
E26A/T60V/V196K/S226T/A331V
A16S/E31K/T60V/D174K/R195L/P210E/E318Q/A331V
+
+
+++

411/412
D17H/V46R/V196K/H212A/H274Q/A331V
A16S/D17H/A26E/E31K/V46R/D174K/R195L/P210E/H212A/T226S/H274Q/E318Q/A331V
+

+++

413/414
H212S
A16S/A26E/E31K/D174K/R195L/K196V/P210E/H212S/T226S/E318Q
++

++

415/416
D17H/V196K/S226T
A16S/D17H/A26E/E31K/D174K/R195L/P210E/E318Q
++
+
+++

417/418
H13P/D17H/E26A/H212A/A331V
H13P/A16S/D17H/E31K/D174K/R195L/K196V/P210E/H212A/T226S/E318Q/A331V
+

+++

419/420
H13P/D17H/H212S/S226T/A331V
H13P/A16S/D17H/A26E/E31K/D174K/R19SL/K196V/P210E/H212S/E318Q/A331V
++

++

421/422
V196K/H212S/A331V
16S/A26E/E31K/D174K/R195L/P210E/H212S/T226S/E318Q/A331V
++
+
+++

423/424
H13P/D17H/V196K/A331V
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/E318Q/A331V
++

+++

425/426
H13P/D17H/E26A/H212A/S226T/A331V
H13P/A16S/D17H/E31K/D174K/R195L/K196V/P210E/H212A/E318Q/A331V
+

++

427/428
D17H/T60R/V196K/H212A/S226T/A331V
A16S/D17H/A26E/E31K/T60R/D174K/R195L/P210E/H212A/E318Q/A331V
+

+++

429/430
H13P/D17H/H212S
H13P/A16S/D17H/A26E/E31K/D174K/R19SL/K196V/P210E/H212S/T226S/E318Q
++

++

431/432
H13P/D17H/E26A/A331V
H13P/A16S/D17H/E31K/D174K/R195L/K196V/P210E/T226S/E318Q/A331V
++

++

433/434
H13P/D17H
H13P/A16S/D17H/A26E/E31K/D174K/R19SL/K196V/P210E/T226S/E318Q
++

++

435/436
H13P/D17H/E26A/V196K
H13P/A16S/D17H/E31K/D174K/R195L/P210E/T226S/E318Q
+

++

437/438
V46R/V196K/H212S/S226T
A16S/A26E/E31K/V46R/D174K/R195L/P210E/H212S/E318Q
+

++

439/440
D17H/T60R/V196K
A16S/D17H/A26E/E31K/T60R/D174K/R195L/P210E/T226S/E318Q
++
+
+++

441/442
D17H/V196K/A331V
A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/E318Q/A331V
++
+
+++

443/444
H13P/D17H/V46R/S226T/A331V
H13P/A16S/D17H/A26E/E31K/V46R/D174K/R195L/K196V/P210E/E318Q/A331V
++

++

445/446
D17H/V196K/S226T/A331V
A16S/D17H/A26E/E31K/D174K/R195L/P210E/E318Q/A331V
+
+
+++

447/448
D17H/T60V/V196K/S226T
A16S/D17H/A26E/E31K/T60V/D174K/R195L/P210E/E318Q
++

+++

449/450
D17H/V46R/V196K/H212S/A331V
A16S/D17H/A26E/E31K/V46R/D174K/R195L/P210E/H212S/T226S/E318Q/A331V
++

++

451/452
H13P/D17H/H212A
H13P/A16S/D17H/A26E/E31K/D174K/R19SL/K196V/P210E/H212A/T226S/E318Q
+

++

453/454
D17H/V46R/H212S/A331V
A16S/D17H/A26E/E31K/V46R/D174K/R19SL/K196V/P210E/H212S/T226S/E318Q/A331V
++

+

455/456
D17H/V196K
A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/E318Q
++
+
+++

457/458
H13P/D17H/H212S/S226T
H13P/A16S/D17H/A26E/E31K/D174K/R19SL/K196V/P210E/H212S/E318Q
++

++

459/460
D17H/H212S
A16S/D17H/A26E/E31K/D174K/R195L/K196V/P210E/H212S/T226S/E318Q
+++

+++

461/462
D17H/E26A/H212S
A16S/D17H/E31K/D174K/R195L/K196V/P210E/H212S/T226S/E318Q
+++

+++

463/464
H13P/D17H/S226T/A331V
H13P/A16S/D17H/A26E/E31K/D174K/R19SL/K196V/P210E/E318Q/A331V
++

+++

465/466
D17H/V196K/H212S/A331V
A16S/D17H/A26E/E31K/D174K/R195L/P210E/H212S/T226S/E318Q/A331V
++

+++

467/468
D17H/V196K/H212S/S226T/A331V
A16S/D17H/A26E/E31K/D174K/R195L/P210E/H212S/E318Q/A331V
++

+++

469/470
H13P/D17H/E26A/S226T/A331V
H13P/A16S/D17H/E31K/D174K/R195L/K196V/P210E/E318Q/A331V
++

++

471/472
V46R/T60V/V196K/S226T/A331V
A16S/A26E/E31K/V46R/T60V/D174K/R195L/P210E/E318Q/A331V
++
+
+++

473/474
D17H/V46R/V196K/A331V
A16S/D17H/A26E/E31K/V46R/D174K/R195L/P210E/T226S/E318Q/A331V
++
++
++

475/476
D17H/H212A/A331V
A16S/D17H/A26E/E31K/D174K/R195L/K196V/P210E/H212A/T226S/E318Q/A331V

++

477/478
H13P/D17H/V46R/V196K
H13P/A16S/D17H/A26E/E31K/V46R/D174K/R195L/P210E/T226S/E318Q
+

++

479/480
V196K/H212S
A16S/A26E/E31K/D174K/R195L/P210E/H212S/T226S/E318Q
+

++

481/482
H13P/D17H/E26A
H13P/A16S/D17H/E31K/D174K/R195L/K196V/P210E/T226S/E318Q
++

+++

483/484
D17G/T83E/D263S
A16S/D17G/A26E/E31K/T83E/D174K/R19SL/K196V/P210E/T226S/D263S/E318Q
++
+
++

485/486
D17G/T83E/K173Q/R227E
A16S/D17G/A26E/E31K/T83E/K173Q/D174K/R195L/K196V/P210E/T226S/R227E/E318Q
++

+++

487/488
D17G/R227E/V301I
A16S/D17G/A26E/E31K/D174K/R195L/K196V/P210E/T226S/R227E/V301I/E318Q
++

++

489/490
D17G/T83E/R227E/D263S
A16S/D17G/A26E/E31K/T83E/D174K/R19SL/K196V/P210E/T226S/R227E/D263S/E318Q
+

++

491/492
D17G/T83E/P125E
A16S/D17G/A26E/E31K/T83E/P125E/D174K/R195L/K196V/P210E/T226S/E318Q
++
+
+++

493/494
D17G/T83E/K173Q
A16S/D17G/A26E/E31K/T83E/K173Q/D174K/R195L/K196V/P210E/T226S/E318Q
+++
++
+++

495/496
D17G/D263S/V301I
A16S/D17G/A26E/E31K/D174K/R195L/K196V/P210E/T226S/D263S/V301I/E318Q
++
+
++

497/498
D17G/P125E
A16S/D17G/A26E/E31K/P125E/D174K/R195L/K196V/P210E/T226S/E318Q
++
+
++

499/500
D17G/T83E/V301I
A16S/D17G/A26E/E31K/T83E/D174K/R19SL/K196V/P210E/T226S/V301I/E318Q
+++
++
+++

501/502
D17G/K173Q
A16S/D17G/A26E/E31K/K173Q/D174K/R195L/K196V/P210E/T226S/E318Q
++
+
+++

503/504
D17G/T83E/R227E
A16S/D17G/A26E/E31K/T83E/D174K/R19SL/K196V/P210E/T226S/R227E/E318Q
++

+++

505/506
D17G/R227E
A16S/D17G/A26E/E31K/D174K/R195L/K196V/P210E/T226S/R227E/E318Q
+

++

507/508
D17G
A16S/D17G/A26E/E31K/D174K/R195L/K196V/P210E/T226S/E318Q
++
+
+++

509/510
D17G/P125E/R227E
A16S/D17G/A26E/E31K/P125E/D174K/R195L/K196V/P210E/T226S/R227E/E318Q
+

++

511/512
D17G/T83E
A16S/D17G/A26E/E31K/T83E/D174K/R19SL/K196V/P210E/T226S/E318Q
+++
++
+++

513/514
D17G/D263S/V301T
A16S/D17G/A26E/E31K/D174K/R195L/K196V/P210E/T226S/D263S/V301T/E318Q
++
+
++

515/516
F128A
A16S/A26E/E31K/F128A/D174K/R195L/K196V/P210E/T226S/E318Q

+

517/518
A76S
A16S/A26E/E31K/A76S/D174K/R195L/K196V/P210E/T226S/E318Q
+
+
+

519/520
G141Q
A16S/A26E/E31K/G141Q/D174K/R195L/K196V/P210E/T226S/E318Q

+

521/522
F110V
A16S/A26E/E31K/F110V/D174K/R195L/K196V/P210E/T226S/E318Q

+

523/524
A79S
A16S/A26E/E31K/A79S/D174K/R195L/K196V/P210E/T226S/E318Q
++
++
++

525/526
M117A
A16S/A26E/E31K/M117A/D174K/R195L/K196V/P210E/T226S/E318Q
+
++

527/528
A79G
A16S/A26E/E31K/A79G/D174K/R195L/K196V/P210E/T226S/E318Q
++
++
+++

529/530
L61M
A16S/A26E/E31K/L61M/D174K/R195L/K196V/P210E/T226S/E318Q
+
+

All activities were determined relative to the reference polypeptide of SEQ ID NO: 320. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” > 1.1, “+++” > 1.5

Example 7
Screening Results of ODC Variants Based on SEQ ID NO: 396

Based on the results from Table 4-3, SEQ ID NO: 396 was chosen as the backbone. Beneficial mutations identified from Table 4-1 and Table 4-3, and diversity from homologs were recombined into the backbone. Additionally, to generate new diversity, variants with mutagenesis at additional positions were also constructed on SEQ ID NO: 396. The variants were assayed in triplicate for ODC activity at pH 2.4 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.4 mg/mL pepsin, pH 2.6) and unchallenged ODC activity at pH 2.4 and 5.5 with 4 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 396 are listed in Table 4-4.

TABLE 4-4

Oxalate decarboxylase activity Relative to SEQ ID NO: 396

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 396)
Amino Acid Differences (Relative to SEQ ID NO: 4)
FIOP Unchallenged Activity at pH 2.4 Relative to SEQ ID NO: 396
FIOP Unchallenged Activity at pH 5.5 Relative to SEQ ID NO: 396
FIOP Pepsin Challenge Relative to SEQ ID NO: 396

531/532
P13H/H17G/A79G
A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
++++
++++
+++

533/534
P13H/L14V/H17G/T60V/A79G
L14V/A16S/D17G/A26E/E31K/T60V/A79G/D174K/R195L/P210E/T226S/E318Q
+++
++++
+++

535/536
P13H/H17G/A79G/T83E
A16S/D17G/A26E/E31K/A79G/T83E/D174K/R195L/P210E/T226S/E318Q
++++
++++
+++

537/538
P13H/H17G/A79G/V301I
A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/V3011/E318Q
++++
++++
+++

539/540
P13H/L14V/H17G/T60V/A79G/H212S
L14V/A16S/D17G/A26E/E31K/T60V/A79G/D174K/R195L/P210E/H212S/T226S/E318Q
+++
+++
+++

541/542
H17G/T60V/L197E
H13P/A16S/D17G/A26E/E31K/T60V/D174K/R195L/L197E/P210E/T226S/E318Q
+++
+++
+

543/544
P13H/T60V/H212S
A16S/D17H/A26E/E31K/T60V/D174K/R195L/P210E/H212S/T226S/E318Q
+++
+++
+++

545/546
P13H/L14V/H17G/T60V/A79G/T83E/V301I
L14V/A16S/D17G/A26E/E31K/T60V/A79G/T83E/D174K/R195L/P210E/T226S/V301I/E318Q
+++
++++
+++

547/548
P13H/H17G/T60V/A79G
A16S/D17G/A26E/E31K/T60V/A79G/D174K/R195L/P210E/T226S/E318Q
++++
++++
+++

549/550
H17G/T60V/V301I
H13P/A16S/D17G/A26E/E31K/T60V/D174K/R195L/P210E/T226S/V301I/E318Q
+++
+++
+++

551/552
L14V/H17G/A79G
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

553/554
H17G/T60V/T83E
H13P/A16S/D17G/A26E/E31K/T60V/T83E/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

555/556
H17G/A79G
H13P/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

557/558
P13H/H17G/T60V/A79G/T83E/V301I
A16S/D17G/A26E/E31K/T60V/A79G/T83E/D174K/R195L/P210E/T226S/V301I/E318Q
++++
++++
+++

559/560
T342P
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/E318Q/T342P
++
++
+++

561/562
K52T/A190Q
H13P/A16S/D17H/A26E/E31K/K52T/D174K/A190Q/R195L/P210E/T226S/E318Q
+
++
+

563/564
Q94N/A190Q
H13P/A16S/D17H/A26E/E31K/Q94N/D174K/A190Q/R195L/P210E/T226S/E318Q
++
++
+

565/566
A190Q/T342P/Q351E
H13P/A16S/D17H/A26E/E31K/D174K/A190Q/R195L/P210E/T226S/E318Q/T342P/Q351E
++
++
+

567/568
P13H/A79G
A16S/D17H/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
+++
++++
+++

569/570
P13H/A96L
A16S/D17H/A26E/E31K/A96L/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

571/572
S273A
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/S273A/E318Q
+++
++
++

573/574
A126T
H13P/A16S/D17H/A26E/E31K/A126T/D174K/R195L/P210E/T226S/E318Q
+
++
++

575/576
A96K
H13P/A16S/D17Ή/A26E/E31K/A96K/D174K/R195L/P210E/T226S/E318Q
++
++
++

577/578
A79S
HI3P/A16S/D17H/A26E/E31K/A79S/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

579/580
T269S
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/T269S/E318Q
++
++
++

581/582
G40S
H13P/A16S/D17H/A26E/E31K/G40S/D174K/R195L/P210E/T226S/E318Q
++
+
+++

583/584
A96L
H13P/A16S/D17H/A26E/E31K/A96L/D174K/R195L/P210E/T226S/E318Q
+++
++
+++

585/586
T44S
H13P/A16S/D17H/A26E/E31K/T44S/D174K/R195L/P210E/T226S/E318Q
+
++
+

587/588
A96R
H13P/A16S/D17H/A26E/E31K/A96R/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

589/590
A79G
H13P/A16S/D17H/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
++
++
++

591/592
S267R
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/S267R/E318Q
++
++
++

593/594
1266V
H13P/A16S/D17Ή/A26E/E31K/D174K/R195L/P210E/T226S/I266V/E318Q
++
++
++

595/596
N160T
H13P/A16S/D17Ή/A26E/E31K/N160T/D174K/R195L/P210E/T226S/E318Q
+
+
+

597/598
Y277T
H13P/A16S/D17Ή/A26E/E31K/D174K/R195L/P210E/T226S/Y277T/E318Q
++
++
+

599/600
I149T
H13P/A16S/D17H/A26E/E31K/I149T/D174K/R195L/P210E/T226S/E318Q
++
++
+

601/602
T22R
H13P/A16S/D17H/T22R/A26E/E31K/D174K/R195L/P210E/T226S/E318Q
++
++
++

603/604
V234L
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/V234L/E318Q
+++
++
++

605/606
I54L
H13P/A16S/D17H/A26E/E31K/I54L/D174K/R195L/P210E/T226S/E318Q
++
++
++

607/608
S297Y
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/S297Y/E318Q
++
++
+++

609/610
Y277A
H13P/A16S/D17H/A26E/E31K/D174K/R195L/P210E/T226S/Y277A/E318Q
+++
++
++

611/612
D106C
H13P/A16S/D17H/A26E/E31K/D106C/D174K/R195L/P210E/T226S/E318Q
++
++
++

613/614
P13H/A43C
A16S/D17H/A26E/E31K/A43C/D174K/R195L/P210E/T226S/E318Q
+++
+++
+++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 396. Levels of increased activity are defined as follows: “+” 0.5 to 0.9, “++” > 0.9, “+++” > 1.1,“++++”> 2

Example 8
Screening Results of ODC Variants Based on SEQ ID NO: 670

Based on the results from Table 4-4, SEQ ID NO: 552 was selected, codon optimized, and 6xHis-tag removed to generate SEQ ID NO: 670, which was used as the next backbone. To generate new diversity, variants with mutagenesis at different positions were constructed into the backbone. The variants were assayed in triplicate for ODC activity at pH 2.5 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.8 mg/mL pepsin, pH 2.6) and unchallenged ODC activity at pH 2.5 and 5.5 with 4 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 670 are listed in Table 4-5.

TABLE 4-5

Oxalate decarboxylase activity Relative to SEQ ID NO: 670

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 670)
Amino Acid Differences (Relative to SEQ ID NO: 2)
FIOP Unchallenged Activity at pH 2.5 Relative to SEQ ID NO: 670
FIOP Unchallenged Activity at pH 5.5 Relative to SEQ ID NO: 670
FIOP Pepsin Challenge Relative to SEQ ID NO: 670

615/616
K37G
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/E318Q
++
++
+++

617/618
P125F
H13P/L14V/A16S/D17G/A26E/E31K/A79G/P125F/D174K/R195L/P210E/T226S/E318Q
+
+
+++

619/620
G162K
H13P/L14V/A16S/D17G/A26E/E31K/A79G/G162K/D174K/R195L/P210E/T226S/E318Q
++
++
+++

621/622
Q94T
H13P/L14V/A16S/D17G/A26E/E31K/A79G/Q94T/D174K/R195L/P210E/T226S/E318Q
++
++
+++

623/624
L195W
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195W/P210E/T226S/E318Q
++
++
+++

625/626
H85R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/H85R/D174K/R195L/P210E/T226S/E318Q
++
++
+++

627/628
G103S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/G103S/D174K/R195L/P210E/T226S/E318Q
++
++
+++

629/630
K232T
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/K232T/E318Q
++
++
+++

631/632
A189R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/A189R/R195L/P210E/T226S/E318Q
++
++
+++

633/634
A186N
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/A186N/R195L/P210E/T226S/E318Q
++
++
+++

635/636
A155R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/A155R/D174K/R195L/P210E/T226S/E318Q
++
++
+++

637/638
Q193A
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/Q193A/R195L/P210E/T226S/E318Q
++
++
+++

639/640
T342A
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/T342A
++
++
+++

641/642
K196V
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/K196V/P210E/T226S/E318Q
++
+
+++

643/644
P63S
H13P/L14V/A16S/D17G/A26E/E31K/P63S/A79G/D174K/R195L/P210E/T226S/E318Q
+
++
+++

645/646
K33R
H13P/L14V/A16S/D17G/A26E/E31K/K33R/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++

647/648
Q351H
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/Q351H
++
++
++

649/650
Q193G
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/Q193G/R195L/P210E/T226S/E318Q
++
++
++

651/652
P125S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/P125S/D174K/R195L/P210E/T226S/E318Q
++
++
++

653/654
T314S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/T314S/E318Q
++
++
++

655/656
T187R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/T187R/R195L/P210E/T226S/E318Q
+++
++
++

657/658
D303T
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/D303T/E318Q
++
++
++

659/660
G103V
H13P/L14V/A16S/D17G/A26E/E31K/A79G/G103V/D174K/R195L/P210E/T226S/E318Q
++
++
++

661/662
G103Q
H13P/L14V/A16S/D17G/A26E/E31K/A79G/G103Q/D174K/R195L/P210E/T226S/E318Q
++
++
++

663/664
A155V
H13P/L14V/A16S/D17G/A26E/E31K/A79G/A155V/D174K/R195L/P210E/T226S/E318Q
+
+
++

665/666
P164R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/P164R/D174K/R195L/P210E/T226S/E318Q
+
++
++

667/668
P153S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/P153S/D174K/R195L/P210E/T226S/E318Q
+
++
++

669/670

H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
++
++
++

671/672
K37R
H13P/L14V/A16S/D17G/A26E/E31K/K37R/A79G/D174K/R195L/P210E/T226S/E318Q
++
++
++

673/674
E346Q
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/E346Q
++
++
++

675/676
E346G
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/E346G
++
++
++

677/678
Q351A
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/Q351A
++
++
++

679/680
V183K
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/V183K/R195L/P210E/T226S/E318Q
++
++
++

681/682
D19C
H13P/L14V/A16S/D17G/D19C/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q
+
++
++

683/684
T187L
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/T187L/R195L/P210E/T226S/E318Q
++
+
++

685/686
D123S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D123S/D174K/R195L/P210E/T226S/E318Q
++
+
++

687/688
H85G
H13P/L14V/A16S/D17G/A26E/E31K/A79G/H85G/D174K/R195L/P210E/T226S/E318Q
++
++
++

689/690
T187S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/T187S/R195L/P210E/T226S/E318Q
++
++
++

691/692
V183C
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/V183C/R195L/P210E/T226S/E318Q
+
++
++

693/694
D123G
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D123G/D174K/R195L/P210E/T226S/E318Q
++
++
++

695/696
K350R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/K350R
++
++
++

697/698
H85E
H13P/L14V/A16S/D17G/A26E/E31K/A79G/H85E/D174K/R195L/P210E/T226S/E318Q
++
++
++

699/700
E205A
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/E205A/P210E/T226S/E318Q
+++
++
+

701/702
K284R
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/K284R/E318Q
++
++
+

703/704
P199V
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P199V/P210E/T226S/E318Q
+
++
+

705/706
E346W
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/E346W
+
++
+

707/708
S343W
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/S343W
+
++
+

709/710
P164V
H13P/L14V/A16S/D17G/A26E/E31K/A79G/P164V/D174K/R195L/P210E/T226S/E318Q
+
++
+

711/712
L200N
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/L200N/P210E/T226S/E318Q
+
++
+

713/714
T342E
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/E318Q/T342E
++
++
+

715/716
E208G
H13P/L14V/A16S/D1G/A26E/E31K/A79G/D174K/R195L/E208G/P210E/T226S/E318Q
++
++
+

717/718
D169A
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D169A/D174K/R195L/P210E/T226S/E318Q
+
++
+

719/720
A190G
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/A190G/R195L/P210E/T226S/E318Q
+
++
+

721/722
K284A
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/P210E/T226S/K284A/E318Q
++
++
+

723/724
P199G
H13P/L14V/A16S/D1G/A26E/E31K/A79G/D174K/R195L/P199G/P210E/T226S/E318Q
+
++
+

725/726
L121W
H13P/L14V/A16S/D17G/A26E/E31K/A79G/L121W/D174K/R195L/P210E/T226S/E318Q
++
++
+

727/728
K196S
H13P/L14V/A16S/D17G/A26E/E31K/A79G/D174K/R195L/K196S/P210E/T226S/E318Q
+
+
+

All activities were determined relative to the reference polypeptide of SEQ ID NO: 670. Levels of increased activity are defined as follows: “+” 0.5 to 0.9, “++” > 0.9, “+++” > 1.1,“++++”> 2

Example 9
Screening Results of ODC Variants Based on SEQ ID NO: 616

Based on the results from Table 4-5, SEQ ID NO: 616 was chosen as the backbone. To generate new diversity, beneficial mutations from Table 4-3 and Table 4-5 variants were recombined into the backbone, along with reversion mutations of neutral and deleterious mutations from Tables 4-1, 4-2, and 4-4. Additionally, to generate new diversity, variants with mutagenesis at different positions were constructed into the backbone. The variants were assayed in triplicate for ODC activity at pH 2.5 with 4 mM potassium oxalate after a 1-2 hour simulated gastric challenge (0.8 mg/mL pepsin, pH 2.5 or pH 2.4) and unchallenged ODC activity at pH 2.5 and 5.5 with 4 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 616 are listed in Table 4-6.

TABLE 4-6

Oxalate decarboxylase activity Relative to SEQ ID NO: 616

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 616)
Amino Acid Differences (Relative to SEQ ID NO: 2)
FIOP Unchallenged Activity at pH 2.5 Relative to SEQ ID NO: 616
FIOP Unchallenged Activity at pH 5.5 Relative to SEQ ID NO: 616
FIOP Pepsin Challenge (1 hr) Relative to SEQ ID NO: 616
FIOP Pepsin Challenge (2 hrs) Relative to SEQ ID NO: 616

729/730
V14L/E26A/T60V/Q318E
H13P/A16S/D17G/E31K/K37G/T60V/A79G/D174K/R195L/P210E/T226S
+
+
++
++++

731/732
V14L/E26A/T60V/Q94T/G162K/H212S
H13P/A16S/D17G/E31K/K37G/T60V/A79G/Q94T/G162K/D174K/R195L/P210E/H212S/T226S/E318Q
+

++
++++

733/734
T60V/G162K/S226T
H13P/L14V/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/E318Q
+
+
++
+++

735/736
T60V
H13P/L14V/A16S/D17G/A26E/E31K/K37G/T60V/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++
++

737/738
V14L/P125F/G162K/H212S/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/P125F/G162K/D174K/R195L/P210E/H212S/T226S

++
+++

739/740
T60V/P125F/S226T
H13P/L14V/A16S/D17G/A26E/E31K/K37G/T60V/A79G/P125F/D174K/R195L/P210E/E318Q

++
++

741/742
V14L/P125F/G162K/S226T
H13P/A16S/D17G/A26E/E31K/K37G/A79G/P125F/G162K/D174K/R195L/P210E/E318Q

+
++
+++

743/744
E26A/T60V/G162K/S226T/Q318E
H13P/L14V/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E
+
+
++
+++

745/746
V14L/E26A/Q94T/G162K/H212S/S226T
H13P/A16S/D17G/E31K/K37G/A79G/Q94T/G162K/D174K/R195L/P210E/H212S/E318Q
+

++
+++

747/748
V14L/T60V/G162K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/T226S/E318Q
+
+
++
++++

749/750
V14L/T60V/G162K/S226T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/E318Q
+
+
++
++++

751/752
V14L/Q94T/G162K/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/Q94T/G162K/D174K/R195L/P210E/T226S

+
++
+++

753/754
V14L/Q94T/G162K
H13P/A16S/D17G/A26E/E31K/K37G/A79G/Q94T/G162K/D174K/R195L/P210E/T226S/E318Q
+
+
++

755/756
V14L/T60V/S226T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/D174K/R195L/P210E/E318Q
+
+
++

757/758
V14L/T60V/Q94T/H212S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/Q94T/D174K/R195L/P210E/H212S/T226S/E318Q
+

++
++++

759/760
V14L/T60V/G162K/H212S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/H212S/T226S/E318Q
+

++
++++

761/762
V14L/G162K
H13P/A16S/D17G/A26E/E31K/K37G/A79G/G162K/D174K/R195L/P210E/T226S/E318Q
+
+
++
+++

763/764
V14L/Q94T/H212S/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/Q94T/D174K/R195L/P210E/H212S/T226S

++

765/766
V14L/T60V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++
++++

767/768
V14L/E26A/G162K
H13P/A16S/D17G/E31K/K37G/A79G/G162K/D174K/R195L/P210E/T226S/E318Q
+
+
++
++++

769/770
V14L
H13P/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++
+++

771/772
V14L/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S

++
++

773/774
V14L/G162K/S226T/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/G162K/D174K/R195L/P210E

++

775/776
V14L/P125F/H212S
H13P/A16S/D17G/A26E/E31K/K37G/A79G/P125F/D174K/R195L/P210E/H212S/T226S/E318Q

++
++

777/778
V14L/P125F/S226T/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/P125F/D174K/R195L/P210E

++

779/780
V14L/E26A/T60V/G162K
H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/T226S/E318Q
+
+
++
+++

781/782
V14L/P125F/G162K/H212S/S226T
H13P/A16S/D17G/A26E/E31K/K37G/A79G/P125F/G162K/D174K/R195L/P210E/H212S/E318Q

++
++

783/784
V14L/Q94T/P125F/G162K/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/Q94T/P125F/G162K/D174K/R195L/P210E/T226S
+
+
++
+++

785/786
V14L/P125F/S226T
H13P/A16S/D17G/A26E/E31K/K37G/A79G/P125F/D174K/R195L/P210E/E318Q

++
++

787/788
Y277R
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/Y277R/E318Q

+
++

789/790
Y277C
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/Y277C/E318Q
+
+
++
++

791/792
Y277T
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/Y277T/E318Q
+
+
++
++

793/794
S166R
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/S166R/D174K/R195L/P210E/T226S/E318Q
+
+
++
++

795/796
T233H
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/T233H/E318Q

++
+

797/798
T83R
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/T83R/D174K/R195L/P210E/T226S/E318Q
+
+
++
+

799/800
T60M
H13P/L14V/A16S/D17G/A26E/E31K/K37G/T60M/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++
++

801/802
Y277I
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/Y277I/E318Q
+
+
++
++

803/804
Y277M
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/Y277M/E318Q
+
+
++
+

805/806
T5S
TSS/H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++
++

807/808
T342R
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/E318Q/T342R
+
+
++
+

809/810
Y277V
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/Y277V/E318Q
+
+
++
++

811/812
T60R
H13P/L14V/A16S/D17G/A26E/E31K/K37G/T60R/A79G/D174K/R195L/P210E/T226S/E31Q

++
++

813/814
A96R
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/A96R/D174K/R195L/P210E/T226S/E31Q
++
+
++
++

815/816
K284S
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/D174K/R195L/P210E/T226S/K284S/E318Q
+
+
++
+

817/818
E26R
H13P/L14V/A16S/D17G/A26R/E31K/K37G/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++
++

819/820
E26N
H13P/L14V/A16S/D17G/A26N/E31K/K37G/A79G/D174K/R195L/P210E/T226S/E318Q
+
+
++

821/822
T83W
H13P/L14V/A16S/D17G/A26E/E31K/K37G/A79G/T83W/D174K/R195L/P210E/T226S/E318Q

++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 616. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” > 1.1, “+++” > 2.0,“++++”>3.0

Example 10
Screening Results of ODC Variants Based on SEQ ID NO: 750

Based on the results from Table 4-6, SEQ ID NO: 750 was chosen as the backbone. Beneficial mutations identified from Table 7 were recombined into the backbone, along with reversions of mutations from Table 4-6. The variants were assayed for ODC activity at pH 2.4 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.8 mg/mL pepsin, pH 2.2), and unchallenged ODC activity at pH 2.4 and 5.5 with 4 mM potassium oxalate as described in Example 3. Analysis of the data relative to SEQ ID NO: 750 are listed in Table 4-7.

TABLE 4-7

Oxalate decarboxylase activity Relative to SEQ ID NO: 750

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 750)
Amino Acid Differences (Relative to SEQ ID NO: 2)
FIOP Unchallenged Activity at pH 2.5 Relative to SEQ ID NO: 750
FIOP Unchallenged Activity at pH 5.5 Relative to SEQ ID NO: 750
FIOP Pepsin Challenge Relative to SEQ ID NO: 750

823/824
T5S/E26A/T83W/T342R
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/E318Q/T342R
+

+++

825/826
T5S/E26A/Y277T
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q

++++

827/828
T5S/A96L/Y277T
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/A96L/G162K/D174K/R195L/P210E/Y277T/E318Q
++
+
++++

829/830
T5S/T83W/Y277T/T342R
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
+

+++

831/832
E26A/T83W/Y277T
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277T/E318Q
+

++++

833/834
T5S/E26A/T83W/Y277V
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

835/836
T5S/Y277T/T342R
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
+

++++

837/838
T5S/T83W/Y277V/T342R
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++

+++

839/840
T5S/E26A/S166R/Y277T
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/S166R/D174K/R195L/P210E/Y277T/E318Q
++

+++

841/842
T83W/Y277T/T342R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
++

++++

843/844
T83R/A96L/Y277V/T342R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++
+
+++

845/846
T83W/A96L/Y277T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277T/E318Q
++

++++

847/848
E26A/T83W/Y277V/T342R
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++

++++

849/850
T5S/E26A/T83W/Y277V/T342R
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++

+++

851/852
T5S/T83R/Y277T/T342R
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
++

+++

853/854
T5S/E26A/T83W/Y277T/T342R
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
++

+++

855/856
T5S/T83R/Y277V
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277V/E318Q
+
+
+++

857/858
E26A/Y277T
H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q
+

+++

859/860
T83W/Y277T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277T/E318Q
+

+++

861/862
T5S/I35V/Y277T
T5S/H13P/A16S/D17G/A26E/E31K/I35V/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q

+++

863/864
T5S/E26A/T83R/A96R/Y277V
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83R/A96R/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
+++

865/866
T5S/E26A/A96L/Y277T
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/A96L/G162K/D174K/R195L/P210E/Y277T/E318Q
++
+
+++

867/868
T5S/Y277T
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q
+
+
+++

869/870
T5S/E26A/T83R/Y277T
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277T/E318Q
++
+
+++

871/872
T5S/T83R/Y277T
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277T/E318Q
+

++++

873/874
E26A/T83R/Y277V
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
+++

875/876
T5S/E26A/Y277V
T5S/H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277V/E318Q
+

+++

877/878
T5S/S166R/Y277T
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/S166R/D174K/R195L/P210E/Y277T/E318Q
++
+
+++

879/880
E26A/T83W/Y277V
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q
+
+
+++

881/882
E26A/T83W/A96L/Y277V/T342R
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++
+
++

883/884
Y277T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q
+
+
++++

885/886
E26A/Y277T/T342R
H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
++
+
+++

887/888
T83W/Y277V/T342R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++

+++

889/890
T83R/Y277V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277V/E318Q
+
+
++++

891/892
T5S
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/E318Q
+
+
+++

893/894
E26A/Y277V
H13P/A16S/D17G/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

895/896
T83R/Y277T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/G162K/D174K/R195L/P210E/Y277T/E318Q
+
+
++++

897/898
T5S/T83W/A96R/Y277T/T342R
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96R/G162K/D174K/R195L/P210E/Y277T/E318Q/T342R
++

+++

899/900
T5S/Y277V
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

901/902
Y277V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277V/E318Q
+
+
++++

903/904
T5S/Y277V/T342R
T5S/H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/Y277V/E318Q/T342R
++
+
++++

905/906
T83W/A96L/Y277V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

907/908
T83W/Y277V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 750. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” > 1.1, “+++” > 2.0,“++++”>3.0

Example 11
Screening Results of ODC Variants Based on SEQ ID NO: 906

Single point mutations were constructed on the ODC polypeptide of SEQ ID NO: 906 through site saturation mutagenesis at the 16 positions that differ from the ODC polypeptide of SEQ ID NO: 2. Additionally, reversion mutations at these 16 positions were recombined into SEQ ID NO: 906. The variants were assayed for ODC activity at pH 2.8 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.3 mg/mL pepsin, pH 3.2), and at pH 2.5 with 4 mM potassium oxalate after a 1 hour simulated gastric challenge (0.8 mg/mL pepsin, pH 2.8) as described in Example 3. Unchallenged ODC activity was assayed at pH 2.8 and 5.5 with 4 mM potassium oxalate as described in Example 3. Analysis of the activity data in reference to both ODC polypeptides of SEQ ID NO: 906 and SEQ ID NO: 2, are listed in Tables 5-1 and 5-2.

TABLE 5-1

Oxalate decarboxylase activity Relative to SEQ ID NO: 4 or SEQ ID NO: 906

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 906)
Amino Acid Differences (Relative to SEQ ID NO: 2)
FIOP Unchallenged Activity pH 2.8 relative to SEQ ID NO: 2
FIOP Unchallenged Activity pH 4.5 relative to SEQ ID NO: 2
FIOP 0.3 g/L Pepsin Challenge relative to SEQ ID NO: 2
FIOP Unchallenged Activity pH 2.8 relative to SEQ ID NO: 906
FIOP Unchallenged Activity pH 4.5 relative to SEQ ID NO: 906
FIOP 0.8 g/L Pepsin Challenge relative to SEQ ID NO: 906

½
P13H/S16A/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E

+
+
+

+

905/906

H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

909/910
E26L
H13P/A16S/D17G/A26L/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+

911/912
K31W
H13P/A16S/D17G/A26E/E31W/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

913/914
L96K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96K/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

915/916
V60R
H13P/A16S/D17G/A26E/E31K/K37G/T60R/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+
+

917/918
Q318D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318D
++

++++

919/920
E26G
H13P/A16S/D17G/A26G/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

921/922
E210R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210R/Y277V/E318Q
++

++++

923/924
K31R
H13P/A16S/D17G/A26E/E31R/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+

925/926
V277F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277F/E318Q
++
+
++++
+
+

927/928
G37M
H13P/A16S/D17G/A26E/E31K/K37M/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

929/930
L96P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96P/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

931/932
S16V
H13P/A16V/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

933/934
V60A
H13P/A16S/D17G/A26E/E31K/K37G/T60A/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

935/936
E26R
H13P/A16S/D17G/A26R/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

937/938
K31G
H13P/A16S/D17G/A26E/E31G/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

939/940
L195G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195G/P210E/Y277V/E318Q
++

++++

941/942
Q318R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318R
++

++++

943/944
E26V
H13P/A16S/D17G/A26V/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

945/946
G79M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79M/T883W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

947/948
S16M
H13P/A16M/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

949/950
E210A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210A/Y277V/E318Q
++
+
+++
+
+

951/952
L96R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96R/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
++
++

953/954
G37E
H13P/A16S/D17G/A26E/E31K/K37E/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

955/956
S16G
H13P/A16G/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
++

957/958
G17W
H13P/A16S/D17W/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

959/960
P13G
H13G/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

961/962
G79E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79E/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

963/964
V277T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277T/E318Q
+++
+
++++
+
+
++

965/966
V60Q
H13P/A16S/D17G/A26E/E31K/K37G/T60Q/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
+

967/968
K31L
H13P/A16S/D17G/A26E/E31L/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

969/970
L96G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96G/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

971/972
L96T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96T/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
++
++

973/974
V60G
H13P/A16S/D17G/A26E/E31K/K37G/T60G/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

975/976
S16W
H13P/A16W/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

977/978
P13T
H13T/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

979/980
G79R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79R/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

981/982
E26Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

983/984
E26S
H13P/A16S/D17G/A26S/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+

985/986
L195R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/P210E/Y277V/E318Q
+++
+
++++
+
+

987/988
G17R
H13P/A16S/D17R/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

989/990
P13V
H13V/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

991/992
V277E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277E/E318Q
++

++++

993/994
G37L
H13P/A16S/D17G/A26E/E31K/K37L/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

995/996
E210G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210G/Y277V/E318Q
++

++++

997/998
V60L
H13P/A16S/D17G/A26E/E31K/K37G/T60L/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

999/1000
P13L
H13L/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1001/1002
W83Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83Y/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++
++

1003/1004
G37W
H13P/A16S/D17G/A26E/E31K/K37W/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+++

1005/1006
V277G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277G/E318Q
+

++++

1007/1008
W83R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83R/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
+

1009/1010
V60Y
H13P/A16S/D17G/A26E/E31K/K37G/T60Y/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1011/1012
E210S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210S/Y277V/E318Q
++

++++

+

1013/1014
Q318S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318S
++

++++

1015/1016
G17I
H13P/A16S/D17I/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1017/1018
G37S
H13P/A16S/D17G/A26E/E31K/K37S/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1019/1020
G79L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79L/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1021/1022
S16R
H13P/A16R/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1023/1024
L195S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195S/P210E/Y277V/E318Q
++
+
++++

+

1025/1026
Q318G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318G
+

++++

1027/1028
E26C
H13P/A16S/D17G/A26C/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+

1029/1030
P13R
H13R/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1031/1032
L195Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195Y/P210E/Y277V/E318Q
++

++++

1033/1034
V60E
H13P/A16S/D17G/A26E/E31K/K37G/T60E/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+

1035/1036
K162V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162V/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+

1037/1038
S16C
H13P/A16C/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1039/1040
L96V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96V/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1041/1042
W83M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83M/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
++

1043/1044
K31M
H13P/A16S/D17G/A26E/E31M/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+
+

1045/1046
K174V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174V/R195L/P210E/Y277V/E318Q
++

++++

1047/1048
V277L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277L/E318Q
++

++++

1049/1050
E26M
H13P/A16S/D17G/A26M/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1051/1052
Q318M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318M
++

++++

1053/1054
E210N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210N/Y277V/E318Q
++

+++

1055/1056
E26A
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
+

1057/1058
V277R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277R/E318Q
++

++++

1059/1060
L96F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96F/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
+

1061/1062
K174G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174G/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1063/1064
S16Q
H13P/A16Q/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1065/1066
V277A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277A/E318Q
++

++++

1067/1068
P13Q
H13Q/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1069/1070
G79F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79F/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1071/1072
S16L
H13P/A16L/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1073/1074
L195N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195N/P210E/Y277V/E318Q
++
++
++++
+
++
+

1075/1076
G79V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79V/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1077/1078
G17P
H13P/A16S/D17P/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1079/1080
V60P
H13P/A16S/D17G/A26E/E31K/K37G/T60P/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1081/1082
G37P
H13P/A16S/D17G/A26E/E31K/K37P/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

++

1083/1084
L96W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96W/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1085/1086
K162T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162T/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1087/1088
L195M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195M/P210E/Y277V/E318Q
++
+
++++
+
++

1089/1090
Q318W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318W
+

++++

1091/1092
G37R
H13P/A16S/D17G/A26E/E31K/K37R/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1093/1094
S16T
H13P/A16T/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1095/1096
E210M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210M/Y277V/E318Q
++

+++

1097/1098
K162Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162Y/D174K/R195L/P210E/Y277V/E318Q
++

++++
+

+

1099/1100
S16P
H13P/A16P/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1101/1102
W83E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83E/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1103/1104
V277M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277M/E318Q
+++
+
++++
+
+

1105/1106
K174L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174L/R195L/P210E/Y277V/E318Q
++

++++

1107/1108
G17V
H13P/A16S/D17V/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1109/1110
G17E
H13P/A16S/D17E/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
+++
+
+

1111/1112
G17L
H13P/A16S/D17L/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1113/1114
L96M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96M/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1115/1116
Q318V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318V
++

++++

1117/1118
K174R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174R/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++

1119/1120
P13S
H13S/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+

1121/1122
K174T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174T/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
+

1123/1124
P13Y
H13Y/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1125/1126
L195A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195A/P210E/Y277V/E318Q
++
+
++++

++

1127/1128
V277W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277W/E318Q
++
+
++++
+
+

1129/1130
V60M
H13P/A16S/D17G/A26E/E31K/K37G/T60M/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+
+

1131/1132
G17A
H13P/A16S/D17A/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1133/1134
Q318A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318A
++

++++

1135/1136
K174H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174H/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1137/1138
W83S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83S/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++

1139/1140
V277I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277I/E318Q
+++

++++
+
+

1141/1142
P13E
H13E/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1143/1144
G17K
H13P/A16S/D17K/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1145/1146
E26K
H13P/A16S/D17G/A26K/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1147/1148
L96A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++

+
+

1149/1150
K174D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1151/1152
E210V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210V/Y277V/E318Q
++

+++

1153/1154
K31S
H13P/A16S/D17G/A26E/E31S/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1155/1156
L195C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195C/P210E/Y277V/E318Q
++
+
++++

+

1157/1158
G79K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79K/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1159/1160
V60W
H13P/A16S/D17G/A26E/E31K/K37G/T60W/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1161/1162
K174S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174S/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1163/1164
P13I
H13I/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1165/1166
V60H
H13P/A16S/D17G/A26E/E31K/K37G/T60H/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1167/1168
K31E
H13P/A16S/D17G/A26E/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
++
++++
++
++

1169/1170
L96E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96E/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
++

1171/1172
W83K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83K/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++
+

1173/1174
K31Q
H13P/A16S/D17G/A26E/E31Q/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++

++++
+
+

1175/1176
S16K
H13P/A16K/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1177/1178
K174M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174M/R195L/P210E/Y277V/E318Q
++

++++

1179/1180
G37V
H13P/A16S/D17G/A26E/E31K/K37V/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+++

1181/1182
G79D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79D/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1183/1184
Q318T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318T
++

++++

1185/1186
L195F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195F/P210E/Y277V/E318Q
+++
+
++++
+
+

1187/1188
E26T
H13P/A16S/D17G/A26T/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1189/1190
K174Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174Q/R195L/P210E/Y277V/E318Q
+++
+
+++
+
+
++

1191/1192
W83L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83L/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1193/1194
L96I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96I/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1195/1196
P13C
H13C/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1197/1198
P13A
H13A/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1199/1200
L96Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96Q/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+
+
++

1201/1202
P13H
A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1203/1204
W83T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
++
++++
++
++
+

1205/1206
K31Y
H13P/A16S/D17G/A26E/E31Y/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1207/1208
P13W
H13W/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+++

1209/1210
V277P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277P/E318Q

++

1211/1212
L195T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195T/P210E/Y277V/E318Q
++
+
++++

++

1213/1214
V60S
H13P/A16S/D17G/A26E/E31K/K37G/T60S/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1215/1216
E26Q
H13P/A16S/D17G/A26Q/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
+++
++
++
+

1217/1218
E210P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/Y277V/E318Q
++
+
+++

+

1219/1220
K174N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174N/R195L/P210E/Y277V/E318Q

1221/1222
W83A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83A/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++
+

1223/1224
K162E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162E/D174K/R195L/P210E/Y277V/E318Q
+++
+
+++
++
+
++

1225/1226
G37F
H13P/A16S/D17G/A26E/E31K/K37F/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

+++

1227/1228
G37Q
H13P/A16S/D17G/A26E/E31K/K37Q/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1229/1230
K31T
H13P/A16S/D17G/A26E/E31T/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1231/1232
K174A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174A/R195L/P210E/Y277V/E318Q
++

++++
+
+
+

1233/1234
W83F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83F/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++
++

1235/1236
V60T
H13P/A16S/D17G/A26E/E31K/K37G/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+

1237/1238
L96S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96S/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
++
+

1239/1240
W83V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83V/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
++
++++
++
++
++

1241/1242
E210I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210I/Y277V/E318Q
++

++++

1243/1244
S16D
H13P/A16D/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1245/1246
G37C
H13P/A16S/D17G/A26E/E31K/K37C/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1247/1248
W83G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83G/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++

1249/1250
P13N
H13N/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1251/1252
K31V
H13P/A16S/D17G/A26E/E31V/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++

++++
+
+

1253/1254
G79S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79S/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1255/1256
L96Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96Y/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1257/1258
G17F
H13P/A16S/D17F/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1259/1260
G17M
H13P/A16S/D17M/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1261/1262
S16A
H13P/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+

1263/1264
W83P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83P/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+

1265/1266
Q318L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318L
++

++++

1267/1268
G79Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79Q/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1269/1270
G17S
H13P/A16S/D17S/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1271/1272
E26W
H13P/A16S/D17G/A26W/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1273/1274
L195D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195D/P210E/Y277V/E318Q
++
+
++++

+

1275/1276
L195W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195W/P210E/Y277V/E318Q
++

++++

1277/1278
V277S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277S/E318Q
++

++++
+
+

1279/1280
K174E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174E/R195L/P210E/Y277V/E318Q
+++
++
++++
++
++
++

1281/1282
S16E
H13P/A16E/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++

+

1283/1284
K162F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162F/D174K/R195L/P210E/Y277V/E318Q

1285/1286
G79Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79Y/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1287/1288
G79A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1289/1290
G17C
H13P/A16S/D17C/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1291/1292
G17Y
H13P/A16S/D17Y/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1293/1294
L195Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195Q/P210E/Y277V/E318Q
+

++++

1295/1296
E26P
H13P/A16S/D17G/A26P/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1297/1298
L1951
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195I/P210E/Y277V/E318Q
++
+
++++

+
+

1299/1300
V60F
H13P/A16S/D17G/A26E/E31K/K37G/T60F/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1301/1302
E26I
H13P/A16S/D17G/A26I/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1303/1304
G17D
H13P/A16S/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1305/1306
Q318K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318K
++

++++

1307/1308
V60N
H13P/A16S/D17G/A26E/E31K/K37G/T60N/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+
+

1309/1310
S161
H13P/A16I/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1311/1312
Q318H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318H
++

++++

1313/1314
G37A
H13P/A16S/D17G/A26E/E31K/K37A/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1315/1316
L195V
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195V/P210E/Y277V/E318Q
++
+
++++
+
++
++

1317/1318
E210Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210Y/Y277V/E318Q
++

+++

1319/1320
L195E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195E/P210E/Y277V/E318Q
++

++++

+

1321/1322
E26H
H13P/A16S/D17G/A26H/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1323/1324
P13D
H13D/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1325/1326
K31P
H13P/A16S/D17G/A26E/E31P/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1327/1328
E210W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210W/Y277V/E318Q
+

++++

1329/1330
Q318P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318P

1331/1332
W83C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83C/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++
+

1333/1334
K174P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174P/R195L/P210E/Y277V/E318Q
++

++++

+

1335/1336
E210T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210T/Y277V/E318Q
++

++++

1337/1338
G17T
H13P/A16S/D17T/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1339/1340
G79T
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79T/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

++

1341/1342
L96H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96H/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
++
+

1343/1344
G79W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79W/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1345/1346
K162L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162L/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1347/1348
Q318E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V
++
+
++++
+
+
++

1349/1350
K31A
H13P/A16S/D17G/A26E/E31A/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1351/1352
E210C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210C/Y277V/E318Q
++

++++

1353/1354
G17N
H13P/A16S/D17N/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++

+

1355/1356
L195K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195K/P210E/Y277V/E318Q
++

++++

1357/1358
Q318Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Y
+

++++

1359/1360
G17H
H13P/A16S/D17H/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
+++
+
+

1361/1362
P13M
H13M/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1363/1364
V60D
H13P/A16S/D17G/A26E/E31K/K37G/T60D/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1365/1366
V277K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277K/E318Q
++

++++

1367/1368
S16N
H13P/A16N/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1369/1370
G79C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79C/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+++

1371/1372
P13K
H13K/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1373/1374
V60K
H13P/A16S/D17G/A26E/E31K/K37G/T60K/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
+

1375/1376
G37N
H13P/A16S/D17G/A26E/E31K/K37N/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1377/1378
V60I
H13P/A16S/D17G/A26E/E31K/K37G/T60I/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
++

1379/1380
K162D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162D/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++
+

1381/1382
K162I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162I/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1383/1384
S16Y
H13P/A16Y/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1385/1386
K162S
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162S/D174K/R195L/P210E/Y277V/E318Q
++

++++
+
+
++

1387/1388
L96C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96C/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
++
++++
+
++
++

1389/1390
E210K
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210K/Y277V/E318Q
++

+++

1391/1392
K174I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174I/R195L/P210E/Y277V/E318Q
++

++++

1393/1394
E210L
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210L/Y277V/E318Q
++

++++

1395/1396
E26D
H13P/A16S/D17G/A26D/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1397/1398
E26N
H13P/A16S/D17G/A26N/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1399/1400
E210D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210D/Y277V/E318Q
++

++++

1401/1402
P13F
H13F/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1403/1404
Q318N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318N
++

++++

1405/1406
V277Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/E318Q
++
+
++++
+
+

1407/1408
K174W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174W/R195L/P210E/Y277V/E318Q
++

++++

1409/1410
K31H
H13P/A16S/D17G/A26E/E31H/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1411/1412
K31D
H13P/A16S/D17G/A26E/E31D/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1413/1414
G79N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79N/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1415/1416
G37K
H13P/A16S/D17G/A26E/E31K/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1417/1418
K31I
H13P/A16S/D17G/A26E/E311/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1419/1420
G37H
H13P/A16S/D17G/A26E/E31K/K37H/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1421/1422
Q318C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318C
++

++++

1423/1424
K31F
H13P/A16S/D17G/A26E/E31F/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1425/1426
G79P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79P/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1427/1428
L195P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195P/P210E/Y277V/E318Q
++

++++

1429/1430
Q318I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318I
++

++++

1431/1432
K162A
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162A/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1433/1434
V277Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210EIY277Q/E318Q
++

++++
+
+

1435/1436
E26F
H13P/A16S/D17G/A26F/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1437/1438
V277C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210EIY277C/E318Q
++

++++

+

1439/1440
G79H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79H/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1441/1442
K31C
H13P/A16S/D17G/A26E/E31C/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1443/1444
G79I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79I/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

1445/1446
G37I
H13P/A16S/D17G/A26E/E31K/K37I/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q

+++

1447/1448
G17Q
H13P/A16S/D17Q/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
++

1449/1450
K31N
H13P/A16S/D17G/A26E/E31N/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1451/1452
V277N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277N/E318Q
++

++++

1453/1454
G37D
H13P/A16S/D17G/A26E/E31K/K37D/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1455/1456
V277H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277H/E318Q
++

++++
+
+

1457/1458
V277D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277D/E318Q
+

++++

1459/1460
L96D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96D/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
++

1461/1462
K174C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174C/R195L/P210E/Y277V/E318Q
++

++++

1463/1464
K162P
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162P/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1465/1466
Q318F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318F
+

++++

1467/1468
W83N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83N/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
+

1469/1470
V60C
H13P/A16S/D17G/A26E/E31K/K37G/T60C/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++
+

1471/1472
W83I
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83I/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1473/1474
K162M
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162M/D174K/R195L/P210E/Y277V/E318Q
+++

++++
+
+
++

1475/1476
E210H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210H/Y277V/E318Q
++

++++

1477/1478
L195H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195H/P210E/Y277V/E318Q
++
++
++++
+
++

1479/1480
K174Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174Y/R195L/P210E/Y277V/E318Q
++

++++

1481/1482
G37Y
H13P/A16S/D17G/A26E/E31K/K37Y/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

+++

1483/1484
E210Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210Q/Y277V/E318Q
++
+
++++
+
+
++

1485/1486
G37T
H13P/A16S/D17G/A26E/E31K/K37T/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

+++

1487/1488
L96N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96N/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

+

1489/1490
W83H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83H/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
+

1491/1492
S16H
H13P/A16H/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++

++++

1493/1494
K174F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174F/R195L/P210E/Y277V/E318Q
++

++++

1495/1496
W83D
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83D/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1497/1498
W83Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83Q/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
+

1499/1500
K162H
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162H/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
+

1501/1502
K162W
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162W/D174K/R195L/P210E/Y277V/E318Q
++

+++

1503/1504
K162N
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162N/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+
++

1505/1506
S16F
H13P/A16F/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V/E318Q
+

++++

1507/1508
K162Q
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162Q/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
++

1509/1510
K162G
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/D174K/R195L/P210E/Y277V/E318Q
++

++++
+
+
+

1511/1512
K162R
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162R/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
+
+
+

1513/1514
E210F
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210F/Y277V/E318Q
+

++++

1515/1516
K162C
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162C/D174K/R195L/P210E/Y277V/E318Q
+++
+
++++
++
+
++

Activities were determined relative to either the reference polypeptide of SEQ ID NO: 4 or SEQ ID NO: 906. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” > 1.1, “+++” > 2.0,“++++”>3.5

TABLE 5-2

Oxalate decarboxylase activity Relative to SEQ ID NO: 4 or SEQ ID NO: 906

SEQ ID NO: (nt/aa)
Amino Acid Differences (Relative to SEQ ID NO: 906)
Amino Acid Differences (Relative to SEQ ID NO: 2)
FIOP Unchallenged Activity at pH 2.8 relative to SEQ ID NO: 2
FIOP Unchallenged Activity at pH 4.5 relative to SEQ ID NO: 2
FIOP 0.3 g/L Pepsin Challenge relative to SEQ ID NO: 2
FIOP Unchallenged Activity at pH 4.5 relative to SEQ ID NO: 906
FIOP Unchallenged Activity at pH 2.8 relative to SEQ ID NO: 906
FIOP 0.8 g/L Pepsin Challenge relative to SEQ ID NO: 906

905/906

H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D 174K/R195L/P210E/Y277V/E318Q
++

+++
+
+
+

½
P13H/S16A/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174 D/L195R/E210P/V277Y/Q318E

+
+
+

++

1517/1518
K174D/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/R 195L/P210E/Y277V
+++
++
++++
++
++
++

1519/1520
E26A/L96A
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/G162K/D174K/R195L/P210E/Y277V/E318Q
++
+
+++

++
+

1521/1522
V60T/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V
+++
+
+++
++
++

1523/1524
K31E/Q318E
H13P/A16S/D17G/A26E/K37G/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E/Y277V
+++
+
+++
++
++

1525/1526
K162G/K174D/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/R195L/P 210E/Y277V
+++
+
+++
++
++
++

1527/1528
K174D/V277Y/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/R 195L/P210E
+++
+
+++
++
++
++

1529/1530
E26A/L195R/Q318 E
H13P/A16S/D17G/E31K/K37G/T60V/A79G/T83W/A96L/G162K/D174K/P210E/Y277V
++

+++
+
+
+

1531/1532
V60T/K162G/L195 R
H13P/A16S/D17G/A26E/E31K/K37G/A79G/T83W/A96L/D174K/P210E/Y277V/E318Q
+++
+
+++
++
++

1533/1534
V60T/K162G/Q318E
H13P/A16S/D17G/A26E/E31K/K37G/A79G/T83W/A96L1D174K/R195L1 P210E/Y277V
+++
+
+++
++
+

1535/1536
K174D/L195R/V277Y
H13P/A16S/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/P 210E/E318Q
++
+
+++
++
++

1537/1538
S16A/G17D/K174D
H13P/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1539/1540
V60T/L195R/V277Y
H13P/A16S/D17G/A26E/E31K/K37G/A79G/T83W/A96L/G162K/D174K/P210E/E318Q
++
+
+++
+
+

1541/1542
G37K/V277Y/Q318E
H13P/A16S/D17G/A26E/E31K/T60V/A79G/T83W/A96L/G162K/D174K/R195L/P210E
++
+
++++

+

1543/1544
S16A/K162G/L195 R/V277Y
H13P/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/D174K/P210E/E318Q
++

+++
+
+

1545/1546
E26A/G79A/W83T/K162G
H13P/A16S/D17G/E31K/K37G/T60V/A96L/D174K/R195L/P210E/Y277V/E318Q
++

++++

1547/1548
E26A/K31E/V60T/Q318E
H13P/A16S/D17G/K37G/A79G/T83W/A96L/G162K/D174K/R195L/P210 E/Y277V
+++
++
++++
++
++

1549/1550
S16A/K174D/L195 R/V277Y
H13P/D17G/A26E/E31K/K37G/T60V/A79G/T83W/A96L/G162K/P210E/E318Q
++
+
++++
+
+

1551/1552
P13H/G37K/W83T/K162G
A16S/D17G/A26E/E31K/T60V/A79G/A96L/D174K/R195L/P210E/Y277V/E318Q
++
+
++++
+
+

1553/1554
S16A/G17D/V60T/L195R
H13P/A26E/E31K/K37G/A79G/T83W/A96L/G162K/D174K/P210E/Y277V/E318Q
+++
+
+++
++
+

1555/1556
P13H/G17D/G37K/W83T
A16S/A26E/E31K/T60V/A79G/A96L/G162K/D174K/R195L/P210E/Y277 V/E318Q
++
+
+++

+

1557/1558
K31E/G37K/K162G/K174D/Q318E
H13P/A16S/D17G/A26E/T60V/A79G/T83W/A96L/R195L/P210E/Y277V
+++
+
+++
++
++

1559/1560
P13H/S16A/K31E/W83T/K162G
D17G/A26E/K37G/T60V/A79G/A96L/D174K/R195L/P210E/Y277V/E318 Q
+++
++
++++
+
++

1561/1562
S16A/V60T/K174D/L195R/Q318E
H13P/D17G/A26E/E31K/K37G/A79G/T83W/A96L/G162K/P210E/Y277V
+++
+
+++
+
+

1563/1564
G17D/V60T/L96A/K162G/L195R/V27 7Y
H13P/A16S/A26E/E31K/K37G/A79G/T83W/D174K/P210E/E318Q
+++
+
+++
+
++

1565/1566
P13H/E26A/V60T/W83T/K162G/K174D
A16S/D17G/E31K/K37G/A79G/A96L/R195L/P210E/Y277V/E318Q
+++
++
+++
++
++

1567/1568
G17D/K31E/V60T/K162G/K174D/V277Y
H13P/A16S/A26E/K37G/A79G/T83W/A96L/R195L/P210E/E318Q
+++
++
+++
++
++

1569/1570
S16A/G17D/E26A/W83T/L96A/V277Y
H13P/E31K/K37G/T60V/A79G/G162K/D174K/R195L/P210E/E318Q
++
++
++++

++

1571/1572
G17D/E26A/G37K/K174D/V277Y/Q318E
H13P/A16S/E31K/T60V/A79G/T83W/A96L/G162K/R195L/P210E
++
+
++++

++

1573/1574
P13H/S16A/K31E/V60T/K162G/K174 D
D17G/A26E/K37G/A79G/T83W/A96L/R195L/P210E/Y277V/E318Q
++
+
++++
+
++

1575/1576
P13H/G17D/K31E/W83T/V277Y/Q318E
A16S/A26E/K37G/T60V/AT9G/A96L/G162K/D174K/R195L/P210E
++
+
+++

++

1577/1578
P13H/S16A/G17D/K31E/W83T/K162 G/Q318E
A26E/K37G/T60V/A79G/A96L/D174K/R195L/P210E/Y277V
++
++
++++
+
++

1579/1580
P13H/S16A/V60T/W83T/K162G/L195R/Q318E
D17G/A26E/E31K/K37G/A79G/A96L/D174K/P210E/Y277V
++
++
++++

++

1581/1582
P13H/S16A/G17D/K31E/G37K/W83T/L96A/K162G
A26E/T60V/A79G/D174K/R195L/P210E/Y277V/E318Q
++
+
+++

++

1583/1584
P13H/G17D/E26A/G37K/V60T/W83T/K162G/E210P/Q318E
A16S/E31K/A79G/A96L/D174K/R195L/Y277V
++

++++

+
++++

1585/1586
P13H/S16A/E26A/G37K/W83T/K162 G/K174D/L195R/Q 318E
D17G/E31K/T60V/A79G/A96L/P210E/Y277V
+++
+
+++
++
++

1587/1588
P13H/S16A/E26A/K31E/W83T/L96A/K162G/K174D/Q31 8E
D17G/K37G/T60V/A79G/R195L/P210E/Y277V
++
++
+++
+
++

1589/1590
P13H/S16A/G17D/K31E/V60T/W83T/L96A/K162G/K174 D/L195R/Q318E
A26E/K37G/A79G/P210E/Y277V
+++
++
+++
+
++

1591/1592
P13H/S16A/E26A/ G37K/V60T/W83T/K162G/K174D/L19 5R/E210P/Q318E
D17G/E31K/A79G/A96L/Y277V
++
+
++++
+
++

1593/1594
P13H/S16A/G37K/G79A/W83T/L96A/K162G/K174D/L19 5R/E210P/V277Y
D17G/A26E/E31K/T60V/E318Q
++

++++

+

1595/1596
P13H/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/E210P
A16S/R195L/Y277V/E318Q
++

++++

+

1597/1598
P13H/S16A/G17D/E26A/G37K/G79A/W83T/L96A/K174 D/L195R/E210D/Q 318E
E31K/T60V/G162K/P210D/Y277V
++

++++

+

1599/1600
P13H/E26A/K31E/G37K/V60T/W83T/L96A/K162G/K174 D/L195R/E210P/V277Y/Q318E
A16S/D17G/A79G
++
+
++++

++

1601/1602
P13H/S16A/G17D/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L19 5R/E210P/Q318E
A26E/Y277V
++
+
+++

++

1603/1604
P13H/S16A/G17D/K31E/V60T/G79A/W83T/L96A/K162 G/K174D/L195R/E 210P/V277Y/Q318 E
A26E/K37G
+
+
+++

++

1605/1606
P13H/S16A/G17D/E26A/G37K/V60T/G79A/L96A/K162G/K174D/L195R/E210P/V277Y/Q318E
E31K/T83W

++

1607/1608
P13H/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162 G/K174D/L195R/E 210P/V277Y/Q318 E
A16S/D17G
++

+++

+

1609/1610
P13H/S16A/G17D/E26A/K31E/G37K/V60T/L96A/K162G /K174D/L195R/E210P/V277Y/Q318E
A79G/T83W
+
+
++

++

1611/1612
P13H/S16A/G17D/E26A/K31E/G37K/G79A/W83T/L96A/K162G/K174D/L19 5R/E210P/Q318E
T60V/Y277V
++
+
+++

++

1613/1614
P13H/S16A/G17D/E26A/K31E/G37K/V60T/G79A/L96A/K162G/K174D/E210P/V277Y/Q318E
T83W/R195L

++

1615/1616
P13H/G17D/E26A/K31E/G37K/V60T/G79A/W83T/L96A/K162G/K174D/L19 5R/E210P/V277Y/Q318E
A16S
++
+
+++

+

1617/1618
P13H/S16A/E26A/ K31E/G37K/V60T/ G79A/W83T/L96A/ K162G/K174D/L19 5R/E210P/V277Y/Q318E
D17G
++
+
+++

++

1619/1620
S16A/G17D/E26A/ K31E/G37K/V60T/ G79A/W83T/L96A/ K162G/K174D/L19 5R/E210P/V277Y/Q318E
H13P
++

+++

1621/1622
P13H/S16A/G17D/ E26A/K31E/G37K/ V60T/G79A/W83T/ L96A/K162G/K174 D/L195R/E210P/V277H/Q318E
Y277H
++
++
+++

++

Activities were determined relative to either the reference polypeptide of SEQ ID NO: 4 or SEQ ID NO: 906. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” > 1.1, “+++” > 2.0,“++++”>3.5

Example 12
In Vivo Dose Response in an Enteric Hyperoxaluria Mini Pig Model With Oral Treatment
Preparation of Engineered Oxalate Decarboxylase

The expression construct for SEQ ID NO: 905 was fermented at high cell density in a fed-batch format. The fermentation process consisted of an inoculum preparation phase and three main fermentation phases: batch, fed-batch growth, and fed-batch expression. Cells were harvested by bucket centrifugation, and the pellets were stored at -80° C. until further use.

Cell pellets were thawed, resuspended, and lysed by a homogenizer. The whole cell lysate was clarified by flocculation followed by centrifugation. The clarified lysate was concentrated and underwent an acid precipitation step to remove whole cell proteins. Upon centrifugation, the target enzyme in the clarified lysate was purified by cation exchange chromatography using a Tosoh GigaCap S 650 M column. The column eluate was concentrated, and buffer exchanged into processing buffer (25 mM Tris, 150 mM NaCl, pH 8.0) by tangential flow filtration against a 100 kDa PES membrane (Repligen). The buffer exchanged protein was then batch sterile filtered through a 0.2 µm PES filter (Sartorius Sartopore 2 XLG) and lyophilized to obtain a dried yellow powder.

In Vivo Studies

A validated model of enteric hyperoxaluria (HyOx) was produced in naive Gottingen minipigs (n=36 females, 15 kg average body weight, aged 7-8 months) and carried forward into in vivo studies. The study objective was to evaluate the ability of an orally administered engineered oxalate decarboxylase (OxDC) enzyme (SEQ ID NO: 906) to break down dietary oxalate in the GI tract, primarily the stomach, thereby lowering urine oxalate levels (UOx).

On days 0-6, all animals were fed a low oxalate diet (LOD: Research Diets Product D22071205 (0 g sodium oxalate/ kg feed)) to establish a baseline. Animals were then switched to a high oxalate diet during days 7-20 (HOD: Research Diets Product D22071206 (1.52 g sodium oxalate/ kg feed)) to simulate a disease state. On days 14-20, treatment (SEQ ID NO: 906) was admixed into HOD immediately prior to feeding. Animals were fed a set meal ration twice daily (AM and PM), approximately 8 hours apart, throughout the study. Animals were dosed with either 5,139 Units, 10,122 Units or 41,029 Units per meal, dependent upon group assignment. One unit of the engineered oxalate decarboxylase (SEQ ID NO: 906) is defined as the amount of engineered oxalate decarboxylase polypeptide which catalyzes the substrate and generates 1.0 µmol of formate per minute at 25° C. The unit of specific activity of SEQ ID NO: 906 is U/mg which is equivalent to µmol/min/mg.

Urine was collected for a total of 24 hours on each collection day, (days 5, 6, 12, 13, 19 and 20) for a total of 6 collections per animal. During urine collection, animals were single housed, and all urine excreted in a 24 hour period was collected into individual vessels. Urine volume was recorded, and aliquots were frozen at -70° C. for later UOx analysis using the oxalate analysis kit from Trinity BioSciences (591D-1KT).

Urine samples were prepared according to the kit directions (Trinity Biosciences, 591D-1KT), employing a minor modification to scale volumes for a plate reader in place of a cuvette. Data analysis was carried out using GraphPad Prism 9 (GraphPad Software, San Diego, CA). Individual UOx values (normalized as mg oxalate/24 hours) as well as percent change in UOx were examined.

To evaluate efficacy of SEQ ID NO: 906 on urine oxalate, a minimum threshold reduction of 20% in UOx from HOD to HOD + Treatment was set. This threshold value is a relevant endpoint for models of Hyperoxaluria because this reduction is believed to reduce the number of future kidney stone occurrences. The response to treatment appears to be dose dependent, as 6 of 6 individual animals responded with a 20% or greater reduction in UOx in the high dose group (FIG. 1). A reduction in UOx with treatment was observed in all groups, however this reduction was statistically significant in the highest dose group only (FIG. 2). In groups that received vehicle (PBS) during the treatment phase, there was no difference between HOD and HOD + Treatment.

ENGINEERED OXALATE DECARBOXYLASES

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

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Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)