ENGINEERED BRANCHED CHAIN KETOACID DECARBOXYLASES

FIELD OF THE INVENTION

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file CX7-170W02_ST25.txt, created on Jun. 26, 2019, with a size of 1,830,912 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Maple syrup urine disease (MSUD), also referred to as “branched chain ketoaciduria,” branched chain alpha-ketoacid dehydrogenase deficiency,” and “BCKD deficiency,” is a rare inherited aminoacidopathy secondary to dysfunction in the branched chain keto acid dehydrogenase (BCKDH) complex that is involved in the catabolic pathway of leucine, isoleucine and valine (i.e., branched chain amino acids). It was first described in 1954 by Menkes et al. (Menkes et al., Pediatrics 14:462-467 [1954]) and named due to the distinctive, sweet odor of the urine of affected newborns. It is also characterized by poor feeding, vomiting, lethargy, abnormal movements (e.g., hyper or hypotonia), and delayed development. Without treatment, the disease can progress to encephalopathy, seizures, coma, permanent neurologic damage, and death. Later in life, developmental delays, learning problems, seizures, and motor difficulties are common. There are four common forms that are classified based on the signs and symptoms of disease. The most common and severe type is the “classic” type, which becomes apparent within two weeks after birth. The other types are intermediate MSUD, intermittent MSUD, and thiamine-responsive MSUD. In the classic form, the disease becomes apparent after the newborn has ingested milk containing protein. This results in an increase in isoleucine, leucine, and valine in the body, which becomes toxic to the brain. In the intermittent form, brain damage occurs during times of physical stress (e.g., infection, fever or not eating for a prolonged period), which leads to metabolic decompensation.

Diagnostic testing for MSUD in newborns includes blood and urine amino acid tests to determine the leucine, isoleucine, alloioleucine, and valine concentrations in these fluids. If MSUD is identified, there will be signs of ketosis and acidosis. Upon diagnosis and during symptomatic episodes, treatment involves eating a protein-free diet and correction of the metabolic consequences associated with the elevated amino acid levels. Use of a special intravenous solution decreases the leucine level (the most toxic) and corrects energy deficits.

Current treatment involves dietary restriction of branched-chain amino acids (BCAAs). Deficient levels of BCKDH complex enzymes results in toxic accretion of BCAAs and their related metabolites in the cerebrospinal fluid, blood, and tissues. Without treatment or constant attentive care, this leads to numerous and serious side effects (e.g., neurological dysfunction, seizures, and infant death). Although some BCAA turnover via renal clearance (resulting in the typical sweet, maple syrup smell of affected patients' urine), it is not sufficient to provide relief from the accumulation of toxic amino acid levels in the body (See, Schadewalt and Wendel, Eur. J. Pediatr., 156(Suppl. 1): S62-66 [1997]; and Skvorak, J. Inherit. Metab. Dis., 32(2):229-46 [2009]).

SUMMARY OF THE INVENTION

The present invention provides engineered branched chain ketoacid decarboxylase (KdcA) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered branched chain ketoacid decarboxylase (KdcA) polypeptides. In some embodiments, the engineered KdcA polypeptides are optimized to provide enhanced catalytic activity, as well as improved serum stability. The invention also relates to the use of the compositions comprising the engineered KdcA polypeptides for therapeutic and industrial purposes. In some embodiments, the present invention is directed to engineered branched chain ketoacid decarboxylase (KdcA) polypeptides and biologically active fragments and analogs thereof having improved properties such an increased storage stability and/or reduced sensitivity to proteolysis.

The present invention is directed to engineered KdcA polypeptides and biologically active fragments and analogs thereof having improved properties when compared to a wild-type KdcA enzyme or a reference KdcA polypeptide under essentially the same conditions. The invention is further directed to methods of using the engineered KdcA polypeptides and biologically active fragments and analogs thereof in therapeutic and/or industrial compositions and methods of using such compositions for therapeutic and/or industrial purposes.

The present invention provides engineered ketoacid decarboxylase polypeptides comprising amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to at least one of SEQ ID NO:2, 46, and/or 58, wherein the amino acid positions of said amino acid sequences are numbered with reference to the amino acid sequence of SEQ ID NO: 2, 46, or 58. In some embodiments, the engineered ketoacid decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2, and wherein the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 28, 37, 121/331, 124/442, 144/184, 153/264, 153/442, 174/453, 329, 355, 388, 419, 437, 442, 471, 500, and 510, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from 28F, 37A, 121L/331S, 124E/442A, 144P/184N, 153A/264E, 153A/442A, 174D/453T, 329K, 355L, 388P, 419D, 437S, 442A, 471P, 500I, and 510F, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from N28F, S37A, K121L/K331S, M124E/S442A, Y144P/S184N, L153A/1264E, L153A/S442A, E174D/I453T, E329K, S355L, F388P, K419D, Q437S, S442A, S471P, R500I, and K510F, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2.

In some additional embodiments, the engineered ketoacid decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 46, and wherein the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 42/124/136/204/321/328/437/471/510, 42/124/136/204/437/471, 42/124/136/204/437/471/510, 42/124/136/331/437/471, 42/124/204/321/471, 42/124/204/471, 42/124/321/355/471, 42/136/204/321/328/331/355/471, 42/136/204/328/331/355/471, 42/136/328/331/471, 42/136/355/471/510, 42/204/321/328/342/355/471/510, 42/204/321/355/437/471/510, 42/204/328/342/437/471, 42/321/331/437/471, 42/321/437/471, 42/321/471, 42/328/471, 42/355/471/510, 42/471, 107/124/233/260/325/328/331/355/388/471, 107/124/233/355/446/471, 107/124/348/388/471, 107/124/471, 107/183/185/233/328/331/351/388/419/471, 107/183/233/260/328/348/351/388/471, 107/183/233/260/328/348/351/471, 107/183/233/260/331/348/351/419/471, 107/183/233/260/331/419/471, 107/183/260/328/348/351/471, 107/183/348/351/471, 107/185/233/328/351/388/471, 107/185/348/351/388/471, 107/233/260/328/331/351/471, 107/233/260/328/348/351/388/471, 107/233/348/471, 107/260/328/471, 107/325/328/348/388/471, 107/325/328/348/446/471, 107/325/331/446/471, 107/328/331/388/471, 107/328/331/471, 107/348/355/388/446/471, 107/348/388/471, 107/355/471, 107/388/471, 124/136/204/355/437/471, 124/136/321/355/471, 124/136/328/331/355/437/471/510, 124/136/331/471, 124/136/471, 124/260/331/355/446/471, 124/321/328/471/510, 124/321/331/342/471/510, 124/325/331/471, 124/325/471, 124/328/331/338/471, 124/331/338/342/355/471, 124/331/342/355/471, 124/338/342/471, 124/355/471, 136/204/328/331/355/437/471, 136/204/437/471, 136/321/471/510, 136/331/338/342/471, 136/437/471, 183/233/351/388/471, 185/233/260/328/331/419/471, 185/328/331/471, 185/471, 204/321/338/471, 233/325/328/331/348/355/471, 233/331/388/446/471, 260/325/331/446/471, 321/328/331/471, 325/328/331/348/355/388/471, 325/328/348/355/388/471, 325/331/388/471, 325/331/446/471, 328/331/355/471, 328/471, 338/342/355/471, 338/342/471, 338/342/471/510, 342/355/437/471, 342/355/471, 351/471, and 388/471, wherein the amino acid positions are numbered with reference to SEQ ID NO: 46. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from 42R/124R/136H/204W/321S/328L/437S/471P/510F, 42R/124R/136H/204W/437S/471P, 42R/124R/136H/204W/437S/471P/510F, 42R/124R/136H/331N/437S/471P, 42R/124R/204W/321S/471P, 42R/124R/204W/471P, 42R/124R/321S/355M/471P, 42R/136H/204W/321S/328L/331N/355L/471P, 42R/136H/204W/328L/331T/355L/471P, 42R/136H/328L/331T/471P, 42R/136H/355M/471P/510F, 42R/204W/321S/328L/342R/355L/471P/510F, 42R/204W/321S/355M/437S/471P/510F, 42R/204W/328L/342R/437S/471P, 42R/321S/331N/437S/471P, 42R/321S/437S/471P, 42R/321S/471P, 42R/328L/471P, 42R/355M/471P/510F, 42R/471P, 107R/124W/233H/260S/325F/328N/331L/355L/388P/471P, 107R/124W/233H/355L/446R/471P, 107R/124W/348D/388P/471P, 107R/124W/471P, 107R/183Y/185P/233R/328N/331L/351V/388P/419D/471P, 107R/183Y/233H/260S/328N/348D/351V/471P, 107R/183Y/233H/260S/331L/419D/471P, 107R/183Y/233R/260S/328N/348D/351V/388P/471P, 107R/183Y/233R/260S/331L/348D/351V/419D/471P, 1078/183Y/260S/328N/348D/351V/471P, 107R/183Y/348D/351V/471P, 1078/185P/233H/328N/351V/388P/471P, 107R/185P/348D/351V/388P/471P, 1078/233H/260S/328N/331L/351V/471P, 107R/233H/260S/328N/348D/351V/388P/471P, 107R/233H/348D/471P, 107R/260S/328N/471P, 107R/325F/328N/348D/388P/471P, 107R/325F/328N/348D/446R/471P, 107R/325F/331L/446R/471P, 107R/328N/331L/388P/471P, 107R/328N/331L/471P, 107R/348D/355L/388P/446R/471P, 107R/348D/388P/471P, 107R/355L/471P, 107R/388P/471P, 124R/136H/204W/355M/437S/471P, 124R/136H/321S/355L/471P, 124R/136H/328L/3311/355M/437S/471P/510F, 124R/136H/331N/471P, 124R/136H/471P, 124R/321S/328L/471P/510F, 124R/321S/331T/342R/471P/510F, 124R/328L/331T/338E/471P, 124R/331N/338E/342R/355L/471P, 124R/331T/342R/355L/471P, 124R/338E/342R/471P, 124R/355M/471P, 124W/260S/331L/355L/446R/471P, 124W/325F/331L/471P, 124W/325F/471P, 136H/204W/328L/331N/355M/437S/471P, 136H/204W/437S/471P, 136H/321S/471P/510F, 136H/331T/338E/342R/471P, 136H/437S/471P, 183Y/233H/351V/388P/471P, 185P/233H/260S/328N/331L/419D/471P, 185P/328N/331L/471P, 185P/471P, 204W/321S/338E/471P, 233H/325F/328N/331L/348D/355L/471P, 233H/331L/388P/446R/471P, 260S/325F/331L/446R/471P, 321S/328L/331T/471P, 325F/328N/331L/348D/355L/388P/471P, 325F/328N/348D/355L/388P/471P, 325F/331L/388P/471P, 325F/331L/446R/471P, 328L/331T/355M/471P, 328N/471P, 338E/342R/355L/471P, 338E/342R/471P, 338E/342R/471P/510F, 342R/355L/437S/471P, 342R/355M/471P, 351V/471P, and 388P/471P, wherein the amino acid positions are numbered with reference to SEQ ID NO: 46. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from K42R/M124R/L136H/N204W/R321S/S328L/Q437S/S471P/K510F, K42R/M124R/L136H/N204W/Q437S/S471P, K42R/M124R/L136H/N204W/Q437S/S471P/K510F, K42R/M124R/L136H/K331N/Q437S/S471P, K42R/M124R/N204W/R321S/S471P, K42R/M124R/N204W/S471P, K42R/M124R/R321S/S355M/S471P, K42R/L136H/N204W/R321S/S328L/K331N/S355L/5471P, K42R/L136H/N204W/S328L/K331T/S355L/S471P, K42R/L136H/S328L/K331T/S471P, K42R/L136H/S355M/S471P/K510F, K42R/N204W/R321S/S328L/K342R/S355L/S471P/K510F, K42R/N204W/R321S/S355M/Q437S/S471P/K510F, K42R/N204W/S328L/K342R/Q437S/S471P, K42R/R321S/K331N/Q437S/S471P, K42R/R321S/Q437S/S471P, K42R/R321S/S471P, K42R/S328L/S471P, K42R/S355M/S471P/K510F, K42R/S471P, D107R/M124W/K233H/K260S/5325F/S328N/K331L/5355L/F388P/5471P, D107R/M124W/K233H/5355L/K446R/5471P, D107R/M124W/1348D/F388P/S471P, D107R/M124W/S471P, D107R/E183Y/S185P/K233R/S328N/K331L/S351V/F388P/K419D/S471P, D107R/E183Y/K233H/K260S/S328N/1348D/5351V/5471P, D107R/E183Y/K233H/K260S/K331L/K419D/S471P, D107R/E183Y/K233R/K260S/S328N/1348D/S351V/F388P/S471P, D107R/E183Y/K233R/K260S/K331L/1348D/S351V/K419D/S471P, D107R/E183Y/K260S/S328N/1348D/S351V/S471P, D107R/E183Y/1348D/S351V/S471P, D107R/S185P/K233H/S328N/S351V/F388P/S471P, D107R/S185P/1348D/S351V/F388P/S471P, D107R/K233H/K260S/S328N/K331L/S351V/S471P, D107R/K233H/K260S/S328N/1348D/S351V/F388P/S471P, D107R/K233H/1348D/S471P, D107R/K260S/S328N/S471P, D107R/S325F/S328N/1348D/F388P/S471P, D107R/S325F/S328N/1348D/K446R/S471P, D107R/S325F/K331L/K446R/S471P, D107R/S328N/K331L/F388P/S471P, D107R/S328N/K331L/S471P, D107R/1348D/S355L/F388P/K446R/S471P, D107R/1348D/F388P/S471P, D107R/S355L/S471P, D107R/F388P/S471P, M124R/L136H/N204W/S355M/Q437S/S471P, M124R/L136H/R321S/S355L/S471P, M124R/L136H/S328L/K331T/S355M/Q437S/S471P/K510F, M124R/L136H/K331N/S471P, M124R/L136H/S471P, M124R/R321S/S328L/S471P/K510F, M124R/R321S/K331T/K342R/S471P/K510F, M124R/S328L/K331T/Q338E/S471P, M124R/K331N/Q338E/K342R/S355L/S471P, M124R/K331T/K342R/S355L/S471P, M124R/Q338E/K342R/S471P, M124R/S355M/S471P, M124W/K260S/K331L/S355L/K446R/S471P, M124W/S325F/K331L/S471P, M124W/S325F/S471P, L136H/N204W/S328L/K331N/S355M/Q437S/S471P, L136H/N204W/Q437S/S471P, L136H/R321S/S471P/K510F, L136H/K331T/Q338E/K342R/S471P, L136H/Q437S/S471P, E183Y/K233H/S351V/F388P/S471P, S185P/K233H/K260S/S328N/K331L/K419D/S471P, S185P/S328N/K331L/S471P, S185P/S471P, N204W/R321S/Q338E/S471P, K233H/S325F/S328N/K331L/I348D/S355L/S471P, K233H/K331L/F388P/K446R/S471P, K260S/S325F/K331L/K446R/S471P, R321S/S328L/K331T/S471P, S325F/S328N/K331L/I348D/S355L/F388P/S471P, S325F/S328N/I348D/S355L/F388P/S471P, S325F/K331L/F388P/S471P, S325F/K331L/K446R/S471P, S328L/K331T/S355M/S471P, S328N/S471P, Q338E/K342R/S355L/S471P, Q338E/K342R/S471P, Q338E/K342R/S471P/K510F, K342R/S355L/Q437S/S471P, K342R/S355M/S471P, S351V/S471P, and F388P/S471P, wherein the amino acid positions are numbered with reference to SEQ ID NO: 46.

In some further embodiments, the engineered ketoacid decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 58, and wherein the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 18/189/193/260/328, 179, 189, 189/203, 249, 314, 334, 346, 368, 369, 392, 507, 511, 516, 527, and 529, wherein the amino acid positions are numbered with reference to SEQ ID NO: 58. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from 18D/189E/193E/260S/328L, 179V, 189E, 189E/203Q, 249A, 314A, 314S, 314T, 334D, 334G, 334L, 334Q, 346R, 346V, 368E, 369A, 369M, 369R, 369T, 392A, 392G, 392M, 392R, 392S, 507A, 507G, 507K, 511K, 511L, 511V, 516A, 527G, 527R, 529C, 529N, 529R, and 529W, wherein the amino acid positions are numbered with reference to SEQ ID NO: 58. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from E18D/T189E/V193E/K260S/N328L, S179V, T189E, T189E/K203Q, S249A, V314A, V314S, V314T, E334D, E334G, E334L, E334Q, E346R, E346V, Q368E, S369A, S369M, S369R, S369T, N392A, N392G, N392M, N392R, N392S, S507A, S507G, S507K, E511K, E511L, E511V, V516A, E527G, E527R, E529C, E529N, E529R, and E529W, wherein the amino acid positions are numbered with reference to SEQ ID NO: 58.

In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from 37A, 37A/144T/229I/233N/320L/388P, 37A/144T/351D/352E, 37A/351D/388P, 37A/388P, 37D, 37G, 38H, 38S, 124E/153A/442A, 124E/442A, 124K, 144R, 144T, 144T/233N/388P, 144T/394H, 151K, 151Q, 151W, 153A/264E, 153A/264E/275L/394H/442A, 153A/275L, 153A/442A, 229I, 229L, 231K/232F/275L/281A/442A, 233L, 233N/350K/351D/355T/388P/394H, 234A, 250D, 252H, 252H/275L/442A, 253H, 253Y, 254A, 254G, 254S, 256T, 256V, 262T, 278C, 290G/541I, 321E, 321L, 321S, 325F, 325H, 325L, 347L, 347M, 348C, 350K/351D/352E/355T/388P/394H, 350T, 350Y, 352I, 352R, 352T, 352W, 353E, 355E, 355F, 355K, 355V, 355W, 388D, 388N, 388P/394H, 388W, 441T, 442A, 443V, 443W, 446H, 446L, and 541I, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from S37A, S37A/Y144T/V229I/K233N/F320L/F388P, S37A/Y144T/S351D/A352E, S37A/S351D/F388P, S37A/F388P, S37D, S37G, R38H, R38S, M124E/L153A/S442A, M124E/S442A, M124K, Y144R, Y144T, Y144T/K233N/F388P, Y144T/R394H, S151K, S151Q, S151W, L153A/1264E, L153A/1264E/F275L/R394H/S442A, L153A/F275L, L153A/S442A, V229I, V229L, E231K/T232F/F275L/V281A/S442A, K233L, K233N/S350K/S351D/S355T/F388P/R394H, L234A, L250D, S252H, S252H/F275L/S442A, F253H, F253Y, L254A, L254G, L254S, I256T, I256V, S262T, M278C, A290G/L541I, R321E, R321L, R321S, S325F, S325H, S325L, F347L, F347M, I348C, S350K/S351D/A352E/S355T/F388P/R394H, S350T, S350Y, A352I, A352R, A352T, A352W, P353E, S355E, S355F, S355K, S355V, S355W, F388D, F388N, F388P/R394H, F388W, L441T, S442A, I443V, 1443W, K446H, K446L, and L541I, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2.

In some embodiments, the engineered ketoacid decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 46, and wherein the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 38/124/355, 38/229/253/256/355, 38/325/355, 38/355, 124/152/229/233/321/328/352/446, 124/152/229/233/321/350, 124/152/229/321/348/350/388/482, 124/152/254/328/350/388/446, 124/152/321/348/350/388, 124/152/388, 124/152/388/446, 124/229/256/321/325/355, 124/233/254/328/348/352/388, 124/254/328/348/350, 124/321/325/355, 124/321/348/388, 124/328, 124/328/348/350/352/388, 124/328/348/352/388, 124/328/350/352/446, 124/328/352/388, 124/348/352, 152/229/233/348/350/352, 152/254/321/348, 152/328/388/446, 229/321/446, 233/254/348, 233/325/388/443/446, 253/254/321/325/355, 275/325/446, 321/350/352/446, 328, and 353, wherein the amino acid positions are numbered with reference to SEQ ID NO: 46. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from 38H/124K/355E, 38H/229A/253H/256V/355E, 38H/325F/355L, 38H/355E, 124K/229A/256V/321E/325F/355E, 124K/321E/325F/355L, 124W/152R/229A/233H/321S/328L/352Y/446R, 124W/152R/229A/233H/321S/350G, 124W/152R/229A/321S/348D/350G/388P/482R, 124W/152R/254G/328N/350G/388P/446R, 124W/152R/321S/348D/350G/388P, 124W/152R/388P, 124W/152R/388P/446R, 124W/233H/254G/328L/348D/352N/388P, 124W/254G/328N/348D/350G, 124W/321S/348D/388P, 124W/328L/348D/350G/352Y/388P, 124W/328L/348D/352N/388P, 124W/328L/350G/352N/446R, 124W/328L/352Y/388P, 124W/328N, 124W/348D/352N, 152R/229A/233H/348D/350G/352N, 152R/254G/321S/348D, 152R/328N/388P/446R, 229A/321S/446R, 233H/254G/348D, 233L/325L/388D/443W/446E, 253Y/254A/321E/325F/355E, 275Q/325F/446E, 321S/350G/352N/446R, 328L, and 353D, wherein the amino acid positions are numbered with reference to SEQ ID NO: 46. In some additional embodiments, the polypeptide sequence of said engineered ketoacid decarboxylase polypeptide comprises at least one substitution or substitution set selected from R38H/M124K/S355E, R38H/V229A/F253H/1256V/5355E, R38H/S325F/S355L, R38H/S355E, M124K/V229A/1256V/R321E/S325F/S355E, M124K/R321E/S325F/S355L, M124W/Q152R/V229A/K233H/R321S/S328L/A352Y/K446R, M124W/Q152R/V229A/K233H/R321S/5350G, M124W/Q152R/V229A/R321S/1348D/5350G/F388P/K482R, M124W/Q152R/L254G/S328N/5350G/F388P/K446R, M124W/Q152R/R321S/I348D/5350G/F388P, M124W/Q152R/F388P, M124W/Q152R/F388P/K446R, M124W/K233H/L254G/S328L/1348D/A352N/F388P, M124W/L254G/S328N/1348D/S350G, M124W/R321S/1348D/F388P, M124W/S328L/1348D/5350G/A352Y/F388P, M124W/S328L/1348D/A352N/F388P, M124W/S328L/S350G/A352N/K446R, M124W/S328L/A352Y/F388P, M124W/S328N, M124W/1348D/A352N, Q152R/V229A/K233H/1348D/5350G/A352N, Q152R/L254G/R321S/1348D, Q152R/S328N/F388P/K446R, V229A/R321S/K446R, K233H/L254G/I348D, K233L/S325L/F388D/I443W/K446E, F253Y/L254A/R321E/S325F/S355E, F275Q/S325F/K446E, R321S/S350G/A352N/K446R, S328L, and P353D, wherein the amino acid positions are numbered with reference to SEQ ID NO: 46.

In some additional embodiments, the engineered ketoacid decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2, 46, and/or 58, or a functional fragment thereof. In some additional embodiments, the engineered ketoacid decarboxylase polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 46, and/or 58, or a functional fragment thereof. In some additional embodiments, the engineered ketoacid decarboxylase polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 46, and/or 58, or a functional fragment thereof. In some embodiments, the engineered ketoacid decarboxylase polypeptide is a variant ketoacid decarboxylase polypeptide provided in any of Table 4.1, Table 4.2, Table 5.1, Table 6.1, Table 6.2, Table 7.1, and/or Table 7.2. In some further embodiments, the engineered ketoacid decarboxylase polypeptide is a Lactococcus lactis variant enzyme. In some embodiments, the ketoacid decarboxylase exhibits at least one improved property as compared to wild-type Lactococcus lactis ketoacid decarboxylase. In yet some additional embodiments, the engineered ketoacid decarboxylase polypeptide is more thermostable than wild-type Lactococcus lactis ketoacid decarboxylase. In yet some further embodiments, the engineered ketoacid decarboxylase polypeptide more resistant to proteolysis than wild-type Lactococcus lactis ketoacid decarboxylase. In some additional embodiments, the engineered ketoacid decarboxylase polypeptide is less immunogenic than wild-type Lactococcus lactis ketoacid decarboxylase. In still some additional embodiments, the engineered ketoacid decarboxylase polypeptide is more serum stable than wild-type Lactococcus lactis ketoacid decarboxylase. In some embodiments, the engineered ketoacid decarboxylase polypeptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any of the even-numbered sequences of SEQ ID NOS: 2-524. In some embodiments, the engineered ketoacid decarboxylase polypeptide comprises a sequence at least 90%, identical to any of the even-numbered sequences of SEQ ID NOS: 2-524. In some further embodiments, the engineered ketoacid decarboxylase polypeptide comprises any of the even-numbered sequences of SEQ ID NOS: 2-524. In some additional embodiments, the engineered ketoacid decarboxylase polypeptide is purified. The present invention also provides compositions comprising at least one engineered ketoacid decarboxylase polypeptide provided herein. The present invention also provides compositions comprising an engineered ketoacid decarboxylase polypeptide provided herein.

The present invention also provides engineered polynucleotide sequences encoding at least one engineered ketoacid decarboxylase polypeptide provided herein. In some embodiments, the engineered polynucleotide sequence encodes an engineered ketoacid decarboxylase polypeptide provided herein. In some embodiments, the engineered polynucleotide sequence comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any of the odd-numbered sequences of SEQ ID NOS: 1-523. In some additional embodiments, the engineered polynucleotide sequence comprises a sequence at least 90% or more identical to any of the odd-numbered sequences of SEQ ID NOS: 1-523. In some further embodiments, the engineered polynucleotide sequence comprises any of the odd-numbered sequences of SEQ ID NOS: 1-523. In some additional embodiments, the engineered polynucleotide sequence is operably linked to a control sequence. In some embodiments, the engineered polynucleotide sequence is codon-optimized.

The present invention also provides expression vectors comprising at least one engineered polynucleotide sequence provided herein. In some embodiments, the expression vectors further comprise at least one control sequence. In some embodiments, the control sequence comprises a promoter. In some further embodiments, the promoter is a heterologous promoter.

The present invention also provides host cells transformed with at least one polynucleotide sequence and/or comprising an expression vector provided herein. In some embodiments, the host cells are transformed with at least one polynucleotide sequence provided herein. In some embodiments, the host cells are transformed with a polynucleotide sequence provided herein. In some additional embodiments, the host cell comprise at least one expression vector provided herein. In some further embodiments, the host cells comprise an expression vector provided herein. In some embodiments, the host cell is E. coli.

The present invention also provides methods of producing an engineered ketoacid decarboxylase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide encoding at least one engineered ketoacid decarboxylase polypeptide provided herein, and/or at least one polynucleotide sequence provided herein, and/or at least one expression vector provided herein, under suitable culture conditions, such that at least one engineered ketoacid decarboxylase polypeptide is produced. In some embodiments, the methods of producing an engineered ketoacid decarboxylase polypeptide in a host cell comprise culturing a host cell comprising at least one polynucleotide encoding at least one engineered ketoacid decarboxylase polypeptide provided herein, under suitable culture conditions, such that at least one engineered ketoacid decarboxylase polypeptide is produced. In some additional embodiments, the methods of producing an engineered ketoacid decarboxylase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide sequence provided herein, under suitable culture conditions, such that at least one engineered ketoacid decarboxylase polypeptide is produced. In some embodiments, the methods of producing an engineered ketoacid decarboxylase polypeptide in a host cell comprise culturing a host cell comprising at least one expression vector provided herein, under suitable culture conditions, such that at least one engineered ketoacid decarboxylase polypeptide is produced. In some embodiments, the methods further comprise recovering at least one engineered ketoacid decarboxylase polypeptide from the culture and/or host cells. In some additional embodiments, the methods further comprise the step of purifying said at least one engineered ketoacid decarboxylase polypeptide.

The present invention also provides compositions comprising at least one engineered polynucleotide provided herein. In some embodiments, the compositions comprise at least one engineered ketoacid decarboxylase polynucleotide provided herein. In some embodiments, the compositions comprise at least one engineered polynucleotide encoding at least one engineered ketoacid decarboxylase provided herein. In some embodiments, the compositions comprise at least one engineered polynucleotide encoding at least one engineered ketoacid decarboxylase polypeptide provided herein. In some embodiments, the composition is a pharmaceutical composition. In some additional embodiments, the composition further comprises at least one pharmaceutically acceptable excipient and/or carrier. In some embodiments, the composition is suitable for the treatment of maple syrup urine disease. In some additional embodiments, the composition is suitable for use in gene therapy. In still some further embodiments, the composition is suitable for use in gene therapy to treat maple syrup urine disease, and/or elevated blood levels of isoleucine, leucine, alloioleucine and/or valine. In some additional embodiments, the composition is suitable for use in mRNA therapy. In yet some additional embodiments, the composition is suitable for oral administration to a human. In some embodiments, the composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. In some further embodiments, the pill, tablet, capsule, or gelcap further comprises an enteric coating. In yet some additional embodiments, the composition is suitable for parenteral injection into an animal. In yet some additional embodiments, the composition is suitable for parenteral injection into a human. In some embodiments, the injections are administered on a daily, weekly, or monthly basis. In some additional embodiments, composition is coadministered with at least one additional therapeutically effective compound. In some embodiments, the composition comprises at least one additional therapeutically effective compound.

The present invention also provides methods for treating and/or preventing the symptoms of maple syrup urine disease in a subject, comprising providing a subject having maple syrup urine disease, and providing the composition provided herein to said subject. In some embodiments, the symptoms of maple syrup urine disease are ameliorated. In some additional embodiments, the subject is able to eat a diet that is less restricted in its in isoleucine, leucine, alloioleucine, and/or valine content than diets required by subjects who have not been provided at least one composition comprising at least one engineered ketoacid decarboxylase polypeptide and/or polynucleotide provided herein. In some embodiments, the subject is able to eat a diet that is less restricted in its in isoleucine, leucine, alloioleucine, and/or valine content than diets required by subjects who have not been provided at least one composition comprising at least one engineered ketoacid decarboxylase polypeptide provided herein. In some further embodiments, the subject is able to eat a diet that is less restricted in its in isoleucine, leucine, alloioleucine, and/or valine content than diets required by subjects who have not been provided at least one composition comprising at least one engineered ketoacid decarboxylase polynucleotide provided herein. In some embodiments, the subject is an infant, child, young adult, or adult. In some embodiments, the subject is an infant. In some embodiments, the subject is a child.

In some embodiments, the subject is a young adult. In some embodiments, the subject is an adult. The present invention also provides for the use of the compositions provided herein. In some embodiments, the compositions comprise at least one engineered ketoacid decarboxylase polypeptide and/or polynucleotide provided herein. In some embodiments, the compositions comprise at least one engineered ketoacid decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise an engineered ketoacid decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise at least one engineered ketoacid decarboxylase polynucleotide provided herein. In some embodiments, the compositions comprise an engineered ketoacid decarboxylase polynucleotide provided herein. In some embodiments, the compositions comprise at least one polynucleotide encoding at least one engineered ketoacid decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise at least two polynucleotides encoding at least two engineered ketoacid decarboxylase polypeptides provided herein. I n some embodiments, the compositions comprise at least one polynucleotide encoding at least one engineered ketoacid decarboxylase provided herein. In some embodiments, the compositions comprise at least one polynucleotide encoding at least two engineered ketoacid decarboxylases provided herein. In some embodiments, the compositions comprise a polynucleotide encoding an engineered ketoacid decarboxylase polypeptide provided herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the extracellular level of KIC observed in in normal fibroblast and MSUD fibroblast cells treated with the three KdcA variants.

FIG. 2 is a graph showing the plasma clearance profiles of SEQ ID NO:46 and WT KdcA (SEQ ID NO: 2).

DESCRIPTION OF THE INVENTION

The present invention provides engineered KdcA polypeptides, mutants, biologically active fragments and analogues thereof, and pharmaceutical and industrial compositions comprising the same.

The invention provides engineered branched chain ketoacid decarboxylase (KdcA) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered (KdcA) polypeptides. In some embodiments, the engineered KdcA polypeptides are optimized to provide enhanced catalytic activity, as well as enhanced stability. The present invention also provides methods for the use of compositions comprising the engineered KdcA polypeptides for therapeutic and industrial purposes.

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. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention 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. Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

Also, 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.

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. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

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).

“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.

“NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

As used herein, the term “branched chain ketoacid decarboxylase (KdcA) polypeptide” refers to a member of the branched-chain-3-oxoacid decarboxylase enzyme class (EC 4.1.1.72). These enzymes use Mg2+ and the cofactor thiamine diphosphate to catalyze the decarboxylation of various 2-oxo acids. KdcA is a dimeric ˜50 kDa protein that deccarboxylates the branched chain ketoacids generated from valine, leucine, and isoleucine to generate carbon dioxide and the corresponding aldehyde (See e.g., Smit et al., Appl. Environ. Microbiol., 71:303-311 [2005]).

“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).

“Polynucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides.

“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. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes, as indicated The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Are or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gln or Q), 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).

The term “engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refers 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.

As used herein, “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.

The term “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., 2:482 [1981]), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), 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., 215: 403-410 [1990]; and Altschul et al., Nucleic Acids Res., 3389-3402 [1977]). 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 89:10915 [1989]). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), 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 polypeptide 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:4 having a valine at the residue corresponding to X39” refers to a reference sequence in which the corresponding residue at position X39 in SEQ ID NO:4 (e.g., an alanine), 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 KDCA, 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.

“Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide 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 X91 as compared to SEQ ID NO:4” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 91 of SEQ ID NO:4. Thus, if the reference polypeptide of SEQ ID NO:4 has a alanine at position 91, then a “residue difference at position X91 as compared to SEQ ID NO:4” refers to an amino acid substitution of any residue other than alanine at the position of the polypeptide corresponding to position 91 of SEQ ID NO:4. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified 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 (e.g., in the Tables in the Examples), the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. 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., X307G/X307Q or X307G/Q). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions.

The terms “amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 1, 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 variant KdcA polypeptides listed in any of the Tables in the Examples.

“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 transaminase 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.

The terms “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 KDCA 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 KDCA 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 KDCA polypeptides provided herein are isolated polypeptides.

“Substantially pure polypeptide” refers to a composition in which the polypeptide 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 KdcA 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 KdcA polypeptides are substantially pure polypeptide compositions.

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

“Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered KdcA 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 KdcA) as compared to the reference KdcA enzyme (e.g., wild-type KdcA and/or another engineered KdcA). 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 KdcA or another engineered KdcA from which the KdcA polypeptides were derived.

In some embodiments, the engineered KdcA polypeptides have a kcat of at least 0.1/sec, at least 0.2/sec, at least 0.3/sec, at least 0.5/sec, at least 1.0/sec, and in some preferred embodiments greater than 2.0/sec. In some embodiments, the Km is in the range of about 1 μm to about 5 mM; in the range of about 5 μm to about 2 mM; in the range of about 10 μm to about 2 mM; or in the range of about 10 μm to about 1 mM. In some specific embodiments, the engineered KdcA 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 KdcA enzyme. KdcA activity can be measured by any standard assay known in the art (e.g., by monitoring changes in spectrophotometric properties of reactants or products). In some embodiments, the amount of products produced or the amount of substrate consumed is measured by High-Performance Liquid Chromatography (HPLC) separation combined with UV absorbance or mass spectra detection and Gas Chromatography (GC) directly or following 3-methylbutanal derivatization. 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.

The phrase “increased storage stability” means that an engineered KdcA polypeptide according to the invention will retain more activity compared to a reference KdcA in a standard assay (e.g., as described in the Examples) after it has been produced in a dried form (e.g., by lyophilization or spray-drying), and stored for a period of time ranging from a few days to multiple months at a temperature above room temperature (e.g., 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 a KDCA 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. 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 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M 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 5×SSC containing 0.1% (w:v) SDS at 65° C. and washing in 0.1×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 KdcA 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” is defined herein as 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 encoding a 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 KdcA polypeptide. “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the KdcA polypeptide on the substrate.

As used herein the term “culturing” refers to the growing of a population of microbial cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

Recombinant polypeptides (e.g., KdcA enzyme variants) can be produced using any suitable methods known the art. For example, there is a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available to make specific substitutions at defined amino acids (site-directed), specific or random mutations in a localized region of the gene (regio-specific), or random mutagenesis over the entire gene (e.g., saturation mutagenesis). Numerous suitable methods are known to those in the art to generate enzyme variants, including but not limited to site-directed mutagenesis of single-stranded DNA or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: U.S. Pat. Nos. 6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393; 7,783,428; and 8,383,346. After the variants are produced, they can be screened for any desired property (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermal stability, and/or acidic pH stability, etc.). In some embodiments, “recombinant KdcA polypeptides” (also referred to herein as “engineered KdcA polypeptides,” “variant KdcA enzymes,” and “KdcA variants”) find use.

As used herein, a “vector” is 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.

As used herein, the term “expression” 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.

As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is “heterologous” to another sequence with which it is operably linked if the two sequences are not associated in nature.

As used herein, the terms “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 KdcA 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.

The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, analogues include non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

The term “therapeutic” refers to a compound administered to a subject who shows signs or symptoms of pathology having beneficial or desirable medical effects.

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

The term “gene therapy” is used in reference to the use of genes (i.e., genetic material) to treat and/or prevent disease in a mammalian subject (e.g., human). In some embodiments, the genetic material is introduced directly into at least some cells of the mammalian subject. It is not intended that the present invention be limited to any specific method(s) or composition(s) useful for gene therapy.

The term “mRNA therapy” is used in reference to the use of messenger RNA (mRNA) to treat and/or prevent disease in a mammalian subject (e.g., human). In some embodiments, the genetic material is introduced directly into at least some cells of the mammalian subject. It is not intended that the present invention be limited to any specific method(s) or composition(s) useful for mRNA therapy.

The term “effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.

The term “subject” encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). It is intended that the term encompass females as well as males.

As used herein, the term “patient” means any subject that is being assessed for, treated for, or is experiencing disease.

The term “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. A “very low birth weight infant” is an infant weighing less than 1500 g at birth.

As used herein, the term “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.

As used herein, the term “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.

As used herein, “composition” and “formulation” encompass products comprising at least one engineered KdcA of the present invention, intended for any suitable use (e.g., pharmaceutical compositions, dietary/nutritional supplements, feed, etc.).

The terms “administration” and “administering” a composition mean providing a composition of the present invention to a subject (e.g., to a person suffering from the effects of MSUD).

The term “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.

The term “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.

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

The term “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 KdcA 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., MSUD). 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.

The term “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.

It is intended that the terms “treating,” “treat” and “treatment” encompass preventative (e.g., prophylactic), as well as palliative treatment.

As used herein, the term “at least one” 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.

Engineered KDCA Polypeptides:

The parent KDCA polypeptides from which the engineered KdcA polypeptides of the invention are derived from include bacterial strains such as Lactococcus lactis.

Furthermore, when a particular KdcA variant (i.e., an engineered KDCA polypeptide) is referred to by reference to modification of particular amino acids residues in the sequence of a wild-type KDCA or reference KDCA it is to be understood that variants of another KDCA modified in the equivalent position(s) (as determined from the optional amino acid sequence alignment between the respective amino acid sequences) are encompassed herein.

In some embodiments, engineered KDCA polypeptides are produced by cultivating a microorganism comprising at least one polynucleotide sequence encoding at least one engineered KDCA polypeptide under conditions which are conducive for producing the engineered KDCA polypeptide. In some embodiments, the engineered KDCA polypeptide is subsequently recovered from the resulting culture medium and/or cells.

The present invention provides exemplary engineered KDCA polypeptides having KDCA activity. The Examples provide Tables showing sequence structural information correlating specific amino acid sequence features with the functional activity of the engineered KDCA polypeptides. This structure-function correlation information is provided in the form of specific amino acid residue differences relative to the reference engineered polypeptide of SEQ ID NO: 2, as well as associated experimentally determined activity data for the exemplary engineered KDCA polypeptides.

In some embodiments, the engineered KDCA polypeptides of the present invention having KdcA activity comprise a) an amino acid sequence having at least 85% sequence identity to reference sequence SEQ ID NO:2; b) an amino acid residue difference as compared to SEQ ID NO:2 at one or more amino acid positions; and c) which exhibits an improved property selected from i) enhanced serum stability, ii) enhanced catalytic activity, iii) reduced proteolytic sensitivity, iv) reduced aggregation, v) increased stability as a lyophilized preparation to elevated temperatures, vi) reduced immunogenicity, or a combination of any of i), ii), iii), iv), v), or vi) as compared to the reference sequence.

In some embodiments, the present invention provides functional fragments of engineered KdcA polypeptides. In some embodiments, functional 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 KdcA polypeptide from which it was derived (i.e., the parent engineered KdcA). In some embodiments, functional 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 KdcA. In some embodiments the functional 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, and less than 50 amino acids.

In some embodiments, the present invention provides functional fragments of engineered KDCA polypeptides. In some embodiments, functional fragments comprise at least about 95%, 96%, 97%, 98%, or 99% of the activity of the engineered KDCA polypeptide from which it was derived (i.e., the parent engineered KDCA). In some embodiments, functional fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered KDCA. In some embodiments the functional 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, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.

In some embodiments, the engineered KDCA polypeptide comprises an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOS: 2, 46, and/or 58, or the amino acid sequence of any variant (e.g., those provided in the Examples). In some embodiments, the reference sequence is selected from SEQ ID NOS: 2, 46, and/or 58.

In some embodiments, the engineered KDCA polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to reference sequence SEQ ID NO: 2, 46, and/or 58, and one or more residue differences as compared to SEQ ID NO: 4, 46, and/or 58.

Polynucleotides Encoding Engineered Polypeptides. Expression Vectors and Host Cells:

The present invention provides polynucleotides encoding the engineered KDCA polypeptides described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. In some embodiments, expression constructs containing at least one heterologous polynucleotide encoding the engineered KDCA polypeptide(s) is introduced into appropriate host cells to express the corresponding KDCA 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 KDCA polypeptide. Thus, the present invention provides methods and compositions for the production of each and every possible variation of KDCA polynucleotides that could be made that encode the KDCA 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 the various Tables).

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. Consequently, codon optimized polynucleotides encoding the engineered KDCA 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.

In some embodiments, the KDCA polynucleotide encodes an engineered polypeptide having KDCA activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOS: 2, 46, and/or 58, or the amino acid sequence of any variant (e.g., those provided in the Examples), and one or more residue differences as compared to the reference polynucleotide of SEQ ID NOS: 1, 45, and/or 57, or the amino acid sequence of any variant as disclosed in the Examples (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In some embodiments, the reference sequence is selected from SEQ ID NOS: 2, 46, and/or 58.

In some embodiments, the polynucleotide encoding the engineered KDCA polypeptides comprises a polynucleotide sequence selected from a polynucleotide sequence selected from SEQ ID NOS: 1, 45, and/or 57. In some embodiments, the polynucleotide encoding an engineered KDCA polypeptide has at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide residue identity to SEQ ID NOS: 1, 45, and/or 57.

In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NOS: 2, 45, and/or 57, or a complement thereof, or a polynucleotide sequence encoding any of the variant KdcA polypeptides provided herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a KDCA polypeptide comprising an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 2, 46, and/or 58.

In some embodiments, an isolated polynucleotide encoding any of the engineered KdcA polypeptides herein is manipulated in a variety of ways to facilitate expression of the KdcA polypeptide. In some embodiments, the polynucleotides encoding the KDCA polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the KDCA polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. 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 75:3727-3731 [1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25 [1983]). 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 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the KDCA polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. 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).

In some embodiments, the control sequence is also a suitable leader sequence (i.e., a non-translated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the KDCA polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. 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 are 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).

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., 15:5983-5990 [1995]).

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 NC1B 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., 57:109-137 [1993]). 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.

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 another aspect, the present invention is directed to a recombinant expression vector comprising a polynucleotide encoding an engineered KDCA 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. In some embodiments, the various nucleic acid and control sequences described herein are joined together to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the KDCA polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

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 KDCA polynucleotide sequence. 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 invention provides a host cell comprising at least one polynucleotide encoding at least one engineered KDCA polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered KDCA enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention 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).

Accordingly, in another aspect, the present invention provides methods of producing the engineered KDCA polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered KDCA polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the steps of isolating and/or purifying the KDCA polypeptides, as described herein.

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 KDCA 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.

Engineered KDCA polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered KDCA 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 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 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., 16:258-261 [1998]), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140 [1994]), and cassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]).

Mutagenesis and directed evolution methods can be readily applied to KDCA-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., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; 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 (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatments or other suitable assay conditions. Clones containing a polynucleotide encoding a KDCA polypeptide are then isolated from the gene, 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 (e.g., standard biochemistry techniques, such as HPLC analysis).

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., 22:1859-69 [1981]; and Matthes et al., EMBO J., 3:801-05 [1984]), 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 KDCA polypeptide can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant as described herein, and (b) expressing the KDCA polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have 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.

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

In some embodiments, any of the engineered KDCA polypeptides expressed in a host cell are recovered from the cells and/or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography.

Chromatographic techniques for isolation of the KDCA polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depends, 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 improved KDCA enzymes. For affinity chromatography purification, any antibody that specifically binds a KDCA polypeptide of interest may find use. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injection with a KDCA polypeptide, or a fragment thereof. In some embodiments, the KDCA polypeptide or fragment 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.

In some embodiments, the engineered KDCA polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., an E. coli strain) comprising a polynucleotide sequence encoding an engineered KDCA polypeptide as described herein under conditions conducive to the production of the engineered KDCA polypeptide and recovering the engineered KDCA polypeptide from the cells and/or culture medium. In some embodiments, the host cell produces more than one engineered KDCA polypeptide.

In some embodiments, the present invention provides a method of producing an engineered KDCA polypeptide comprising culturing a recombinant bacterial cell comprising a polynucleotide sequence encoding an engineered KDCA polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to reference sequences SEQ ID NO: 2, 46, and/or 58, and one or more amino acid residue differences as compared to SEQ ID NO: 2, 46, and/or 58, as provided herein, under suitable culture conditions to allow the production of the engineered KDCA polypeptide and optionally recovering the engineered KDCA polypeptide from the culture and/or cultured bacterial cells. In some embodiments, the host cell produces more than one engineered KDCA polypeptide.

In some embodiments, once the engineered KDCA polypeptides are recovered from the recombinant host cells and/or culture medium, they are further purified by any suitable method(s) known in the art. In some additional embodiments, the purified KDCA polypeptides are combined with other ingredients and compounds to provide compositions and formulations comprising the engineered KDCA polypeptide as appropriate for different applications and uses (e.g., pharmaceutical compositions).

Compositions:

The present invention provides engineered KDCA polypeptides suitable for use in numerous compositions. These compositions find use in many fields, including but not limited to pharmaceuticals, dietary/nutritional supplements, food, feed, and fine chemical production. For example, in some embodiments, the present invention provides food and/or feeds comprising at least one engineered KDCA variant and/or at least one polynucleotide sequence encoding at least one KDCA variant. In some embodiments, the present invention provides beverages comprising at least one engineered KDCA variant.

In some embodiments, the engineered KDCA variant in food, feed, and/or nutritional/dietary supplement is glycosylated. Furthermore, the engineered KDCA variants find use in any suitable edible enzyme delivery matrix. In some embodiments, the engineered KDCA variants are present in an edible enzyme delivery matrix designed for rapid dispersal of the KDCA variant within the digestive tract of an animal upon ingestion of the variant.

The present invention also provides engineered KDCA polypeptides suitable for use in production of fine chemicals and other industrially important compounds (See e.g., US Pat. Appln. Nos. 2013/0340119, 2013/0005012, and 2005/0260724, and WO 2012/122333).

Pharmaceutical and Other Compositions:

The present invention provides engineered KDCA polypeptides suitable for use in pharmaceutical and other compositions, such as dietary/nutritional supplements.

Depending on the mode of administration, these compositions comprising a therapeutically effective amount of an engineered KDCA according to the invention are 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. Details on techniques for formulation and administration are well known in the art and described in the literature.

In some embodiments, the engineered KDCA polypeptides are formulated for use in oral pharmaceutical compositions. Any suitable format for use in delivering the engineered KDCA 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 KDCA polypeptides are provided in a format suitable for injection (i.e., in an injectable formulation). In some embodiments, the engineered KDCA polypeptides are provided in biocompatible matrices such as sol-gels, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the engineered KDCA polypeptides are encapsulated. In some alternative embodiments, the engineered KDCA polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres, liposomes, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery formulation and/or means of delivery. It is intended that the engineered KDCA 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 KDCA 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 29:283-287 [2005]; U.S. Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,553,653; US Pat. Appln. Publ. Nos. 2013/0039898, 2012/0177722, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery method and/or mechanism.

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

In some embodiments, compositions comprising the engineered KDCA 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 KdcA 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 KdcA polypeptide are be 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 KdcA polypeptides are administered to subjects in combination with other compounds used in the treatment of MSUD, as well as any other suitable compounds.

In some embodiments, the present invention provides engineered KdcA polypeptides suitable for use in decreasing, ameliorating, or eliminating the signs and/or symptoms of MSUD. The dosage of engineered KdcA polypeptide(s) administered to a patient depends upon the genotype of the patient, the general condition of the patient, and other factors known to those in the art. In some embodiments, the compositions are intended for single or repeat administration to a patient. In some embodiments, it is contemplated that the concentration of engineered KdcA polypeptide(s) in the composition(s) administered to a patient is sufficient to effectively treat, ameliorate and/or prevent the symptoms of the disease. In some embodiments, the engineered KdcA polypeptides are administered in combination with other pharmaceutical and/or dietary compositions.

Industrial Compositions:

It is contemplated that the engineered KdcA polypeptides of the present invention will find use in industrial compositions, including such areas as food flavorings (e.g., cheese; See, Smit et al., supra).

In some embodiments, the engineered KdcA polypeptides are formulated for use in the food and/or feed industries. In some embodiments, the engineered KdcA polypeptides are formulated in granulated or pelleted products which are mixed with animal feed components such as additional enzymes (for example, cellulases, laccases, and amylases). In some alternative embodiments, the engineered KDCA polypeptides are used in liquid animal feed compositions (e.g., aqueous or oil based slurries). Thus, in some embodiments, the engineered KdcA variants of the present invention are sufficiently thermotolerant and thermostable to withstand the treatment used to produce pellets and other processed feed/foods.

The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

EXPERIMENTAL

The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

In the experimental disclosure below, the following abbreviations apply: 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 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and um (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, Conn.); HTP (high throughput); HPLC (high pressure liquid chromatography); LC (liquid chromatography); MS (mass spectroscopy); CFSE (carboxyfluorescein succinimidyl ester); KIC (ketoisocaproate); IPTG (isopropyl β-D-1-thiogalactopyranoside); PES (polyethersulfone); BSA (bovine serum albumin); PBMC (peripheral blood mononuclear cells); MSUD (maple syrup urine disease); MHC (major histocompatibility complex); HLA (human leukocyte antigen); HLA-DR (an MHC Class II cell surface receptor encoded by the HLA complex on chromosome #6); FIOPC (fold improvements over positive control); LB (Luria broth); TB (Terrific broth); Innovative Research (Innovative Research, Novi, Mich.); Microfluidics (Microfluidics Corp., Newton, Mass.); Thermotron (Thermotron, Holland, Mich.); Infors (Infors AG, Bottmingen, Switzerland); Waters (Waters Corp., Milford, Mass.); Infors (Infors AG, Bottmingen, Switzerland); Coriell Institute for Medical Research (Coriell Institute for Medical Research, Camden, N.J.); GE Healthcare (GE Healthcare Bio-Sciences, Pittsburgh, Pa.); Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, N.Y.), Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); Thermo Scientific (part of Thermo Fisher Scientific, Waltham, Mass.); Gibco (Gibco part of Thermo Fisher Scientific, Waltham, Mass.); Corning (Corning, Inc., Palo Alto, Calif.); and AB Sciex (AB Sciex, Framingham, Mass.).

Example 1
KdcA Gene Acquisition and Construction of Expression Vectors

A synthetic gene (SEQ ID NO: 1) encoding the branched-chain keto acid decarboxylase (kdcA) from Lactococcus lactis (SEQ ID NO: 2) was synthesized with codon optimization for expression in E. coli. The synthetic kdcA gene (SEQ ID NO: 1) was cloned into the E. coli expression vector pCK100900i (See, U.S. Pat. No. 9,714,437, which is hereby incorporated by reference). This 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., U.S. Pat. No. 8,383,346 and WO2010/144103) as well as KdcA derivatives.

Example 2
Preparation of HTP KdcA-Containing Cell Lysates

To lyse the cells, 200 μl lysis buffer containing 50 mM KiPO4, pH 6.5, 1 mg/mL lysozyme, and 0.5 mg/mL PMBS was added to the cell paste. The cells were incubated at room temperature for 1.5 hours with shaking on a bench top shaker. The plate was then centrifuged for 15 minutes at 4000 rpm and 4° C.

Example 3
Preparation of Lyophilized Lysates from Shake Flask (SF) Cultures

A single colony containing the desired gene picked from an LB agar plates with 1% glucose and 30 μg/ml CAM, and incubated overnight at 37° C. was transferred to 6 ml of LB with 1% glucose and 30 μg/ml CAM. The culture was grown for 18 h at 30° C., 250 rpm, and subcultured approximately 1:50 into 250 ml of TB containing 30 μg/ml CAM, to a final OD₆₀₀of about 0.05. The subculture was grown for approximately 195 minutes at 30° C., 250 rpm, to an OD₆₀₀between 0.6-0.8, and induced with 1 mM IPTG. The subculture was then grown for 20 h at 30° C., 250 rpm. The subculture was centrifuged at 4000 rpm for 20 min. The supernatant was discarded, and the pellet was resuspended in 35 ml of 50 mM potassium phosphate buffer, pH 6.5, 2.5 mM magnesium sulfate, and 0.1 mM thiamine pyrophosphate. The cells were lysed using a MICROFLUIDIZER® processor system (Microfluidics) at 18,000 psi. The lysate was pelleted (10,000 rpm×60 min) at 4° C., and the supernatant was frozen and lyophilized to generate shake flake (SF) enzyme powder.

Example 4
KdcA Variants with Improved Activity and Serum Stability Relative to SEQ ID NO: 2

The wild type KdcA (SEQ ID NO: 2) was chosen as the parent enzyme. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1, and the clarified lysates were generated as described in Example 2.

For HTP activity assays, 10 μL of the clarified supernatant from the lysis as described in Example 2 were added to 240 μL of 50 mM KiPO4, pH 6.5 (25× dilution) and the diluted lysates were used in subsequent biocatalytic reactions described below. HTP reactions were carried out in 96 well plates containing 100 μL of 50 mM KiPO4, pH 6.5, 80% of pooled normal human AB serum (Innovative Research; cat IPLA-SERAB), 400 μM ketoisocaproate (KIC) and 10 μl of above diluted HTP supernatant. The HTP plates were incubated in a THERMOTRON® shaker (3 mm throw, model # AJ185, Infors) at 37° C., 200 rpm, for 2 hours. 20 μL of the reaction mixture was transferred to a 96 well plate and quenched with 180 μl of 0.1% formic acid in acetonitrile containing 50 μM d3-KIC (used as an internal control) and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and supernatant was loaded to LC-MS instrument. Mass signal of KIC substrate and internal control d3-KIC was analyzed.

Activity relative to SEQ ID NO: 2 was reported as fold improvement over positive control (FIOP), which is calculated as the normalized mass signal of KIC by d3-KIC in the corresponding backbone per the normalized mass signal of KIC by d3-KIC in the product under the specified reaction conditions. Active variants and their associated FIOP values relative to SEQ ID NO: 2 are listed in Table 4.1.

Beneficial mutations were identified in variants from HTP assays and subsequently another library of engineered genes was produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). These engineered genes have the same parent (SEQ ID NO: 2) as those described in Table 4.1. For HTP activity assays with these variants, a more stringent serum challenge assay was implemented, in which variants were pre-incubated with serum for 1 h before activity was measured. Briefly, 20 uL of the clarified supernatant from the lysis as described in Example 2 was added to 180 uL of 50 mM KiPO4, pH 6.5 (10× dilution) and the diluted lysates were used in subsequent biocatalytic reactions. HTP reactions were carried out in 96 well plates. Then, 10 uL of the above diluted HTP lysate was pre-exposed to 80 uL of pooled normal human AB serum (Innovative Research, IPLA-SERAB; this serum was used in all of the Examples herein in which serum was utilized) for 1 hr at 37° C. Then, 10 uL of 40 mM stock KIC in 50 mM KiPO4, pH 6.5 were added to the well, to produce a final total of 100 uL of reaction mixture containing 4 mM of KIC and 80% of serum in the wells. The HTP plates were incubated in THERMOTRON® shaker (3 mm throw, model # AJ185, Infors) at 37° C., 200 rpm, for another hour. Then, 20 uL of the reaction mixture were transferred to a 96 well plate and quenched with 180 μl of 0.1% formic acid in acetonitrile containing 10 uM d3-KIC (used as an internal control) and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and supernatant was loaded to LC-MS instrument. Mass signal of KIC substrate and internal control d3-KIC were analyzed. Active variants and their associated FIOP values relative to SEQ ID NO: 2 are listed in Table 4.2

TABLE 4.1

Activity of kdcA Variants (Relative to SEQ ID NO: 2)

SEQ ID NO:
Amino Acid Differences
Primary FIOP by KIC/KIC-d3

(nt/aa)
(Relative to SEQ ID NO: 2)
(Relative to SEQ ID NO: 2)¹

1/2

+

3/4
S471P
+++

5/6
N28F
+

7/8
L153A/I264E
++

9/10
M124E/S442A
++

11/12
S37A
+

13/14
R500I
+

15/16
S442A
++

17/18
L153A/S442A
+++

19/20
S355L
+++

21/22
E329K
+

23/24
Q437S
+

25/26
F388P
++

27/28
E174D/I453T
+++

29/30
Y144P/S184N
+

31/32
K419D
+

33/34
K510F
+

35/36
K121L/K331S
++

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 2, and defined as follows:

““+”” = 1.00 to 1.10 (first 50%);

““++”” > 1.10 (next 30%); and

““+++”” > 1.19 (top 20%).

TABLE 4.2

Activity of kdcA Variants Relative to SEQ ID NO: 2

FIOP by KIC/d3-KIC

(Pre-Serum Exposure,

SEQ ID

Followed by 4 mM

NO:
Amino Acid Differences
KIC Challenge) (Relative

(nt/aa)
(Relative to SEQ ID NO: 2)
to SEQ ID NO: 2)¹

1/2

+

37/38
S37A/Y144T/S442A
+

39/40
T187L/Q338E/F388P/S442A
++

41/42
S37A/Y144T/T187L
+

43/44
S37A/S442A
++

45/46
S37A/Y144T/T187L/S286I/S442A
+++

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 2, and defined as follows:

““+”” = 1.00 to 1.17 (first 50%),

““++”” > 1.17 (next 30%); and

““+++”” > 1.28 (top 20%).

Example 5
KdcA Variants with Improved Activity and Serum Stability Relative to SEQ ID NO: 46

The kdcA variant SEQ ID NO: 46 was chosen as the parent enzyme. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1, and the clarified lysates were generated as described in Example 2.

For HTP assays, 10 uL of the clarified supernatant from the lysis prepared as described in Example 2 were added to 240 uL of 50 mM KiPO4, pH 6.5 (25× dilution) and the diluted lysates were used in subsequent biocatalytic reactions. HTP reactions were carried out as described in Example 4. Fold improvement over positive control (FIOP) is calculated as the normalized mass signal of KIC by d3-KIC in the corresponding backbone per the normalized mass signal of KIC by d3-KIC in the product under the specified reaction conditions. Active variants with corresponding fold-improvement over positive control (FIOP) relative to SEQ ID NO: 46 are listed in Table 5.1.

TABLE 5.1

Activity of kdcA Variants Relative to SEQ ID NO: 46

FIOP by

SEQ ID

KIC/KIC-d3

NO:
Amino Acid Differences
(Relative to SEQ

(nt/aa)
(Relative to SEQ ID NO: 46)
ID NO: 46)¹

45/46

+

47/48
D107R/M124W/I348D/F388P/S471P
+

49/50
D107R/K260S/S328N/S471P
+++

51/52
D107R/S328N/K331L/F388P/S471P
+++

53/54
D107R/M124W/K233H/K260S/S325F/S328N/K331L/S355L/F388P/
++

S471P

55/56
D107R/M124W/S471P
++

57/58
D107R/E183Y/S185P/K233R/S328N/K331L/S351V/F388P/K419D/
+

S471P

59/60
D107R/S355L/S471P
+++

61/62
D107R/F388P/S471P
+

63/64
D107R/E183Y/K233H/K260S/S328N/I348D/S351V/S471P
+

65/66
Q338E/K342R/S471P
++

67/68
D107R/E183Y/K233H/K260S/K331L/K419D/S471P
+

69/70
K42R/N204W/S328L/K342R/Q437S/S471P
+

71/72
M124R/L136H/N204W/S355M/Q437S/S471P
+++

73/74
S328N/S471P
+

75/76
D107R/K233H/K260S/S328N/I348D/S351V/F388P/S471P
+++

77/78
K42R/R321S/S471P
+

79/80
D107R/K233H/K260S/S328N/K331L/S351V/S471P
+

81/82
D107R/S185P/I348D/S351V/F388P/S471P
++

83/84
Q338E/K342R/S355L/S471P
+

85/86
S185P/S471P
++

87/88
D107R/S325F/S328N/I348D/F388P/S471P
++

89/90
M124W/K260S/K331L/S355L/K446R/S471P
+

91/92
M124R/L136H/S328L/K331T/S355M/Q437S/S471P/K510F
++

93/94
K42R/S328L/S471P
+

95/96
F388P/S471P
+

97/98
Q338E/K342R/S471P/K510F
+

99/100
L136H/N204W/S328L/K331N/S355M/Q437S/S471P
+

101/102
D107R/E183Y/I348D/S351V/S471P
+

103/104
K42R/M124R/L136H/N204W/R321S/S328L/Q437S/S471P/K510F
+

105/106
M124R/S355M/S471P
++

107/108
L136H/K331T/Q338E/K342R/S471P
+

109/110
D107R/S185P/K233H/S328N/S351V/F388P/S471P
++

111/112
S328L/K331T/S355M/S471P
+

113/114
M124R/K331T/K342R/S355L/S471P
+++

115/116
K42R/M124R/N204W/R321S/S471P
+

117/118
N204W/R321S/Q338E/S471P
+++

119/120
D107R/E183Y/K260S/S328N/I348D/S351V/S471P
++

121/122
D107R/E183Y/K233R/K260S/S328N/I348D/S351V/F388P/S471P
+++

123/124
M124W/S325F/K331L/S471P
+

125/126
M124R/L136H/R321S/S355L/S471P
+

127/128
K42R/S355M/S471P/K510F
+

129/130
K42R/M124R/L136H/N204W/Q437S/S471P
+

131/132
K42R/M124R/N204W/S471P
+

133/134
R321S/S328L/K331T/S471P
+++

135/136
E183Y/K233H/S351V/F388P/S471P
+

137/138
M124R/Q338E/K342R/S471P
++

139/140
K42R/R321S/K331N/Q437S/S471P
+

141/142
S351V/S471P
+

143/144
K42R/M124R/R321S/S355M/S471P
++

145/146
M124W/S325F/S471P
+

147/148
K42R/N204W/R321S/S355M/Q437S/S471P/K510F
+

149/150
K42R/M124R/L136H/K331N/Q437S/S471P
+

151/152
K233H/K331L/F388P/K446R/S471P
+

153/154
K342R/S355L/Q437S/S471P
++

155/156
K42R/M124R/L136H/N204W/Q437S/S471P/K510F
+++

157/158
M124R/L136H/S471P
+

159/160
D107R/S328N/K331L/S471P
++

161/162
K42R/L136H/S355M/S471P/K510F
+

163/164
L136H/R321S/S471P/K510F
+

165/166
S185P/K233H/K260S/S328N/K331L/K419D/S471P
+

167/168
D107R/K233H/I348D/S471P
++

169/170
M124R/K331N/Q338E/K342R/S355L/S471P
+++

171/172
M124R/S328L/K331T/Q338E/S471P
++

173/174
L136H/N204W/Q437S/S471P
+

175/176
K42R/L136H/S328L/K331T/S471P
++

177/178
K42R/R321S/Q437S/S471P
+

179/180
K233H/S325F/S328N/K331L/I348D/S355L/S471P
+

181/182
M124R/L136H/K331N/S471P
++

183/184
D107R/I348D/F388P/S471P
+++

185/186
S325F/S328N/K331L/I348D/S355L/F388P/S471P
++

187/188
L136H/Q437S/S471P
++

189/190
K42R/L136H/N204W/R321S/S328L/K331N/S355L/S471P
++

191/192
D107R/S325F/K331L/K446R/S471P
++

193/194
K260S/S325F/K331L/K446R/S471P
+

195/196
S325F/S328N/I348D/S355L/F388P/S471P
++

197/198
K42R/L136H/N204W/S328L/K331T/S355L/S471P
+++

199/200
K42R/S471P
+++

201/202
D107R/I348D/S355L/F388P/K446R/S471P
+++

203/204
S185P/S328N/K331L/S471P
+

205/206
S325F/K331L/F388P/S471P
+

207/208
S325F/K331L/K446R/S471P
+

209/210
D107R/S325F/S328N/I348D/K446R/S471P
+++

211/212
M124R/R321S/K331T/K342R/S471P/K510F
++

213/214
K42R/N204W/R321S/S328L/K342R/S355L/S471P/K510F
+

215/216
D107R/E183Y/K233R/K260S/K331L/I348D/S351V/K419D/S471P
++

217/218
K342R/S355M/S471P
++

219/220
D107R/M124W/K233H/S355L/K446R/S471P
++

221/222
M124R/R321S/S328L/S471P/K510F
+++

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 58, and defined as follows:

““+”” = 1.00 to 1.49 (first 50%);

““++”” > 1.49 (next 30%); and

““+++”” > 1.84 (top 20%).

Example 6
KdcA Variants with Improved Activity and Serum Stability Relative to SEQ ID NO: 58

The kdcA variant SEQ ID NO: 58 was chosen as the parent enzyme. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1, and the clarified lysates were generated as described in Example 2.

For HTP assays, 80 uL of the clarified supernatant from the lysis as described in Example 2 were added to 160 uL of 50 mM KiPO4, pH 6.5 (3× dilution) and the diluted lysates were used in the subsequent biocatalytic reactions described below. HTP reactions were carried out in 96 well plates containing 100 μL of 50 mM KiPO4, pH 6.5, 80% of human serum, 1.5 mM KIC, and 10 μl of above diluted HTP supernatant. The HTP plates were incubated in THERMOTRON® shakers (3 mm throw, model # AJ185, Infors) at 37° C., 200 rpm, for 1 hour. Then, 20 uL of the reaction mixture was transferred to a 96 well plate and quenched with 180 μl of 0.1% formic acid in acetonitrile containing 50 uM d3-KIC (used as an internal control) and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and supernatant was loaded to LC-MS instrument. Mass signal of KIC substrate and internal control d3-KIC were analyzed. Active variants with corresponding fold-improvement over positive control (FIOP) relative to SEQ ID NO: 58 are listed in Table 6.1. FIOP is calculated as the normalized mass signal of KIC by d3-KIC in the corresponding backbone per the normalized mass signal of KIC by d3-KIC in the product under the specified reaction conditions.

Some variants were tested with a more stringent serum challenge assay, previously described in Example 4 with some modifications. In these HTP assays, 80 uL of the clarified supernatant from the lysis as described in Example 2 was added to 160 uL of 50 mM KiPO4, pH 6.5 (3× dilution) and the diluted lysates were used in subsequent biocatalytic reactions described below. HTP reactions were carried out in 96 well plates. In these assays, 10 uL of the above diluted HTP lysate were pre-exposed to 80 uL of human serum for 1 hr at 37° C., and followed by adding 10 uL of 40 mM stock KIC in 50 mM KiPO4, pH 6.5 to final total of 100 uL of reaction mixture containing 4 mM of KIC and 80% of human serum. The HTP plates were incubated in THERMOTRON® shakers (3 mm throw, model # AJ185, Infors) at 37° C., 200 rpm, for another hour. Then, 20 uL of the reaction mixture were transferred to a 96 well plate and quenched with 180 μl of 0.1% formic acid in acetonitrile containing 10 uM d3-KIC (used as an internal control) and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and supernatant was loaded to LC-MS instrument. Mass signal of KIC substrate and internal control d3-KIC were analyzed. Active variants with corresponding fold-improvement over positive control (FIOP) relative to SEQ ID NO: 58 are listed in Table 6.2. FIOP is calculated as the normalized mass signal of KIC by d3-KIC in the corresponding backbone per the normalized mass signal of KIC by d3-KIC in the product under the specified reaction conditions.

TABLE 6.1

Activity of kdcA Variants Relative to SEQ ID NO: 58

SEQ ID

FIOP by KIC/KIC-d3

NO:
Amino Acid Differences
(Relative to

(nt/aa)
(Relative to SEQ ID NO: 58)
SEQ ID NO: 58)¹

57/58

+

223/224
E529W
+++

225/226
E529R
+++

227/228
S507A
+++

229/230
E334L
+++

231/232
E527G
+++

233/234
E529C
+++

235/236
Q368E
+++

237/238
V314T
+++

239/240
E527R
+++

241/242
S507A
++

243/244
E529N
++

245/246
S369A
++

247/248
E334L
++

249/250
N392M
++

251/252
T189E
++

253/254
V516A
++

255/256
E346V
++

257/258
N392A
++

259/260
N392G
++

261/262
V314S
++

263/264
S249A
++

265/266
E18D/T189E/V193E/K260S/N328L
++

267/268
S507G
+

269/270
V314A
+

271/272
N392G
+

273/274
N392S
+

275/276
S369M
+

277/278
E346V
+

279/280
S369T
+

281/282
E511L
+

283/284
S369R
+

285/286
E346R
+

287/288
S369T
+

289/290
E511K
+

291/292
N392R
+

293/294
E334D
+

295/296
S369A
+

297/298
S179V
+

299/300
E334Q
+

301/302
S507K
+

303/304
E511V
+

305/306
E334G
+

307/308
T189E/K203Q
+

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 58, and defined as follows:

““+”” = 1.00 to 1.49 (first 50%);

““++”” > 1.49 (next 30%); and

““+++”” > 1.84 (top 20%).

TABLE 6.2

Activity of kdcA Variants Relative to SEQ ID NO: 58

SEQ

FIOP by KIC/KIC-d3

ID NO:
Amino Acid Differences
in serum 1 hr

(nt/aa)
(Relative to SEQ ID NO: 58)
(Relative to SEQ ID NO: 58)¹

57/58

+

309/310
K203Q
+

311/312
T189E/V193A/K342R/E527D
+

313/314
L295F
+++

315/316
H13K/E18D
+

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 58, and defined as follows:

““+”” = 1.00 to 1.5; and

““++”” > 1.5.

Example 7
Extracellular MC Depletion by Purified KdcA Variants Using In Vitro MSUD Patient-Derived Fibroblast Cells

N-terminal His-tag including 6 histidine amino acids was constructed into three KdcA variants (SEQ ID NO: 2, SEQ ID NO: 46, and SEQ ID NO: 58) (L. lactis). Crude lysate of these variants were obtained as described in Example 3. Purification of the lysate was carried out in HisPur™ Ni-NTA column (ThermoFisher Scientific, cat #88229) in ÄKTA Start Chromatograph System (GE Healthcare), per the manufacturer's instructions. The purified KdcA variants harvested from the column were further purified by removal of endotoxin using standard protocols known in the art. The purified, endotoxin-free KdcA variants were stored in 50 mM potassium phosphate buffer, pH 6.5, 2.5 mM magnesium sulfate, and 0.1 mM thiamine pyrophosphate. The protein concentrations were calculated from a BSA standard curve, as known in the art.

The extracellular levels of KIC in MSUD fibroblast cells treated by the purified and endotoxin free KdcA variants (SEQ ID NO: 46 and SEQ ID NO: 58) described above were compared to a reference enzyme (His-tagged purified and endotoxin-free WT KdcA; SEQ ID NO: 2). In addition, the KdcA in vitro efficacies of these variants were determined. Normal fibroblast cells (GM00730; these cells were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research) and MSUD patient-derived fibroblast cells (GM00297; Coriell Institute for Medical Research) were seeded at 10,000 cell/well into a 96-well plate with 200 uL of supplemented Minimal Essential Media (MEM; Gibco #11095-080, with 1% non-essential amino acids [NEAA; Gibco #11140-050], and 15% fetal bovine serum [Corning #35-016-CV]) and allowed to grow at 37° C. with 5% CO₂. After 24 hr growth, 50 ul of medium was sampled from each well and used as Day 1 samples. The rest of the medium was removed, and replaced with fresh MEM medium with FBS and NEAA with or without 0.005 mg/mL of the three KdcA variants (n=3), added at 200 uL/well. Then, 50 uL/well of medium was sampled (stored at −20° C.), and 50 ul of fresh medium was added each following day for three days. To measure the sample KIC level, a 20 uL aliquot of the collected samples was quenched by 0.1% formic acid in acetonitrile with 10 uM of d3-KIC. The KIC and d3-KIC levels were analyzed by LC-MS, as described in Example 10. The KIC levels in the samples were interpreted as KIC/d3-KIC. FIG. 1 provides a graph of the extracellular level of KIC observed in in normal fibroblast and MSUD fibroblast cells treated with the three KdcA variants.

Example 8
Serum Pharmacokinetics of Purified KdcA Variants

Purified and endotoxin-free KdcA variants produced as described in Example 7, were assessed for stability in the serum of live rats. WT KdcA (SEQ ID NO:2) or SEQ ID NO:46 stock (6.6 mg/ml in 50 mM potassium phosphate buffer, pH 6.5, 2.5 mM magnesium sulfate, and 0.1 mM thiamine pyrophosphate) were administered intravenously at adjusted volumes, to achieve 4.6 mg/kg to naïve jugular vein cannulated Sprague-Dawley rats (7-8 weeks old; n=4 per group). Prior to administration and at 5, 15, 30, 60, 120, 240, 360 minutes post-administration, 300 μL of blood was collected from each rat in an EDTA tube and centrifuged at 4° C. and 6000 rpm to generate ˜250 μL of plasma per sample. Samples were frozen and stored on dry ice prior to analysis. For analysis, a 40 μL aliquot of plasma was mixed with 5 μL of stock KIC (2 M, in 10× assay buffer containing 50 mM KiPO4, pH 7, 0.1 mM thiamine pyrophosphate, 2.5 mM MgCl₂) at final concentration of 200 mM, and 5 μL of assay buffer to make final volume of 50 μL, and incubated at room temperature for 2h. Samples were quenched with ethyl acetate at ˜40× dilution factor. After centrifugation, the supernatants were analyzed by gas chromatography for 3-methylbutanal quantification. The peak area of 3-methylbutanal was calculated as an indication of KdcA enzyme activity for each plasma sample. The total amount of enzyme activity injected to each rat was accessed similarly by assaying the variant stock solution (6.6 mg/mL) using rat plasma collected prior to administration and the same KIC and buffer condition. The percentage of injected dose per mL (% inj/mL) was determined by the enzyme activity in each rat plasma sample normalized by the total amount of activity injected per rat. The plasma clearance profiles demonstrated a longer plasma half-life on SEQ ID NO: 46 than that of WT KdcA SEQ ID NO: 2 (See, FIG. 2).

Example 9
Analytic Detection of KIC

Data described in Examples 5-7 were collected using analytical methods described for Tables 4.1, 4.2, 5.1, 6.1, and 6.2 and FIG. 1. The methods provided herein all find use in analyzing the variants produced using the present invention. Samples for LC analysis were prepared as dilutions in 0.1% formic acid in acetonitrile. The dilution level was dependent upon the starting conditions for the assay. Analysis of reaction samples was completed using an ACQUITY® Ultra Performance Liquid Chromatography system (Waters) equipped with a binary pump and tunable UV detector (TUV UV detector) and 3200 QTRAP® mass spectrometer (AB Sciex). For each run, 10 μL of quenched reaction supernatant was injected onto an ECLIPSE™ C18 column (50×4.6 mm, 1.8μ; Agilent) kept at a constant temperature of 30° C. Samples were eluted with water (buffer A) and acetonitrile (buffer B) with a constant flow rate of 0.75 mL/min. The following gradient was used: time 0 minutes, 50% buffer B; time 0.9 minutes, 90% buffer B; time 1.2 minutes, 90% buffer B; time 1.25 minutes, 50% buffer B; hold 50% buffer B until a final run time of 2 minutes. Eluting peaks were monitored with MS detection in negative mode looking for the Q3 ions m/z 129→129 for ketoisocaproic acid (KIC) and m/z 132→132 for ketoisocaproic acid-d3 (KIC-d3). For the 3200 QTRAP® Ultra Performance Liquid Chromatography system, the source temperature was 550° C., ionspray voltage was −4500, and ion source gas 1 and 2 were set to 40. The declustering potential was −45, the entrance potential was −10, the collision energy was −5, and the collision cell exit potential was −3. KIC and KIC-d3 eluted at 0.95 minutes.

Example 10
Analytic Detection of 3-Methylbutanal

Data described in Example 8 and shown in FIG. 2 were collected using the gas chromatograph (GC) analytical method described for 3-methylbutanal detection. Samples were prepared as dilutions in ethyl acetate, and 3-methylbutanal was further extracted in the supernatant as described in Example 8. Calibration was performed by measuring several dilutions of 3-methylbutanal spiked in plasma at concentrations ranging from 1 to 50 mM. Analysis of the reaction samples was completed using an Agilent GC system equipped with a CTC PAL auto-sampler and flame ionization detector (FID). For each run, 10 μL of the prepared sample was injected using a split factor of 10:1 onto a DB-5 ms column (J&W 122-5532, 30 m×250 μm×0.25 μm nominal, 7 inch cage; Agilent) and eluted by increasing column temperature with a constant helium gas flow rate of 1.8 mL/minutes. The inlet and FID temperatures were at 250° C. and 300° C., respectively. The oven temperature was initially held at 40° C. for 3 minutes post injection, increased at a rate of 60° C./minutes until warmed to 150° C., and then held at 150° C. for 1 min longer. The total run time per sample was 5.8 minutes. Elution peaks were monitored with the FID, and 3-methylbutanal eluted at 2.5 min using this method.

Example 11
Active KdcA Variants with Reduced in Silico T-Cell Epitopes

In this example, experiments conducted to identify diversity that would remove T-cell epitopes from kdcA are described.

Identification of Deimmunizing Diversity

Computational methods were employed to identify amino acids that, when mutated, could remove predicted T-cell epitopes. In parallel, the experimental search for neutral or beneficial mutations was also conducted. The predicted immunogenicity was then calculated for each active variant.

Computational Identification of Putative T-Cell Epitopes in a Variant kdcA

Putative T-cell epitopes in kdcA (SEQ ID NO. 2) identified using the Immune Epitope Database (IEDB; Immune Epitope Database and Analysis Resource website) tools, as known in the art and proprietary statistical analysis tools (See e.g., iedb.org; and Vita et al., Nucl. Acids Res., 38(Database issue):D854-62 [2010]. Epub 2009 Nov. 11]). The kdcA variant was parsed into all possible 15-mer analysis frames, with each frame overlapping the last by 14 amino acids. The 15-mer analysis frames were evaluated for immunogenic potential by scoring their 9-mer core regions for predicted binding to eight common Class II HLA-DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) that collectively cover nearly 95% of the human population (See e.g., Southwood et al., J. Immunol., 160:3363-3373 [1998]), using methods recommended on the IEDB website. Potential T-cell epitope clusters contained within the variant kdcA (i.e., sub-regions contained within the variant kdcA which have an unusually high potential for immunogenicity) were identified using statistical analysis tools, as known in the art. The identified T-cell epitope clusters were screened against the IEDB database of known epitopes and the GenBank protein database. These screens identified 11 putative T-cell epitopes in kdcA seq ID NO: 2.

Design of Deimmunizing Libraries:

A combinatorial library containing mutations predicted to reduce immunogenicity across the 11 T-cell epitopes was constructed. In parallel, libraries were designed using saturation mutagenesis to mutagenize positions within these 11 T-cell epitopes. Variants with neutral or beneficial activity were identified from the combinatorial and saturation mutagenesis library and the effects of these mutations on the predicted binding to the eight common Class II HLA-DR alleles were analyzed. Multiple mutations were predicted to remove or reduce several of the eleven identified T cell epitopes (See, Table 7.1).

The best hits from the above combinatorial and saturation mutagenesis libraries were then recombined in another combinatorial library and screened to identify variants with neutral or beneficial diversity that further reduced the predicted TIS and IHC scores (See, Table 7.2).

Construction and Screening of Deimmunizing Libraries

Combinatorial and saturation mutagenesis libraries designed as described above were constructed by methods known in the art, and tested for activity in the screening conditions as described in Example 4. Active variants were identified and sequenced. Their activities and mutations with respect to kdcA SEQ ID NO: 2 and SEQ ID NO: 46, are provided in Table 7.1 and Table 7.2, respectively.

Identification of Deimmunizing Diversity

Active variants were analyzed for their levels of immunogenicity by evaluating their binding to the eight common Class II HLA-DR alleles described above. The total immunogenicity score and immunogenic hit count are indicated in Table 7.1 and Table 7.2. The total immunogenicity score reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity). The immunogenic “hit count” indicates the number of 15-mer analysis frames with an unusually high potential for immunogenicity (i.e., a higher hit count indicates a higher potential for immunogenicity). Mutations with a lower total predicted immunogenicity score and/or an immunogenic hit count less than that of the reference variant were considered to be “deimmunizing mutations.” The deimmunizing mutations that were identified as being the best were recombined to generate a number of variants that are active and predicted to be significantly less immunogenic than the starting reference variant kdcA. In the following Tables, the FIOP results are from the screening assay as described in Example 4; for the total immunogenicity score (TIS) and immunogenic hit count (IHC), the results are indicated for the full length kdcA protein (Table 7.1 and Table 7.2).

TABLE 11.1

Activity, Predicted Total Immunogenicity (TIS) and Immunogenic Hit Count (IHC)

for Variants Targeting Predicted T cell epitopes in kdcA Variants Relative to SEQ ID NO: 2

FIOP by

KIC/KIC-
TIS
IHC

SEQ ID

d3 (Relative
(Relative to
(Relative to

NO:
Amino Acid Differences
to SEQ ID
SEQ ID
SEQ ID

(nt/aa)
(Relative to SEQ ID NO: 2)
NO: 2)¹
NO: 2)
NO: 2)

1/2

+
660
54

17/18
L153A/S442A
+
646
50

9/10
M124E/S442A
+
642
47

317/318
K233N/S350K/S351D/S355T/F388P/R394H
+
603
40

319/320
S350K/S351D/A352E/S355T/F388P/R394H
+
614
44

321/322
S37A/S351D/F388P
+
615
43

7/8
L153A/I264E
+
650
53

15/16
S442A
+
654
50

323/324
S37A/Y144T/S351D/A352E
+
606
41

325/326
S252H/F275L/S442A
+
646
50

327/328
M124E/L153A/S442A
+
634
47

329/330
Y144T/R394H
+
626
42

331/332
A290G/L541I
+
653
49

333/334
F388P/R394H
+
638
49

335/336
L153A/F275L
+
646
54

337/338
V229I
+
645
47

339/340
E231K/T232F/F275L/V281A/S442A
+
642
49

341/342
S37A/Y144T/V229I/K233N/F320L/F388P
+
574
30

343/344
S37A/F388P
+
625
48

345/346
Y144T/K233N/F388P
+
601
38

347/348
L153A/I264E/F275L/R394H/S442A
+
625
44

349/350
S37A
+
646
53

351/352
L250D
+
657
53

353/354
Y144T
+
639
47

355/356
L441T
+
634
49

357/358
S252H
+
658
54

359/360
I256T
+
645
52

361/362
L541I
+
656
50

363/364
S37D
+
646
53

365/366
R38H
+
655
53

367/368
R38S
+
656
53

369/370
S37G
+
650
53

371/372
M124K
+
648
51

373/374
S151Q
+
659
49

375/376
Y144R
+
641
47

377/378
S151K
+
655
49

379/380
S151W
+
651
49

381/382
V229L
+
644
51

383/384
K233L
+
648
47

385/386
L234A
+
653
53

387/388
L254A
+
643
52

389/390
F253H
+
654
52

391/392
F253Y
+
657
53

393/394
L254G
+
643
52

395/396
S262T
+
653
53

397/398
L254S
+
643
52

399/400
I256V
+
650
52

401/402
M278C
+
621
46

403/404
R321E
+
629
47

405/406
S325F
+
634
47

407/408
R321S
+
636
47

409/410
R321L
+
651
52

411/412
S325L
+
645
51

413/414
S325H
+
649
51

415/416
A352I
+
652
49

417/418
P353E
+
639
49

419/420
S355E
+
634
49

421/422
A352W
+
636
49

423/424
S350Y
+
657
53

425/426
F347M
+
644
49

427/428
S355W
+
634
49

429/430
I348C
+
639
49

431/432
S355K
+
646
50

433/434
S350T
+
651
49

435/436
A352T
+
655
49

437/438
A352R
+
648
49

439/440
S355F
+
649
50

441/442
F347L
+
648
49

443/444
S355V
+
648
50

445/446
F388W
+
640
53

447/448
F388D
+
630
49

449/450
F388N
+
641
49

451/452
K446H
+
641
49

453/454
I443W
+
648
49

455/456
K446L
+
648
49

457/458
I443V
+
648
49

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 2, and defined as follows:

““+”” = 0.9 to 1.2.

TABLE 11.2

Activity, Predicted Total Immunogenicity (TIS) and Immunogenic Hit Count (IHC)

for Variants Targeting Predicted T-cell Epitopes in kdcA Variants Relative to SEQ ID NO: 46

FIOP by
TIS
IHC

SEQ ID

KIC/KIC-d3
(Relative
(Relative

NO:
Amino Acid Differences
(Relative to SEQ
to SEQ ID
to SEQ ID

(nt/aa)
(Relative to SEQ ID NO: 46)
ID NO: 46)¹
NO: 46)
NO: 46)

45/46

+
613
40

459/460
S328L
+
612
39

461/462
M124W/Q152R/V229A/R321S/I348D/
+
514
16

S350G/F388P/K482R

463/464
M124W/K233H/L254G/S328L/I348D/
+
534
21

A352N/F388P

465/466
M124W/R321S/I348D/F388P
+
538
21

467/468
M124W/L254G/S328N/I348D/S350G
+
549
26

469/470
M124W/Q152R/R321S/I348D/S350G/
+
551
23

F388P

471/472
M124W/Q152R/L254G/S328N/S350G/
+
546
23

F388P/K446R

473/474
M124W/S328L/S350G/A352N/K446R
+
589
31

475/476
M124W/S328N
+
592
33

477/478
M124W/Q152R/V229A/K233H/R321S/
+
548
21

S350G

479/480
M124W/Q152R/V229A/K233H/R321S/
+
548
21

S328L/A352Y/K446R

481/482
R321S/S350G/A352N/K446R
+
570
29

483/484
Q152R/V229A/K233H/I348D/S350G/
+
571
31

A352N

485/486
K233H/L254G/I348D
+
555
31

487/488
M124W/S328L/I348D/A352N/F388P
+
566
26

489/490
V229A/R321S/K446R
+
559
27

491/492
Q152R/L254G/R321S/I348D
+
559
29

493/494
M124W/S328L/I348D/S350G/A352Y/
+
561
26

F388P

495/496
M124W/S328L/A352Y/F388P
+
561
26

497/498
M124W/I348D/A352N
+
588
33

499/500
Q152R/S328N/F388P/K446R
+
588
33

501/502
M124W/Q152R/F388P/K446R
+
601
35

503/504
M124W/Q152R/F388P
+
602
34

505/506
R38H/V229A/F253H/I256V/S355E
+
538
26

507/508
M124K/R321E/S325F/S355L
+
551
26

509/510
M124K/V229A/I256V/R321E/S325F/
+
497
16

S355E

511/512
F253Y/L254A/R321E/S325F/S355E
+
532
26

513/514
R38H/S355E
+
586
35

515/516
R38H/S325F/S355L
+
574
29

517/518
R38H/M124K/S355E
+
574
32

519/520
P353D
+
590
35

521/522
K233L/S325L/F388D/I443W/K446E
+
537
24

523/524
F275Q/S325F/K446E
+
552
31

¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 46, and defined as follows:

““+”” = 0.9 to 1.2.

While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

For all purposes in the United States of America, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

ENGINEERED BRANCHED CHAIN KETOACID DECARBOXYLASES

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