# US 7,608,432 B2 · Method for producing purine nucleoside by fermentation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,608,432 B2 - Document ID: US7608432B2 - Status: Granted - Kind: B2 - Application number: 11/682,155 - Filed: 2007-03-05 - Published: 2008-02-21 - Granted: 2009-10-27 - Earliest claimed: 1997-07-18 - Assignees: Ajinomoto Co., Inc. - Inventors: Hiroshi Matsui; Hisashi Kawasaki; Megumi Shimaoka; Yasuhiro Takenaka; Osamu Kurahashi - Web page: https://patentgrep.com/patent/US7608432B2 ## CPC - C12N 9/1077: {Pentosyltransferases (2.4.2)} - C12N 1/205: Bacterial isolates - C12P 19/38: Nucleosides - C12P 19/40: having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides - C12R 2001/19: Escherichia coli - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract A microorganism which has a gene encoding an enzyme in which feedback inhibition is desensitized by substitution of one or two amino acids in PRPP amidotransferase encoded by purF of Escherichia coli, a gene encoding a protein which is an inactivated repressor of purine nucleotide biosynthesis encoded by purR, a gene encoding an enzyme which is inactivated purine nucleoside phosphorylase encoded by deoD, a gene encoding an enzyme which is inactivated succinyl-AMP synthase encoded by purA, a gene encoding an enzyme which is inactivated 6-phosphogluconate dehydrase encoded by edd, a gene encoding an enzyme which is inactivated phosphoglucose isomerase encoded by pgi and like is bred and a purine nucleoside is produced by culturing the microorganism. ## Claims ### Claim 1 A method for producing a purine nucleoside by fermentation comprising: culturing an Escherichia coli microorganism in a culture medium to produce and accumulate the purine nucleoside in the medium, and collecting the purine nucleoside, wherein said microorganism has purine nucleoside-producing ability and is modified to inactivate a purine repressor wherein said microorganism has purine nucleoside-producing ability and is modified to inactivate a purine repressor, said purine repressor being encoded by a gene obtained by PCR amplification employing the primer pair of SEQ ID NO: 7 and SEQ ID NO: 8 and Escherichia coli chromosomal DNA as the template wherein said microorganism is further modified to desensitize feedback inhibition of phosphoribosyl pyrophosphate amidotransferase, said phosphoribosyl pyrophosphate amidotransferase being encoded by a gene obtained by PCR amplification employing the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 and Escherichia coli chromosomal DNA as the template, and wherein said microorganism is further modified to block reactions catalyzed by purine nucleoside phosphorylase and adenosine deaminase, said purine nucleoside phosphorylase being encoded by a gene obtained by PCR amplification employing the primer pair of SEQ ID NO: 5 and SEQ ID NO: 6 and Escherichia coli chromosomal DNA as the template, and said adenosine deaminase being encoded by a gene obtained by PCR amplification employing the primer pair of SEO ID NO: 11 and SEO ID NO: 12 and Escherichia coli chromosomal DNA as the template. ### Claim 2 (depends on 1) The method according to claim 1, wherein said microorganism is further modified to increase an activity of an enzyme involved in purine nucleoside biosynthesis in cells of the microorganism. ### Claim 3 (depends on 1) The method according to claim 1, wherein said microorganism is further modified to increase an expression amount of a gene for an enzyme involved in purine nucleoside biosynthesis. ### Claim 4 (depends on 3) The method according to claim 3, wherein the enzyme involved in the purine nucleoside biosynthesis is phosphoribosyl pyrophosphate amidotransferase or phosphoribosyl pyrophosphate synthetase. ### Claim 5 (depends on 1) The method according to claim 1, wherein said microorganism is further modified to block a reaction branching from purine nucleoside biosynthesis and leading to another metabolite. ### Claim 6 (depends on 5) The method according to claim 5, wherein the reaction branching from the purine nucleoside biosynthesis and leading to another metabolite is a reaction catalyzed by an enzyme selected from the group consisting of succinyl-adenosine monophosphate synthase, inosine-guanosine kinase, guanosine monophosphate reductase, 6-phosphogluconoate dehydrase, phosphoglucose isomerase, adenine deaminase, and xanthosine phosphorylase. ### Claim 7 (depends on 1) The method according to claim 1, wherein said microorganism is further modified to weaken incorporation of a purine nucleoside into cells of the microorganism. ### Claim 8 (depends on 7) The method according to claim 7, wherein the incorporation of the purine nucleoside into cells of the microorganism is weakened by blockage of a reaction involved in the incorporation of the purine nucleoside into cells of the microorganism, and the reaction involved in the incorporation of the purine nucleoside into cells of the microorganism is a reaction catalyzed by nucleoside permease. ### Claim 9 (depends on 1) The method according to claim 1, wherein said purine nucleoside is a purine nucleoside selected from the group consisting of inosine and guanosine. ### Claim 10 (depends on 1) The method according to claim 1, wherein said microorganism is further modified to block a reaction catalyzed by succinyl-adenosine monophosphate synthase, said succinyl-adenosine monophosphate synthase being encoded by a gene obtained by PCR amplification employing the primer pair of SEQ ID NO: 3 and SEQ ID NO: 4 and Escherichia coli chromosomal DNA as the template.