Patent Application
20210246476
This application contains a sequence listing in Computer Readable Form (CRF). The CFR file containing the sequence listing entitled “PA572-0001_ST25.txt”, which was created on Feb. 1, 2021, and is 437,797 bytes in size. The information in the sequence listing is incorporated herein by reference in entirety.
The present disclosure is directed generally to methods for the biosynthesis of nicotinamide mononucleotide (NMN), nicotinamide mononucleotide derivatives, or mixtures thereof. More specifically, the present disclosure is directed to enzymes, multiple-enzyme pathways, and methods to adjust reaction conditions and components in order to maximize the conversion of low-cost starting materials towards desired products in high yields.
Nicotinamide mononucleotide (NMN) is a phosphate ester nucleotide derived from nicotinamide riboside (NR). NMN exists in two forms: an oxidized and reduced form, abbreviated as NMN+ and NMNH, respectively. Humans can transport NMN across the cell membrane and then generate NAD, which promotes cellular NAD production and counteract age-associated pathologies associated with a decline in tissue NAD levels. Supplementation of NMN increases arterial SIRT1 activity and reverses age-associated arterial dysfunction and oxidative stress. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age-related arterial dysfunction by decreasing oxidative stress (de Picciotto et al. (2016). “Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice.” Aging cell 15(3): 522-530). Long-term uptake of NMN mitigates age-associated physiological decline in mice. Orally administered NMN was quickly utilized to synthesize NAD in tissues. NMN effectively mitigates age-associated physiological decline in mice. Without any obvious toxicity or deleterious effects, NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies (Mills et al. (2016). “Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice.” Cell Metabolism. 24(6): 795-806).
Nicotinamide adenine dinucleotide (NAD) is a coenzyme that is central to metabolism in all living cells, carrying electrons from one biochemical to another. It consists of two nucleotides (i.e., adenine and nicotinamide) joined through their phosphate groups. NAD exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH, respectively. Nicotinamide adenine dinucleotide phosphate (NADP) is a coenzyme used in anabolic reactions. NADP exists in two forms: an oxidized and reduced form, abbreviated as NADP+ and NADPH, respectively.
Niacin or nicotinic acid (NA) is a simplest form of vitamin B3, an essential human nutrient. Nicotinamide (NAM) is another form of vitamin B3 found in food and used as a dietary supplement and medication. NAM is better than NA. NAM is added into grains (such as rice and wheat flour) in numerous countries, such as USA for the prevention of pellagra (niacin deficiency). Nicotinamide riboside (NR) is be another new form of vitamin B3, a compound that NAM is linked with D-ribose. Their structures and names are present in
Nicotinamide riboside phosphorylase (NRP) can irreversibly synthesize NR from NAM and R1P (Reaction 1).
nicotinamide (NAM)+R1P→nicotinamide riboside (NR)+Pi [1]
where → denotes a reversible reaction, Pi is an inorganic orthophosphate anion. NRP is a kind of purine nucleoside phosphorylase (PNP, EC 2.4.2.1), some of which have a promiscuous NRP activity using nicotinamide although nicotinamide is not a purine. For example, several PNPs from Homo sapiens (human) (Grossman, L. and N. O. Kaplan (1958). “NICOTINAMIDE RIBOSIDE PHOSPHORYLASE FROM HUMAN ERYTHROCYTES: II. NICOTINAMIDE SENSITIVITY.” Journal of Biological Chemistry 231(2): 727-740), Bos taurus (Imai, T. and B. M. Anderson (1987). “Nicotinamide riboside phosphorylase from beef liver: Purification and characterization.” Archives of Biochemistry and Biophysics 254(1): 253-262), Escherdia coli (Wielgus-Kutrowska, B., E. Kulikowska, J. Wierzchowski, A. Bzowska and D. Shugar (1997). “Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.” European Journal of Biochemistry. 243(1-2): 408-414), and Cellulomonas sp. (Velasquez J E, Green P R, Wos J A (2015) Method for preparing nicotinamide riboside. US20170121746A1), have validated NPR activities in vitro.
Nicotinamide riboside kinase (NRK, EC 2.7.1.22) can synthesize NMN from NR and adenosine triphosphate (ATP) (Reaction 2), which can be regenerated by numerous well-known means including an enzymatic ATP regeneration system or ATP-generating permeabilized living microorganisms (e.g., Escherichia coli, Saccharomyces cerevisiae) that continuously make ATP (Chen, H.-G., and Zhang, Y.-H. P. J. (2020) “Enzymatic Regeneration and Conservation of ATP: Challenges and Opportunities” Critical Review in Biotechnology, Epub, doi.org/10.1080/07388551.07382020.01826403).
nicotinamide riboside (NR)+ATP→nicotinamide mononucleotide (NMN)+ADP [2]
where → denotes an irreversible reaction with a Keq (equilibrium constant) value of more than 100.
Hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) can reversibly synthesize 5-phospho-alpha-D-ribose-1-diphosphate (PRPP) from nucleotides, such as inosine monophosphate (IMP) or guanosine monophosphate (GMP) or adenosine monophosphate (AMP), and diphosphate (PPi), cogenerating hypoxanthine or guanosine or adenosine, respectively. Reaction 3 shows a representative biochemical reaction starting with IMP.
inosine monophosphate (IMP)+diphosphate (PPi)hypoxanthine+PRPP [3]
Nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) can reversibly convert PRPP and NAM to NMN and PPi (Reaction 4).
PRPP+NAMNMN+PPi [4]
NR can be synthesized by using organic chemical methods (Sauve, A. A. and T. Yang (2006). Nicotinoyl riboside compositions and methods of use. WO2007061798A2; Felczak K Z (2017) Synthetic methods for the preparation of nicotinamide riboside and related compounds. WO2017218580A1; Gelman, D., A. ZOABI, A. Zeira and R. Abu-Reziq (2017). Process for preparation of nicotinamide riboside (NR) and cosmetic composition comprising NR and a phosphate-binding agent. WO2017145151A1). However, organic synthesis is complex, requiring the addition and removal of protection groups, and using environmentally-unfriendly organic solvents. NR can be synthesized by enzymes pentosyl transferases (Velasquez J E, Green P R, Wos J A (2015) Method for preparing nicotinamide riboside. US20170121746A1), but it requires a lot of methods to shift the equilibrium reactions toward NR for relatively high yields, for example, in situ removal of phosphate ions. It can also be produced by engineered microorganisms (Brenner, C., P. Belenky and K. L. Bogan (2009). Yeast strain and method for using the same to produce nicotinamide riboside. U.S. Pat. No. 8,114,626B2; Lawrence, A. and C. ViAROUGE (2017). Microbial production of nicotinamide riboside. WO2018211051A1), but its titers and yields are low.
NMN can be synthesized from NR by chemical synthesis (Sauve, A. and F. S. Mohammed (2015). Efficient synthesis of nicotinamide mononucleotide. WO2016160524A1), by using an enzyme mutant of phosphoribosylpyrophosphate (PRPP) synthetase (Wu L, Sinclair D A, Meetze K (2015) Enzymatic systems and methods for synthesizing nicotinamide mononucleotide and nicotinic acid mononucleotide. WO2016198948A1.), nicotinamide riboside kinase (Tao A, Fu M, Liang X-L (2016) Method for preparing beta-nicotinamide mononucleotide by using enzymic method. WO2018120069A1, CN201611245619), or a combination of nicotinamide phosphoribosyltransferase, phosphoribose pyrophosphokinase, and ribokinase (Fu R-Z, and Zhang Q (2016) Method for preparing nicotinamide mononucleotide. WO2017185549A1), or by engineered organism containing overexpressed overexpresses an enzyme nicotinamide phosphoribosyltransferase (Sinclair D A, and Ear P H (2014) Biological production of NAD precursors and analogs. US20160287621A1, WO2015069860A1).
NAD was purified from microorganisms but its production costs were very high. It can be overexpressed by fermentation of engineered microorganisms (Sinclair, D A and Ear, P H (2014) Biological production of NAD precursors and analogs. US20160287621A1, WO2015069860A1). A U.S. Pat. No. 4,411,995 (Whitesides, G. and D. R. Walt (1981). Synthesis of nicotinamide cofactors. U.S. Pat. No. 4,411,995) describes an enzymatic process for producing NAD, but such a method, while efficient in its yield, requires carefully controlled conditions and the addition of costly enzymes. Recently, it is mainly produced from NMN and ATP by an NMNAT enzyme (Tao, A., B. Li, X. Ju, X. Liang and J. Zhuang (2013). Method for preparing oxidized coenzyme I. WO2014146250A1) or even one enzyme from a hyper-thermophilic microorganism (Fu, R. Z. (2013). Nicotinamide mononucleotide adenylyltransferase (Nmnat) mutant as well as coding gene and application thereof. CN103710321B). It can also be synthesized from ATP, deamide-NAD and ammonia by the Bacillus stearothermophilus NAD synthetase (EC 6.3.1.5) (Takahashi, M., H. Misaki, S. Imamura and K. Matsuura (1987). “Novel NAD synthetase, assay method using said novel NAD synthetase and a process for production thereof. U.S. Pat. No. 4,921,786A). It also can be synthesized by using an in vitro reconstituted mammalian NAD biosynthesis system comprising of NAMPT and NMNAT from NAM, PRPP and ATP (Imai, S.-I., J. R. Revollo and A. A. Grimm (2005). NAD biosynthesis systems. US20090246803A1. WO2006041624A2). NADP can be synthesized with ATP or polyphosphate and NAD by using NAD kinase (Kawai, S., K. Murata, H. Matsukawa, S. Tomisako, Y. Ando and Y. Matsuo (2001). Process for preparing nicotinamide adenine dinucleotide phosphate (NADP). U.S. Pat. No. 7,863,014B2; Tao, A., B. Li, L. Xie, J. Zhuang, Y. Zhou, C. Zhang and G. Liu (2012). Enzymatic preparation method of oxidized coenzyme II. CN102605027B).
To make NMN and its derivatives NAD and NADP at low costs and in high yields, the present invention was demonstrated to address the below issues:
The present invention utilizes available and low-cost starting materials, isolated enzymes, either freely soluble or immobilized for stability and recoverable for reuse, or microorganisms containing said enzymes, and allows for the integrated multiple-enzyme reactions (called artificial enzymatic pathways in one pot or multiple-enzyme one pot) to push the overall reaction toward the synthesis of desired products in high yields.
In one embodiment, NMN can be synthesized from nicotinamide (NAM) and D-ribose-1-phosphate (R1P) catalyzed by two enzymes—nicotinamide riboside phosphorylase (NRP, EC 2.4.2.1) and nicotinamide riboside kinase (NRK, EC 2.7.1.22), along with ATP regeneration (
In one embodiment, alpha-D-ribose-1-phosphate (R1P) can be generated from purine nucleosides (e.g., inosine, guanosine and adenosine) catalyzed by purine nucleoside phosphorylase (PNP) and/or guanosine nucleoside phosphorylase (GP, 2.4.2.15) (
In one embodiment, R1P can be generated from nucleotides (e.g., inosine monophosphate (IMP), guanosine monophosphate (GMP), or adenosine monophosphate (AMP)) catalyzed by pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10) and phosphopentomutase (PPM, EC 5.4.2.7) (
In one embodiment, R1P can be generated from D-ribose and ATP catalyzed by ribokinase (RK, EC 2.7.1.15) and phosphopentomutase (PPM, EC 5.4.2.7) (
In one embodiment, R1P can be generated from D-xylose, which can be converted to D-ribose by D-xylose isomerase (D-XI, EC 5.3.1.5), pentose 3-epimerase (P3E, EC 5.1.3.31), and D-ribose isomerase (D-RI, EC 5.3.1.20) or mannose 6-phosphate isomerase (MPI, EC 5.3.1.8) (
In one embodiment, R1P can be generated from D-xylose and polyphosphate (
In one embodiment, R1P can be generated from starch and phosphate (
In one embodiment, R1P can be generated from cellodextrin/cellobiose and phosphate (
In one embodiment, R1P can be generated from sucrose and phosphate (
In one embodiment, R1P can be generated from D-glucose and polyphosphate (
In one embodiment, R1P can be generated from D-fructose and polyphosphate (
In one embodiment, R1P can be generated from a mixture of hexoses and phosphate/or polyphosphate, with a mixture of the above enzymes (
In one embodiment, ATP can be regenerated from acetyl phosphate catalyzed by acetate kinase (AK, EC 2.7.2.1) (
In one embodiment, ATP can be recycled for in-depth use by using adenylate kinase (ADK, EC 2.7.4.3) and adenosine kinase (AdK, EC 2.7.1.20) (
In one embodiment, adenosine is hydrolyzed to adenine and ribose catalyzed by adenosine nucleosidase (AN, EC 3.2.2.7) or purine nucleosidase (PN, EC 3.2.2.1).
In one embodiment, ATP can be regenerated from polyphosphate by using polyphosphate kinase (PPK, EC 2.7.4.1) (
In one embodiment, ATP can be regenerated from polyphosphate by using polyphosphate: AMP phosphotransferase (PPT, EC 2.7.4.B2) and adenylate kinase (ADK, EC 2.7.4.3) (
In one embodiment, ATP can be regenerated by using permeabilized cells (e.g., Escherichia coli, Saccharomyces cerevisiae) (
In one embodiment, ATP can be regenerated by using artificial or natural organelles (e.g., light-driven chloroplasts and mitochondria) that continuously make ATP.
In one embodiment, NMN can be synthesized from inosine monophosphate (IMP) and nicotinamide (NAM) catalyzed by two enzymes: hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) and nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) (
IMP+NAMNMN+hypoxanthine [5]
In one embodiment, NMN can be synthesized from guanosine monophosphate (GMP) and nicotinamide (NAM) catalyzed by two enzymes: hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) and nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) (
GMP+NAMNMN+guanosine [6]
In one embodiment, NAD can be synthesized from NMN and ATP catalyzed by nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) with a removal of diphosphate by diphosphatase (DPP, EC 3.6.1.1) (
In one embodiment, NADP can be synthesized from NAD and ATP or polyphosphate catalyzed by ATP-dependent or polyphosphate-dependent NAD kinase (NADK, EC 2.7.1.23) (
In one embodiment, NADP can be synthesized from NMN and ATP/polyphosphate catalyzed by NMNAT, DPP, and NADK (
In one embodiment, NR can be hydrolyzed from NMN, cogenerating Pi, catalyzed by 5′-nucleotidase (EC 3.1.3.5), nucleotide phosphatase (EC 3.1.3.31), or acid phosphatase (EC 3.1.3.2).
In one embodiment, adenosine can be converted to adenine and ribose by using adenosine nucleosidase (AN, EC 3.2.2.7) or purine nucleosidase (PN, EC 3.2.2.1) (
In one embodiment, guanine can be converted to urate by using guanine deaminase (GDA, EC 3.5.4.3), xanthine oxidase (XO, EC 1.17.3.2), and catalase (CA, EC 1.11.1.7, EC 1.11.1.21) (
In one embodiment, hypoxanthine can be converted to xanthine and urate by using XO and CA (
In one embodiment, hypoxanthine can be converted to xanthine by xanthine dehydrogenase (XDH, EC 1.17.1.4), H2O-forming NADH oxidase (NOX, EC 1.6.3.4 or EC 1.6.3.2) (
In one embodiment, diphosphate can be converted to two phosphate ions by (inorganic) diphosphatase (DPP, EC 3.6.1.1).
In one embodiment, NMN can be synthesized from NAM, IMP and acetyl-phosphate catalyzed by five enzymes: inosinate nucleosidase (IMPN, EC 3.2.2.12), PPM, NRP, NRK, and AK (
In one embodiment, NMN can be synthesized from NAM, IMP and ATP catalyzed by eight enzymes: IMPN, PPM, NRP, NRK, ADK, AdK, NO, and CA (
For example, two supplementary enzymes may be xanthine dehydrogenase (XDH) and H2O-forming NADH oxidase (NOX).
In one embodiment, NMN can be synthesized from NAM, IMP and polyphosphate catalyzed by five enzymes: IMPN, PPM, NRP, NRK, PPK (
In one embodiment, NAD can be synthesized from NAM, IMP and ATP catalyzed by IMPN, PPM, NRP, NRK, NMNAT and DPP (
In one embodiment, NMN can be synthesized from IMP and NAM catalyzed by four enzymes: hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8), nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12), xanthine oxidase (XO), and catalase (CA) (
In one embodiment, NMN can be synthesized from IMP and NAM catalyzed by four enzymes: HGPRT, NAMPT, xanthine dehydrogenase (XDH), and H2O-forming NADH oxidase (NOX) (
In one embodiment, NMN can be synthesized from GMP and NAM catalyzed by five enzymes: HGPRT, NAMPT, guanine deaminase (GDA, EC 3.5.4.3), XO, and CA (
In one embodiment, NAD can be synthesized from IMP, ATP, and NAM catalyzed by five enzymes: hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8), nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12), nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1), XDH and NOX (
In one embodiment, NAD can be synthesized from GMP, ATP, and NAM catalyzed by six enzymes: HGPRT, NAMPT, NMNAT, GDA, XO and CA (
In one embodiment, NADP can be synthesized from IMP (or GMP), ATP, NAM and polyphosphate catalyzed by four enzymes: HGPRT, NAMPT, NMNAT, and NADK (
One-pot biosynthesis comprised of multiple enzymes and even artificial enzymatic pathways is an emerging biomanufacturing platform. The integration of numerous enzymes in one pot or bioreactor or vessel consolidate multiple-step bioreactions, having multiple benefits, such as no separation of intermediates, enhanced volumetric productivity. But the careful design of pathways featuring the unidirectional last reaction step or effectively complete removal of byproducts by unidirectional reactions can increase the yields of desired products.
As used herein, “artificial enzymatic pathway” refers to the manmade enzyme mixture, whereas the product of one enzyme is the substrate of another enzyme. Via the intermediates (i.e., the product of one enzyme and the substrate of another enzyme), several enzymes make up the artificial enzymatic pathway.
As used herein, “one pot” or “one vessel” refers to the multiple-step bioreactions catalyzed by multiple enzymes or the artificial enzymatic pathway occurring in one bioreactor or even in one microbial cell (also called a microbioreactor), whereas they work together in trade-off reaction conditions for all of enzyme components. Sometimes, it was called the consolidated bioreactions in one pot.
As used herein, the term “nucleoside” refers to a glycosylamine having a nitrogenous base, such as a purine or pyrimidine, linked to a 5-carbon sugar (e.g. D-ribose or 2-deoxy-D-ribose) via a β-glycosidic linkage. Nucleosides are also referred as “ribonucleosides” when the sugar moiety is D-ribose, for example, inosine, guanosine, adenosine, nicotinamide riboside.
As used herein, the term “purine nucleoside” refers to a nucleoside, wherein the nitrogenous base is a purine.
As used herein, the term “pyrimidine nucleoside” refers to a nucleoside, wherein the nitrogenous base is a pyrimidine.
As used herein, the term “nucleotide”, also known as “nucleoside monophosphate”, refers to a compound having a nucleoside esterified to an orthophosphate group via the hydroxyl group bound to the 5-carbon of the sugar moiety. Nucleotides are also referred as “ribonucleotides”. Non-limiting examples of nucleotide include, but not limited to for example, inosine monophosphate (IMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), nicotinamide mononucleoside (NMN).
As used herein, the term “nitrogenous base”, refers to a compound containing a nitrogen atom that has the chemical properties of a base. Non-limiting examples of nitrogenous bases are compounds comprising pyridine, purine, or pyrimidine moieties, including, but not limited to adenine, guanine, hypoxanthine, thymine, cytosine, uracil, and nicotinamide.
As used herein, the term “inorganic orthophosphate” refers to a compound composed of four oxygen atoms arranged in an almost regular tetrahedral array about a central phosphorus atom. Inorganic orthophosphate or phosphate or phosphate ions may be present in several ionic forms, depending on the pH of the solution, including [PO4]3−, [HPO4]2−, [H2PO4]−, and H3PO4.
As used herein, the term “inorganic diphosphate”, also known as “inorganic pyrophosphate”, “pyrophosphate”, “PPi” refers to a compound containing one P—O—P bond generated by corner sharing of two PO4 tetrahedra. Inorganic diphosphate may be present in several ionic forms, depending on the pH of the solution, including [P2O7]4−, [HP2O7]3−, [H2P2O7]2−, [H3P2O7]−, and H4P2O7.
As used herein, the term “conservative substitution” refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. A conservative substitution involves replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art, and include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
As used herein, the term “converting” refers to a chemical transformation from one molecule to another, primarily catalyzed by an enzyme or enzymes, although other organic or inorganic catalysts may be used.
As used herein, the term “conversion,” in the context of chemical transformations, refers to the ratio in % between the molar amount of the desired product and the molar amount of the limiting reagent.
As used herein, the term “endogenous” refers to polynucleotides, polypeptides, or other compounds that are expressed naturally or originate within an organism or cell. That is, endogenous polynucleotides, polypeptides, or other compounds are not exogenous. For instance, an “endogenous” polynucleotide or peptide is present in the cell when the cell was originally isolated from nature.
As used herein, the term “exogenous” refers to any polynucleotide or polypeptide that is not naturally found or expressed in the particular cell or organism where expression is desired. Exogenous polynucleotides, polypeptides, or other compounds are not endogenous.
As used herein, the term “identical” or percent “identity,” in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection.
As used herein, the term “isolated enzyme” refers to enzymes free of a living organism. Isolated enzymes of the invention may be suspended in solution following lysing of the cell they were expressed in, partially or highly purified, soluble or bound to an insoluble matrix.
As used herein, the term “naturally-occurring” refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. As used herein, “naturally-occurring” and “wild-type” are synonyms.
As used herein, a recombinant gene that is “over-expressed” produces more RNA and/or protein than a corresponding naturally-occurring gene in the microorganism. Methods of measuring amounts of RNA and protein are known in the art. Over-expression can also be determined by measuring protein activity such as enzyme activity. Depending on the embodiment of the invention, “over-expression” is an amount at least 3%, at least 5%, at least 10%, at least 20%, at least 25%, or at least 50% more. An over-expressed polynucleotide is generally a polynucleotide native to the host cell, the product of which is generated in a greater amount than that normally found in the host cell. Over-expression is achieved by, for instance and without limitation, operably linking the polynucleotide to a different promoter than the polynucleotide's native promoter or introducing additional copies of the polynucleotide into the host cell.
As used herein, the term “polynucleotide” refers to a polymer composed of nucleotides. The polynucleotide may be in the form of a separate fragment or as a component of a larger nucleotide sequence construct, which has been derived from a nucleotide sequence isolated at least once in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard molecular biology methods, for example, using a cloning vector. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T”. Put another way, “polynucleotide” refers to a polymer of nucleotides removed from other nucleotides (a separate fragment or entity) or can be a component or element of a larger nucleotide construct, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA and cDNA sequences.
As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues which may or may not contain modifications such as phosphates and formyl groups.
As used herein, “recombinant polynucleotide” refers to a polynucleotide having sequences that are not joined together in nature. A recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host cell.” The polynucleotide is then expressed in the recombinant host cell to produce, e.g., a “recombinant polypeptide”.
As used herein, the term “recombinant expression vector” refers to a DNA construct used to express a polynucleotide that, e.g., encodes a desired polypeptide. A recombinant expression vector can include, for example, a transcriptional subunit comprising: i) an assembly of genetic elements having a regulatory role in gene expression, for example, promoters and enhancers; ii) a structural or coding sequence which is transcribed into mRNA and translated into protein; and iii) appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmid, virus, bacteriophage, and transposon. Possible vectors for use in the invention include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences, e.g., bacterial plasmids; phage DNA; yeast plasmids; and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowl pox, baculovirus, SV40, and pseudorabies.
As used herein, a “recombinant gene” is not a naturally-occurring gene. A recombinant gene is man-made. A recombinant gene includes a protein coding sequence operably linked to expression control sequences. Embodiments include, but are not limited to, an exogenous gene introduced into a microorganism, an endogenous protein coding sequence operably linked to a heterologous promoter (i.e., a promoter not naturally linked to the protein coding sequence) and a gene with a modified protein coding sequence (e.g., a protein coding sequence encoding an amino acid change or a protein coding sequence optimized for expression in the microorganism). The recombinant gene is maintained in the genome of the microorganism, on a plasmid in the microorganism or on a phage in the microorganism.
As used herein, the term “substantially homologous” or “substantially identical” in the context of two nucleic acids or polypeptides, generally refers to two or more sequences or subsequences that have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. The substantial identity can exist over any suitable region of the sequences, such as, for example, a region that is at least about 50 residues in length, a region that is at least about 100 residues, or a region that is at least about 150 residues. In certain embodiments, the sequences are substantially identical over the entire length of either or both comparison biopolymers.
Compounds:
Enzymes:
The current invention provides enzymatic reactions and methods to produce NMN via an intermediate α-D-ribose-1-phosphate and then nicotinamide riboside is converted to NMN catalyzed by nicotinamide riboside kinase (NRK, EC 2.4.1.22) (
RIP Generation from Nucleosides
Inosine, guanosine, and adenosine are purine nucleosides comprising a respective purine base (hypoxanthine, guanine, and adenine) attached to a D-ribose ring via a β-N9-glycosidic bond. They are cheap bulk biochemicals produced by industrial microbial fermentation. Inosine has a higher water solubility of 15.8-21 g/L at 20° C. than guanosine (0.7 g/L at 18° C.). Hypoxanthine has a low water solubility of 0.7 g/L at 20° C. while guanine is water insoluble but is soluble in diluted acid.
In one embodiment, alpha-D-ribose-1-phosphate (R1P) is generated from inosine, guanosine or adenosine catalyzed by purine nucleoside phosphorylase (PNP, EC 2.4.2.1) and/or guanosine nucleoside phosphorylase (GP, 2.4.2.15) (
In one embodiment, alpha-D-ribose-1-phosphate (R1P) is generated from pyrimidine nucleosides and phosphate ions catalyzed by their respective pyrimidine-nucleoside phosphorylase (PyNP, EC 2.4.2.2), uridine phosphorylase (UP, EC 2.4.2.3) or thymidine phosphorylase (TP, EC 2.4.2.4).
RIP Generation from Nucleotides
Inosine monophosphate (IMP) is an ester of phosphoric acid with hypoxanthine. It is widely used as an umami flavor enhancer with E number (food additive) reference E630. It used to be made from chicken byproducts or other meat industry waste but it is produced by microbial fermentation. Guanosine monophosphate (GMP) is another flavor enhancer with E number reference E626. A blend of fermented products GMP, IMP and monosodium glutamate (MSG, E621) is widely used in Asian cuisine. Industrial production of both IMP and GMP was made either by RNA breakdown and nucleotide extraction and is produced by microbial fermentation using different microorganisms such as Corynebacterium, Bacillus, or Escherichia coli. Approximately 22,000 tons of GMP and IMP were produced in year 2010. Adenosine monophosphate (AMP) can be produced from RNA breakdown and nucleotide extraction and is produced by microbial fermentation using different microorganisms. The other NMPs (e.g., cytidine monophosphate, CMP; and uridine monophosphate, UMP) are not as cheap as IMP, GMP and AMP.
In one embodiment, alpha-D-ribose-1-phosphate is generated from a nucleotide inosine monophosphate (IMP) catalyzed by pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10) or inosinate nucleosidase (EC 3.2.2.12) followed by phosphopentomutase (PPM, EC 5.4.2.7) (
In one embodiment, alpha-D-ribose-1-phosphate is generated from a nucleotide guanosine monophosphate (GMP) catalyzed by pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10) and phosphopentomutase (PPM, EC 5.4.2.7) (
In one embodiment, alpha-D-ribose-1-phosphate is generated from a nucleotide adenosine monophosphate (AMP) catalyzed by pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN) or AMP nucleosidase (EC 3.2.2.4) and phosphopentomutase (PPM, EC 5.4.2.7) (
RIP Generation from D-Pentoses
D-ribose is an affordable pentose, which may be produced from glucose by microbial fermentation or converted from D-xylose catalyzed by three cascade enzymes: D-xylose isomerase (D-XI, EC 5.3.1.5), pentose 3-epimerase (P3E, EC 5.1.3.31), and D-ribose isomerase (D-RI, EC 5.3.1.20) or mannose 6-phosphate isomerase (MPI, EC 5.3.1.8).
In one embodiment, alpha-D-ribose-1-phosphate is generated from D-ribose and ATP catalyzed by ribokinase (RK, EC 2.7.1.15) and phosphopentomutase (PPM, EC 5.4.2.7) (
D-xylose is the most abundant pentose in nature. It is produced from hydrolysis of nonfood hemicellulose. It is called wood sugar and its prices were as low as sucrose in 1940s.
In one embodiment, alpha-D-ribose-1-phosphate is generated from D-xylose, which can be converted to D-ribose by D-xylose isomerase (D-XI, EC 5.3.1.5), pentose 3-epimerase (P3E, EC 5.1.3.31), and D-ribose isomerase (D-RI, EC 5.3.1.20) or mannose 6-phosphate isomerase (MPI, EC 5.3.1.8) (
In one embodiment, alpha-D-ribose-1-phosphate is generated from D-xylose and polyphosphate (
RIP Generation from Hexoses
Hexoses are among the least costly natural sugars. Low-cost and representative hexoses can be classified into monosaccharides (e.g., D-glucose and D-fructose), oligosaccharides (e.g., sucrose, cellobiose, cellodextrins, maltose, maltodextrin, and amylodextrin), and polysaccharides (e.g., cellulose, starch and soluble starch). A few oligosaccharides and starch can be converted to glucose 1-phosphate via their respective phosphorylases without ATP. The examples of glucans and their glucan phosphorylases are starch, maltodextrin and amylopectin catalyzed by alpha-glucan phosphorylase (αGP, EC 2.4.1.1); cellodextrin and cellobiose catalyzed by cellodextrin phosphorylase (CDP, EC 2.4.1.49) and cellobiose phosphorylase (CBP, EC 2.4.1.20); sucrose phosphorylase (SP, EC 2.4.1.7), and cellulose catalyzed by engineered cellulose phosphorylase. Then glucose 1-phosphate is converted glucose 6-phosphate and then it is converted to ribose 5-phosphate via a partial pentose phosphate pathway that can convert one glucose 6-phosphate to one ribose 5-phosphate, two reduced NAD(P)H, and one CO2 (
In one embodiment, alpha-D-ribose-1-phosphate is generated from starch, maltodextrin, or amylodextrin and phosphate (
In another embodiment, soluble starch or maltodextrin is debranched to linear amylodextrin by pullulanase (EC 3.2.1.41) and/or isoamylase (EC 3.2.1.68).
In another embodiment, maltose and maltotriose is converted to long-chain amylodextrin catalyzed by 4-alpha-glucanotransferase (EC 2.4.1.25).
In one embodiment, alpha-D-ribose-1-phosphate is generated from cellodextrin/cellobiose and phosphate (
In another embodiment, water soluble cellodextrin and cellobiose is made through the enzymatic hydrolysis of cellulose catalyzed by that beta-glucosidase-free cellulase mixture containing endo-glucanase (EC 3.2.1.4) and cellobiohydrolase (EC 3.2.1.91).
In another embodiment, water soluble cellodextrin and cellobiose is made through the acidic hydrolysis of cellulose, wherein the strong acid may be fuming HCl, a mixture of concentrated HCl and concentrated sulfuric acid, a mixture of concentrated HCl and concentrated phosphoric acid, and/or their mixture, and so on.
In one embodiment, alpha-D-ribose-1-phosphate is generated from sucrose and phosphate (
In one embodiment, alpha-D-ribose-1-phosphate is generated from glucose and polyphosphate (
In one embodiment, alpha-D-ribose-1-phosphate is generated from fructose and polyphosphate (
In one embodiment, alpha-D-ribose-1-phosphate is generated from a mixture of hexoses (e.g. starch, sucrose, glucose, fructose) and phosphate/or polyphosphate, with a mixture of the above enzymes (
The current invention disclosure presents several commonly-used ATP regeneration (
In one embodiment, ATP is regenerated from acetyl phosphate catalyzed by acetate kinase (AK, EC 2.7.2.1) (
In one embodiment, ATP is recycled for in-depth use by using adenylate kinase (ADK, EC 2.7.4.3) and adenosine kinase (AdK, EC 2.7.1.20) (
In one embodiment, adenosine is hydrolyzed to adenine and ribose catalyzed by adenosine nucleosidase (AN, EC 3.2.2.7) or purine nucleosidase (PN, EC 3.2.2.1). Non-limiting examples of adenosine nucleosidase (AN, EC 3.2.2.7) or purine nucleosidase (PN, EC 3.2.2.1) are listed in Table 1. Exemplary amino acid sequences of ANs from Trypanosoma brucei brucei, Bacillus thuringiensis, and Escherichia coli K-12 known in the art are respectively set out in SEQ ID NOs: 95-97. Also, a typical AN can be isolated from Coffee arabica.
In one embodiment, ATP is regenerated from polyphosphate by using polyphosphate kinase (PPK, EC 2.7.4.1) (
In one embodiment, ATP is regenerated from polyphosphate by using polyphosphate: AMP phosphotransferase (PPT, EC 2.7.4.B2) and adenylate kinase (ADK, EC 2.7.4.3) (
In one embodiment, ATP is regenerated by using permeabilized cells (
In one embodiment, ATP is regenerated by using artificial or natural organelles (e.g., light-driven chloroplasts and mitochondria) that can make ATP continuously.
The current invention provides enzymatic reactions and methods to produce nicotinamide mononucleotide via an intermediate 5-phospho-α-D-ribose-1-diphosphate (PRPP). NMN can be synthesized from inosine monophosphate (IMP) or guanosine monophosphate (GMP) and nicotinamide catalyzed by two enzymes: hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) and nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) (
In one embodiment, NMN can be synthesized from inosine monophosphate or guanosine monophosphate and nicotinamide catalyzed by two enzymes HGPRT (EC 2.4.2.8) and NAMPT (EC 2.4.2.12) in one pot. Non-limiting examples of HGPRTs and NAMPTs are listed in Table 1. Exemplary amino acid sequences of HGPRTs from Giardia intestinalis ATCC 50803, Trypanosoma cruzi, Gallus gallus (chick), Clostridium thermocellum, Haloferax volcanii and Halobacterium salinarum known in the art are respectively set out in SEQ ID NOs: 36-45. Exemplary amino acid sequences of NAMPTs from Haemophilus ducreyi, Shewanella oneidensis, Homo sapiens, Meiothermus ruber DSM 1279, Tenacibaculum maritimum, Marivirga tractuosa and Synechocystis sp. PCC 6803 known in the art are respectively set out in SEQ ID NOs: 46-52.
NMN derivatives include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and nicotinamide riboside (NR). NAD is reversibly synthesized from NMN catalyzed by nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1). To increase NAD yield, the byproduct diphosphate is removed by diphosphatase (DPP, EC 3.6.1.1). NADP is reversibly synthesized from NAD and ATP or polyphosphate. To increase NADP yield, it is important to remove the byproduct. NR is synthesized from NMN catalyzed by 5′-nucleotidase (EC 3.1.3.5), nucleotide phosphatase (EC 3.1.3.31), or acid phosphatase (EC 3.1.3.2). This reaction is unidirectional so that the removal of other byproduct is not important to increase its yield.
In one embodiment, NAD is synthesized from NMN and ATP catalyzed by nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) (
In one embodiment, NAD is synthesized from NR and ATP catalyzed by a bifunctional enzyme having activities of nicotinamide riboside kinase (NRK, EC 2.7.1.22) and nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1). Non-limiting examples of these bifunctional enzymes (NRK/NMNATs) are listed in Table 1. Exemplary amino acid sequences of NRK/NMNATs from Clostridium thermocellum, Escherichia coli (strain K12) and Salmonella typhimurium known in the art are respectively set out in SEQ ID NOs: 91-93.
In one embodiment, diphosphate is hydrolyzed to two phosphate ions by diphosphatase (DPP, EC 3.6.1.1) (
In one embodiment, NADP is synthesized from NAD and ATP or polyphosphate catalyzed by ATP-dependent or polyphosphate-dependent NAD kinase (NADK, EC 2.7.1.23) (
In one embodiment, NADP is synthesized from NMN and ATP/polyphosphate catalyzed by NMNAT, DPP, and NADK (
In one embodiment, NR is hydrolyzed from NMN, releasing inorganic phosphate (Pi), catalyzed by 5′-nucleotidase (EC 3.1.3.5), nucleotide phosphatase (EC 3.1.3.31), acid phosphatase (EC 3.1.3.2), the same function enzyme, or their mixture.
To shift the equilibrium reaction toward the formation of desired products, it is useful to in situ remove the byproducts of these equilibrium reactions by using the enzymes.
In one embodiment, adenosine is hydrolyzed to adenine and ribose by adenosine nucleosidase (AN, EC 3.2.2.7) or purine nucleosidase (PN, EC 3.2.2.1) (
In one embodiment, guanine is converted to xanthine by guanine deaminase (GDA, EC 3.5.4.3) (
In one embodiment, xanthine is converted to urate by xanthine oxidase (XO, EC 1.17.3.2) or xanthan dehydrogenase (XDH, EC 1.17.3.4) followed by catalase (CA, EC 1.11.1.6, EC 1.11.1.21, or EC 1.11.1.7) (
In one embodiment, hypoxanthine can be converted to xanthine and then to urate by xanthine oxidase (XO, EC 1.17.3.2) and catalase (
In one embodiment, hypoxanthine can be converted to xanthine by xanthine dehydrogenase (XDH, EC 1.17.1.4) and H2O-forming NADH oxidase (NOX, EC 1.6.3.4 or EC 1.6.3.2) (
In one embodiment, NOX may be replaced with other NAD(P)H-consuming enzymes. For example, hydrogenase replaces a mixture of H2O2 formation NADH oxidase (EC 1.6.3.3) and catalase.
In one embodiment, diphosphate can be converted to two phosphate ions by (inorganic) diphosphatase (DPP, EC 3.6.1.1). The hydrolysis of diphosphate is unidirectional. Non-limiting examples of DPPs are listed in Table 1. Exemplary amino acid sequences of DPPs from Thermoplasma acidophilum and Pyrococcus furiosus known in the art are respectively set out in SEQ ID NOs: 105-106.
Enzyme Variants
Enzymes disclosed in the invention are naturally occurring in various organisms. While specific enzymes with the desired activity are used in the examples, the invention is not limited to these enzymes as other enzymes may have similar activities and can be used. For example, nucleoside phosphorylases catalyze the reversible phosphorolysis of purines and pyrimidines. Specifically, purine nucleoside phosphorylase has been demonstrated to catalyze the similar reaction on nicotinamide riboside, although nicotinamide is neither a purine nor a pyrimidine. It may be discovered that some pyrimidine nucleoside phosphorylases may also catalyze this reaction and is disclosed in this invention. Other reactions described in this invention may be catalyzed by enzymes not described in the embodiment, and are also incorporated into the embodiment.
In certain embodiments, variants of these enzymes in which the catalytic activity has been modified, e.g., to make it more active and stable in acidic conditions, may be used in the invention. Amino acid sequence variants of the polypeptide include substitution, insertion, or deletion variants, and variants may be substantially homologous or substantially identical to the unmodified polypeptides.
In certain embodiments, the variants retain at least some of the biological activity, e.g., catalytic activity, of the polypeptide. Other variants include variants of the polypeptide that retain at least about 50%, preferably at least about 75%, more preferably at least about 90%, of the biological activity.
In certain embodiments, substitutional variants typically exchange one amino acid for another at one or more sites within the protein. Substitutions of this kind can be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. An example of the nomenclature used herein to indicate an amino acid substitution is “S345F ThrA” wherein the naturally occurring serine occurring at position 345 of the naturally occurring ThrA enzyme which has been substituted with a phenylalanine.
A polypeptide or polynucleotide “derived from” an organism contains one or more modifications to the naturally-occurring amino acid sequence or nucleotide sequence and exhibits similar, if not better, activity compared to the native enzyme (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or at least 110% the level of activity of the native enzyme). For example, enzyme activity is improved in some contexts by directed evolution of a parent/naturally-occurring sequence. Additionally or alternatively, an enzyme coding sequence is mutated to achieve less product inhibition.
Forms of the Enzymes
The isolated enzymes used in this invention are water soluble. It is often preferable to use immobilized enzymes. Immobilized enzymes are often more stable and robust, including examples of purine nucleoside phosphorylases (Hori, N., Watanabe, M., Sunagawa, K., Uehara, K., Mikami, Y. (1991) Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859. Journal of Biotechnology 17: 121-131). Immobilized enzymes are also easier to recover and use in multiple catalytic cycles, thus lowering the cost of an industrial process. Multiple means of enzyme immobilization are known in the art, as summarized (Es, I., Vieira, J. D. G., Amaral, A. C., (2015) “Principles, techniques, and applications of biocatalyst immobilization for industrial application” Applied Microbiology and Biotechnology 99:2065-2082). Enzymes may also be crossed linked to form Cross Linked Enzyme Aggregates (CLEAs) (Schoevaart, R., Wolbers, M. W., Golubovic, M., Ottens, M., Kieboom, A. P. G., van Rantwijk, F., van der Wielen, L. A. M., Sheldon, R. A., (2004) “Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs)” Biotechnology and Bioengineering 87 (6): 754-762) which are often more stable and are easier to recover and reuse. Many of these enzymes are found together in organisms which can be used as biocatalysts to produce nucleosides (Trelles, J. A., Fernindez-Lucas, J., Condezo, L. A., Sinisterra, J. V. (2004) “Nucleoside synthesis by immobilized bacterial whole cells” Journal of Molecular Catalysis B: Enzymatic 30: 219-227), but they may also be heterologously expressed in engineered microorganisms, which then can be used as biocatalysts. Methods of immobilization and crosslinking of enzymes catalyzing the reactions disclosed in this invention are incorporated herein. The cell lysates made from said enzyme-overexpressed whole cells, which may be treated by heat or selective filtration to remove other cellular components (e.g., other proteins, membrane), can be mixed to reconstitute the artificial enzymatic pathways to synthesize the desired products.
Several whole cells contained the one or several said enzymes that may be permeabilized by heat, enzymes, or an organic solvent can be reconstituted to make the artificial enzymatic pathways to synthesize the desired products.
The purified enzymes, immobilized enzymes, permeabilized whole cells containing the said enzymes, or cell lysates or whole-cells or permeabilized whole-cells, and mixtures thereof, can be mixed to make the artificial enzymatic pathways to synthesize the desired products.
One whole-cell contained the all said enzymes that may be permeabilized by heat, enzymes, or an organic solvent can be used to make the artificial enzymatic pathways to synthesize the desired products.
Process Optimization
The reactions disclosed herein can be performed simultaneously within one bioreactor or pot or vessel. The bioreactor can be operated in the exposure of air or in anoxic conditions. The anoxic conditions are preferred, whereas the deoxygenated aqueous pH-controlled buffer containing said metal ions is a preferred aqueous solvent. The experimental conditions are optimized to trade off their different optimal conditions. The conditions including, but not limited to temperature, pH, solvent, timing of addition of reactants, length of reaction, concentration, agitation and deoxygenation (by vacuum or heating and nitrogen or CO2 flashing) may be optimized for the consolidated bioreaction in one pot.
Production in Engineered Organisms
Enzymes catalyzing some or all of the reactions described in this invention may be expressed in non-natural, engineered heterologous organisms for the production of desired products. Specifically, genes coding for the enzymatic pathway enzymes may be isolated, inserted into expression vectors used to transform a production organism, may be incorporated into the genome, and direct expression of the enzymes. Protocols used to manipulate organisms are known in the art and explained in publications, such as Current Protocols in Molecular Biology, Online ISBN: 9780471142720, John Wiley and Sons, Inc., and Microbial Metabolic Engineering: Methods and Protocols, Qiong Cheng Ed., Springer.
Those familiar to the art can culture the engineered microbial cells to convert feedstocks such as carbohydrates or other precursors into nicotinamide riboside, and recover the nicotinamide mononucleotide. Guidance and protocols can be found in publications such as Fermentation and Biochemical Engineering Handbook: Principles, Process Design, and Equipment, 2d Edition, Henry C. Vogel and Celeste L. Todaro, Noyes Publications 1997.
NMN, NAD, NADP, or nicotinamide riboside may be separated from enzymes and reactants, and recovered from the reaction medium using a variety of procedures known in the art. These include, but are not limited to, crystallization, adsorption and release from ionic, hydrophobic and size exclusion resins, filtration, microfiltration, extraction, precipitation as salts or with solvents, or combinations thereof. The extent of the separation required may be limited to the removal of the enzymes, and a mixture of some or all of the remaining products and reactants including nicotinamide riboside, nicotinic acid riboside, 1,4-dihydronicotinamide riboside, nicotinamide, nicotinic acid, 1,4-dihydronicotinamide, inorganic orthophosphate, inorganic diphosphate, α-D-ribose-1-phosphate, 5-phospho-α-D-ribose-1-diphosphate, D-ribose-5-phosphate, β-nicotinamide D-ribonucleotide, and nitrogenous bases may be useful without further purification. Recovery of nicotinamide mononucleotide, NAD, NADP and NR with or without other products and reactants is a further embodiment of the invention.
Chemicals and materials. All chemicals were reagent grade or higher, purchased from Sigma-Aldrich (St. Louis, Mo., USA) or Sinopharm (Shanghai, China), unless otherwise noted. Genomic DNA samples of microorganisms, such as Clostridium thermocellum, Thermotoga maritima, Aquifex aeolicus and so on were purchased from the American Type Culture Collection (Manassas, Va., USA), DSMZ—German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), and China General Microbiological Culture Collection Center (CGMCC, Beijing, China), and Japan Collection of Microorganisms (Ibaraki, Japan).
Escherichia coli TOP10 and E. coli DH5alpha (Thermo Fisher Scientific, Maltham, Mass., USA) were used for DNA manipulation and plasmid amplification. E. coli BL21(DE3) (Invitrogen, Carlsbad, Calif., USA) was used for recombinant protein expression. The Luria-Bertani (LB) medium (Miller formula) supplemented with either 100 μg/L ampicillin or 50 μg/L kanamycin was used for E. coli cell cultures and recombinant protein expression. All enzymes for molecular biology experiments were purchased from New England Biolabs (NEB, Ipswich, Mass., USA), unless otherwise used. Super GelRed™ was used as DNA gel stain (US Everbright Inc., Suzhou, China).
Samples containing NAM and NR were injected directly in the Shimadzu HPLC equipped with a Princeton Chromatograph Inc. SPHER-60 AMINO (NH2) column (250×4.6 mm) (Princeton Chromatograph Inc., Cranbury, N.J., USA). The mobile phase was 20 mM KH2PO4. NAM and NR were detected spectroscopically at 261 nm by a Waters 2487 detector (Waters, Milford, Mass., USA) and quantified by comparison to standards from Sigma-Aldrich.
The concentrations of NAM, NMN, NAD, and PRPP in sample can also be measured by HPLC equipped with a Supelco Supelcosil LC-18-T (C18) reversed-phase column (250×4.6 mm; Supelco, Bellefonte, Pa., USA) and a Waters 2487 detector for the absorbance at 261 nm and quantified by comparison to standards. The mobile phase was used by mixing Buffer 1 (0.1 M potassium phosphate buffer, pH 6.0) and Buffer 2 (buffer 1 containing 20% methanol).
500 of the aqueous solution after the enzymatic reaction was quenched by adding 125 μl of 1 M HClO4. After precipitation at 18,000 g, and 500 μL of the supernatant was neutralized with 40 μl of 3 M K2CO3. After centrifugation, the supernatants were used for HPLC assays and enzymatic assays.
The concentrations of NAD (including NAD+ and NADH) were measured by the BioVision Inc. NAD/NADH Quantitation Colorimetric Kit (Catalog K337) (BioVision Inc. Milpitas, Calif., USA). This kit used the NAD-specific dehydrogenase that reduced NAD to NADH. NADH reacts with a colorimetric probe that produces a colored product which can be measured at 450 nm. There was no interference of NADP, NMN and NR.
The concentrations of NADP (including NADP+ and NADPH) were measured by the Cell Biolabs Inc. NADP/NADPH quantitation colorimetric Kit (Catalog MET-5018) (Cell Biolabs Inc., San Diego, Calif. 92126 USA). This kit used the NADP-specific dehydrogenase that reduced NADP+ to NADPH. NADPH reacts with a colorimetric probe that produces a colored product which can be measured at 450 nm. There was no interference of NAD, NMN and NR.
All recombinant enzymes were over-expressed by E. coli BL21(DE3). The pET plasmids encoding corresponding enzymes whose amino acid sequences were referred in SEQ ID. 1-122 were prepared by prolonged overlap extension PCR (You, C., X.-Z. Zhang and Y.-H. P. Zhang (2012). “Simple Cloning: direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis.” Applied and Environmental Microbiology. 78: 1593-1595). Some of their DNA sequences were amplified by PCR based on available genomic DNA templates, especially for bacteria microorganisms, such as Clostridium thermocellum, Bacillus subtilis, E. coli, T. maritima, and so on. The other DNA sequences were synthesized with codon optimization by some gene-synthesized companies, such as GenScript (Piscataway, N.J., USA) and Qinglan Biotech (Wuxi, Jiangsu, China)
All recombinant enzymes overexpressed by E. coli BL21(DE3) were purified based on their His-tags through affinity adsorption on nickel-charged resin, unless otherwise noted. E. coli BL21 cells harboring pET plasmid encoding a target protein were cultivated in 250 mL of the LB medium at 37° C. Protein expression was induced by adding isopropyl-β-d-thiogalacto-pyranoside (IPTG) to a final concentration of 0.1 mM until A600 reached ˜0.6-0.8. Protein expression was cultivated at 37° C. for 6 h or 18° C. for 16 h. Cell pellets were harvested by centrifugation and then were re-suspended in 50 mM HEPES buffer (pH 7.5) containing 0.1 M NaCl and 10 mM imidazole. After sonication and centrifugation, the supernatant was loaded onto the column packed with nickel-charged resins. The purified protein was eluted with 50 mM HEPES buffer (pH 7.5) containing 0.1 M NaCl and 150-500 mM imidazole. Mass concentration of protein was measured by the Bradford assay with bovine serum albumin as the standard. Protein expression and purity of recombinant proteins were examined by SDS-PAGE and analyzed by using densitometry analysis of the Image Lab software (Bio-Rad, Hercules, Calif., USA).
Some of recombinant enzymes that originated from the thermophilic microorganisms were partially purified by simple heat treatment (e.g., 50-80° C. for 10-30 min). After centrifugation, the supernatants of cell lysates containing the said enzymes were used to implement the enzyme-catalyzed biosynthesis in one pot.
One-pot biosynthesis of NMN was conducted at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM NAM, 10 mM R1P, and 10 mM ATP. The recombinant enzymes were 1 U/mL nicotinamide riboside phosphorylase (NRP) and 1 U/mL nicotinamide riboside kinase (NRK). An aliquot of mixed recombinant NRP (E.C. 2.4.2.1, SEQ ID NOs: 25-28) and NRK (EC 2.7.1.22, SEQ ID NOs: 61-63) was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of NMN was generated. NMN concentration was measured by both HPLC and enzymatic assays.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 50 mM inosine and 20 mM inorganic sodium phosphate. The recombinant enzyme was 1 U/mL purine nucleoside phosphorylase (PNP, EC 2.4.2.1, SEQ ID NOs: 29-35). An aliquot of PNP was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 10 mM guanosine and 20 mM inorganic sodium phosphate. The recombinant enzyme was 1 U/mL purine nucleoside phosphorylase (PNP, EC 2.4.2.1) or guanosine phosphorylase (GP, EC 2.4.2.5, SEQ ID NOs: 29-35). An aliquot of PNP or GP was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 50 mM adenosine and 20 mM inorganic sodium phosphate. The recombinant enzyme was 1 U/mL purine nucleoside phosphorylase (PNP, EC 2.4.2.1, SEQ ID NOs: 29-35). An aliquot of PNP was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2 and 20 mM inosine monophosphate. The recombinant enzymes were 1 U/mL pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10, SEQ ID NOs: 98-100) and 1 U/mL phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID NOs: 120-122). An aliquot of mixed PPMPN and PPM was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2 and 20 mM guanosine monophosphate. The recombinant enzymes were 1 U/mL pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10, SEQ ID NOs: 98-100) and 1 U/mL phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID NOs: 120-122). An aliquot of mixed PPMPN and PPM was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2 and 40 mM adenosine monophosphate. The recombinant enzymes were 1 U/mL pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10, SEQ ID NOs: 98-100) and 1 U/mL phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID NOs: 120-122). An aliquot of mixed PPMPN and PPM was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM ATP, and 50 mM ribose. The recombinant enzymes were 1 U/mL ribokinase (RK, EC 2.7.1.15, SEQ ID NOs: 53-55) and 1 U/mL phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID NOs: 120-122). An aliquot of mixed RK and PPM was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM ATP, and 100 mM D-xylose. The recombinant enzymes were 1 U/mL D-xylose isomerase (D-XI, EC 5.3.1.5, SEQ ID NOs: 111-113), pentose 3-epimerase (P3E, EC 5.1.3.31, SEQ ID NOs: 109-110), and D-ribose isomerase (D-RI, EC 5.3.1.20, SEQ ID No: 117) or mannose 6-phosphate isomerase (MPI, EC 5.3.1.8, SEQ ID No: 116), and 1 U/mL phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID Nos: 120-122). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 20 mM polyphosphate, and 50 mM ribose. The recombinant enzymes were 1 U/mL D-xylose isomerase (D-XI, EC 5.3.1.5, SEQ ID Nos: 111-113), 1 U/mL polyphosphate xylulokinase (XK, EC 2.7.1.17, SEQ ID Nos: 56-57), 1 U/mL D-xylulose 5-phosphate 3-epimerase (RuPE, EC 5.1.3.1, SEQ ID Nos: 107-108), 1 U/mL ribose-5-phosphate isomerase (RPI, EC 5.3.1.6, SEQ ID Nos: 114-115), and 1 U/mL phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID Nos: 120-122). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 10 mM phosphate, 5 mM NAD(P)+, and 20 mM starch (maltodextrin). The enzymes added (1 U/mL) were αGP (alpha-glucan phosphorylase, EC 2.4.1.1, SEQ ID Nos: 16-18), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 10 mM phosphate, 5 mM NAD(P)+, and 20 mM starch (maltodextrin). Starch or maltodextrin may be treated by isoamylase or pullulanase before its use. The enzymes added (1 U/mL, each) were αGP (alpha-glucan phosphorylase, EC 2.4.1.1, SEQ ID Nos: 16-18), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. RIP concentration was measured by HPLC. Debranched starch treated by isoamylase or pullulanase resulted in about 10-30% more R1P than non-treated starch.
The generation of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 10 mM phosphate, 5 mM NAD(P)+, and 50 mM sucrose. The enzymes added (1 U/mL, each) were SP (sucrose phosphorylase, EC 2.4.1.7, SEQ ID Nos: 19-21), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. RIP concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 10 mM phosphate, 5 mM NAD(P)+, and 20 mM cellobiose. The enzymes added (1 U/mL each) were CBP (cellobiose phosphorylase, EC 2.4.1.20, SEQ ID Nos: 22 or 24), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 10 mM phosphate, 5 mM NAD+, and 30 mM cellodextrins (including cellobiose and long-chain cellodextrins). The enzymes added (1 U/mL each) were CDP (cellodextrin phosphorylase, EC 2.4.1.49, SEQ ID Nos: 23-24), CBP (cellobiose phosphorylase, EC 2.4.1.20, SEQ ID Nos: 22 or 24), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 20 mM polyphosphate, 5 mM NAD(P)+, and 50 mM glucose. The enzymes added (1 U/mL each) were PPGK (polyphosphate glucokinase, EC 2.7.1.63, SEQ ID No: 70), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 20 mM polyphosphate, 5 mM NAD(P)*, and 50 mM fructose. The enzymes added (1 U/mL each) were D-XI (xylose isomerase, EC 5.3.1.5, SEQ ID Nos: 111-113), PPGK (polyphosphate glucokinase, EC 2.7.1.63, SEQ ID No: 70), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 10 mM phosphate, 20 mM polyphosphate, 5 mM NAD(P)+, 50 mM sucrose and 20 mM polyphosphate. The enzymes added (1 U/mL each) were SP (sucrose phosphorylase, EC 2.4.1.7, SEQ ID Nos: 19-21), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), D-XI (xylose isomerase, EC 5.3.1.5, SEQ ID Nos: 111-113), PPGK (polyphosphate glucokinase, EC 2.7.1.63, SEQ ID No: 70), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The synthesis of R1P was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 10 mM phosphate, 20 mM polyphosphate, 5 mM NAD(P)+, and 50 mM starch (maltodextrin). Starch or maltodextrin may be treated by isoamylase or pullulanase. The enzymes added (1 U/mL, each) were αGP (alpha-glucan phosphorylase, EC 2.4.1.1, SEQ ID Nos: 16-18), PPGK (polyphosphate glucokinase, EC 2.7.1.63, SEQ ID No: 70), PGM (phosphoglucomutase, EC 5.4.2.2, SEQ ID Nos: 118-119), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, SEQ ID Nos: 3-4), 6-phosphogluconolactonase (6PGL, EC 3.1.1.31, SEQ ID No: 94), 6PGDH (6-phosphogluconate dehydrogenase, EC 1.1.1.44, SEQ ID Nos: 1-2), RPI (ribose 5-phosphate isomerase, EC 5.3.1.6, SEQ ID Nos: 114-115), and PPM (phosphopentomutase, EC 5.4.2.7, SEQ ID Nos: 120-122) as well as NAD(P)H oxidase (NOX, EC 1.6.3.4, SEQ ID Nos: 5-7). An aliquot of mixed enzymes was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. until significant amount of R1P was generated. R1P concentration was measured by HPLC.
The regeneration of ATP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2, 2 mM ADP, and 10 mM acetyl phosphate. The enzyme added (1 U/mL, each) was acetate kinase (AK, EC 2.7.2.1, SEQ ID Nos: 71-73). An aliquot of the enzyme was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. ATP net regeneration amount was measured by ATP enzymatic kit. Also, glucose and glucose kinase were added to consume regenerated ATP, wherein the product glucose 6-phosphate was measured by coupled with glucose 6-phosphate dehydrogenase in the presence of NAD+. The absorbency of NADH at 340 nm was used to calculate the amount of ATP net regeneration.
The regeneration of ATP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2 and 0.1 mM ATP. The enzymes added (1 U/mL, each) were adenylate kinase (ADK, EC 2.7.4.3, SEQ ID Nos: 85-87) and adenosine kinase (AdK, EC 2.7.1.20, SEQ ID Nos: 58-60). ATP net generation amount was measured by ATP enzymatic kit. More ATP regeneration catalyzed by ADK and AdK than that by ADK only. Also, glucose and glucose kinase were added to consume regenerated ATP, wherein the product glucose 6-phosphate was measured by coupled by glucose 6-phosphate dehydrogenase in the presence of NAD+. The absorbency of NADH at 340 nm was used to calculate the amount of ATP regeneration.
The regeneration of ATP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 1 mM ADP, and 10 mM polyphosphate. The enzyme added (1 U/mL, each) was polyphosphate kinase (PPK, EC 2.7.4.1, SEQ ID Nos: 74-82). An aliquot of the enzyme was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. ATP generation amount was measured by the ATP enzymatic kit. Also, glucose and glucose kinase were added to consume regenerated ATP, wherein the product glucose 6-phosphate was measured by coupled by glucose 6-phosphate dehydrogenase in the presence of NAD+. The absorbency of NADH at 340 nm was used to calculate the amount of ATP regeneration.
The regeneration of ATP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 1 mM ADP, and 10 mM polyphosphate. The enzymes added (1 U/mL, each) were polyphosphate: AMP phosphotransferase (PPT, EC 2.7.4.B2; SEQ ID Nos: 83-84) and adenylate kinase (ADK, EC 2.7.4.3; SEQ ID Nos: 85-86). An aliquot of the enzyme was added to the solution to initiate the reaction. The biotransformation was incubated at 37° C. ATP generation amount was measured by ATP enzymatic kit. Also, glucose and glucose kinase were added to consume regenerated ATP, wherein the product glucose 6-phosphate was measured by coupled by glucose 6-phosphate dehydrogenase in the presence of NAD+. The absorbency of NADH at 340 nm was used to calculate the amount of ATP regeneration.
The regeneration of ATP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 1 mM ADP, and living baker yeast that was treated by freezing-melting. The living yeast cells were gifted by a Chinese beer factory. The yeast cell added was 50 g/L weight cell weight. ATP generation amount of ATP was measured by ATP enzymatic kit. Also, glucose and glucose kinase were added to consume regenerated ATP, wherein the product glucose 6-phosphate was measured by coupled with glucose 6-phosphate dehydrogenase in the presence of NAD+. The absorbency of NADH at 340 nm was used to calculate the amount of ATP regeneration.
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM inosine monophosphate, 2 mM diphosphate, and 20 mM NAM. The enzymes added (1 U/mL, each) were hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8, SEQ ID Nos: 36-45) and nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12, SEQ ID Nos: 46-52). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NMN was incubated at 37° C. NMN concentrations were measured by both HPLC and enzymatic assay.
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM guanosine monophosphate, 2 mM diphosphate, and 20 mM NAM. The enzymes added (1 U/mL, each) were hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8, SEQ ID Nos: 36-45) and nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12, SEQ ID Nos: 46-52). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NMN was incubated at 37° C. NMN concentrations were measured by both HPLC and enzymatic assay.
The synthesis of NAD was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2, 20 mM NMN and 20 mM ATP. The enzyme added (1 U/mL, each) was nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1, SEQ ID Nos. 88-90). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NAD was incubated at 37° C. NAD concentrations were measured by both HPLC and enzymatic assay.
The synthesis of NAD was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2, 20 mM NMN, and 20 mM ATP. The enzyme added (1 U/mL, each) was nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1, SEQ ID Nos. 88-90) and diphosphate by diphosphatase (DPP, EC 3.6.1.1, SEQ ID Nos. 105-106). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NAD was incubated at 37° C. NAD concentrations were measured by both HPLC and enzymatic assay. More NAD was synthesized when DPP was added.
The synthesis of NADP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM NMN, and 40 mM ATP. The enzymes added (1 U/mL, each) were nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1, SEQ ID Nos. 88-90) and ATP-dependent NAD kinase (NADK, EC 2.7.1.23, SEQ ID Nos. 64-69). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NADP was incubated at 37° C. NADP concentration was measured by both HPLC and enzymatic assay.
The synthesis of NADP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 20 mM NMN, 10 mM ATP and 20 mM polyphosphate. The enzymes added (1 U/mL, each) were nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1, SEQ ID Nos. 88-90) and polyphosphate-dependent NAD kinase (NADK, EC 2.7.1.23, SEQ ID Nos. 64-69). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NADP was incubated at 37° C. NADP concentration was measured by both HPLC and enzymatic assay.
The synthesis of NR was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2 and 20 mM NMN. The enzyme added (1 U/mL, each) was 5′-nucleotidase. An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NR was incubated at 37° C. NR concentration was measured by both HPLC and enzymatic assay.
The in situ selective removal of adenosine was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2 and 5 mM adenosine. The enzyme added (1 U/mL, each) was adenosine nucleosidase (AN, EC 3.2.2.7, SEQ ID No. 97). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The hydrolysis of adenosine was incubated at 37° C. D-Ribose concentration was measured by both HPLC equipped with Bio-Rad 87-H column with a refractive index detector. AN may be replaced with purine nucleosidase (PN, EC 3.2.2.1).
The in situ selective removal of guanine was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2 and 5 mM guanine. The enzymes added (1 U/mL, each) were guanine deaminase (GDA, EC 3.5.4.3, SEQ ID Nos. 101-104), xanthine oxidase (XO, EC 1.17.3.2, SEQ ID Nos. 11-15), and catalase (CA, EC 1.11.1.6, EC 1.11.1.21, or EC 1.11.1.7, SEQ ID Nos. 8-10). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The hydrolysis of adenosine was incubated at 37° C. The concentration of ammonia, a product of GDA, was measured by the ammonia enzymatic kit (Sigma, AA0100-1KT).
The in situ selective removal of guanine was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2, and 10 mM hypoxanthine. The enzymes added (1 U/mL, each) were xanthine oxidase (XO, EC 1.17.3.2, SEQ ID Nos. 11-15) and catalase (CA, EC 1.11.1.6, EC 1.11.1.21, or EC 1.11.1.7, SEQ ID Nos. 8-10). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The hydrolysis of hypoxanthine was incubated at 37° C. The disappearance of hypoxanthine was measured by HPLC.
The in situ selective removal of guanine was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 5 mM MgCl2, and 10 mM hypoxanthine. The enzymes added (1 U/mL, each) were xanthine dehydrogenase (XDH, EC 1.17.3.4, SEQ ID Nos. 11-15), and H2O-forming NADH oxidase (NOX, EC 1.6.3.4, SEQ ID Nos. 5-7). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The hydrolysis of hypoxanthine was incubated at 37° C. The disappearance of hypoxanthine was measured by HPLC.
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM inosine monophosphate, 0.5 mM ATP, and 10 mM acetyl phosphate (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM guanosine monophosphate, 1 mM ATP, and 10 mM acetyl phosphate. The enzymes added (1 U/mL, each) were inosinate nucleosidase (IMPN, EC 3.2.2.12, EC 3.2.2.10, SEQ ID Nos 98-100), phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID Nos 120-122), nicotinamide riboside phosphorylase (NRP, EC 2.4.2.1, SEQ ID Nos 25-35), nicotinamide riboside kinase (NRK, EC 2.7.1.22, SEQ ID Nos 61-63), and acetate kinase (AK, EC 2.7.2.1, SEQ ID Nos 71-73). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. The biotransformation of NMN was incubated at 37° C. NMN concentration was measured by both HPLC and enzymatic assay.
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 30 mM inosine monophosphate, and 5 mM ATP (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 30 mM inosine monophosphate, and 5 mM ATP (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM inosine monophosphate, 20 mM polyphosphate, and 1 mM ATP (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM guanosine monophosphate, 20 mM polyphosphate, and 1 mM ATP. The enzymes added (1 U/mL, each) were inosinate nucleosidase (IMPN, EC 3.2.2.12, EC 3.2.2.10, SEQ ID Nos 98-100), phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID Nos 120-122), nicotinamide riboside phosphorylase (NRP, EC 2.4.2.1, SEQ ID Nos 25-35), nicotinamide riboside kinase (NRK, EC 2.7.1.22, SEQ ID Nos 61-63), and polyphosphate kinase (PPK, EC 2.7.4.1, SEQ ID Nos 74-82). The biotransformation of NMN was incubated at 37° C. An aliquot of the mixed enzymes was added to the solution to initiate the reaction. NMN concentration was measured by both HPLC and enzymatic assay and its synthesis was confirmed.
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM inosine monophosphate, 20 mM polyphosphate, and 1 mM ATP. The enzymes added (1 U/mL, each) were pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10, SEQ ID Nos 98-100), phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID Nos 120-122), nicotinamide riboside phosphorylase (NRP, EC 2.4.2.1, SEQ ID Nos 25-35), nicotinamide riboside kinase (NRK, EC 2.7.1.22, SEQ ID Nos 61-63), polyphosphate kinase (PPK, EC 2.7.4.1, SEQ ID Nos 74-82), xanthine dehydrogenase (XDH, EC 1.17.3.4, SEQ ID Nos. 11-15), and H2O-forming NADH oxidase (NOX, EC 1.6.3.4, SEQ ID Nos. 5-7). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. NMN concentration was measured by both HPLC and enzymatic assay and its synthesis was confirmed. The supplementary addition of XDH and NOX improved NMN yield.
The synthesis of NMN was carried out at 50° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 30 mM inosine monophosphate, 20 mM polyphosphate, and 1 mM ATP (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 30 mM inosine monophosphate, 20 mM polyphosphate, and 10 mM ATP (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 30 mM inosine monophosphate, 20 mM polyphosphate, and 10 mM ATP (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 20 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM guanosine monophosphate, 20 mM polyphosphate, and 10 mM ATP. The enzymes added (1 U/mL, each) were pyrimidine/purine nucleotide 5′-monophosphate nucleosidase (PPMPN, EC 3.2.2.10, SEQ ID Nos: 98-100), phosphopentomutase (PPM, EC 5.4.2.7, SEQ ID Nos 120-122), nicotinamide riboside phosphorylase (NRP, EC 2.4.2.1), a bifunctional enzyme (SEQ ID Nos 91-93) including nicotinamide riboside kinase (NRK, EC 2.7.1.22) and nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1), polyphosphate kinase (PPK, EC 2.7.4.1, SEQ ID Nos 74-82), and diphosphatase (DPP, EC 3.6.1.1, SEQ ID Nos. 105-106). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. NAD concentration was measured by both HPLC and enzymatic assay.
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, and 20 mM inosine monophosphate (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM inosine monophosphate and 1 mM NAD (
The synthesis of NMN was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, and 20 mM guanosine monophosphate (
The synthesis of NAD was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM inosine monophosphate, 10 mM ATP, and 0.05 mM NAD+ (
The synthesis of NAD was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM inosine monophosphate and 10 mM ATP (
The synthesis of NAD was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 10 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM guanosine monophosphate, and 10 mM ATP (
The synthesis of NADP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM polyphosphate, 20 mM inosine monophosphate, and 10 mM ATP (
The synthesis of NADP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM polyphosphate, 20 mM inosine monophosphate and 10 mM ATP (
The synthesis of NADP was carried out at 37° C. in a 100 mM HEPES buffer (pH 7.0) containing 30 mM MgCl2, 0.5 mM MnCl2, 20 mM NAM, 20 mM polyphosphate, 20 mM guanosine monophosphate and 10 mM ATP. The enzymes added (1 U/mL, each) were hypoxanthine/guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8, SEQ ID Nos 36-45), nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12, SEQ ID Nos 46-52), nicotinamide nucleotide adenylyltransferase (NMNAT, EC 2.7.7.1, SEQ ID Nos 88-90), and polyphosphate-dependent NAD kinase (NADK, EC 2.7.1.23, SEQ ID Nos. 64-69). An aliquot of the mixed enzymes was added to the solution to initiate the reaction. NADP concentration was measured by both HPLC and enzymatic assay and its synthesis was confirmed.
Thermotoga maritima
Moorella thermoacetica
Thermotoga maritima
Zymomonas mobilis
Clostridium aminovalericum
Clostridium acetobutylicum
Streptococcus mutans
Thermus brockianus
Bacillus pumilus
Geobacillus stearothermophilus
Bos
taurus (bovine)
Homo sapiens
Rattus norvegicus
E. coli K-12
Blastobotrys
adeninivorans
Clostridium thermocellum
Thermotoga neapolitana
Thermococcus
kodakarensis KOD1
Bifidobacterium adolescentis
Leuconostoc mesenteroides
Thermoanaerobacterium
thermosaccharolyticum
Clostridium thermocellum
Clostridium thermocellum
Thermosipho africanus
Cellulomonas sp.
Bos
taurus (Beef liver)
Homo
sapiens
Escherichia
coli (strain K12)
Bacillus halodurans
Thermus thermophilus
Thermotoga
maritima MSB8
Meiothermus silvanus
Clostridium thermocellum
Geobacillus thermodenitrificans
Deinococcus geothermalis
Giardia
intestinalis ATCC 50803
Trypanosoma
cruzi
Gallus
gallus (chicken)
Clostridium
thermocellum
Haloferax
volcanii
Halobacterium
salinarum
Haemophilus ducreyi
Shewanella oneidensis
Homo sapiens
Meiothermus
ruber DSM 1279
Tenacibaculum
maritimum
Mariyirga
tractuosa
Synechocystis sp. PCC 6803
Escherichia
coli (strain K12)
Thermoanaerobacterium
thermosaccharolyticum
Thermus thermophilus
Thermotoga maritima
Geobacillus stearothermophilus
Myceliophthora thermophila
Thermothielavioides terrestris
Saccharomyces cerevisiae
Saccharomyces cereyisioe
Myceliophthora thermophila
Pseudomonas alcaligenes
Clostridium thermocellum
Thermotoga maritima
Thermococcus kodakarensis
Pyrococcus horikoshii
Thermoanaerobacterium saccharolyticum
Microroccus luteus
Thermobifida fusca
Geobacillus stearothermophilus
Methanosarcina thermophila
Thermotoga maritima
Corynebacterium
glutamicum ATCC
Cytophaga hutchinsonii
Deinococcus radiodurans
Meiothermus ruber
Deinococcus geothermalis
Mycobacterium tuberculosis
Pseudomonas
aeruginosa ATCC 15692
Thermus
thermophilus HB27
Acinetobacter
johnsonii 210A
Myxococcus
xanthus
Clostridium thermocellum
Sulfolobus acidocaldarius
Thermotoga maritima
Methanocaldococcus jannaschii
Saccharomyces cerevisiae
Leishmania braziliensis
Clostridium thermocellum
Escherichia
coli (strain K12)
Salmonella
typhimurium
Thermotoga
maritima
Trypanosoma brucei brucei
Bacillus thuringiensis
Coffea arabica
Escherichia
coli K-12 MG1655
Escherichia
coli K-12 MG1655
Shigella
flexneri
Mannheimia
succiniciproducens
Bacillus
subtilis 168
Haloferax
volcanii
Halobacterium
salinarum
Thermoplasma
acidophilum
Pyrococcus
furiosus
Clostridium thermocellum
Thermotoga maritima
Pseudomonas cichorii
Rhodobacter sphaeroides
Thermus thermophilus
Thermotoga neapolitana
Geobacillus stearothermophilus
Clostridium thermocellum
Thermotoga maritima
Geobacillus thermodenitrificans
Mycobacterium smegmatis
Clostridium thermocellum
Thermus thermophilus
Thermotoga maritima
Thermus thermophilus
Clostridium thermocellum
The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a concentration disclosed as “10 mM” is intended to mean “about 10 mM.”
Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62968606 | Jan 2020 | US |
