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Timestamp: 2014-09-22 15:38:31
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Matched Legal Cases: ['Application No. 2006109062', 'Application No. 2006109063', 'Application No. 2006111808', 'Application No. 2006111809', 'Application No. 2006115067', 'Application No. 2006115068', 'Application No. 2006115070', 'Application No. 2006119216', 'Application No. 2006123751', 'Application No. 2007101437', 'Application No. 2007101440']

Patent US7803584 - Cultivating an Escherichia coli that has been modified to attenuate ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsThe present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to attenuate expression of a gene coding for sRNA....http://www.google.com/patents/US7803584?utm_source=gb-gplus-sharePatent US7803584 - Cultivating an Escherichia coli that has been modified to attenuate expression of the sraL gene and collecting L-threonine and/or L-lysine from the mediumAdvanced Patent SearchPublication numberUS7803584 B2Publication typeGrantApplication numberUS 12/212,743Publication dateSep 28, 2010Filing dateSep 18, 2008Priority dateMar 23, 2006Fee statusPaidAlso published asEP2004803A2, EP2055771A2, EP2055771A3, EP2184348A1, EP2184348B1, EP2186881A1, EP2186881B1, EP2351830A1, EP2351830B1, US8088606, US8227214, US20090098621, US20100311129, US20120070865, WO2007119574A2, WO2007119574A3Publication number12212743, 212743, US 7803584 B2, US 7803584B2, US-B2-7803584, US7803584 B2, US7803584B2InventorsKonstantin Vyacheslavovich Rybak, Aleksandra Yurievna Skorokhodova, Elvira Borisovna Voroshilova, Mikhail Markovich Gusyatiner, Tatyana Viktorovna Leonova, Yury Ivanovich Kozlov, Takuji UedaOriginal AssigneeAjinomoto Co., Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (40), Non-Patent Citations (26), Referenced by (7), Classifications (12), Legal Events (2) External Links: USPTO, USPTO Assignment, EspacenetCultivating an Escherichia coli that has been modified to attenuate expression of the sraL gene and collecting L-threonine and/or L-lysine from the mediumUS 7803584 B2Abstract The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to attenuate expression of a gene coding for sRNA.
collecting said L-amino acid from the medium,
wherein said Escherichia coli has been modified to attenuate expression of the sraL gene, and
wherein said L-amino acid is selected from the group consisting of L-threonine, L-lysine, and combinations thereof.
2. The method according to claim 1, wherein said sraL gene comprises a nucleotide sequence that hybridizes with a complement of the nucleotide sequence of SEQ ID NO: 103 under stringent conditions comprising washing at 0.1�SSC, 0.1% SDS at 60� C.
3. The method according to claim 1, wherein said sraL gene comprises the nucleotide sequence of SEQ ID NO: 103.
This application is a continuation under 35 U.S.C. �120 to PCT Patent Application No. PCT/JP2007/056752, filed on Mar. 22, 2007, which claims priority under 35 U.S.C. �119(a) to Russian Patent Application No. 2006109062, filed on Mar. 23, 2006, Russian Patent Application No. 2006109063, filed on Mar. 23, 2006, Russian Patent Application No. 2006111808, filed on Apr. 11, 2006, Russian Patent Application No. 2006111809, filed on Apr. 11, 2006, Russian Patent Application No. 2006115067, filed on May 4, 2006, Russian Patent Application No. 2006115068, filed on May 4, 2006, Russian Patent Application No. 2006115070, filed on May 4, 2006, Russian Patent Application No. 2006119216, filed on Jun. 2, 2006, Russian Patent Application No. 2006123751, filed on Jul. 4, 2006, Russian Patent Application No. 2007101437, filed on Jan. 16, 2007, and Russian Patent Application No. 2007101440, filed on Jan. 16, 2007, the entireties of which are hereby incorporated by reference. The Sequence Listing filed electronically herewith is also hereby incorporated by reference in its entirety (File Name: US-275_Seq_List; File Size: 30 KB; Date Created: Sep. 18, 2008).
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids. Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing 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 U.S. Pat. Nos. 4,346,170; 5,661,012 and 6,040,160).
Small RNA (sRNA) molecules have gained much interest recently. Many Escherichia coli genes are known to 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 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)).
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 sequence similarity between sraE and iygB, and they are transcribed in the same direction. SraE and rygB, which are 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)).
The dsrA gene encodes DsrA RNA, a small (87-nt) regulatory RNA of E. coli that acts via RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H�NS, a histone-like nucleoid protein and global transcription repressor (Lease R. A., et al, Proc. Natl. Acad. Sci. USA, 95(21):12456-61 (1998)). Genes regulated globally by RpoS and H�NS include stress response proteins and virulence factors for pathogenic E. coli. Genes induced by DsrA have been identified by using transcription profiling via DNA arrays (Lease R. A., et al, J. Bacteriol., 186(18):6179-85 (2004)). Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. Overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant, confirming that DsrA RNA plays a regulatory role in acid resistance.
The micC gene (IS063) encodes a �100-nucleotide small-RNA transcript. The expression of this small RNA is increased at a low temperature and in minimal medium. Twenty-two nucleotides at the 5′ end of this transcript have the potential to form base pairs with the leader sequence of the mRNA encoding the outer membrane protein OmpC. MicC was shown to inhibit ribosome binding to the ompC mRNA leader in vitro and to require the Hfq RNA chaperone to function (Chen, S., et al., J. Bacteriol., 186(20):6679-80 (2004)).
SUMMARY OF THE INVENTION Aspects of the present invention include enhancing the productivity of L-amino acid-producing strains and providing a method for producing an L-amino acid using these strains.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
cultivating the bacterium as described above in a medium, and
collecting said L-amino acid from the medium.
It is a further aspect of the present invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the relative positions of primers P1 (upstream primer) and P2 (downstream primer) on plasmid pMW118-attL-Cm-attR which is used as a template for PCR amplification of the cat gene.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Bacterium of the Present Invention The bacterium of the present invention is an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the a gene coding for sRNA (small RNA).
The phrase �L-amino acid-producing bacterium� means a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.
The term �L-amino acid-producing bacterium� also means a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain of E. coli, such as E. coli K-12, and preferably means that the microorganism is able to cause accumulation in a medium of the target L-amino acid in an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L. The term �L-amino acid� includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
The term �aromatic L-amino acid� includes L-phenylalanine, L-tyrosine, and L-tryptophan. The term �non-aromatic L-amino acid� includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline, and L-arginine are particularly preferred.
The phrase �a bacterium belonging to the genus Escherichia� means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. coli).
The phrase �a bacterium belonging to the genus Pantoea� means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on the nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).
The phrase �a gene coding for sRNA� means a gene encoding an RNA that is not translated into a protein and has a small size, preferably 50 to 500 bases in length.
The phrase �bacterium has been modified to attenuate expression of a gene coding for sRNA� means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the sRNA, as compared with an unmodified bacterium, or is unable to synthesize the sRNA.
The phrase �inactivation of a gene coding for sRNA� means that the modified DNA region is unable to naturally express the gene due to the deletion of a part of the gene or of the gene entirely, or the modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoters, enhancers, attenuators, etc.
The c0067 gene encodes the C0067 RNA. The c0067 gene (nucleotides in positions 238,462 to 238,586; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yafT ORF and the yafU ORF on the chromosome of E. coli K-12. The nucleotide sequence of the c0067 gene is shown in SEQ ID NO: 69.
The c0293 gene encodes the C0293 RNA. The c0293 gene (nucleotides in positions 1,195,937 to 1,196,009; GenBank accession no. NC�000913.2; gi: 49175990) is located between the icd gene and the ymfD ORF on the chromosome of E. coli K-12. The nucleotide sequence of the c0293 gene is shown in SEQ ID NO: 70.
The c0299 gene encodes the C0299 RNA. The c0299 gene (nucleotides in positions 1,229,852 to 1,229,930; GenBank accession no. NC�000913.2; gi: 49175990) is located between the hlyE gene and the umuD gene on the chromosome of E. coli K-12. The nucleotide sequence of the c0299 gene is shown in SEQ ID NO:71.
The c0343 gene encodes the C0343 RNA. The c0343 gene (nucleotides in positions 1,407,387 to 1,407,461; GenBank accession no. NC�000913.2; gi: 49175990) is located between the ydaN ORF and the dbpA gene on the chromosome of E. coli K-12. The nucleotide sequence of the c0343 gene is shown in SEQ ID NO:72.
The c0362 gene encodes the C0362 RNA. The c0362 gene (nucleotides in positions 1,550,025 to 1,550,410; GenBank accession no. NC�000913.2; gi: 49175990) is located between the fdnI gene and the yddM ORF on the chromosome of E. coli K-12. The nucleotide sequence of the c0362 gene is shown in SEQ ID NO: 1.
The c0465 gene encodes the C0465 RNA. The c0465 gene (nucleotides in positions 1,970,763 to 1,970,840; GenBank accession no. NC�000913.2; gi: 49175990) is located between the tar gene and the cheW gene on the chromosome of E. coli K-12. The nucleotide sequence of the c0465 gene is shown in SEQ ID NO: 2.
The c0614 gene encodes the C0614 RNA. The c0614 gene (nucleotides complemented to nucleotides in positions 2,651,474 to 2,651,560; GenBank accession no. NC�000913.2; gi: 49175990) is located between the sseA gene and the IS128 gene, overlapping with the IS128 gene, on the chromosome of E. coli K-12. The nucleotide sequence of the c0614 gene is shown in SEQ ID NO:73.
The c0664 gene encodes the C0664 RNA. The c0664 gene (nucleotides in positions 2,833,077 to 2,833,189; GenBank accession no. NC�000913.2; gi: 49175990) is located between the ygbD gene and the hypF gene on the chromosome of E. coli K-12. The nucleotide sequence of the c0664 gene is shown in SEQ ID NO: 74.
The c0719 gene encodes the C0719 RNA. The c0719 gene (nucleotides in positions 3,119,380 to 3,119,601; GenBank accession no. NC�000913.2; gi: 49175990) is located between the glcA gene and the glcB gene on the chromosome of E. coli K-12. The nucleotide sequence of the c0719 gene is shown in SEQ ID NO: 75.
The csrB gene (synonyms�ECK2787, b4408) encodes the CsrB RNA. The csrB gene (nucleotides complementary to nucleotides in positions 2,922,178 to 2,922,537; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yqcC ORF and the syd gene on the chromosome of E. coli K-12. The nucleotide sequence of the csrB gene is shown in SEQ ID NO: 76.
The dicF gene (synonyms�ECK1568, b1574) encodes the DicF RNA. The dicF gene (nucleotides in positions 1,647,406 to 1,647,458; GenBank accession no. NC�000913.2; gi: 49175990) is located between the rzpQ gene and the dicB gene on the chromosome of E. coli K-12. The nucleotide sequence of the dicF gene is shown in SEQ ID NO: 77.
The dsrA gene (synonym�b1954) encodes DsrA RNA, a global regulator of gene expression. The dsrA gene (nucleotides in positions 2,023,336 to 2,023,250; GenBank accession no. NC�000913.2; gi:49175990) is located between the yodD and yedP genes on the E. coli strain K-12 chromosome. The nucleotide sequence of the dsrA gene is shown in SEQ ID NO: 3.
The ffs gene (synonyms�ECK0449, b0455) encodes the Ffs RNA. The ifs gene (nucleotides in positions 475,672 to 475,785; GenBank accession no. NC�000913.2; gi: 49175990) is located between the ybaZ ORF and the ybaA ORF on the chromosome of E. coli K-12. The nucleotide sequence of the ffs gene is shown in SEQ ID NO: 78.
The gadY gene (synonyms�ECK3500, b4452, IS183) encodes the GadY RNA. The gadY gene (nucleotides in positions 1,647,406 to 1,647,458; GenBank accession no. NC�000913.2; gi: 49175990) is located between the rzpQ gene and the dicB gene on the chromosome of E. coli K-12. The nucleotide sequence of the gadY gene is shown in SEQ ID NO: 79.
The gcvB gene (synonyms: ECK2804, psrA11, IS145, b4443) encodes the GcvB RNA. The gcvB gene (nucleotides in positions 2,940,718 to 2,940,922; GenBank accession no. NC�000913.2; gi: 16130715) is located between the gcvA gene and the ygdI ORF on the chromosome of E. coli K-12. The nucleotide sequence of the gcvB gene is shown in SEQ ID NO: 4.
The IS092 gene (synonyms�ECK1902, b4434) encodes the IS092 RNA. The IS092 gene (nucleotides complementary to nucleotides in positions 1,985,863 to 1,986,022; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yecJ ORF and the yecR ORF on the chromosome of E. coli K-12. The nucleotide sequence of the IS092 gene is shown in SEQ ID NO: 80.
The IS102 gene (synonyms�ECK1992, b4435) encodes the IS102 RNA. The IS102 gene (nucleotides in positions 2,069,339 to 2,069,542; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yeeP ORF and the flu gene on the chromosome of E. coli K-12. The nucleotide sequence of the IS102 gene is shown in SEQ ID NO: 81.
The IS128 gene encodes the IS128 RNA. The IS128 gene (nucleotides in positions 2,651,537 to 2,651,745; GenBank accession no. NC�000913.2; gi: 49175990) is located between the c0614 gene and the ryfA gene, overlapping with the c0614 gene, on the chromosome of E. coli K-12. The nucleotide sequence of the IS128 gene is shown in SEQ ID NO: 82.
The isrA gene (synonyms�ECK1336, b4426, IS061) encodes the IsrA RNA. The isrA gene (nucleotides complemented to nucleotides in positions 1,403,676 to 1,403,833; GenBank accession no. NC�000913.2; gi: 49175990) is located between the abgR gene and the ydaL ORF on the chromosome of E. coli K-12. The nucleotide sequence of the isrA gene is shown in SEQ ID NO: 83.
The micC gene (synonyms: ECK1373, IS063, tke8, b4427) encodes the MicC RNA.
The micC gene (nucleotides in positions 1,435,145 to 1,435,253; GenBank accession no. NC�000913.2; gi: 16127999) is located between the gene ompN and the ORF ydbK on the chromosome of E. coli K-12. The nucleotide sequence of the micC gene is shown in SEQ ID NO: 5.
The micF gene (synonyms: ECK2208, stc, b4439) encodes the MicF RNA. The micFgene (nucleotides in positions 2,311,106 to 2,311,198; GenBank accession no. NC�000913.2; gi: 16127999) is located between the ompC gene and the rcsD gene on the chromosome of E. coli K-12. The nucleotide sequence of the micF gene is shown in SEQ ID NO: 84.
The oxyS gene (synonyms�ECK3952, b4458) encodes the OxyS RNA. The oxyS gene (nucleotides complementary to nucleotides in positions 4,156,308 to 4,156,417; GenBank accession no. NC�000913.2; gi: 49175990) is located between the argH gene and the oxyR gene on the chromosome of E. coli K-12. The nucleotide sequence of the oxyS gene is shown in SEQ ID NO: 85.
The rnpB gene (synonyms�ECK3111, b3123) encodes the RnpB RNA. The rnpB gene (nucleotides complementary to nucleotides in positions 3,268,238 to 3,268,614; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yhaC ORF and the garK gene on the chromosome of E. coli K-12. The nucleotide sequence of the rnpB gene is shown in SEQ ID NO: 86.
The rprA gene (synonyms�ECK1686, psrA5, IS083, b4431) encodes the RprA RNA The rprA gene of E. coli (nucleotides in positions 1,768,395 to 1,768,499; GenBank accession no. NC�000913.2; gi:49175990) is located between the ydiK ORF and the ydiL ORF on the chromosome of E. coli K-12. The nucleotide sequence of the rprA gene is shown in SEQ ID NO:6.
The rybA gene (synonyms: ECK0806, b4416) encodes the RybA RNA. The rybA gene (nucleotides complementary to nucleotides in positions 852,175 to 852,263; GenBank accession no. NC�000913.2; gi:16127999) is located between the yliL ORF and the mntR gene on the chromosome of E. coli K-12. The nucleotide sequence of the rybA gene is shown in SEQ ID NO: 87.
The rybB gene (synonyms: p25, b4417) encodes the RybB RNA. The rybB gene (nucleotides in positions 887,198 to 887,276; GenBank accession no. NC�000913.2; gi:16127999) is located between the ORF ybjK and the ORF ybjL on the chromosome of E. coli K-12. The nucleotide sequence of the rybB gene is shown in SEQ ID NO: 7.
The rydB gene (synonyms: ECK1681, tpe7, IS082, b4430) encodes the RydB RNA. The rydB gene (nucleotides complemented to nucleotides in positions 1,762,737 to 1,762,804; GenBank accession no. NC�000913.2; gi:16127999) is located between the sufA gene and the ydiH ORF on the chromosome of E. coli K-12. The nucleotide sequence of the rydB gene is shown in SEQ ID NO: 88.
The ryeA gene (synonyms: ECK1838, sraC, sraCyeA, psrA8, tkpe79, IS091, b4432) encodes the RyeA RNA. The ryeA gene (nucleotides in positions 1,921,090 to 1,921,338; GenBank accession no. NC�000913.2; gi:16127999) is located between the pphA gene and the yebY ORF, interlapping the ryeB gene which is oriented in the opposite orientation, on the chromosome of E. coli K-12. The nucleotide sequence of the ryeA gene is shown in SEQ ID NO: 89.
The ryeB gene (synonyms: ECK1839, tkpe79, IS091, b4433) encodes the RyeB RNA. The ryeB gene (nucleotides complementary to nucleotides in positions 1,921,188 to 1,921,308; GenBank accession no. NC�000913.2; gi:16127999) is located in the region of the ryeA gene on the chromosome of E. coli K-12. The nucleotide sequence of the ryeB gene is shown in SEQ ID NO: 90.
The ryeC gene (synonyms: ECK2068, QUAD1a, tp11, b4436) encodes the RyeC RNA. The ryeC gene (nucleotides in positions 2,151,299 to 2,151,447; GenBank accession no. NC�000913.2; gi:16127999) is located between the yegL ORF and the ryeD gene on the chromosome of E. coli K-12. The nucleotide sequence of the ryeC gene is shown in SEQ ID NO: 91.
The ryeD gene (synonyms: ECK2069, QUAD1b, tpe60, b4437) encodes the RyeD RNA. The ryeD gene (nucleotides in positions 2,151,634 to 2,151,776; GenBank accession no. NC�000913.2; gi:16127999) is located between the ryeC gene and the mdtA gene on the chromosome of E. coli K-12. The nucleotide sequence of the ryeD gene is shown in SEQ ID NO: 92.
The ryeE gene (synonyms: ECK2078, b4438) encodes the RyeE RNA. The ryeE gene (nucleotides in positions 2,165,136 to 2,165,221; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yegQ ORF and the ogrK gene on the chromosome of E. coli K-12. The nucleotide sequence of the ryeE gene is shown in SEQ ID NO: 8.
The ryfA gene (synonyms: ECK2518, b4440) encodes the RyeE RNA. The ryeE gene (nucleotides in positions 2,651,877-2,652,180; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yegQ ORF and the ogrK gene on the chromosome of E. coli K-12. The nucleotide sequence of the ryfA gene is shown in SEQ ID NO: 93.
The rygB gene (synonyms: ECK2834, PAIR2, t59, b4445, omrB) encodes the RygB RNA. The rygB gene (nucleotides complemented to nucleotides in positions 2,974,332 to 2,974,407; GenBank accession no. NC�000913.2; gi: 49175990) is located between the sraE gene and the galR gene on the chromosome of E. coli K-12. The nucleotide sequence of the rygB gene is shown in SEQ ID NO: 9. The rygB gene may be attenuated together with the adjacent sraE gene.
The rygC gene (synonyms: ECK2908, QUAD1c, t27, b4446) encodes the RygC RNA. The rygC gene (nucleotides in positions 3,054,837 to 3,054,987; GenBank accession no. NC�000913.2; gi: 49175990) is located between the sraE gene and the galR gene on the chromosome of E. coli K-12. The nucleotide sequence of the rygC gene is shown in SEQ ID NO: 94.
The rygD gene (synonyms: ECK3041, tp8, C0730, IS156, QUAD1d, b4447) encodes the RygD RNA. The rygD gene (nucleotides complementary to nucleotides in positions 3,192,773 to 3,192,992; GenBank accession no. NC�000913.2; gi: 49175990) is located between the sraE gene and the galR gene on the chromosome of E. coli K-12. The nucleotide sequence of the rygD gene is shown in SEQ ID NO: 95.
The sgrS gene (synonyms: ECK0071, ryaA, b4577) encodes the SgrS RNA. The sgrS gene (nucleotides in positions 77,367 to 77,593; GenBank accession no. NC�000913.2; gi: 49175990) is located between the sgrR gene and the setA gene on the chromosome of E. coli K-12. The nucleotide sequence of the sgrS gene is shown in SEQ ID NO: 96.
The spf gene (synonyms: ECK3856, b3864, spot42) encodes the Spf RNA. The spf gene (nucleotides in positions 4,047,922 to 4,048,030; GenBank accession no. NC�000913.2; gi: 49175990) is located between the polA gene and the yihA ORF on the chromosome of E. coli K-12. The nucleotide sequence of the spf gene is shown in SEQ ID NO: 97.
The sraA gene (synonyms: psrA3, t15) encodes the SraA RNA. The sraA gene (nucleotides complementary to nucleotides in positions 457,952 to 458,008; GenBank accession no. NC�000913.2; gi: 49175990) is located between the clpX gene and the lon gene on the chromosome of E. coli K-12. The nucleotide sequence of the sraA gene is shown in SEQ ID NO:10.
The sraB gene (synonyms: psrA4, pke20) encodes the SraB RNA. The sraB gene (nucleotides in positions 1,145,811 to 1,145,979; GenBank accession no. NC�000913.2; gi:49175990) is located on the chromosome of E. coli K-12 upstream, but overlapping with the yceD ORF, which is oriented in the opposite direction and located upstream of sraB. The nucleotide sequence of the sraB gene is shown in SEQ ID NO: 11.
The sraD gene (synonyms: micA, ECK2682, psrA10, b4442) encodes the SraD RNA. The sraD gene (nucleotides in positions 2,812,823 to 2,812,897; GenBank accession no. NC�000913.2; gi: 49175990) is located between the luxS gene and the gshA gene on the chromosome of E. coli K-12. The nucleotide sequence of the sraD gene is shown in SEQ ID NO: 98.
The sraE gene (synonyms: ECK2833, psrA12, rygA, PAIR2, t59, b4444, omrA) encodes the SraE RNA. The sraE gene (nucleotides complemented to nucleotides in positions 2,974,124 to 2,974,211; GenBank accession no. NC�000913.2; gi: 49175990) is located between the aas gene and the rygB gene on the chromosome of E. coli K-12. The nucleotide sequence of the rygB gene is shown in SEQ ID NO: 12. The sraE gene may be attenuated together with the adjacent rygB gene.
The sraG gene (synonyms: ECK3153, psrA15, p3, b4449) encodes the SraG RNA. The sraG gene (nucleotides in positions 3,309,247 to 3,309,420; GenBank accession no. NC�000913.2; gi: 49175990) is located between the pnp gene and the rpsO gene, overlapping with the pnp gene, on the chromosome of E. coli K-12. The nucleotide sequence of the sraG gene is shown in SEQ ID NO: 99.
The sraH gene (synonyms: ECK3199, psrA16, ryhA, b4450) encodes the SraH RNA. The sraH gene (nucleotides in positions 3,348,599 to 3,348,706; GenBank accession no. NC�000913.2; gi: 49175990) is located between the elbB gene and the arcB gene on the chromosome of E. coli K-12. The nucleotide sequence of the sraH gene is shown in SEQ ID NO: 13.
The sraI gene (synonyms: ECK3426, psrA18, IS176, b4451, ryhB) encodes the SraI RNA. The sraI gene (nucleotides complemented to nucleotides in positions 3,578,946 to 3,579,039; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yhhX ORF and the yhhY ORF on the chromosome of E. coli K-12. The nucleotide sequence of the sraI gene is shown in SEQ ID NO: 100.
The sraJ gene (synonyms: ECK3795, psrA20, ryiA, k19, b4456) encodes the SraJ RNA. The sraJ gene (nucleotides in positions 3,984,455 to 3,984,626; GenBank accession no. NC�000913.2; gi: 49175990) is located between the asiA gene and the hemY gene on the chromosome of E. coli K-12. The nucleotide sequence of the sraJ gene is shown in SEQ ID NO: 101.
The sraK gene (synonyms: ECK3858, psrA21, ryiB, tpk2, IS198, b4457, csrC) encodes the SraK RNA. The sraK gene (nucleotides in positions 4,049,059 to 4,049,303; GenBank accession no. NC�000913.2; gi: 49175990) is located between the yihA ORF and the yihi ORF on the chromosome of E. coli K-12. The nucleotide sequence of the sraK gene is shown in SEQ ID NO: 102.
The sraL gene (synonyms: ECK4056, psrA24, ryjA, b4459) encodes the SraL RNA. The sraL gene (nucleotides complemented to nucleotides in positions 4,275,950 to 4,276,089; GenBank accession no. NC�000913.2; gi: 49175990) is located between the soxR gene and the yjcD ORF, overlapping with the soxR gene, on the chromosome of E. coli K-12. The nucleotide sequence of the sraL gene is shown in SEQ ID NO: 103.
The sroA gene (synonym tpe79) encodes the SroA RNA. The sroA gene (nucleotides complementary to nucleotides in positions 75,516 to 75,608; GenBank accession no. NC�000913.2; gi: 49175990) is located between the tbpA gene and the sgrR gene on the chromosome of E. coli K-12. The nucleotide sequence of the sroA gene is shown in SEQ ID NO: 104.
The sroB gene encodes the SroB RNA. The sroB gene (nucleotides in positions 506,428 to 506,511; GenBank accession no. NC�000913.2; gi: 49175990) is located between the ybaK ORF and the ybaP ORF, overlapping with the ybaP ORF, on the chromosome of E. coli K-12. The nucleotide sequence of the sroB gene is shown in SEQ ID NO: 105.
The sroC gene (synonym HB�314) encodes the SroC RNA. The sroC gene (nucleotides complemented to nucleotides in positions 685,904 to 686,066; GenBank accession no. NC�000913.2; gi: 49175990) is located between the gltJ gene and the gItI gene, overlapping with the gItI gene, on the chromosome of E. coli K-12. The nucleotide sequence of the sroC gene is shown in SEQ ID NO: 106.
The sroD gene (synonym p24) encodes the SroD RNA. The sroD gene (nucleotides complemented to nucleotides in positions 1,886,041 to 1,886,126; GenBank accession no. NC�000913.2; gi: 49175990) is located between the rnd gene and the fadD gene, overlapping with the fadD gene, on the chromosome of E. coli K-12. The nucleotide sequence of the sroD gene is shown in SEQ ID NO: 107.
The sroE gene (synonym k20) encodes the SroE RNA (synonym k20). The sroE gene (nucleotides complementary to nucleotides in positions 2,638,617 to 2,638,708; GenBank accession no. NC�000913.2; gi: 49175990) is located between the hisS gene and the ispG gene on the chromosome of E. coli K-12. The nucleotide sequence of the sroE gene is shown in SEQ ID NO: 14.
The sroF gene (synonyms: ECK2554, b4441, tke1) encodes the SroF RNA. The sroF gene (nucleotides complementary to nucleotides in positions 2689362 to 2689214); GenBank accession no. NC�000913.2; gi: 49175990) is located between the yfhK ORF and the purL gene on the chromosome of E. coli K-12. The nucleotide sequence of the sroF gene is shown in SEQ ID NO: 108.
The sroG gene (synonym HB�456) encodes the SroG RNA. The sroG gene (nucleotides complemented to nucleotides in positions 3,182,592 to 3,182,740; GenBank accession no. NC�000913.2; gi: 49175990) is located between the ribB gene and the yqiC ORF on the chromosome of E. coli K-12. The nucleotide sequence of the sroG gene is shown in SEQ ID NO: 109.
The sroH gene encodes the SroH RNA. The sroH gene (nucleotides complemented to nucleotides in positions 4,188,350 to 4,188,510; GenBank accession no. NC�000913.2; gi: 49175990) is located between the htrC gene and the thiH gene, overlapping with the htrC gene, on the chromosome of E. coli K-12. The nucleotide sequence of the sroH gene is shown in SEQ ID NO: 110.
The ssrA gene (synonyms: ECK2617, b262, sipB) encodes the SsrA RNA. The ssrA gene (nucleotides in positions 2,753,615 to 2,753,977; GenBank accession no. NC�000913.2; gi: 49175990) is located between the smpB and intA genes on the chromosome of E. coli K-12. The nucleotide sequence of the ssrA gene is shown in SEQ ID NO: 111.
The ssrS gene (synonyms: ECK2906, b2911, ssr) encodes the SsrS RNA. The ssrS gene (nucleotides in positions 3,054,005 to 3,054,187; GenBank accession no. NC�000913.2; gi: 49175990) is located between the zapA gene and the ygfA ORF on the chromosome of E. coli K-12. The nucleotide sequence of the ssrS gene is shown in SEQ ID NO: 112.
The tff gene (synonyms: ECK0167, b4414, T44) encodes the Tff RNA. The tff gene (nucleotides in positions 189,712 to 189,847; GenBank accession no. NC�000913.2; gi: 49175990) is located between the map gene and the rsp gene on the chromosome of E. coli K-12. The nucleotide sequence of the tff gene is shown in SEQ ID NO: 113.
The tp2 gene encodes the Tp2 RNA. The tp2 gene (nucleotides complemented to nucleotides in positions 122,697 to 122,857; GenBank accession no. NC�000913.2; gi: 49175990) is located between the pdhR gene and the aceE gene, overlapping with the pdhR gene, on the chromosome of E. coli K-12. The nucleotide sequence of the tp2 gene is shown in SEQ ID NO: 114.
The tpke11 gene encodes the Tpke11 RNA. The tpke11 gene (nucleotides complemented to nucleotides in positions 14,080 to 14,168; GenBank accession no. NC�000913.2; gi: 49175990) is located between the dnaK gene and the dnaJ gene on the chromosome of E. coli K-12. The nucleotide sequence of the tpke11 gene is shown in SEQ ID NO: 115.
The tpke70 gene encodes the Tp70 RNA. The tpke70 gene (nucleotides complementary to nucleotides in positions 2,494,216 to 2,494,651; GenBank accession no. NC�000913.2; gi: 49175990) is located between the lpxP gene and the yfdZ ORF, overlapping with the lpxP gene, on the chromosome of E. coli K-12. The nucleotide sequence of the tpke70 gene is shown in SEQ ID NO: 116.
Therefore, a gene coding for sRNA may be a variant which hybridizes under stringent conditions with a complement of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 69-116, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional sRNA prior to inactivation, or provided that attenuation of expression of the gene in a host bacterium leads to improvement of L-amino acid-producing ability of the host bacterium. �Stringent conditions� include those under which a specific hybrid, for example, a hybrid having homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 97%, and most preferably not less than 98%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1�SSC, 0.1% SDS, preferably 0.1�SSC, 0.1% SDS at 60� C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Hybond� N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times. The length of the probe may be suitably selected, depending on the hybridization conditions, in this specific case it may be about 100 bp.
For example, the following methods may be employed to introduce a mutation by gene recombination. A mutant gene is prepared, and a bacterium is transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected. Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as �Red-driven integration� (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication (U.S. Pat. No. 6,303,383 or JP 05-007491A). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid lacking the ability to replicate in the host.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E. F., and Maniatis, T., �Molecular Cloning: A Laboratory Manual, Second Edition�, Cold Spring Harbor Laboratory Press (1989).
L-Δmino Acid-Producing Bacteria
The strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSF110-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine. The strain B-3996 was deposited on Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Apr. 7, 1987 under the accession number VKPM B-3996.
the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
the thrB gene which codes for homoserine kinase;
the thrC gene which codes for threonine synthase;
the rhtA gene which codes for a putative transmembrane protein;
the asd gene which codes for aspartate-α-semialdehyde dehydrogenase; and
the aspC gene which codes for aspartate aminotransferase (aspartate transaminase);
The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession NC�000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession NC�000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC�000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K-12. All three genes functions as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808, WO2003/097839).
The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession NC�000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.
Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession NC�000913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.
Examples of parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases (U.S. Pat. No. 6,218,168, Russian patent application 2003121601); E. coli W3110 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Pat. No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JP11155571A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307A1), and the like.
Examples of parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121)) or leucine analogs including P3-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the gene engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.
Examples of parent strains for deriving L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli A180/pFM201 (U.S. Pat. No. 6,258,554) and the like.
Examples of parent strains for deriving L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (his H), histidinol phosphate aminotransferase (his C), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth.
Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of such genes include genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfJk, pfkB), and glucose phosphate isomerase (pgi).
E. coli W3110sucA::KmR is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as �sucA gene�) of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.
Examples of parent strains for deriving L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Pat. No. 5,354,672); E. coli MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat. No. 4,407,952). Also, as aparent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488-424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Pat. No. 4,371,614). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). The tryptophan synthase consists of a and 13 subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).
2. Method of the Present Invention The method of the present invention is a method for producing an L-amino acid comprising cultivating the bacterium of the present invention in a culture medium to produce and excrete the L-amino acid into the medium, and collecting the L-amino acid from the medium.
The cultivation is preferably performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40� C., preferably 30 to 38� C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
EXAMPLES The present invention will be more concretely explained below with reference to the following non-limiting Examples.
Example 1 Construction of a Strain with an Inactivated Gene Coding for sRNA 1. Deletion of a Target Gene
A strain having deletion of a gene coding for sRNA can be constructed by the method initially developed by Datsenko, K. A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called �Red-driven integration�. The DNA fragment containing the CmR marker encoded by the cat gene can be obtained by PCR, using primers P1 (upstream primer) and P2 (downstream primer) and plasmid pMW118-attL-Cm-attR (WO 05/010175) as a template. Primer P1 contains both a region complementary to the 35/36-nt region located at the 3′ end of a target gene and a region complementary to the attL region in the plasmid pMW118-attL-Cm-attR. Primer P2 contains both a region complementary to the 35/36-nt region located at the 5′ end of a target gene and a region complementary to the attR region in the plasmid pMW118-attL-Cm-attR. The nucleotide sequences of the region complementary to the attL region in the plasmid pMW118-attL-Cm-attR and the region complementary to the attR region in the plasmid pMW118-attL-Cm-attR are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
Conditions for PCR can be as follows: denaturation step for 3 min at 95� C.; profile for two first cycles: 1 min at 95� C., 30 sec at 50� C., 40 sec at 72� C.; profile for the last 25 cycles: 30 sec at 95� C., 30 sec at 54� C., 40 sec at 72� C.; final step: 5 min at 72� C.
Electrocompetent cells can be prepared as follows: E. coli MG1655/pKD46 can be grown overnight at 30� C. in LB medium containing ampicillin (100 mg/l), and the culture can be diluted 100 times with 5 ml of SOB medium (Sambrook et al, �Molecular Cloning: A Laboratory Manual, Second Edition�, Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (1 mM). The cells can be grown with aeration at 30� C. to an OD600 of ≈0.6 and then can be made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation can be performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation can be incubated with 1 ml of SOC medium (Sambrook et al, �Molecular Cloning: A Laboratory Manual, Second Edition�, Cold Spring Harbor Laboratory Press, 1989) at 37� C. for 2.5 hours and then can be plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37� C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42� C. can be performed and the colonies can be tested for sensitivity to ampicillin.
The mutants having a gene coding for sRNA deleted and marked with the Cm resistance gene can be verified by PCR. Locus-specific primers P3 and P4 can be used in PCR for the verification. Conditions for PCR verification can be as follows: denaturation step for 3 min at 94� C.; profile for 30 cycles: 30 sec at 94� C., 30 sec at 54� C., 1 min at 72� C.; final step: 7 min at 72� C. The PCR product obtained in the reaction with the cells of parental strain MG1655 as a template and the PCR product obtained in the reaction with the cells of mutant strain as the template should differ in length (FIG. 2). Mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be obtained in this manner.
Length of PCR product
SEQ ID of upstream(P1)
obtained using as a
and downstream(P2)
template chromosome of:
P1 - SEQ ID NO: 17
P3 - SEQ ID NO: 19
P2 - SEQ ID NO: 18
P4 - SEQ ID NO: 20
P1 - SEQ ID NO: 21
P3 - SEQ ID NO: 23
P2 - SEQ ID NO: 22
P4 - SEQ ID NO: 24
P1 - SEQ ID NO: 25
P3 - SEQ ID NO: 27
P2 - SEQ ID NO: 26
P4 - SEQ ID NO: 28
P1 - SEQ ID NO: 29
P3 - SEQ ID NO: 31
P2 - SEQ ID NO: 30
P4 - SEQ ID NO: 32
P1 - SEQ ID NO: 33
P3 - SEQ ID NO: 35
P2 - SEQ ID NO: 34
P4 - SEQ ID NO: 36
P1 - SEQ ID NO: 37
P3 - SEQ ID NO: 39
P2 - SEQ ID NO: 38
P4 - SEQ ID NO: 40
P1 - SEQ ID NO: 41
P3 - SEQ ID NO: 43
P2 - SEQ ID NO: 42
P4 - SEQ ID NO: 44
P1 - SEQ ID NO: 45
P3 - SEQ ID NO: 47
P2 - SEQ ID NO: 46
P4 - SEQ ID NO: 48
P1 - SEQ ID NO: 49
P3 - SEQ ID NO: 51
P2 - SEQ ID NO: 50
P4 - SEQ ID NO: 52
P1 - SEQ ID NO: 53
P3 - SEQ ID NO: 55
P2 - SEQ ID NO: 54
P4 - SEQ ID NO: 56
P4 - SEQ ID NO: 57
P3 - SEQ ID NO: 59
P2 - SEQ ID NO: 58
P4 - SEQ ID NO: 60
P1 - SEQ ID NO: 61
P3 - SEQ ID NO: 63
P2 - SEQ ID NO: 62
P4 - SEQ ID NO: 64
P1 - SEQ ID NO: 65
P3 - SEQ ID NO: 67
P2 - SEQ ID NO: 66
P4 - SEQ ID NO: 68
Example 2 Production of L-Threonine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on threonine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the threonine-producing E. coli strain VKPM B-3996 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain B-3996-Δtarget gene. The strain B-3996 was deposited on Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street, 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Apr. 7, 1987 under the accession number B-3996. The strains B-3996-Δc0362, B-3996-Δc0465, B-3996-ΔdsrA, B-3996-ΔgcvB, B-3996-ΔmicC, B-3996-ΔrprA, B-3996-ΔrybB, B-3996-ΔryeE, B-3996-ΔrygB-sraE, B-3996-ΔsraB and B-3996-ΔsraH were obtained in this manner.
Strains B-3996 and each of strains B-3996-Δc0362, B-3996-Δc0465, B-3996-ΔdsrA, B-3996-ΔgcvB, B-3996-ΔmicC, B-3996-ΔrprA, B-3996-ΔrybB, B-3996-ΔryeE, B-3996-ΔrygB-sraE, B-3996-ΔsraB or B-3996-ΔsraH, were separately grown for 18-24 hours at 37� C. on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32� C. for 18 hours in 20�200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20�200-mm test tubes. Cells were grown for 65 hours at 32� C. with shaking at 250 rpm.
OD540 Amount of L-threonine, g/l
24.7 � 0.3
24.7 � 0.7
26.8 � 1.6
29.5 � 0.5
29.4 � 0.7
27.6 � 0.6
25.0 � 1.1
29.0 � 0.4
25.0 � 0.5
31.0 � 0.7
28.3 � 0.3
29.7 � 0.6
28.5 � 0.7
32.8 � 0.9
32.2 � 0.5
21.9 � 0.6
22.9 � 1.5
25.5 � 0.5
18.5 � 2.0
25.9 � 0.7
30.3 � 1.2
28.0 � 1.1
28.5 � 1.0
29.9 � 1.2
27.1 � 0.9
24.4 � 0.4
26.8 � 0.5
24.5 � 1.2
27.6 � 0.9
19.3 � 0.8
20.2 � 0.9
19.8 � 0.9
MnSO4 5H2O
Glucose and magnesium sulfate were sterilized separately. CaCO3 was sterilized by dry-heat at 180� C. for 2 hours. The pH was adjusted to 7.0. The antibiotic was introduced into the medium after sterilization.
Example 3 Production of L-Lysine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on lysine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the lysine-producing E. coli strain AJ11442 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain AJ11442-Δtarget gene. AJ11442 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and received an accession number of FERM BP-1543. Both E. coli strains, AJ11442 and AJ11442-Δtarget gene, can be cultured in L-medium at 37� C., and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500-ml flask. The cultivation can be carried out at 37� C. for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm. After the cultivation, the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.
The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115� C. for 10 min. Glucose and MgSO47H2O are sterilized separately. CaCO3 is dry-heat sterilized at 180� C. for 2 hours and added to the medium for a final concentration of 30 μl.
Example 4 Production of L-Cysteine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-cysteine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the E. coli L-cysteine-producing strain JM15(ydeD) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain JM15(ydeD)-Δtarget gene.
Example 5 Production of L-Leucine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-leucine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain 57-Δtarget gene. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386.
Both E. coli strains, 57 and 57-Δtarget gene, can be cultured for 18-24 hours at 37� C. on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32� C. for 18 hours in 20�200-mm test tubes containing 2 ml of L-broth supplemented with 4% sucrose. Then, the fermentation medium can be inoculated with 0.21 ml of seed material (10%). The fermentation can be performed in 2 ml of a minimal fermentation medium in 20�200-mm test tubes. Cells can be grown for 48-72 hours at 32� C. with shaking at 250 rpm. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol-acetic acid-water=4:1:1).
Example 6 Production of L-Histidine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-histidine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the histidine-producing E. coli strain 80 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain 80-Δtarget gene. The strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Oct. 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on Jul. 12, 2004.
Both E. coli strains, 80 and 80-Δtarget gene, can each be cultured in L-broth for 6 h at 29� C. Then, 0.1 ml of obtained culture can be inoculated into 2 ml of fermentation medium in a 20�200-mm test tube and cultivated for 65 hours at 29� C. with shaking on a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase consisting of n-butanol: acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.
0.2 of as total nitrogen
FeSO4 7H20
Example 7 Production of L-Glutamate by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-glutamate production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the E. coli L-glutamate-producing strain VL334thrC+ (EP 1172433) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain VL334thrC+-Δtarget gene. The strain VL334thrC+ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Dec. 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on Dec. 8, 2004.
Both strains, VL334thrC+ and VL334thrC+-Δtarget gene, can be grown for 18-24 hours at 37� C. on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2 ml of fermentation medium. The fermentation medium contains glucose (60 g/l), ammonium sulfate (25 μl), KH2PO4 (2 g/l), MgSO4 (1 μl), thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCO3 (25 μl). The pH is adjusted to 7.2. Glucose and CaCO3 are sterilized separately. Cultivation can be carried out at 30� C. for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl2.
Example 8 Production of L-Phenylalanine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-phenylalanine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain AJ12739-Δtarget gene. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Nov. 6, 2001 under accession no. VKPM B-8197 and then converted to a deposit under the Budapest Treaty on Aug. 23, 2002.
Both strains, AJ12739-Δtarget gene and AJ12739, can be cultivated at 37� C. for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20�200-mm test tube and cultivated at 37� C. for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. The 10�15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing no fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol:ethylacetate:25% aqueous ammonia:water=40:40:7:16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.
Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180� for 2 hours. The pH is adjusted to 7.0.
Example 9 Production of L-Tryptophan by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-tryptophan production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the tryptophan-producing E. coli strain SV164 (pGH5) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain SV164 (pGH5)-Δtarget gene. The strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine. The strain SV164 (pGH5) was described in detail in U.S. Pat. No. 6,180,373 or European patent 0662143. The strains SV164 (pGH5)-ΔgcvB::cat, SV164 (pGH5)-ΔrprA::cat, SV164 (pGH5)-ΔsraA::cat, SV164 (pGH5)-ΔsraB::cat, SV164 (pGH5)-ΔsraH::cat and SV164 (pGH5)-ΔsroE::cat were obtained in this manner.
Strains SV164(pGH5) and each of strains SV164 (pGH5)-ΔgcvB::cat, SV164 (pGH5)-ΔrprA::cat, SV164 (pGH5)-ΔsraA::cat, SV164 (pGH5)-ΔsraB::cat, SV164 (pGH5)-ΔsraH::cat or SV164 (pGH5)-ΔsroE::cat, were separately cultivated with shaking at 32� C. for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (10 mg/ml, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) were inoculated into 3 ml of a fermentation medium containing tetracycline (10 mg/ml) in 20�200-mm test tubes, and cultivated at 32� C. for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium was determined by TLC as described in Example 8. The results of ten (for SV164 (pGH5)-ΔgcvB::cat, SV164 (pGH5)-ΔrprA::cat and SV164 (pGH5)-ΔsraB::cat)/seven (for SV164 (pGH5)-ΔsraA::cat and SV164 (pGH5)-ΔsraH::cat)/five (for SV164 (pGH5)-ΔsroE::cat) independent test tube fermentations are shown in Table 4. As follows from Table 4, SV164 (pGH5)-ΔgcvB::cat, SV164 (pGH5)-ΔrprA::cat, SV164 (pGH5)-ΔsraA::cat, SV164 (pGH5)-ΔsraB::cat, SV164 (pGH5)-ΔsraH::cat and SV164 (pGH5)-ΔsroE::cat produced a higher amount of L-tryptophan, as compared to SV164 (pGH5).
34.6 � 1.6
34.8 � 1.4
32.9 � 0.9
38.5 � 0.3
34.1 � 1.1
4.3 � 0.9
34.2 � 1.8
28.6 � 0.5
26.3 � 1.1
6.6 � 0.1
28.7 � 0.5
ZnSO4 7 H2O
Example 10 Production of L-Proline by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-proline production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the proline-producing E. coli strain 702ilvA by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain 702ilvA-Δtarget gene. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Jul. 18, 2000 under accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
Both E. coli strains, 702ilvA and 702ilvA-Δtarget gene, can be grown for 18-24 hours at 37� C. on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 7.
Example 11 Production of L-Arginine by E. coli Strain Having a Gene Coding for sRNA Deleted To test the effect of inactivation of a gene coding for sRNA on L-arginine production, DNA fragments from the chromosome of the above-described mutant E. coli MG1655 having a gene coding for sRNA (target gene) deleted and marked with the Cm resistance gene can be transferred to the arginine-producing E. coli strain 382 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain 382-Δtarget gene. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Apr. 10, 2000 under accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001. The strains 382-ΔgcvB::cat, 382-ΔrprA::cat, 382-ΔrygB-sraE::cat and 382-ΔsraH::cat were obtained in this manner.
Strains 382 and each of strains 382-ΔgcvB::cat, 382-ΔrprA::cat, 382-ΔrygB-sraE::cat or 382-ΔsraH::cat were separately cultivated with shaking at 37� C. for 18 hours in 3 ml of nutrient broth, and 0.3 ml of the obtained cultures were inoculated into 2 ml of a fermentation medium in 20�200-mm test tubes and cultivated at 32� C. for 48 hours on a rotary shaker.
After the cultivation, the amount of L-arginine which had accumulated in the medium was determined by paper chromatography using the following mobile phase: butanol: acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-arginine was cut out, L-arginine was eluted with 0.5% water solution of CdCl2, and the amount of L-arginine was estimated spectrophotometrically at 540 nm. The results of ten (for 382-ΔgcvB::cat and 382-ΔrprA::cat)/nine (for 382-ΔrygB-sraE::cat and 382-ΔsraH::cat) independent test tube fermentations are shown in Table 5. As follows from Table 5, 382-ΔgcvB::cat, 382-ΔrprA::cat, 382-ΔrygB-sraE::cat and 382-ΔsraH::cat produced a higher amount of L-arginine, as compared to 382.
OD540 Amount of L-arginine, g/l
13.0 � 2.2
9.7 � 1.3
12.9 � 1.6
13.8 � 0.7
13.4 � 0.7
15.0 � 0.9
11.9 � 0.7
14.0 � 0.7
12.6 � 0.4
Glucose and magnesium sulfate were sterilized separately. CaCO3 was dry-heat sterilized at 180� C. for 2 hours. The pH was adjusted to 7.0.
Example 12 Elimination of Cm Resistance Gene (Cat Gene) from the Chromosome of L-Amino Acid-Producing E. coli Strains The Cm resistance gene (cat gene) can be eliminated from the chromosome of the L-amino acid-producing strain using the int-xis system. For that purpose, an L-amino acid-producing strain having DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δtarget gene::cat transferred by P1 transduction can be transformed with plasmid pMWts-Int/Xis. Transformant clones can be selected on the LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30� C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37� C. (at that temperature repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of CmSApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P3 and P4 (see Table 1) can be used in PCR for the verification. Conditions for PCR verification can be as described above. The PCR product obtained in reaction with cells having the eliminated cat gene as a template should be much shorter (�0.3-0.5 kbp in length) then strain with cat gene.
Example 13 Production of L-Lysine Using an Escherichia Bacterium in which Expression of rygB-sraE Gene is Attenuated In order to evaluate the effect of rygB-sraE gene attenuation on L-lysine yield, a rygB-sraE-disrupted strain was constructed. The whole genome sequence of Escherichia coli K-12 strain has been disclosed (Science, 277, 1453-1474 (1997)). The nucleotide sequence of rygB-sraE gene is also disclosed, and the strain can be constructed based on the sequence.
The rygB-sraE gene was disrupted according to the method called �Red-driven integration� that was developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645). �Red-driven integration� makes it possible to construct a gene-disrupted strain in one step by employing a PCR product obtained by using as primers synthetic oligonucleotides designed to have a part of the targeted gene on the 5′-ends and a part of an antibiotic-resistance gene on the 3′-ends. According to this method, primers which are complementary to a region proximal to the rygB-sraE gene and to a region proximal to a gene conferring antibiotic resistance to a template plasmid, respectively, were designed. PCR was performed using the oligonucleotides of SEQ ID NOS: 117 and 118 and the plasmid pMW118-att-cat as a template.
The obtained PCR product was purified by using an agarose gel, and used to transform the E. coli WC196LC strain that carries a plasmid pKD46 that has temperature-sensitive replication ability. pKD46 contains a DNA fragment of 2,154 nucleotides derived from λ phage which contains the Red recombinase-encoding genes (γ, β, and exo genes) of the λ Red homologous recombination system, which is controlled by an arabinose-inducible ParaB promoter (GenBank/EMBL Accession No. J02459, nucleotide numbers 31088 to 33241). pKD46 is necessary to integrate the PCR product into the chromosome of the WC196LC strain. The WC196LC strain (WO2006/038695) is an L-lysine-producing strain obtained by disrupting lysine decarboxylase genes (cadA and ldcC) in the WC1-96 strain (FERM BP-5252).
Competent cells for electroporation were prepared as follows. E. coli WC196LC/pKD46 strain was cultured overnight at 30� C. in LB medium that contained 100 mg/L of ampicillin, and then was diluted to 100-fold with 5 ml of SOB medium (Molecular Cloning Laboratory Manual 2nd Edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) that contained ampicillin and L-arabinose (1 mM). The diluted cells were grown at 30� C. with aeration until OD600 became approximately 0.6, and then concentrated to 100-fold and washed with ice-cold 10% glycerol solution three times, which were then used for electroporation. Electroporation was performed using 40 μL of the competent cells and about 100 ng of the PCR product. After electroporation, the cells were added to 1 mL of SOC medium (Molecular Cloning Laboratory Manual 2nd Edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) and cultured at 37� C. for 1 hour, and then cultured overnight at 37� C. on L-agar medium containing chloramphenicol to select a chloramphenicol-resistant recombinant strain. Then, in order to cure the pKD46 plasmid, the strain was subjected to passage culture twice at 42� C. on L-agar medium containing chloramphenicol, and the ampicillin-resistance of the obtained strains was tested to select an ampicillin-sensitive strain due to curing of the pKD46 plasmid.
According to a conventional method, the WC196LC strain and WC196LCArygB-sraE strain were transformed with the plasmid pCABD2 (WO01/53459), which is a plasmid for L-lysine production containing the dapA gene, dapB gene, lysC gene and ddh gene, to prepare a WC196LC/pCABD2 strain and WC196LCArygB-sraE/pCABD2 strain.
The WC196LC/pCABD2 strain and WC196LCArygB-sraE/pCABD2 strain were cultured at 37� C. in L medium contains 20 mg/L of streptomycin until OD600 became approximately 0.6, and then added to an equal volume of 40% glycerol solution and mixed. Then, the mixture was dispensed in an appropriate volume and stocked at −80� C., which was used as a glycerol stock.
The glycerol stocks of these strains were thawed, and 100 μl of each of the strains was spread uniformly over an L plate containing 20 mg/L of streptomycin and cultured at 37� C. for 24 hours. About one eighth of the cells of each strain on the plate was inoculated into 20 mL of the fermentation medium that has the composition shown below and further contains 20 mg/L of streptomycin in 500 mL-volume Sakaguchi flask, and cultured at 37� C. for 24 hours with reciprocal shaker. The amount of L-lysine that accumulated in the medium was analyzed by using Biotech Analyze AS210 (Sakura Seiki).
MnSO4�5H2O
The medium was adjusted to pH 7.0 with potassium hydroxide and sterilized by steam at 115� C. for 10 minutes.
Calcium carbonate (Official grade) was separately sterilized by heating at 180� C.
accumulation (g/L)
Example 14 Production of L-Lysine Using an Escherichia Bacterium in which Expression of gcvB Gene is Attenuated In order to evaluate the effect of gcvB gene attenuation on L-lysine yield, a gcvB-disrupted strain was constructed. The whole genome sequence of Escherichia coli K-12 strain has been disclosed (Science, 277, 1453-1474 (1997)). The nucleotide sequence of gcv gene is also disclosed, and the strain can be constructed based on the sequence.
The gcvB gene was disrupted according to the method called �Red-driven integration� that was developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645). According to this method, primers which are complementary to a region proximal to the gcvB gene and to a region proximal to a gene conferring antibiotic resistance to a template plasmid, respectively, were designed. PCR was performed using the oligonucleotides of SEQ ID NOS: 119 and 120 and the plasmid pMW118-att-cat as a template.
Competent cells for electroporation were prepared as follows. E. coli WC196LC/pKD46 strain was cultured overnight at 30� C. in LB medium that contained 100 mg/L of ampicillin, and then was diluted to 100-fold with 5 ml of SOB medium that contained ampicillin and L-arabinose (1 mM). The diluted cells were grown at 30� C. with aeration until OD600 became approximately 0.6, and then concentrated to 100-fold and washed with ice-cold 10% glycerol solution three times, which were used for electroporation. Electroporation was performed using 40 μL of the competent cells and about 100 ng of the PCR product. After electroporation, the cells were added to 1 mL of SOC medium and cultured at 37� C. for 1 hour, and then cultured overnight at 37� C. on L-agar medium containing chloramphenicol to select a chloramphenicol-resistant recombinant strain. Then, in order to cure the pKD46 plasmid, the strain was subjected to passage culture twice at 42� C. on L-agar medium containing chloramphenicol, and the ampicillin-resistance of the obtained strains was tested to select an ampicillin-sensitive strain due to curing of the pKD46 plasmid.
According to a conventional method, the WC196LC strain and WC196LCΔgcvB strain were transformed with the plasmid pCABD2 to prepare a WC196LC/pCABD2 strain and WC196LCAgcvB/pCABD2 strain.
The WC196LC/pCABD2 strain and WC196LCΔgcvB/pCABD2 strain were cultured at 37� C. in L medium containing 20 mg/L of streptomycin until OD600 became approximately 0.6, and then added to an equal volume of 40% glycerol solution and mixed. Then, the mixture was dispensed in an appropriate volume and stocked at −80� C., which was used as a glycerol stock.
The glycerol stocks of these strains were thawed, and 100 μl of each of the strains was spread uniformly over an L plate containing 20 mg/L of streptomycin and cultured at 37� C. for 24 hours. About one eighth of the cells of each strain on the plate was inoculated into 20 mL of the fermentation medium that has a composition shown below and further contains 20 mg/L of streptomycin in 500 mL-volume Sakaguchi flask, and cultured at 37� C. for 24 hours with reciprocal shaker. The amount of L-lysine that accumulated in the medium was analyzed by using Biotech Analyze AS210 (Sakura Seiki).
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Acids Res. 2007;35(3):1018-1037.Referenced byCiting PatentFiling datePublication dateApplicantTitleUS8273562Jan 5, 2012Sep 25, 2012Ajinomoto Co., Inc.Method for producing 4-hydroxy-L-isoleucineUS8367381Jan 5, 2012Feb 5, 2013Ajinomoto Co., Inc.Method for producing 4-hydroxy-L-isoleucineUS8367382Jan 5, 2012Feb 5, 2013Ajinomoto Co., Inc.Method for producing 4-hydroxy-L-isoleucineUS8703446Jan 7, 2013Apr 22, 2014Ajinomoto Co., Inc.Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae familyUS8722370Nov 28, 2012May 13, 2014Ajinomoto Co., Inc.Method for producing an L-amino acid using a bacterium of the enterobacteriaceae family, having attenuated expression of gene(s) encoding peptidaseUS8728774Feb 22, 2011May 20, 2014Ajinomoto Co., Inc.Method for producing L-amino acids using bacteria of the enterobacteriaceae familyUS8785161May 8, 2012Jul 22, 2014Ajinomoto Co., Inc.Method for producing L-amino acids using bacteria of the enterobacteriaceae familyClassifications U.S. Classification435/106, 536/23.1, 435/252.3, 435/320.1, 435/183International ClassificationC12N9/00, C12P13/04, C12N1/20, C12N15/00, C07H21/02Cooperative ClassificationC12P13/04European ClassificationC12P13/04Legal EventsDateCodeEventDescriptionFeb 26, 2014FPAYFee paymentYear of fee payment: 4Dec 29, 2008ASAssignmentOwner name: AJINOMOTO CO., INC., JAPANFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RYBAK, KONSTANTIN VYACHESLAVOVICH;SKOROKHODOVA, ALEKSANDRA YURIEVNA;VOROSHILOVA, ELVIRA BORISOVNA;AND OTHERS;REEL/FRAME:022033/0452;SIGNING DATES FROM 20081125 TO 20081201Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RYBAK, KONSTANTIN VYACHESLAVOVICH;SKOROKHODOVA, ALEKSANDRA YURIEVNA;VOROSHILOVA, ELVIRA BORISOVNA;AND OTHERS;SIGNING DATES FROM 20081125 TO 20081201;REEL/FRAME:022033/0452RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google