MICROBIAL PRODUCTION OF NICOTINIC ACID RIBOSIDE

Abstract

The present invention relates to a novel method, expression vectors, and host cells for producing nicotinic acid riboside by regulating the pathways that lead to the production of nicotinic acid riboside.

Claims

1-19. (canceled)

20. A genetically modified bacterium capable of producing nicotinic acid riboside (NaR), wherein the bacterium comprises at least one modification selected from a group consisting of: a) blocking or reducing the activity of a protein which functions to repress NAD+ biosynthesis by repressing transcription of nadA, nadB, nadC genes or combinations thereof; b) adding or increasing the transcription of a gene which encodes L-aspartate oxidase, quinolate synthase, quinolate phoshoribosyltransferase, or combinations thereof; and c) blocking or reducing the activity of a protein which functions as a nicotinic acid mononucleotide adenyltransferase; wherein the bacterium with said at least one modification produces an increased amount of NaR than the bacterium without any of said modifications; and further comprising one or more additional modifications selected from the group consisting of: d) blocking or reducing the activity of a protein which functions as a nicotinic acid riboside phosphorylase; e) blocking or reducing the activity of a protein which functions as a nicotinic acid riboside kinase; f) blocking or reducing the activity of a protein which functions as a nicotinic acid riboside transport protein; g) blocking or reducing the activity of a protein which functions as a nicotinic acid phosphoribosyl transferase; h) adding or increasing the activity of a protein which functions as a nicotinamide mononucleotide amidohydrolase; and i) adding or increasing the activity of a protein which functions as a nicotinic acid mononucleotide hydrolase.

21. The bacterium of claim 20, wherein said protein which functions to repress NAD+ biosynthesis is a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 1, 2 or 3 or a variant thereof, wherein said polypeptide has an activity for repressing NAD+ biosynthesis.

22. The bacterium of claim 20, wherein said L-aspartate oxidase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 26 or 27 or a variant thereof, wherein said polypeptide has an activity for converting aspartic acid to iminosuccinic acid in an FAD dependent reaction.

23. The bacterium of claim 20, wherein said quinolate synthase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 23, 24 or 25 or a variant thereof, wherein said polypeptide has an activity for converting iminosuccinic acid and dihydroxyacetone phosphate to quinolate and phosphate.

24. The bacterium of claim 20, wherein said quinolate phosphoribosyltransferase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 28, 29 or 30 or a variant of said polypeptide, wherein said polypeptide has an activity for converting quinolate and phosphoribosylpyrophosphate to nicotinic acid mononucleotide and carbon dioxide.

25. The bacterium of claim 20, wherein the nicotinic acid mononucleotide adenyltransferase protein is a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 4, 5, or 6 or a variant of said polypeptide, wherein said polypeptide has a nicotinic acid mononucleotide adenyltransferase activity for converting nicotinic acid mononucleotide to nicotinic acid adenine dinucleotide.

26. The bacterium of claim 20, wherein the nicotinic acid riboside phosphorylase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 7, 8, 18, or 19 or a variant of said polypeptide, wherein said polypeptide has a nucleoside cleavage activity for converting nicotinic acid riboside to nicotinic acid and ribose phosphate.

27. The bacterium of claim 20, wherein said nicotinic acid riboside kinase is a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or a variant of said polypeptide, wherein said polypeptide has an activity for converting nicotinic acid riboside to nicotinic acid mononucleotide.

28. The bacterium of claim 20, wherein the nicotinic acid riboside transporter is a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 9, 10, or 11 or a variant of said polypeptide, wherein said polypeptide has a nicotinic acid riboside transport activity for importing nicotinic acid riboside.

29. The bacterium of claim 20, wherein the nicotinic acid phosphoribosyl transferase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 15, 16, or 17 or a variant of said polypeptide, wherein said polypeptide has a nicotinic acid phosphoribosyl transferase activity for converting nicotinic acid, 5-phospho-ribose 1-diphosphate, and adenosine triphosphate to nicotinic acid mononucleotide, adenosine diphosphate, diphosphate and phosphate.

30. The bacterium of claim 20, wherein the nicotinamide mononucleotide amidohydrolase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 20, 21, or 22 or a variant of said polypeptide, wherein said polypeptide has a nicotinamide mononucleotide amidohydrolase activity for converting nicotinamide mononucleotide to nicotinic acid mononucleotide.

31. The bacterium of claim 20, wherein the nicotinic acid mononucleotide hydrolase is a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 12, 13, or 14 or a variant of said polypeptide, wherein said polypeptide has a nicotinic acid mononucleotide hydrolase activity for converting nicotinic acid mononucleotide to nicotinic acid riboside.

32. The bacterium of claim 1, wherein said bacterium is selected from a group consisting of: E. coli, B. subtilis, C. glutamicum, A. baylyi and R. eutropha.

33. A method for producing NaR, comprising: culturing a bacterium cell under conditions effective to produce NaR and recovering NaR from the medium and thereby producing NaR, wherein the host microorganism comprises at least one modification selected from the group consisting of: a) blocking or reducing the activity of a protein which functions to repress NAD+ biosynthesis by repressing transcription of nadA, nadB, nadC genes or combinations thereof; b) adding or increasing the transcription of a gene which encodes L-aspartate oxidase, quinolate synthase, quinolate phosphoribosyltransferase, or combinations thereof; and c) blocking or reducing the activity of a protein which functions as a nicotinic acid mononucleotide adenyltransferase.

34. The method of claim 33, wherein the bacterium cell further comprises at least one modification selected from the group consisting of: d) blocking or reducing the activity of a protein which functions as a nicotinate riboside phosphorylase; e) blocking or reducing the activity of a protein which functions as a nicotinic acid riboside kinase; f) blocking or reducing the activity of a protein which functions as a nicotinic acid riboside transport protein; g) blocking or reducing the activity of a protein which functions as a nicotinic acid phosphoribosyl transferase; h) adding or increasing the activity of a protein which functions as a nicotinamide mononucleotide amidohydrolase; and i) adding or increasing the activity of a protein which functions as a nicotinic acid mononucleotide hydrolase.

Description

FIGURES

[0093] FIG. 1. Biochemical pathways for synthesizing quinolate from aspartate and dihydroxyacetone phosphate in the presence of NadA and NadB enzymes using E. coli nomenclature.

[0094] FIG. 2. Biochemical pathways and enzymes for synthesizing nicotinamide adenine dinucleotide using E. coli nomenclature.

[0095] FIG. 3. Biochemical pathways useful for the production of nicotinic acid riboside from intermediates of NAD+ biosynthesis using E. coli nomenclature.

[0096] FIG. 4. biochemical pathways with undesirable activities for nicotinic acid riboside production using E. coli nomenclature. ATP: adenosine triphosphate; pRpp: 5-phospho-alpha-D-ribose 1-diphosphate; PPi: diphosphate; Pi: phosphate.

[0097] FIG. 5. Nicotinic acid riboside levels during fed-batch fermentation of strain ME517.

OVERVIEW OF THE SEQUENCE LISTING

[0098] The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviation for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. In the accompanying sequence listing:

[0099] SEQ ID NO: 1 is the amino acid sequence encoding the trifunctional Escherichia coli NadR enzyme (NMN synthetase, NaR kinase, negative regulator of NAD+ biosynthesis), which is a repressor protein.

[0100] SEQ ID NO: 2 is the amino acid sequence encoding the Bacillus subtilis YxrA enzyme, which is a repressor protein.

[0101] SEQ ID NO: 3 is the amino acid sequence encoding the Corynebacterium glutamicum CgR_1153 enzyme, which is a repressor protein.

[0102] SEQ ID NO: 4 is the amino acid sequence encoding the Escherichia coli NadD enzyme, which is a nicotinic acid mononucleotide adenyltransferase.

[0103] SEQ ID NO: 5 is the amino acid sequence encoding the Bacillus subtilis NadD enzyme, which is a nicotinic acid mononucleotide adenyltransferase.

[0104] SEQ ID NO: 6 is the amino acid sequence encoding the Corynebacterium glutamicum NadD Cg2584 enzyme, which is a nicotinic acid mononucleotide adenyltransferase.

[0105] SEQ ID NO: 7 is the amino acid sequence encoding the Escherichia coli DeoD enzyme, which is a nicotinic acid riboside phosphorylase.

[0106] SEQ ID NO:8 is the amino acid sequence encoding the Bacillus subtilis DeoD enzyme, which is a nicotinic acid riboside phosphorylase.

[0107] SEQ ID NO: 9 is the amino acid sequence encoding the Acinetobacter baylyi PnuC enzyme, which is a NaR transporter protein.

[0108] SEQ ID NO: 10 is the amino acid sequence encoding the Corynebacterium glutamicum PnuC enzyme, which is a NaR transporter protein.

[0109] SEQ ID NO: 11 is the amino acid sequence encoding the Escherichia coli PnuC enzyme, which is a NaR transporter protein.

[0110] SEQ ID NO: 12 is the amino acid sequence encoding the Escherichia coli UshA enzyme, which is a nicotinic acid mononucleotide hydrolase.

[0111] SEQ ID NO: 13 is the amino acid sequence encoding the Bacillus subtilis YfkN enzyme, which is a nicotinic acid mononucleotide hydrolase.

[0112] SEQ ID NO: 14 is the amino acid sequence encoding the Corynebacterium glutamicum Cg0397 enzyme, which is a nicotinic acid mononucleotide hydrolase.

[0113] SEQ ID NO: 15 is the amino acid sequence encoding the Escherichia coli PncB enzyme, which is a nicotinic acid phosphoribosyl transferase.

[0114] SEQ ID NO: 16 is the amino acid sequence encoding the Bacillus subtilis YueK enzyme, which is a nicotinic acid phosphoribosyl transferase.

[0115] SEQ ID NO: 17 is the amino acid sequence encoding the Corynebacterium glutamicum cg2774 enzyme, which is a nicotinic acid phosphoribosyl transferase.

[0116] SEQ ID NO:18 is the amino acid sequence encoding the Bacillus subtilis PupG enzyme, which is a nicotinic acid riboside phosphorylase.

[0117] SEQ ID NO:19 is the amino acid sequence encoding the Bacillus subtilis Pdp enzyme, which is a nicotinic acid riboside phosphorylase.

[0118] SEQ ID NO:20 is the amino acid sequence encoding the Escherichia coli PncC enzyme, which is a nicotinamide mononucleotide amidohydrolase.

[0119] SEQ ID NO:21 is the amino acid sequence encoding the Bacillus subtilis CinA enzyme, which is a nicotinamide mononucleotide amidohydrolase.

[0120] SEQ ID NO:22 is the amino acid sequence encoding the Corynebacterium glutamicum cg2153 enzyme, which is a nicotinamide mononucleotide amidohydrolase.

[0121] SEQ ID NO:23 is the amino acid sequence encoding the Escherichia coli NadA enzyme, which is a quinolate synthase.

[0122] SEQ ID NO:24 is the amino acid sequence encoding the Bacillus subtilis NadA enzyme, which is a quinolate synthase.

[0123] SEQ ID NO:25 is the amino acid sequence encoding the Corynebacterium glutamicum NadA enzyme, which is a quinolate synthase.

[0124] SEQ ID NO:26 is the amino acid sequence encoding the Escherichia coli NadB enzyme, which is a L-aspartate oxidase.

[0125] SEQ ID NO:27 is the amino acid sequence encoding the Bacillus subtilis NadB enzyme, which is a L-aspartate oxidase.

[0126] SEQ ID NO:28 is the amino acid sequence encoding the Escherichia coli NadC enzyme, which is a quinolate phosphoribosyl transferase.

[0127] SEQ ID NO:29 is the amino acid sequence encoding the Bacillus subtilis NadC enzyme, which is a quinolate phosphoribosyl transferase.

[0128] SEQ ID NO:30 is the amino acid sequence encoding the Corynebacterium glutamicum NadC enzyme, which is a quinolate phosphoribosyl transferase.

[0129] The following examples are intended to illustrate the invention without limiting its scope in any way.

EXAMPLES

Example 1: Disruption of the Negative Regulator of NAD+ Biosynthesis

[0130] All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. or Ausubel et al. (J. Sambrook, E. F. Fritsch, T. Maniatis (eds). 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York; and F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (eds.). 1998. Current Protocols in Molecular Biology. Wiley: New York).

[0131] Deletion of the gene encoding the negative regulator of NAD+ biosynthesis (nadR) is accomplished by lambda red mediated recombineering. An antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, is PCR amplified using oligonucleotides that have flanks of 20-50 bps that are is homologous to the region upstream and downstream of the native nadR open reading frame. Alternatively, these flanks may be within the open reading frame, resulting in translation of a non-functional protein. The host strain, for example BL21(DE3), is prepared for lambda red recombineering by transformation with and induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR for successful disruption of the targeted gene.

Example 2: Enhancement of Conversion from L-aspartic acid, dihydroxyacetone phosphate and 1--D-ribosylpyrophosphate to nicotinic acid mononucleotide

[0132] Aspartic acid is oxidized to iminosuccinic acid by the L-aspartate oxidase encoded in E. coli by the nadB gene. This example describes the construction of E. coli strains with alterations to the expression of the native nadB gene. The nadB gene is placed under the control of a strong constitutive promoter. DNA fragments encoding E. coli nadB gene are obtained either by PCR cloning or de novo DNA synthesis. In the case that DNA is obtained by synthesis, codon usage is optimized for expression in E. coli. DNA synthesis and optimization is carried out by GenScript, Inc. The nadB gene is expressed in E. coli under control of an inducible promoter. For example, the open reading frame is cloned into XhoI/NdeI-digested pET24, resulting in plasmid pET24-nadB. Transformation into a strain harboring the T7 polymerase, such as BL21(DE3), allows for IPTG induction of the nadB gene in order to promote NaR synthesis.

[0133] Alternatively, expression of the native nadB gene can also be altered by placing the nadB gene under the control of an inducible promoter. DNA fragments encoding inducible or constitutive promoters, such as the arabinose inducible pBAD or the constitutive pLac promoters, are obtained either by PCR cloning or de novo DNA synthesis. The promoter is fused downstream of an antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, by fusion PCR. This marker-promoter cassette will contain flanks of 20-50 bps that are homologous to the region upstream of the native nadB promoter and to the first nucleotides of the nadB open reading frame. The host strain, for example BL21(DE3) is prepared for lambda red recombineering by transformation with and induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR for successful incorporation of the altered promoter sequence.

[0134] Quinolate synthase, which contains an iron-sulfur cluster, subsequently carries out the condensation and cyclization of iminosuccinic acid with dihydroxyacetone phosphate yielding quinolate and is encoded in E. coli by the nadA gene. This example describes the construction of E. coli strains with alterations to the expression of the native nadA gene.

[0135] The nadA gene is placed under the control of a strong constitutive promoter. DNA fragments encoding E. coli nadA gene are obtained either by PCR cloning or de novo DNA synthesis. In the case that DNA is obtained by synthesis, codon usage is optimized for expression in E. coli. DNA synthesis and optimization is carried out by GenScript, Inc. The nadA gene is expressed in E. coli under control of an inducible promoter. For example, the open reading frame is cloned into XhoI/NdeI-digested pET24, resulting in plasmid pET24-nadA. Transformation into a strain harboring the T7 polymerase, such as BL21(DE3), allows for IPTG induction of the nadA gene in order to promote NaR synthesis.

[0136] Alternatively, expression of the native nadA gene can also be altered by placing the nadB gene under the control of an inducible promoter. DNA fragments encoding inducible or constitutive promoters, such as the arabinose inducible pBAD or the constitutive pLac promoters, are obtained either by PCR cloning or de novo DNA synthesis. The promoter is fused downstream of an antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, by fusion PCR. This marker-promoter cassette will contain flanks of 20-50 bps that are homologous to the region upstream of the native nadB promoter and to the first nucleotides of the nadA open reading frame. The host strain, for example BL21(DE3) is prepared for lambda red recombineering by transformation with and induction of pKD46 as described (Datsenko and Wanner, 2000, Proc. Natl. Acad. U.S.A 97(12):6640-5.). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR for successful incorporation of the altered promoter sequence.

[0137] Quinolate phosphoribosyltransferase transfers the phosphoribosyl moiety from phosphoribosylpyrophosphate to the quinolate nitrogen and catalyzes the subsequent decarboxylation of the intermediate to produce nicotinic acid mononucleotide and is encoded in E. coli by the nadC gene. This example describes the construction of E. coli strains with alterations to the expression of the native nadC gene.

[0138] The nadC gene is placed under the control of a strong constitutive promoter. DNA fragments encoding E. coli nadC gene are obtained either by PCR cloning or de novo DNA synthesis. In the case that DNA is obtained by synthesis, codon usage is optimized for expression in E. coli. DNA synthesis and optimization is carried out by GenScript, Inc. The nadC gene is expressed in E. coli under control of an inducible promoter. For example, the open reading frame is cloned into XhoI/NdeI-digested pET24, resulting in plasmid pET24-nadC. Transformation into a strain harboring the T7 polymerase, such as BL21(DE3), allows for IPTG induction of the nadC gene in order to promote NAR synthesis.

[0139] Alternatively, expression of the native nadC gene can also be altered by placing the nadC gene under the control of an inducible promoter. DNA fragments encoding inducible or constitutive promoters, such as the arabinose inducible pBAD or the constitutive pLac promoters, are obtained either by PCR cloning or de novo DNA synthesis. The promoter is fused downstream of an antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, by fusion PCR. This marker-promoter cassette will contain flanks of 20-50 bps that are homologous to the region upstream of the native nadC promoter and to the first nucleotides of the nadC open reading frame. The host strain, for example BL21(DE3) is prepared for lambda red recombineering by transformation with and induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR for successful incorporation of the altered promoter sequence.

[0140] Alternatively, expression nadA, nadB, and nadC is accomplished by expression in an operon. DNA fragments encoding E. coli nadA, nadB and nadC gene are obtained either by PCR cloning or de novo DNA synthesis. In the case that DNA is obtained by synthesis, codon usage is optimized for expression in E. coli. DNA synthesis and optimization is carried out by GenScript, Inc. Each gene is linked to a 5 ribosome binding site and a 3 terminator sequence. The operon is expressed in E. coli under control of an inducible promoter. For example, the open reading frame is cloned into XhoI/NdeI-digested pET24, resulting in plasmid pET24-nadABC. Transformation into a strain harboring the T7 polymerase, such as BL21(DE3), allows for IPTG induction of the nadABC gene in order to promote NAR synthesis.

Example 3: Blockage or Reduction of Conversion from Nicotinic Acid Mononucleotide (NaMN) to Nicotinic Acid Adenine Dinucleotide (NaAD)

[0141] In E. coli and B. subtilis, NaMN is adenylated by the enzyme NadD. The enzymatic activity is essential for viability as all salvage and de novo pathways to NAD+ require this adenylation activity, however, accumulation of high levels of NaMN is desirable for NaR production. Replacement of the nadD gene with an inducible promoter would prevent these competing reactions, facilitating NaMN overproduction. Alternatively, alleles of the nadD gene with reduced enzyme activity have been characterized. Replacement of the native nadD gene with an allele with lower substrate affinity will decrease the effect of NadD enzyme activity on NaMN levels.

[0142] Many inducible promoters have been described in E. coli and are well characterized In this example, the IPTG inducible Lad promoter from E. coli is fused downstream of an antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, by fusion PCR. This marker-pLac cassette will contain flanks of 20-50 bps that are homologous to the region surrounding the native nadD promoter. The host strain, for example BL21(DE3), is prepared for lambda red recombineering by transformation with induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR and sequencing for successful incorporation of the Lac promoter sequence in place of the native nadD promoter.

[0143] Alleles of E. coli nadD gene with lower activity but that are still able to support growth on minimal medium have been described, for example N40A or T11A. These mutations serve to increase the Km of NadD for NaMN, thereby increasing intracellular NaMN concentrations. Point mutations are introduced in vitro to the nadD gene via the Stragene QuickChange site mutagenesis kit. The mutated gene is fused downstream of an antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, by fusion PCR. This marker-nadD* cassette will contain flanks of 20-50 bps that are homologous to the region surrounding the native nadD gene. The host strain, for example BL21(DE3), is prepared for lambda red recombineering by transformation with induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR and sequencing for successful incorporation of the altered nadD sequence.

Example 4: Enhancement of Conversion from Nicotinic Acid Mononucleotide (NaMN) to Nicotinic Acid Riboside (NaR)

[0144] Secreted NaMN is dephosphorylated to NaR via the periplasmic acid phosphatase encoded in E. coli by the ushA gene. This example describes the construction of E. coli strains with alterations to the expression of the native ushA gene.

[0145] To ensure NMN is dephosphorylated, the ushA gene is placed under the control of a strong constitutive promoter. DNA fragments encoding E. coli ushA gene are obtained either by PCR cloning or de novo DNA synthesis. In the case that DNA is obtained by synthesis, codon usage is optimized for expression in E. coli. DNA synthesis and optimization is carried out by GenScript, Inc. The ushA gene is expressed in E. coli under control of an inducible promoter. For example, the open reading frame is cloned into XhoI/NdeI-digested pET24, resulting in plasmid pET24-ushA. Transformation into a strain harboring the T7 polymerase, such as BL21 (DE3), allows for IPTG induction of the ushA gene in order to promote NAR synthesis.

[0146] Alternatively, expression of the native ushA gene can also be altered by placing the ushA gene under the control of an inducible promoter. DNA fragments encoding inducible or constitutive promoters, such as the arabinose inducible pBAD or the constitutive pLac promoters, are obtained either by PCR cloning or de novo DNA synthesis. The promoter is fused downstream of an antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, by fusion PCR. This marker-promoter cassette will contain flanks of 20-50 bps that are homologous to the region upstream of the native ushA promoter and to the first nucleotides of the ushA open reading frame. The host strain, for example BL21(DE3) is prepared for lambda red recombineering by transformation with and induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR for successful incorporation of the altered promoter sequence.

Example 5: Disruption of the Nicotinamide Adenine Dinucleotide (NAD) Salvage Pathway

[0147] Deletion of the gene encoding the nucleoside phosphorylase (deoD), the nicotinic acid/nicotinamide kinase (nadR) and the gene encoding the nicotinamide riboside uptake transporter (pnuC), either singly or in combination, is accomplished by lambda red mediated recombineering. An antibiotic gene, for example kanamycin resistance or chloramphenicol resistance, is PCR amplified using oligonucleotides that have flanks of 20-50 bps that are homologous to the region upstream and downstream of the native deoD, nadR, or pnuC open reading frame. Alternatively, these flanks may be within the open reading frame, resulting in translation of a non-functional protein. The host strain, for example BL21 (DE3), is prepared for lambda red recombineering by transformation with and induction of pKD46 as described (Datsenko and Wanner, 2000). Prepared cells are transformed by electroporation or chemical transformation and transformants are screened by PCR for successful disruption of the targeted gene. These knockouts may be combined with knockout of nadR (as disclosed in Example 1) or alterations to the NadD activity (as disclosed in Example 2) by assembly of the antibiotic gene with expression cassettes for these genes as described above for the promoter swap.

Example 6: Cell Growth Condition and Protocols

[0148] E. coli strains engineered for the production of NaR are inoculated in LB medium with appropriate antibiotics and grown overnight at 37 C. Washed cells are resuspended in M9 medium with 5% glucose and grown for 3 days at 37 C. Where appropriate, IPTG is added to a concentration of 10-50 uM for induction.

Example 7: Construction of a B. subtilis Strain with Increased Levels of NaR Production

[0149] Cassettes for the precise deletion of nadR, deoD, and pupG were constructed by long flanking PCR (LF-PCR). Flanking regions for each gene were obtained by amplification of BS168 genomic DNA (Roche High Pure PCR template preparation kit) with primers in Table 5, which were designed such that sequences homologous to the 5 or 3 region of the appropriate antibiotic resistance gene (spectinomycin, tetracycline, and neomycin, respectively, SEQ ID NOs: 48 to 50) were incorporated into the PCR product (Phusion Hot Start Flex DNA Polymerase, 200 nM each primer, initial denaturation 2 min @ 95 C, 30 cycles of: 30 sec @ 95 C; 20 sec @ 50 C; 60 sec @ 72 C, final hold 7 min at 72 C). Antibiotic resistance genes were similarly amplified with primers to incorporate sequences homologous to the 5 and 3 flanking regions. PCR products were gel purified and used for LF-PCR with appropriate primers (Table 5) (Phusion Hot Start Flex DNA Polymerase, 200 nM each primer, 150 ng each PCR product, initial denaturation 30 sec @ 98 C, 35 cycles of: 30 sec @ 98 C; 30 sec @ 55 C; 360 sec @ 72 C). LF-PCR product was purified and used for transformation of B. subtilis strains.

[0150] BS168 was transformed with LF-PCR product via natural transformation (Molecular Biological Methods for Bacillus. 1990. Edited by C. R Harwood and S. M. Cutting. John Wiley and Sons) yielding BS6209 (nadR::spe), ME479 (deoD::tet), and ME492 (pupG::neo). Genomic DNA (prepared as above) from ME492 was used to transform BS6209, yielding ME496 (nadR::spe pupG::neo). Genomic DNA (prepared as above) from ME479 was used to transform ME496, yielding ME517 (nadR::spe pupG::neo deoD::tet).

Example 8: Production of Nicotinic Acid Riboside

[0151] ME517 was grown for 8 hours at 37 C in a 500 mL baffled flask containing 50 mL of Seed (per liter: 30 g yeal infusion broth, 5 g bacto yeast extract, 10 g sorbitol, 1 drop Basildon 86/013 antifoam) medium. 1 mL of preculture was used to inoculate 300 mL of Seed medium in 2 L baffled flask and grown 16 hours at 37 C. 80 mL of this seed fermentation was used to inoculate production vessel (NBS) containing 1.2 L batch medium (1096 g H2O, 26.4 g dextrose, 9.6 g KH2PO4, 3.6 g MgSO4*7 H2O, 0.24 g CaCl.sub.2)*H2O, 2.5 g L-tryptophan, 0.036 g MnCl2, 18 g MH4NO3, 0.12 g NaCitrate, 0.24 mL Clerol antifoam, 12 mg Na2EDTA*2 H2O, 57.5 mg ZnSO4*7 H2O, 3.2 mg MnSO4*H2O, 3.2 mg CuSO4, 4.8 mg Na2MoO4*2 H2O, CoCl2*6 H2O, 28 mg FeSO4*7 H2O). Agitation was initially set at 400 rpm, pH was maintained at 6.8 with addition of NH.sub.4OH, and temperature was set at 37 C. During consumption of batch carbon, dO was maintained above 60% by increasing agitation as needed, and following consumption of batch carbon, dO was maintained at 60% by glucose feed. Nicotinic acid riboside was quantified as described below and results are shown in FIG. 5.

Example 9: Detection of Nicotinic Acid Riboside in Production Cultures

[0152] Production cultures were diluted 10 fold in 20% acetonitrile, 0.1% formic acid in water and centrifuged.

[0153] NaR and intermediates were analyzed by liquid chromatography/mass spectrometry (LCMS). 20 l of broth was diluted 1:50 in aqueous 5 mM ammonium acetate at pH 9.8 with 70% acetonitrile prior to centrifugation (5000g, 10 m). The supernatant was removed and injected in 5 l portions onto a HILIC HPLC column (Waters Atlantis C18, 2.1150 mm). Compounds were eluted at a flow rate of 50 uL min-1, using a linear gradient from 5 mM ammonium acetate at pH 9.8 with 70% acetonitrile (A) to 5 mM ammonium acetate at pH 9.8 (B) over 20 minutes followed by a 5 minute hold in B and 10 minutes re-equilibration in A. Eluting compounds were detected with a triple quadropole mass spectrometer using positive electrospray ionization. The instrument is operated in MRM mode to detect NaR. NaR is quantified by comparison with standards (Sigma Aldrich) injected under the identical condition.