Patent application title: Yeast Strain and Method for Using the Same to Produce Nicotinamide Riboside
Charles M. Brenner (Iowa City, IA, US)
Peter Belenky (Brighton, MA, US)
Katrina L. Bogan (Iowa City, IA, US)
IPC8 Class: AC12N119FI
Class name: Fermentation processes alcoholic beverage production or treatment to result in alcoholic beverage of fruit or fruit material
Publication date: 2012-06-28
Patent application number: 20120164270
The present invention embraces a fungal strain deficient in nicotinamide
riboside import and salvage and use thereof for producing nicotinamide
riboside. Methods for producing nicotinamide riboside and a nicotinamide
riboside-supplemented food product using the strain of the invention are
1. An isolated fungal strain deficient in nicotinamide riboside import
2. The fungal strain of claim 1, wherein said strain does not express Nicotinamide Riboside Kinase 1 (Nrk1), Uridine Hydrolase 1 (Urh1), Purine Nucleoside Phosphorylase (Pnp1), and Nicotinamide Riboside Transporter 1 (Nrt1).
3. The fungal strain of claim 1, wherein said strain secretes at least 8 mg/L nicotinamide riboside.
4. The fungal strain of claim 1, wherein said fungus is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluveromyres, Aspergillus and Pichia.
5. The fungal strain of claim 1, wherein said fungus is Saccharomyces cerevisiae.
6. A method for producing nicotinamide riboside comprising culturing the fungal strain of claim 1 in culture medium and recovering nicotinamide riboside from the medium thereby producing nicotinamide riboside.
7. The method of claim 6, wherein the culture medium comprises nicotinic acid or nicotinamide.
8. The method of claim 6, wherein the fungal strain is cultured to an optical density of at least 3.
9. The method of claim 6, wherein the nicotinamide riboside is recovered by solubilizing nicotinamide riboside from the medium with methanol and subjecting the nicotinamide riboside to column chromatography.
10. A method for producing a nicotinamide riboside-supplemented food product comprising providing a fermentable substrate and fermenting the fermentable substrate in the presence of the fungal strain of claim 1 thereby producing a nicotinamide riboside supplemented food product.
11. A nicotinamide riboside supplemented food product fermented in the presence of the fungal strain of claim 1.
12. The product of claim 11, wherein said food product is wine, beer, cider, kvass, root beer, soy sauce or bread.
BACKGROUND OF THE INVENTION
 Nicotinic acid (NA), nicotinamide (Nam) and nicotinamide riboside (NR) constitute three salvageable NAD.sup.+ precursor vitamins in yeast. NA is imported by the high affinity major facilitator superfamily (MSF) type transporter Tna1 (Llorente & Dujon (2000) FEBS Lett. 475:237-41; Klebl, et al. (2000) FEBS Lett. 481:86-7). However, not all NA import is Tna1-dependent and at concentrations above 1 μM NA, Tna1-independent import is detectable (Llorente & Dujon (2000) supra). NA is converted to NAD.sup.+ via the 3-step Preiss-Handler pathway (Preiss & Handler (1958) J. Biol. Chem. 233:488-92; Preiss & Handler (1958) J. Biol. Chem. 233:493-500). Nam is converted to NA by the nicotinamidase (Pncl) (Ghislain, et al. (2002) Yeast 19:215-24; Anderson, et al. (2003) Nature 423:181-5), for entry into Preiss-Handler salvage. A Nam transporter has not been identified.
SUMMARY OF THE INVENTION
 The present invention features an isolated fungal strain deficient in nicotinamide riboside import and salvage. In one embodiment, the strain does not express Nicotinamide Riboside Kinase 1 (Nrk1), Uridine Hydrolase 1 (Urh1), Purine Nucleoside Phosphorylase (Pnp1), and Nicotinamide Riboside Transporter 1 (Nrt1). In another embodiment, the strain secretes at least 8 mg/L nicotinamide riboside. In a further embodiment, the fungus is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluveromyces, Aspergillus and Pichia. In a specific embodiment, the fungus is Saccharomyces cerevisiae.
 The present invention also embraces a method for producing nicotinamide riboside by culturing the fungal strain of the invention in culture medium and recovering nicotinamide riboside from the medium. In one embodiment, the culture medium further includes nicotinic acid or nicotinamide. In another embodiment, the fungal strain is cultured to an optical density of at least 3. In a particular embodiment, the nicotinamide riboside is recovered by solubilizing nicotinamide riboside from the medium with methanol and subjecting the nicotinamide riboside to column chromatography.
 A method for producing a nicotinamide riboside-supplemented food product is also provided. According to this method, a fermentable substrate is fermented in the presence of the fungal strain of the invention thereby producing a nicotinamide riboside supplemented food product. A nicotinamide riboside supplemented food product fermented in the presence of the fungal strain of the invention is also provided. In some embodiments, the food product is wine, beer, cider, kvass, root beer, soy sauce or bread.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 shows the purification of NR from PAB076-conditioned media. Media collected from PAB076 grown to optical density (OD) 60 in 2xYPD and supplemented with 5 mM NA was cleaned and concentrated by lyophilization followed by resuspension in cold methanol. This material was then loaded directly onto the SP-SEPHADEX resin. FIG. 1 shows the measured absorbance of fractions collected from preparative SP-SEPHADEX chromatography. Salt concentration is depicted below the x-axis. An HPLC chromatogram of each fraction was obtained and selected traces are included as the eight smallest inlays. NR eluted between fraction 27 and 36.
DETAILED DESCRIPTION OF THE INVENTION
 NR is converted into NAD.sup.+ through two distinct pathways. The first pathway utilizes the NR kinase, Nrk1, to produce nicotinamide mononucleotide, which is then converted into NAD.sup.+. The second pathway cleaves NR into Nam and a ribose, by exploiting two independently acting enzymes uridine hydrolase 1 (Urh1) and purine nucleoside phosphorylase (Pnp1). Jointly these pathways are described as the NR salvage pathways and they feed into the NAD.sup.+ cycle in two places.
 It has now been shown that mutants which are deficient in NR salvage (i.e., nrk1 urh1 pnp1) can export NR in an Nrt-independent manner and support the growth of the NR auxotroph, qns1. More significantly, deletion of Nrt1 in a nrk1 urh1 pnp1 strain actually leads to increased extracellular NR accumulation. Moreover, NA or nicotinamide supplementation of a nrk1 urh1 pnp1 nrt1 strain increases NR yield from the strain. Accordingly, the present invention embraces a fungal strain deficient in the salvage and import of NR and use of said strain as a source for the production of NR. In addition, the invention provides a simple and scalable extraction method for inexpensively obtaining NR. Fungal strains of the present invention find application in large-scale production of NR as well as in the processes for fermenting of bread, soy, wine, beer, cider, kvass, root beer and other beverages, thereby providing added value of high nicotinamide riboside content. The nicotinamide riboside produced and isolated according to the present invention finds use in dietary supplement and pharmaceutical compositions for the prevention and treatment of a disease or condition associated with the nicotinamide riboside kinase pathway of NAD+ biosynthesis.
 As indicated, the present invention embraces an isolated fungal strain deficient in nicotinamide riboside import and salvage. For the purposes of the present invention, a "fungal strain deficient in nicotinamide riboside import and salvage" is a strain that fails to import nicotinamide into the cytoplasm and also fails to utilize nicotinamide riboside as a NAD.sup.+ precursor. In one embodiment, the fungal strain is produced by destroying or deleting by knocking out one or more genes involved in import and salvage of NR. Such gene deletions or disruptions are routinely practiced in the art and any conventional method, including those exemplified herein, can be employed.
 In accordance with particular embodiments, the fungal strain of the invention does not express Nicotinamide Riboside Kinase 1 (Nrk1), Uridine Hydrolase 1 (Urh1), Purine Nucleoside Phosphorylase 1 (Pnp1), and Nicotinamide Riboside Transporter 1 (Nrt1). Genes encoding these proteins are known in the art and available from databases such as NCBI Entrez Nucleotide database, the Saccharomyces Genome Database, and the Schizosaccharomyces pombe genome project. For example, Nrk1 is provided under GENBANK accession nos. NP--014270 (S. cerevisiae), NP--595603 (S. pombe), XP--456163 (Kluveromyces lactis), XP--001820220 (Aspergillus oryzae), and XP--001386700 (Pichia stipitis). Similarly, Urh1 is provided under GENBANK accession nos. NP--010688 (S. cerevisiae), NP--593725 (S. pombe), XP--452497 (K. lactis), XP--001816861 (A. oryzae), and XP--001384876 (P. stipitis). Pnp1 is provided under GENBANK accession nos. NP--013310 (S. cerevisiae), NP--593927 (S. pombe), and XP--452943 (K. lactis). In addition, Nrt1 is provided under GENBANK Accession Nos. NP--014714 (S. cerevisiae), NP--595061 (S. pombe), XP--453096 (K. lactis), XP--001821563 (A. oryzae), and XP--001383412 (P. stipitis). Using these known sequences, the skilled artisan can readily disrupt or knockout the genes of interest to obtain a fungal strain deficient in NR transport and salvage. Strains with the desired gene knockouts or deletions can be identified by routine screens including, but not limited to, Southern blot analysis, RT-PCR, northern blot analysis, western blot analysis and the like.
 In certain embodiments, the fungal strain of the present invention is used in the production of pharmaceuticals or in food fermentation, e.g., in the production of bread, wine, beer, cider, kvass, root beer, cheese, or soy sauce. In accordance with such embodiments, the fungal strain of the invention is selected from the genus Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia, or Aspergillus (e.g., A. oryzae or A. sojae). In particular embodiments, the fungal strain is a yeast, e.g., a fungus of the genus Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. boulardii, S. pastorianus, S. rouxii and S. uvarum), Schizosaccharomyces (e.g., S. pombe), Kluveromyces (e.g., K. lactis and K. fragilis) and Pichia. In particular embodiments, the fungus is Saccharomyces cerevisiae.
 Unexpectedly, by blocking NR uptake and salvage, the strain of this invention secretes at least 4.0 μM or 8 mg/L of nicotinamide riboside into the culture medium; a 40-fold increase over production of nicotinamide riboside in a wild-type strain. Furthermore, supplementation of the culture medium with either nicotinic acid or nicotinamide increases nicotinamide riboside production to as much as 7-8 μM, wherein even higher amounts of nicotinamide riboside are produced when the cells are cultured to extremely high densities. For example, S. cerevisiae grown to an optical density (600 nm) of 60 in 2× YPD+5 mM NA was capable of producing 28 pM nicotinamide riboside.
 Thus, given the significant amount of nicotinamide riboside secreted by a fungal strain deficient in NR transport and salvage, the present also features a method for producing nicotinamide riboside by culturing the fungal strain of the invention in growth medium and recovering the methanol-solubilized nicotinamide riboside from the medium. In accordance with this method, the fungal strain is cultured in a fermentation, culture, or growth medium for production of nicotinamide riboside. An appropriate, or effective, culture medium refers to any medium in which a fungal strain of the present invention, when cultured, is capable of producing nicotinamide riboside. Such a medium is typically an aqueous medium composed of assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients. It should be recognized, however, that a variety of fermentation conditions are suitable and can be selected by those skilled in the art based upon art recognized culture conditions and the teachings of the present disclosure. In this regard, particular embodiments embrace the addition of nicotinamide or nicotinic acid to the culture medium. In other embodiments, the culture medium is formulated to support extremely high densities of cells, i.e., an OD600 nm of at least 3.
 Depending on the result to be achieved, the fungus can be cultured under anaerobic (deficient in oxygen) as well as aerobic (oxygenated) conditions. Under aerobic conditions, microorganisms such as yeast cells can break down sugars to end products such as CO2 and H2O. Under anaerobic conditions, yeast cells utilize an alternative pathway to produce CO2 and ethanol. The fermentation reaction of the present invention is preferably anaerobic, i.e., partially or completely deficient in oxygen. Fermentation can also be used to refer to the bulk growth of microorganisms on a growth medium where no distinction is made between aerobic and anaerobic metabolism.
 Fungal strains of the present invention can be cultured in conventional fermentation modes, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous. In a fed-batch mode, when during fermentation some of the components of the medium are depleted, it may be possible to initiate the fermentation with relatively high concentrations of such components so that growth is supported for a period of time before additions are required. The preferred ranges of these components are maintained throughout the fermentation by making additions as levels are depleted by fermentation. Levels of components in the fermentation medium can be monitored by, for example, sampling the fermentation medium periodically and assaying for concentrations. Alternatively, once a standard fermentation procedure is developed, additions can be made at timed intervals corresponding to known levels at particular times throughout the fermentation. The additions to the fermentor may be made under the control of a computer in response to fermentor conditions or by a preprogrammed schedule. Moreover, to avoid introduction of foreign microorganisms into the fermentation medium, addition is performed using aseptic addition methods, as are known in the art. In addition, a small amount of anti-foaming agent may be added during the fermentation, or anti-foaming device may be employed.
 In particular embodiments, recovery of the nicotinamide riboside from the culture medium is achieved by a simple, inexpensive process. The process involves solubilizing the nicotinamide riboside from the medium with methanol leaving behind a methanol-insoluble pellet; and subjecting the nicotinamide riboside to column chromatography to isolate the nicotinamide riboside from other contaminants. To facilitate the solubilization step, the culture medium can be concentrated, e.g., by lyophilization (freeze-drying) or rotoevaporation. In addition to the SP-SEPHADEX column chromatography exemplified herein, nicotinamide riboside can alternatively or also be purified by solid phase extraction, porous graphitic carbon or hydrophilic interaction chromatography. It is contemplated that the number and types of chromatographic columns employed will be dependent on the final use of the nicotinamide riboside and the level of purification desired.
 In so far as yeast and other fungi are routinely used in the production of food products, the present invention also embraces a method for producing a nicotinamide riboside supplemented food product by providing a fermentable substrate and fermenting the fermentable substrate in the presence of the fungal strain of the invention. Food products, which can be produced in accordance with the method of this invention include, but are not limited to, bread, cheese, wine, beer, cider, kvass, root beer, or other beverages. As such, a fermentable substrate is intended to include any substratem which, when fermented, produces the above-referenced food products. Fermentable substrates include, but are not limited to, vegetables, oat, wheat, barley, millet, rice, rye, sorghum, potato, fruits, fruit juices, and the like.
 Nicotinic acid is an effective agent in controlling low-density lipoprotein cholesterol, increasing high-density lipoprotein cholesterol, and reducing triglyceride and lipoprotein (a) levels in humans (see, e.g., Miller (2003) Mayo Clin. Proc. 78(6):735-42). Though nicotinic acid treatment effects all of the key lipids in the desirable direction and has been shown to reduce mortality in target populations, its use is limited because of a side effect of heat and redness termed flushing, which is significantly effected by the nature of formulation. Further, nicotinamide protects against stroke injury in model systems, due to multiple mechanisms including increasing mitochondrial NAD+ levels and inhibiting PARP (Klaidman, et al. (2003) Pharmacology 69(3):150-7). Altered levels of NAD+ precursors have been shown to effect the regulation of a number of genes and lifespan in yeast (Anderson, et al. (2003) Nature 423(6936):181-5).
 NAD+ administration and NMN adenylyltransferase (Nmnatl) expression have also been shown to protect neurons from axonal degeneration (Araki, et al. (2004) Science 305:1010-1013). Because nicotinamide riboside is a soluble, transportable nucleoside precursor of NAD.sub.+, nicotinamide riboside can be used to protect against axonopathies such as those that occur in Alzheimer's Disease, Parkinson's Disease and Multiple Sclerosis. As such administration of nicotinamide riboside or a nicotinamide riboside supplemented-food product could also protect against axonal degeneration.
 NMN adenylytransferase overexpression has been shown to protect neurons from the axonopathies that develop with ischemia and toxin exposure, including vincristine treatment (Araki, et al. (2004) Science 305:1010-1013). Vincristine is one of many chemotherapeutic agents whose use is limited by neurotoxicity. Thus, administration of nicotinamide riboside or a nicotinamide riboside supplemented-food product could be used to protect against neurotoxicity before, during or after cytotoxic chemotherapy.
 Further, conversion of benign Candida glabrata to the adhesive, infective form is dependent upon the expression of EPA genes encoding adhesins whose expression is mediated by NAD+limitation, which leads to defective Sir2-dependent silencing of these genes (Domergue, et al. (March 2005) Science, 10.1126/science.1108640). Treatment with nicotinic acid reduces expression of adhesins and increasing nicotinic acid in mouse chow reduces urinary tract infection by Candida glabrata. Thus, nicotinamide riboside or a nicotinamide riboside-supplemented food product can be used in the treatment of fungal infections, in particular, those of Candida species by preventing expression of adhesins.
 Accordingly, the nicotinamide riboside or a nicotinamide riboside-supplemented food product of this invention could have therapeutic value in improving plasma lipid profiles, preventing stroke, providing neuroprotection with chemotherapy treatment, treating fungal infections, preventing or reducing neurodegeneration, or in prolonging health and well-being. Thus, the present invention is further a method for preventing or treating a disease or condition associated with the nicotinamide riboside kinase pathway of NAD+ biosynthesis by administering an effective amount of a nicotinamide riboside composition. Diseases or conditions which typically have altered levels of NAD+ or NAD+ precursors or could benefit from increased NAD+ biosynthesis by treatment with nicotinamide riboside include, but are not limited to, lipid disorders (e.g., dyslipidemia, hypercholesterolaemia or hyperlipidemia), stroke, neurodegenerative diseases (e.g., Alzheimer's, Parkinsons and Multiple Sclerosis), neurotoxicity as observed with chemotherapies, Candida glabrata infection, and the general health declines associated with aging. Such diseases and conditions can be prevented or treated by diet supplementation or providing a therapeutic treatment regime with a nicotinamide riboside composition.
 An effective amount of nicotinamide riboside is one which prevents, reduces, alleviates or eliminates the signs or symptoms of the disease or condition being prevented or treated and will vary with the disease or condition. Such signs or symptoms can be evaluated by the skilled clinician before and after treatment with the nicotinamide riboside to evaluate the effectiveness of the treatment regime and dosages can be adjusted accordingly.
 The nicotinamide riboside produced in accordance with the method of the invention can be conveniently used or administered in a composition containing the active agent in combination with a pharmaceutically acceptable carrier. Such compositions can be prepared by methods and contain carriers which are well-known in the art. A generally recognized compendium of such methods and ingredients is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000. A carrier, pharmaceutically acceptable carrier, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, is involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
 Examples of materials which can serve as carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
 Nicotinamide riboside produced in accordance with the method of the invention can be administered via any route include, but not limited to, oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into the brain for delivery to the central nervous system). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular compound which is being used.
 A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required for prevention or treatment in an animal subject such as a human, agriculturally-important animal, pet or zoological animal.
 In addition to the specific fungal strains disclosed herein, it is expected that these fungal strains may be further manipulated to achieve other desirable characteristics, or even higher specific yields of fermentation products. For example, selection of strains by passaging the strains of the present invention on medium containing a particular substrate of interest may result in improved fungi with enhanced fermentation rates.
 The invention is described in greater detail by the following non-limiting examples.
Materials and Methods
 Yeast Strains and Medium. All Saccharomyces cerevisiae strains used in this study were derivatives of the common wild-type strain, BY4742. Construction of single deletion strains was according to established methods (Winzeler, et al. (1999) Science 285:901-6). Additional deletions were created by direct transformation with PCR products (Brachmann, et al. (1998) Yeast 14:115-32). Primers employed in the PCR reactions are listed in Table 1.
TABLE-US-00001 TABLE 1 Primer Sequence (5' to 3') SEQ ID NO: 14050 gctctagaCAGACAAGTGGTATGCATATCC 1 14051 cggggtaccGATGTGCTGTGACTGGG 2 14060 gccgctcgagCTTCCCGCTATGTAATAAATAGAGG 3 14061 cgcggatccGCATCATCTGTCAATTTCCTTG 4 14121 NRT1 GAATTTATATTATTCTTTATTGTACTGATATCCCCA 5 Deletion F TTATAACTATCAAAAAAAGGACTTCAGCACCTGTGC GGTATTTCACACCG 14122 NRT1 CTGTACAGATTTTCAAATGAAGCGTTGAAGTTTCCT 6 Deletion R CTTTGTATATTTGAGATCTTCATTTTATCAGATTGT ACTGAGAGTGCA 14124 NRT1 CTAGTGTTGCTACCGCTATTTGTTCTTCG 7 Diagnostic F 14124 NRT1 GCAGTCGAGGATCGATCTGGTAGTATTC 8 Diagnostic R 4750 AATAGCGTGCAAAAGCTATCGAAGTGTGAGCTAGAG 9 TAGAACCTCAAAATAGATTGTACTGAGAGTGCA 4751 CTAATCCTTACAAAGCTTTAGAATCTCTTGGCACAC 10 CCAGCTTAAAGGTCTGTGCGGTATTTCACACCG 14113 CTCTCCGAGCTCGGATTCTTTGTCATCAGACAACTT 11 GTTGAGTGG 14112 GTGCCCAAGCTTGTGTGCCAATGTAGCGTGGTTGCA 12 TG
 pPAB01 was constructed by amplifying the PNP1 gene from wild-type yeast genomic DNA with primers 14061 and 14060. The PCR product was inserted into pRS416 with XhoI and BamHI. pPAB02 was constructed by amplifying the URH1 gene using primers 14051 and 14050. The PCR product was inserted into pRS416 with KpnI and XbaI. Plasmids were confirmed by DNA sequencing and used for construction of deletion strains.
 A yeast strain carrying disruption of the NRK1 locus was made by transformation of the strain BY165-1d with the HIS3 marker introduced into disruption cassette by PCR with primers 4750 and 4751.
 Plasmid pNRT1, carrying NRT1 under the control of its own promoter, was created by amplifying the gene from BY4742 DNA using primers 14112 and 14113. After digestion with SacI and HindIII, the product was inserted into pRS317.
 Strains generated and used herein are listed in Table 2.
TABLE-US-00002 TABLE 2 Name Genotype B4742a MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 PAB011 BY4742 nrt1Δ::kanMX4 PAB038 BY4742 pnp1Δ::kanMX4 urh1::NAT nrk1Δ::HIS3 PAB075 BY4742 nrt1Δ::kanMX4 fun26Δ::URA3 PAB076 BY4742 pnp1Δ::kanMX4 urh1::NAT nrk1Δ::HIS3 nrt1Δ::URA3 PY165-d qns1::URA3 pB175 aBrachmann, et al. (1998) Yeast 14: 115-32.
 NA-free synthetic dextrose complete media (SDC) and its vitamin supplemented forms are described in the art (Wickerham (1946) J. Bacteriol. 52:293-301). 2×SDC and 2×YPD were prepared as the more concentrated forms of the conventional preparation.
 qns1 Bioassay. Strain BY165-1d, the chromosomal deletion of qns1 carrying the QNS1 plasmid pB175 (Bieganowski, et al. (2003) J. Biol. Chem. 278:33049-33055), was plated on 5-FOA plates supplemented with NR to remove pB175. The resulting strain was cultured on NR containing media at all times. Conditioned media was prepared by incubating the specified yeast strain in the appropriate media. After 18 hours the cells were removed by centrifugation followed by filtration. The conditioned media was retained and mixed in a 1 to 1 ratio with fresh 2× SDC. BY165-1d with no pB175 was incubated in the resulting media and growth was measured spectroscopically.
 MALDI-MS NR Quantification. NR content in conditioned media was measured using MALDI-MS. Prior to measurement, [18O] NR was added to the media to a final concentration of 10 μM as an internal standard. One microliter of the [18O] spiked samples was mixed with 1 μl 2,5-Dihydroxy benzoic acid (DHB) matrix, and the mixture was allowed to air dry. The DHB matrix was composed of 50% acetonitrile saturated with DHB. MS spectra were collected on the ABI Voyager-DE Pro MALDI-TOF mass spectrometer and the ratio of the labeled standard to the unlabeled NR was used to determine the NR concentration.
 HPLC Measurements. NA, Nam and NR were also measured using HPLC. Media samples were injected directly onto a Princeton SPHER-60 SAX 60A u (250×4.6mm) column and separated by an isocratic run of 20 mM KH2PO4. Metabolites were detected spectroscopically at 260 nm and quantified by comparison to a standard curve.
 NR Extraction. NR was extracted from 2× YPD. PAB076 was incubated in 500 ml of 2× YPD to an OD600 nm of 60 (˜60 hours). The media was divided into 150 ml portions and frozen at -80° C. As the first step in the purification process, the samples were lyophilized and resuspended in 25 ml of cold methanol. Cold methanol solubilized the NR but left the majority of the contaminants as a pellet after centrifugation. The methanol samples were then lyophilized again and resuspended in 5 ml of water. The aqueous samples were then run over a 10 ml SP-SEPHADEX column, and eluted using a stepped NaCl gradient. NR eluted at 25-50 mM NaCl. Fractions were analyzed using HPLC, and NR was confirmed using MALDI-MS and a biological NAD.sup.+ assay.
 Biological NAD.sup.+ Assay. Yeast cultures were grown with agitation in 0.5 L cultures. During growth, the OD600 nm of 1:10 diluted cells were recorded and 20 ml cultural volumes were pelleted, washed with water, repelleted, and frozen at -80° C. Cell pellets were extracted in 250 ml of ice-cold 1 M formic acid saturated with butanol. After 30 minutes, 62.5 ml of 100% (w/v) trichloroacetic acid was added to each extract, and the samples were allowed to precipitate on ice for 15 minutes. Samples were microcentrifuged for 5 minutes, and the acid soluble supernatants were recovered. Pellets were washed with 125 ml of 20% TCA and repelleted. First and second supernatants were pooled and measured volumetrically. In three 1 ml cuvettes, reactions were assembled containing 10 ml 5 mg/ml alcohol dehydrogenase (two samples) or 10 ml water (control sample), and this was followed by addition of 840 ml 360 mM Tris (pH 9.7), 240 mM lysine, 0.24% (v/v) EtOH, and 150 ml extract. After a 5 minute incubation at room temperature, the spectrophotometer was zeroed against the control sample for determining the alcohol dehydrogenase-dependent increase in absorbance at 340 nm of the duplicate reactions. Mean net absorbances were converted to molar NAD.sup.+ with the extinction coefficient of NADH(6220M-1cm-1) . Molar NAD.sup.+ in the cuvette was converted to molar NAD.sup.+ in the extract by a factor of 6.67. Moles of NAD.sup.+ in the extract were determined from the fraction of the extract assayed. To determine the intracellular volumes corresponding to the extracts and the corresponding intracellular NAD.sup.+ concentrations, a nonlinear conversion between the 1:10 diluted OD600 nm values and the cell number was used (Burke, et al. (2000) Methods in Yeast Genetics, Cold Spring Harbor, N.Y.: Cold Spring Harbor Press) and took the volume of a haploid cell to be 7×10-14 (Sherman (1991) Methods Enzymol. 194:3-21). For cells grown in media containing nicotinic acid, NAD.sup.+ concentrations were determined, in duplicate, 6 to 18 times during the growth of a liquid culture. For cells grown in media without nicotinic acid, the cells were taken with 1:10 diluted OD600 nm values of 0.095-0.105, and the NAD.sup.+ concentrations were determined, in duplicate, from three to eight independent cultures.
NR Export is Nrt1-Independent
 In yeast, NR has activity as a qns1-bypassing and lifespan extending vitamin. It has also been found that NR is an intracellular and extracellular metabolite. On the basis of the discovery of the specific NR transporter, Nrt1 (YOR071C gene), it was of interest to determine whether this importer is responsible for the observed NR export activity.
 The NR-non-salvaging genotype nrk1 urh1 pnp1 (strain PAB038) exhibits reduced NAD.sup.+ levels and exports NR. To test whether Nrt1 is required for the export of NR, NRT1 was deleted in the PAB038 strain through homologous recombination using the URA3 marker to replace NRT1.
 Extracellular NR is detectable using a qns1 bioassay that relies on the NR auxotrophy of the qns1 strain. In this assay, the strains being tested for NR export are grown overnight in SDC medium, at which point the cells are removed and the conditioned media is retained. The qns1 strain is then incubated in medium containing equal measures of conditioned media and fresh 2× SDC. In this assay, the extent of qns1 growth is proportional to the extracellular concentration of NR. Based on qns1 growth, the nrt1 deletion does not reduce extracellular NR. On the contrary NR levels are actually elevated. By comparison to SDC supplemented with purified NR, it was estimated that the NR-non-salvaging strain, PAB038, produced 1 μM extracellular NR when incubated to an OD of 3, whereas the NR-non-salvaging and NR-non-importing strain, nrk1 urh1 pnp1 nrt1 (PAB076), produced 2 μM extracellular NR under the same growth conditions. The excess of extracellular NR in the nrt1 mutant was apparently due to the fact that NR export was Nrt1-independent. The results of this analysis indicated that in strain PAB076, NR can be exported but not reabsorbed, resulting in higher accumulation of extracellular NR by the PAB076 strain.
Increases in NR Yield
 NR has potential to become an important vitamin for daily dietary supplementation and at higher levels a drug for the treatment of disorders like dyslipidemia. One of the hurdles to the development of NR as a product for human consumption has been the difficulty and expense of enzymatic or chemical synthesis. Nicotinamide riboside is costly to produce, largely because of the cost of blocked (i.e., acetylated or benzoylated) ribose used in its organic synthesis (Tanimori, et al. (2002) Bioorg. Med. Chem. 12:1135-1137). As such, improved NR export from yeast may provide a clean and simple biological alternative to the current modes of NR production. It was contemplate that one possible way to upregulate NR export would be to supplement yeast with the inexpensive NAD.sup.+ precursors NA or Nam. Niacin supplementation would have two potentially beneficial effects: first it would help replenish NAD lost in the synthesis of NR and second it would lead to the over expression of NR producing 5' nucleotidases.
 Assaying the content of NR in media conditioned by PAB076 in the presence of 1 mM NA or Nam revealed that supplementation substantially increased the amount of NR produced as assayed by qns1 growth. The extent of qns1 growth was higher than the growth provide by 3 μM NR, indicating that the concentration of NR in the conditioned media was at least 6 μM.
 The qns1 bioassay is an effective method of detecting the presence of low amounts of NR in conditioned media but becomes nonlinear at high concentrations. To more accurately measure the extracellular concentration of NR, MALDI-MS was employed with an internal standard of [18O] NR at a concentration of 10 μM. The concentration of NR in the media was determined from the ratio of the labeled standard to the unlabeled NR.
 Using MS quantification, it was found that wild-type yeast had 0.120±0.4 μM NR, PAB038 (pnp1 urh1 nrk1) had 1.2 μM±0.4 μM NR and PAB076 (pnp1 urh1 nrk1 nrt1) had 4.0±0.9 μM NR, in conditioned medium from cells grown in SDC to an OD of 3 (Table 3). Adding 1 mM NA, increased the extracellular NR produced by both PAB076 and PAB038 to a concentration of 7.7±1.1 μM and 3.9±1.5 μM respectively. Changing the niacin to Nam or supplementing with both niacins did not further improve the NR yield from the PAB076.
TABLE-US-00003 TABLE 3 Strain and Condition [NR] μM Wild-type SDC (OD 3) 0.12 ± 0.4 PAB038 SDC (OD 3) 1.20 ± 0.4 PAB038 SDC + 1 mM NA (OD 3) 3.90 ± 1.5 PAB076 SDC (OD 3) 4.06 ± 0.9 PAB076 SDC + 1 mM NA (OD 3) 7.70 ± 1.1 PAB076 SDC + 1 mM Nam (OD 3) 7.17 ± 0.2 PAB076 SDC + 1 mM Nam & NA (OD 3) 7.30 ± 0.3 PAB076 YPD + 1 mM NA (OD 15) 10.60 ± 5.6 PAB076 2X YPD + 1 mM NA (OD 21) 21.20 ± 4.6 PAB076 SDC + 5 mM NA (OD 7) 16.80 ± 0.3 PAB076 2X SDC + 5 mM NA (OD 13) 20.80 ± 4.2 PAB076 2X YPD + 5 mM NA (OD 60) 28.15 ± 8.5
 By adding NA or Nam, the amount of extracellular NR produced could be doubled. To further increase the yield, cells were cultured to extremely high densities. PAB076 was incubated in YPD, 2× YPD, SDC or 2× SDC and growth was measured over a period of 31 hours. Surprisingly, PAB076 was able to grow to an unusually high density in all three media formulations (Table 4). For example, this strain attained an OD of 29 when grown in YPD and an OD of 35 when grown in 2× YPD. To determine the genetic cause of this phenotype, the growth of other related strains was assayed (Table 4). Only one other strain, nrt1 fun26 (PAB75), had this unusual ability to grow to high cell density. The common element present in these two strains and absent in the others was an intact URA3 gene. URA3 was used to knock out nrt1 in the PAB076 stain and fun26 in PAB075. Other nonrelated strains chosen from lab stocks also had the same URA3-dependent high growth phenotype.
TABLE-US-00004 TABLE 4 Strain and Condition OD at 31 hours nrk1 urh1 pnp1 nrt1 URA3 2X YPD 35.0 nrk1 urh1 pnp1 nrt1 URA3 YPD 29.0 nrk1 urh1 pnp1 nrt1 URA3 SDC 7.0 nrk1 urh1 pnp1 ura3 2X YPD 12.9 nrk1 urh1 pnp1 ura3 YPD 8.1 nrk1 urh1 pnp1 ura3 SDC 6.4 Wild-type (ura3) 2X YPD 12.2 Wild-type (ura3) SDC 5.5 nrt1 ura3 2X YPD 13.0 nrt fun26 URA3 2X YPD 33.7 nrt fun26 URA3 2X YPD 33.2 nrk1 urh1 pnp1 nrt1 URA3 2X YPD 36.1
 Growing cells to extremely high cultural density dramatically increased extracellular NR accumulation (Table 3). Cells incubated in 2× SDC (5 mM NA) to an OD of 13 and cells incubated in 2× YPD (5 mM NA) to an OD of 60 produced the highest amounts of extracellular NR, 20.2±4.3 μM and 28.1±8 μM extracellular NR, respectively. Cells that were incubated in 2× YPD, but did not reach stationary phase produced somewhat less extracellular NR than cells grown to an OD of 60. Similarly, cells incubated in 1× SDC or 1× YPD produced significantly less NR than the cells incubated in the 2× formulations. From this data, it appears that the final concentration of NR is both a function final cell number and whether or not the culture reached stationary.
Purification of NR from PAB076-Conditioned Media
 Cultures of PAB076 (500 mL) were grown in 2× SDC or 2× YPD with 5 mM NA, to an OD of 13 and 60, respectively. To extract NR from this medium, a two step process was implemented that first concentrated NR by lyophilization and methanol extraction, and then separated NR from contaminants using SP-SEPHADEX chromatography. The SP-SEPHADEX fractions were analyzed by HPLC. NA and the majority of the media components eluted in the first 100 ml of the run that contained no salt (FIG. 1). NR was retained by the resin and eluted between 20 and 50 mM NaCl in fractions 27-36. The majority of these fractions were more than 98% pure NR, although the early fractions contained trace amounts of NA. Each fraction was concentrated by lyophilization and the concentration of NR was determined by absorbance at 259 nm. The total yield was ˜700 μg of NR from 150 ml of the media or 5.6 mg/L. Based on MALDI-MS measurements, the concentration of NR in the conditioned 2× YPD prior to extraction was ˜8 mg/L. It was found that NR from fraction 28 and from pooled fractions 31-34 (added at 10 μM) was capable of increasing intracellular NAD.sup.+ in wild-type yeast as efficiently as chemically or enzymatically synthesized NR.
 In so far as 2× SDC media could not be effectively fractionated by SP-SEPHADEX because of the high salt content of this media, conditioned 2× SDC medium would require de-salting (e.g., with a disposable C18 spin columns) prior to chromatography.
 In addition to the above-described approaches, other improvements are contemplated for increasing the yield of NR. These include the use of a chemostat fermenter and the use of industrial scale preparative HPLC chromatography; and genetically engineering a PAB076 strain that also overexpresses the major NMN 5' nucleotidase thereby increasing extracellular NR production and lowering the concentration of NA supplementation. The recommended daily allowance of niacin is 15 mg, and with only slight improvements made possible by industrialization, one liter or less of yeast would be able to produce the daily Niacin requirement in the form of NR.
12130DNAArtificial sequenceSynthetic oligonucleotide 1gctctagaca gacaagtggt atgcatatcc 30226DNAArtificial sequenceSynthetic oligonucleotide 2cggggtaccg atgtgctgtg actggg 26335DNAArtificial sequenceSynthetic oligonucleotide 3gccgctcgag cttcccgcta tgtaataaat agagg 35431DNAArtificial sequenceSynthetic oligonucleotide 4cgcggatccg catcatctgt caatttcctt g 31586DNAArtificial sequenceSynthetic oligonucleotide 5gaatttatat tattctttat tgtactgata tccccattat aactatcaaa aaaaggactt 60cagcacctgt gcggtatttc acaccg 86684DNAArtificial sequenceSynthetic oligonucleotide 6ctgtacagat tttcaaatga agcgttgaag tttcctcttt gtatatttga gatcttcatt 60ttatcagatt gtactgagag tgca 84729DNAArtificial sequenceSynthetic oligonucleotide 7ctagtgttgc taccgctatt tgttcttcg 29828DNAArtificial sequenceSynthetic oligonucleotide 8gcagtcgagg atcgatctgg tagtattc 28969DNAArtificial sequenceSynthetic oligonucleotide 9aatagcgtgc aaaagctatc gaagtgtgag ctagagtaga acctcaaaat agattgtact 60gagagtgca 691069DNAArtificial sequenceSynthetic oligonucleotide 10ctaatcctta caaagcttta gaatctcttg gcacacccag cttaaaggtc tgtgcggtat 60ttcacaccg 691145DNAArtificial sequenceSynthetic oligonucleotide 11ctctccgagc tcggattctt tgtcatcaga caacttgttg agtgg 451238DNAArtificial sequenceSynthetic oligonucleotide 12gtgcccaagc ttgtgtgcca atgtagcgtg gttgcatg 38
Patent applications by Charles M. Brenner, Iowa City, IA US
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