Production of NMN and its derivatives via microbial processes

Abstract

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

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 020052, filed May 5, 2020, under 35 U.S.C. § 119(e), and entitled “PRODUCTION OF NMN AND ITS DERIVATIVES VIA MICROBIAL PROCESSES,” the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The field of the invention relates to the production of nicotinamide mononucleotide (NMN) and its derivatives, such as nicotinamide riboside (NR) and nicotinamide adenine dinucleotide (NAD), via microbial processes. [Background technology]

[0003] In recent years, nicotinamide adenine dinucleotide (NAD) has evolved from a simple metabolic cofactor to a therapeutic agent in healthcare applications (Katsyuba et al. 2020 - NAD + homeostasis in health and disease. Nature Metabolism 2: 9-31). Similarly, two other compounds, both closely related to NAD, namely nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), are currently recognized as dietary supplements with potential for anti-aging and longevity applications. NMN and NR are currently commercially produced using chemical methods, and there is a need to develop bioprocessing technologies to manufacture these compounds on a commercial scale in a cost-effective manner.

[0004] The bacterium Corynebacterium stathionis (ATCC6872 and variously described as Brevibacterium ammoniagenes, Corynebacterium ammoniagenes and Brevibacterium stathionis) has been found to be a prolific producer of NMN and related compounds when grown under restrictive conditions. Exemplary restrictive conditions include manganese starvation, addition of specific chemical inhibitors, and cultivation at elevated temperatures with specific temperature-sensitive mutants. This stress-induced microbial production of NMN includes nicotinic acid mononucleotide (NAMN), a compound related to NMN, when cells are grown under restrictive conditions and fed either nicotinic acid (NA) or nicotinamide (Nam). There remains a need in the art for a method of producing NMN that increases the amount of NMN produced while minimizing or eliminating the formation of the by-product NAMN. Summary of the Invention

[0005] The present invention addresses the above needs by providing Corynebacterium glutamicum with genetic modifications that enable it to produce nicotinamide mononucleotide (NMN) instead of nicotinic acid mononucleotide (NAMN), as well as NMN-derived molecules such as NR and NAD.

[0006] The present invention relates to engineered host cells with genetic modifications that allow the production of NMN and its derivatives NR and NAD.In this invention, a method for genetically engineering microbial host cells that can selectively produce either NMN or NR or NAD is also provided.Feedstocks useful for the production of NMN, NR and NAD according to the present invention include nicotinamide (Nam) and nicotinic acid (NA).In a preferred embodiment of the present invention, Nam is used as the feedstock for the production of NMN, NAR or NAD.

[0007] Microbial host cells useful for the production of NMN, NR and / or NAD according to the present invention include, but are not limited to, microbial cells such as bacterial cells, fungal cells and yeast cells that are suitable for genetic modification. In one embodiment of the present invention, Corynebacterium glutamicum is used as a preferred microbial host cell. In a preferred embodiment of the present invention, a Corynebacterium glutamicum cell useful for the present invention is first subjected to a genetic modification that alters its restriction-modification system, making it suitable for other genetic modifications that allow the bacterial cell to be used to produce NMN, NR and NAD using Nam or NA as a feedstock. The engineered host cell, for example, Corynebacterium glutamicum, can include a genetic modification to introduce a ribonucleotide reductase, for example, a ribonucleotide reductase encoded by the NrdHIEJ operon, that is conditionally active to block cell division and biomass accumulation without affecting protein synthesis and normal metabolic activity.

[0008] In one embodiment of the present invention, genetic modifications suitable for the production of NMN, NR and / or NAD include, but are not limited to, the introduction of exogenous genes that express enzyme proteins not naturally present in the microbial host cell, increase the relative expression of endogenous genes encoding enzyme proteins leading to increased activity of the corresponding enzyme proteins, and / or reduce or completely eliminate the expression of endogenous genes encoding enzyme proteins causing reduced or completely eliminated activity of the corresponding enzyme proteins.

[0009] In one aspect of the invention, when an engineered microbial host cell lacks an endogenous gene encoding an enzyme capable of converting Nam to NMN, an exogenous gene, nadV, encoding a nicotinamide phosphoribosyltransferase enzyme is introduced into the engineered microbial host cell. In one exemplary embodiment of the invention, sources of the nadV gene encoding a nicotinamide phosphoribosyltransferase enzyme include, but are not limited to, Stenotrophomonas maltophilia, Chromobacterium violaceum, Deinococcus radiodurans, Synechocystis sp. PCC6803, Pseudonocardia dioxanivorans CB1190, Shewanella oneidensis MR-1, and Ralstonia solanacearum GMI1000. In a most preferred aspect of the invention, a nadV gene from any one of the above microbial species is isolated and introduced into a strain of Corynebacterium glutamicum using one or more genetic engineering techniques well known in the art. In a preferred embodiment, the foreign gene nadV is codon-optimized prior to its introduction into the engineered microbial host cell to ensure optimal expression of the nicotinamide phosphoribosyltransferase enzyme in the microbial host cell. The foreign gene in the Corynebacterium glutamicum cell may be under an inducible promoter, such as the lacZ promoter, and can be induced using lactose or IPTG.

[0010] In another aspect of the invention, the NAMPT gene from Haemophilus ducreyi is introduced into engineered microbial host cells as a source of nicotinamide phosphoribosyltransferase enzyme activity. In yet another aspect of this embodiment, in those microbial host cells that already have an endogenous gene encoding the nicotinamide phosphoribosyltransferase enzyme, the activity of this enzyme can be further enhanced by introducing an exogenous nadV gene (optionally codon-optimized and obtained from any of the sources listed in the entire paragraph) or an optionally codon-optimized NAMPT gene from Haemophilus ducreyi.

[0011] In another aspect of this embodiment, the microbial host cell selected for producing NMN using Nam as a feedstock and having a codon-optimized exogenous gene encoding a nicotinamide phosphoribosyltransferase enzyme further includes additional genetic modifications to ensure the availability of phosphoribosyl pyrophosphate (also known as PRPP, 5-phospho-ribose 1-diphosphate), which serves as a co-substrate in the enzymatic reaction leading to the conversion of Nam to NMN. Additional genetic modifications include the introduction of an exogenous gene, such as prsA, which encodes phosphoribosyl pyrophosphate synthetase, or improving the performance of the endogenous prsA gene by increasing its expression or improving the performance of the phosphoribosyl pyrophosphate synthetase enzyme PrsA encoded by the prsA gene. In a preferred aspect of this invention, a prsA gene encoding a feedback-resistant phosphoribosyl pyrophosphate synthetase enzyme is used.

[0012] In another embodiment of this invention, other pathways that use the PRPP pool in the cell are blocked as a way to preserve PRPP, which serves as a co-substrate in the production of NMN from Nam. In one aspect of the invention, the pyrE gene is mutated, leading to the inactivation of the orotate phosphoribosyltransferase enzyme, which is responsible for catalyzing the transfer of the ribosyl phosphate group from PRPP to orotate (leading to the formation of orotidine).

[0013] In another embodiment of the present invention, a microbial host cell selected for producing NMN using Nam as a feedstock and harboring a codon-optimized exogenous gene encoding a nicotinamide phosphoribosyltransferase enzyme further comprises additional genetic modifications that ensure conversion of most Nam to produce NMN, while blocking other biochemical pathways for Nam utilization, such as conversion of Nam to nicotinic acid. In one aspect of this embodiment, the pncA gene is mutated to delete the nicotinamidase enzyme function involved in the deamidation of Nam to nicotinic acid (NA).

[0014] In another embodiment of the present invention, the microbial host cell selected for producing NMN using Nam as a feedstock and having a codon-optimized exogenous gene encoding a nicotinamide phosphoribosyltransferase enzyme further comprises an additional genetic modification to ensure the storage of NMN produced within the cell using Nam as a feedstock, the additional genetic modification blocking other biochemical pathways that consume NMN. In one aspect of this embodiment, the ushA gene is deleted, which encodes an enzyme capable of converting NMN to NR. In another aspect of this embodiment, the pncC gene is deleted, resulting in the loss of nicotinamide-nucleotide amidohydrolase enzyme activity capable of converting NMN to nicotinic acid mononucleotide (NAMN). In yet another aspect of this invention, the genes cgl1364, cgl1977 and cgl2835 are deleted, leading to the elimination of putative nucleosidases capable of converting NMN to Nam and ribose-5-phosphate. In another embodiment of the invention, the gene nadD is genetically modified such that the activity of the enzyme capable of converting NMN to NAD is blocked.

[0015] In another aspect of this embodiment, the microbial host cell selected for producing NMN using Nam as a feedstock and having a codon-optimized exogenous gene encoding nicotinamide phosphoribosyltransferase enzyme further comprises an additional genetic modification, resulting in knockout or impairment of the pgi gene, thereby redirecting D-glucose-6-phosphate from glycolysis to the pentose phosphate pathway (PPP) and increasing PRPP availability.However, it has been found that the deletion of pgi causes growth retardation in some microbial species in certain media.To at least partially restore normal growth, the microbial host cell can further be genetically modified to express a heterologous ZWF enzyme that has a higher ability to overcome feedback inhibition relative to the native ZWF of the microbial host cell.

[0016] In non-limiting example embodiments, the transgenic ZWF enzyme may be: (i) a feedback resistant mutant polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the amino acid sequence set forth in SEQ ID NO:28; (ii) a Leuconostoc mesenteroides mutant polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the amino acid sequence set forth in SEQ ID NO:30; or (iii) a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the Zymomonas mobilis (zmZWF) amino acid sequence set forth in SEQ ID NO:32.

[0017] It has also been found that in microbial species lacking a native pyridine nucleotide transhydrogenase, growth can also be rescued by overcoming NADPH accumulation. This can be further achieved by genetically modifying the host cell to contain one or more soluble or membrane-bound transhydrogenases. In a non-limiting example, UdhA from E. coli is a soluble transhydrogenase that has been reported to favor the reduction of NAD+ by NADPH, and PntAB from E. coli is a membrane-bound transhydrogenase that has been reported to favor the reduction of NADP+ by NADH.

[0018] In one exemplary embodiment, the recombinant ZWF and transhydrogenase are paired in a cgDVS expression vector.

[0019] In various embodiments of the present invention, the microbial host cell selected for producing NMN using Nam as a feedstock and having a codon-optimized exogenous gene encoding a nicotinamide phosphoribosyltransferase enzyme can include any combination of the various genetic modifications described in the previous paragraph. Thus, the microbial host cell according to the present invention can include, for example, at least one, at least two, at least three, at least four, or at least five genetic modifications described in the previous paragraph.

[0020] In a further 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 encoding a nicotinamide phosphoribosyltransferase enzyme further comprises an additional genetic modification in the nadD gene, resulting in an upregulated NAD(+) synthetase enzyme involved in the conversion of NMN to NAD. In yet another aspect of this embodiment, the gene nudC is mutated so that the enzyme involved in the conversion of NAD back to NMN is blocked.

[0021] 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 encoding a nicotinamide phosphoribosyltransferase enzyme further comprises a functionally active dephosphorylation enzyme encoded by the ushA gene. In another aspect of this invention, the ushA gene is genetically modified to obtain a dephosphorylation enzyme with enhanced activity for the conversion of NMN to NR. In yet another aspect of this invention, the genes cgl1364, cgl1977 and cgl2835 are deleted, leading to the elimination of purine nucleosidases involved in the conversion of NR to NA and ribose.

[0022] Accordingly, in one aspect, the present invention provides a method for producing NMN, such method comprising: feeding Nam to a culture of a genetically modified Corynebacterium glutamicum strain, said strain comprising at least one genetic modification selected from the group consisting of: (a) heterologous expression of a nadV gene encoding a nicotinamide phosphoribosyltransferase capable of converting Nam to NMN; (b) deletion or modification of one or more genes capable of enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of enzymatic conversion of NMN to NR; (d) deletion or modification of one or more genes capable of producing PRPP. (e) modification or deletion of a biochemical pathway requiring PRPP other than the biochemical pathway for conversion of Nam to NMN; (f) deletion or modification of a gene capable of enzymatic conversion of NMN to NAMN; (g) deletion or modification of a gene capable of conversion of NMN to NAD; (h) deletion or modification of one or more genes encoding NMN nucleosidase capable of 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 combination thereof. In various embodiments, the genetically modified strain having at least one of the modifications produces increased amounts of NMN compared to a strain having none of the modifications.

[0023] Thus, in another aspect, the present invention provides a method for producing NR, such method comprising: feeding Nam to a culture of a genetically modified Corynebacterium glutamicum strain, said strain comprising at least one genetic modification selected from the group consisting of: (a) heterologous expression of a nadV gene encoding a nicotinamide phosphoribosyltransferase capable of converting 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, including an ushA gene, capable of the enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing phosphoribosyl pyrophosphate (PRPP); (e) modification of one or more genes capable of producing phosphoribosyl pyrophosphate (PRPP) for the conversion of Nam to NMN. Modification or deletion of biochemical pathways that require PRPP other than the biochemical pathway for NMN; (f) deletion or modification of genes capable of enzymatic conversion of NMN to NAMN; (g) deletion or modification of genes capable of conversion of NMN to NAD; (h) deletion or modification of one or more genes encoding NMN nucleosidases capable of conversion of NMN to Nam and ribose-5-phosphate; (i) deletion or modification of one or more genes encoding nucleosidases capable of 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 combination thereof. In various embodiments, the genetically modified strains having at least one of the modifications produce increased amounts of NR compared to strains that do not have any of the modifications. In some embodiments, at least one of the one or more genes encoding an NMN nucleosidase capable of converting NMN to Nam and ribose-5 phosphate can be the same gene(s) as at least one of the one or more genes encoding a nucleosidase capable of converting NR to Nam and ribose-sugar.

[0024] Thus, in yet another aspect, the present invention provides a method for producing NAD, such method comprising: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, said strain comprising at least one genetic modification selected from the group consisting of: (a) heterologous expression of a nadV gene encoding a nicotinamide phosphoribosyltransferase capable of converting Nam to NMN; (b) deletion or modification of one or more genes capable of enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification of Nam to NMN. Modification or deletion of a biochemical pathway requiring PRPP other than the biochemical pathway for conversion to MN; (f) deletion or modification of a gene capable of the enzymatic conversion of NMN to NAMN; (g) modification of a 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 encoding NMN 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 combination thereof. In various embodiments, the genetically modified strain having at least one of the modifications produces increased amounts of NAD compared to a strain having none of the modifications. In some embodiments, at least one of the one or more genes encoding an NMN nucleosidase capable of converting NMN to Nam and ribose-5-phosphate can be the same gene(s) as at least one of the one or more genes encoding a nucleosidase capable of converting NR to Nam and ribose-sugars.

[0025] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and hereinafter described in detail, It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiments disclosed, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

[0026] Other features and advantages of the present invention will become apparent in the following detailed description of preferred embodiments of the invention, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0027] [Figure 1] The chemical structures of the desired products nicotinamide adenine dinucleotide (NAD+), nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR) are shown. In this figure, the chemical structures of the by-products, namely nicotinic acid adenine dinucleotide (NAAD), nicotinic acid mononucleotide (NAMN), nicotinic acid riboside (NAR), and the substrates nicotinamide (Nam) and nicotinic acid (NA) are also shown. The by-products are eliminated by genetic modification according to the present invention. The structures of NA, NAR, NAMN, and NAAD differ from Nam, NR, NMN, and NAD by the terminal group C(O)OH versus C(O)NH2, respectively. [Diagram 2]Certain genetic modifications are shown for increasing the production of NR, NAD and NMN according to the present teachings. Such genetic modifications may include one or more of the following: (a) heterologous expression of a nadV gene encoding a nicotinamide phosphoribosyltransferase capable of converting 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 a biochemical pathway that requires PRPP other than the biochemical pathway for the conversion of Nam to NMN; (f) deletion or modification of a gene involved in the enzymatic conversion of NMN to NAMN; (g) deletion or modification of a gene involved in the conversion of NMN to NAD; and (h) deletion or modification of one or more genes encoding a promiscuous NMN nucleosidase capable of converting NMN to Nam and ribose-5-phosphate. [Diagram 3] FIG. 1 shows a pathway for the production of NMN from Nam as a feedstock in an example genetically modified microbial host cell according to the present invention. [Figure 4] The mechanism of action of purine nucleosidases known in the art and the unexpected observation of the same enzyme functioning as a nicotinamide mononucleotide nucleosidase are presented. [Diagram 5] The method used to delete a target gene using the suicide plasmid pDEL is shown. As shown in this figure, the upstream and downstream flanking regions of the target gene to be deleted were obtained using PCR and cloned into the pDEL plasmid. C. glutamicum cells were transformed with the pDEL plasmid carrying the upstream and downstream flanking regions of the gene targeted for deletion. After transformation, the cells were plated on kanamycin plates to select for transformants. In a second round of screening, the transformants were plated on sucrose-containing plates to select for double recombinants with deletion of the target gene. [Figure 6]1 shows the production of NMN in two different Corynebacterium glutamicum strains constructed according to the present invention. Strain NMN4 has the genotype Δcgl1777-8 ΔpncA ΔpncC ΔushA ΔpyrE, and strain NR5 has the genotype Δcgl1777-8 ΔpncA ΔpncC ΔpnuC Δcgl1364 Δcgl1977 Δcgl2835. [Figure 7] FIG. 1 shows the production of NMN in four different Corynebacterium glutamicum strains constructed according to the present invention. [Figure 8] 1 shows a pathway for enhancing PRPP production from D-glucose-6-phosphate in an example genetically modified microbial host cell according to the invention. [Figure 9] Figure 2 shows the growth of a number of Corynebacterium glutamicum strains transformed with ZWF and transhydrogenase genes in CGXII medium. [Figure 10] Figure 1 shows NMN production in a number of ΔPGI Corynebacterium glutamicum strains containing the indicated plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] 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 a feedstock, and methods for producing NMN, NR and NAD using these genetically modified host cells. In a preferred embodiment, Nam is used as a feedstock for the production of NMN, NR and NAD.

[0029] The genetically modified host cells useful in the present invention can be microbial cells, such as bacterial cells, fungal cells and yeast cells.Bacterial cells useful in the present invention include, but are not limited to: 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., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Coma spp., Pseudomonas ... Monas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidothiobacillus spp., Micrornatus 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.

[0030] Yeast cells of the present disclosure include, but are not limited to, engineered Saccharomyces spp., Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Candida boidinii, and Pichia. In accordance with the present disclosure, the yeasts claimed herein are unicellular microorganisms classified as eukaryotes, members of the fungal kingdom. Although yeasts are unicellular organisms that evolved from a multicellular ancestor, some species useful in the present disclosure are capable of expressing multicellular properties by forming strings of connected budding cells known as pseudohyphae or pseudohyphae.

[0031] Figure 1 shows the chemical structures of the desired products NAD, NMN and NR. In this figure, the chemical structures of the by-products, namely NAAD, NAMN, NAR, and substrates Nam and NA are also shown. The by-products are eliminated by genetic modification according to the present invention. The structures of NA, NAR, NAMN and NAAD differ from Nam, NR, NMN and NAD by the terminal group C(O)OH vs. C(O)NH2, respectively.

[0032] Figure 2 shows some of the metabolic pathways in C. glutamicum cells that are relevant to the present invention. The objective of the present invention is to produce NMN, NR and NAD using Nam or NA as a feedstock while eliminating the associated by-products such as NAR, NAMN and NAAD.

[0033] In a preferred embodiment, Nam is used as a feedstock.As shown in Figure 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) contains significantly more enzyme steps than the pathway from Nam to NMN, and NR and NAD (via NMN).Therefore, Nam is considered to be a preferred feedstock for producing NMN, NR and NAD.

[0034] When Nam is used as the preferred feedstock, it is advantageous to address the conversion of Nam to NA by the deamidase enzyme encoded by the pncA gene. One method to eliminate the conversion of Nam to NA is to inactivate the pncA gene. Inactivation of the pncA gene can be achieved using a number of genetic engineering techniques. A preferred genetic engineering of the pncA gene is to delete the nucleotide sequence from the chromosomal DNA of the microbial host cell.

[0035] Using various tools available for genetic engineering, a person skilled in the art of microbial strain construction would be able to design metabolic pathways to produce each of the desired compounds, i.e., NMN, NR and NDA. As shown in Figure 2, the production of NMN from Nam is achieved in a single enzymatic step in a microbial host cell. Once NMN is accumulated in a microbial host cell, the other two compounds of interest, i.e., NDA and NR, can be obtained as derived products from NMN through subsequent enzymatic reactions.

[0036] Tables 1-3 list the various genes and enzymes involved in the biochemical pathways shown in Figure 2. Table 4 provides sequence information for the genes and enzymes involved in the operation of the biochemical pathways shown in Figure 2.

[0037] A number of genetic modifications can be made to Corynebacterium glutamicum or related organisms to provide a more direct pathway for the production of nicotinamide NMN, NR, and NAD. In some embodiments, such genetic modifications include heterologous expression of a gene (nadV) encoding nicotinamide phosphoribosyltransferase. Nicotinamide phosphoribosyltransferase is an activity not naturally found in Corynebacterium. However, it is found in many other bacteria and eukaryotic microorganisms. Table 2 lists certain preferred sources of such genes. In some embodiments, the genetic modification can include deletion or modification of a gene (pncA) capable of converting nicotinamide to nicotinic acid, resulting in a loss or reduction in enzyme activity. In some embodiments, the genetic modification can include modification of the prsA gene capable of producing phosphoribosyl pyrophosphate (PRPP), an important 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) can be used in accordance with the present invention.In some embodiments, the modification can include deletion or modification of the gene (pncC) involved in the conversion of NMN to NaMN, resulting in loss or reduction of enzyme activity. In some embodiments, the modification can include the creation and incorporation of a conditionally active ribonucleotide reductase using a) temperature-sensitive (ts) mutation(s) in the protein coding sequence, b) ts 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, which is required for DNA production and replication.

[0038] Engineered pathway for NMN production As shown in Figures 2 and 3, NMN production from Nam can be achieved in engineered host cells through a single enzymatic step involving the nicotinamide phosphoribosyltransferase enzyme encoded by the nadV gene. In microorganisms lacking an endogenous gene encoding a phosphoribosyltransferase enzyme, such as C. glutamicum, it is necessary to introduce a foreign gene encoding a phosphoribosyltransferase enzyme using techniques well known in the field of genetic engineering. In a preferred embodiment of the present invention, the source of the nadV gene encoding the nicotinamide phosphoribosyltransferase enzyme can be selected from the group consisting of Stenotrophomonas maltophilia, Chromobacterium violaceum, Deinococcus radiodurans, Synechocystis sp. PCC6803, Pseudonocardia dioxanivorans CB1190, Shewanella oneidensis MR-1, and Ralstonia solanacearum GMI1000. In an exemplary embodiment of the present invention, the nadV gene from any one of the above microbial species is isolated and introduced into a strain of Corynebacterium glutamicum using genetic engineering techniques well known in the art. In a preferred embodiment, the foreign gene nadV is codon-optimized prior to its transfer into the selected microbial host cell to ensure optimal expression of the nicotinamide phosphoribosyltransferase enzyme in the microbial host cell.

[0039] In phosphoribosyltransferase-mediated enzyme reactions, phosphoribosyl pyrophosphate (PRPP) acts as a co-substrate, and it is necessary to ensure that there is a sufficient amount of PRPP available in the cell to facilitate the phosphoribosyltransferase enzyme-mediated reaction. The pool size of PRPP in a microorganism can be increased by enhancing the expression of PRPP synthetase activity. Enhanced expression of PRPP synthetase activity can be achieved by expressing the foreign gene prsA. In addition, when there is feedback inhibition on PrsA enzyme activity, it is preferable to use the prsA gene encoding a feedback-resistant variant of the PrsA enzyme.

[0040] In another embodiment of this invention, as one way to preserve the PRPP pool that serves as a co-substrate in the production of NMN from Nam, additional genetic modifications can be introduced to block other pathways that use the PRPP pool in the cell. In one aspect of the invention, the pyrE gene is mutated, leading to the inactivation of the orotate phosphoribosyltransferase enzyme, which can catalyze the transfer of a ribosyl phosphate group from PRPP to orotate, which in turn leads to the formation of orotidine.

[0041] To achieve the production target for NMN production, apart from ensuring that appropriate phosphoribosyltransferase and phosphoribosylpyrophosphate (PRPP) synthetase enzymes are present and sufficient amounts of PRPP are present within the microbial cells for the production of NMN from Nam, it is advantageous to block or inactivate possible NMN utilization / degradation pathways within the microbial cells.

[0042] For example, a microbial host cell selected for producing NMN using Nam as a feedstock and having a codon-optimized exogenous gene encoding a nicotinamide phosphoribosyltransferase enzyme can further include additional genetic modifications to ensure the preservation of NMN produced within the cell, which additional genetic modifications block other biochemical pathways that consume NMN. In one aspect of this embodiment, the ushA gene is deleted, which encodes an enzyme capable of converting NMN to NR. In another aspect of this embodiment, the gene pncC is deleted, which results in the loss of nicotinamide-nucleotide amidohydrolase enzyme activity capable of converting NMN to nicotinic acid mononucleotide (NAMN). In yet another aspect of this invention, the genes cgl1364, cgl1977, and cgl2835 are deleted, which leads to the elimination of putative nucleosidases capable of converting NMN to Nam and ribose-5-phosphate. In another aspect of this invention, the gene nadD is genetically modified such that the activity of an enzyme capable of converting NMN to NAD is blocked.

[0043] Accordingly, in one aspect, the present invention provides a method for producing NMN, such method comprising: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, said strain comprising at least one genetic modification selected from the group consisting of: (a) heterologous expression of a gene encoding a nicotinamide phosphoribosyltransferase capable of converting Nam to NMN; (b) deletion or modification of one or more genes capable of enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of enzymatic conversion of NMN to NR; (d) deletion or modification of one or more genes capable of producing PRPP. (e) modification or deletion of a biochemical pathway requiring PRPP other than the biochemical pathway for conversion of Nam to NMN; (f) deletion or modification of a gene capable of enzymatic conversion of NMN to NAMN; (g) deletion or modification of a gene capable of conversion of NMN to NAD; (h) deletion or modification of one or more genes encoding promiscuous nucleosidase reactions capable of 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 combination thereof. In various embodiments, the genetically modified strain having at least one of the modifications produces increased amounts of NMN compared to a strain having none of the modifications.

[0044] Altered pathway for NAD production 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 encoding a nicotinamide phosphoribosyltransferase enzyme can further include an additional genetic modification in the nadD gene, resulting in an upregulated NAD(+) synthetase enzyme capable of converting NMN to NAD. In yet another aspect of this embodiment, the gene nudC is mutated to block the enzyme capable of converting NAD back to NMN.

[0045] Thus, in yet another aspect, the present invention provides a method for producing NAD, such method comprising: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, said strain comprising at least one genetic modification selected from the group consisting of: (a) heterologous expression of a gene encoding a nicotinamide phosphoribosyltransferase capable of converting Nam to NMN; (b) deletion or modification of one or more genes capable of enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification of Nam to NMN. Modification or deletion of a biochemical pathway that requires PRPP other than the biochemical pathway for conversion to MN; (f) deletion or modification of a gene capable of the enzymatic conversion of NMN to NAMN; (g) modification of a 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 encoding nucleosidases 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 combination thereof. In various embodiments, the genetically modified strains having at least one of the modifications produce increased amounts of NMN compared to strains that do not have any of the modifications.

[0046] Engineered pathway for NR production 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 encoding a nicotinamide phosphoribosyltransferase enzyme can further comprise a functionally active dephosphorylation enzyme encoded by the ushA gene. In another aspect of this invention, the ushA gene is genetically modified to obtain a dephosphorylase enzyme with enhanced activity for converting NMN to NR.

[0047] Thus, in another aspect, the present invention provides a method for producing NR, such method comprising: feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, said strain comprising at least one genetic modification selected from the group consisting of: (a) heterologous expression of a gene encoding a nicotinamide phosphoribosyltransferase capable of converting Nam to NMN; (b) deletion or modification of one or more genes capable of enzymatic conversion of Nam to NA; (c) modification of one or more genes capable of enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification of PR other than the biochemical pathway for conversion of Nam to NMN. Modification or deletion of biochemical pathways that require PP; (f) deletion or modification of genes capable of enzymatic conversion of NMN to NAMN; (g) deletion or modification of genes capable of conversion of NMN to NAD; (h) deletion or modification of one or more genes encoding nucleosidase enzymes capable of conversion of NMN to Nam and ribose-5-phosphate; (i) deletion or modification of one or more genes encoding nucleosidase enzymes capable of 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 combination thereof. In various embodiments, the genetically modified strain with at least one of the modifications produces increased amounts of NR compared to a strain that does not have any of the modifications.

[0048] Modified pathway to increase NMN titer The production of NMN from NAM, catalyzed by NadV enzymes, depends at least in part on the rate at which the microbial host cell produces the PRPP co-substrate. The oxidative branch of the pentose phosphate pathway ("PPP") is shown in Figure 8, in which D-glucose-6-phosphate is converted to D-ribulose-5-phosphate and then to PRPP. A strategy to increase the flux through the PPP involves knocking out the pgi gene, thus redirecting D-glucose-6-phosphate from glycolysis to the PPP, increasing PRPP availability. However, a problem associated with this approach in Corynebacterium glutamicum is that growth is significantly slowed on minimal media such as CGXII. Without wishing to be bound by any particular theory, it is believed that the native ZWF protein, i.e. the glucose 6-phosphate dehydrogenase enzyme that catalyzes the first step of the PPP pathway, is inhibited by ATP and PEP (phosphoenolpyruvate) accumulation during growth on glucose, thus significantly reducing carbon flow through the PPP. Moreover, assuming that another ZWF could be identified that increases carbon flow, a further problem would appear, possibly 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., soluble UdhA or transmembrane PntAB, to address this shortcoming.

[0049] Thus, in one embodiment, the present invention provides a recombinant Δpgi mutant of Corynebacterium glutamicum, where expression of a transgenic ZWF enzyme in combination with pyridine nucleotide transhydrogenase rescues growth while improving production of PRPP as reflected in higher titers of NMN. Thus, in a related aspect, the invention provides a method of producing NMN, the method comprising feeding nicotinamide to a culture of a genetically modified Corynebacterium glutamicum strain, the strain comprising a genetic modification comprising: (a) heterologous expression of a gene encoding a nicotinamide phosphoribosyltransferase capable of converting Nam to NMN; (b) deletion or modification of one or more genes capable of enzymatic conversion of Nam to NA; (c) deletion or modification of one or more genes capable of enzymatic conversion of NMN to NR; (d) modification of one or more genes capable of producing PRPP; (e) modification or deletion of a biochemical pathway requiring PRPP other than the biochemical pathway for conversion of Nam to NMN; (f) deletion or modification of a gene capable of enzymatic conversion of NMN to NAMN; (g) deletion or modification of a gene capable of conversion of NMN to NAD; (h) deletion or modification of a gene capable of enzymatic conversion of NMN to NAD; (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, the combination of which is capable of compensating for a 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; (l) incorporation of a ribonucleotide reductase that is conditionally active to block cell division and biomass accumulation without affecting protein synthesis and normal metabolic activity; and (m) any combination thereof.

[0050] Thus, PRPP production is improved as reflected in higher titers of NMN relative to strains without such modifications.

[0051] Nicotinamide compounds (NMN, NR, NAD) produced according to the present disclosure can be used in any of a variety of applications, such as to exploit their biological or therapeutic properties (e.g., to control low density lipoprotein cholesterol, increase high density lipoprotein cholesterol, etc.) For example, according to the present disclosure, nicotinamide ribose can be used in pharmaceuticals, food products, dietary supplements, and the like.

[0052] Nicotinamide mononucleotide (NMN) produced by the method disclosed in this invention can have therapeutic value in improving plasma lipid profile, preventing stroke, providing neuroprotection with chemotherapy treatment, treating fungal infection, 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 cells described above for treating diseases or conditions associated with the nicotinamide riboside kinase pathway of NAD+ biosynthesis by administering an effective amount of the nicotinamide riboside composition.

[0053] Typically, diseases or conditions that have altered levels of NAD+ or NAD+ precursors or that can 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 disease, Parkinson's disease and multiple sclerosis), neurotoxicity observed with chemotherapy, Candida glabrata infection, and overall decline in health associated with aging. Such diseases and conditions can be prevented or treated by providing dietary supplementation or a therapeutic treatment regime with a nicotinamide riboside composition.

[0054] It will be appreciated that the nicotinamide compounds isolated from the genetically modified bacteria of the present invention can be reformulated into a final product. In some other embodiments of the present disclosure, the nicotinamide riboside compounds produced by the genetically modified host cells described herein are incorporated into a final product (e.g., a dietary supplement or feed, a pharmaceutical product, etc.) in association with the host cells. For example, the host cells can be lyophilized, freeze-dried, frozen or otherwise inactivated, and then the whole cells can be incorporated into or used as a final product. The host cells can also be treated prior to incorporation into the product to increase bioavailability (e.g., by lysis).

[0055] In some embodiments of the present disclosure, the nicotinamide riboside compound produced is incorporated into a food or feed ingredient (e.g., dietary supplement).The type of food into which the nicotinamide riboside compound can be incorporated according to the present disclosure is not particularly limited, and includes beverages such as milk, water, soft drinks, energy drinks, tea, and juice; confectionery such as jellies and biscuits; fat-containing foods and beverages such as dairy products; processed foods such as rice, bread, breakfast cereals, etc. In some embodiments, the nicotinamide riboside compound produced is incorporated into dietary supplements, such as, for example, multivitamins.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred materials and methods are described below.

[0057] The present disclosure will be more fully understood in consideration of the following non-limiting examples. It should be understood that these examples, while showing preferred embodiments of the subject technology, are provided by way of example only. From the above discussion and these examples, those skilled in the art can ascertain the essential characteristics of the subject technology, and can make various changes and modifications to the subject technology to adapt it to various applications and conditions without departing from its spirit and scope.

[0058] Working Example Example 1 Strain construction Various engineered C. glutamicum strains were constructed with one or more genetic modifications described in Table 5. To perform the gene deletions in C. glutamicum summarized in Table 5, the pDEL plasmid (a suicide plasmid lacking an origin of replication for C. glutamicum) was used. The pDEL plasmid contained homologous regions (approximately 500-800 bp each) upstream and downstream of the gene targeted for deletion. As shown in Figure 5, the upstream and downstream regions flanking the gene targeted for deletion were obtained using PCR and cloned into the pDEL plasmid containing the SacB gene and an antibiotic marker.

[0059] C. glutamicum cells were transformed with such plasmids and plated on CASO medium containing kanamycin (25 μg / mL) overnight. The resulting colonies were plated on medium containing 6% sucrose to select for double recombinants that had the targeted gene deletion and had not integrated any part of the pDEL plasmid into their chromosomal DNA.

[0060] The above engineered strains were subjected to further genetic modification using the plasmids described in Table 6 to introduce exogenous NadV and prsA gene variants / mutations.

[0061] Example 2 Production of NMN from Nam Strains constructed according to Example 1 were grown overnight in BHI medium containing kanamycin. Cells were pelleted, washed, and resuspended in CGXII medium for 2-3 hours for recovery, as C. glutamicum cells have a long lag phase when transferred from one medium to another. To initiate the fermentation assay, cells were resuspended in CGXII medium (Table 7) at 0.2 OD 600 The cells were inoculated at 30°C and maintained on a rotary shaker (250 rpm). 600 Upon reaching 100%, the cells were induced with 0.4 mM IPTG. Nam was fed to the cell cultures and samples were collected every 24 hours up to 72 hours to confirm NMN production.

[0062] Specifically, samples were obtained from culture supernatants and centrifuged at 4000×G for 5 min. The clear supernatants were mixed with an equal volume of methanol and vortexed for 30 s or shaken on a plate shaker at 600 rpm for 10 min and centrifuged at >4000×G for 5 min to remove any additional debris, and the resulting supernatants were analyzed by HPLC method using a Phenomenex Luna 3 μm NH2 100 Å operated in HILAC mode with a maximum pressure of 400 bar. 2 μL injections were monitored at 261 nm and calibration curves were 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.

[0063] The NMN production titers by the NMN4 and NR5 strains (both containing the foreign genes smaOP and prsAL136I), respectively, are shown in FIG.

[0064] FIG. 7 shows the titers of NMN production by the NMN3 strain containing the foreign genes cviHA and prsA L136I, the NMN4 strain containing the foreign genes cviHA and prsAL136I, the NMN4 strain containing the foreign genes smaOP and prsA L136I, and the NR5 strain containing the foreign genes smaOP and prsA L136I, respectively.

[0065] As shown in Figures 6 and 7, the NR5 strain, characterized by the deletion of endogenous genes cgl1364 encoding purine nucleosidase iunH3, cgl1977 encoding purine nucleosidase iunH2, and cgl2835 for purine nucleosidase iunH1, appears to have the highest NMN production titer. Without wishing to be bound by any particular theory, it is believed that the deletion of one or more of the genes may lead to a significant reduction in the degradation of NMN back to nicotinamide (Nam) and ribose-5P. This observation was very surprising in view of the fact that it was previously known that the deletion of one or more of cgl1364, cgl1977, and cgl2835 only had the effect of reducing the degradation of NR to NA and ribose.

[0066] Example 3 Proliferative rescue and augmentation of the PPP pathway As predicted above, knocking out the pgi gene redirects D-glucose-6-phosphate from glycolysis to the PPP, thus increasing PRPP availability. However, the Δpgi mutant of C. glutamicum suffers from severely 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 that catalyzes the first step of the PPP pathway, is inhibited by ATP and PEP (phosphoenolpyruvate) accumulation during growth on glucose, thus significantly reducing carbon flow through the PPP.

[0067] Three recombinant ZWFs were screened for their ability to overcome the inhibition exhibited by the native C. glutamicum ZWF: (i) a feedback-resistant mutant (A243T) of the C. glutamicum ZWF (SEQ ID NO:28); (ii) an engineered mutant (R46E / Q47E) of the ZWF from Leuconostoc mesenteroides (lmZWF, SEQ ID NO:32) that has mixed NADH and NADPH usage; and (iii) a ZWF from Zymomonas mobilis (zmZWF) that prefers NADH usage.

[0068] Two transhydrogenases were screened for their ability to overcome NADPH accumulation: (i) UdhA from E. coli, a soluble transhydrogenase that has been reported to favor the reduction of NAD+ by NADPH; and (ii) PntAB from E. coli, a membrane-bound transhydrogenase that has been reported to favor the reduction of NADP+ by NADH.

[0069] The recombinant ZWF and transhydrogenase were paired in a constitutive expression or cumic acid inducible configuration in the cgDVS expression vector as follows: Constitutive expression: (i)cgDVS.pSOD.lmZWF.pntAB(S.lmZWF.pntAB) (ii)cgDVS.pSOD.lmZWF.udhA(S.lmZWF.udhA) (iii)cgDVS.pSOD.zmZWF.pntAB(S.zmZWF.pntAB) (iv)cgDVS.pSOD.zmZWF.udhA(S.zmZWF.udhA) Cumic acid-induced expression: (v) cgDVS.pCT5.cgZWF * .udhA (S.pCT5.cg.udhA)

[0070] Each of the above vectors (i)-(v) was 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 one of two control vectors, cgDVS, expressing the red fluorescent protein mCherry from either the min3 constitutive promoter (S.min3.mCH) or the pCT5 inducible promoter (S.pCT5.mCH). The transformed strains were then grown on CGXII medium to identify successful rescue from the growth deficiency in NMN11.

[0071] As shown in FIG. 9, time is measured in hours on the x-axis and OD 600 On the y-axis, the BASE strain, min3.mCherry and pCT5.mCherry showed almost zero growth, as expected. The pCT5.mCherry strain eventually gave a signal at a wavelength of 630 nm, but still showed no significant visible growth or mCherry concentration by visual inspection, which would explain the subsequent increase in emission at 630 nm, since mCherry still absorbs near such wavelength. The zmZWF.udhA (dark blue) and lmZWF.PntAB (yellow) strains showed the best rescue of the growth defect. The second best result was obtained from lmZWF.udhA, while zmZWF.pntAB showed a modest initial growth that gradually decreased. Initial production tests performed in flasks revealed that strain zmZWF.udhA produced the most NMN.

[0072] The following strains were transformed with the indicated vectors: NMN4:Δcgl1777-8Δcgl2487Δcgl1963Δcgl0328Δcgl2773 (1) No control: cgDVK.ptac.mcherry (mCherry) (2) Generation control: cgDVK.ptac.smaOP.prsA (K.smaOP.PRS) NMN13:Δcgl1777-8Δcgl2487Δcgl1963Δcgl0328Δcgl0851 (3)cgDVK.ptac.smaOP.prsA (4)cgDVK.ptac.smaOP.prsA+cgDVS.pSOD.zmZWF.udhA(S.ZWF.udhA) NMN11:Δcgl1777-8Δcgl2487Δcgl1963Δcgl0328Δcgl0851Δcgl2773 (5)cgDVK.ptac.smaOP.prsA (6)cgDVK.ptac.smaOP.prsA+cgDVS.pSOD.zmZWF.udhA

[0073] Two strains were also generated with the pSOD.zmZWF.udhA construct inserted into the pgi locus to eliminate the need for cgDVS and the spectinomycin antibiotic: NMN29:Δcgl1777-8Δcgl2487Δcgl1963Δcgl0328Δcgl0851:pSODzmZWF.udhA (7)cgDVK.ptac.smaOP.prsA NMN30:Δcgl1777-8Δcgl2487Δcgl1963Δcgl0328Δcgl0851:pSODzmZWF.udhAΔcgl2773 (8)cgDVK.ptac.smaOP.prsA

[0074] The amount of NMN produced by each strain was assessed after 48 and 72 hours of cell culture growth, respectively, and the results are shown in Figure 10. [Table 1] [Table 2] [Table 3] [Table 4] TIFF0007597396000005.tif177165 [Table 5] [Table 6] [Table 7] Target sequence SEQ ID NO:1;PRT;Chromobacterium violaceum;Nicotinamide phosphoribosyltransferase-cviNadVha (cviHA) MTAPKAAQDKNQLVPFNLADFYKTGHPAMYPRETTRLVANFTPRSAKYAQVLPQLFDDKVVWFGLQGFIQEYLIDLFNREFFQRPKADAVRRYQRRMDTALGAGAVDGGRLEALHDLGHLPLEIRSLPEGARVDIKVPPV TFSNTHPDFPWVATYFETLFSCESWKPSTVATIAFEFRKLLSYFAALTGAPQDFVAWQGHDFSMRGMSGVHDAMRCGAGHLLSFTGTDTIPALDYLEDHYGADAERELVGGSIPASEHSVMALRILLTQQRLARMPAHQG LDDKALRRLAEREVVREFVTRDYPAGMVSIVSDTFDFWNVLTVIARELKDDIQARRPDALGNAKVVFRPDSGDPVRILAGYRDDELQFDDAGNCTARDDGRPVSAAERKGAVECLWDIFGGTVTERGYRVLDSHVGLIYG DSITLPRARDILLRLAEKGYASCNVVFGIGSFVYGMNSRDTFGYALKAVYAEVAGEAVDIYKDPATDDGTKKSARGLLRVEEENGRYALYQQQTPAEAEGGALRPVFRDGELLVKQTLAEIRQRLQASWTCPEAGSIVWNA SEQ ID NO:2;PRT;Stenotrophomonas maltophilia;Nicotinamide phosphoribosyltransferase-smaNadVop (smaOP) MHYLDNLLLNTDSYKASHWLQYPPGTDATFFYVESRGGLHDRTVFFGLQAILKDALARPVTHADIDDAAAVFAAHGEPFNEAGWRDIVDRLGGHLPVRIRAVPEGSVVPTHQALMTIESTDPAAFWVPSYLETLLLRVWYPVTVATISWHARQTIAAFLQQTSDDPQGQLPFKLHDFGARGVSSLESAALGGAAHLVNFLGTDTVSALCLARAHYHAPMAGYSIPAAEHSTITSWGREREVDAYRNMLRQFGKPGSIVAVVSDSYDIYRAISEHWGTTLRDDVIASGATLVIRPDSGDPVEVVAESLRRLDEAFGHAINGKGYRVLNHVRVIQGDGINPDTIRAILQRITDDGYAADNVAFGMGGALLQRLDRDTQKFALKCSAARVEGEWIDVYKDPVTDAGKASKRGRMRLLRRLDDGSLHTVPLPANGDDTLPDGFEDAMVTVWENGHLLYDQRLDDIRTRAAVGH SEQ ID NO:3;PRT; Codon-optimized PRPP synthetase variant, CYL77_RS04805-prsA (PRS) MTAHWKQNQKNLMLFSGRAHPELAEAVAKELDVNVTPMTARDFANGEIYVRFEESVRGSDCFVLQSHTQPLNKWLMEQLLMIDALKRGSAKRITAILPFYPYARQDKKHRGREPISARLIADLMLTAGADRIVSVDLHTDQIQGFFDGPVDHMHAMPILTDHIKENYNLDNICVVSPDAGRVKVAEKWANTLGDAPMAFVHKTRSTEVANQVVANRVVGDVDGKDCVLLDDMIDTGGTIAGAVGVLKKAGAKSVVIACTHGVFSDPARERLSACGAEEVITTDTLPQSTEGWSNLTVLSIAPLLARTINEIFENGSVTTLFEGEA SEQ ID NO:4;PRT; Feedback-resistant PRPP synthetase variant, prsAL136I (PRSL136I) MTAHWKQNQKNLLMFSGRAHPELAEAVAKELDVNVTPMTARDFANGEIYVRFEESVRGSDCFVLQSHTQPLNKWLMEQLLMIDALKRGSAKRITAILPFYPYARQDKKHRGREPISARLIADLMLTAGADRIVSVDIHTDQIQGFFDGPVDHHAMPILTDH IKENYNLDNICVVSPDAGRVKVAEKWANTLGDAPMAFVHKTRSTEVANQVVANRVVGDVDGKDCVLLDDMIDTGGTIAGAVGVLKKAGAKSVVIACTHGVFSDPARERLSACGAEEVITTDTLPQSTEGWSNLTVLSIAPLLARTINEIFENGSVTTLFEGEA SEQ ID NO.:5;DNA;ニコチンアミダズpncA(CGL_RS12350、ncgl2401、cgl2487) ATGGCACGCGCACTCATTCTGGTTGATGTTCAAAAAAGACTTCTGCCCCGGTGGCAGCCTAGCCACCGAACGAGGCGATGAAGTGGCGGGAAAAATCGGTGCCTATCAGCTGTCCCACGGCTCAGAGTACGACGTCGTTGTGGCGACCCAAGATTGGCACATCGATCCAGGCGAGCACTTTTCAGAAACCCACGACTTTAAAAACTCCTGGCCAATCCACTGCGTCGCGATTCCGATGGTGCGCCATGCATGACCGCATCAACACCGATTCAATCGATG AGTTCTTCCGCAAAGGCCATTACACCGCGGCGTATTCCGGGTTCGAGGGAACTGCAGTCAGTGAAGAACTCCTCATGTCTCCATGGCTGAAGAACAAGGGAGTCACTGATGTAGACATCGTAGGGATCGCTACGGATCACTGCGTTCGAGCCAGCACTTGATGCTCTCAAGGAGGCTTCAACGTCTCATTTTGACGTCGATGTGTTCTGCGGTGATTTCCATGCGGGAGACCACGCTTTTGGAGGAACTACATGAAGCCGGGGCGATTCTGATTTAA SEQ ID No.: 6; PRT; Nicotinamidase pncA (CGL_RS12350, ncgl2401, cgl2487) MARALILVDVQKDFCPGGSLATERGDEVAGKIGAYQLSHGSEYDVVVATQDWHIDPGEHFSETPDFKNSWPIHCVADSDGAAMHDRINTDSIDEFFRKGHYTAAYSGFEGTAVSEELLMSPWLKNKGVTDVDIVGIATDHCVRATALDALKEGFNVSILTSMCSAVDFHAGDHALEELHEAGAILI SEQ ID NO:7; DNA; Nicotinamide-nucleotide amidohydrolase pncC (CGL_RS09770, ncgl1888, cgl1963) ATGTCGGAGAATCTGGCGGGGCGAGTGGTGGAGCTGTTGAAATCGCGCGGTGAAACGCTGGCGTTTTGTGAATCCCTCACCGCCGGCCTTGCCAGTGCGACGATCGCAGAGATCCCCGGCGCCTCAGTG GTACTTAAAGGCGGGCTGGTCACCTATGCCACCGAGCTTAAGGTTGCGCTTGCCGGTGTGCCGCAGGAGCTTATCGACGCGCACGGCGTTGTTTCCCCGCAGTGCGCCCGTGCGATGGCAACGGGGCCG CACACAGATGCCAGGCAGATTGGGCGGTTTCGCTCACGGGCGTTGCTGGCCCAGCAAACAAGATGGTCATCCGGTGGGGGAAGTGTGGATCGGAGTGGCTGGTCCTGCGCATTTTGGGGCGTCGGGAAC AATTGACGCGTATCGTGCGTTTGAAAGTGAACAACAGGTAATATTGGCTGAATTGGGACGGCATCATATTAGAGAGTCTGCTGTGCAGCAAAGCTTTCGCCTGCTGATTGACCATATTGAGTCGCAGTGA SEQ ID NO:8; PRT; Nicotinamide-nucleotide amidohydrolase pncC (CGL_RS09770, ncgl1888, cgl1963) MSENLAGRVVELLKSRGETLAFCESLTAGLASATIAEIPGASVVLKGGLVTYATELKVALAGVPQELIDAHGVVSPQCARAMATGAAHRCQADWAVSLTGVAGPSKQDGHPVGEVWIGVAGPAHFGASGTIDAYRAFESEQQVILAELGRHHIRESAVQQSFRLLIDHIESQ SEQ ID No.:9; DNA; Restriction modification system; RM = cgl1777 (CYL77_RS08985) SEQ ID NO:10;PRT;Restriction-modification system;RM=cgl1777 (CYL77_RS08985) MKPTVNVVFNAHHPKDTQPLDKFFDKELKDTHHLDITVGYISEKSLQYLLLIAGTHPDLTITLTCGMHAREGMTAAQLHHARVLHDYLSDHDRGGVFVIPRLRYHGKIYLFHKNQHTDPIAYIGSANLSAIVPGYTSTFETGVILDPAPEDLVLHLNRDVVPLCVPIDTAHVPIIKDQE SPMKHVAEATAVSTSDVVAIMSSPFTYSFDLKLKATASSNLNAHNSGGGARKQKNGSFLARNWYEGEIIVGVETTRLPGYPQNKSEFTAVTDDGWSFVCKISGGNGKNLRSKGDLSILGTWLKSRFIEQGALEYGEDATQENIDRFGRTHMTMRYHPDFDVWSFDLSQTPKPSTQIGQD SEQ ID NO:11;DNA;Restriction-modification system;RM=cgl1778 (CYL77_RS08990) SEQ ID NO:12;PRT;Restriction-modification system;RM=cgl1778 (CYL77_RS08990) MSHHTHLNNYITSLSDNADLREKVTATVDAFRHTVMDDFDYISDQQVLLYGDVQSGKTSHMLGIIADCLDSTFHTIVILTSPNTRLVQQTYDRVAQAFPDTLVCDRDGYNDFRANQKSLTPRKSIVVVGKIPAVLGNWLRVFNDSGALSGHPVLIDD EADATSLNTKVNQSDVSTINHQLTSIRDLATGCIYLQVTGTPQAVLLQSDDSNWAAEHVLHFAPGESYIGGQLFFSELNNPYLRLFANTQFDEDSRFSDAIYTYLLTAALFKLRGESLCTMLIHPSHTASSHRDFAQEARLQLTFAFERFYEPMIQHN FQRAYEQLAQTDSNLPPLRKILNILGGMEDDFSIHIVNSDNPTVEEDWADGYNIIVGGNSLGRGLTFNNLQTVFYVRESKRPQADTLWQHARMFGYKRHKDTMRVFMPATIAQTFQEVYLGNEAIKNQLDHGTHINDIRVILGDGVAPTRANVLDKRK VGNLSGGVNYFAADPRIKNVEALDKKLLAYLDKHGEDSTIGMRAIITILNAFTVDPNDLDLATFKAALLDFERNQPHLTARMVLRTNRKVNQGTGALLSPTDQALSRAEVAHPLLILYRIEGVNDAAAQRGEPTWSSDPIWVPNIKLPGQRQFWCVDG SEQ ID NO:13;DNA;orotate phosphoribosyltransferase pyrE;CGL_RS13820, ncgl2676, cgl2773 ATGTCATCTAATTCCATTAACGCAGAAGCGCGCGCTGAGCTTGCTGAACTGATCAAAGAGCTAGCTGTCGTCCACGGTGAAGTCACCTTGTCTTCGGGCAAGAAGGCTGATTACTACATCGATGTCCGTCGTGCCACCTTGCACGCGCGCGCATCTCGCCTGATCGGTCAGCTGCTGCGCGAAGCCACGCCTGACTGGGACTATGACGCAGTTGGCGGCCTGACCTTGGGCGCTGACCGGTTGCCACCGCCATCATGCACGCCGACGGCCGCGATA TCAACGCGTTGTGGTGCGCAAGGAGGCCAAGAAGCACGGCATGCAGCGTCGCATTGAGGGCCCTGACCTGACGGGCAAGAAGGTGCTCGTGGTGGAAGATACCACCACCACCGGAAATTCCCCTCTGACAGCTGTTGCCGCGTTGCGTGAAGCTGGCATTGAGGTTGGGCGTTGCCACCGTGGTCGATCGCGCAACCGGTGCAGATGAGGTTATCGCAGCGGAAGGCCTTCCTTACCGCAGCTTGGGACTTTCTGATCTTGGACTCACTAA SEQ ID NO: 14;PRT; MSSNSINAEARAELAELIKELAVVHGEVTLSSGKKADYYIDVRRATLHARASRLIGQLLREATADWDYDAVGGLTLLGADPVATAIMHADGRDINAFVVRKEAKKHGMQRRIEGPDLTGKKVLVVEDTTTTGNSPLTAVAALREAGIEVVGVATVVDRATGADEVIAAEGLPYRSLLGLSDLGLN SEQ ID NO:15;DNA;5'-ヌクレオチダキushA;cgl0328-CGL_RS01710、ncgl0322、cg0397 SEQ ID NO:16;PRT;5'-ヌクレオチダキushA;cgl0328-CGL_RS01710、ncgl0322、cg0397 MKRLSRAALAVVATTAVSFSALAVPAFADEASNVELNILGVTDFHGHIEQKAVKDDKGVITGYSEMGASGVACYVDAERADNPNTRFITVGDNIGGSPFVSSILKDEPTLQALSAIGVDASALGNHEFDQGYSDLVNRVSLDGSGSAKFPYLGANVEGGTPAPAKSEIIEMDGVKIAYVGAVTEETATLVSPAGIEGITFTGDIDAINAEADRVIEAGEADVVIALIHAEAAPTDLFSNNVDVVFSGHTHFDYVAEGEARGDKQPLVVIQGHEYGKVISDVEISYDREAGKITNIEAKNVSATDVVENCETPNTAVDAIVAAAVEAAEEAGNEVVATIDNGFYRGAD EEGTTGSNRGVESSLSNLIAEAGLWAVNDATILNADIGIMNAGGVRADLEAGEVTFADAYATQNFSNTYGVREVSGAQFKEALEQQWKETGDRPRLALGLSSNVQYSYDETREYGDRITHITFNGEPMDMKETYRVTGSSFLAGGDSFTAFAEGGPIAETGMVDIDLFNNYIAAHPDAPIRANQSSVGIALSGPAVAEDGTLVPGEELTVDLSSLSYTGPEAKPTTVEVTVGTEKKTADVDNTIVPQFDSTGKATVTLTVPEGATSVKIATDNGTTFELPVTVNGEGNNDDDDKEQQSSGSSDAGSLVAVLGVLGALGGLVAFFLNSAQGAPFLAQLQAMFAQFM SEQ ID NO:17;DNA;ニコチンデメーリボーストランスポーターpnuC;ncgl0063、CGL_RS00355、cgl0064 ATGAATCCTATAACCGAATTATTAGACGCAACACTATGGATCGGCGGAGTTCCGATTCTGTGGCGCGAAATCATCGGCAACGTTTTCGGATTATTTAGCGCGTGGGCAGGAATGCGACGCATCGTGTGGGCATGGCCCATCGGCATCATAGGCAACGGCCTGCTGTTCACAGT ATTTATGGGCGGCCTTTTCCACACTCCACAAAAACCTCGATCTCTACGGCCAAGCGGTGCGCCAGATCATGTTCATCATCGTCAGTGGTTATGGCTGGTACCAATGGTCGGCCGCAAAACGTCGCGCACTCACCCAGAAAATGCAGTAGCAGTGGTTCCTCGCTGGGCAAGCA CCAAAGAACGCGCCGGCATTGTGATTGCGGCGGTGTGGGAACACTCAGCTTTGCCTGGATTTTCCAAGCACTCGGCTCCTGGGGGCCATGGCCGACGCGTGGATTTTCGTCGGCTCAATCCTGGCTACCTACGGAATGGCTCGCGGATGGACAGAGTTCTGGCTGATCTG ATCGCCGTCGACATAGTTGGCGTTCCTCTACTTTTGACTGCTGCTACTACCCCATCCGCGGTGCTTTACCTGGTGTACGGTGCGTTTGTCAGCGGGGATTTGTCGTGTGGCTGCGGGTGCAAAAAGCAGACAAGGCTCGTGCGCTGGAAGCTCAGGAGTCTGTGACAGTCTGGA SEQ ID NO:18;PRT;ニコチンデメーリボーストランスポーターpnuC;ncgl0063、CGL_RS00355、cgl0064 MNPITELLDATLWIGGVPILWREIIGNVFGLFSAWAGMRRIVWAWPIGIIGNALLFTVFMGGLFHTPQNLDLYGQAGRQIMFIIVSGYGWYQWSAAKRRALTPENAVAVVPRWAS TKERAGIVIAAVVGTLSFAWIFQALGSWGPWADAWIFVGSILATYGMARGWTEFWLIWIAVDIVGVPLLLTAGYYPSAVLYLVYGAFVSWGFVVWLRVQKADKARALEAQESVTV SEQ ID NO:19; DNA; Purine nucleosidase iunH3; cgl1364 (CGL_RS06810, ncgl1309, cg1543, iunH3) ATGACCACCAAGATCATCCTCGACTGCGATCCAGGACACGACGACGCTGTAGCCATGCTGCTCGCAGCCGGCAGCCCAGAAATTGAACTGCTTGGAATCACCACGGTCGGCGGCAACCAGACCTTGGACAAGGTCACCCACAATACGCAGGTCGTAGCCACCATCGCTGATATCAATGCGCCCATCTACCGCGGTGTCACCCGACCATTGGTGCGCCCCGTTGAGGTAGCCGAAGATATCCACGGCGATACCGGCATGGAAATCCACAAGTACGAACTGCCTGAACCAACCAAGCAGGTAGAAGACACCCACGCGGTGGATTTCATCATCGATACCATCATGAATAACGAGCCCGGCAGCGTAGCGCTGGTTCCCACCGGACCACTGACCAACATCGCGCTGGCAGTCCGGAAAGAACCACGCATCGCCGAGCGAGTCAAGGAAGTTGTCCTCATGGGCGGGGGCTACCACGTAGGAAACTGGACCGCCGTAGCTGAATTCAACATCAAGATCGACCCCGAAGCAGCCCACATCGTATTCAACGAAAAGTGGCCACTGACTATGGTCGGCCTCGACCTTACCCACCAGGCGCTCGCAACACCTGAGATCGAAGCCAAGTTCAACGAGCTGGGCACCGACGTCGCCGACTTCGTCGTCGCGCTTTTCGACGCTTTCCGCAAGAATTACCAGGACGCACAGGGTTTTGATAACCCACCAGTACACGACCCTTGTGCTGTTGCATACCTTGTTGACCCAACCGTATTCACCACCCGCAAAGCACCACTCGATGTGGAGCTGTACGGCGCACTCACCACAGGCATGACCGTTGCTGATTTCCGCGCACCGGCTCCAGCAGATTGCACCACCCAAGTAGCTGTTGACCTGGACTTTGATAAATTCTGGAACATGGTGATCGATGCAGTAAAGCGCATCGGATAG SEQ ID NO:20; PRT; Purine nucleosidase iunH3; cgl1364 (CGL_RS06810, ncgl1309, cg1543, iunH3) MTTKIILDCDPGHDDAVAMLLAAGSPEIELLGITTVGGNQTLDKVTHNTQVVATIADINAPIYRGVTRPLVRPVEVAEDIHGDTGMEIHKYELPEPTKQVEDTHAVDFIIDTIMNNEPGSVALVPTGPLTNIALAVRKEPRIAERVKEVVLMGGY HVGNWTAVAEFNIKIDPEAAHIVFNEKWPLTMVGLDLTHQALATPEIEAKFNELGTDVADFVVALFDAFRKNYQDAQGFDNPPVHDPCAVAYLVDPTVFTTRKAPLDVELYGALTTGMTVADFRAPAPADCTTQVAVDLFDKFWNMVIDAVKRIG SEQ ID NO:21;DNA;purine nucleosidase iunH2;cgl1977(cg2168, iunH2, CYL77_RS09970) ATGAGCAAAAAAGCCATCCTTGATATCGACACCGGCATCGATGATGCCCTCGCACTTGCCTACGCACTGGGCTCACCTGAACTAGAGCTCATTGGTGTCACCACCACCTACGGTAACGTGCTACTCGAAACCGGTGCAGTCAATGACCTGGCACTGCTTGATCTGTTCGGTGCACCAGAAGTACCTGTGTACTTGGGTGAGCCACACGCACAGACCAAGGATGGCTTTGAAGTTCTTGAGATCTCCGCGTTCATTCACGGACAAAACGGCATCGGCGAAGTCGAGCTGCCAGCAAGCGAGTCAAAGGCACTCCCCGGCGCAGTGGATTTCCTCATTGATTCCGTCAACACCCACGGCGATGACCTGGTGATCATCGCAACTGGTCCCATGACCAACCTGTCTGCGGCAATCGCAAAGGATCCAAGCTTTGCTTCCAAGGCTCACGTGGTCATCATGGGTGGCGCCTTGACTGTCCCAGGCAACGTCAGCACATGGGCAGAAGCAAACATCAACCAGGACCCAGATGCAGCAAACGATCTGTTCCGTTCCGGTGCAGATGTCACCATGATCGGTCTTGATGTCACCCTGCAGACCCTTCTTACCAAGAAGCACACTGCGCAGTGGCGCGAACTGGGCACTCCAGCTGCTATCGCACTGGCCGACATGACTGATTACTACATCAAGGCATATGAGACCACCGCACCACACCTGGGCGGTTGCGGCCTGCACGACCCACTGGCAGTAGGCGTTGCAGTGGACCCAAGCCTGGTCACTTTGCTCCCCATCAACCTCAAGGTAGACATTGAGGGCGAGACCCGTGGACGCACCATTGGCGATGAAGTCCGCCTCAACGATCCAGTGCGCACCTCCCGCGCAGCTGTCGCCGTAGACGTGGATCGTTTCCTTTCTGAATTCATGACCCGCATCGGCCGAGTCGCAGCACAGCAGTAA SEQ ID NO:22;PRT;プリンヌクレオシダエiunH2;cgl1977(cg2168、iunH2、CYL77_RS09970) MSKKAILDIDTGIDDALALAYALGSPELELIGVTTTYGNVLLETGAVNDLALLDLFGAPEPVVYLGEPHAQTKDGFEVLEISAFIHGQNGIGEVELPASESKALPGAVDFLIDSVNTHGDDLVIIATGPMTNLSAAIAKDPSFASKAHVVIMGGALTVPGNVSTWAEANINQDPDAANDLFRSGADVTMIGLDVTLQTLLTKKHTAQWRELGTPAAIALADMTDYYIKAYETTAPHLGGCGLHDPLAVGVAVDPSLVTLLPINLKVDIEGETRGRTIGDEVRLNDPVRTSRAAVAVDVDRFLSEFMTRIGRVAAQQ SEQ ID No.:23;DNA;プリンヌクレオシダーズiunH1;cgl2835(cg3137、iunH1、CYL77_RS14340)ATGATTCCTGTTCTCATCGACTGCGACACCGGCATCGACGACGCCCTGCCCCTGATCTACCTGGTTGCTTTGCATAAACGTGGTGAAATCCAACTTTTTGGAGCAACGACCACCGAGGAAATGTTGATGGAAACAAACCGCTCAATCCAGGTGGGTGTTGGATCAGTGTGGATTA GCGGACATCCCGGTCCTCGCAGGACAACCTGAACCAAAGCACGTGCCGCTAGTGACTACTCCAGAAACACACGGCGACCATGGCCTTGGTTATATAAACCCAGGTCACGTCGAAATTCCAGAAGGTGACTGGAAGCAGCTGTGGAAAGAACACCTCAGTAACCCAGAAACTAAGCTGATTGTCACCGGGCCCGCCAACCTTGCGGAATTCGGGCCAGTGGAAAACGTCACGCTGATGGG TGGCACCTACCTTTATCCAGGCAACACCACTCCAACGGCAGAATGGAATACCTGGGTTGATCCACACGGAGCTAAAGAAGCATTCGCGGCAGCCCAAAGCCCATTACGGTGTGTTCCTTGGGCGTGACCGAGCAGTTTACGCTGAACCCGGACATCCTTTCTACACTTATCAACACGCTTGGCAGCCAACCCATCGCAGAGCATTTACCTGAGATGCTGCGCTTTACTTTGAATTTCAC AAGTGCAGGGCGAAGGTTACCTTGCTCAAATTCATGACCTGCTGACCTGCATGATTGCCTTGGATAAAATCCCATTTCAGGCCGTGAAGTAACCGTGGACGTGGAGGCTGATTCGCCTTGATGCGTGGCACCACTGTTGCAGATATTCGCGGACATTGGGGCAAGCCAGCTAACGCATTTCTTGTGGAAACCGCAGACATTGAGGCCGCCCACGCGGAACTTCTAAGAGCAGTGGAAT SEQ ID No.:24;PRT;Purine nucleosidase iunH1;cgl2835(cg3137, iunH1, CYL77_RS14340) MIPVLIDCDTGIDDALALIYLVALHKRGEIQLFGATTTAGNVDVKQTAINTRWVLDQCGLADIPVLAGQPEPKHVPLVTTPETHGDHGLGYINPGHVEIPEGDWKQLWKEHLSNPETKLIVTGPATNLAEFGPVENVTLMGGTYLYPGNT TPTAEWNTWVDPHGAKEAFAAAQKPITVCSLGVTEQFTLNPDILSTLINTLGSQPIAEHLPEMLRFYFEFHEVQGEGYLAQIHDLLTCMIALDKIPFSGREVTVDVEADSPLMRGTTVADIRGHWGKPANAFLVETADIEAAHAELLRAVE SEQ ID No. 25;DNA;glucose-6P isomerase pgi;cgl0851(ncgl0817) SEQ ID No. 26; PRT; Glucose-6P isomerase PGI; cgl0851 (ncgl0817) MADISTTQVWQDLTDHYSNFQATTLRELFKEENRAEKYTFSAAGLHVDLSKNLLDDATLTKLLALTEESGLRERIDAMFAGEHLNNTEDRAVLHTALRLPAEADLSVDGQDVAADVHEVLGRMRDFATALRSGNW LGHTGHTIKKIVNIGIGGSDLGPAMATKALRAYATAGISAEFVSNVDPADLVSVLEDLDAESTLFVIASKTFTTQETLSNARAARAWLVEKLGEEAVAKHFVAVSTNAEKVAEFGIDTDNMFGFWDWVGGRYSVD SAVGLSLMAVIGPRDFMRFLGGFHAMDEHFRTTKFEENVPILMALLGVWYSDFYGAETHAVLPYSEDLSRFAAYLQQLTMESNGKSVHRDGSPVSTGTGEIYWGEPGTNGQHAFFQLIHQGTRLVPADFIGFARP KQDLPAGERTMHDLLMSNFFAQTKVLAFGKNAEEIAAEGVAPELVNHKVMPGNRPTTTILAEELTPSILGALIALYEHIVMVQGVIWDINSFDQWGVELGKQQANDLAPAVSGEEDVDSGDSSTDSLIKWYRANR SEQ ID No. 27; DNA; cgZWF codon-optimized mutant (A243T) of glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum (cg1778, Cgl1576, NCgl1514) SEQ ID No. 28; DNA; cgZWF mutant (A243T) of glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum (cg1778, Cgl1576, NCgl1514) MSTNTTPSSWTNPLRDPQDKRLPRIAGPSGMVIFGVTGDLARKKLLPAIYDLANRGLLPPGFSLVGYGRREWSKEDFEKYVRDAASAGARTEFRENVWERLAEGMEFVRGNFDDDAAFDNLAATLKRIDKTRGTAGNWAYYLSIPPDSFTAVCHQLERSGMAESTEEAWRRVIIEKPFGHNLESAHELNQLVNAVFPESSVFRIDHY LGKETVQNILALRFANQLFEPLWNSNYVDHVQITMAEDIGLGGRAGYYDGIGAARDVIQNHLIQLLALVAMEEPISFVPAQLQAEKIKVLSATKPCYPLDKTS ARGQYAAGWQGSELVKGLREEDGFNPESTTETFAACTLEITSRRWAGVPFYLRTGKRLGRRVTEIAVVFKDAPHQPFDGDMTVSLGQNAIVIRVQPDEGVLIRF GSKVPGSAMEVRDVNMDFSYSESFTEESPEAYERLILDALLDESLFPTNEEVELSWKILDPILEAWDADGEPEDYPAGTWGPKSADEMLSRNGHTWRRP * SEQ ID No. 29; DNA; Codon-optimized mutant (R46E / Q47E) of the gene for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, accession number M64446.1; lmZWF SEQ ID No.30;PRT;Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase mutant (R46E / Q47E);lmZWF MVSEIKTLVTFFGGTGDLAKRKLYPSVFNLYKKGYLQKHFAIVGTAEEALNDDEFKQLVRDSIKDFTDDQAQAEAFIEHFSYRAHDVTDAASYAVLKEAIEEAADKFDIDGNRIFYMSVAP RFFGTIAKYLKSEGLLADTGYNRLMIEKPFGTSYDTAAELQNDLENAFDDNQLFRIDHYLGKEMVQNIAALRFGNPIFDAAWNKDYIKNVQVTLSEVLGVEERAGYYDTAGALLDMIQNHTM QIVGWLAMEKPESFTDKDIRAAKNAAFNALKIYDEAEVNKYFVRAQYGAGDSADFKPYLEELDVPADSKNNTFIAGELQFDLPRWEGVPFYVRSGKRLAAKQTRVDIVFKAGTFNFGSEQE AQEAVLSIIIDPKGAIELKLNAKSVEDAFNTRTIDLGWTVSDEDKKNTPEPYERMIHDTMNGDGGSNFADWNGVSIAWKFVDAISAVYTADKAPLETYKSGSMGPEASDKLLAANGDAWVFKG SEQ ID No.31; DNA; Codon-optimized variant of glucose-6-phosphate dehydrogenase from Zymomonas mobilis strain ATCC 10988 (Zmob_0908) SEQ ID No. 32; PRT; Codon-optimized variant of glu6-phosphate dehydrogenase from Zymomonas mobilis strain ATCC 10988 (NCBI-Protein ID: AEH62743.1) MTNTVSTMILFGSTGDLSQRMLLPSLYGLDADGLLADDLRIVCTSRSEYDTDGFRDFAEKALDRFVASDRLNDDAKAKFLNKLFYATVDITDPTQFGKLADLCGPVEKGIAIYLSTAPSLF EGAIAGLKQAGLAGPTSRLALEKPLGQDLASSDHINDAVLKVFSEKQVYRIDHYLGKETVQNLLTLRFGNALFEPLWNSKGIDHVQISVAETVGLEGRIGYFDGSGSLRDMVQSHILQLVA LVAMEPPAHMEANAVRDEKVKVFRALRPINNDTVFTHTVTGQYGAGVSGGKEVAGYIDELGQPSDTETFVAIKAHVDNWRWQGVPFYIRTGKRLPARRSEIVVQFKPVPHSIFSSSGGILQ PNKLRIVLQPDETIQISMMVKEPGLDRNGAHMREVWLDLSLTDVFKDRKRRIAYERLMLDLIEGDATLFVRRDEVEAQWVWIDGIREGWKANSMKPKTYVSGTWGPSTAIALAERDGVTWYD * 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) MPHSYDYDAIVIGSGPGGEGAAMGLVKQGARVAVIERYQNVGGGCTHWGTIPSKALRHAVSRIIEFNQNPLYSDHSRLLRSSFADILNHADNVINQQTRMRQGFYERNHCEILQGN ARFVDEHTLALDCLDGSVETLTAEKFVIACGSRPYHPTDVDFTHPRIYDSDSILSMHHEPRHVLIYGAGVIGCEYASIFRGMDVKVDLINTRDRLLAFLDQEMSDSLSYHFWNSGVV IRHNEEYEKIEGCDDGVIMHLKSGKKLKADCLLYANGRTGNTDSLALQNIGLETDSRGQLKVNSMYQTAQPHVYAVGDVIGYPSLASAAYDQGRIAAQALVKGEATAHLIEDIPTG IYTIPEISSVGKTEQQLTAMKVPYEVGRAQFKHLARAQIVGMNVGTLKILFHRETKEILGIHCFGERAAEIIHIGQAIMEQKGGGNTIEYFVNTTFNYPTMAEAYRVAALNGLNRLF SEQ ID No. 35; DNA; "A" subunit of membrane-bound pyridine nucleotide transhydrogenase (pnt) from Escherichia coli MG1655; ECK1598 (variant g1342a, NP_416120.1) SEQ ID No. 36; PRT; "A" subunit of membrane-bound pyridine nucleotide transhydrogenase (pnt) from Escherichia coli; P07001.2 (A434T variant) MRIGIPRERLTNETRVAATPKTVEQLLKLGFTVAVESGAGQLASFDDKAFVQAGAEIVEGNSVWQSEIILKVNAPLDDEIALLNPGTTLVSFIWPAQNPELMQKLAERNVTVMAMDSVPRISRAQSL DALSSMANIAGYRAIVEAAHEFGRFFTGQITAAGKVPPAKVMVIGAGVAGLAAIGAANSLGAIVRAFDTRPEVKEQVQSMGAEFLELDFKEEAGSDGYAKVMSDAFIKAEMELFAAQAKEVDIIVTT ALIPGKPAPKLITREMVDSMKAGSVIVDLAAQNGGNCEYTVPGEIFTTENGVKVIGYTDLPGRLPTQSSQLYGTNLVNLLKLLCKEKDGNITVDFDDVVIRGVTVIRAGEITWPAPPIQVSAQPQAA QKAAPEVKTEEKCTCSPWRKYALMALAIILFGWMASVAPKEFLGHFTVFALTCVVGYYVVWNVSHALHTPLMSVTNAISGIIVVGALLQIGQGGWVSFLSFIAVLIASINIFGGFTVTQRMLKMFRKN 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; "B" subunit of membrane-bound pyridine nucleotide transhydrogenase (pntB) from Escherichia coli; P0AB69.1 MSGGLVTAAYIVAAILFIFSLAGLSKHETSRQGNNFGIAGMAIALIATIFGPDTGNVGWILLAMVIGGAIGIRLAKKVEMTEMPELVAILHSFVGLAAVLVGFNSYLHHDAGMAP ILVNIHLTEVFLGIFIGAVTFTGSVVAFGKLCGKISSKPLMLPNRHKMNLAALVVSFLLLIVFVRTDSVGLQVLALLIMTAIALVFGWHLVASIGGADMPVVVSMLNSYSGWAAAA AGFMLSNDLLIVTGALVGSSGAILSYIMCKAMNRSFISVIAGGFGTDGSSTGDDQEVGEHREITAEETAELLKNSHSVIITPGYGMAVAQAQYPVAEITEKLRARGINVRFGIHP VAGRLPGHMNVLLAEAKVPYDIVLEMDEINDDFADTDTVLVIGANDTVNPAAQDDPKSPIAGMPVLEVWKAQNVIVFKRSMNTGYAGVQNPLFFKENTHMLFGDAKASVDAILKAL

Claims

1. 1. A genetically modified microbial cell capable of producing nicotinamide mononucleotide and / or nicotinamide adenine dinucleotide from nicotinamide, comprising: The genetically modified microbial cell is Corynebacterium glutamicum; The genetically modified microbial cell comprises mutations in endogenous genes cgl1364 (SEQ ID NO: 19), cgl1977 (SEQ ID NO: 21), and cgl2835 (SEQ ID NO: 23), each of which encodes a nucleosidase enzyme; The mutation is a deletion, frameshift or point mutation that reduces or eliminates nucleosidase activity. Genetically modified microbial cells.

2. The genetically modified microbial cell of claim 1 , wherein the genetically modified microbial cell is Corynebacterium glutamicum ATCC 13032.

3. 3. The genetically modified microbial cell of any one of claims 1 to 2, further comprising a mutation in the pncA gene, such mutation leading to inactivation or reduced activity of an enzyme capable of deamidating nicotinamide to nicotinic acid.

4. 4. The genetically modified microbial cell of claim 3, wherein the mutation is a deletion, frameshift or point mutation in the pncA gene that reduces or eliminates deamidation activity.

5. 3. The genetically modified microbial cell of any one of claims 1 to 2, further comprising an exogenous gene nadV encoding a nicotinamide phosphoribosyltransferase capable of converting nicotinamide to nicotinamide mononucleotide.

6. 6. The genetically modified microbial cell of claim 5, wherein the exogenous gene nadV is derived from Stenotrophomonas maltophilia.

7. 7. The genetically modified microbial cell of claim 6, wherein the exogenous gene nadV from Stenotrophomonas maltophilia is codon-optimized for expression in Corynebacterium glutamicum.

8. The genetically modified microbial cell of claim 5 , wherein the exogenous gene nadV is derived from Chromobacterium violaceum.

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

10. 6. The genetically modified microbial cell of claim 5, further comprising a prsA gene variant or mutant encoding phosphoribosyl pyrophosphate synthetase.

11. 11. The genetically modified microbial cell of claim 10, wherein the prsA gene variant or mutant is a codon-optimized variant of the prsA gene from Corynebacterium glutamicum ATCC 13032.

12. 11. The genetically modified microbial cell of claim 10, wherein the prsA gene variant or mutant encodes a feedback resistant mutant of phosphoribosyl pyrophosphate synthetase.

13. 6. The genetically modified microbial cell of claim 5, wherein the microbial cell further comprises a genetic modification of the endogenous pyrE gene, leading to reduced or eliminated expression of a phosphoribosyltransferase enzyme.

14. 6. The genetically modified microbial cell of claim 5, further comprising a mutation in the endogenous ushA gene that causes inactivation or reduced activity of a purine nucleosidase enzyme capable of converting nicotinamide mononucleotide to nicotinamide riboside.

15. 6. The genetically modified microbial cell of claim 5, further comprising a genetic modification in the endogenous pncC gene encoding a nicotinamide nucleotide amidase enzyme capable of converting nicotinamide mononucleotide to nicotinic acid mononucleotide, causing inactivation or reduced activity.

16. 6. The genetically modified microbial cell of claim 5, further comprising a genetic modification in the endogenous nadD gene that leads to upregulation of a nicotinic acid-nucleotide adenyltransferase enzyme capable of converting nicotinamide mononucleotide to nicotinamide adenine dinucleotide.

17. 6. The genetically modified microbial cell of claim 5, further comprising expression of a heterologous gene encoding a nicotinic acid-nucleotide adenyltransferase enzyme capable of converting nicotinamide mononucleotide to nicotinamide adenine dinucleotide.

18. 20. The genetically modified microbial cell of claim 17, further comprising a genetic modification in the endogenous nudC gene that leads to reduced or eliminated expression of NADH pyrophosphatase capable of converting NAD(+) to nicotinamide mononucleotide.

19. 8. The genetically modified microbial cell of claim 7, further comprising a conditionally active ribonucleotide reductase encoded by the NrdHIEJ operon.

20. 3. The genetically modified microbial cell of any one of claims 1 to 2, further comprising a mutation in the pgi gene, such mutation leading to inactivation or reduced activity of an enzyme capable of converting D-glucose-6-phosphate into D-ribulose-5-phosphate.

21. 21. The genetically modified microbial cell of claim 20, wherein the mutation is a deletion, frameshift or point mutation in the pgi gene that reduces or eliminates the conversion of D-glucose-6-phosphate to D-ribulose-5-phosphate.

22. 21. The genetically modified microbial cell of claim 20, further comprising an exogenous gene encoding a soluble transhydrogenase.

23. 23. The genetically modified microbial cell of claim 22, wherein the exogenous gene is the udhA gene from Escherichia coli.

24. 24. The genetically modified microbial cell of claim 23, wherein the udhA gene from Escherichia coli is codon-optimized for expression in Corynebacterium glutamicum.

25. 21. The genetically modified microbial cell of claim 20, further comprising an exogenous gene encoding a membrane-bound transhydrogenase.

26. 26. The genetically modified microbial cell of claim 25, wherein the exogenous gene is the pntAB gene from E. coli.

27. 27. The genetically modified microbial cell of claim 26, wherein the pntAB genes from Escherichia coli are codon-optimized for expression in Corynebacterium glutamicum.

28. 28. A method for producing nicotinamide mononucleotide comprising culturing the genetically modified microbial cell of any one of claims 1-27 in the presence of nicotinamide for a period of time sufficient to allow conversion of nicotinamide to nicotinamide mononucleotide.

29. 20. A method for producing nicotinamide adenine dinucleotide comprising culturing the genetically modified microbial cell of claim 16 or claim 18 in the presence of nicotinamide for a period of time sufficient to allow conversion of nicotinamide to nicotinamide adenine dinucleotide.

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