# Patent

## Title
Method for the preparation of nicotinic acid

## Abstract

A method for the preparation of nicotinic acid, which includes the step of obtaining a culture solution containing quinolinic acid by incubating a microorganism having an ability to produce quinolinic acid, and the step of adding an acid to the culture solution and conducting a decarboxylation reaction.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is the U.S. national phase of PCT Application No. PCT/KR2012/000370 filed on Jan. 16, 2012, which claims priority to Korean Patent Application No. 10-2011-0004093 filed on Jan. 14, 2011, the disclosures of which are incorporated in their entirety by reference herein.
SEQUENCE LISTING
The text file is Sequence_Listing_PCT_KR2012_000370.txt, created Jul. 5, 2013, and of size 26 KB, filed therewith, is hereby incorporated by reference.
TECHNICAL FIELD
The present invention relates to a method for preparing nicotinic acid via incubation of microorganisms having an ability to produce quinolinic acids and decarboxylation of the quinolinic acids obtained therefrom.
BACKGROUND ART
Nicotinic acid is an oxide of nicotine and is extensively present in animal and plant bodies as a water-soluble vitamin, which is also called vitamin B complex, niacin or vitamin B3. Deficiency of nicotinic acid may result in pellagra disease or neuropathies. Nicotinic acid is generally present in the form of nicotinic acid amide co-enzyme (NAD, NADP) in the living body, and participates in the oxidation-reduction reaction.
Nicotinic acid as usefully utilized in food and medicinal products can be prepared by means of chemical synthetic method or biological producing method. Chemical synthesis of nicotinic acid has been generally accomplished through oxidation using 3-picolne as an oxidizing catalyst. Specifically, 2-methylpentanediamine (MPDA) is subjected to hyperthermal reaction (280 to 360° C.) by means of a catalyst to synthesize 3-picoline, and then 3-picoline is subjected to ammoxidation to produce 3-cyanopyrine, which is then hydrolyzed to synthesize niacinamide or nicotinic acid. Alternatively, nicotinic acid can be directly synthesized from 3-picoline through selective oxidation (Applied Catalysis A: General 280 (2005) 75-82). However, because chemical synthesis results in large quantities of toxic wastes including the catalyst, there is a need of thorough management and great expenses are required for disposal of wastes. In order to solve such problem the method for synthesizing niacin from 3-cyanopyridine using an enzyme has been developed. However, this method also has similar problems due to the use of 3-cyanopyrine which causes a generation of wastes in large quantities. Further, because pyrimidine used as a precursor has various derivatives, and thus, suffers from a great fluctuation in the supply and price thereof, this method may cause instability of niacin price.
In addition, other methods for producing nicotinic acid from quinolinic acid have been disclosed. Chinese Patent CN101353322C discloses the method for synthesis of nicotinic acid using quinolinic acid as the substrate through hydrothermal decarboxylation. The method for producing nicotinic acid proceeds by mixing quinolinic acid with deionized hot water in the ratio of 2:1 to 5:1 and then allowing the mixture to react at a high temperature of 150 to 250° C. and high pressure of 1 to 2 MPa for 5 to 60 minutes (Ind. Eng. Chem. Res. 2009, 48, 10467-10471). This method has an advantage in that no side product of the catalyst is produced, while it has also the problems that the reaction conditions are high temperature and high pressure of 150 to 250° C. and 2 MPa require high energy. All the established chemical synthetic methods use non-renewable materials derived from petroleum as the raw material, and therefore, are greatly influenced by environmental problems or the unit price of petroleum extraction.
In order to solve such problems involved in the chemical synthesis methods, methods for biologically producing nicotinic acid by means of renewable carbohydrate-derived materials has been studied. Biological production of nicotinic acid has been accomplished mainly through two kinds of synthetic pathway. The first one is a pathway to produce quinolinic acid from tryptophan as a starting material, and then biologically synthesize nicotinic acid from the quinolinic acid, and the other is a pathway to produce quinolinic acid from aspartic acid as a starting material, and then biologically synthesize nicotinic acid from the quinolinic acid. In general, eukaryotes biologically synthesize nicotinic acid through the pathway to synthesize nicotinic acid from tryptophan as the starting material, while prokaryotes utilize the pathway to synthesize nicotinic acid from aspartic acid as the starting material as the main pathway. Both pathways comprise quinolinic acid as the intermediate, and synthesize nicotinic acid by the action of quinolinate phosphoribosyltransferase (nadC), nicotinate-mononucleotide adenylyltransferase (nadD), NAD synthetase (nadE), NMN adenylyltransferase (nadR) and nicotinamidase (pncA) from quinolinic acid.
The method for biological production of nicotinic acid utilizing recombinant Escherichia coli or Corynebacterium glutamicum, which produce nicotinic acid through the aspartic acid pathway, has been reported. U.S. Pat. Nos. 6,692,946 and 6,689,587 disclose the methods for producing nicotinic acid by separating the nadA gene and nadC gene, which encode quinolinate synthetase and quinolinate phosphoribosyltransferase, respectively, from the Corynebacterium glutamicum (ATCC 13032) strain, and then, incubating host cells which over-express such genes. The amount of nicotinic acid produced by the methods for biological production of nicotinic acid as disclosed in said US patents is very low, below 100 mg/L. It is considered that the causes of this low production include transcriptional suppression by NadR, which is an NAD-related transcriptional repressor of nadB as the gene coding for aspartate oxidase and nadA as the gene coding for quinolinate synthetase (Gerasimova AV (2005). J Bioinform Comput Biol 3(4); 1007-19.), feedback inhibition of aspartate oxidase and NAD synthetase with NAD (Biol Chem Hoppe Seyler. 1990 March; 371(3):239-48), complexity of the reaction including the steps by NadB, NadA, NadC, as well as NadD, NadE, NadR and PncA, and the like.
The methods for biological production of nicotinic acid have the disadvantages in that the production yield of nicotinic acid is very low due to inhibition of the expression of enzymes involved in said biosynthetic pathways, feedback inhibition and complexity of reaction.
DISCLOSURE OF INVENTION
Technical Problem
In light of the above-mentioned technical challenges, the present inventors have conducted a study to solve the problems involved in the chemical synthesis and biological production methods for nicotinic acid, and to improve the production yield of nicotinic acid, and thus, complete the present method for producing nicotinic acid in a high yield through the combination of a biological production method and a chemical synthesis method.
Solution to Problem
The purpose of the present invention is to provide a method for the preparation of nicotinic acid, which comprises the step of obtaining a culture solution containing quinolinic acid by incubating a microorganism having an ability to produce quinolinic acid, and the step of adding an acid to the culture solution and conducting a decarboxylation reaction.
Advantageous Effects of Invention
By replacing the prior process for preparing nicotinic acid via chemical synthesis with the process for producing quinolinic acid via fermentation, and providing the process for converting quinolinic acid into nicotinic acid through the addition of acid to the culture solution containing quinolinic acid and decarboxylation reaction, the present invention can solve the problems involved in the prior methods, including the catalyst side products, the requirement of high energy and environmental problem caused by use of non-renewable resources in chemical synthesis, and the low yield in biological production, thereby producing nicotinic acid in a more environmental-friendly and efficient manner.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a pathway for the preparation of nicotinic acid in the method for preparing nicotinic acid according to one embodiment of the present invention.
FIG. 2 shows the construction of pPro-nadBA as the expression plasmid of genes coding for aspartate oxidase and quinolinate synthetase.
FIG. 3 shows the result of HPLC to identify quinolinic acid in the culture solution and nicotinic acid obtained after decarboxylation reaction of said culture solution, according to one embodiment of the present invention.



BEST MODE FOR CARRYING OUT THE INVENTION
In one aspect, the present invention provides a method for the preparation of nicotinic acid, which comprises the step of obtaining a culture solution containing quinolinic acid by incubating a microorganism having an ability to produce quinolinic acid, and the step of adding an acid to the culture solution and conducting a decarboxylation reaction.
As used herein, the term “microorganism having an ability to produce quinolinic acid” denotes microorganisms which can produce quinolinic acid from a carbon source in a culture medium and accumulate them.
As used herein, the term “decarboxylation” denotes a reaction to produce nicotinic acid by decarboxylation of the reactant, i.e. quinolinic acid.
In one embodiment of the present invention, the microorganism having an ability to produce quinolinic acid can be microorganism whose ability to produce quinolinic acid is improved through weakening or removal of quinolinate phosphoribosyltransferase activity and enhancement of aspartate oxidase and quinolinate synthetase activities.
In another embodiment of the present invention, quinolinate phosphoribosyl-transferase can have the amino acid sequence equivalent to SEQ ID NO: 21 or a sequence having a high homology thereto; aspartate oxidase can have the amino acid sequence equivalent to SEQ ID NO: 19 or a sequence having a high homology thereto; and quinolinate synthetase can have the amino acid sequence equivalent to SEQ ID NO: 20 or a sequence having a high homology thereto.
To improve the ability to produce quinolinic acid, it is required that microorganisms produce large quantities of quinolinic acid and quinolinic acid thus produced can be accumulated without being used in another pathway. Therefore, in the present invention, microorganism having an improved ability to produce quinolinic acid can be prepared by the way to remove or weaken the activity of quinolinate phosphoribosyltransferase, which acts on the decomposition pathway of quinolinic acid, the way to enhance the expression of aspartate oxidase and quinolinate synthetase, which act on the synthetic pathway of quinolinic acid, or the combination thereof.
In still another embodiment of the present invention, said weakening or removal of quinolinate phosphoribosyltransferase activity can be achieved by one or more way selected from the way to replace an endogenous gene coding for quinolinate phosphoribosyltransferase with a modified gene whose enzyme activity is weakened or removed, the way to replace an endogenous promoter for said gene with a promoter whose activity is weaker than that of the endogenous promoter, or the way to delete said gene from chromosome.
In still another embodiment of the present invention, said enhancement of aspartate oxidase and quinolinate synthetase activities can be achieved by one or more way selected from the way to increase the genomic copy number of intracellular genes coding for aspartate oxidase and quinolinate synthetase, the way to modify expression regulatory sequences of said genes, and the way to replace said gene with a modified gene whose enzyme activity is enhanced.
In yet another embodiment of the present invention, in order that quinolinic acid can be accumulated in the culture solution of microorganisms, the promoter portion of nadB as the gene coding for aspartate oxidase protein is substituted with a constitutive promoter, pPro, of SEQ ID NO: 16 to construct the constitutive expressible nadB gene, which is not suppressed by NadR as the transcriptional repressor to suppress the expression of nadB gene with intracellular NAD level, in the form of a plasmid, and said plasmid is then introduced into microorganisms to induce over-expression of aspartate oxidase.
In another embodiment of the present invention, aspartate oxidase can have the amino acid sequence of SEQ ID NO: 19. The sequence of gene nadB encoding said enzyme can be obtained from the genome sequence (gi: GI:89109380) of Escherichia coli as disclosed in Mol Syst Biol. 2006; 2:2006.0007. Epub 2006 Feb. 21, or the database available from the National Center for Biotechnology Information (NCBI) and the DNA Data Bank for Japan (DDBJ).
Aspartate oxidase has an activity to oxidize aspartic acid to iminosuccinic acid, as shown in the following reaction scheme:

L-Aspartate+Fumarate<=>α-Iminosuccinate+Succinate+H+

L-Aspartate+Oxygen<=>Hydrogen Peroxide+α-Iminosuccinate+H+

Therefore, if the activity of aspartate oxidase is enhanced, accumulation of iminosuccinic acid, as the precursor of quinolinic acid in cells can be increased, thereby increasing the production of quinolinic acid.
In still another embodiment of the present invention, in order to increase the accumulation of quinolinic acid, the promoter of the gene coding for quinolinate synthetase protein is substituted with a stronger promoter, pCysK of SEQ ID NO: 17, to construct the constitutive expressible nadA gene, which is not suppressed by NadR, as the transcriptional repressor to suppress the expression of nadA gene with intracellular NAD level, in the form of a plasmid, and said plasmid is then introduced into microorganisms to induce over-expression of quinolinate synthetase.
In still another embodiment of the present invention, quinolinate synthetase can have the amino acid sequence of SEQ ID NO: 20. The sequence of gene nadA encoding said enzyme can be obtained from the genome sequence (gi: GI: 89107601) of Escherichia coli as published in Mol Syst Biol. 2006; 2:2006.0007. Epub 2006 Feb. 21, or the database available from the National Center for Biotechnology Information (NCBI) and the DNA Data Bank for Japan (DDBJ).
Quinolinate synthetase has an activity to synthesize quinolinic acid from iminosuccinic acid, as shown in the following reaction scheme:

α-Iminosuccinic acid+Dihydroxyacetone phosphate<=>Quinolinate+Phosphate+2H2O

Therefore, if the expression of the gene encoding quinolinate synthetase or the activity of said enzyme is enhanced, the production of quinolinic acid in cells can be increased.
In microorganism having the ability to produce quinolinic acid, the activities of aspartate oxidase and quinolinate synthetase can be enhanced by substituting the endogenous promoters of genes coding for aspartate oxidase and quinolinate synthetase with a stronger promoter, or by introducing a mutation in the promoters to increase the activity thereof or increasing the copy number of said genes, respectively. For substitution with said stronger promoter, those generally known as being stronger promoters, including pTac, pTrc, pPro, pR, pL, pCJ1, pCysK, etc., can be used.
In still another embodiment of the present invention, the promoters of genes nadB and nadA participating in the biosynthesis of quinolinic acid can be substituted with a stronger promoter pPro or pCysK to prepare microorganism strains, which overexpress said genes thereby having an improved ability to produce quinolinic acid. As the promoter substituting for the endogenous promoter to increase the expression of said genes, promoters pPro and pCysK of SEQ ID NOs: 17 and 18, respectively, or a portion thereof can be used.
In addition, in order that microorganism can further accumulate quinolinic acid, the activity of quinolinate phosphoribosyltransferase, as the enzyme to convert quinolinic acid into nicotinate mononucleotide, which is located on the genome of microorganisms having the ability to produce quinolinic acid, can be removed. For this purpose, nadC as the gene coding for quinolinate phosphoribosyltransferase can be removed from the genome of microorganism by means of homologous recombination. The sequence of the gene nadC can be obtained from the genome sequence (gi: GI: 89106990) of Escherichia coli as published in Mol Syst Biol. 2006; 2:2006.0007. Epub 2006 Feb. 21, or the database available from the National Center for Biotechnology Information (NCBI) and the DNA Data Bank for Japan (DDBJ).
In still another embodiment of the present invention, said phosphoribosyltransferase can have the amino acid sequence of SEQ ID NO: 21.
Quinolinate phosphoribosyltransferase has an activity to synthesize nicotinate mononucleotide from quinolinic acid, as shown in the following reaction scheme. Therefore, if the gene having said activity is removed or the expression thereof is weakened, the production of quinolinic acid in cells can be increased.

5-Phospho-α-D-ribose 1-diphosphate+Quinolinate+2H+<=>CO2+diphosphate+Nicotinate mononucleotide

In still another embodiment of the present invention, microorganisms having an ability to produce quinolinic acid can be prokaryotic and eukaryotic microorganism strains. Said microorganisms having an ability to produce quinolinic acid can include, but are not be limited to, those belonging to Enterbacter genus, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus or Brevibacterium genus.
Preferably, the microorganisms having an ability to produce quinolinic acid can be those belonging to Escherichia genus and more preferably, Escherichia coli. 
In still another embodiment of the present invention, E. coli variant strain, TF4076 (KFCC 10718, Korean Patent Publication No. 92-8365), which produces L-threonine can be used as the parent strain for improving the ability to produce quinolinic acid. Escherichia coli TF4076 requires methionine or is resistant to threonine analogues (AHV: α-amino-β-hydroxy valeric acid), lysine analogues (AEC: S-(2-aminoethyl)-L-cysteine), isoleucine analogues (α-aminobutyric acid), methionine analogues (ethionine), etc.
Said Escherichia coli strain TF4076 can be modified to enhance the activities of aspartate oxidase and quinolinate synthetase, and to remove the activity of quinolinate phosphoribosyltransferase, thereby preparing the microorganism having an improved ability to produce quinolinic acid.
In yet another embodiment of the present invention, the microorganism having an ability to produce quinolinic acid can be Escherichia coli, which has the enhanced pathway for biosynthesis of quinolinic acid, through enhancement of the expression of genes coding for aspartate oxidase and quinolinate synthetase and removal and lowering of the activity of quinolinate phosphoribosyltransferase from the threonine-producing strain, TF4076 (KFCC 10718, Korean Patent Publication No. 92-8365), which has the enhanced pathway for biosynthesis of aspartic acid.
In still another embodiment of the present invention, microorganisms having an ability to produce quinolinic acid can be strains derived from lysine, threonine, isoleucine or methionine-producing microorganism strains of which the biosynthesis pathway for aspartic acid is enhanced.
A quinolinic acid-producing strain, the Escherichia coli CV01-0009 strain, which is prepared through enhancement of the expression of genes coding for aspartate oxidase and quinolinate synthetase and the removal and lowering of the activity of quinolinate phosphoribosyltransferase from Escherichia coli TF4076 strain, was deposited under the Budapest Treaty at the Korean Culture Center of Microorganisms (KCCM, located on Hongjae 1-Dong, Seodaemun-Gu, Seoul, Korea) with Accession No. KCCM11165P on Jan. 10, 2011.
The method for preparing nicotinic acid according to the present invention comprises the step of incubating the microorganism having an ability to produce quinolinic acid to obtain the culture solution containing quinolinic acid.
The incubation of microorganisms having an ability to produce quinolinic acid can be accomplished using a suitable culture medium under suitable culture conditions are as well-known in the relevant technical field. Such incubation procedures can be used by a person skilled in the relevant technical field and are readily adjusted according to the selected microorganism. The methods for incubation include, but are not limited to, batch, continuous and fed-batch cultures. Various methods for incubation of microorganisms have been disclosed in, for example, “Biochemical Engineering” by James M. Lee, Prentice-Hall International Editions, pp 138-176.
The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are to be found in the handbook [“Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981)]. The said culture media contains various carbon source, nitrogen source and microelement.
The useful carbon source may include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, groundnut oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. Those substances may be used individually or in the form of a mixture.
The useful nitrogen source may include organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or in the form of a mixture.
The useful phosphorus source may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium can also contain metal salts such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, may be used in addition to the above-mentioned substances. Suitable precursors may be also added to the culture medium. The mentioned substances may be added to the culture by continuous or batch type in a suitable manner during the cultivation.
Further, in order to control the pH value of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia or acid compounds such as phosphoric acid or sulfuric acid, are expediently used. In order to control the development of foam, an anti-foaming agent such as fatty acid polyglycol esters may be used. In order to maintain aerobic conditions, oxygen or oxygen-containing gas such as air is introduced into the culture. The temperature of the culture is normally from 20° C. to 45° C., and preferably from 25° C. to 40° C. The culture is continued until the expectative amount of the quinolinic acid has formed. That aim is normally achieved within a period of from 10 hours to 160 hours.
The method for preparing nicotinic acid according to the present invention comprises the step of conducting decarboxylation by adding the acid to the culture solution containing quinolinic acid.
More specifically, the culture solution containing quinolinic acid obtained from incubation of the microorganism having an ability to produce quinolinic acid is subjected to centrifugation or membrane filtration to remove the microorganisms. Then, to accelerate the decarboxylation reaction, the acid to provide hydrogen group is added to the culture solution containing quinolinic acid. Any acid can be used without restriction on the kind, as long as it can provide hydrogen group to the culture solution.
In one embodiment of the present invention, the culture solution containing quinolinic acid can be utilized without purification.
In another embodiment of the present invention, acids added to said culture solution can be hydrochloric acid or sulfuric acid.
In yet another embodiment of the present invention, after the addition of said acid, the culture solution can have the pH value of 5 or less.
In still another embodiment of the present invention, after the addition of said acid, the culture solution can have the pH value of 2 to 3.
In still another embodiment of the present invention, the decarboxylation of the culture solution can be conducted at a temperature ranges 100° C. to 150° C.
In still another embodiment of the present invention, the decarboxylation of the culture solution can be conducted at a temperature of 135° C.
In still another embodiment of the present invention, the decarboxylation of the culture solution can be conducted at a pressure ranges 0.1 MPa to 0.5 MPa.
In still another embodiment of the present invention, the decarboxylation of the culture solution can be conducted at a pressure of 0.2 MPa.
Upon conducting the decarboxylation under the high temperature and high pressure conditions for 1 to 3 hours after adding the acid to the fermentation solution containing quinolinic acid, quinolinic acid present in the culture solution is converted into nicotinic acid as shown in the following reaction scheme:

Quinolinate+2H+<=>CO2+Nicotinate

The method for preparing nicotinic acid according to the present invention can further comprise steps for recovering and purifying nicotinic acid.
In the present invention, the recovery of nicotinic acid can be accomplished by any conventional method as known in the technical field to which the present invention belongs and which comprises the procedures for filtrating and crystallizing the culture solution.
Mode for the Invention
Hereinafter, it is intended to more specifically explain the present invention through Examples and Experimental Examples. However, these Examples are provided only to illustrate the present invention more in detail and the scope of the present invention is not limited by these Examples.
EXAMPLE 1
Preparation of Quinolinic Acid-Producing Strain
1-1. Construction of Plasmid for Expression of Aspartate Oxidase
The gene nadB coding for aspartate oxidase was obtained through PCR using chromosomal DNA of Escherichia coli W3110 as the template. On the basis of the base sequence for the nadB gene (NCBI Registration No. “GI:89109380”) of SEQ ID NO: 13 obtained from the GenBank of the National Institute of Health (NIH GenBank), the ATG region and ORF region containing the TAA in nadB gene could be amplified, and primers of SEQ ID NOs: 1 and 2 having the recognition sites of restriction enzymes NdeI and BamHI were synthesized.
PCR was conducted using chromosomal DNA of Escherichia coli W3110 as the template and oligonucleotides of SEQ ID NOs: 1 and 2 as the primer. PfuUltraTM DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted by repeating the cycle 30 times comprising denaturation at 96° C. for 30 seconds, annealing at 50° C. for 30 seconds and extension at 72° C. for 2 minutes. Thus, amplified gene of about 1.9 kb, which contains nadB gene and the recognition sites of restriction enzymes NdeI and BamHI was obtained.
The nadB gene obtained through said PCR procedures was treated with restriction enzymes NdeI and BamHI, and is then cloned by ligating into pProLar (CloneTech) vector treated with restriction enzymes NdeI and BamHI to ultimately construct pPro-nadB recombinant vector into which the nadB gene, of which the expression is controlled under pPro promoter as the constitutive promoter, is cloned.
1-2. Construction of Plasmid for Expression of Aspartate Oxidase and Quinolinate Synthetase
The gene nadA coding for quinolinate synthetase was obtained through PCR using chromosomal DNA of Escherichia coli W3110 as the template. On the basis of the base sequence for the nadA gene (NCBI Registration No. “GI:89107601”) of SEQ ID NO: 14 obtained from the GenBank of the National Institute of Health (NIH GenBank), the ATG region and ORF region containing TAA in nadA gene could be amplified, and primers of SEQ ID NOs: 3 and 4 having the recognition sites of restriction enzymes ApaI and NotI were synthesized.
PCR was conducted using chromosomal DNA of Escherichia coli W3110 as the template and oligonucleotides of SEQ ID NOs: 3 and 4 as the primer. PfuUltraTM DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted by repeating the cycle 30 times comprising denaturation at 96° C. for 30 seconds, annealing at 50° C. for 30 seconds and extension at 72° C. for 2 minutes. Thus, amplified gene of about 1.0 kb, which contains nadA gene and the recognition sites of restriction enzymes ApaI and NotI was obtained.
cysK promoter was obtained through PCR using chromosomal DNA of Escherichia coli W3110 as the template. On the basis of the base sequence information (SEQ ID NO: 17) for promoter located within upstream 0.3 kb of cysK gene obtained from the GenBank of the National Institute of Health (NIH GenBank), primers of SEQ ID NOs: 5 and 6 having the recognition sites of restriction enzyme BamHI and ApaI were synthesized for ligating cysK promoter with said amplified nadA gene.
PCR was conducted using chromosomal DNA of Escherichia coli W3110 as the template and oligonucleotides of SEQ ID NOs: 5 and 6 as the primer. PfuUltraTM DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted by repeating the cycle 30 times comprising denaturation at 96° C. for 30 seconds, annealing at 50° C. for 30 seconds and extension at 72° C. for 1 minute. Thus, amplified gene of about 0.3 kb, which contains cysK promoter and the recognition sites of restriction enzymes BamHI and ApaI was obtained.
The nadA gene obtained through said PCR procedures was treated with restriction enzymes ApaI and NotI, and amplified cysK promoter fragment was treated with ApaI and BamHI. The restriction enzyme treated nadA and cysK promoter fragments were cloned by ligating into the NotI and BamHI-treated pPro-nadB vector obtained from the above 1-1 to ultimately construct pPro-nadBA recombinant vector into which the nadB gene, of which the expression is controlled under pPro promoter as the constitutive promoter, and the nadA gene of which the expression is controlled by cysk gene promoter, are cloned. Constructed pPro-nadBA has the sequence of SEQ ID NO: 18. FIG. 2 shows the construction of pPro-nadBA as the expression plasmid of genes coding for aspartate oxidase and quinolinate synthetase.
1-3. Construction of Quinolinate Phosphoribosyltransferase-Deficient Strain
In the present example, the nadC gene involved in the decomposition pathway of quinolinic acid was obtained through PCR using chromosomal DNA of Escherichia coli TF4076 as the template. On the basis of the base sequence information of the nadC gene (NCBI Registration No. “GI:89106990”) obtained from the GenBank of the National Institute of Health (NIH GenBank), primers of SEQ ID NOs: 7 and 8 to amplify the downstream region of nadC gene, primers of SEQ ID NOs: 9 and 10 to amplify the upstream and downstream regions of nadC gene and loxpCm, and primers of SEQ ID NOs: 11 and 12 to amplify the upstream region of nadC gene, were synthesized.
PCR was conducted using chromosomal DNA of Escherichia coli TF4076 as the template and oligonucleotides of SEQ ID NOs: 7 and 8, and 11 and 12 as the primer to amplify the downstream and upstream regions of nadC gene of 0.5 kb and 0.3 kb, respectively. In addition, PCR was conducted using the plasmid vector containing loxpCm, pLoxpCat2 vector as the template, and oligonucleotides of SEQ ID NOs: 9 and 10 as the primer to amplify loxpCm gene having the sequence homologous to nadC gene on both ends of 1.0 kb. PfuUltraTM DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted by repeating the cycle 30 times comprising denaturation at 96° C. for 30 seconds, annealing at 53° C. for 30 seconds and extension at 72° C. for 1 minute. Then, nadC-upstream fragment, nadC-downstream fragment, and loxpCm section obtained from said PCR reactions were used as template to conduct PCR under PCR conditions including 10 repetition of the cycle comprising denaturation at 96° C. for 60 seconds, annealing at 50° C. for 60 seconds and extension at 72° C. for 1 minute, and 20 repetition of said cycle after addition of primers of SEQ ID NOs: 7 and 12. Thus, a nadC deficient cassette containing the upstream region of nadC gene-loxpCm-downstream region of nadC gene of 1.8 kb was obtained.
Escherichia coli TF4076 containing pKD46 as lambda red recombinase expression vector was transformed with the nadC deficient cassette by means of electroporation, and then the strain was smeared on LB (Luria-Bertani) plating medium (tryptone 10 g, yeast extract 5 g, NaCl 10 g, and agar 1.5%/L) containing chloramphenicol as the selective marker and incubated at 37° C. overnight to select microorganism strains displaying a resistance against chloramphenicol.
Strains selected as the template were directly subjected to PCR using primers of SEQ ID NO: 7 and 12 under the same conditions, and then the deletion of nadC gene was confirmed by identifying the gene size in wild strain and nadC-deficient strain to be 1.0 kb and 1.8 kb, respectively, on 1.0% agarose gel. In addition, nadC gene was also removed from E. coli W3110 as the wild strain according to the same method as above.
1-4. Preparation of Quinolinic Acid-Producing Strain
The pPro-nadBA plasmid constructed in Example 1-3 was used through CaCl2 method to transform TF4076ΔnadC strain and W3110ΔnadC, as constructed in Example 1-3, which were then smeared on LB-Km plating medium (yeast extract 10 g/L, NaCl 5 g/L, tryptone 10 g/L, kanamycin 25 μg/L) and incubated at 37° C. overnight. Then, kanamycin-resistant 10 colonies were selected. The prepared strain for producing quinolinic acid thus constructed was designated as CV01-0009.
EXAMPLE 2
Preparation of Nicotinic Acid
2-1. Production of Quinolinic Acid
The strain producing quinolinic acid as prepared in Example 1 was incubated in LB-Km plating medium within the incubator at 37° C. overnight to obtain a single colony, which was then inoculated on 25 ml of quinolinic acid titer medium by 1 platinum loop and incubated with 250 rpm at 37° C. for 24 to 72 hours. The following Table 1 shows the composition of the medium for producing quinolinic acid.










TABLE 1







Composition
Conc. (per L)




















Glucose
70
g



Ammonium Sulfate
17
g



KH2PO4
1.0
g



MgSO4•7H2O
0.5
g



FeSO4•7H2O
5
mg



MnSO4•8H2O
5
mg



ZnSO4
5
mg



Calcium carbonate
30
g



Yeast extract
2
g



Methionine
0.15
g










Quinolinic acid in the culture solution was analyzed by HPLC. The result of analysis is shown in the following Table 2, and indicates the ability of the strain to produce quinolinic acid.











TABLE 2







Strains
Plasmids
Quinolinic acid (g/L)









W3110
—
Not Detected (ND)




pPro-nadBA
ND



W3110ΔnadC
—
ND




pPro-nadBA
0.4



TF4076
—
ND




pPro-nadBA
ND



TF4076ΔnadC
—
0.1




pPro-nadBA
5.5










As shown in Table 2, when the expression of genes coding for aspartate oxidase and quinolinate synthetase was enhanced through promoter substitution in TF4076 derived from threonine-producing strain and W3110 strain as wild E. coli, quinolinic acid was not produced in both strains. The reason thereof is that all quinolinic acid produced is consumed in the NAD synthesis pathway due to the activity of quinolinate phosphoribosyltransferase.
On the contrary, the W3110ΔnadC strain, which was produced by removing quinolinate phosphoribosyltransferase to inhibit the decomposition of quinolinic acid produced in cells with quinolinate phosphoribosyltransferase and enhancing the expression of aspartate oxidase and quinolinate synthetase, produced quinolinic acid in an amount of 0.4 g/L. And the TF4076ΔnadC derived from threonine-producing strain, which was produced by removing quinolinate phosphoribosyltransferase to inhibit the decomposition of quinolinic acid produced in cells with quinolinate phosphoribosyltransferase and enhancing the expression of aspartate oxidase and quinolinate synthetase, produced quinolinic acid in an amount of 5.5 g/L, which is 13-times higher than the wild strain W3110ΔnadC, due to the biosynthetic pathway enhanced for aspartic acid, which is inherent in the strain itself. That is, it was confirmed that the strain modified by the combination of enhancement of the expression of aspartate oxidase and quinolinate synthetase, removal of the activity of quinolinate phosphoribosyltransferase, and enhancement of the biosynthetic pathway for producing aspartic acid can produce quinolinic acid at a higher efficiency than the existing strains.
2-2. Production of Nicotinic Acid Through Decarboxylation
Decarboxylation reaction was conducted to convert quinolinic acid in the culture solution of quinolinic acid-producing strain, CV02-0009, which contains 5.5 g/L of quinolinic acid into nicotinic acid under high temperature and high pressure conditions. First, the culture solution containing quinolinic acid was centrifuged at 3000 to 4000 rpm for 10 to 30 minutes to remove cells in the culture solution. The supernatant containing quinolinic acid as obtained after centrifugation was used as the sample for decarboxylation reaction. The decarboxylation reaction was carried out under the conditions of 135° C. and 0.2 MPa for 3 hours, and the conditions of the sample as used are as shown in the following Table 3. Quinolinic acid comprised in deionized water as the experiment for the control group was the standard available from Sigma-Aldrich, and pH of aqueous quinolinic acid solution was titrated with sodium hydroxide, ammonia water, hydrochloric acid or sulfuric acid. The following Table 3 shows the rate of conversion of quinolinic acid into nicotinic acid by the reaction under high temperature and high pressure conditions. FIG. 3 shows the result of HPLC to identify quinolinic acid in the culture solution and nicotinic acid obtained after decarboxylation reaction of said culture solution.









TABLE 3









Yield












Quinolinic

pH
Nicotinic

Molar


Acid
Solution
(acid)
Acid
g/g
basis





5.5 g/L
deionized
—
3.8 g/L
70%
95%



water



Culture
6-7
ND
—
—



solution
2
4.0 g/L
73%
99%




(HCl)




3
3.4 g/L
63%
85%




(HCl)




2
3.6 g/L
66%
90%




(H2SO4)




3
3.0 g/L
55%
75%




(H2SO4)









The experiment to convert quinolinic acid into nicotinic acid using deionized water as the aqueous solution under the temperature and pressure conditions including 135° C. and 0.2 MPa, which are lower than 150 to 250° C. and 2 MPa conditions as disclosed in the prior reference Chinese Patent No. CN101353322C was conducted for 3 hours to obtain the result as shown in the above Table 3. This demonstrates that quinolinic acid was converted into nicotinic acid at up to 95% even under lower temperature and pressure conditions than those disclosed in said prior reference.
The experiment was conducted to convert quinolinic acid into nicotinic acid using the same method as that disclosed in the prior reference except that the fermentation culture solution was used as the aqueous solution of quinolinic acid. As a result, it could be identified that quinolinic acid was converted into nicotinic acid in deionized water, but not converted into nicotinic acid in the culture solution. The reason is that various ions present in the culture solution prevent the approach of hydrogen ion to the carboxyl group, and the movement of hydrogen ion as the requirements for decarboxylation of quinolinic acid. In order to solve this problem, it was intended to confirm whether the decarboxylation reaction occure or not in the fermentation culture solution containing quinolinic acid, through the method for increasing the chances to contact hydrogen ion with quinolinic acid in the fermentation culture solution by elevating the level of hydrogen ion. For this purpose, the pH value of the culture solution containing quinolinic acid was titrated from pH 6 to 7 to the range of pH 2 to 3 at which hydrogen ion can be maintained on high level. Said titration was conducted using hydrochloric acid or sulfuric acid.
As a result, the conversion rate of quinolinic acid into nicotinic acid was 85% to 99% when the ph value of the culture solution was titrated to pH 2 to 3 with the addition of hydrochloric acid, and 75% to 90% with the addition of sulfuric acid. According to this, nicotinic acid could be efficiently produced by addition of the acid and decarboxylation under mild temperature and pressure conditions as compared to the prior references, without additional purification of the culture solution obtained after the incubation of the microorganism strains.

## Claims

1. A method for the preparation of nicotinic acid, which comprises the steps of:
obtaining a culture solution containing quinolinic acid by incubating a microorganism having an ability to produce quinolinic acid, in which the activity of quinolinate phosphoribosyltransferase is decreased relative to the wild-type or deleted and the activities of aspartate oxidase and quinolinate synthetase are increased relative to the wild-type; and
adding an acid to the culture solution to decarboxylate quinolinic acid,
wherein the microorganism having an ability to produce quinolinic acid belongs to Enterbacter genus, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus or Brevibacterium genus.

2. The method according to claim 1, wherein the activity of quinolinate phosphoribosyltransferase of the microorganism is decreased relative to the wild-type or deleted by substituting a gene encoding the quinolinate phosphoribosyltransferase with a gene mutated to decrease the activity of quinolinate phosphoribosyltransferase, replacing an endogenous promoter for the gene with a promoter having weakened activity, or deleting the gene from chromosome.

3. The method according to claim 1, wherein the activities of aspartate oxidase and/or quinolinate synthetase of the microorganism are increased relative to the wild-type by increasing the intracellular copy number of genes encoding the aspartate oxidase and/or quinolinate synthetase, modifying expression regulatory sequences of the genes, or substituting the genes encoding the aspartate oxidase and/or quinolinate synthetase with genes mutated to increase the activities of aspartate oxidase and/or quinolinate synthetase.

4. The method according to claim 1, wherein the microorganism is a strain belonging to Escherichia genus.

5. The method according to claim 1, wherein the microorganism is Escherichia coli.

6. The method according to claim 1, wherein the microorganism is Escherichia coli deposited under accession number KCCM11165P.

7. The method according to claim 1, wherein the acid added to the culture solution is hydrochloric acid or sulfuric acid.

8. The method according to claim 1, wherein after addition of an acid, the culture solution has a pH value of 5 or less.

9. The method according to claim 1, wherein the decarboxylation reaction is conducted at a temperature ranges from 100° C. to 150° C.

10. The method according to claim 9, wherein the decarboxylation reaction is conducted at a temperature of 135° C.

11. The method according to claim 1, the decarboxylation reaction is conducted at a pressure ranges 0.1 MPa to 0.5 MPa.

12. The method according to claim 11, the decarboxylation reaction is conducted at a pressure of 0.2 MPa.

13. The method according to claim 1, further comprising the step of recovering and purifying nicotinic acid.