Process for the preparation of L-amino acids by fermentation and nucleotide sequences coding for the

专利摘要:
PURPOSE: Provided are a method for the fermentative preparation of L-amino acids and nucleotide sequences coding for the accDA gene by using Coryneform bacteria. CONSTITUTION: Cloned corynebacterium glutamicum DNA is replicable in coryneform microorganisms and contains at least nucleotide sequence coding accDNA gene of SEQ ID NO:1. Coryneform microorganisms transformed with one or more copies of the cloned corynebacterium glutamicum DNA. The shuttle vector pZ1accDA is contained in corynebacterium glutamicum (DSM 12785). The process for producing L-amino acids, comprises the step of culturing a coryneform bacterium that overexpresses the accDA gene.
公开号:KR20010049429A
申请号:KR1020000028677
申请日:2000-05-26
公开日:2001-06-15
发明作者:틸크이폰네；에겔링로타르；아이크만스베른하르트；삼헤르만；멕켈베티나
申请人:데구사-휠스 악티엔게젤샤프트；아달베르트 베. 플라텐타이히, 하；포르슝스젠트룸 율리히 게엠베하；
IPC主号:

专利说明:

Process for the preparation of L-amino acids by fermentation and nucleotide sequences coding for the accDA gene}
〈110〉 Degussa-Huls Aktiengesellschaft
Forschungszentrum Julich GmbH
120 Process for the preparation of L-amino acids by fermentation and
nucleotide sequences coding for the accDA gene
130 5-1999-024333-4; 5-1998-082611-0
〈150〉 DE 199 24 365.4
(151) 1999-05-27
〈160〉 3
〈170〉 KOPATIN 1.5
〈210〉 1
<211> 2123
<212> DNA
213 Corynebacterium glutamicum
〈220〉
<221> gene
222 (508) .. (1980)
<223> accDA
<400> 1
ctcgagcggg agtcggtgat cggccactct ctaagcaatg ccggctttaa aataaagcaa 60
cttatatgtt tctcaccaca tctggccgac gaccacgaag tatgttgtcg atcacagcta 120
aacgtgtgaa tgtgaagtta cctaactcac attgcaatgc gatagcgatt tggaaaactc 180
actcccccca atatcttaac ttaaacttaa aagtagtgtt ttacctgcat ttataaaagt 240
tcccgatcta ccccctcttt accccgaaat accccttttg caaagattgc aaacacaaca 300
gtgcaatagt taacgggctt cacacgtcac cattctgtcc ggttttaggc tatgttcggg 360
acgtctaggc aaaaagtagt tttgtgagat gaaacgcata atccgtcatt ttttacgcaa 420
tcgatagcct aaattgggct tagatcttcc gcctctaaat aggtatgcag agacattcga 480
attaattaac aaagccattt ttcggccgtg gagaagcgtt ttccgactat ggtgtggggc 540
atggaacaca cttcagcatt gacgctcata gactcggttt tggaccctga cagcttcatt 600
tcttggaatg aaactcccca atatgacaac ctcaatcaag gctatgcaga gaccttggag 660
cgggctcgaa gcaaggccaa atgcgatgaa tcggtaatta ctggagaagg caccgtggag 720
ggcattccgg tagccgttat tttgtccgat ttttccttcc tcggcggttc tttgggcacg 780
gtcgcgtcgg tgcgcatcat gaaggcgatt caccgcgcca cagagctgaa actcccactg 840
ctggtctccc ctgcttccgg tggtgcgcgc atgcaggaag acaatcgagc ttttgtcatg 900
atggtgtcca taaccgcggc tgtgcagcgt caccgcgagg cgcatttgcc gttcctggtg 960
tatttgcgca atcccacgat gggtggcgcc atggcctcgt ggggttcatc tgggcatctc 1020
acttttgcgg aacccggcgc gcagataggt ttcctgggtc ctcgcgtggt ggagttaacc 1080
actgggcatg cgcttccaga cggtgtgcag caggcggaga atttggtgaa aactggtgtg 1140
attgatggaa ttgtgtcgcc actccaattg cgtgcagcgg tggcaaaaac cctcaaggtt 1200
attcagccgg tagaggcaac ggatcgtttt tctccaacaa ctcctggcgt ggcacttccg 1260
gtgatggagg cgattgcgcg ttctcgtgac ccgcagaggc ctggaatcgg ggagattatg 1320
gaaacgttgg gggcagacgt cgtcaagctt tctggtgcgc gtgctggcgc attgagcccg 1380
gctgtgcgcg ttgccctggc gcgcatcggg ggccggcccg tggtgctgat tgggcaggat 1440
cgccgcttca cgcttgggcc gcaggagctg cgttttgcgc gtcgtggcat ttcgctggcg 1500
cgcgagctaa acctgccgat cgtgtccatc atcgacacct ccggcgccga attgtcgcag 1560
gcggctgagg agctcggcat cgcaagctcg attgcgcgca ccttgtccaa gcttatcgac 1620
gctcccctcc ccaccgtttc ggtcattatt ggtcagggcg ttggcggtgg cgcgctggcc 1680
atgctgcccg ccgatctggt ctacgcggcc gaaaacgcgt ggctgtccgc attgccacca 1740
gagggcgcct cggccatcct cttccgcgac accaaccacg ccgcggaaat catagagcga 1800
caaggcgtgc aggcgcacgc acttttaagc caagggctta tcgacgggat cgtcgccgaa 1860
accgagcact ttgttgaaga aattctcggc acaatcagca acgccctctc cgaattggat 1920
aacaatccgg agagggcggg acgcgacagt cgcttcacac gatttgagcg tttagcgcag 1980
taaagaaaat tatgcgctga tcaaatcgat gatgaacacc agggtacggc cagacagtgg 2040
gtggccggaa ccctcagggc cgtaagcagc ctctggcgga atggtcagct gacgacgtcc 2100
gccgaccttc atgcctggaa ttc 2123
〈210〉 2
<211> 1473
<212> DNA
213 Corynebacterium glutamicum
〈220〉
<221> CDS
222 (1) .. (1473)
<223> accDA
<400> 2
gtg gag aag cgt ttt ccg act atg gtg tgg ggc atg gaa cac act tca 48
Val Glu Lys Arg Phe Pro Thr Met Val Trp Gly Met Glu His Thr Ser
1 5 10 15
gca ttg acg ctc ata gac tcg gtt ttg gac cct gac agc ttc att tct 96
Ala Leu Thr Leu Ile Asp Ser Val Leu Asp Pro Asp Ser Phe Ile Ser
20 25 30
tgg aat gaa act ccc caa tat gac aac ctc aat caa ggc tat gca gag 144
Trp Asn Glu Thr Pro Gln Tyr Asp Asn Leu Asn Gln Gly Tyr Ala Glu
35 40 45
acc ttg gag cgg gct cga agc aag gcc aaa tgc gat gaa tcg gta att 192
Thr Leu Glu Arg Ala Arg Ser Lys Ala Lys Cys Asp Glu Ser Val Ile
50 55 60
act gga gaa ggc acc gtg gag ggc att ccg gta gcc gtt att ttg tcc 240
Thr Gly Glu Gly Thr Val Glu Gly Ile Pro Val Ala Val Ile Leu Ser
65 70 75 80
gat ttt tcc ttc ctc ggc ggt tct ttg ggc acg gtc gcg tcg gtg cgc 288
Asp Phe Ser Phe Leu Gly Gly Ser Leu Gly Thr Val Ala Ser Val Arg
85 90 95
atc atg aag gcg att cac cgc gcc aca gag ctg aaa ctc cca ctg ctg 336
Ile Met Lys Ala Ile His Arg Ala Thr Glu Leu Lys Leu Pro Leu Leu
100 105 110
gtc tcc cct gct tcc ggt ggt gcg cgc atg cag gaa gac aat cga gct 384
Val Ser Pro Ala Ser Gly Gly Ala Arg Met Gln Glu Asp Asn Arg Ala
115 120 125
ttt gtc atg atg gtg tcc ata acc gcg gct gtg cag cgt cac cgc gag 432
Phe Val Met Met Val Ser Ile Thr Ala Ala Val Gln Arg His Arg Glu
130 135 140
gcg cat ttg ccg ttc ctg gtg tat ttg cgc aat ccc acg atg ggt ggc 480
Ala His Leu Pro Phe Leu Val Tyr Leu Arg Asn Pro Thr Met Gly Gly
145 150 155 160
gcc atg gcc tcg tgg ggt tca tct ggg cat ctc act ttt gcg gaa ccc 528
Ala Met Ala Ser Trp Gly Ser Ser Gly His Leu Thr Phe Ala Glu Pro
165 170 175
ggc gcg cag ata ggt ttc ctg ggt cct cgc gtg gtg gag tta acc act 576
Gly Ala Gln Ile Gly Phe Leu Gly Pro Arg Val Val Glu Leu Thr Thr
180 185 190
ggg cat gcg ctt cca gac ggt gtg cag cag gcg gag aat ttg gtg aaa 624
Gly His Ala Leu Pro Asp Gly Val Gln Gln Ala Glu Asn Leu Val Lys
195 200 205
act ggt gtg att gat gga att gtg tcg cca ctc caa ttg cgt gca gcg 672
Thr Gly Val Ile Asp Gly Ile Val Ser Pro Leu Gln Leu Arg Ala Ala
210 215 220
gtg gca aaa acc ctc aag gtt att cag ccg gta gag gca acg gat cgt 720
Val Ala Lys Thr Leu Lys Val Ile Gln Pro Val Glu Ala Thr Asp Arg
225 230 235 240
ttt tct cca aca act cct ggc gtg gca ctt ccg gtg atg gag gcg att 768
Phe Ser Pro Thr Thr Pro Gly Val Ala Leu Pro Val Met Glu Ala Ile
245 250 255
gcg cgt tct cgt gac ccg cag agg cct gga atc ggg gag att atg gaa 816
Ala Arg Ser Arg Asp Pro Gln Arg Pro Gly Ile Gly Glu Ile Met Glu
260 265 270
acg ttg ggg gca gac gtc gtc aag ctt tct ggt gcg cgt gct ggc gca 864
Thr Leu Gly Ala Asp Val Val Lys Leu Ser Gly Ala Arg Ala Gly Ala
275 280 285
ttg agc ccg gct gtg cgc gtt gcc ctg gcg cgc atc ggg ggc cgg ccc 912
Leu Ser Pro Ala Val Arg Val Ala Leu Ala Arg Ile Gly Gly Arg Pro
290 295 300
gtg gtg ctg att ggg cag gat cgc cgc ttc acg ctt ggg ccg cag gag 960
Val Val Leu Ile Gly Gln Asp Arg Arg Phe Thr Leu Gly Pro Gln Glu
305 310 315 320
ctg cgt ttt gcg cgt cgt ggc att tcg ctg gcg cgc gag cta aac ctg 1008
Leu Arg Phe Ala Arg Arg Gly Ile Ser Leu Ala Arg Glu Leu Asn Leu
325 330 335
ccg atc gtg tcc atc atc gac acc tcc ggc gcc gaa ttg tcg cag gcg 1056
Pro Ile Val Ser Ile Ile Asp Thr Ser Gly Ala Glu Leu Ser Gln Ala
340 345 350
gct gag gag ctc ggc atc gca agc tcg att gcg cgc acc ttg tcc aag 1104
Ala Glu Glu Leu Gly Ile Ala Ser Ser Ile Ala Arg Thr Leu Ser Lys
355 360 365
ctt atc gac gct ccc ctc ccc acc gtt tcg gtc att att ggt cag ggc 1152
Leu Ile Asp Ala Pro Leu Pro Thr Val Ser Val Ile Gly Gln Gly
370 375 380
gtt ggc ggt ggc gcg ctg gcc atg ctg ccc gcc gat ctg gtc tac gcg 1200
Val Gly Gly Gly Ala Leu Ala Met Leu Pro Ala Asp Leu Val Tyr Ala
385 390 395 400
gcc gaa aac gcg tgg ctg tcc gca ttg cca cca gag ggc gcc tcg gcc 1248
Ala Glu Asn Ala Trp Leu Ser Ala Leu Pro Pro Glu Gly Ala Ser Ala
405 410 415
atc ctc ttc cgc gac acc aac cac gcc gcg gaa atc ata gag cga caa 1296
Ile Leu Phe Arg Asp Thr Asn His Ala Ala Glu Ile Ile Glu Arg Gln
420 425 430
ggc gtg cag gcg cac gca ctt tta agc caa ggg ctt atc gac ggg atc 1344
Gly Val Gln Ala His Ala Leu Leu Ser Gln Gly Leu Ile Asp Gly Ile
435 440 445
gtc gcc gaa acc gag cac ttt gtt gaa gaa att ctc ggc aca atc agc 1392
Val Ala Glu Thr Glu His Phe Val Glu Glu Ile Leu Gly Thr Ile Ser
450 455 460
aac gcc ctc tcc gaa ttg gat aac aat ccg gag agg gcg gga cgc gac 1440
Asn Ala Leu Ser Glu Leu Asp Asn Asn Pro Glu Arg Ala Gly Arg Asp
465 470 475 480
agt cgc ttc aca cga ttt gag cgt tta gcg cag 1473
Ser Arg Phe Thr Arg Phe Glu Arg Leu Ala Gln
485 490
〈210〉 3
<211> 491
<212> PRT
213 Corynebacterium glutamicum
<400> 3
Val Glu Lys Arg Phe Pro Thr Met Val Trp Gly Met Glu His Thr Ser
1 5 10 15
Ala Leu Thr Leu Ile Asp Ser Val Leu Asp Pro Asp Ser Phe Ile Ser
20 25 30
Trp Asn Glu Thr Pro Gln Tyr Asp Asn Leu Asn Gln Gly Tyr Ala Glu
35 40 45
Thr Leu Glu Arg Ala Arg Ser Lys Ala Lys Cys Asp Glu Ser Val Ile
50 55 60
Thr Gly Glu Gly Thr Val Glu Gly Ile Pro Val Ala Val Ile Leu Ser
65 70 75 80
Asp Phe Ser Phe Leu Gly Gly Ser Leu Gly Thr Val Ala Ser Val Arg
85 90 95
Ile Met Lys Ala Ile His Arg Ala Thr Glu Leu Lys Leu Pro Leu Leu
100 105 110
Val Ser Pro Ala Ser Gly Gly Ala Arg Met Gln Glu Asp Asn Arg Ala
115 120 125
Phe Val Met Met Val Ser Ile Thr Ala Ala Val Gln Arg His Arg Glu
130 135 140
Ala His Leu Pro Phe Leu Val Tyr Leu Arg Asn Pro Thr Met Gly Gly
145 150 155 160
Ala Met Ala Ser Trp Gly Ser Ser Gly His Leu Thr Phe Ala Glu Pro
165 170 175
Gly Ala Gln Ile Gly Phe Leu Gly Pro Arg Val Val Glu Leu Thr Thr
180 185 190
Gly His Ala Leu Pro Asp Gly Val Gln Gln Ala Glu Asn Leu Val Lys
195 200 205
Thr Gly Val Ile Asp Gly Ile Val Ser Pro Leu Gln Leu Arg Ala Ala
210 215 220
Val Ala Lys Thr Leu Lys Val Ile Gln Pro Val Glu Ala Thr Asp Arg
225 230 235 240
Phe Ser Pro Thr Thr Pro Gly Val Ala Leu Pro Val Met Glu Ala Ile
245 250 255
Ala Arg Ser Arg Asp Pro Gln Arg Pro Gly Ile Gly Glu Ile Met Glu
260 265 270
Thr Leu Gly Ala Asp Val Val Lys Leu Ser Gly Ala Arg Ala Gly Ala
275 280 285
Leu Ser Pro Ala Val Arg Val Ala Leu Ala Arg Ile Gly Gly Arg Pro
290 295 300
Val Val Leu Ile Gly Gln Asp Arg Arg Phe Thr Leu Gly Pro Gln Glu
305 310 315 320
Leu Arg Phe Ala Arg Arg Gly Ile Ser Leu Ala Arg Glu Leu Asn Leu
325 330 335
Pro Ile Val Ser Ile Ile Asp Thr Ser Gly Ala Glu Leu Ser Gln Ala
340 345 350
Ala Glu Glu Leu Gly Ile Ala Ser Ser Ile Ala Arg Thr Leu Ser Lys
355 360 365
Leu Ile Asp Ala Pro Leu Pro Thr Val Ser Val Ile Gly Gln Gly
370 375 380
Val Gly Gly Gly Ala Leu Ala Met Leu Pro Ala Asp Leu Val Tyr Ala
385 390 395 400
Ala Glu Asn Ala Trp Leu Ser Ala Leu Pro Pro Glu Gly Ala Ser Ala
405 410 415
Ile Leu Phe Arg Asp Thr Asn His Ala Ala Glu Ile Ile Glu Arg Gln
420 425 430
Gly Val Gln Ala His Ala Leu Leu Ser Gln Gly Leu Ile Asp Gly Ile
435 440 445
Val Ala Glu Thr Glu His Phe Val Glu Glu Ile Leu Gly Thr Ile Ser
450 455 460
Asn Ala Leu Ser Glu Leu Asp Asn Asn Pro Glu Arg Ala Gly Arg Asp
465 470 475 480
Ser Arg Phe Thr Arg Phe Glu Arg Leu Ala Gln
485 490
The present invention provides a method for producing L-amino acids, in particular L-lysine, by fermentation using a nucleotide sequence encoding the accDA gene and corynebacteria in which the accDA gene has been amplified.
L-amino acids, in particular L-lysine, are used in the animal feed, human pharmaceutical and pharmaceutical industries.
As is known, these amino acids are produced by fermentation of Corynebacteria strains, in particular Corynebacterium glutamicum. Because of their enormous importance, attempts have been made to improve the manufacturing method. Improvements to this method include fermentation techniques (eg, stirring and oxygenation), composition of the nutrient medium (eg, concentration of sugars during fermentation), post-treatment to product form (eg, ion exchange chromatography), or microorganisms themselves. It relates to means associated with the inherent productivity features of.
The productivity characteristics of these microorganisms, using mutagenesis, screening and mutant selection, are nutritional factors for regulatory amino acids that are resistant or important to antimetabolites such as lysine homologue S- (2-aminoethyl) cysteine. It is a constitution and can be improved by obtaining a strain that produces L-amino acid.
Recombinant DNA technology has been used for many years to amplify each amino acid biosynthetic gene and study its effects on L-lysine production to improve L-amino acid production-Corynebacterium strains. The results of this study are described in particular in Kinoshita, "Glutamic Acid Bacteria" in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Chummings, London, UK, 1985, 115-142; Hilliger (BioTec 2, 40-44 (1991); Eggeling (Amino Acids 6, 261-272 (1994); Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995); Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996).
The enzyme acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. The enzyme from Escherichia coli consists of four subunits. The accB gene encodes a biotin carboxyl carrier protein, the accC gene encodes a biotin carboxylase, and the accA and accD genes encode transcarboxylase. Cronan and Rock, Biosynthesis of Membrane Lipids, in: Escherichia coli and Salmonella typhimurium (ed. FC Neidhardt), 1996, pp. 612-636, American Scociety for Microbiology. Because of the nature of the enzyme that carboxylates an acyl group in acyl-CoA form, it is called acyl-CoA carboxylase.
The nucleotide sequence of the accBC gene from Corynebacterium glutamicum was determined by Jager et al. (Archives of Microbiology 166, 76-82 (1996)), and is generally located in Heidelberg, Germany. Available under Accession No. U35023 from the Data Bank of Molecular Biologies Laboratories (EMBL). The accBC gene encodes a subunit of acetyl-CoA carboxylase carrying a biotin carboxyl carrier protein domain and a biotin carboxylase domain.
It is an object of the present inventors to provide a novel method of improving the preparation of L-amino acids, in particular L-lysine, by fermentation.
1 is a map of plasmid pZ1accDA.
2 is a map of the plasmid pEK0accBCaccDA.
L-amino acids are used in the animal feed, human medicine and pharmaceutical industries. Therefore, it is generally important to provide new methods that improve the preparation of L-amino acids.
Where L-lysine or lysine is mentioned in the context of the present application below, it should be understood to mean not only bases but also salts such as lysine monohydrochloride or lysine sulfate.
Preferably, the present invention provides recombinant DNA originating from Corynebacterium, having the ability to replicate in Coryneform microorganisms and comprising at least the nucleotide sequence encoding the accDA gene shown in SEQ ID NO: 1.
The present invention also relates to (i) the nucleotide sequence shown in SEQ ID NO: 1, (ii) one or more sequences corresponding to said sequence (i) in the degenerate region of the genetic code, or (iii) said sequence (i) or (ii) One or more sequences capable of hybridizing with a sequence complementary to), and optionally (vi) a DNA having the replication capacity as claimed in claim 1, comprising (vi) a neutral sense mutation in sequence (i) above. .
The present invention also provides a transformed Coryneform microorganism, in particular the Corynebacterium genus, by introducing said DNA with replication capacity.
The present invention further relates to a method for producing L-amino acids by fermentation, in particular using corynebacteria, which produce L-amino acids and which nucleotides encoding the accDA gene have been amplified, in particular overexpressed.
Finally, the present invention provides a method for amplifying acyl-CoA carboxylase in Corynebacteria, using co-overexpression of the novel accDA gene and known accBC gene according to the present invention.
As used herein, the term " amplification " is used by appropriate DNA, e.g., by using a strong promoter or by using a gene encoding a suitable enzyme of high activity, or by optionally combining these means to increase the number of copies of that gene. By increasing the intracellular activity of one or more enzymes encoded in the microorganism.
Microorganisms provided by the present invention can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch or cellulose, or from glycerol and ethanol. Representative examples of such microorganisms are Corynebacteria, in particular Corynebacteria. Among the genera Corynebacterium, mention may be made of the species Corynebacterium glutamicum, which is known to those skilled in the art, in particular for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular Corynebacterium glutamicum species, are known wild type strains, namely Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum (lactofermentum) ATCC 13869, Brevibacterium divaricatum ATCC 14020, and L-amino acid-producing mutants or strains prepared therefrom, such as Corynebacterium glutamicum FERM-P 1709, Brevibacterium plaboom FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463 and Corynebacterium glutamicum FERM-P 6464.
We have successfully isolated a new accDA gene from Corynebacterium glutamicum. This accDA or other gene is isolated into Corynebacterium glutamicum by first constructing a genetic library of this microorganism in E. coli. Construction of gene libraries is generally provided in well-known textbooks and papers. Examples that may be mentioned are textbooks [author: Winnacker, titled: From Genes to Clones, Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990)] or paper [author: Sambrook et al., Titled: Molecular Cloning, A] Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well known gene library is a library of E. coli K-12 strain W3110 constructed in a λ vector by Kohara et al. (Cell 50, 495-508 (1987)). Bathe et al., Molecular and General Genetics 252, 255-265 (1996), describe the cosmid vector SuperCos I (WahI et al., Proceedings of the National Academy of Science USA 84, 2160-2164 (1987)). Gene library of Corynebacterium glutamicum ATCC 13032 constructed in E. coli K-12 strain NM554 (Raleigh et al., Nucleic Acids Research 16, 1563-1575 (1988)) is described. It is. In addition, Bormann et al. (Molecular Microbiology 6 (3), 317-326) describes a gene library of Corynebacterium glutamicum ATCC 13032 using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). In addition, the gene library of Corynebacterium glutamicum in E. coli can be expressed by pBR322 [Bolivar, Life Science 25, 807-818 (1979)] or pUC9 [Viera et al., Gene 19, 259-268. (1982), such as restriction- and recombinant-defective E. coli strains are particularly suitable strains, examples of which are described in Grant et al., Proceedings of the National Academy of Science USA 87 , 4645-4649 (1990), and then subcloned into a common vector suitable for sequencing long-chain DNA fragments colonized using cosmids, followed by Sanger et al. , Proceedings of the National of Science of the United States of America USA 74, 546 The sequence is analyzed as described in 3-5467 (1997).
A novel DNA sequence encoding the accDA gene from Corynebacterium glutamicum is obtained by the method described above, which is part of the present invention as SEQ ID NO: 1. The coding region (cds) of the accDA gene is shown in SEQ ID NO: 2. In addition, the amino acid sequence of the corresponding protein is derived from the DNA sequence of the present invention by the method described above. The resulting amino acid sequence of the accDA gene product is shown in SEQ ID NO: 3.
Because of the degeneracy of the genetic code, the DNA coding sequence obtained from SEQ ID NO: 1 is also part of the present invention. Similarly, DNA sequences that hybridize with SEQ ID NO: 1 or fragments of SEQ ID NO: 1 are also part of the present invention. In addition, conservative amino acid exchange, such as alanine and glycine exchange in a protein or glutamic acid and aspartic acid, is known to those skilled in the art as sense mutations that do not change the basis of protein activity, ie neutral. It is also known that changes in the N and / or C terminus of a protein may not substantially impair or even stabilize the function of the protein. Those skilled in the art are particularly well known in Ben-Bassat et al., Journal of Bacteriology 169, 751-757 (1987); O'Regan et al., Gene 77, 237-251 (1989), Sahin-Toth et al., Protein science 3, 240-247 (1994), Hochuli et al., Bio / Technology 6, 1321-1325 (1988 )] And the well-known genetics and molecular biology textbooks can be found on the subject. Amino acid sequences correspondingly obtained from SEQ ID NO: 3 are also part of the present invention.
We have found that overexpression of the accDA gene in Corynebacteria improves L-lysine production.
Overexpression can be achieved by increasing the number of copies of appropriate genes or by mutating ribosomal binding sites or promoters and regulatory regions located on top of the structural genes. Expression cassettes inserted on top of the structural genes work in the same way. In addition, by the inducible promoter, the expression can be increased during the course of L-lysine production by fermentation. Means to extend the life of the mRNA may also increase the expression thereof. In addition, enzyme activity can be increased by preventing degradation of enzyme proteins. The gene or gene construct may be located in a variety of copy number plasmids or integrated into the chromosome and amplified. Alternatively, overexpression of the gene of interest can be achieved by changing the composition of the medium and the culture technique.
One of ordinary skill in the art will, in particular, see Martin et al., Bio / Technology 5, 137-146 (1987); Guerrero et al., Gene 138, 35-41 1994); Tsuchiya and Morinaga Bio / Technology 6, 428-430 (1988); Eikmanns et al., Gene 102, 93-98 (1991); EP 0 472 869; US 4,601,893; Schwarzer and Puhler, Bioy / Technology 9, 84-87 (1991), Reinscheid et al., Applied and Environmental Microbiology 60, 126-132 (1994); LaBarre et al., Journal of Bacteriology 175, 1001-1007 (1993); WO 96/15246; Malumbres et al., Gene 134, 15-24 (1993); JP-A-10-229891; Jensen and Hammer, Biotechnology and Bioengineering 58, 191-195 (1998), Makrides, Microbiological Reviews 60, 512-538 (1996)] and textbooks on genetics and molecular biology can be found.
An example of a plasmid in which the accDA gene can be overexpressed is pZ1accDA (FIG. 1), which is included in strain MH20-22B / pZ1accDA. The plasmid pZ1accDA has two. E. coli-Corynebacterium glutamicum round trip vector, which is based on the plasmid pZ1 (Menkel et al., Applied and Environmental Microbiology 55 (3), 684-688 (1989)) with the accDA gene. Other plasmid vectors that can be replicated in Corynebacterium glutamicum, for example pEKEx1 [Eikmanns et al., Gene 102, 93-98 (1991)] or pZ8-1 [EP 0 375 889] in the same way Can be used.
In addition, the inventors have found that overexpression of known accBC genes in addition to the novel accDA gene according to the present invention in Corynebacteria may enhance acyl-CoA carboxylase production. An example of a plasmid in which the accDA gene and accBC gene can be overexpressed together is pEK0accBCaccDA (FIG. 2). Plasmid pEK0accBCaccDA is an E. coli-Corynebacterium glutamicum round trip vector, which carries the accBC and accDA genes and is based on plasmid pEK0 [Eikmanns et al., Gene 102, 93-98 (1991)]. Other plasmid vectors that can be replicated in Corynebacterium glutamicum, for example pEKEx1 [Eikmanns et al., Gene 102, 93-98 (1991)] or pZ8-1 [EP 0 375 889] in the same way Can be used.
In addition, overexpressing one or more enzymes in the biosynthetic pathway as well as the accDA gene may be advantageous for the production of L-amino acids. Thus, for the production of L-lysine, for example, simultaneously overexpressing the dapA gene [EP-B 0 197 335] encoding dihydrodipicolinate synthase, or S- (2-aminoethyl) cysteine resistance DNA fragments [EP-A 0 088 166] can be amplified at the same time.
Moreover, overexpressing the accDA gene, as well as blocking undesirable secondary reactions, may be advantageous for the production of L-amino acids, especially L-lysine. See Nakayama, "Breeding of Amino Acid Producing Micro-organism" in Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982.
The microorganisms prepared according to the invention can be cultured continuously or discontinuously by a batch process, a feed batch process or a repeated feed batch process, in order to produce L-lysine. A summary of known culture methods is found in the textbooks of Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Bioprocess Technology 1. Introduction to Bioengineering) (Gustav Fischer Verlag, Stuttgart, 1991) and Storhas textbooks. Bioreactors und periphere Einrichtungen (Bioreactors and Peripheral Equipment) (View Verlag, Brunswick / Wiesbaden, 1994).
The culture medium to be used must suitably meet the needs of the particular strain. Techniques for culture media of various microorganisms can be found in the "Manual of Methods for General Bacteriology", the American Society for Bacteriology (Washington DC, USA, 1981). Carbon sources that can be used include sugars and carbohydrates (e.g. glucose, sucrose, lactose fructose, maltose, molasses, starch and cellulose), oils and fats (e.g. soybean oil, sunflower oil, peanut oil and coconut fat), Fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids (acetic acid). These materials can be used individually or as a mixture. Nitrogen sources that can be used are organic nitrogen-containing compounds (e.g. peptone, yeast extract, meat extract, malt extract, corn deposit, soy flour, urea), or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, carbonic acid) Ammonium and ammonium nitrate). Nitrogen sources can be used individually or as a mixture. Phosphorus sources that can be used are phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium salts. In addition, the culture medium should contain metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Finally, essential growth-promoting substances such as amino acids and vitamins can be used in addition to the substances mentioned above. Appropriate precursors can be added to the culture medium. The feed materials may be added all at once to the medium, or may be appropriately supplied during the culture.
The pH of the medium is adjusted by suitably using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled using antifoams such as fatty acid polyglycol esters. The stability of the plasmid can be maintained by adding a suitably selectively acting substance, such as an antibiotic, to the medium. Aerobic conditions can be maintained by introducing oxygen or an oxygen-containing gas mixture (eg air) into the medium. The temperature of the medium is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until the formation of the desired L-amino acid reaches a maximum. This goal can typically be achieved in 10 to 160 hours.
L-lysine can be analyzed via anion exchange chromatography followed by ninhydrin derivatization (Spackman et al., Analytical Chemistry 30, 1190 (1958)).
The following microorganisms have been deposited under the Treaty of Budapest: Deutsche Sammlung fur Mikrorganismen und Zellkulturen (DSMZ) in Braunschweig, Germany:
Corynebacterium glutamicum strain DSM5715 / pZ1accDA (Accession Number: DSM 12785)
Corynebacterium glutamicum strain DSM5715 / pEK0accBCaccDA (Accession Number: DSM 12787).
The process according to the invention is carried out to prepare L-amino acids, in particular L-aspartic acid, L-asparagine, L-homoserine, L-threonine, L-isoleucine and L-methionine by fermentation of Corynebacteria. Used. It is especially used for the production of L-lysine.
Example
The invention will be explained in more detail by the following examples.
Example 1
Cloning and Sequencing the accDA Gene
The gene library of Corynebacterium glutamicum ATCC 13032 is described in Cosmid pHC79 [Hohn and Collins, Gene 11, 291-298] as described in Bormann et al., Molecular Microbiology 6 (3), 317-326. (1980)].
The selected cosmids are digested using the restriction enzymes EcoRI and XhoI as directed by the manufacturer of these restriction enzymes (Boehringer Mannheim). The formed DNA fragment was mixed with the vector pUC18 (Norrander et al., Gene 26, 101-106 (1983)) already treated with the restriction enzymes EcoRI and XhoI, and then treated with T4 DAN ligase. Cloned into E. coli strain DH5α mcr [Grant et al., Proceedings of the National Academy of Science USA 87, 4645-4645 (1990)]. Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring. Harbor Laboratories]. Transformants are selected on LB agar containing 50 μg / ml of ampicillin (Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories). Plasmid DNA is isolated from the transformants and is called pUCaccDA. Subclones are then prepared via exonuclease III digestion using a kit (Erase-a-Base) provided by Promega (Heidelberg, Germany) for this purpose. The subclone is sequenced by dideoxy chain termination (Sanger et al. Proceedings of the National Academy of Science USA 74, 5463-5467 (1997)). This is done using an Auto-Read Sequencing Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Gel electrophoresis analysis is performed using an automated laser fluorescence (A.L.F.) sequencer (Amersham Pharmacia Biotech (Uppsala, Sweden)). The obtained nucleotide sequence is analyzed using HUSAR software package (Release 4.0, EMBL, Heidelberg, Geremany). The nucleotide sequence is shown in SEQ ID NO: 1. Analysis of the nucleotide sequence reveals an open reading frame of 1473 base pairs, which is termed the accDA gene. The accDA gene from Corynebacterium glutamicum encodes a polypeptide of 484 amino acids.
Example 2
Expression of accDA Gene in Corynebacterium glutamicum
The accDA gene is subcloned into the vector pZ1 [Menkel et al., Applied and Envirnmental Microbiology 55, 684-688 (1989)] for expression in Corynebacterium glutamicum. This is done by cleaving the plasmid pUCaccDA (see Example 1) with the restriction enzyme ClaI. The resulting 1.6 kb fragment is isolated as in Example 1, treated with Klenow polymerase and alkaline phosphatase and bound to a pZ1 vector previously linearized with ScaI. Using a binding compound. E. coli DH5αmcr [Grant et al., Proceedings of the National Academy of Science USA 87, 4645-4645 (1990)] and transformants were selected from LB agares containing kanamycin (50 μg / ml), A 7.7 kb round trip vector pZ1accDA (FIG. 1) is obtained. This is described by Haynes, FEMS Microbiol. Letters 61, 329-334 (1989)] were inserted into strain DSM 5715 by electroporation and the transformants were introduced into LBHIS agar [Liebl et al., FERMS Microbiology Letters 65, 299-304 (1989)]. Screening on yields Corynebacterium glutamicum strain DSM5715 / pZ1accDA.
Example 3
Preparation of L-Lysine Using Strain DSM5715 / pZ1accDA
After preculturing in medium CgIII [Keilhauer et al., Journal of Bacteriology 175, 5595-5603 (1993)], strain DSM5715 / pZ1accDA was produced and produced in medium CgXII [Keilhauer et al., Journal of Bacteriology 175, 5595-5603 (1993). )]. Glucose 4 and 50 mg / l of kanamycin sulfate are added.
After incubation for 48 hours, the absorbance at 660 nm and the concentration of L-lysine formed are measured. The experimental results are given in Table 1.
StrainODL-lysine (g / l) DSM571531.47.2 DSM5715 / pZ1accDA43.18.0
Example 4
Co-expression of accBC and accDA
(i) Construction of the expression vector pEK0accBCaccDA
Plasmid pWJ71 containing accBC (Jager et al., Archives of Microbiology 166, 76-82 (1996)) is digested with restriction enzymes AgeI and SmaI and then treated with Klenow polymerase and alkaline phosphatase. In parallel, the plasmid pUCaccDA is digested with EcoRI / XhoI and then treated with Klenow polymerase and alkaline phosphatase. A 2.1 kb fragment carrying accDA is isolated from the agarose gel by preliminary separation (Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories). The fragment is bound to the vector pWJ71 already prepared as above. The 4.6 kb fragment with accBCaccDA is cleaved from the resulting plasmid by KpnI / SalI digestion and separated again by preparative agarose gel electrophoresis. This fragment is Corynebacterium glutamicum / I. To bind to the coli reciprocating vector pEK0 (Eikmanns et al., Gene 102, 93-98 (1991)), pEK0 is digested with restriction enzymes KpnI and SalI and then treated with klenow polymerase and alkaline phosphatase. In this way the prepared vector is bound to a 4.6 kb fragment with accBCaccDA. The resulting vector pEK0accBCaccDA is shown in FIG. 2. This vector was electroporated as described in Example 2 [Haynes, FERM Microbiol. Letters 61, 329-334 (1989)] to obtain Corynebacterium glutamicum ATCC13032 / pEK0accBCaccDA.
(ii) Determination of Acyl-CoA Carboxylase Activity
After preincubation in medium CGIII [Keilhauer et al., Journal of Bacteriology 175, 5595-5603 (1993)], strain Corynebacterium glutamicum ATCC13032 / pEK0accBCaccDA was transferred to medium CGXII (Keilhauer et al., Jouranl). of Bacteriology 175, 5595-5603 (1993). Cells are harvested by centrifugation, and the cell pellet is washed once with 60 mM Tris-HCl, pH 7.2, and then resuspended in the same buffer. Cells are digested with 10 min sonication [Branson sonifier W-250, Branson Sonic Power Co., Danbury, USA]. Cell by-products are then separated by centrifugation at 4 ° C. for 30 minutes and the supernatant is used as crude extract in the enzyme test. The reaction mixture for enzyme testing consisted of 4 mg crude extract in 1 ml reaction volume, 60 mM Tris-HCl (pH 7.2), 65 mM KHCO3, 1 mM ATP, 1.5 mM MgCl ₂ , and 4 mM acyl-CoA (acetyl-CoA or propionyl-CoA). Optional). The test mixture was incubated at 30 ° C. and 100 μl samples were taken after 15, 30, 45 and 60 seconds, and the concentration of double malonyl-CoA or methylmalonyl-CoA was analyzed by HPLC [Kimura et al., Journal of Bacteriology 179, 7098-7102 (1997). As given in Table 2, strain Corynebacterium glutamicum ATCC13032 / pEK0accBCaccDA has high acyl-CoA carbosylase activity against both acetyl-CoA and propionyl-CoA, whereas the control strain has acetyl-CoA and propy It has low acyl-CoA carbosylase activity against both O'Neill-CoA.
Acyl-CoA Carboxylase Activity with Substrates StrainAcetyl-CoAPropionyl-CoA ATCC13032 / pEK0accBCaccDA0.0480.124 ATCC13032 / pEKO0.0110.018
The present invention allows the production of L-amino acids, in particular L-lysine, which can be used in animal feed, human pharmaceutical and pharmaceutical industries.

权利要求:
Claims (16)
[1" claim-type="Currently amended] Preferred recombinant DNA derived from Corynebacterium, which can be replicated in Coryneform microorganisms and comprises at least the nucleotide sequence encoding the accDA gene of SEQ ID NO: 1.
[2" claim-type="Currently amended] The nucleotide sequence of claim 1, wherein: (i) the nucleotide sequence of SEQ ID NO: 1
(ii) one or more sequences corresponding to sequence (i) in the degenerate region of the genetic code, or
(iii) one or more sequences that hybridize with the sequence complementary to sequence (i) or (ii), and optionally
(vi) DNA having replication capability comprising a neutral sense mutation in sequence (i).
[3" claim-type="Currently amended] A protein having the amino acid sequence of SEQ ID NO: 3 derived from the nucleotide sequence claimed in claim 1.
[4" claim-type="Currently amended] Coryneform microorganisms, in particular of the genus Corynebacterium, which have been transformed by introducing one or more DNA having the ability to replicate as claimed in claim 1.
[5" claim-type="Currently amended] A round trip vector pZ1accDA with a restriction map as shown in FIG. 1 deposited under accession number DSM12785 in Corynebacterium glutamicum.
[6" claim-type="Currently amended] A method for producing L-amino acids, in particular L-lysine, by fermentation of corynebacteria, in which the accDA gene or nucleotide sequence encoding it is amplified, in particular overexpressed.
[7" claim-type="Currently amended] The method of claim 5, wherein a bacterium is used in which other genes of the biosynthetic pathway of the desired L-amino acid are added and amplified.
[8" claim-type="Currently amended] The method of claim 5, wherein a bacterium is used wherein at least partly the metabolic pathways that reduce the formation of the desired L-amino acids are blocked.
[9" claim-type="Currently amended] 8. The method according to any one of claims 5 to 7, wherein a strain transformed with a plasmid vector carrying a nucleotide sequence encoding an accDA gene is used.
[10" claim-type="Currently amended] The method of claim 8, wherein a bacterium transformed by plasmid vector pZ1accDA deposited under accession number DSM12785 in Corynebacterium glutamicum is used.
[11" claim-type="Currently amended] The method according to any one of claims 6 to 10, wherein corynebacteria are used to produce L-aspartic acid, L-asparagine, L-homoserine, L-threonine, L-isoleucine and L-methionine. .
[12" claim-type="Currently amended] The method according to any one of claims 5 to 10, wherein corynebacteria, which produce L-lysine, are used.
[13" claim-type="Currently amended] The method of claim 5, wherein the accBC gene is overexpressed in addition to the accDA gene.
[14" claim-type="Currently amended] The method of claim 5, wherein the dapA gene encoding dihydrodipicolinate synthase is simultaneously overexpressed.
[15" claim-type="Currently amended] The method of claim 5, wherein the DNA fragments conferring S- (2-aminoethyl) cysteine resistance are simultaneously amplified.
[16" claim-type="Currently amended] The fermentation step according to any one of claims 6 to 15, wherein a) the fermentation of Corynebacterium producing the desired L-amino acid, at least the accDA gene is amplified,
b) concentration of the desired L-amino acid in the medium or bacterial cell, and
c) A process for producing L-amino acid by fermentation, by performing a step of separating the desired L-amino acid.

类似技术:

---

公开号 | 公开日 | 专利标题

---

EP1094111B1|2006-08-30|Coryneform Bacteria with a deletion of Phosphoenolpyruvate-carboxykinase and their use

---

US7435584B2|2008-10-14|L-lysine-producing corynebacteria and process for the preparation of L-lysine

---

US7074607B2|2006-07-11|Polynucleotide sequences which encode the zwa1 gene

---

US7135313B2|2006-11-14|Method for producing L-lysine or L-lysine containing feed additives with a cornebacteria containing a mutated lysC

---

US7504242B2|2009-03-17|Process for the fermentative production of amino acids using coryneform bacteria in which OpcA gene expression is amplified

---

US6200785B1|2001-03-13|L-lysine-producing corynebacteria and process for the preparation of l-lysine

---

US20020192674A1|2002-12-19|Nucleotide sequence coding for the OtsA protein

---

US20050196848A1|2005-09-08|Process for the fermentative production of L-amino acids by attenuation of the poxB gene

---

US8445241B2|2013-05-21|Process for the preparation of L-amino acids

---

ES2203384T3|2004-04-16|Sequences of nucleotides coding the gen thre and procedure for the fermentative preparation of l-treonine with corineform bacteria.

---

US6872553B2|2005-03-29|Nucleotide sequences which code for the pck gene

---

US6861246B2|2005-03-01|L-lysine-producing corynebacteria and process for the preparation of lysine

---

US20060014259A9|2006-01-19|Process for the preparation of L-amino acids with amplification of the zwf gene

---

US20020058277A1|2002-05-16|Nucleotide sequences which code for the Gap2 protein

---

US6713289B2|2004-03-30|Nucleotide sequences which code for the eno gene

---

ES2344248T3|2010-08-23|Sequences of nucleotides coding the dapc gene, and procedure for the preparation of l-lisine.

---

US20030175911A1|2003-09-18|Process for the preparation of L-amino acids with amplification of the zwf gene

---

WO2002074944A1|2002-09-26|Process for the preparation of l-amino acids by using coryneform bacteria

---

KR20040014489A|2004-02-14|Process for the production of l-amino acids by fermentation using coryneform bacteria

---

EP1096010B1|2010-09-15|Nucleotide sequences coding for branched-chain amino acids export proteins, method for isolating them and their use

---

EP1103611A1|2001-05-30|Nucleotide sequences coding for the genes sucC and sucD

---

US7105302B2|2006-09-12|Nucleotide sequences coding for the pknB gene

---

US6783967B2|2004-08-31|Nucleotide sequences which code for the rpoB gene

---

US20020106748A1|2002-08-08|Novel nucleotide sequences encoding the zwa2 gene

---

ES2247987T3|2006-03-16|Corineform bacteria producers of l-lisina, and procedure for the preparation of lisina.

同族专利:

---

公开号 | 公开日

---

EP1055725A2|2000-11-29|

---

CN1275620A|2000-12-06|

---

ZA200002658B|2000-11-29|

---

EP1055725B1|2003-10-22|

---

US6808914B2|2004-10-26|

---

JP2001008693A|2001-01-16|

---

ES2208178T3|2004-06-16|

---

AU3644900A|2001-02-01|

---

CA2307327A1|2000-11-27|

---

HU0002022A2|2003-02-28|

---

ID26143A|2000-11-30|

---

SK7442000A3|2000-12-11|

---

DE19924365A1|2000-11-30|

---

US20020142405A1|2002-10-03|

---

EP1055725A3|2000-12-20|

---

US6361986B1|2002-03-26|

---

BR0002493A|2001-05-08|

---

HU0002022D0|2000-08-28|

---

AT252635T|2003-11-15|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
1999-05-27|Priority to DE19924365A

---

1999-05-27|Priority to DE19924365.4

---

2000-05-26|Application filed by 데구사-휠스 악티엔게젤샤프트, 아달베르트 베. 플라텐타이히, 하, 포르슝스젠트룸 율리히 게엠베하

---

2001-06-15|Publication of KR20010049429A

优先权:

---

申请号 | 申请日 | 专利标题

---

DE19924365A|DE19924365A1|1999-05-27|1999-05-27|Process for the fermentative production of L-amino acids and nucleotide sequences coding for the accDA gene|

---

DE19924365.4|1999-05-27|