PRODUCTION OF NMN AND ITS DERIVATIVES VIA MICROBIAL PROCESSES

Abstract

The present invention relates to microbial production of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide adenine dinucleotide (NAD) using a genetically modified bacterium.

Claims

1. A genetically modified microbial cell capable of producing nicotinamide mononucleotide and/or nicotinamide adenine dinucleotide from nicotinamide, said cell comprising a mutation in one or more endogenous genes selected from the group consisting of cgl1364, cgl1977, cgl2835, iunH1, iunH2, and iunH3, wherein each of said one or more genes codes for a nucleosidase enzyme.

2. The genetically modified microbial cell as in claim 1, wherein the mutation is a deletion, frameshift or point mutation decreasing or eliminating nucleosidase activity.

3. The genetically modified microbial cell as in claim 1, wherein the microbial cell is Corynebacteriun glutamicum.

4. The genetically modified microbial cell as in claim 1, wherein the microbial cell is Corynebacterium glutamicum ATCC 13034.

5. The genetically modified microbial cell as in claim 1, further comprising a mutation in the pncA gene, wherein such mutation leads to the inactivation or reduced activity of an enzyme capable of deamidation of nicotinamide to nicotinic acid.

6. The genetically modified microbial cell as in claim 5, wherein the mutation is the deletion, frameshift or point mutation of the pncA gene decreasing or eliminating deamidation activity.

7. The genetically modified microbial cell as in claim 1, further comprising an exogenous gene nadV coding for a nicotinamide phosphoribosyl transferase capable of the conversion of nicotinamide to nicotinamide mononucleotide.

8. The genetically modified microbial cell as in claim 7, wherein: (i) the exogenous gene nadV is from Stenophomonas maltophilia; (ii) the exogenous gene nadV is from Chromobacterium violaceum; (iii) the genetically modified microbial cell further comprises a prsA gene variant or mutant encoding a phosphoribosyl pyrophosphate synthelase; (iv) the genetically modified microbial cell further comprises a genetic modification to an endogenous pyrE gene, leading to the reduced or eliminated expression of a phosphoribosyltransferase enzyme; (v) a mutation in an endogenous ushA gene causing inactivation or reduction in activity of a purine nucleosidase enzyme capable of the conversion of nicotinamide mononucleotide to nicotinamide riboside; (vi) a genetic modification in an endogenous pncC gene coding for a nicotinamide nucleotide amidase enzyme capable of the conversion of nicotinamide mononucleotide to nicotinate mononucleotide, causing inactivation or reduction in activity; (vii) a genetic modification in an endogenous nadD gene, leading to the upregulation of a nicotinate-nucleotide adenyl transferase enzyme capable of the conversion of nicotinamide mononucleotide to nicotinamide adenine dinucleotide; or (viii) the expression of a heterologous gene encoding a nicotinate-nucleotide adenyl transferase enzyme capable of the conversion of nicotinamide mononucleotide to nicotinamide adenine dinucleotide.

9. The genetically modified microbial cell as in claim 8, wherein the gene nadV from Stenophomonas maltophilia is codon optimized for expression in Corynebacterium glutamicum.

10. (canceled)

11. The genetically modified microbial cell as in claim 8, wherein the gene nadV from Chromobacterium violaceum is codon optimized for expression in Corynebacterium glutamicum.

12. (canceled)

13. The genetically modified microbial cell as in claim 8, wherein: (i) the prsA gene variant or mutant is a codon optimized variant of the prsA gene from Corynebacterium glutamicum ATCC 13032; or (ii) the prsA gene variant or mutant encodes a feedback resistant mutant of phosphoribosyl pyrophosphate synthetase.

14.-19. (canceled)

20. The genetically modified microbial cell as in claim 8, further comprising a genetic modification in an endogenous nudC gene, leading to the reduced or eliminated expression of an NADH pyrophosphatase capable of the conversion of NAD(+) to nicotinamide mononucleotide.

21. The genetically modified microbial cell as in claim 9, further comprising a conditionally active ribonucleotide reductase coded by a NrdHIEJ operon.

22. The genetically modified microbial cell as in claim 1, further comprising a mutation in the pgi gene, wherein such mutation leads to the inactivation or reduced activity of an enzyme capable of converting D-glucose-6-phosphate to D-ribulose-5-phosphate.

23. The genetically modified microbial as in claim 22, wherein: (i) the mutation is the deletion, frameshift or point mutation of the pgi gene decreasing or eliminating conversion of D-glucose-6-phosphate to D-ribulose-5-phosphate; (ii) the genetically modified microbial cell further comprises an exogenous gene coding for a soluble transhydrogenase; or (iii) the genetically modified microbial cell further comprises an exogenous gene coding for a membrane-bound transhydrogenase.

24. (canceled)

25. The genetically modified microbial cell as in claim 23, wherein; (i) the exogenous gene is a udhA gene from E. coli; or (ii) the exogenous gene is a pntAB gene from E. coli.

26. The genetically modified microbial cell as in claim 25, wherein the gene udhA from E. coli is codon optimized for expression in Corynebacterium glutamicum.

27.-28. (canceled)

29. The genetically modified microbial cell as in claim 25, wherein the gene pntAB from E. coli is codon optimized for expression in Corynebacterium glutamicum.

30. A method for producing nicotinamide mononucleotide comprising: culturing a genetically modified microbial cell as in claim 1 in the presence nicotinamide for a sufficient period of time to allow conversion of nicotinamide to nicotinamide mononucleotide.

31. A method for producing nicotinamide adenine dinucleotide comprising: culturing a genetically modified microbial cell as in claim 8 in the presence of nicotinamide for a sufficient period of time to allow conversion of nicotinamide to nicotinamide adenine dinucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1. Chemical structures of desired products nicotinamide adenine dinucleotide (NAD.sup.+), nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR). Also shown in this figure are the chemical structure of side products namely nicotinate adenine dinucleotide (NAAD), nicotinate mononucleotide (NAMN), nicotinate riboside (NAR), as well as substrates nicotinamide (Nam) and nicotinic acid (NA). The side products are eliminated by genetic modifications according to the present invention. The structures of NA, NAR, NAMN and NAAD are different from Nam, NR, NMN, and NAD respectively by the end group C(O)OH versus C(O)NH.sub.2.

[0028] FIG. 2. Certain genetic modifications for increased production of NR, NAD and NMN according to the present teachings. Such genetic modifications may include one or more of: (a) heterologous expression of nadV gene encoding nicotinamide phosphoribosyltransferase capable of the conversion of Nam to NMN; (b) deletion or modification of one or more genes capable of the enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of the enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification or deletion of PRPP requiring biochemical pathways other than the biochemical pathway for the conversion of Nam to NMN; (f) deletion or modification of the gene responsible for the enzymatic conversion of NMN to NAMN; (g) deletion or modification of the gene responsible for the conversion of NMN to NAD; and (h) deletion or modification of one or more genes coding for a promiscuous NMN nucleosidase capable of the conversion of NMN to Nam and ribose-5-phosphate.

[0029] FIG. 3. Pathway for NMN production from Nam as a feedstock in an example genetically modified microbial host cell according to the present invention.

[0030] FIG. 4. Mechanism of action of purine nucleosidase known in the art and the unexpected observation of the same enzyme functioning as nicotinamide mononucleotide nucleosidase.

[0031] FIG. 5. Method used for deleting a target gene using the suicidal plasmid pDEL. As shown in this illustration, the upstream and downstream flanking regions of a target gene to be deleted were obtained using PCR and cloned into a pDEL plasmid. C. glutamicum cells were transformed with a pDEL plasmid with upstream and downstream flanking regions of the gene targeted for deletion. After transformation, the cells were plated in a kanamycin plate to select the transformants. In the second round of screening, the transformants were plated on a sucrose containing plates to select the double recombinants with the deletion of target gene.

[0032] FIG. 6. Production of NMN in two different Corynebacterium glutamicum strains constructed according to the present invention. The strain NMN4 has the genotype of cgl1777-8 pncA pncC ushA pyrE and the strain NR5 has the genotype of cgl1777-8 pncA pncC pnuC cgl1364 cgl1977 cgl2835.

[0033] FIG. 7. Production of NMN in four different Corynebacterium glutamicum strains constructed according to the present invention.

[0034] FIG. 8. Pathway for enhancing PRPP production from D-glucose-6-phosphate in an example genetically modified microbial host cell according to the present invention.

[0035] FIG. 9 Growth of a number of Corynabacterium glutamicum strains transformed with ZWF and transhydrogenase genes in CGXII medium.

[0036] FIG. 10 NMN production in a number of PGI Corynabacterium glutamicum strains containing indicated plasmids.

DETAILED DESCRIPTION

[0037] The present invention relates to the construction of genetically modified host cells useful in the biological production of NMN, NR and NAD using Nam or NA as feedstock and the method of producing NMN, NR and NAD using those genetically modified host cells. In a preferred embodiment, Nam is used as the feedstock for the production of NMN. NR and NAD.

[0038] Genetically modified host cells useful in the present invention may be microbial cells such as bacterial cells, fungal cells and yeast cells. Bacterial cells useful in the present invention include, without limitation, Escherichia spp., Streptomyces spp., Zymomonas spp. Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp. Lactobacillus spp. Lactococcus spp. Bacillus spp., Alcaligenes spp., Pseudononas spp., Aerononas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp. Gluconobacter spp., Ralstonia spp. Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp. Serratia spp., Saccharopolyspora spp. Thermus spp., Stenotrophomonas spp. Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp., Pantoea spp., and Vibrio natriegens. In a preferred embodiment, Corynebacterium glutamicum is used.

[0039] Yeast cells of the present disclosure include, without limitation, engineered Saccharomyces spp., Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Candida boidinii, and Pichia. According to the current disclosure, a yeast as claimed herein are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Yeasts are unicellular organisms which evolved from multicellular ancestors but with some species useful for the current disclosure being those that have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudo hyphae or false hyphae.

[0040] FIG. 1 shows the chemical structures of desired products NAD, NMN and NR. Also shown in this figure are the chemical structure of side products namely NAAD, NAMN, NAR, as well as substrates Nam and NA. The side products are eliminated by genetic modifications according to the present invention. The structures of NA, NAR, NAMN and NAAD are different from Nam, NR, NMN, and NAD respectively by the end group C(O)OH versus C(O)NH.sub.2.

[0041] FIG. 2 shows the portions of the metabolic pathway inside a C. glutamicum cell which is relevant to the present invention. The objective of the present invention is to produce NMN, NR and NAD using Nam or NA as the feedstock while eliminating related side products such as NAR, NAMN and NAAD.

[0042] In a preferred embodiment Nam is used as the feedstock. As illustrated in FIG. 2, the metabolic pathway from Nam to NMN, NR and NAD is different from the metabolic pathway from NA to NAD, NMN and NR. The pathway from NA to NAD (via NAMN and NAAD), NMN (via NAMN), and NR (via NAMN and NMN) involves significantly more enzymatic steps than the pathway from Nam to NMN, and NR and NAD (via NMN). Therefore, Nam is considered as the preferred feedstock for producing NMN, NR, and NAD.

[0043] When Nam is used as the preferred feedstock, it is advantageous to address the conversion of Nam into NA by the deamidase enzyme coded by the pncA gene. One way to eliminate the conversion of Nam into NA is to inactivate the pncA gene. The inactivation of the pncA gene can be accomplished using a number of genetic manipulation techniques. The preferred genetic manipulation of pncA gene is to delete the nucleotide sequence from the chromosomal DNA of the microbial host cell.

[0044] Using a variety of tools available for genetic manipulations, a person skilled in the art of microbial strain construction will be able to design the metabolic pathways for producing each of the desired compounds namely NMN, NR and NDA. As illustrated in FIG. 2, the production of NMN from Nam is accomplished in a single enzymatic step within the microbial host cell. Once NMN is accumulated within the microbial host cell, the other two compounds of interest namely, NDA and NR can be obtained from NMN as a derivative product through subsequent enzymatic reactions.

[0045] Tables 1-3 lists the various genes and enzymes involved in the biochemical pathways illustrated in FIG. 2. Table 4 provides the sequence information for the genes and enzymes involved in the operation of the biochemical pathways illustrated in FIG. 2.

[0046] To provide a more direct route for the production of nicotinamide NMN, NR, and NAD, a number of genetic modifications can be made to Corynebacterium glutamicum or a related organism. In some embodiments, such genetic modifications can include heterologous expression of a gene encoding nicotinamide phosphoribosyltransferase (nadV). Nicotinamide phosphoribosyltransferase is an activity not natively found in Corynebacterium. However, it is found in a number of other bacterial and eukaryotic microorganisms. Table 2 lists certain preferred sources of such gene. In some embodiments, the genetic modifications can include deletion or modification of the gene (pncA) capable of the conversion of nicotinamide to nicotinic acid resulting in the loss or decrease in enzyme activity. In some embodiments, the genetic modifications can include modification of the prsA gene capable of the production of phosphoribosyl pyrophosphate (PRPP), a key precursor to NMN, NR, and NAD, such that higher levels of PRPP are available. Such modifications can include upregulation of gene expression and/or introduction of protein variants that lead to increased levels of enzyme activity under production conditions. Modifications to the prs gene such as those described in Marinescu et al., Beta-nicotinamide mononucleotide (NMN) production in Escherichia coli, Sci. Rep., 8: 12278 (2018), and Zakataeva et al., Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from Bacillus amyloliquefaciens: purification, characterization, and application to increase purine nucleoside production,Appl. Microbiol. Biotechnol., 93: 2023-33 (2012), may be used according to the present invention. In some embodiments, the modifications can include deletion or modification of the gene (pncC) responsible for the conversion of NMN to NaMN, resulting in loss or decrease in enzyme activity. In some embodiments, the modifications can include generation and incorporation of a conditionally active ribonucleotide reductase using a) temperature-sensitive (ts) mutation(s) in the protein coding sequence, b) is self-splicing intein, c) ligand-dependent gene expression, and/or d) ligand-dependent enzyme inactivation. The purpose of the modification is to block cell division and biomass accumulation while maintaining protein synthesis and metabolic activity by inhibiting deoxyribonucleotide synthesis required for DNA production and replication.

Modified Pathway for NMN Production

[0047] As shown in FIGS. 2 and 3, NMN production from Nam can be achieved within an engineered host cell through a single enzymatic step involving nicotinamide phosphoribosyl transferase enzyme coded by an nadV gene. In those microorganisms lacking an endogenous gene coding for a phosphoribosyl transferase enzyme such as C. glutamicum, it is necessary to introduce an exogenous gene coding for a phosphoribosyl transferase enzyme using well-known techniques in the field of genetic engineering. In a preferred aspect of the present invention, the source of nadV gene coding for nicotinamide phosphoribosyl transferase enzyme can be selected from the group consisting of Stenotrophomonas maltophilia, Chromobacterium violaceum, Deinococcus radiodurans, Synechocystis sp. PCC 6803. Pseudonocardia dioxanivorans CB1190, Shewanella oneidensis MR-1, and Ralstonia solanacearum GMI1000. In an exemplary embodiment of the present invention, an nadV gene from any one of the foregoing microbial species is isolated and introduced into a strain of Corynebacterium glutamicum using genetic engineering techniques well-known in the art. In a preferred aspect, the exogenous gene nadV is codon optimized before its transfer into a selected microbial host cell to assure optimal expression of the nicotinamide phosphoribosyl transferase enzyme in the microbial host cell.

[0048] In the phosphoribosyl transferase mediated enzyme reaction, phosphoribosyl pyrophosphate (PRPP) acts as a co-substrate and it is necessary to make sure that there is a sufficient amount of PRPP available within the cell to facilitate the phosphoribosyl transferase enzyme-mediated reaction. The pool size of PRPP within the microbial may be increased by means of enhancing the expression of the activity of a PRPP synthetase. The enhanced expression of PRPP synthetase activity can be achieved by means of expressing an exogenous gene prsA. In addition, if there is any feedback inhibition on the PrsA enzyme activity, it is preferable to use a prsA gene that encodes a feedback-resistant variant of the PrsA enzyme.

[0049] In another embodiment of this invention, as a way of conserving the PRPP pool serving as the co-substrate in the production of NMN from Nam, additional genetic modifications can be introduced to block other pathways utilizing PRPP pool within the cell. In one aspect of the present invention, the pyrE gene is mutated leading to the inactivation of the orotate phosphoribosyl transferase enzyme capable of catalyzing the transfer of a ribosyl phosphate group from PRPP to orotate, which in turn leads to the formation of orotidine.

[0050] In order to achieve the production target for NMN production, besides from ensuring that the appropriate phosphoribosyl transferase and phosphoribosyl pyrophosphate (PRPP) synthetase enzymes are present and sufficient amount of PRPP are present within the microbial cell for production of NMN from Nam, it is advantageous to block or inactivate possible NMN utilization/degradation pathways within the microbial cell.

[0051] For example, the microbial host cell selected for producing NMN using Nam as a feedstock and having a codon optimized exogenous gene coding for nicotinamide phosphoribosyl transferase enzyme can further comprise additional genetic modifications to ensure the conservation of the NMN produced within the cell, wherein the additional genetic modifications block other biochemical pathways that consume NMN. In one aspect of this embodiment, the ushA gene coding for an enzyme capable of the conversion of NMN to NR is deleted. In another aspect of this embodiment, the gene pncC is deleted resulting in the loss of nicotinamide-nucleotide amidohydrolase enzyme activity capable of the conversion of NMN into nicotinate mononucleotide (NAMN). In yet another aspect of this invention, the genes cgl1364, cgl1977 and cgl2835 are deleted leading to the elimination of a putative nucleosidase capable of the conversion of NMN into Nam and ribose-5 phosphate. In another aspect of this invention, the gene nadD is genetically modified so that the activity of the enzyme capable of the conversion of NMN to NAD is blocked.

[0052] Accordingly, in one aspect, the present invention provides methods of producing NMN, wherein such method comprises: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, where said strain comprises at least one genetic modification selected from the group consisting of: (a) heterologous expression of a gene encoding nicotinamide phosphoribosyltransferase capable of the conversion of Nam to NMN; (b) deletion or modification of one or more genes capable of the enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of the enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification or deletion of PRPP-requiring biochemical pathways other than the biochemical pathway for the conversion of Nam to NMN; (f) deletion or modification of a gene capable of the enzymatic conversion of NMN to NAMN; (g) deletion or modification of a gene capable of the conversion of NMN to NAD; (h) deletion or modification of one or more genes coding for a promiscuous nucleosidase reaction capable of the conversion of NMN to Nam and ribose-5-phosphate; (i) incorporation of a conditionally active ribonucleotide reductase to block cell division and biomass accumulation without affecting protein synthesis and normal metabolic activity; and (j) any combinations thereof. In various embodiments, the present genetically modified strain with said at least one modification produces an increased amount of NMN compared to a strain without any of said modifications.

Modified Pathway for NAD Production

[0053] In yet another embodiment, the present invention provides a method for producing NAD using Nam as a feedstock. In one aspect of this embodiment, the microbial host cell selected for producing NMN using Nam and having a codon optimized exogenous gene coding nicotinamide phosphoribosyl transferase enzyme can further comprise an additional genetic modification in the nadD gene yielding an upregulated NAD(+) synthetase enzyme capable of the conversion of NMN to NAD. In yet another aspect of this embodiment, the gene nudC is mutated so that enzyme capable of the conversion of NAD back to NMN is blocked.

[0054] Accordingly, in yet another aspect, the present invention provides methods of producing NAD, wherein such method comprises: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, where said strain comprises at least one genetic modification selected from the group consisting of: (a) heterologous expression of a gene encoding a nicotinamide phosphoribosyltransferase capable of the conversion of Nam to NMN; (b) deletion or modification of one or more genes capable of the enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of the enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification or deletion of PRPP requiring biochemical pathways other than the biochemical pathway for the conversion of Nam to NMN; (f) deletion or modification of the gene capable of the enzymatic conversion of NMN to NAMN; (g) modification of the gene capable of the conversion of NMN to NAD; (h) deletion or modification of one or more genes capable of the conversion of NAD back to NMN; (i) deletion or modification of one or more genes coding for a nucleosidase capable of the conversion of NMN to Nam and ribose-5-phosphate; (j) incorporation of a conditionally active ribonucleotide reductase to block cell division and biomass accumulation without affecting protein synthesis and normal metabolic activity; and (k) any combinations thereof. In various embodiments, the present genetically modified strain with said at least one modification produces an increased amount of NMN compared to a strain without any of said modifications.

Modified Pathway for NR Production

[0055] In yet another embodiment, the present invention provides a method for producing NR using Nam as a feedstock. In one aspect of this embodiment, the microbial host cell selected for producing NMN using Nam and having a codon optimized exogenous gene coding nicotinamide phosphoribosyl transferase enzyme can further comprise a functionally active dephosphorylation enzyme coded by a ushA gene. In another aspect of this invention, the ushA gene is genetically modified yielding a dephosphorylase enzyme with enhanced activity for the conversion of NMN to NR.

[0056] Accordingly, in another aspect, the present invention provides methods of producing NR, wherein such method comprises: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, where said strain comprises at least one genetic modification selected from the group consisting of: (a) heterologous expression of a gene encoding a nicotinamide phosphoribosyltransferase capable of the conversion of Nam to NMN; (b) deletion or modification of one or more genes capable of the enzymatic conversion of Nam to NA; (c) modification of one or more genes capable of the enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification or deletion of PRPP requiring biochemical pathways other than the biochemical pathway for the conversion of Nam to NMN; (f) deletion or modification of the gene capable of the enzymatic conversion of NMN to NAMN; (g) deletion or modification of the gene capable of the conversion of NMN to NAD; (h) deletion or modification of one or more genes coding for a nucleosidase enzyme capable of the conversion of NMN to Nam and ribose-5-phosphate; (i) deletion or modification of one or more genes coding for a nucleosidase enzyme capable of the conversion of NR to Nam and ribose-sugar, (j) incorporation of a conditionally active ribonucleotide reductase to block cell division and biomass accumulation without affecting protein synthesis and normal metabolic activity; and (k) any combinations thereof. In various embodiments, the present genetically modified strain with said at least one modification produces an increased amount of NR compared to a strain without any of said modifications.

Modified Pathway for Increasing NMN Titer

[0057] The production of NMN from NAM as catalyzed by the NadV enzyme depends at least in part upon the rate at which microbial host cells produce the PRPP co-substrate. Illustrated in FIG. 8 is the oxidative branch of the pentose phosphate pathway (PPP) in which D-glucose-6-phosphate is converted to D-ribulose-5-phosphate and then PRPP. A strategy to increase flux through the PPP entails knocking out the pgi gene, thereby redirecting the D-glucose-6-phosphate from glycolysis to the PPP and increasing PRPP availability. However, a problem associated with this approach in Corynabacterium gluamicum is that growth is greatly retarded on minimal media such as CGXII. Without wishing to be bound to any particular theory, it is believed that the native ZWF protein, i.e., the glucose 6-phosphate dehydrogenase enzyme catalyzing the first step of the PPP pathway, is inhibited by ATP and PEP (phosphoenolpyruvate) accumulation during growth on glucose, thereby greatly reducing carbon flux through the PPP. Moreover, assuming that an alternative ZWF can be identified that allows carbon flux to increase, a further problem emerges likely due to the accumulation of redox equivalents in the form of NADPH instead of NADH, and wild-type Corynebacterium lacks a pyridine nucleotide transhydrogenase, e.g. a soluble UdhA or a transmembrane PntAB, to address this shortcoming.

[0058] Accordingly, in one aspect, the present invention provides a recombinant pgi mutant of Corynebacteriun glutamicum where the expression of a transgenic ZWF enzyme in combination with a pyridine nucleotide transhydrogenase rescues growth while improving production of PRPP as reflected in higher titers of NMN. Hence, in a related aspect, the present invention provides a method of producing NMN, wherein the method comprises: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, wherein said strain comprises genetic modifications including: (a) heterologous expression of a gene encoding nicotinamide phosphoribosyltransferase capable of the conversion of Nam to NMN; (b) deletion or modification of one or more genes capable of the enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of the enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification or deletion of PRPP-requiring biochemical pathways other than the biochemical pathway for the conversion of Nam to NMN; (f) deletion or modification of a gene capable of the enzymatic conversion of NMN to NAMN; (g) deletion or modification of a gene capable of the conversion of NMN to NAD; (h) deletion or modification of one or more genes coding for a promiscuous nucleosidase reaction capable of the conversion of NMN to Nam and ribose-5-phosphate; (i) deletion or modification of a gene capable of the enzymatic conversion of D-glucose-6-phosphate to D-fructose-6-phosphate; (j) incorporation of a recombinant ZWF; (k) incorporation of a recombinant pyridine nucleotide transhydrogenase, where the combination of the recombinant ZWF and the recombinant pyridine nucleotide transhydrogenase allows for compensating the growth defect associated with the deletion or modification of a gene capable of the enzymatic conversion of D-glucose-6-phosphate to D-fructose-6-phosphate; (1) incorporation of a conditionally active ribonucleotide reductase to block cell division and biomass accumulation without affecting protein synthesis and normal metabolic activity; and (m) any combinations thereof.

[0059] The production of PRPP is thereby improved as reflected in higher titers of NMN relative to a strain without such modifications.

[0060] Nicotinamide compounds (NMN, NR, NAD) produced according to the present disclosure can be utilized in any of a variety of applications, for example, exploiting their biological or therapeutic properties (e.g., controlling low-density lipoprotein cholesterol, increasing high-density lipoprotein cholesterol, etc.). For example, according to the present disclosure, nicotinamide ribose may be used in pharmaceuticals, foodstuffs, and dietary supplements, etc.

[0061] The nicotinamide mononucleotide (NMN) produced by the method disclosed in this invention could have therapeutic value in improving plasma lipid profiles, preventing stroke, providing neuroprotection with chemotherapy treatment, treating fungal infections, preventing or reducing neurodegeneration, or in prolonging health and well-being. Thus, the present invention is further directed to the nicotinamide riboside compounds obtained from the genetically modified bacterial cell described above, for treating a disease or condition associated with the nicotinamide riboside kinase pathway of NAD+ biosynthesis by administering an effective amount of a nicotinamide riboside composition.

[0062] Diseases or conditions which typically have altered levels of NAD+ or NAD+ precursors or could benefit from increased NAD+ biosynthesis by treatment with nicotinamide riboside include, but are not limited to, lipid disorders (e.g., dyslipidemia, hypercholesterolemia or hyperlipidemia), stroke, neurodegenerative diseases (e.g., Alzheimer's, Parkinsons and Multiple Sclerosis), neurotoxicity as observed with chemotherapies, Candida glabrata infection, and the general health declines associated with aging. Such diseases and conditions can be prevented or treated by diet supplementation or providing a therapeutic treatment regime with a nicotinamide riboside composition.

[0063] It will be appreciated that, the nicotinamide compounds isolated from the genetically modified bacteria of this invention can be reformulated into a final product. In some other embodiments of the disclosure, nicotinamide riboside compounds produced by genetically modified host cells as described herein are incorporated into a final product (e.g., food or feed supplement, pharmaceutical, etc.) in the context of the host cell. For example, host cells may be lyophilized, freeze dried, frozen or otherwise inactivated, and then whole cells may be incorporated into or used as the final product. The host cell may also be processed prior to incorporation in the product to increase bioavailability (e.g., via lysis).

[0064] In some embodiments of the disclosure, the produced nicotinamide riboside compounds are incorporated into a component of food or feed (e.g., a food supplement). Types of food products into which nicotinamide riboside compounds can be incorporated according to the present disclosure are not particularly limited, and include beverages such as milk, water, soft drinks, energy drinks, teas, and juices; confections such as jellies and biscuits; fat-containing foods and beverages such as dairy products; processed food products such as rice, bread, breakfast cereals, or the like. In some embodiments, the produced nicotinamide riboside compound is incorporated into a dietary supplement, such as, for example, a multivitamin.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

[0066] The disclosure will be more fully understood upon consideration of the following non-limiting Examples. It should be understood that these examples, while indicating preferred embodiments of the subject technology, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of the subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the subject technology to adapt it to various uses and conditions.

EXAMPLES

Example 1

Strain Construction

[0067] Various engineered C. glutamicum strains were constructed with one or more genetic modifications as described in Table 5. In carrying out the genetic deletions in C. glutamicum summarized in Table 5, pDEL plasmids (a suicidal plasmid which lacks an origin of replication for C. glutamicum) were used. The pDEL plasmid contained homologous regions (approximately 500-800 bps each) upstream and downstream of the gene targeted for deletion. As shown in FIG. 5, the upstream and downstream regions flanking the gene targeted for deletion were obtained using PCR and cloned into the pDEL plasmid containing an SacB gene and an antibiotic marker.

[0068] C. glutamicum cells were transformed with such plasmids and plated in CASO medium containing kanamycin (25 ug/mL) and left overnight. The resulting colonies were plated in a medium containing 6% sucrose to select for double recombinants which have target gene deletion and does not have any portion of the pDEL plasmid integrated into its chromosomal DNA.

[0069] The above engineered strains were subjected to further genetic modifications using plasmids described in Table 6 to introduce an exogenous NadV gene and a prsA gene variant/mutant.

Example 2

NMN Production from Nam

[0070] Strains constructed according to Example 1 were grown overnight in a BHI medium containing kanamycin. The cells were pelleted down, washed and resuspended in CGXI1 medium for 2-3 hrs for recovery as the C. glutamicum cells have a long lag phase when moved from one medium to another medium. To start the fermentation assay, cells were inoculated in a CGXII medium (Table 7) at 0.2 OD.sub.600, at 30 C. and kept on a rotary shaker (250 rpm). The cells were induced with 0.4 mM IPTG when cell density reaches 0.6-0.8 OD.sub.600. Nam was fed to the cell culture, and samples were collected every 24 h for up to 72 h to confirm NMN production.

[0071] Specifically, samples were obtained from the culture supernatant and centrifuged at 0.4000 G for 5 minutes. The clear supernatant was mixed with equal volume of methanol, vortexed for 30 seconds or shaken at 600 rpm on a plate shaker for 10 minutes and centrifuged at >4000 G for 5 minutes to remove any additional debris and the resultant supernatant was analyzed by HPLC method using a Phenomenex Luna 3 m NH2 100 operating in HILAC mode with a maximum pressure of 400 Barr. 2 L injections were monitored at 261 nm with standard curves generated using standards of NAM, NR, NMN, and NAD+. The aqueous buffer was 5 mM ammonium acetate pH 9.9 (A) while the organic buffer was acetonitrile (B). The HPLC column was run at 0.1 mL/min with the following gradient: 0 min-95% B, 1 min-95% B, 15 min-0% B, 20 min-0% B, 20.01 min-95% B, 30 min-95% B.

[0072] The production titers of NMN by the NMN4 and NR5 strains (both containing exogenous genes smaOP and prsA L136I), respectively, are shown in FIG. 6.

[0073] The production titers of NMN by an NMN3 strain containing exogenous genes cviHA and prsA L136I, an NMN4 strain containing exogenous genes cviHA and prsA L136I, an NMN4 strain containing exogenous genes smaOP and prsA L136I, and an NR5 strain containing exogenous genes smaOP and prsA L136I, respectively, are shown in FIG. 7.

[0074] As shown in FIG. 6 and FIG. 7, the NR5 strain characterized by the deletion of endogenous genes cgl1364 coding for purine nucleosidase iunH3, cgl1977 coding for purine nucleosidase iunH2, and cgl2835 for purine nucleosidase iunH1 appears to have the highest NMN production titers. Without wishing to be bound by any particular theory, it is believed that deletion of one or more of the foregoing genes can lead to a significant decrease in the degradation of NMN back into nicotinamide (Nam) and ribose-5P. This observation was highly surprising given the fact that the deletion of one of more of cgl1364, cgl1977, and cgl2835 was previously known only to have the effect of reducing the degradation of NR into NA and ribose.

Example 3

Growth Rescue and Increase in the PPP Pathway

[0075] As anticipated above, the pgi gene may be knocked out to redirect the D-glucose-6-phosphate away from glycolysis and into the PPP, thereby increasing PRPP availability. However, the pgi mutant of C. glutamicum is negatively affected by greatly impaired growth. Without being bound to any particular theory, it is believed that the native ZWF protein, i.e., the glucose 6-phosphate dehydrogenase enzyme catalyzing the first step of the PPP pathway, is inhibited by ATP and PEP (phosphoenolpyruvate) accumulation during growth on glucose, thereby greatly reducing carbon flux through the PPP.

[0076] Three recombinant ZWFs were screened for their ability to overcome the inhibition displayed by native C. glutamicum ZWF: (i) a feedback resistant mutant (A243T) of C. glutamicum ZWF (SEQ ID NO: 28); (ii) an engineered mutant (R46E/Q47E) of the ZWF from Leuconostoc mesenteroides (lmZWF, SEQ ID NO: 32) which has mixed use of NADH and NADPH; and (iii) the ZWF from Zymomonas mobilis (zmZWF) which favors NADH use.

[0077] Two transhydrogenases were screened for their ability to overcome NADPH accumulation: (i) UdhA from E. coli is a soluble transhydrogenase that is reported to favor the reduction of NAD+ by NADPH; and (ii) PntAB from E. coli is a membrane-bound transhydrogenase that is reported to favor the reduction of NADP+ by NADH.

[0078] The recombinant ZWFs and the transhydrogenase were paired in the cgDVS expression vector in constitutively expressed or cumate-induced configurations, as follows:

Constitutive Expression:

[0079] (i) cgDVS.pSOD.lmZWF.pntAB (S.lmZWF.pntAB) [0080] (ii) cgDVS.pSOD.lmZWF.udhA (S.lmZWF.udhA) [0081] (iii) cgDVS.pSOD.zmZWF.pntAB (S.zmZWF.pntAB) [0082] (iv) cgDVS.pSOD.zmZWF.udhA (S.zmZWF.udhA)

Cumate-Induced Expression:

[0083] (v) cgDVS.pCT5.cgZWF*.udhA (S.pCT5.cg.udhA)

[0084] The above vectors (i)-(v) were each independently transformed into the BASE strain, i.e., the NMN11 strain previously transformed with the cgDVK.ptac.smaOP.prsA vector. The BASE strain was also transformed with either of two control vectors cgDVS expressing red fluorescent protein mCherry from either the min3 constitutive promoter (S.min3.mCH) or the pCT5-induced promoter (S.pCT5.mCH). The transformed strains were then grown on CGXII medium to identify instances of successful rescue from growth deficit in NMN11.

[0085] As illustrated in FIG. 9, where the time is measured in hours on the x-axis and the OD.sub.600 is shown on the y-axis, the BASE strain, min3.mCherry and pCT5.mCherry exhibited almost zero growth, as expected. The pCT5.mCherry strain eventually emitted a signal at a wavelength of 630 nm, but still showed no major visible growth or mCherry concentration by visual examination, which would explain the later rise in emission at 630 nm as mCherry is still absorbing in the vicinity of such wavelength. The zmZWF.udhA (dark blue) and lmZWF.PntAB (yellow) strains exhibited the best rescues of the growth defect. The second-best results were obtained from lmZWF.udhA, while zmZWF.pntAB showed a modest early growth that trailed off. Initial production tests carried out in flasks revealed that strain zmZWF.udhA produced the most NMN.

[0086] The following strains were transformed with the vectors as indicated: [0087] NMN4: cgl1777-8 cgl2487 cgl1963 cgl0328 cgl2773 [0088] (1) No control: cgDVK.ptac.mcherry (mCherry) [0089] (2) Production control: cgDVK.ptac.smaOP.prsA (K.smaOP.PRS) [0090] NMN13: cgl1777-8 cgl2487 cgl963 cgl0328 cgl0851 [0091] (3) cgDVK.ptac.smaOP.prsA [0092] (4) cgDVK.ptac.smaOP.prsA+cgDVS.pSOD.zmZWF.udhA (S.ZWF.udhA) [0093] NMN11: cgl1777-8 cgl2487 cgl9%3 cgl0328 cgl0851 cgl2773 [0094] (5) cgDVK.ptac.smaOP.prsA [0095] (6) cgDVK.ptac.smaOP.prsA+cgDVS.pSOD.zmZWF.udhA

[0096] Two strains were also manufactured with the pSOD.zmZWF.udhA construct inserted into the pgi location to do away with the need for cgDVS and the spectinomycin antibiotic: [0097] NMN29: cgl1777-8 cgl2487 cgl1963 cgl0328 cgl0851: pSODzmZWF.udhA [0098] (7) cgDVK.ptac.smaOP.prsA [0099] NMN30: cgl1777-8 cgl2487 cgl1963 cgl0328 cgl0851: pSODzmZWF.udhA cgl2773 [0100] (8) cgDVK.ptac.smaOP.prsA

[0101] The amount of NMN produced by each strain was assessed after 48 hours and 72 hours of cell culture growth, respectively. The results are illustrated in FIG. 10.

TABLE-US-00001 TABLE 1 Genes and enzymes used in the present invention Corynebacterium Gene glutamicum ATCC 13032 EC name NCBI gene symbol Name of the enzyme Number pncA NCgl2401 Nicotinamidase/ 3.5.1.19 pyrazinamidase prs NCgl0905 Ribose-phosphate 2.7.6.1 pyrophosphokinase or PRPP synthase pncB NCgl2431 Nicotinate 6.3.4.21 phosphoribosyltransferase pncC NCgl1888 Nicotinamide-nucleotide 3.5.1.42 amidohydrolase nadD NCgl2270 Nicotinate-nucleotide 2.7.7.18 adenylyltransferase nadE NCgl2446 NH(3)-dependent 6.3.1.5 NAD(+) synthetase nudC NCgl0744 NADH pyrophosphatase 3.6.1.22 cgl0328 NCgl0322 5-nucleotidase 3.1.3.5 cgl1364 NCgl1309 Nicotinamide riboside 3.2.2.1 hydrolase nadV Not present in Nicotinamide 2.4.2.12 Corynebacterium phosphoribosyltransferase glutamicum ATCC 13032.

TABLE-US-00002 TABLE 2 Preferred sources and genes for nadV coding for nicotinamide phosphoribosyltransferase activity Source of nadV gene NCBI gene ID Chromobacterium violaceum ATCC 12472 CV_RS00140 Deinococcus radiodurans R1 DR_0294 Synechocystis sp. PCC 6803 slr0788 Pseudonocardia dioxanivorans CB1190 Psed_2146 Shewanella oneidensis MR-1 SO_1981 Ralstonia solanacearum GMI1000 RSp0836 Stenotrophomonas maltophilia B2FI76 Haemophilus ducreyi NAMPT gene

TABLE-US-00003 TABLE 3 Additional genes and proteins used in the present invention Gene Accession Gene Number: Protein Details cviHA AAQ57714 cviNadVha Codon harmonized variant of the nicotinamide phosphoribosyltransferase (NadV) from Chromobacterium violaceum smaOP SQG65624 smaNadVop Codon optimized variant of the nicotinamide phosphoribosyltransferase (NadV) from Stenotrophomonas maltophilia prsA PrsA (PRS) Codon optimized variant of the PRPP synthase from Corynebacterium glutamicum ATCC 13032 prsA PrsA L136I Feedback resistant mutant of PrsA L136I (PRS L136I) zmZWF zmZWF Codon optimized variant of the glucose-6-phosphate dehydrogenase from Zymomonas mobilis strain ATCC 10988, Zmob_0908 lmZWF lmZWF Codon optimized mutant R46E/Q47E (R46E/Q47E) of the glucose-6- phosphate dehydrogenase from Leuconoston mesenteroides cgZWF* cgZWF Codon optimized mutant (A243T) A243T of the of the glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum udhA udbA Soluble Pyridine Nucleotide Transhydrogenase from Escherichia coli pntAB pntA.B Membrane-bound Pyridine Nucleotide Transhydrogenase from Escherichia coli

TABLE-US-00004 TABLE 4 Sequence Information SEQ ID No. 1 PRT; Chromobacterium violaceum; nicotinamide phosphoribosyltransferase-cviNadVha (cviHA) SEQ ID No. 2 PRT; Stenotrophomonas maltophilia; nicotinamide phosphoribosyltransferase-smaNadVop (smaOP) SEQ ID No. 3 PRT; codon-optimized PRPP synthase variant, CYL77_RS04805-prsA (PRS) SEQ ID No. 4 PRT; feedback resistant PRPP synthase mutant, prsA L136I (PRS L136I) SEQ ID No. 5 DNA; nicotinamidase pncA (CGL_RS12350, ncgl2401, cgl2487 SEQ ID No. 6 PRT; nicotinamidase pncA (CGL_RS12350, ncgl2401, cgl2487 SEQ ID No. 7 DNA; Nicotinamide-nucleotide amidohydrolase pncC (CGL_RS09770, ncgl1888, cgl1963) SEQ ID No. 8 PRT; Nicotinamide-nucleotide amidohydrolase pncC (CGL_RS09770, ncgl1888, cgl1963) SEQ ID No. 9 DNA; restriction modification system; RM = cgl1777 (CYL77_RS08985) SEQ ID No. 10 PRT; restriction modification system; RM = cgl1777 (CYL77_RS08985) SEQ ID No. 11 DNA; restriction modification system; RM = cgl1778 (CYL77_RS08990) SEQ ID No. 12 PRT; restriction modification system; RM = cgl1778 (CYL77_RS08990) SEQ ID No. 13 DNA; orotate phosphoribosyltransferase pyrE; CGL_RS13820, ncgl2676 SEQ ID No. 14 PRT; orotate phosphoribosyltransferase pyrE; CGL_RS13820, ncgl2676, cgl2773 SEQ ID No. 15 DNA; 5-nucleotidase ushA; cgl0328-CGL_RS01710, ncgl0322, cg0397 SEQ ID No. 16 PRT; 5-nucleotidase ushA; cgl0328-CGL_RS01710, ncgl0322, cg0397 SEQ ID No. 17 DNA; nicotinamide ribose transporter pnuC; ncgl0063, CGL_RS00355, cgl0064 SEQ ID No. 18 PRT; nicotinamide ribose transporter pnuC; ncgl0063, CGL_RS00355, cgl0064 SEQ ID No. 19 DNA; purine nucleosidase iunH3; cgl1364 (CGL_RS06810, ncgl1309, cgl543, iunH3) SEQ ID No. 20 PRT; purine nucleosidase iunH3; cgl1364 (CGL_RS06810, ncgl1309, cg1543, iunH3) SEQ ID No. 21 DNA; purine nucleosidase iunH2; cgl1977 (cg2168, iunH2, CYL77_RS09970) SEQ ID No. 22 PRT; purine nucleosidase iunH2; cgl1977 (cg2168, iunH2, CYL77_RS09970) SEQ ID No. 23 DNA; purine nucleosidase iunH1; cgl2835 (cg3137, iunH1, CYL77_RS14340) SEQ ID No. 24 PRT; purine nucleosidase iunH1; cgl2835 (cg3137, iunH1, CYL77_RS14340) SEQ ID No. 25 DNA; glucose-6P isomerase pgi; cgl0851 (ncgl0817) SEQ ID No. 26 PRT; glucose-6P isomerase PGI; cgl0851 (ncgl0817) SEQ ID No. 27 DNA; codon-optimized mutant (A243T) of the of the glucose-6- phosphate dehydrogenase from Corynebacterium glutamicum; cgZWF SEQ ID No. 28 PRT; codon-optimized mutant (A243T) of the of the glucose-6- phosphate dehydrogenase from Corynebacterium glutamicum; cgZWF SEQ ID No. 29 DNA; glucose-6-phosphate dehydrogenase of Leuconoston.mesenteroides, codon optimized mutant (R46E/Q47E) of the gene having accession number M64446.1; lmZWF SEQ ID No. 30 RNA; glucose-6-phosphate dehydrogenase of Leuconoston.mesenteroides, codon optimized mutant (R46E/Q47E); lmZWF SEQ ID No. 31 DNA; codon-optimized variant of the glu6-phosphate dehydrogenase from Zymomonas mobilis strain ATCC 10988, Zmob_0908 SEQ ID No. 32 PRT; codon-optimized variant of the glu6-phosphate dehydrogenase from Zymomonas mobilis strain ATCC 10988, NCBI-ProteinID: AEH62743.1 SEQ ID No. 33 DNA; soluble Pyridine Nucleotide Transhydrogenase from Escherichia coli; udhA NP_418397.2, EG11428 SEQ ID No. 34 PRT; soluble Pyridine Nucleotide Transhydrogenase from Escherichia coli; udhA AAC76944 SEQ ID No. 35 DNA; A subunit of Membrane bound Pyridine Nucleotide Transhydrogenase (pntA) from Escherichia coli MG1655; ECK1598, variant g1342a, NP_416120.1 SEQ ID No. 36 PRT; A subunit of Membrane bound Pyridine Nucleotide Transhydrogenase (pntB) from Escherichia coli; P07001.2 A434T variant SEQ ID No. 37 DNA; B subunit of Membrane bound Pyridine Nucleotide Transhydrogenase (pntB) from Escherichia coli MG1655; ECK1597, NP_416119.1 SEQ ID No. 38 PRT; B subunit of Membrane bound Pyridine Nucleotide Transhydrogenase (pntB) from Escherichia coli; P0AB69.1

TABLE-US-00005 TABLE 5 Strains constructed according to the present invention Strain name Genotype/Description of genetic modification ATCC 13032 Corynebacterium glutamicum ATCC 13032 Cgl1 ATCC 13032 cgl1777 cgl1778-Deletion of restriction modification (RM) system NMN1 Cgl1 cgl2487-Deletion of nicotinamidase (pncA) to prevent conversion of NA to NAM NMN2 NMN1 cgl1963-Deletion of nicotinamide-nucleoside amidase (pncC) to prevent conversion of NMN to NaMN. NMN3 NMN2 cgl0328-Deletion of 5-nucleotidase (ushA) to prevent conversion of NMN to NR. NMN4 NMN3 cgl2773-Deletion of orotate phosphoribosyltransferase (pyrE) to prevent conversion of PRPP to Orotidine-5-P; Uracil auxotroph. RM pncA pncC ushA pyrE NR1 NMN2 cgl1364-deletion of purine nucleosidase (iunH3) to prevent conversion of NR to NAM. NR2 NR1 cgl0064-Deletion of nicotinamide ribose transporter (pnuC) to inhibit NR uptake NR3 NR2 cgl2835-Deletion of purine nucleosidase (iunH1) to prevent conversion of NR to NAM NR5 NR3 cgl1977-deletion of purine nucleosidase (iunH2) to prevent conversion of NR to NAM RM (9, 11) pncA (5) pncC (7) pnuC (17) cgl1364 cgl1977 cgl2835 (19, 21, 23) NMN11 NMN4 cgl0851-Deletion of glucose-6P isomerase (PGI) to push carbon flux towards PPP NMN13 NMN3 cgl0851-Deletion of glucose-6P isomerase (PGI) to push carbon flux towards PPP NMN16 NR5 cgl0328-Deletion of 5-nucleotidase (ushA) to prevent conversion of NMN to NR NMN17 NMN16 cgl2773-Deletion of orotate phosphoribosyltransferase (pyrE) to prevent conversion of PRPP to Orotidine-5-P; Uracil auxotroph NMN29 NMN11 cgl0851:zmZWF.udhA-Insertion of zmZWF.udhA into PGI locus to increase carbon flux towards PPP NMN30 NMN13 cgl0851:zmZWF.udhA-Insertion of zmZWF.udhA into PGI locus to increase carbon flux towards PPP

TABLE-US-00006 TABLE 6 Plasmids for introducing exogenous genes Plasmid Code Plasmid Name Description cgDVK E. coli/C. glut. Shuttle vector ColE1 ori/pHM1519 ori KanR cviHA.prsA(L136I) cgDVK.pTAC.cviHA.prsA(L136I) cgDVK constitutive LacI production, IPTG induced expression of cviNadVha and PrsA(L136I) smaOP.prsA(L136I) cgDVK.pTAC.smaOP.prsA(L136I) cgDVK constitutive LacI production, IPTG induced expression of smaNadVop and PrsA(L136I) cgDVS E. coli/C. glut shuttle vector pBL1/ori SpecR zmZWF.udhA cgDVS.pSOD.zmZWF.udhA cgDVS constitutive pSOD expression of zmZWF.udhA zmZWF.pntAB cgDVS.pSOD.zmZWF.pntAB cgDVS constitutive pSOD expression of zmZWF.pntAB lmZWF.udhA cgDVS.pDOS.lmZWF.udbA cgDVS constitutive pSOD expression of lmZWF.udhA lmZWF.pntAB cgDVS.pSOD.lmZWF.pntAB cgDVS constitutive pSOD expression of lmZWF.pntAB cgZWF*.udhA cgDVS.pCT5.cgZWF*.udhA cgDVS constitutive CymR, Cumate induced expression of cgZWF*.udhA

TABLE-US-00007 TABLE 7 Chemical Composition of CGXII Growth medium Component Conc./L NH.sub.4SO.sub.4 20 g KH.sub.2PO.sub.4 1 g K.sub.2HPO.sub.4 1 g Urea (300 gl) 5 g MgSO.sub.4 * 7H.sub.2O (1M) 0.25 g CaCl.sub.2*2H.sub.2O (1M) 10 mg CG TE 50x See paper PCA (50 gl in 57% etoh) 30 mg Biotin (10 gl) 0.2 mg Thiamine HCl (20 gl) 1 mg MOPS 42 g Glucose (50%) 30 g

TABLE-US-00008 SequencesofInterest SEQIDNO:1;PRT;Chromobacteriumviolaceum;nicotinamide phosphoribosyltransferase-cviNadVha(cviHA) MTAPKAAQDKNQLVPFNLADFYKTGHPAMYPRETTRLVANFTPRSAKYAQVLPQLF DDKVVWFGLQGFIQEYLIDLFNREFFQRPKADAVRRYQRRMDTALGAGAVDGGRLE ALHDLGHLPLEIRSLPEGARVDIKVPPVTFSNTHPDFPWVATYFETLFSCESWKPSTVA TIAFEFRKLLSYFAALTGAPQDFVAWQGHDFSMRGMSGVHDAMRCGAGHLLSFTGT DTIPALDYLEDHYGADAERELVGGSIPASEHSVMALRILLTQQRLARMPAHQGLDDK ALRRLAEREVVREFVTRDYPAGMVSIVSDTFDFWNVLTVIARELKDDIQARRPDALGN AKVVFRPDSGDPVRILAGYRDDELQFDDAGNCTARDDGRPVSAAERKGAVECLWDIF GGTVTERGYRVLDSHVGLIYGDSITLPRARDILLRLAEKGYASCNVVFGIGSFVYGMN SRDTFGYALKAVYAEVAGEAVDIYKDPATDDGTKKSARGLLRVEEENGRYALYQQQ TPAEAEGGALRPVFRDGELLVKQTLAEIRQRLQASWTCPEAGSIVWNA SEQIDNO:2;PRT;Stenotrophomonasmaltophilia;nicotinamide phosphoribosyltransferase-smaNadVop(smaOP) MHYLDNLLLNTDSYKASHWLQYPPGTDATFFYVESRGGLHDRTVFFGLQAILKDALA RPVTHADIDDAAAVFAAHGEPFNEAGWRDIVDRLGGHLPVRIRAVPEGSVVPTHQAL MTIESTDPAAFWVPSYLETLLLRVWYPVTVATISWHARQTIAAFLQQTSDDPQGQLPF KLHDFGARGVSSLESAALGGAAHLVNFLGTDTVSALCLARAHYHAPMAGYSIPAAEH STITSWGREREVDAYRNMLRQFGKPGSIVAVVSDSYDIYRAISEHWGTTLRDDVIASG ATLVIRPDSGDPVEVVAESLRRLDEAFGHAINGKGYRVLNHVRVIQGDGINPDTIRAIL QRITDDGYAADNVAFGMGGALLQRLDRDTQKFALKCSAARVEGEWIDVYKDPVTDA GKASKRGRMRLLRRLDDGSLHTVPLPANGDDTLPDGFEDAMVTVWENGHLLYDQRL DDIRTRAAVGH SEQIDNO:3;PRT;Codon-optimizedPRPPsynthasevariant, CYL77_RS04805-prsA(PRS) MTAHWKQNQKNLMLFSGRAHPELAEAVAKELDVNVTPMTARDFANGEIYVRFEESV RGSDCFVLQSHTQPLNKWLMEQLLMIDALKRGSAKRITAILPFYPYARQDKKHRGREP ISARLIADLMLTAGADRIVSVDLHTDQIQGFFDGPVDHMHAMPILTDHIKENYNLDNIC VVSPDAGRVKVAEKWANTLGDAPMAFVHKTRSTEVANQVVANRVVGDVDGKDCV LLDDMIDTGGTIAGAVGVLKKAGAKSVVIACTHGVFSDPARERLSACGABEVITTDTL PQSTEGWSNLTVLSIAPLLARTINEIFENGSVTTLFEGEA SEQIDNO:4;PRT;FeedbackresistantPRPPsynthasemutant, prsAL136I(PRSL136I) MTAHWKQNQKNLMLFSGRAHPELAEAVAKELDVNVTPMTARDFANGEIYVRFEESV RGSDCFVLQSHTQPLNKWLMEQLLMIDALKRGSAKRITAILPFYPYARQDKKHRGREP ISARLIADLMLTAGADRIVSVDIHTDQIQGFFDGPVDHMHAMPILTDHIKENYNLDNIC VVSPDAGRVKVAEKWANTLGDAPMAFVHKTRSTEVANQVVANRVVGDVDGKDCV LLDDMIDTGGTIAGAVGVLKKAGAKSVVIACTHGVFSDPARERLSACGAEEVITTDTL PQSTEGWSNLTVLSIAPLLARTINEIFENGSVTTLFEGEA SEQIDNO.:5;DNA;NicotinamidasepncA(CGL_RS12350, ncgl2401,cgl2487) ATGGCACGCGCACTCATTCTGGTTGATGTTCAAAAAGACTTCTGCCCCGGTGGCAG CCTAGCCACCGAACGAGGCGATGAAGTGGCGGGAAAAATCGGTGCCTATCAGCTG TCCCACGGCTCAGAGTACGACGTCGTTGTGGCGACCCAAGATTGGCACATCGATC CAGGCGAGCACTTTTCAGAAACCCCAGACTTTAAAAACTCCTGGCCAATCCACTGC GTCGCGGATTCCGATGGTGCCGCCATGCATGACCGCATCAACACCGATTCAATCG ATGAGTTCTTCCGCAAAGGCCATTACACCGCGGCGTATTCCGGGTTCGAGGGAACT GCAGTCAGTGAAGAACTCCTCATGTCTCCATGGCTGAAGAACAAGGGAGTCACTG ATGTAGACATCGTAGGGATCGCTACGGATCACTGCGTTCGAGCCACAGCACTTGA TGCTCTCAAGGAGGGCTTCAACGTCTCCATTTTGACGTCGATGTGTTCTGCGGTGG ATTTCCATGCGGGAGACCACGCTTTGGAGGAACTACATGAAGCCGGGGCGATTCT GATTTAA SEQIDNo.:6;PRT;NicotinamidasepncA(CGL_RS12350, ncgl2401,cgl2487) MARALILVDVQKDFCPGGSLATERGDEVAGKIGAYQLSHGSEYDVVVATQDWHIDP GEHFSETPDFKNSWPIHCVADSDGAAMHDRINTDSIDEFFRKGHYTAAYSGFEGTAVS EELLMSPWLKNKGVTDVDIVGIATDHCVRATALDALKEGFNVSILTSMCSAVDFHAG DHALEELHEAGAILI SEQIDNO:7;DNA;Nicotinamide-nucleotideamidohydrolase pncC(CGL_RS09770,ncgl1888,cgl1963) ATGTCGGAGAATCTGGCGGGGCGAGTGGTGGAGCTGTTGAAATCGCGCGGTGAAA CGCTGGCGTTTTGTGAATCCCTCACCGCCGGCCTTGCCAGTGCGACGATCGCAGAG ATCCCCGGCGCCTCAGTGGTACTTAAAGGCGGGCTGGTCACCTATGCCACCGAGCT TAAGGTTGCGCTTGCCGGTGTGCCGCAGGAGCTTATCGACGCGCACGGCGTTGTTT CCCCGCAGTGCGCCCGTGCGATGGCAACGGGGGCCGCACACAGATGCCAGGCAGA TTGGGCGGTTTCGCTCACGGGCGTTGCTGGCCCCAGCAAACAAGATGGTCATCCG GTGGGGGAAGTGTGGATCGGAGTGGCTGGTCCTGCGCATTTTGGGGCGTCGGGAA CAATTGACGCGTATCGTGCGTTTGAAAGTGAACAACAGGTAATATTGGCTGAATT GGGACGGCATCATATTAGAGAGTCTGCTGTGCAGCAAAGCTTTCGCCTGCTGATTG ACCATATTGAGTCGCAGTGA SEQIDNO:8;PRT;Nicotinamide-nucleotideamidohydrolase pncC(CGL_RS09770,ncgl1888,cgl1963) MSENLAGRVVELLKSRGETLAFCESLTAGLASATIAEIPGASVVLKGGLVTYATELKV ALAGVPQELIDAHGVVSPQCARAMATGAAHRCQADWAVSLTGVAGPSKQDGHPVG EVWIGVAGPAHFGASGTIDAYRAFESEQQVILAELGRHHIRESAVQQSFRLLIDHIESQ SEQIDNo.:9;DNA;Restrictionmodificationsystem; RM=cgl1777(CYL77_RS08985) ATGAAGCCCACCGTTAATGTTGTGTTCAATGCGCATCACCCCAAAGATACGCAGCC GTTGGATAAGTTCTTCGATAAAGAACTTAAAGACACACATCATCTCGATATAACG GTGGGTTATATCAGTGAGAAATCACTACAATATTTGCTTCTTATTGCAGGCACTCA CCCCGACCTCACCATCACACTCACCTGTGGAATGCACGCTCGTGAAGGCATGACTG CTGCCCAACTGCATCATGCGCGAGTGCTCCATGACTACTTAAGCGACCATGATCGA GGCGGGGTGTTCGTTATTCCCCGATTGCGTTATCACGGCAAAATCTATCTTTTCCA CAAGAACCAGCACACAGATCCTATTGCTTATATCGGTAGCGCTAACCTCTCAGCCA TCGTTCCTGGGTACACCTCTACATTCGAGACCGGCGTCATCTTAGACCCCGCACCT GAAGATCTCGTGCTTCATCTCAACCGTGATGTCGTACCCCTATGTGTCCCCATTGA CACCGCGCATGTCCCCATCATTAAAGATCAAGAATCCCCGATGAAGCACGTCGCT GAAGCAACAGCTGTGTCCACCTCTGATGTTGTTGCCATCATGTCCAGCCCATTTAC TTATAGTTTTGACCTTAAACTCAAAGCCACTGCCAGCAGCAACCTCAATGCTCATA ACTCAGGCGGTGGCGCGCGCAAACAGAAAAACGGTAGCTTCCTTGCACGCAATTG GTATGAGGGCGAAATCATTGTCGGTGTCGAGACAACAAGACTCCCAGGTTACCCA CAAAACAAATCCGAATTCACTGCGGTCACTGATGACGGCTGGTCATTTGTTTGCAA AATCAGCGGAGGAAACGGAAAGAACCTACGCAGCAAAGGTGACCTGTCCATCCTC GGTACGTGGTTAAAGTCTCGATTCATTGAACAAGGTGCCCTGGAATACGGCGAGG ATGCCACCCAAGAAAACATCGACCGTTTTGGGAGAACACATATGACCATGCGCTA TCACCCAGATTTCGATGTGTGGTCATTCGATCTCAGCCAAACCCCGAAGCCTTCGA CACAGATTGGGCAGGATTAA SEQIDNO:10;PRT;Restrictionmodificationsystem; RM=cgl1777(CYL77_RS08985) MKPTVNVVFNAHHPKDTQPLDKFFDKELKDTHHLDITVGYISEKSLQYLLLIAGTHPD LTITLTCGMHAREGMTAAQLHHARVLHDYLSDHDRGGVFVIPRLRYHGKIYLFHKNQ HTDPIAYIGSANLSAIVPGYTSTFETGVILDPAPEDLVLHLNRDVVPLCVPIDTAHVPIIK DQESPMKHVAEATAVSTSDVVAIMSSPFTYSFDLKLKATASSNLNAHNSGGGARKQK NGSFLARNWYEGEIIVGVETTRLPGYPQNKSEFTAVTDDGWSFVCKISGGNGKNLRSK GDLSILGTWLKSRFIEQGALEYGEDATQENIDRFGRTHMTMRYHPDFDVWSFDLSQTP KPSTQIGQD SEQIDNO:11;DNA;Restrictionmodificationsystem; RM=cgl1778(CYL77_RS08990) RTCACCTCAATAACTACATCACGAGCTTGAGTGATAACGCTGATCTCCGTGAAAAA GTCACCGCAACCGTAGACGCTTTCCGCCATACCGTCATGGATGACTTCGACTACAT CAGTGATCAACAAGTCCTGCTTTATGGCGATGTCCAAAGCGGTAAAACCTCACAC ATGCTGGGAATTATCGCAGATTGCCTCGACAGTACGTTTCACACCATTGTTATTCT GACCTCGCCTAACACACGGCTCGTGCAACAAACATACGACCGTGTTGCCCAAGCA TTTCCAGATACTTTGGTGTGCGACCGTGACGGATACAATGATTTCCGTGCGAATCA AAAGAGCCTCACCCCGCGAAAATCTATCGTAGTCGTCGGAAAAATACCTGCAGTT CTTGGTAATTGGTTACGCGTCTTTAACGACAGTGGCGCACTTTCTGGACACCCTGT ACTCATTATTGATGACGAAGCAGATGCGACAAGTCTCAACACCAAAGTAAATCAG TCTGATGTTTCGACCATTAACCACCAGCTCACTAGCATAAGAGACCTTGCCACAGG ATGCATCTACCTTCAGGTCACAGGTACACCTCAAGCGGTGCTTCTTCAAAGCGACG ATAGCAACTGGGCAGCGGAACATGTGCTTCACTTCGCACCTGGTGAGAGCTACAT CGGTGGTCAACTTTTCTTTTCTGAGCTCAACAACCCTTATCTACGACTTTTCGCTAA TACCCAATTTGACGAGGATTCTCGCTTCAGCGACGCCATTTACACCTATCTCTTAA CCGCAGCACTGTTCAAACTTCGCGGTGAAAGCTTGTGTACCATGCTCATTCACCCC AGCCACACTGCATCCAGTCATAGAGACTTCGCGCAAGAAGCCCGCCTCCAACTCA CTTTCGCCTTCGAGCGATTCTATGAACCAATGATTCAGCACAATTTCCAACGTGCT TATGAACAGCTCGCACAAACTGACAGCAACCTGCCACCCTTGAGAAAAATTCTTA ACATTCTTGGTGGCATGGAAGATGACTTCTCCATCCACATCGTCAATAGCGACAAC CCGACTGTTGAGGAAGATTGGGCTGATGGTTATAACATTATTGTCGGTGGCAACTC GCTTGGGCGCGGTTTAACATTCAACAACTTGCAAACCGTTTTCTACGTGCGCGAAT CCAAGCGACCACAAGCAGACACCCTGTGGCAGCACGCCCGCATGTTTGGCTACAA ACGCCACAAAGACACCATGCGTGTGTTCATGCCGGCCACTATTGCTCAAACCTTCC AAGAGGTCTATCTCGGCAACGAAGCTATTAAAAATCAGCTCGATCATGGCACGCA TATCAACGACATTCGGGTCATTTTAGGTGATGGCGTCGCACCTACTCGTGCCAATG TTCTCGACAAACGCAAAGTTGGAAACCTCAGCGGTGGCGTCAACTACTTTGCCGCT GATCCTAGAATCAAGAATGTCGAAGCACTCGACAAAAAACTCTTGGCCTACTTAG ACAAGCACGGTGAGGACTCCACCATCGGTATGCGCGCGATAATCACCATTCTCAA CGCCTTTACTGTAGACCCCAACGATCTCGACCTCGCGACCTTCAAGGCTGCGCTCC TTGACTTTGAACGCAACCAACCTCATCTCACAGCACGTATGGTGCTGCGAACAAAC CGCAAAGTCAATCAGGGTACAGGCGCCCTGCTCTCCCCTACTGATCAAGCTCTCAG CCGTGCAGAAGTCGCACACCCATTATTGATCCTATACCGCATTGAAGGTGTTAACG ATGCTGCTGCGCAACGAGGTGAACCTACGTGGTCAAGCGACCCTATCTGGGTGCC TAATATTAAACTCCCTGGTCAACGTCAATTCTGGTGCGTAGACGGCTAA SEQIDNO:12;PRT;Restrictionmodificationsystem; RM=cgl1778(CYL77_RS08990) MSHHTHLNNYITSLSDNADLREKVTATVDAFRHTVMDDFDYISDQQVLLYGDVQSG KTSHMLGIIADCLDSTFHTIVILTSPNTRLVQQTYDRVAQAFPDTLVCDRDGYNDFRA NQKSLTPRKSIVVVGKIPAVLGNWLRVENDSGALSGHPVLIIDDEADATSLNTKVNQS DVSTINHQLTSIRDLATGCIYLQVTGTPQAVLLQSDDSNWAAEHVLHFAPGESYIGGQ LFFSELNNPYLRLFANTQFDEDSRFSDAIYTYLLTAALFKLRGESLCTMLIHPSHTASSH RDFAQEARLQLTFAFERFYEPMIQHNFQRAYEQLAQTDSNLPPLRKILNILGGMEDDES IHIVNSDNPTVEEDWADGYNIIVGGNSLGRGLTFNNLQTVFYVRESKRPQADTLWQH ARMFGYKRHKDTMRVFMPATIAQTFQEVYLGNEAIKNQLDHGTHINDIRVILGDGVA PTRANVLDKRKVGNLSGGVNYFAADPRIKNVEALDKKLLAYLDKHGEDSTIGMRAII TILNAFTVDPNDLDLATFKAALLDFERNQPHLTARMVLRTNRKVNQGTGALLSPTDQ ALSRAEVAHPLLILYRIEGVNDAAAQRGEPTWSSDPIWVPNIKLPGQRQFWCVDG SEQIDNO:13;DNA;OrotatephosphoribosyltransferasepyrE; CGL_RS13820,ncgl2676,cgl2773 ATGTCATCTAATTCCATTAACGCAGAAGCGCGCGCTGAGCTTGCTGAACTGATCAA AGAGCTAGCTGTCGTCCACGGTGAAGTCACCTTGTCTTCGGGCAAGAAGGCTGATT ACTACATCGATGTCCGTCGTGCCACCTTGCACGCGCGCGCATCTCGCCTGATCGGT CAGCTGCTGCGCGAAGCCACCGCTGACTGGGACTATGACGCAGTTGGCGGCCTGA CCTTGGGCGCTGACCCGGTTGCCACCGCCATCATGCACGCCGACGGCCGCGATATC AACGCGTTTGTGGTGCGCAAGGAGGCCAAGAAGCACGGCATGCAGCGTCGCATTG AGGGCCCTGACCTGACGGGCAAGAAGGTGCTCGTGGTGGAAGATACCACCACCAC CGGAAATTCCCCTCTGACAGCTGTTGCCGCGTTGCGTGAAGCTGGCATTGAGGTTG TGGGCGTTGCCACCGTGGTCGATCGCGCAACCGGTGCAGATGAGGTTATCGCAGC GGAAGGCCTTCCTTACCGCAGCTTGCTGGGACTTTCTGATCTTGGACTCAACTAA SEQIDNO:14;PRT;OrotatephosphoribosyltransferasepyrE; CGL_RS13820,ncgl2676,cgl2773 MSSNSINAEARAELAELIKELAVVHGEVTLSSGKKADYYIDVRRATLHARASRLIGQL LREATADWDYDAVGGLTLGADPVATAIMHADGRDINAFVVRKEAKKHGMQRRIEGP DLTGKKVLVVEDTTTTGNSPLTAVAALREAGIEVVGVATVVDRATGADEVIAAEGLP YRSLLGLSDLGLN SEQIDNO:15;DNA;5-nucleotidaseushA;cgl0328-CGL_RS01710, ncgl0322,cg0397 ATGAAGAGGCTTTCCCGTGCAGCCCTCGCAGTGGTCGCCACCACCGCAGTTAGCTT CAGCGCACTCGCAGTTCCAGCTTTCGCAGACGAAGCAAGCAATGTTGAGCTCAAC ATCCTCGGTGTCACCGACTTCCACGGACACATCGAGCAGAAGGCTGTTAAAGATG ATAAGGGAGTAATCACCGGTTACTCAGAAATGGGTGCCAGTGGCGTTGCCTGCTA CGTCGACGCTGAACGCGCGGACAACCCAAACACCCGCTTCATCACCGTTGGTGAC AACATTGGTGGATCCCCATTCGTGTCCTCCATCCTGAAGGATGAGCCAACCTTGCA AGCCCTCAGCGCCATCGGTGTTGACGCATCCGCACTGGGCAATCACGAATTCGAC CAGGGCTACTCAGACCTGGTGAACCGCGTTTCCCTCGACGGCTCCGGCAGCGCAA AGTTCCCATACCTCGGCGCAAACGTTGAAGGTGGCACCCCAGCACCTGCAAAGTC TGAAATCATCGAGATGGACGGCGTCAAGATCGCTTACGTCGGCGCAGTAACCGAG GAGACCGCAACCTTGGTCTCCCCAGCAGGCATCGAAGGCATCACCTTCACCGGCG ACATCGACGCTATCAACGCAGAAGCAGATCGCGTCATTGAGGCAGGCGAAGCAGA CGTAGTCATCGCATTGATCCACGCTGAAGCCGCTCCAACCGATCTATTCTCCAACA ACGTTGACGTTGTATTCTCCGGACACACCCACTTCGACTACGTTGCTGAAGGCGAA GCACGTGGCGACAAGCAGCCACTCGTTGTCATCCAGGGCCACGAATACGGCAAGG TCATCTCCGACGTGGAGATCTCCTACGACCGCGAAGCAGGCAAGATCACCAACAT TGAGGCGAAGAATGTCTCTGCTACTGACGTTGTGGAAAACTGTGAGACTCCAAAC ACAGCAGTCGACGCAATCGTTGCAGCTGCTGTTGAGGCCGCTGAAGAAGCAGGTA ATGAAGTTGTTGCAACCATTGACAACGGCTTCTACCGTGGGGCGGATGAAGAGGG TACGACCGGCTCCAACCGTGGTGTTGAGTCTTCCCTGAGCAACCTCATCGCAGAAG CTGGACTGTGGGCAGTCAACGACGCGACCATCCTGAACGCTGACATCGGCATCAT GAACGCAGGCGGCGTGCGTGCGGACCTCGAAGCAGGCGAAGTTACCTTCGCAGAT GCATACGCAACCCAGAACTTCTCCAACACCTACGGCGTACGTGAAGTGTCTGGTG CGCAGTTCAAAGAAGCACTGGAACAGCAGTGGAAGGAAACCGGCGACCGCCCAC GTCTGGCATTGGGACTGTCCAGCAACGTCCAGTACTCCTACGACGAGACCCGCGA ATACGGCGACCGCATCACCCACATCACCTTCAACGGTGAGCCAATGGATATGAAG GAGACCTACCGCGTCACAGGATCATCCTTCCTGCTCGCAGGTGGCGACTCCTTCAC TGCATTCGCTGAAGGCGGCCCAATCGCTGAAACCGGCATGGTTGACATTGACCTGT TCAACAACTACATCGCAGCTCACCCAGATGCACCAATTCGTGCAAATCAGAGCTC AGTAGGCATCGCCCTTTCCGGCCCGGCAGTTGCAGAAGACGGAACTTTGGTCCCTG GTGAAGAGCTGACCGTCGATCTTTCTTCCCTCTCCTACACCGGACCTGAAGCTAAG CCAACCACCGTTGAGGTGACCGTTGGTACTGAGAAGAAGACTGCGGACGTCGATA ACACCATCGTTCCTCAGTTTGACAGCACCGGCAAGGCAACTGTCACCCTGACTGTT CCTGAGGGAGCTACCTCTGTCAAGATCGCAACTGACAATGGCACTACCTTTGAACT GCCAGTAACCGTAAACGGTGAAGGCAACAATGATGACGATGATGATAAGGAGCA GCAGTCCTCCGGATCCTCCGACGCCGGTTCCCTTGTAGCAGTTCTCGGTGTTCTTG GAGCACTCGGTGGCCTGGTGGCGTTCTTCCTGAACTCTGCGCAGGGCGCACCATTC TTGGCTCAGCTTCAGGCTATGTTTGCGCAGTTCATGTAA SEQIDNO:16;PRT;5-nucleotidaseushA;cgl0328-CGL_RS01710, ncgl0322,cg0397 MKRLSRAALAVVATTAVSFSALAVPAFADEASNVELNILGVTDFHGHIEQKAVKDDK GVITGYSEMGASGVACYVDAERADNPNTRFITVGDNIGGSPFVSSILKDEPTLQALSAI GVDASALGNHEFDQGYSDLVNRVSLDGSGSAKFPYLGANVEGGTPAPAKSEIIEMDG VKIAYVGAVTEETATLVSPAGIEGITFTGDIDAINAEADRVIEAGEADVVIALIHAEAAP TDLFSNNVDVVFSGHTHFDYVAEGEARGDKQPLVVIQGHEYGKVISDVEISYDREAG KITNIEAKNVSATDVVENCETPNTAVDAIVAAAVEAAEEAGNEVVATIDNGFYRGAD EEGTTGSNRGVESSLSNLIAEAGLWAVNDATILNADIGIMNAGGVRADLEAGEVTFAD AYATQNFSNTYGVREVSGAQFKEALEQQWKETGDRPRLALGLSSNVQYSYDETREY GDRITHITFNGEPMDMKETYRVTGSSFLLAGGDSFTAFAEGGPIAETGMVDIDLFNNYI AAHPDAPIRANQSSVGIALSGPAVAEDGTLVPGEELTVDLSSLSYTGPEAKPTTVEVTV GTEKKTADVDNTIVPQFDSTGKATVTLTVPEGATSVKIATDNGTTFELPVTVNGEGNN DDDDDKEQQSSGSSDAGSLVAVLGVLGALGGLVAFFLNSAQGAPFLAQLQAMFAQF M SEQIDNO:17;DNA;NicotinamideribosetransporterpnuC; ncgl0063,CGL_RS00355,cgl0064 ATGAATCCTATAACCGAATTATTAGACGCAACACTATGGATCGGCGGAGTTCCGA TTCTGTGGCGCGAAATCATCGGCAACGTTTTCGGATTATTTAGCGCGTGGGCAGGA ATGCGACGCATCGTGTGGGCATGGCCCATCGGCATCATAGGCAACGCGCTGCTGT TCACAGTATTTATGGGCGGCCTTTTCCACACTCCACAAAACCTCGATCTCTACGGC CAAGCGGGTCGCCAGATCATGTTCATCATCGTCAGTGGTTATGGCTGGTACCAATG GTCGGCCGCAAAACGTCGCGCACTCACCCCAGAAAATGCAGTAGCAGTGGTTCCT CGCTGGGCAAGCACCAAAGAACGCGCCGGCATTGTGATTGCGGCGGTTGTGGGAA CACTCAGCTTTGCCTGGATTTTCCAAGCACTCGGCTCCTGGGGGCCATGGGCCGAC GCGTGGATTTTCGTCGGCTCAATCCTGGCTACCTACGGAATGGCTCGCGGATGGAC AGAGTTCTGGCTGATCTGGATCGCCGTCGACATAGTTGGCGTTCCTCTACTTTTGA CTGCTGGCTACTACCCATCCGCGGTGCTTTACCTGGTGTACGGTGCGTTTGTCAGC TGGGGATTTGTCGTGTGGCTGCGGGTGCAAAAAGCAGACAAGGCTCGTGCGCTGG AAGCTCAGGAGTCTGTGACAGTCTGA SEQIDNO:18;PRT;NicotinamideribosetransporterpnuC; ncgl0063,CGL_RS00355,cgl0064 MNPITELLDATLWIGGVPILWREIIGNVFGLFSAWAGMRRIVWAWPIGIIGNALLFTVF MGGLFHTPQNLDLYGQAGRQIMFIIVSGYGWYQWSAAKRRALTPENAVAVVPRWAS TKERAGIVIAAVVGTLSFAWIFQALGSWGPWADAWIFVGSILATYGMARGWTEFWLI WIAVDIVGVPLLLTAGYYPSAVLYLVYGAFVSWGFVVWLRVQKADKARALEAQESV TV SEQIDNO:19;DNA;PurinenucleosidaseiunH3;cgl1364 (CGL_RS06810,ncgl1309,cgl543,iunH3) ATGACCACCAAGATCATCCTCGACTGCGATCCAGGACACGACGACGCTGTAGCCA TGCTGCTCGCAGCCGGCAGCCCAGAAATTGAACTGCTTGGAATCACCACGGTCGG CGGCAACCAGACCTTGGACAAGGTCACCCACAATACGCAGGTCGTAGCCACCATC GCTGATATCAATGCGCCCATCTACCGCGGTGTCACCCGACCATTGGTGCGCCCCGT TGAGGTAGCCGAAGATATCCACGGCGATACCGGCATGGAAATCCACAAGTACGAA CTGCCTGAACCAACCAAGCAGGTAGAAGACACCCACGCGGTGGATTTCATCATCG ATACCATCATGAATAACGAGCCCGGCAGCGTAGCGCTGGTTCCCACCGGACCACT GACCAACATCGCGCTGGCAGTCCGGAAAGAACCACGCATCGCCGAGCGAGTCAAG GAAGTTGTCCTCATGGGGGGGGCTACCACGTAGGAAACTGGACCGCCGTAGCTG AATTCAACATCAAGATCGACCCCGAAGCAGCCCACATCGTATTCAACGAAAAGTG GCCACTGACTATGGTCGGCCTCGACCTTACCCACCAGGCGCTCGCAACACCTGAG ATCGAAGCCAAGTTCAACGAGCTGGGCACCGACGTCGCCGACTTCGTCGTCGCGC TTTTCGACGCTTTCCGCAAGAATTACCAGGACGCACAGGGTTTTGATAACCCACCA GTACACGACCCTTGTGCTGTTGCATACCTTGTTGACCCAACCGTATTCACCACCCG CAAAGCACCACTCGATGTGGAGCTGTACGGCGCACTCACCACAGGCATGACCGTT GCTGATTTCCGCGCACCGGCTCCAGCAGATTGCACCACCCAAGTAGCTGTTGACCT GGACTTTGATAAATTCTGGAACATGGTGATCGATGCAGTAAAGCGCATCGGATAG SEQIDNO:20;PRT;PurinenucleosidaseiunH3;cgl1364 (CGL_RS06810,ncgl1309,cg1543,iunH3) MTTKIILDCDPGHDDAVAMLLAAGSPEIELLGITTVGGNQTLDKVTHNTQVVATIADI NAPIYRGVTRPLVRPVEVAEDIHGDTGMEIHKYELPEPTKQVEDTHAVDFIIDTIMNNE PGSVALVPTGPLTNIALAVRKEPRIAERVKEVVLMGGGYHVGNWTAVAEFNIKIDPEA AHIVFNEKWPLTMVGLDLTHQALATPEIEAKFNELGTDVADFVVALFDAFRKNYQDA QGFDNPPVHDPCAVAYLVDPTVFTTRKAPLDVELYGALTTGMTVADFRAPAPADCTT QVAVDLDFDKFWNMVIDAVKRIG SEQIDNO:21;DNA;PurinenucleosidaseiunH2;cgl1977 (cg2168,iunH2,CYL77_RS09970) ATGAGCAAAAAAGCCATCCTTGATATCGACACCGGCATCGATGATGCCCTCGCAC TTGCCTACGCACTGGGCTCACCTGAACTAGAGCTCATTGGTGTCACCACCACCTAC GGTAACGTGCTACTCGAAACCGGTGCAGTCAATGACCTGGCACTGCTTGATCTGTT CGGTGCACCAGAAGTACCTGTGTACTTGGGTGAGCCACACGCACAGACCAAGGAT GGCTTTGAAGTTCTTGAGATCTCCGCGTTCATTCACGGACAAAACGGCATCGGCGA AGTCGAGCTGCCAGCAAGCGAGTCAAAGGCACTCCCCGGCGCAGTGGATTTCCTC ATTGATTCCGTCAACACCCACGGCGATGACCTGGTGATCATCGCAACTGGTCCCAT GACCAACCTGTCTGCGGCAATCGCAAAGGATCCAAGCTTTGCTTCCAAGGCTCAC GTGGTCATCATGGGTGGCGCCTTGACTGTCCCAGGCAACGTCAGCACATGGGCAG AAGCAAACATCAACCAGGACCCAGATGCAGCAAACGATCTGTTCCGTTCCGGTGC AGATGTCACCATGATCGGTCTTGATGTCACCCTGCAGACCCTTCTTACCAAGAAGC ACACTGCGCAGTGGCGCGAACTGGGCACTCCAGCTGCTATCGCACTGGCCGACAT GACTGATTACTACATCAAGGCATATGAGACCACCGCACCACACCTGGGCGGTTGC GGCCTGCACGACCCACTGGCAGTAGGCGTTGCAGTGGACCCAAGCCTGGTCACTT TGCTCCCCATCAACCTCAAGGTAGACATTGAGGGCGAGACCCGTGGACGCACCAT TGGCGATGAAGTCCGCCTCAACGATCCAGTGCGCACCTCCCGCGCAGCTGTCGCC GTAGACGTGGATCGTTTCCTTTCTGAATTCATGACCCGCATCGGCCGAGTCGCAGC ACAGCAGTAA SEQIDNO:22;PRT;PurinenucleosidaseiunH2;cgl1977 (cg2168,iunH2,CYL77_RS09970) MSKKAILDIDTGIDDALALAYALGSPELELIGVTTTYGNVLLETGAVNDLALLDLFGAP EVPVYLGEPHAQTKDGFEVLEISAFIHGQNGIGEVELPASESKALPGAVDFLIDSVNTH GDDLVIIATGPMTNLSAAIAKDPSFASKAHVVIMGGALTVPGNVSTWAEANINQDPDA ANDLFRSGADVTMIGLDVTLQTLLTKKHTAQWRELGTPAAIALADMTDYYIKAYETT APHLGGCGLHDPLAVGVAVDPSLVTLLPINLKVDIEGETRGRTIGDEVRLNDPVRTSR AAVAVDVDRFLSEFMTRIGRVAAQQ SEQIDNo.:23;DNA;PurinenucleosidaseiunH1;cgl2835 (cg3137,iunH1,CYL77_RS14340) ATGATTCCTGTTCTCATCGACTGCGACACCGGCATCGACGACGCC CTCGCCCTGATCTACCTGGTTGCTTTGCATAAACGTGGTGAAATCCAACTTTTTGG AGCAACGACCACCGCAGGAAATGTTGATGTGAAACAAACCGCCTCAATACCAGGT GGGTGTTGGATCAGTGTGGATTAGCGGACATCCCGGTCCTCGCAGGACAACCTGA ACCAAAGCACGTGCCGCTAGTGACTACTCCAGAAACACACGGCGACCATGGCCTT GGTTATATAAACCCAGGTCACGTCGAAATTCCAGAAGGTGACTGGAAGCAGCTGT GGAAAGAACACCTCAGTAACCCAGAAACTAAGCTGATTGTCACCGGGCCCGCCAC CAACCTTGCGGAATTCGGGCCAGTGGAAAACGTCACGCTGATGGGTGGCACCTAC CTTTATCCAGGCAACACCACTCCAACGGCAGAATGGAATACCTGGGTTGATCCAC ACGGAGCTAAAGAAGCATTCGCGGCAGCCCAAAAGCCCATTACGGTGTGTTCCTT GGGCGTGACCGAGCAGTTTACGCTGAACCCGGACATCCTTTCTACACTTATCAACA CGCTTGGCAGCCAACCCATCGCAGAGCATTTACCTGAGATGCTGCGCTTTTACTTT GAATTTCACGAAGTGCAGGGCGAAGGTTACCTTGCTCAAATTCATGACCTGCTGAC CTGCATGATTGCCTTGGATAAAATCCCATTTTCAGGCCGTGAAGTAACCGTGGACG TGGAGGCTGATTCGCCCTTGATGCGTGGCACCACTGTTGCAGATATTCGCGGACAT TGGGGCAAGCCAGCTAACGCATTTCTTGTGGAAACCGCAGACATTGAGGCCGCCC ACGCGGAACTTCTAAGAGCAGTGGAATGA SEQIDNo.:24;PRT;PurinenucleosidaseiunH1;cgl2835 (cg3137,iunH1,CYL77_RS14340) MIPVLIDCDTGIDDALALIYLVALHKRGEIQLFGATTTAGNVDVKQTAINTRWVLDQC GLADIPVLAGQPEPKHVPLVTTPETHGDHGLGYINPGHVEIPEGDWKQLWKEHLSNPE TKLIVTGPATNLAEFGPVENVTLMGGTYLYPGNTTPTAEWNTWVDPHGAKEAFAAA QKPITVCSLGVTEQFTLNPDILSTLINTLGSQPIAEHLPEMLRFYFEFHEVQGEGYLAQI HDLLTCMIALDKIPFSGREVTVDVEADSPLMRGTTVADIRGHWGKPANAFLVETADIE AAHAELLRAVE SEQIDNo.25;DNA;glucose-6Pisomerasepgi;cgl0851 (ncgl0817) ATGGCGGACATTTCGACCACCCAGGTTTGGCAAGACCTGACCGATCATTACTCAA ACTTCCAGGCAACCACTCTGCGTGAACTTTTCAAGGAAGAAAACCGCGCCGAGAA GTACACCTTCTCCGCGGCTGGCCTCCACGTCGACCTGTCGAAGAATCTGCTTGACG ACGCCACCCTCACCAAGCTCCTTGCACTGACCGAAGAATCTGGCCTTCGCGAACGC ATTGACGCGATGTTTGCCGGTGAACACCTCAACAACACCGAAGACCGCGCTGTCC TCCACACCGCGCTGCGCCTTCCTGCCGAAGCTGATCTGTCAGTAGATGGCCAAGAT GTTGCTGCTGATGTCCACGAAGTTTTGGGACGCATGCGTGACTTCGCTACTGCGCT GCGCTCAGGCAACTGGTTGGGACACACCGGCCACACGATCAAGAAGATCGTCAAC ATTGGTATCGGTGGCTCTGACCTCGGACCAGCCATGGCTACGAAGGCTCTGCGTGC ATACGCGACCGCTGGTATCTCAGCAGAATTCGTCTCCAACGTCGACCCAGCAGAC CTCGTTTCTGTGTTGGAAGACCTCGATGCAGAATCCACATTGTTCGTGATCGCTTC GAAAACTTTCACCACCCAGGAGACGCTGTCCAACGCTCGTGCAGCTCGTGCTTGGC TGGTAGAGAAGCTCGGTGAAGAGGCTGTCGCGAAGCACTTCGTCGCAGTGTCCAC CAATGCTGAAAAGGTCGCAGAGTTCGGTATCGACACGGACAACATGTTCGGCTTC TGGGACTGGGTCGGAGGTCGTTACTCCGTGGACTCCGCAGTTGGTCTTTCCCTCAT GGCAGTGATCGGCCCTCGCGACTTCATGCGTTTCCTCGGTGGATTCCACGCGATGG ATGAACACTTCCGCACCACCAAGTTCGAAGAGAACGTTCCAATCTTGATGGCTCTG CTCGGTGTCTGGTACTCCGATTTCTATGGTGCAGAAACCCACGCTGTCCTACCTTA TTCCGAGGATCTCAGCCGTTTTGCTGCTTACCTCCAGCAGCTGACCATGGAATCAA ATGGCAAGTCAGTCCACCGCGACGGCTCCCCTGTTTCCACTGGCACTGGCGAAATT TACTGGGGTGAGCCTGGCACAAATGGCCAGCACGCTTTCTTCCAGCTGATCCACCA GGGCACTCGCCTTGTTCCAGCTGATTTCATTGGTTTCGCTCGTCCAAAGCAGGATC TTCCTGCCGGTGAGCGCACCATGCATGACCTTTTGATGAGCAACTTCTTCGCACAG ACCAAGGTTTTGGCTTTCGGTAAGAACGCTGAAGAGATCGCTGCGGAAGGTGTCG CACCTGAGCTGGTCAACCACAAGGTCATGCCAGGTAATCGCCCAACCACCACCAT TTTGGCGGAGGAACTTACCCCTTCTATTCTCGGTGCGTTGATCGCTTTGTACGAAC ACATCGTGATGGTTCAGGGCGTGATTTGGGACATCAACTCCTTCGACCAATGGGGT GTTGAACTGGGCAAACAGCAGGCAAATGACCTCGCTCCGGCTGTCTCTGGTGAAG AGGATGTTGACTCGGGAGATTCTTCCACTGATTCACTGATTAAGTGGTACCGCGCA AATAGGTAG SEQIDNo.26;PRT;glucose-6PisomerasePGI;cgl0851 (ncgl0817) MADISTTQVWQDLTDHYSNFQATTLRELFKEENRAEKYTFSAAGLHVDLSKNLLDDA TLTKLLALTEESGLRERIDAMFAGEHLNNTEDRAVLHTALRLPAEADLSVDGQDVAA DVHEVLGRMRDFATALRSGNWLGHTGHTIKKIVNIGIGGSDLGPAMATKALRAYATA GISAEFVSNVDPADLVSVLEDLDAESTLFVIASKTFTTQETLSNARAARAWLVEKLGEE AVAKHFVAVSTNAEKVAEFGIDTDNMFGFWDWVGGRYSVDSAVGLSLMAVIGPRDF MRFLGGFHAMDEHFRTTKFEENVPILMALLGVWYSDFYGAETHAVLPYSEDLSRFAA YLQQLTMESNGKSVHRDGSPVSTGTGEIYWGEPGINGQHAFFQLIHQGTRLVPADFIG FARPKQDLPAGERTMHDLLMSNFFAQTKVLAFGKNABEIAAEGVAPELVNHKVMPG NRPTTTILAEELTPSILGALIALYEHIVMVQGVIWDINSFDQWGVELGKQQANDLAPAV SGEEDVDSGDSSTDSLIKWYRANR SEQIDNo.27;DNA;cgZWFcodon-optimizedmutant(A243T) oftheoftheglucose-6-phosphatedehydrogenasefrom Corynebacteriumglutamicum(cg1778,Cgl1576,NCgl1514) ATGAGTACCAACACCACCCCGTCAAGCTGGACAAATCCATTGCGCGACCCCCAGG ATAAGCGCTTGCCCCGCATCGCAGGACCCTCCGGCATGGTCATTTTTGGGGTGACC GGCGATCTGGCACGCAAGAAACTGCTACCAGCCATCTATGACTTGGCAAATCGCG GCTTACTGCCACCTGGCTTCTCTCTCGTGGGCTATGGTCGCCGTGAATGGTCTAAG GAGGACTTCGAAAAGTACGTTCGTGATGCAGCGTCCGCGGGAGCCCGAACGGAAT TTCGTGAAAACGTCTGGGAACGCCTTGCAGAAGGCATGGAATTTGTCCGCGGAAA TTTTGATGATGACGCCGCATTCGACAACTTGGCGGCGACGCTGAAGCGCATCGAT AAGACGAGAGGCACTGCTGGTAACTGGGCGTACTATCTGTCCATCCCACCGGACT CCTTTACGGCGGTGTGCCACCAGCTAGAGCGTTCCGGCATGGCTGAGTCCACCGA AGAGGCATGGCGCCGAGTGATCATTGAAAAGCCATTCGGGCACAACCTGGAATCG GCACACGAGCTCAACCAACTGGTCAACGCCGTTTTCCCGGAGTCATCAGTGTTTAG AATCGATCACTACCTGGGTAAAGAAACCGTGCAGAATATCCTCGCGCTGCGATTC GCAAATCAACTTTTTGAACCCCTTTGGAACAGCAACTATGTCGATCACGTCCAAAT TACCATGACTGAAGATATTGGCTTGGGAGGACGCGCGGGTTATTATGATGGAATC GGAGCAGCGCGCGACGTCATCCAGAATCACCTCATTCAGCTGTTGGCGCTGGTAG CGATGGAGGAACCCATTAGCTTTGTGCCTGCTCAGCTGCAAGCAGAAAAGATCAA AGTTCTGAGCGCTACCAAACCTTGTTACCCTCTGGATAAGACCTCAGCTCGCGGTC AATATGCTGCTGGCTGGCAAGGATCTGAGCTGGTCAAGGGCCTTCGTGAAGAGGA CGGTTTCAACCCCGAGAGCACCACGGAAACCTTCGCCGCATGTACCCTTGAAATC ACAAGTCGCCGCTGGGCCGGCGTCCCATTCTACCTGCGTACTGGCAAGAGACTCG GCCGACGAGTTACAGAGATCGCTGTTGTGTTTAAAGATGCTCCCCACCAGCCGTTT GATGGAGACATGACCGTTTCCCTTGGCCAAAATGCGATCGTAATTCGCGTACAACC AGACGAGGGTGTTCTTATCCGCTTTGGTTCCAAGGTGCCCGGTTCCGCTATGGAGG TTCGTGACGTTAATATGGACTTCAGCTATAGCGAATCCTTCACCGAAGAGTCACCT GAAGCATACGAACGCCTGATCCTGGATGCCCTCCTGGACGAGTCCAGCTTGTTTCC AACCAACGAGGAAGTGGAACTGTCTTGGAAAATCCTGGACCCAATTCTGGAAGCT TGGGATGCCGATGGCGAACCGGAGGACTACCCAGCTGGGACCTGGGGGCCAAAAT CGGCGGATGAGATGTTATCCCGTAACGGCCACACATGGCGCCGACCTTGA SEQIDNo.28;DNA;cgZWFmutant(A243T)oftheofthe glucose-6-phosphatedehydrogenasefromCorynebacterium glutamicum(cgl778,Cgl1576,NCgl1514) MSTNTTPSSWTNPLRDPQDKRLPRIAGPSGMVIFGVTGDLARKKLLPAIYDLANRGLL PPGFSLVGYGRREWSKEDFEKYVRDAASAGARTEFRENVWERLAEGMEFVRGNFDD DAAFDNLAATLKRIDKTRGTAGNWAYYLSIPPDSFTAVCHQLERSGMAESTEEAWRR VIIEKPFGHNLESAHELNQLVNAVFPESSVFRIDHYLGKETVQNILALRFANQLFEPLW NSNYVDHVQITMAEDIGLGGRAGYYDGIGAARDVIQNHLIQLLALVAMEEPISFVPAQ LQAEKIKVLSATKPCYPLDKTSARGQYAAGWQGSELVKGLREEDGENPESTTETFAA CTLEITSRRWAGVPFYLRTGKRLGRRVTEIAVVFKDAPHQPFDGDMTVSLGQNAIVIR VQPDEGVLIRF GSKVPGSAMEVRDVNMDFSYSESFTEESPEAYERLILDALLDESSLFPTNEEVELSWKI LDPILEAWDADGEPEDYPAGTWGPKSADEMLSRNGHTWRRP* SEQIDNo.29;DNA;glucose-6-phosphatedehydrogenaseof Leuconostonmesenteroides,codonoptimizedmutant(R46E/Q47E) ofthegenehavingaccessionnumberM64446.1;lmZWF ATGGTTTCCGAAATAAAGACCCTCGTTACTTTCTTTGGCGGCACCGGTGACCTTGC AAAACGCAAGCTCTACCCCTCTGTATTCAACCTGTACAAAAAAGGGTATCTGCAA AAACACTTCGCCATTGTTGGTACCGCTGAAGAGGCGCTAAACGACGACGAGTTCA AACAGCTTGTCCGTGATTCCATTAAAGACTTCACCGATGACCAAGCCCAGGCAGA GGCCTTCATCGAACATTTTTCTTATCGAGCACACGATGTGACCGATGCCGCATCGT ATGCAGTCCTGAAGGAAGCGATCGAGGAGGCGGCCGATAAGTTCGATATTGACGG TAACCGCATATTTTACATGTCGGTGGCACCACGCTTCTTCGGTACCATCGCTAAAT ATCTGAAGTCCGAGGGTCTGCTTGCTGATACTGGCTACAATCGGCTGATGATTGAA AAGCCTTTTGGAACCTCTTATGACACAGCCGCAGAACTACAGAATGACTTGGAGA ACGCTTTCGATGATAATCAGCTTTTCCGTATCGATCATTATCTGGGTAAAGAAATG GTCCAGAATATCGCAGCTCTGCGCTTCGGAAACCCTATATTCGACGCTGCGTGGAA CAAGGATTACATCAAGAACGTTCAAGTTACACTCTCCGAAGTGTTGGGGGTTGAA GAGCGAGCCGGCTACTATGACACCGCCGGAGCTCTACTTGATATGATCCAGAACC ACACGATGCAGATCGTTGGCTGGCTCGCCATGGAAAAACCAGAGTCCTTCACCGA TAAAGACATCCGCGCGGCTAAGAACGCCGCTTTTAATGCCCTGAAAATCTACGAC GAGGCTGAAGTGAACAAATATTTTGTGCGTGCCCAATATGGTGCTGGAGATTCTGC CGATTTCAAACCTTACTTAGAGGAACTCGATGTCCCAGCAGATTCCAAAAACAAC ACCTTCATTGCAGGCGAATTACAGTTTGATCTTCCACGTTGGGAAGGTGTGCCTTT TTACGTGCGCAGCGGTAAACGACTCGCAGCCAAACAGACTCGCGTCGATATTGTTT TCAAGGCTGGCACCTTTAATTTCGGGTCTGAACAGGAAGCTCAAGAGGCCGTTCTG TCCATCATCATTGATCCGAAGGGAGCAATCGAACTGAAGCTCAATGCAAAATCTG TGGAAGATGCTTTCAACACCCGCACTATCGACTTGGGCTGGACCGTGAGCGATGA GGACAAGAAAAATACGCCAGAACCTTATGAAAGGATGATCCACGATACGATGAA CGGTGACGGCAGCAACTTCGCAGATTGGAATGGCGTGAGCATTGCGTGGAAGTTC GTAGATGCTATTTCTGCGGTATATACGGCCGATAAGGCCCCGCTTGAAACCTACAA GTCGGGCTCCATGGGACCCGAAGCCAGCGACAAATTGCTCGCCGCAAACGGAGAT GCATGGGTATTCAAGGGGTAG SEQIDNo.30;PRT;mutant(R46E/Q47E)oftheglucose-6- phosphatedehydrogenaseofLeuconoston.mesenteroides;lmZWF MVSEIKTLVTFFGGTGDLAKRKLYPSVFNLYKKGYLQKHFAIVGTAEEALNDDEFKQ LVRDSIKDFTDDQAQAEAFIEHFSYRAHDVTDAASYAVLKEAIEEAADKFDIDGNRIFY MSVAPRFFGTIAKYLKSEGLLADTGYNRLMIEKPFGTSYDTAAELQNDLENAFDDNQL FRIDHYLGKEMVQNIAALRFGNPIFDAAWNKDYIKNVQVTLSEVLGVEERAGYYDTA GALLDMIQNHTMQIVGWLAMEKPESFTDKDIRAAKNAAFNALKIYDEAEVNKYFVR AQYGAGDSADFKPYLEELDVPADSKNNTFIAGELQFDLPRWEGVPFYVRSGKRLAAK QTRVDIVFKAGTFNFGSEQEAQEAVLSIIIDPKGAIELKLNAKSVEDAFNTRTIDLGWT VSDEDKKNTPEPYERMIHDTMNGDGSNFADWNGVSIAWKFVDAISAVYTADKAPLE TYKSGSMGPEASDKLLAANGDAWVFKG SEQIDNo.31;DNA;Codonoptimizedvariantoftheglucose- 6-phosphatedehydrogenasefromZymomonasmobilisstrain ATCC10988(Zmob_0908) ATGACTAATACTGTTTCTACCATGATCCTTTTCGGCAGCACCGGAGATCTCTCGCA GCGCATGCTTCTTCCCTCGCTGTACGGGCTGGATGCAGACGGTCTACTCGCCGACG ACCTCCGCATTGTGTGTACCTCTCGTTCCGAGTACGATACCGACGGATTTCGTGAT TTTGCTGAGAAGGCACTGGACCGTTTCGTTGCCTCCGACAGACTTAATGATGATGC AAAAGCGAAGTTCCTCAACAAGCTTTTCTACGCAACGGTTGACATCACCGATCCA ACCCAATTTGGAAAGCTCGCAGACCTCTGCGGTCCAGTCGAAAAGGGCATTGCAA TCTACCTTTCCACAGCACCATCCTTGTTCGAAGGCGCAATTGCTGGCTTGAAACAG GCGGGCCTGGCCGGCCCGACCTCCCGCCTTGCATTGGAAAAGCCCTTGGGTCAAG ATCTTGCTTCCTCTGATCACATCAACGACGCAGTGCTGAAGGTTTTTTCCGAAAAA CAAGTATACCGTATCGACCACTATCTTGGGAAAGAAACCGTCCAGAATCTCCTAA CACTCCGCTTTGGAAATGCATTGTTCGAGCCGTTGTGGAACTCAAAGGGGATTGAC CACGTGCAGATCTCCGTCGCTGAGACAGTGGGACTCGAAGGACGCATCGGCTACT TTGACGGCTCCGGCTCCCTGCGAGACATGGTGCAGTCTCACATCCTGCAATTGGTT GCCCTTGTAGCTATGGAGCCCCCGGCTCACATGGAAGCAAACGCGGTCCGCGACG AAAAGGTTAAGGTGTTCCGTGCACTTCGTCCCATTAACAACGACACTGTTTTCACA CACACCGTGACTGGCCAATACGGCGCCGGCGTGTCGGGGGGAAAGGAAGTTGCAG GCTACATCGATGAGCTTGGACAACCGAGTGATACTGAAACCTTTGTTGCAATTAAA GCACACGTGGATAACTGGCGCTGGCAGGGAGTTCCCTTCTACATCCGCACTGGTA AACGGCTCCCTGCCCGCCGTTCAGAGATCGTCGTTCAGTTCAAACCAGTTCCCCAC TCCATTTTTTCAAGCTCAGGAGGAATCCTTCAGCCTAATAAATTGCGCATTGTCCT GCAACCAGACGAAACCATCCAAATCTCAATGATGGTCAAGGAACCAGGTCTTGAC AGAAATGGTGCACACATGCGTGAGGTCTGGCTGGATCTCTCTTTGACCGACGTGTT CAAAGATCGAAAGCGCCGGATTGCTTACGAGCGCCTTATGCTCGATCTGATTGAG GGTGACGCAACCCTCTTCGTGCGCCGCGACGAGGTCGAGGCACAGTGGGTTTGGA TCGACGGTATCCGGGAAGGCTGGAAGGCTAATAGCATGAAGCCTAAAACCTATGT CTCCGGCACCTGGGGACCCTCCACCGCTATTGCATTGGCAGAGCGCGATGGCGTC ACCTGGTACGACTAA SEQIDNo.32;PRT;codon-optimizedvariantoftheglu6- phosphatedehydrogenasefromZymomonasmobilisstrain ATCC10988(NCBI-ProteinID:AEH62743.1) MTNTVSTMILFGSTGDLSQRMLLPSLYGLDADGLLADDLRIVCTSRSEYDTDGFRDFA EKALDRFVASDRLNDDAKAKFLNKLFYATVDITDPTQFGKLADLCGPVEKGIAIYLST APSLFEGAIAGLKQAGLAGPTSRLALEKPLGQDLASSDHINDAVLKVFSEKQVYRIDH YLGKETVQNLLTLRFGNALFEPLWNSKGIDHVQISVAETVGLEGRIGYFDGSGSLRDM VQSHILQLVALVAMEPPAHMEANAVRDEKVKVFRALRPINNDTVFTHTVTGQYGAG VSGGKEVAGYIDELGQPSDTETFVAIKAHVDNWRWQGVPFYIRTGKRLPARRSEIVVQ FKPVPHSIFSSSGGILQPNKLRIVLQPDETIQISMMVKEPGLDRNGAHMREVWLDLSLT DVFKDRKRRIAYERLMLDLIEGDATLFVRRDEVEAQWVWIDGIREGWKANSMKPKT YVSGTWGPSTAIALAERDGVTWYD* SEQIDNo.33;DNA;solublePyridineNucleotideTranshydrogenase fromEscherichiacoli;udhA(NP_418397.2,EG11428) ATGCCACATTCCTACGATTACGATGCCATAGTAATAGGTTCCGGCCCCGGCGGCGA AGGCGCTGCAATGGGCCTGGTTAAGCAAGGTGCGCGCGTCGCAGTTATCGAGCGT TATCAAAATGTTGGCGGCGGTTGCACCCACTGGGGCACCATCCCGTCGAAAGCTCT CCGTCACGCCGTCAGCCGCATTATAGAATTCAATCAAAACCCACTTTACAGCGACC ATTCCCGACTGCTCCGCTCTTCTTTTGCCGATATCCTTAACCATGCCGATAACGTGA TTAATCAACAAACGCGCATGCGTCAGGGATTTTACGAACGTAATCACTGTGAAAT ATTGCAGGGAAACGCTCGCTTTGTTGACGAGCATACGTTGGCGCTGGATTGCCTGG ACGGCAGCGTTGAAACACTAACCGCTGAAAAATTTGTTATTGCCTGCGGCTCTCGT CCATATCATCCAACAGATGTTGATTTCACCCATCCACGCATTTACGACAGCGACTC AATTCTCAGCATGCACCACGAACCGCGCCATGTACTTATCTATGGTGCTGGAGTGA TCGGCTGTGAATATGCGTCGATCTTCCGCGGTATGGATGTAAAAGTGGATCTGATC AACACCCGCGATCGCCTGCTGGCATTTCTCGATCAAGAGATGTCAGATTCTCTCTC CTATCACTTCTGGAACAGTGGCGTAGTGATTCGTCACAACGAAGAGTACGAGAAG ATCGAAGGCTGTGACGATGGTGTGATCATGCATCTGAAGTCGGGTAAAAAACTGA AAGCTGACTGCCTGCTCTATGCCAACGGTCGCACCGGTAATACCGATTCGCTGGCG TTACAGAACATTGGGCTAGAAACTGACAGCCGCGGACAGCTGAAGGTCAACAGCA TGTATCAGACCGCACAGCCACACGTTTACGCGGTGGGCGACGTGATTGGTTATCCG AGCCTGGCGTCGGCGGCCTATGACCAGGGGCGCATTGCCGCGCAGGCGCTGGTAA AAGGCGAAGCCACCGCACATCTGATTGAAGATATCCCTACCGGTATTTACACCATC CCGGAAATCAGCTCTGTGGGCAAAACCGAACAGCAGCTGACCGCAATGAAAGTGC CATATGAAGTGGGCCGCGCCCAGTTTAAACATCTGGCACGCGCACAAATCGTCGG CATGAACGTGGGCACGCTGAAAATTTTGTTCCATCGGGAAACAAAAGAGATTCTG GGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGTCAGGCGAT TATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACCACCTTT AACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTAAACC GCCTGTTTTAA SEQIDNo.34;PRT;solublePyridineNucleotideTranshydrogenase fromEscherichiacoli;udhA(AAC76944) MPHSYDYDAIVIGSGPGGEGAAMGLVKQGARVAVIERYQNVGGGCTHWGTIPSKAL RHAVSRIIEFNQNPLYSDHSRLLRSSFADILNHADNVINQQTRMRQGFYERNHCEILQG NARFVDEHTLALDCLDGSVETLTAEKFVIACGSRPYHPTDVDFTHPRIYDSDSILSMHH EPRHVLIYGAGVIGCEYASIFRGMDVKVDLINTRDRLLAFLDQEMSDSLSYHFWNSGV VIRHNEEYEKIEGCDDGVIMHLKSGKKLKADCLLYANGRTGNTDSLALQNIGLETDSR GQLKVNSMYQTAQPHVYAVGDVIGYPSLASAAYDQGRIAAQALVKGEATAHLIEDIP TGIYTIPEISSVGKTEQQLTAMKVPYEVGRAQFKHLARAQIVGMNVGTLKILFHRETKE ILGIHCFGERAAEIIHIGQAIMEQKGGGNTIEYFVNTTFNYPTMAEAYRVAALNGLNRL F SEQIDNo.35;DNA;AsubunitofMembraneboundPyridine NucleotideTranshydrogenase(pnt)fromEscherichiacoli MG1655;ECK1598(variantg1342a,NP_416120.1) ATGCCACATTCCTACGATTACGATGCCATAGTAATAGGTTCCGGCCCCGGCGGCGA AGGCGCTGCAATGGGCCTGGTTAAGCAAGGTGCGCGCGTCGCAGTTATCGAGCGT TATCAAAATGTTGGCGGCGGTTGCACCCACTGGGGCACCATCCCGTCGAAAGCTCT CCGTCACGCCGTCAGCCGCATTATAGAATTCAATCAAAACCCACTTTACAGCGACC ATTCCCGACTGCTCCGCTCTTCTTTTGCCGATATCCTTAACCATGCCGATAACGTGA TTAATCAACAAACGCGCATGCGTCAGGGATTTTACGAACGTAATCACTGTGAAAT ATTGCAGGGAAACGCTCGCTTTGTTGACGAGCATACGTTGGCGCTGGATTGCCTGG ACGGCAGCGTTGAAACACTAACCGCTGAAAAATTTGTTATTGCCTGCGGCTCTCGT CCATATCATCCAACAGATGTTGATTTCACCCATCCACGCATTTACGACAGCGACTC AATTCTCAGCATGCACCACGAACCGCGCCATGTACTTATCTATGGTGCTGGAGTGA TCGGCTGTGAATATGCGTCGATCTTCCGCGGTATGGATGTAAAAGTGGATCTGATC AACACCCGCGATCGCCTGCTGGCATTTCTCGATCAAGAGATGTCAGATTCTCTCTC CTATCACTTCTGGAACAGTGGCGTAGTGATTCGTCACAACGAAGAGTACGAGAAG ATCGAAGGCTGTGACGATGGTGTGATCATGCATCTGAAGTCGGGTAAAAAACTGA AAGCTGACTGCCTGCTCTATGCCAACGGTCGCACCGGTAATACCGATTCGCTGGCG TTACAGAACATTGGGCTAGAAACTGACAGCCGCGGACAGCTGAAGGTCAACAGCA TGTATCAGACCGCACAGCCACACGTTTACGCGGTGGGCGACGTGATTGGTTATCCG AGCCTGGCGTCGGCGGCCTATGACCAGGGGCGCATTGCCGCGCAGGCGCTGGTAA AAGGCGAAGCCACCGCACATCTGATTGAAGATATCCCTACCGGTATTTACACCATC CCGGAAATCAGCTCTGTGGGCAAAACCGAACAGCAGCTGACCGCAATGAAAGTGC CATATGAAGTGGGCCGCGCCCAGTTTAAACATCTGGCACGCGCACAAATCGTCGG CATGAACGTGGGCACGCTGAAAATTTTGTTCCATCGGGAAACAAAAGAGATTCTG GGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGTCAGGCGAT TATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACCACCTTT AACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTAAACC GCCTGTTTTAA SEQIDNo.36;PRT;AsubunitofMembraneboundPyridine NucleotideTranshydrogenase(pnt)fromEscherichiacoli; P07001.2(A434Tvariant) MRIGIPRERLTNETRVAATPKTVEQLLKLGFTVAVESGAGQLASFDDKAFVQAGAEIV EGNSVWQSEIILKVNAPLDDEIALLNPGTTLVSFIWPAQNPELMQKLAERNVTVMAM DSVPRISRAQSLDALSSMANIAGYRAIVEAAHEFGRFFTGQITAAGKVPPAKVMVIGA GVAGLAAIGAANSLGAIVRAFDTRPEVKEQVQSMGAEFLELDFKEEAGSGDGYAKV MSDAFIKAEMELFAAQAKEVDIIVTTALIPGKPAPKLITREMVDSMKAGSVIVDLAAQ NGGNCEYTVPGEIFTTENGVKVIGYTDLPGRLPTQSSQLYGTNLVNLLKLLCKEKDGN ITVDFDDVVIRGVTVIRAGEITWPAPPIQVSAQPQAAQKAAPEVKTEEKCTCSPWRKY ALMALAIILFGWMASVAPKEFLGHFTVFALTCVVGYYVVWNVSHALHTPLMSVTNAI SGIIVVGALLQIGQGGWVSFLSFIAVLIASINIFGGFTVTQRMLKMFRKN SEQIDNo.37;DNA;BsubunitofMembraneboundPyridine NucleotideTranshydrogenase(pntB)fromEscherichiacoli (MG1655;ECK1597;NP_416119.1) ATGTCTGGAGGATTAGTTACAGCTGCATACATTGTTGCCGCGATCCTGTTTATCTTC AGTCTGGCCGGTCTTTCGAAACATGAAACGTCTCGCCAGGGTAACAACTTCGGTAT CGCCGGGATGGCGATTGCGTTAATCGCAACCATTTTTGGACCGGATACGGGTAAT GTTGGCTGGATCTTGCTGGCGATGGTCATTGGTGGGGCAATTGGTATCCGTCTGGC GAAGAAAGTTGAAATGACCGAAATGCCAGAACTGGTGGCGATCCTGCATAGCTTC GTGGGTCTGGCGGCAGTGCTGGTTGGCTTTAACAGCTATCTGCATCATGACGCGGG AATGGCACCGATTCTGGTCAATATTCACCTGACGGAAGTGTTCCTCGGTATCTTCA TCGGGGCGGTAACGTTCACGGGTTCGGTGGTGGCGTTCGGCAAACTGTGTGGCAA GATTTCGTCTAAACCATTGATGCTGCCAAACCGTCACAAAATGAACCTGGCGGCTC TGGTCGTTTCCTTCCTGCTGCTGATTGTATTTGTTCGCACGGACAGCGTCGGCCTGC AAGTGCTGGCATTGCTGATAATGACCGCAATTGCGCTGGTATTCGGCTGGCATTTA GTCGCCTCCATCGGTGGTGCAGATATGCCAGTGGTGGTGTCGATGCTGAACTCGTA CTCCGGCTGGGCGGCTGCGGCTGCGGGCTTTATGCTCAGCAACGACCTGCTGATTG TGACCGGTGCGCTGGTCGGTTCTTCGGGGGCTATCCTTTCTTACATTATGTGTAAG GCGATGAACCGTTCCTTTATCAGCGTTATTGCGGGTGGTTTCGGCACCGACGGCTC TTCTACTGGCGATGATCAGGAAGTGGGTGAGCACCGCGAAATCACCGCAGAAGAG ACAGCGGAACTGCTGAAAAACTCCCATTCAGTGATCATTACTCCGGGGTACGGCA TGGCAGTCGCGCAGGCGCAATATCCTGTCGCTGAAATTACTGAGAAATTGCGCGC TCGTGGTATTAATGTGCGTTTCGGTATCCACCCGGTCGCGGGGCGTTTGCCTGGAC ATATGAACGTATTGCTGGCTGAAGCAAAAGTACCGTATGACATCGTGCTGGAAAT GGACGAGATCAATGATGACTTTGCTGATACCGATACCGTACTGGTGATTGGTGCTA ACGATACGGTTAACCCGGCGGCGCAGGATGATCCGAAGAGTCCGATTGCTGGTAT GCCTGTGCTGGAAGTGTGGAAAGCGCAGAACGTGATTGTCTTTAAACGTTCGATG AACACTGGCTATGCTGGTGTGCAAAACCCGCTGTTCTTCAAGGAAAACACCCACA TGCTGTTTGGTGACGCCAAAGCCAGCGTGGATGCAATCCTGAAAGCTCTGTAA SEQIDNo.38;BsubunitofMembraneboundPyridine NucleotideTranshydrogenase(pntB)fromEscherichiacoli; P0AB69.1 MSGGLVTAAYIVAAILFIFSLAGLSKHETSRQGNNFGIAGMAIALIATIFGPDTGNVGWI LLAMVIGGAIGIRLAKKVEMTEMPELVAILHSFVGLAAVLVGFNSYLHHDAGMAPILV NIHLTEVFLGIFIGAVTFTGSVVAFGKLCGKISSKPLMLPNRHKMNLAALVVSFLLLIVF VRTDSVGLQVLALLIMTAIALVFGWHLVASIGGADMPVVVSMLNSYSGWAAAAAGF MLSNDLLIVTGALVGSSGAILSYIMCKAMNRSFISVIAGGFGTDGSSTGDDQEVGEHRE ITAEETAELLKNSHSVIITPGYGMAVAQAQYPVAEITEKLRARGINVRFGIHPVAGRLP GHMNVLLAEAKVPYDIVLEMDEINDDFADTDTVLVIGANDTVNPAAQDDPKSPIAGM PVLEVWKAQNVIVFKRSMNTGYAGVQNPLFFKENTHMLFGDAKASVDAILKAL