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A Method for Producing an L-Amino Acid Using Bacterium of the Enterobacteriaceae Family with Attenuated Expression of a Gene Coding for Small

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A Method for Producing an L-Amino Acid Using Bacterium of the Enterobacteriaceae Family with Attenuated Expression of a Gene Coding for Small (19) TZZ ¥_¥Z_T (11) EP 2 351 830 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 1/20 (2006.01) C12P 13/04 (2006.01) 23.04.2014 Bulletin 2014/17 (21) Application number: 11153815.3 (22) Date of filing: 22.03.2007 (54) A method for producing an L- amino acid using bacterium of the Enterobacteriaceae family with attenuated expression of a gene coding for small RNA Methode zur Produktion einer L-Aminosäure unter Verwendung eines Bakteriums der Enterobacteriaceae Familie mit einer abgeschwächten Expression eines Gens kodierend für sRNA Procede permettant de produire un acide amine à l’aide d’une bacterie appartenant a la famille des enterobacteries ayant une expression attenuee d’un gene codant un sRNA (84) Designated Contracting States: (73) Proprietor: Ajinomoto Co., Inc. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Tokyo HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE 104-8315 (JP) SI SK TR (72) Inventors: (30) Priority: 23.03.2006 RU 2006109062 • Rybak, Konstantin Vyacheslavovich 23.03.2006 RU 2006109063 117149 Moscow (RU) 11.04.2006 RU 2006111808 • Skorokhodova, Aleksandra Yurievna 11.04.2006 RU 2006111809 115304 Moscow (RU) 04.05.2006 RU 2006115067 • Voroshilova, Elvira Borisovna 04.05.2006 RU 2006115068 117648 Moscow (RU) 04.05.2006 RU 2006115070 • Gusyatiner, Mikhail Markovich 02.06.2006 RU 2006119216 117648 Moscow (RU) 04.07.2006 RU 2006123751 • Leonova, Tatyana Viktorovna 16.01.2007 RU 2007101437 123481 Moscow (RU) 16.01.2007 RU 2007101440 • Kozlov, Yury Ivanovich Moscow (RU) (43) Date of publication of application: • Ueda, Takuji 03.08.2011 Bulletin 2011/31 Kawasaki-shi, Kanagawa 210-8681 (JP) (83) Declaration under Rule 32(1) EPC (expert (74) Representative: HOFFMANN EITLE solution) Patent- und Rechtsanwälte Arabellastrasse 4 (62) Document number(s) of the earlier application(s) in 81925 München (DE) accordance with Art. 76 EPC: 10151962.7 / 2 186 881 (56) References cited: 08171633.4 / 2 055 771 WO-A-03/046184 WO-A-2006/123763 07740190.9 / 2 004 803 US-B1- 6 228 638 • FAUBLADIER M ET AL: "ESCHERICHIA COLI CELL DIVISION INHIBITOR DICF-RNA OF THE DICB OPERON", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 212, no. 3, 1990, pages 461-471, XP008066900, ISSN: 0022-2836 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 351 830 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 351 830 B1 • MAJDALANI NADIM ET AL: "Bacterial small RNA • ANDERSEN J ET AL: "The isolation and regulators.", CRITICAL REVIEWS IN characterization of RNA coded by the micF gene BIOCHEMISTRY AND MOLECULAR BIOLOGY in Escherichia coli.", NUCLEIC ACIDS 2005 MAR-APR, vol. 40, no. 2, March 2005 RESEARCH 11 MAR 1987 LNKD- PUBMED: (2005-03), pages 93-113, XP002460439, ISSN: 2436145, vol. 15, no. 5, 11 March 1987 1040-9238 (1987-03-11) , pages 2089-2101, XP001525866, • HERSHBERG RUTH ET AL: "A survey of small ISSN: 0305-1048 RNA-encoding genes in Escherichia coli.", • STORZ GISELA ET AL: "An abundance of RNA NUCLEIC ACIDS RESEARCH 1 APR 2003, vol. 31, regulators.", ANNUAL REVIEW OF no. 7, 1 April 2003 (2003-04-01), pages 1813-1820, BIOCHEMISTRY 2005, vol. 74, 2005, pages XP002460256, ISSN: 1362-4962 199-217, XP002460440, ISSN: 0066-4154 • DELIHAS N ET AL: "MicF: an antisense RNA gene • URBAN JOHANNES H ET AL: "Translational involved in response of Escherichia coli to global controland targetrecognition by Escherichia coli stress factors", JOURNAL OF MOLECULAR small RNAs in vivo.", NUCLEIC ACIDS BIOLOGY, LONDON, GB, vol. 313, no. 1, 12 RESEARCH 2007, vol. 35, no. 3, 2007, pages October 2001 (2001-10-12), pages 1-12, 1018-1037, XP002460257, ISSN: 1362-4962 XP004466184, ISSN: 0022-2836, DOI: DOI: 10.1006/JMBI.2001.5029 • BEGIC SANELA ET AL: "Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH", MICROBIOLOGY (READING), vol. 152, no. Part 2, February 2006 (2006-02), pages 485-491, XP002639866, ISSN: 1350-0872 2 EP 2 351 830 B1 Description Technical Field 5 [0001] The present invention relates to the microbiological industry, and specifically to a method for producing L- threonine using Escherichia coli which has been modified to attenuate expression of the micF gene. Background Art 10 [0002] Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microor- ganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids. [0003] Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganism with recombinant DNA (see, for example, US patent No. 4,278,765). Other techniques for enhancing 15 production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or US patent Nos. 4,346,170; 5,661,012 and 6,040,160). [0004] WO 03/046184 A1 discloses a process for the preparation of non-aromatic L-amino acids, in particular L- threonine, in which the following steps are carried out: a) fermentation of the microorganisms of the Enterobacteriaceae 20 family which produce the desired L-amino acid and in which the csrA gene or nucleotide sequences which code for it are enhanced, in particular over-expressed, b) concentration of the desired L-amino acid in the medium or in the cells of the bacteria, and c) isolation of the desired L-amino acid. [0005] Another way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, genes which are responsible for diverting the precursors 25 of the target L-amino acid from the L-amino acid biosynthetic pathway, genes which are involved in the redistribution of carbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.. [0006] Small, untranslated RNAs are present in many different organisms, ranging from bacteria to mammals. These RNAs carry out a variety of biological functions. Many of them can function as regulators of gene expression at the posttranscriptional level, either by acting as antisense RNAs, by binding to complementary sequences of target tran- 30 scripts, or by interacting with proteins. Regulatory RNAs are involved in the control of a large variety of processes such as plasmid replication, transposition in pro- and eukaryotes, phage development, viral replication, bacterial virulence, global circuits in bacteria in response to environmental changes, or developmental control in lower eukaryotes (Argaman L.et.al., Current Biology, 11: 941-50 (2001)). [0007] Small RNA (sRNA) molecules have gained much interest recently. Many Escherichia coli genes are known to 35 code for sRNAs: c0067, c0293, c0299, c0343, c0362, c0465, c0614, c0664, c0719, csrB, dicF, dsrA, ffs, gadY, gcvB, is092, is102, is128, isrA, micC, micF, oxyS, rnpB, rprA, rybA, rybB, rydB, ryeA, ryeB, ryeC, ryeD, ryeE, ryfA, rygB, rygC, rygD, sgrS, spf, sraA, sraB, sraD, sraE, sraG, sraH, sraI, sraJ, sraK, sraL, sroA, sroB, sroC, sroD, sroE, sroF, sroG, sroH, ssrA, ssrS, t44(tff), tp2, tpke11, tpke70 (Hershberg, R., et.al., Nucleic Acids Res., 31(7):1813-20 (2003) and Vogel, J., et al, Nucleic Acids Res., 31(22): 6435-43(2003)). Most of these genes are still uncharacterized and their cellular 40 roles are unknown. Traditionally, most RNA molecules were thought to function as mediators that carry the information from the gene to the translational machinery. Exceptions were the transfer RNAs and ribosomal RNAs that had long been known to have functions of their own, associated also with translation. However, it is now widely acknowledged that other types of untranslated RNA molecules (sRNA) exist that are involved in a diverse range of functions, from structural through regulatory to catalytic (Hershberg, R.,et al., Nucleic Acids Res. 31 (7): 1813-1820 (2003)). 45 [0008] The sraE and rygB genes encode small, untranslated RNAs - SraE and RygB of approximately 89 nt and 83 nt in length, respectively, which are encoded within the same inter-ORF region of the genome. Interactions between the SraE RNA and Hfq protein and between the RygB RNA and Hfq have been detected, SraE RNA and RygB RNA bound Hfq quite efficiently (>30% bound) (Wassarman, K.M. et al, Genes Dev. 1; 15(13):1637-51(2001)). There is some se- quence similarity between sraE and rygB, and they are transcribed in the same direction.SraE and rygB, which are 50 located in the same intergenic region between aas and galR, show significant sequence similarity of 77% identity over 84 nt (Hershbserg, R., et.al., Nucleic Acids Res., 31(7):1813-20 (2003)). Despite this high sequence similarity, these two sRNAs exhibit an almost mutually exclusive expression pattern: RygB levels increase around the onset of the stationary phase and decrease thereafter (Vogel, J., et al, Nucleic Acids Res., 31(22): 6435-43(2003)), whereas SraE is produced as stationary phase progresses (Argaman, L. et al, Current Biology, 11: 941-50 (2001)). 55 [0009] The sroE gene encodes a small, untranslated RNA called SroE. SroE sRNA was shown to be processed from a longer transcript, that is, the upstreamgcpE gene.
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