RU2832588C1 - Recombinant strain for production of l-amino acid, method for construction and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a bacterium for producing L-amino acid. Bacterium has improved expression of the polynucleotide for encoding the protein represented by SEQ ID NO: 3, and improved expression of a polynucleotide for encoding a protein represented by SEQ ID NO: 31, and/or has base mutations in positions −45 bp and −47 bp promoter region represented by SEQ ID NO: 57.

EFFECT: invention enables to obtain amino acids, including lysine, with a high degree of efficiency.

19 cl, 8 tbl, 16 ex

Description

This application claims priority under Patent Application 202011105063.5, filed with the National Intellectual Property Administration of China on October 15, 2020, entitled "Recombinant Strain for Producing L-Amino Acid, a Method for Constructing It, and a Use Thereof"; Patent Application 202010790887.4, filed with the National Intellectual Property Administration of China on August 7, 2020, entitled "Recombinant Strain for Producing L-Amino Acid, a Method for Constructing It, and a Use Thereof"; and Patent Application 202010514037.1, filed with the National Intellectual Property Administration of China on June 8, 2020, entitled "Recombinant Strain for Modifying lysc Gene, a Method for Constructing It, and a Use Thereof". All three of these prior applications are incorporated herein by reference in their entireties.

Field of technology

This invention relates to the field of genetic engineering and microbial technologies, in particular relates to a recombinant strain producing an L-amino acid, to a method for constructing it and to its use.

Prior art

L-lysine has a wide range of applications, including medicine, nutrition, feed and other aspects. Among them, L-lysine used as a food additive accounts for more than 90% of the total. At present, China is the second largest consumer market and the largest producer of L-lysine.

At present, L-lysine is mainly produced by direct fermentation, using strains with a complete L-lysine biosynthesis pathway, and using waste molasses, starch hydrolysate, etc. as substrates to produce L-lysine through aerobic fermentation. This method currently accounts for 2/3 of the world's L-lysine production, and its process is well established. This method is mainly applied to yeast, bacteria, and mold, and is widely represented in microorganisms. At present, the production strains used for L-lysine fermentation in industry are mainly mutant strains of the Corynebacterium and Brevibacterium genera propagated through mutagenesis. With the development of metabolic engineering and genetic engineering, gene mutations have become controllable. Therefore, in the process of constructing the parent strain by metabolic engineering, it is possible to accurately identify the key enzyme genes for producing L-lysine in the metabolic process, and then improve the expression of such key enzyme genes to achieve increased L-lysine production.

L-glutamic acid is mainly used in the production of monosodium glutamate and spices, and is used as a substitute for salt, food additive and biochemical reagent and the like. L-glutamic acid itself can be used as a medicine to participate in the metabolism of protein and sugar in the brain to promote the oxidation process. This product combines with ammonia in the body to synthesize non-toxic glutamine, which can reduce the amount of ammonia in the blood and relieve the symptoms of hepatic coma. In the past, the production of monosodium glutamate was mainly carried out by hydrolysis of wheat gluten (glutenin), and now the microbial fermentation method is used for large-scale production.

Brief summary of the invention

The object of the present invention is to develop a new strain with the ability to produce L-amino acid, thereby providing a method for efficiently producing L-amino acid.

In order to achieve the above objective, the inventor discovered through research that the gene and/or gene capable of producing amino acids through fermentation can have a highly efficient ability to produce L-amino acids by modifying this gene or improving its expression, which was unknown in the prior art; in addition, the inventor also found that mutation of a certain promoter sequence can also improve the ability of the corresponding microorganisms to produce L-amino acids. Based on the obtained results, the invention was completed.

The invention provides a bacterium producing L-amino acids, wherein the expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 is improved, and/or the expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 is improved, and/or the bases at positions 45 bp and -47 bp of the promoter region, presented in SEQ ID NO: 57, are mutated. The invention also provides a method for producing an L-amino acid by using such a microorganism.

According to the invention, the improvement of expression consists in that the expression of the polynucleotide is enhanced, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 31 has point mutations, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 31 has point mutations and the expression is enhanced.

In the first aspect of the invention, a bacterium is provided that produces an L-amino acid in which the expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 is improved. Preferably, the L-amino acid is L-lysine or L-glutamic acid.

The amino acid sequence SEQ ID NO: 3 represents the protein encoded by the gene .

The bacterium has an increased ability to produce L-amino acid.

The bacterium having the ability to produce an L-amino acid may be a bacterium that can accumulate the target L-amino acid in a culture medium in an amount preferably exceeding 0.5 g/L, more preferably exceeding 1.0 g/L.

Polynucleotides may encode amino acid sequences with a sequence homology of about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, about 99% or more to the amino acid sequence of SEQ ID NO: 3.

In one specific embodiment of the invention, the polynucleotide with improved expression comprises the nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the invention, improving expression means that the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 has point mutations such that arginine at position 334 of the amino acid sequence of SEQ ID NO: 3 is replaced by a terminator.

According to the invention, the amino acid sequence in which arginine at position 334 of the amino acid sequence shown in SEQ ID NO: 3 is replaced by a terminator is presented in SEQ ID NO: 4.

In one embodiment of the invention, the polynucleotide sequence with a point mutation is formed by mutating the 1000th base of the polynucleotide sequence shown in SEQ ID NO: 1.

According to the invention, the mutation comprises a mutation of the 1000th base of the polynucleotide sequence presented in SEQ ID NO: 1 from cytosine (C) to thymine (T).

In one embodiment of the invention, the polynucleotide sequence with a point mutation comprises the polynucleotide sequence shown in SEQ ID NO: 2.

The invention also provides a bacterium that produces L-amino acids that have improved expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31. Preferably, the L-amino acid is L-lysine. Preferably, the bacterium is a bacterium that belongs to the genus Corynebacterium.

The amino acid sequence SEQ ID NO: 31 represents the protein encoded by the gene

The microorganism has an increased ability to produce L-lysine compared to the wild type or parent strain.

Polynucleotides may encode amino acid sequences with about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology to the amino acid sequence of SEQ ID NO: 31.

In one specific embodiment of the invention, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 29.

In one embodiment of the invention, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 has point mutations such that tyrosine at position 592 of the amino acid sequence of SEQ ID NO: 31 is replaced by other amino acids.

According to the invention, it is preferred that tyrosine at position 592 is replaced by phenylalanine.

According to the invention, an amino acid sequence in which tyrosine (Y) at position 592 of the amino acid sequence shown in SEQ ID NO: 31 is replaced by phenylalanine (F) is shown in SEQ ID NO: 32.

In one embodiment of the invention, the polynucleotide sequence with a point mutation is formed by mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29.

According to the invention, the mutation comprises a mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29 from adenine (A) to thymine (T).

In one embodiment of the invention, the polynucleotide sequence with a point mutation comprises the polynucleotide sequence shown in SEQ ID NO: 30.

According to the invention, the bacterium may be a microorganism that belongs to the genus Corynebacterium, such as Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium pekinense.

In one embodiment of the invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YP97158, with the deposition number CGMCC No.12856, which was deposited on August 16, 2016, the depository is the General Microbiology Center of China Microbial Species Conservation and Management Commission, No. 3, Yard. 1 Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355, recorded in Chinese Patent Application CN106367432A (filing date: September 1, 2016, publication date: February 1, 2017).

In one embodiment of the invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC 13869.

Expression of polynucleotides can be enhanced by the following means: substitution or mutation in expression regulatory sequences, introduction of mutation into polynucleotide sequences, increase in the number of copies of polynucleotides introduced via a chromosomal insertion or vector, or a combination thereof.

The expression regulation sequences of the polynucleotides can be modified. The expression regulation sequences control the expression of the polynucleotides to which they are functionally linked and can include, for example, promoters, terminators, enhancers, silencers, and the like. The polynucleotides can have changes in the start codon. The polynucleotides can be incorporated into specific sites on chromosomes to increase the copy number. Here, specific sites can include, for example, transposon sites or intergenic sites. In addition, the polynucleotides can be incorporated into an expression vector, and the expression vector can be introduced into host cells to increase the copy number.

In one embodiment of the invention, the copy number is increased by incorporating polynucleotides or polynucleotides with point mutations into specific sites on microbial chromosomes.

In one embodiment of the invention, the nucleic acid sequence is overexpressed by incorporating polynucleotides with promoter sequences or polynucleotides with promoter sequences and point mutations into specific sites on microbial chromosomes.

In one embodiment of the invention, the copy number is increased by incorporating polynucleotides or polynucleotides with point mutations into expression vectors and introducing these expression vectors into host cells.

In one embodiment of the invention, the amino acid sequence is overexpressed by incorporating polynucleotides with promoter sequences or polynucleotides with promoter sequences and point mutations into expression vectors and introducing these expression vectors into host cells a.

In one specific embodiment of the invention, the promoter is a polynucleotide promoter (gene ), encoding the amino acid sequence of SEQ ID NO: 3.

In one specific embodiment of the invention, the promoter is a polynucleotide promoter (gene ), encoding the amino acid sequence of SEQ ID NO: 31.

In some specific embodiments of the invention, the vectors used are the pK18mobsacB plasmid and the pXMJ19 plasmid.

According to the invention, the bacterium may also have other improvements associated with an increase in the production of L-amino acids.

In a second aspect of the invention, there is provided a polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector comprising the polynucleotide sequence, and a recombinant strain comprising the polynucleotide sequence.

According to the invention, the polynucleotide sequence has improved expression and the improvement includes point mutations of the polynucleotide encoding the polypeptide containing the amino acid sequence presented in SEQ ID NO: 3, wherein arginine at position 334 of the amino acid sequence is replaced by a terminator.

According to the invention, the amino acid sequence in which arginine at position 334 of the amino acid sequence presented in SEQ ID NO: 3 is replaced by a terminator is presented in SEQ ID NO: 4.

According to the invention, a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence presented in SEQ ID NO: 3 comprises the polynucleotide sequence presented in SEQ ID NO: 1.

In one embodiment of the invention, the mutant polynucleotide sequence presented in the invention is formed by mutation of the 1000th base of the polynucleotide sequence presented in SEQ ID NO: 1.

According to the invention, the mutation comprises a mutation of the 1000th base of the polynucleotide sequence shown in SEQ ID NO: 1 from cytosine (C) to thymine (T).

In one embodiment of the invention, the mutant polynucleotide sequence comprises the polynucleotide sequence presented in SEQ ID NO: 2.

According to the invention, the amino acid sequence with substitution comprises the amino acid sequence presented in SEQ ID NO: 4.

According to the invention, the polynucleotide sequence comprises a polynucleotide encoding polypeptides containing the amino acid sequence presented in SEQ ID NO: 31, where tyrosine at position 592 is replaced by other amino acids.

According to the invention, preferably, tyrosine at position 592 is replaced by phenylalanine.

According to the invention, the amino acid sequence in which tyrosine (Y) at position 592 of the amino acid sequence shown in SEQ ID NO: 31 is replaced by phenylalanine (F) is presented in SEQ ID NO: 32.

According to the invention, preferably the polynucleotide

a sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 31 comprises the polynucleotide sequence shown in SEQ ID NO: 29.

In one embodiment of the invention, the polynucleotide sequence is formed by mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29.

According to the invention, the mutation comprises a mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29 from adenine (A) to thymine (T).

In one embodiment of the invention, the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO: 30.

According to the invention, the amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 32.

According to the invention, mutation refers to a change in a base/nucleotide of a site. The mutation method can be selected from at least one of mutagenesis, a PCR site-directed mutation method, and/or a homologous recombination method. In the present invention, the PCR site-directed mutation method and/or the homologous recombination method are preferred.

According to the invention, a recombinant vector is constructed by introducing a polynucleotide sequence into a plasmid.

In one embodiment of the invention, the plasmid is plasmid pK18mobsacB.

In another embodiment of the invention, the plasmid is plasmid pXMJ19.

In particular, the polynucleotide sequence and plasmid can be constructed as a recombinant vector using the NEBuider recombination system.

According to the invention, the recombinant strain contains a polynucleotide sequence.

As one embodiment of the invention, the parent strain of the recombinant strain is YP97158.

As one embodiment of the invention, the parent strain of the recombinant strain is ATCC 13869.

In a third aspect of the invention, a method for constructing a recombinant strain for producing an L-amino acid is also provided.

According to the invention, the design method includes the following stages:

Modification of the polynucleotide sequence wild type shown in SEQ ID NO: 1 in a host strain to mutate its 1000th base, to obtain a recombinant strain containing the mutated encoding gene

According to the method of construction according to the invention, the modification comprises at least one of a mutagenesis method, a PCR site-directed mutation method and/or a homologous recombination method.

According to the construction method of the invention, the mutation refers to a mutation of the 1000th base in SEQ ID NO: 1 from cytosine (C) to thymine (T). In particular, a polynucleotide sequence containing a mutant coding gene shown in SEQ ID NO: 2. In addition, the method of construction includes the following steps:

(1) Modification of the nucleotide sequence of a gene wild type, as shown in SEQ ID NO: 1, to mutate its 1000th base, to obtain a mutant polynucleotide sequence of the gene

(2) Linking the mutant polynucleic acid sequence to a plasmid to construct a recombinant vector; and

(3) Introduction of the recombinant vector into the host strain to obtain a recombinant strain containing the mutant coding gene .

According to the method of construction according to the invention, step (1) comprises: constructing a gene with point mutation: synthesis of two pairs of primers P1 and P2, P3 and P4 for amplification of gene fragments based on the genome sequence of the unmodified strain, and the introduction of a point mutation into the sequence of SEQ ID NO: 1 gene wild type by the PCR site-directed mutation method, to obtain the nucleotide sequence SEQ ID NO: 2 of the gene with a point mutation, which is fixed as

In one embodiment of the invention, the genome of the unmodified strain may be derived from strain ATCC13032 and its genome sequence may be obtained from the NCBI website.

In one embodiment of the invention, in step (1), the primers are:

In one embodiment of the invention, PCR amplification is performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 40 seconds at 72°C (30 cycles), and extension for 10 minutes at 72°C.

In one embodiment of the invention, PCR amplification with overlapping primers is performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 60 seconds at 72°C (30 cycles), and extension for 10 minutes at 72°C.

According to the method of construction according to the invention, step (2) comprises constructing a recombinant plasmid, comprising: combining isolated and purified plasmids and pK18mobsacB via the NEBuider recombination system to produce a recombinant plasmid.

According to the method of construction according to the invention, step (3) includes construction of a recombinant strain: transformation of a recombinant plasmid into a host strain to obtain a recombinant strain.

In one embodiment of the invention, the transformation in step (3) is an electrical transformation method.

In one embodiment of the invention, the host strain is YP97158. In one embodiment of the invention, recombination is achieved by homologous recombination.

In a fourth aspect of the invention, a method for constructing a recombinant strain for producing an L-amino acid is also provided.

According to the invention, the method of construction includes the following stages: amplification of upstream and downstream fragments of the homologous arm of the gene , coding region of the gene and the sequence of its promoter region, or amplification of the coding region of the gene or and the sequence of its promoter region, followed by the introduction of the gene or into the host strain genome through homologous recombination to obtain overexpression of the gene or in the strain.

In one embodiment of the invention, primers for amplifying the upstream portion of the homologous arm are:

In one embodiment of the invention, primers for amplifying the downstream portion of the homologous arm are:

In one embodiment of the invention, primers for amplifying the sequence of the coding region of a gene and its promoter region are:

In one embodiment of the invention, the above P7-P12 are used as primers and the upstream homologous fragment, the downstream homologous fragment and the fragment or with its own promoter obtained by amplification are mixed as amplification templates to obtain an integrated fragment of the homologous arm.

In one embodiment of the invention, the PCR system used is: 10×Ex Taq buffer 5 μl, dNTP mix (deoxynucleotides) (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume: 50 μl; PCR amplification is performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 60 seconds at 72°C (30 cycles), and extension for 10 minutes at 72°C.

In one embodiment of the invention, the PK18mobsacB shuttle plasmid is combined with the upstream and downstream homology arm fragments, coding region fragments of the gene and the promoter region, using the NEBuider recombination system, to produce an integrated plasmid.

In one embodiment of the invention, the integrated plasmid is transfected into a host strain and the gene or are introduced into the genome of the host strain by means of homologous recombination.

In one embodiment of the invention, the host strain is YP97158.

In one embodiment of the invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 2.

In a fifth aspect of the invention, a method for constructing a recombinant strain for producing an L-amino acid is also provided.

According to the invention, the design method includes the following stages:

Amplification of the coding region of a gene and the sequence of the promoter region, or coding region of the gene and promoter region sequences, construction of an overexpression plasmid vector, and transfection of the vector into a host strain to obtain overexpression or in the strain.

In one embodiment of the invention, primers for amplifying the sequence of the coding region of a gene and its promoter region are:

In one embodiment of the invention, the PCR system comprises: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 units/μl) 0.25 μl, total volume 50 μl; PCR amplification is performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 60 seconds at 72°C (30 cycles), and extension for 10 minutes at 72°C.

In one embodiment of the invention, the shuttle plasmid pXMJ19 is combined with fragments And with their own promoters by using the NEBuider recombination system to produce an overexpression plasmid.

In one embodiment of the invention, the host strain is YP97158. In one embodiment of the invention, the host strain is ATCC 13869.

In one embodiment of the invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 2.

The invention also provides a method for constructing a recombinant strain of corynebacterium.

According to the invention, the design method includes the following stages:

modification of the polynucleotide sequence wild type in the host strain as shown in SEQ ID NO: 29 to mutate its 1775th base, to obtain a recombinant Corynebacterium strain containing the mutant encoding gene NCgl1575.

According to the method of construction according to the invention, the modification comprises at least one method of mutagenesis, a PCR site-directed mutation method and/or a homologous recombination method.

According to the construction method of the invention, the mutation refers to a mutation of the 1775th base in SEQ ID NO: 29 from adenine (A) to thymine (T); in particular, the polynucleotide sequence containing the mutant encoding gene NCgl1575 is shown in SEQ ID NO: 30.

In addition, the design method includes the following stages:

(1) modifying the nucleotide sequence of the wild-type NCgl1575 gene shown in SEQ ID NO: 29 by mutating its 1775th base to obtain a mutant polynucleotide sequence of the NCgl1575 gene;

(2) linking the mutant polynucleic acid sequence to a plasmid to construct a recombinant vector; and

(3) introducing the recombinant vector into the host strain to obtain a recombinant Corynebacterium strain containing the mutant NCgl1575 encoding gene.

According to the method of construction according to the invention, step (1) comprises: construction of the NCgl1575 gene with a point mutation: synthesis of two pairs of primers P1' and P2', as well as P3' and P4' for amplification of fragments of the NCgl1575 gene, based on the sequence of the Corynebacterium glutamicum genome, and introduction of a point mutation into SEQ ID NO: 29 of the wild-type NCgl1575 gene using the PCR site-directed mutation method, with obtaining the nucleotide sequence SEQ ID NO: 30 of the NCgl1575 gene with a point mutation, which is registered as NCgl1575 ^A1775T .

In one embodiment of the invention, the Corynebacterium glutamicum genome may be derived from strain ATCC13032 and its genome sequence may be obtained from the NCBI website.

In one embodiment of the invention, in step (1), the primers are:

In one embodiment of the invention, PCR amplification is performed as follows: denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, and elongation for 40 seconds at 72°C (30 cycles).

In one embodiment of the invention, PCR amplification with overlapping primers is performed as follows: denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, and elongation for 90 seconds at 72°C (30 cycles).

According to the construction method of the invention, step (2) comprises constructing a recombinant plasmid, comprising: combining the isolated and purified plasmids NCgl1575 ^A1775T and pK18mobsacB using the NEBuider recombination system to obtain the recombinant plasmid pK18-NCgl1575 ^A1775T .

According to the method of construction according to the invention, step (3) includes construction of a recombinant strain: transformation of the recombinant plasmid pK18-NCgl1575 ^A1775T into the host strain to obtain a recombinant strain.

In one embodiment of the invention, the transformation in step (3) is an electrical transformation method.

In one embodiment of the invention, the host strain is YP97158.

In one embodiment of the invention, recombination is accomplished by homologous recombination.

The invention also provides a method for constructing a recombinant strain of Corynebacterium.

According to the invention, the design method includes the following stages:

amplifying the upstream and downstream fragments of the homologous arm of the NCgl1575 gene, the coding region of the NCgl1575 gene and the sequence of its promoter region, or the coding region of the NCgl1575 ^A1775T gene and the sequence of its promoter region, and introducing the NCgl1575 or NCgl1575 ^A1775T gene into the genome of the host strain by homologous recombination to achieve overexpression of the NCgl1575 or NCgl1575 ^A17751 gene in the strain.

In one embodiment of the invention, primers for amplifying the upstream portion of the homologous arm are:

In one embodiment of the invention, primers for amplifying the downstream portion of the homologous arm are:

In one embodiment of the invention, primers for amplifying the sequence of the coding region of a gene and its promoter region are:

In one embodiment of the invention, the above-mentioned P77P12' are used as primers, and the upstream homologous fragment, the downstream homologous fragment, and the NCgl1575 or NCgl1575 ^A1775T fragment with its own promoter obtained by amplification are mixed as amplification templates to obtain an integrated homologous arm fragment.

In one embodiment of the invention, the PCR system used is: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl; PCR amplification is performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 180 sec at 72°C (30 cycles) and extension for 10 minutes at 72°C.

In one embodiment of the invention, the PK18mobsacB shuttle plasmid is combined with the integrated homologous arm fragment using the NEBuider recombinant system to produce the integrated plasmid.

In one embodiment of the invention, the integrated plasmid is transfected into a host strain and the NCgl1575 or NCgl1575 ^A1775T gene is introduced into the host strain genome by homologous recombination.

In one embodiment of the invention, the host strain is YP97158.

In one embodiment of the invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 30.

The invention also provides a method for constructing a recombinant strain of Corynebacterium.

According to the invention, the design method includes the following stages:

amplifying the coding region of the NCgl1575 gene and the promoter region sequence, or the coding region of the NCgl1575 ^A1775T gene and the promoter region sequence, constructing an overexpression plasmid vector, and transfecting the vector into a host strain to achieve overexpression of the NCgl1575 or NCgl1575 ^A1775T gene in the strain.

In one embodiment of the invention, the primers for amplifying the sequence of the coding region of the gene and the promoter region are:

In one embodiment of the invention, the PCR system is: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl; PCR amplification is performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 120 sec at 72°C (30 cycles) and extension for 10 minutes at 72°C.

In one embodiment of the invention, the shuttle plasmid pXMJ19 is combined with fragments of NCgl1575 or NCgl1575 ^A1775T with their own promoters using the NEBuider recombinant system to produce an overexpression plasmid.

In one embodiment of the invention, the host strain is YP97158.

In one embodiment of the invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 30.

Another aspect of the invention is to provide a nucleotide sequence of a promoter, which comprises a nucleotide sequence formed by mutation of bases at positions -45 bp and 47 bp in the promoter region shown in SEQ ID NO: 57.

According to the invention, the nucleotide guanine (G) at position -45 bp is mutated to adenine (A), and the nucleotide guanine (G) at position -47 bp is mutated to thymine (T) in the promoter region shown in SEQ ID NO: 57.

According to the invention, the nucleotide sequence of the promoter is as follows:

(a) the nucleotide sequence shown in SEQ ID NO: 58; or

(b) a nucleotide sequence having a sequence identity of more than 90%, preferably more than 95%, 98% with the nucleotide sequence shown in SEQ ID NO: 58 and retaining the increased activity of the promoter (a), while retaining the adenine (A) at position 45 bp and the thymine (T) at position 47 bp.

The invention also provides an expression cassette comprising the above promoter, comprising the promoter and a coding sequence that can be operably linked downstream of the promoter. In one embodiment of the invention, the coding sequence is the coding sequence of a gene .

The invention also provides a recombinant vector containing the nucleotide sequence of the promoter of the invention.

According to the invention, a recombinant vector is constructed by linking the nucleotide sequence of the promoter of the invention to a shuttle plasmid; as one embodiment of the invention, the shuttle plasmid is the pK18mobsacB plasmid.

The invention also provides a recombinant strain containing a nucleotide sequence of a promoter or the above-mentioned recombinant vector.

The recombinant strain of the invention comprises the nucleotide sequence shown in SEQ ID NO: 58. The nucleotide sequence shown in SEQ ID NO: 58 is the promoter region of the gene . In addition, the nucleotide sequence shown in SEQ ID NO: 58 is linked to the coding sequence of the gene . In particular, the recombinant strain may comprise an expression cassette or a recombinant vector as described in the invention above. In particular, the recombinant strain of the invention is obtained by transforming the expression cassette or the recombinant vector. The recombinant strain of the invention is formed by introducing the nucleotide sequence of the above-mentioned mutant promoter into a host strain for recombination; the host strain may be selected from strains that produce an L-amino acid, especially L-lysine, as known in the art, for example at least a strain selected from Corynebacterium. The Corynebacterium may be Corynebacterium glutamicum, Corynebacterium flavum, Corynebacterium crenatum and Corynebacterium pekinene; Corynebacterium glutamicum is preferred. As one embodiment of the invention, the host strain is YP97158.

The recombinant strain according to the invention uses the plasmid pK18mobsacB as a vector.

The recombinant strain of the invention may further comprise other modifications.

The invention also provides a method for constructing a recombinant strain producing L-lysine, which includes the following steps:

(1) modifying the promoter region shown in SEQ ID NO:57 by mutation at positions -45bp and -47bp to obtain a nucleotide sequence comprising a mutated promoter region.

According to the invention, the mutation refers to a mutation of the nucleotide guanine (g) at position -45 bp to adenine (a) and the nucleotide guanine (g) at position -47 bp to thymine (T) in the promoter region shown in SEQ ID NO: 57. In particular, the nucleotide sequence of the mutant promoter region is shown in SEQ ID NO: 58. In addition, the construction method further comprises the following steps:

(2) Linking the nucleotide sequence of the mutant promoter region to the plasmid to construct a recombinant vector; and

(3) Introduction of the recombinant vector into the host strain to obtain a recombinant strain producing L-lysine containing a mutant promoter region.

According to the invention, the mutation method in step (1) comprises mutagenesis, PCR site-directed mutation or homologous recombination, preferably PCR site-directed mutation.

According to the invention, stage (1) comprises:

Design of two pairs of primers for amplification of the promoter region of a gene followed by obtaining the nucleotide sequence of the mutant promoter region using PCR technology.

In one embodiment of the present invention, the primers in step (1) are:

In one embodiment of the present invention, step (1) comprises: using Corynebacterium glutamicum ATCC13032 as a template and using primers P1'' and P2'', P3'' and P4'' respectively to perform PCR amplification to obtain two DNA fragments; using the above two DNA fragments as templates and P1'' and P4'' as primers to obtain a DNA fragment comprising the nucleotide sequence of the promoter region (SEQ ID NO: 58) of the present invention by PCR amplification with overlapping primers.

According to the invention, in step (1), the DNA fragment obtained by PCR amplification with overlapping primers contains EcoRI and SphI enzymatic digestion sites at both ends, respectively.

According to the invention, step (2) comprises: agarose gel electrophoresis of the product amplified by the PCR reaction with overlapping primers and separation and purification, joining the fragment by double enzymatic digestion (EcoR I/Sph I) with the shuttle plasmid by the same digestion with two enzymes ((EcoR I/Sph I)) to obtain a recombinant vector with an allelic substitution.

According to the invention, the shuttle plasmid is the pK18mobsacB plasmid; and the constructed recombinant vector is

In one embodiment of the present invention, the recombinant plasmid has a kanamycin resistance marker.

In one embodiment of the present invention, the transformation step (3) is an electrical transformation method; for example, in step (3), the recombinant vector is transformed into the YP97158 strain.

The above various recombinant strains obtained by the invention can be used in fermentation to produce L-amino acids individually or in combination, or can be mixed with other L-amino acid-producing bacteria for fermentation to produce L-amino acids.

In another aspect of the invention, a method for producing L-amino acids is provided, which comprises culturing bacteria; and obtaining L-amino acids from the culture.

The bacteria can be cultured in a suitable medium under culture conditions known in the art. The culture medium can contain a carbon source, a nitrogen source, trace elements and combinations thereof. During the culture, the pH of the culture can be adjusted. In addition, during the culture, prevention of bubble formation can be included, for example by using antifoaming agents to prevent bubble formation. In addition, during the culture, introduction of a gas into the culture can be included. Gases can include any gas capable of maintaining aerobic conditions in the culture. During the culture, the temperature of the culture can be from 20 to 45°. The formed L-amino acids can be extracted from the culture, i.e. the culture is treated with sulfuric or hydrochloric acid, etc., followed by a combination of methods such as anion exchange chromatography, concentration, crystallization and precipitation at the isoelectric point.

In the invention:

SEQ ID NO:1: ORF (open reading frame) sequence wild type:

SEQ ID NO:2: ORE sequence NCgl0609 ^334* :

SEQ ID NO:3: Amino acid sequence of the protein encoded by wild-type NCg10609:

SEQ ID NO:4: amino acid sequence of the protein encoded by NCg10609 ^R334* :

SEQ ID NO:29: ORF sequence of wild-type NCgl1575:

SEQ ID NO:30: ORF sequence NCgl1575 ^A1775T :

SEQ ID NO:31: protein sequence encoded by wild-type NCgl1575:

SEQ ID NO:32: protein sequence encoded by NCgl1575 ^Y592F :

SEQ ID NO:57: wild-type promoter sequence:

SEQ ID NO:58: mutant promoter sequence:

Definition of terms:

In the present invention, the term "bacterium with an ability to produce L-amino acids" refers to an ability to produce and accumulate L-amino acids of interest in a culture medium and/or in bacterial cells to a value specified below, so that the bacterium producing the L-amino acid can be collected when the bacterium is cultured in the culture medium. The bacterium with an ability to produce L-amino acids may be a bacterium that can accumulate L-amino acids of interest in a culture medium and/or in bacterial cells in an amount that is greater than that obtainable by an unmodified strain.

Examples of L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline; aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and glycine; amino acids such as hydroxy monoamino carboxylic acids such as L-threonine and L-serine; cyclic amino acids such as L-proline; aromatic amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan; sulfur-containing amino acids such as L-cysteine, L-cystine, and L-methionine; acidic amino acids such as L-glutamate and L-aspartate; and amino acids with amide groups in the side chain such as L-glutamine and L-asparagine. Specific examples of L-amino acids include L-glutamic acid, L-lysine, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, and L-cysteine. More specific examples of L-amino acids include L-glutamate, L-lysine, l-threonine, and L-tryptophan. Even more specific examples of L-amino acids include L-glutamate and L-lysine.

In the present invention, unless otherwise specified, the term "amino acid" refers to an L-amino acid. In the present invention, unless otherwise specified, the term "L-amino acid" refers to an L-amino acid in free form, a salt thereof, or a mixture thereof.

The term "unmodified strain" refers to a control strain that has not been modified to have specific characteristics. That is, examples of unmodified strains include wild-type strains and parental strains.

The term "homology" refers to the percent identity of two polynucleotides or two polypeptide elements. Sequence homology of one element to another element can be determined using methods known in the art. For example, such sequence homology can be determined using the BLAST algorithm.

The term "functional linking" refers to the functional linking of a regulatory sequence to a polynucleotide sequence, wherein the regulatory sequence controls the transcription and/or translation of the polynucleotide sequence. The regulatory sequence may be a strong promoter that can improve the expression level of the polynucleotides. The regulatory sequence may be a promoter derived from a microorganism belonging to the genus Corynebacterium or may be a promoter derived from other microorganisms. For example, the promoter may be a trc promoter, a gap promoter, a tac promoter, a T7 promoter, a lac promoter, a trp promoter, an araBAD promoter, or a cj7 promoter.

The term "vector" refers to a polynucleotide construct that contains a gene regulatory sequence and a gene sequence and is configured to express a target gene in a suitable host cell. Alternatively, a vector may also refer to a polynucleotide construct that contains sequences that can be used for homologous recombination, so that, due to the vector being introduced into the host cell, the regulatory sequence of an endogenous gene in the host cell genome can be changed, or a target gene that can be expressed can be introduced into a specific site in the host genome. In this case, the vector used in the present invention may further comprise a selectable marker for determining the introduction of the vector into the host cell or the integration of the vector into the chromosome of the host cell. Selectable markers may include markers that confer selectable phenotypes such as drug resistance, auxotrophic type, resistance to cytotoxic agents or expression of surface proteins. In media treated with selective agents, transformed cells can be selected because cells expressing only selectable markers may survive or exhibit different phenotypic characteristics.

The term "transformation" as used herein refers to the introduction of polynucleotides into host cells, so that the polynucleotides can be used as extragenomic elements or integrated into the host cell genome for replication. The method of transforming the vectors used in the present invention may include a method of introducing nucleic acids into cells. In addition, as described in the related technology, the electrical impulse method can be implemented based on host cells.

Beneficial effects

In this invention, it was discovered that the product encoded by the gene affects the level of amino acid production as a result of weakening or inactivating the gene or the NCgl1575 gene. The recombinant strain was obtained by introducing point mutations into the coding sequence or increasing the copy number or overexpressing the gene. Compared with the wild-type strain, the obtained strain resulted in the production of high concentrations of amino acids. In addition, the recombinant strain was obtained by introducing point mutations into the promoter region of the lysC gene. Compared with the wild-type strain, the obtained strain could also significantly improve the production of L-lysine, in addition, improve the generation efficiency, reduce the generation cost, and facilitate popularization and application.

Detailed embodiments

Below, the technical solution of the present invention will be further described in detail in conjunction with specific examples. It should be understood that the following examples are only illustrative and illustrative of the present invention and should not be considered as limiting the scope of the invention. All technologies implemented on the basis of the above-mentioned content of the present invention fall within the scope of the present invention. Unless otherwise indicated, all raw materials and reagents used in the following examples are commercially available products or can be obtained by known methods. All operations are known in the art or are carried out in accordance with the user manual of commercially available products.

In the following examples, the basic medium used for culturing the strains has the same composition, and sucrose, kanamycin or chloramphenicol, etc. are added to such a basic medium composition, if necessary. The composition of the basic medium is as follows:

The preparation and conditions of PAGE (polyacrylamide gel electrophoresis) with SSCP (single-stranded conformational polymorphism analysis) in the following examples were as follows:

In the following examples, the fermentation medium and fermentation process of L-lysine are shown in Table 1 and 2 below:

In the following examples, the fermentation medium and fermentation process of L-glutamate are shown in Table 3 and 4 below:

Example 1: Constructing a transformed vector , containing the coding region of the NCgl0609 gene with a point mutation

Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were designed and synthesized to amplify the coding region sequence of the NCgl0609 gene. A point mutation was introduced into the coding region of the gene (SEQ ID NO: 1, and the corresponding amino acid sequence encoding the proteins is SEQ ID NO: 3) based on the YP97158 strain [deposit number: CGMCC No. 12856, deposition date: August 16, 2016, depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355, recorded in Chinese Patent Application CN106367432A (filing date: September 1, 2016, and publication date: February 1, 2017), and it was confirmed through sequencing that the gene wild type was retained in the chromosome of the strain] by allelic substitution, and thus the nucleotide sequence of the gene at position 1000 was replaced from C to T (SEQ ID NO: 2), and the corresponding amino acid sequence encoding proteins at position 334 was replaced from arginine to terminator (SEQ ID NO: 4: ). The primers were designed as follows

(synthesized by Shanghai Invitrogen):

Construction method: Corynebacterium glutamicum ATCC13032 was used as a template, and primers P1 and P2, P3 and P4 were used, respectively, for PCR amplification. PCR system: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) each 2 μl, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl. PCR amplification was performed as follows: pre-denaturation for 5 minutes at 94°C, (denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 40 seconds at 72°C, 30 cycles), and extension for 10 minutes at 72°C, and then two DNA fragments containing the coding region of the gene were obtained. , with a size of 698 bp and 648 bp, respectively ( above and (See below). After separation of the two DNA fragments and purification by agarose gel electrophoresis, the two DNA fragments as template were amplified into 1317 bp fragments by overlapping PCR using P1 and P4 as primers.

PCR system: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl. PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, (denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 60 sec at 72°C, 30 cycles), and extension for 10 min at 72°C. In this DNA fragment, a cytosine (C) substitution occurred at position 1000 in the coding region of the YP97158 gene to thymine (T), and finally in the substitution of the 334th amino acid ^of the encoded protein from arginine (R) to a terminator. This DNA fragment was purified by agarose gel electrophoresis and combined with the plasmid pK18mobsacB (purchased from Addgene, digested with two enzymes I/BamH I, respectively), which was digested with two enzymes and then purified using NEBuilder enzyme (purchased from NEB) at 50°C for 30 min, and the positive vector was obtained from a monoclone grown after transformation of the fusion product by PCR identification, and this plasmid contained a kanamycin resistance marker. Vector with the correct enzyme cleavage were sent to a sequencing company for sequencing and identification, and the vector containing the correct point mutation (C-T) was stored for use.

Example 2: Construction of engineered strains , containing a point mutation.

Construction method: plasmid with an allelic substitution was transformed into the L-lysine-producing strain YP97158 by electric shock (see WO2014121669A1 for the construction method; sequencing confirmed that the coding region of the gene wild type is retained in the chromosome of this strain). A single colony obtained by culturing was identified using primer P1 and universal primer M13R, and the strain that could amplify bands of 1375 bp was a positive strain. The positive strain was cultured in a medium containing 15% sucrose, a single colony obtained by culturing was grown on a medium containing kanamycin and on a medium without kanamycin, respectively, and the strains that grew on a medium without kanamycin but did not grow on a medium containing kanamycin were then identified by PCR using the following primers (synthesized by Shanghai mvitrogen):

The above PCR amplification product was 264 bp and was denatured at 95°C for 10 min and exposed to an ice bath for 5 min, followed by sscp electrophoresis (the amplified plasmid fragment (used as a positive control, the amplified fragment of YP97158 was used as a negative control, and water was used as a blank control). Due to the different structures of the fragments and positions on electrophoresis, strains with electrophoretic positions different from those of the negative control fragments and identical to those of the positive control fragments represent strains with successful allelic substitution. Fragment The positive strain was PCR amplified using the P5/P6 primer and combined with the PMD19-T vector for sequencing. According to the sequence alignment, the strain with the mutation (C-T) in the original sequence was a positive strain with a successful allelic substitution and was named YPL-4-041.

Example 3: Construction of engineered strains that overexpress genes And in the genome.

Based on the genome sequence of the wild-type Corynebacterium glutamicum ATCC13032 published by NCBI, three pairs of primers were designed and synthesized to amplify the upstream and downstream fragments of the homologous arm and the coding region and promoter region sequences of the gene And , and gene or was introduced into strain YP97158 by homologous recombination.

Primers were designed as follows (synthesized by Shanghai Invitrogen):

Construction method: Corynebacterium glutamicum ATCC13032 or YPL-4-041 were used as a template, respectively, for PCR amplification with primers P7/P8, P9/P10, P11/P12 to obtain the 768 bp upstream homologous arm fragment of the gene or and its 1626 bp promoter fragment and 623 bp downstream homologous arm fragment. After completion of the PCR reaction, the three amplified fragments were isolated by gel column electrophoresis using a DNA extraction kit, respectively. The three isolated fragments were fused with pK18mobsacB plasmid (purchased from Addgene Company, digested with two enzymes I/BamH I, respectively), which was digested with two enzymes and then purified with NEBuilder enzyme (purchased from NEB Company) at 50°C for 30 minutes, and a positive integrated plasmid was obtained from the monoclone grown after transformation of the fused product by PCR identification. This plasmid contained a kanamycin resistance marker, and a recombinant with the plasmid integrated into the genome could be obtained by kanamycin selection.

PCR system: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl. PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 60 sec at 72°C (30 cycles), and extension for 10 min at 72°C. The correctly sequenced integrated plasmid was electrotransformed into the L-lysine-producing strain YP97158. A single colony obtained by culturing was identified by PCR using primers P13/P14. The strain with a 1317 bp fragment amplified by PCR was the positive strain, and the strain without the fragment in the amplification was the original strain. The positive strain was cultured in a medium containing 15% sucrose and a single colony obtained by culturing was then identified by PCR using primers P15/P16. Bacteria amplifying a 1352 bp fragment are positive strains with the gene or , integrated into the YP97158 genome, and they were named YPL-4-042 (without mutation site) and YPL-4-043 (with mutation site).

Example 4: Construction of engineered strains that overexpress genes or plasmid.

Based on the genome sequence of the wild-type Corynebacterium glutamicum ATCC13032 published by NCBI, a pair of primers was designed and synthesized to amplify the coding region and promoter region sequences of the gene or The primers were designed as follows (synthesized by Shanghai Invitrogen):

Construction method: Corynebacterium glutamicum ATCC 13032 and YPL-4-041 were used as templates, respectively, for PCR amplification with primers P17/P18 to obtain genes And and their 1582 bp promoter fragments. The amplified products were electrophoresed and purified using a gel column DNA extraction kit. The isolated DNA fragment and shuttle plasmid pXMJ19 obtained by digestion with EcoRI enzyme were combined at 50°C with NEBuilder enzyme (purchased from NEB) for 30 minutes, and the positive overexpression plasmids pXMJ19- and pXMJ19- were obtained from monoclones grown after transformation of the related products by PCR identification with primer M13, and these plasmids were then sent for sequencing. Since the plasmid contains a chloramphenicol resistance marker, chloramphenicol can be used to determine whether the plasmid has been transformed into the strain.

PCR system: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl. PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 60 sec at 72°C (30 cycles), and extension for 10 min at 72°C.

Correctly sequenced plasmids pXMJ19- and pXMJ19- were electrotransformed into L-lysine-producing strain YP97158, respectively. A single colony obtained by culturing was identified by PCR with primers M13F/P18. Strains with PCR amplification of a 1585 bp fragment were positive strains, and they were named YPL-4-044 (without mutation site) and YPL-4-045 (with mutation site).

Example 5: Construction of engineered strains with a gene , removed from the genome.

Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were designed and synthesized to amplify fragments at both ends of the coding region of the gene. , as the upstream and downstream fragments of the homologous arm. The primers were designed as follows (synthesized by Shanghai Invitrogen):

Construction method: Corynebacterium glutamicum ATCC 13032 was used as a template for PCR amplification with primers P19/P20 and P21/P22, respectively, to generate the 661 bp upstream homologous arm fragment and the 692 bp downstream homologous arm fragment. Then, primers P19/P22 were used for overlapping PCR to generate the entire 1334 bp homologous arm fragment. The amplified products were electrophoresed and purified using a gel column DNA extraction kit. The isolated DNA fragments were fused with pK18mobsacB plasmid (purchased from Addgene Company) digested with two enzymes I/BamH I, respectively), which was digested with the two enzymes and then purified using NEBuilder enzyme (purchased from NEB Company) at 50°C for 30 minutes. Positive knockout vector was obtained from monoclones grown after transformation with the combined products by PCR identification using primer M13, and then these plasmids were sequenced. The plasmid had kanamycin resistance as a marker for selection.

PCR system: 10×Ex Taq buffer 5 µl, dNTP mix (2.5 mM each) 4 µl, Mg2 ⁺ (25 mM) 4 µl, primers (10 pM) 2 µl each, Ex Taq (5 units/µl) 0.25 µl, total volume 50 µl.

PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 90 sec at 72°C (30 cycles), and extension for 10 min at 72°C.

Correctly sequenced knockout plasmid was electrotransformed into the lysin-producing strain YP97158, and a single colony obtained by culturing was identified by PCR with the following primers (synthesized by Shanghai Invitrogen Company):

Strains with simultaneous amplification of the 1334 bp and 1788 bp bands by the above PCR were positive strains, and strains with only the 1788 bp band amplification were the parental strains. After selection on 15% sucrose medium, positive strains were cultured in kanamycin-containing medium and in kanamycin-free medium, respectively. Strains that grew in kanamycin-free medium but did not grow in kanamycin-free medium were then identified by PCR using primers P23/P24. Strains with amplification of the 1334 bp band were positive strains, in which the coding region of the gene was knocked out. Again, a fragment of the positive strain was PCR amplified with primers P23/P24 and coupled to the PMD19-T vector for sequencing. The correctly sequenced strain was named YPL-4-046.

Example 6: L-lysine fermentation experiment

The strains constructed in Examples 2 to 5 and the original strain YP97158 were subjected to a fermentation experiment in a BLBIO-5GC-4-H fermentation tank (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the culture medium shown in Table 1 and the control process shown in Table 2. The experiment with each strain was repeated three times, and the results are shown in Table 5.

The results are shown in Table 5. Point mutation NCgl0609R334* and overexpression of the coding region of the gene in Corynebacterium glutamicum promotes increased L-lysine production and growth rate, while attenuation or knockout of the gene does not promote L-lysine accumulation and reduces the growth rate of the strain.

Example 7: Introduction of gene overexpression in a glutamate-producing strain or point mutation and overexpression in the coding region of the gene and performing fermentation experiments.

According to the methods of Examples 1-5, using the same primers and experimental conditions, Corynebacterium ATCC 13 869 was used as a parent bacterium and the bacterium ATCC 13869 was used as an expression bacterium to obtain glutamate-producing engineered strains YPG-013 containing with a point mutation, glutamate-producing engineered strains YPG-014 and YPG-015, overexpressing genes And in the genome of glutamate-producing engineered strains YPG-016 and YPG-017, overexpressing genes And in a plasmid, and a glutamate-producing engineered strain YPG-018 lacking the gene in the genome.

The strains constructed in the Examples and the original strain were subjected to a fermentation experiment (with ATCC 13869 as the expression bacterium) in a BLBIO-5GC-4-H fermentation tank (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the culture medium shown in Table 3 and the control process shown in Table 4. The experiment with each strain was repeated three times, and the results are shown in Table 6.

The results are shown in Table 6. Point mutation and overexpression of the coding region of the gene in Corynebacterium glutamicum it promotes increased L-glutamate production and growth rate, while weakening or inactivation of the gene does not promote L-glutamic acid accumulation and reduces the growth rate of the strain.

Example 8: Construction of the pK18-NCgl1575 ^A1775T transformation vector containing the coding region of the NCgl1575 gene with a point mutation

Based on the genome sequence of the wild-type Corynebacterium glutamicum ATCC 13032 published by NCBI, two pairs of primers were designed and synthesized to amplify the coding region sequence of the NCgl1575 gene. A point mutation was introduced into the coding region of the NCgl1575 gene (SEQ ID NO:29) based on the YP97158 strain (it was confirmed by sequencing that the wild-type NCgl1575 gene was retained in the chromosome of the strain) by allelic replacement. The corresponding amino acid sequence encoding the proteins was SEQ ID NO:31, and in the nucleotide sequence of the NCgl1575 gene at position 1775, nucleotide A was replaced by T (SEQ ID NO:30: NCgl1575 ^A1775T ), and in the corresponding amino acid sequence encoding the proteins, at position 592, tyrosine was replaced by phenylalanine (SEQ ID NO:32: NCgl1575 Y592F).

The primers were designed as follows (synthesized by Shanghai mvitrogen Company):

Construction method: Corynebacterium glutamicum ATCC13032 was used as a template for PCR amplification with primers P1' and P2', P3' and P4', respectively.

PCR system: 10×Ex Taq buffer 5 µl, dNTP mix (2.5 mM each) 4 µl, Mg2 ⁺ (25 mM) 4 µl, primers (10 pM) 2 µl each, Ex Taq (5 units/µl) 0.25 µl, total volume 50 µl.

PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 40 sec at 72°C (30 cycles), and extension for 10 min at 72°C. Two DNA fragments containing the coding region of the NCgl1575 gene of 766 bp and 759 bp, respectively (NCgl1575 upstream and NCgl1575 downstream), were obtained.

After the above two DNA fragments were isolated and purified by agarose gel electrophoresis, the above two DNA fragments were used as templates, and P1' and P4' were used as primers, to amplify a fragment of approximately 1495 bp by PCR with overlapping primers.

PCR system: 10×Ex Taq buffer 5 µl, dNTP mix (2.5 mM each) 4 µl, Mg2 ⁺ (25 mM) 4 µl, primers (10 pM) 2 µl each, Ex Taq (5 units/µl) 0.25 µl, total volume 50 µl.

PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 90 sec at 72°C (30 cycles), and extension for 10 min at 72°C.

This DNA fragment (NCgl1575 ^A1775T ) was obtained by replacing adenine (A) at position 1775 in the coding region of the YP97158 NCgl1575 gene with thymine (T), and finally resulting in a change of amino acid at position 592 of the coding protein from tyrosine (Y) to phenylalanine (F).

NCgl1575 ^A1775T isolated and purified by agarose gel electrophoresis and pK18mobsacB (purchased from Addgene) isolated by Xba I digestion were combined using the NEBuider recombinant system to generate the pK18-NCgl1575 ^A1775T vector and the plasmid contained the kanamycin resistance marker. The pK18-NCgl1575 ^A1775T vector was submitted to the company for sequencing and identification, and the pK18-NCgl1575 ^A1775T _vector containing the correct point mutation (A-T) was retained for use.

Example 9: Construction of engineered strains containing NCgl1575 ^A1775T point mutation

Construction method: The allelic substitution plasmid pK18-NCgl1575 ^A1775T was transformed into L-lysine-producing strain YP97158 by electric shock. A single colony obtained by culturing was identified by primer P1' and universal primer M13R, respectively. The strain that can amplify the 1502 bp band is a positive strain. The positive strains were cultured in a medium containing 15% sucrose, and a single colony obtained by culturing was grown in a medium containing kanamycin and a medium without kanamycin, respectively. Strains that grew in the medium without kanamycin but did not grow in the medium containing kanamycin were then identified by PCR with the following primers (synthesized by Shanghai mvitrogen Company):

And

The above PCR amplified product was 256 bp in size and was denatured at high temperature and placed in an ice bath, followed by sscp electrophoresis (the amplified fragment of pK18-NCgl1575 ^A1775T plasmid was used as a positive control, the amplified fragment of YP97158 was used as a negative control, and water was used as a blank control). Due to the different fragment structures and electrophoresis positions, the strains whose electrophoresis positions were different from those of the negative control fragments and the same as those of the positive control fragments were the strains with successful allelic substitutions. The fragment of interest from the strains with successful allelic substitution was PCR amplified again using primer P5' and P6' and fused into the PMD19-T vector for sequencing. Through sequence alignment, the allelic replacement of the strain was confirmed to be successful for the sequence in which the base sequence was mutated and it was named YPL-4-023.

Example 10: Construction of engineered strains overexpressing NCgl1575 or NCgl1575 ^A1775T genes in the genome.

Based on the genome sequence of wild-type Corynebacterium glutamicum ATCC 13032 published by NCBI, three pairs of primers were designed and synthesized to amplify the upstream and downstream fragments of the homologous arm and the coding region sequences of the NCgl1575 or NCgl1575 ^A17751 gene and the promoter region, and the NCgl1575 or NCgl1575 ^A1775T gene was introduced into strain YP97158 through homologous recombination.

The primer was designed as follows (synthesized by Shanghai mvitrogen Company):

Construction method: Corynebacterium glutamicum ATCC 13032 or YPL-4-023 were used as a template, respectively, for PCR amplification with primers P7'/P8', P9'/P10', P11'/P12', to obtain the 802 bp upstream homologous arm fragment of the NCgl1575 gene and its 2737 bp promoter fragment, or the NCgl1575 ^A1775T gene and its 2737 bp promoter fragment and the 647 bp downstream homologous arm fragment. Then, the above three amplified fragments (the upstream fragment of the homologous arm, the NCgl1575 gene and its promoter fragment and the downstream fragment of the homologous arm; or the upstream fragment of the homologous arm, the NCgl1575 ^A1775T gene and its promoter fragment and the downstream fragment of the homologous arm) were mixed as an amplification template with primers P7'/P12' to obtain an integrated homologous arm fragment of 4111 bp.

After completion of the PCR reaction, the amplified product was electrophoretically isolated, and the desired DNA fragment of 4111 bp was isolated using a gel column DNA isolation kit (TIANGEN), and combined with the shuttle plasmid PK18mobsacB isolated by digestion with Xba I enzyme using the NEBBuider recombination system to obtain an integrated plasmid PK18mobsacB-NCgl1575 or PK18mobsacB- ^{NCgl1575A1775T} . A plasmid containing a kanamycin resistance marker and a recombinant with the plasmid integrated into the genome can be obtained through kanamycin selection.

PCR system: 10×Ex Taq buffer 5 µl, dNTP mix (2.5 mM each) 4 µl, Mg2 ⁺ (25 mM) 4 µl, primers (10 pM) 2 µl each, Ex Taq (5 units/µl) 0.25 µl, total volume 50 µl.

PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 180 sec at 72°C (30 cycles), and extension for 10 min at 72°C.

The two pooled plasmids were electrotransformed into L-lysine-producing strain YP97158, respectively, and a single colony obtained by culturing was identified by PCR with primers P13'/P14'. The strain with PCR-amplified fragments of 1778 bp was the positive strain, and the strain without amplified fragments was the parent strain. Positive strains were selected on 15% sucrose medium and then cultured on kanamycin-containing medium and without kanamycin, respectively. Strains that grew on kanamycin-free medium but did not grow on kanamycin-containing medium were then identified by PCR with primers P15'/P16'. Bacteria with amplified fragment of 1756 bp were identified by PCR with primers P15'/P16'. were strains with the NCgl1575 or NCgl1575 ^A1775T gene integrated into the YP97158 genome, which were named YPL-4-024 (without mutation site) and YPL-4-025 (with mutation site), respectively.

Example 11: Construction of engineered strains overexpressing the NCgl1575 or NCgl1575 ^A1775T gene in a plasmid.

Based on the genome sequence of the wild-type Corynebacterium glutamicum ATCC 13032 published by NCBI, a pair of primers was designed and synthesized to amplify the coding region and promoter region sequences of the NCgl1575 or NCgl1575 ^{A1775T gene.} The primers were designed as follows (synthesized by Shanghai mvitrogen Company):

Construction method: ATCC13032 and YPL-4-023 were used as templates, respectively, for PCR amplification with primers P17'/P18' to obtain the NCgl1575 or NCgl1575 ^A1775T gene and their 2749 bp promoter fragments. The amplified products were isolated by electrophoresis. The desired 2749 bp DNA fragments were isolated by a gel column DNA isolation kit and fused with the shuttle plasmid pXMJ19 isolated by digestion with EcoRI enzyme using the NEBuider recombinant system to obtain the overexpression plasmids pXMJ19-NCgl1575 and pXMJ19-NCgl1575 ^A1775T . Plasmids containing chloramphenicol resistance markers can be obtained by chloramphenicol resistance and transformed into strains.

PCR system: 10×Ex Taq buffer 5 µl, dNTP mix (2.5 mM each) 4 µl, Mg2 ⁺ (25 mM) 4 µl, primers (10 pM) 2 µl each, Ex Taq (5 units/µl) 0.25 µl, total volume 50 µl.

PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 120 sec at 72°C (30 cycles), and extension for 10 min at 72°C.

Plasmids pXMJ19-NCgl1575 and pXMJ19-NCgl1575 ^A1775T were electrotransformed into L-lysine-producing strain YP97158, respectively. A single colony obtained by culturing was identified by PCR with primers M13(-48) and P18'. A single colony with a PCR-amplified fragment of 2752 bp was transformed into strains that were named YPL-4-026 (without mutation site) and YPL-4-027 (with mutation site).

Example 12: Construction of engineered strains with the NCgl1575 gene deleted from the genome

Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify fragments at both ends of the coding region of the NCgl1575 gene as the upstream and downstream fragments of the homologous arm. The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

Corynebacterium glutamicum ATCC 13032 was used as a template for PCR amplification with primers P19'/P20' and P21'/P22', respectively, to generate the 775 bp upstream homologous arm fragment and the 807 bp downstream homologous arm fragment. They were then subjected to overlapping PCR with primers P19'/P22' to generate the entire 1545 bp homologous arm fragment. After completion of the PCR reaction, the amplified product was electrophoretically isolated and the desired 1545 bp homologous arm fragment was purified. The DNA fragment was extracted using a gel column DNA extraction kit and fused with the shuttle plasmid pk18mobsacB, isolated by digestion with Xba I, using the NEBuider recombinant system to obtain a knockout plasmid. This plasmid contained a kanamycin resistance marker.

The knockout plasmid was electrotransformed into the lysin-producing strain YP97158, and a single colony obtained by culturing was identified by PCR with the following primers (synthesized by Shanghai Invitrogen):

The strains with amplified bands of 1471 bp and 4150 bp by the above-described PCR were positive strains, and the strains with amplified band of 4150 only were parent bacteria. After selection in 15% sucrose medium, the positive strains were cultured in kanamycin-containing medium and in kanamycin-free medium, respectively, and the strains that grew in kanamycin-free medium but did not grow in kanamycin-containing medium were then identified by PCR using primers P237P24'. The strain with amplified band of 1471 bp was an engineered strain with the coding sequence of the NCgl1575 gene deleted, which was named YPL-4-028.

Example 13: L-lysine fermentation experiment

The constructed strains of Examples 9 to 12 and the original strain YP97158 were subjected to a fermentation experiment in a BLBIO-5GC-4-H fermentation tank (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the culture medium shown in Table 1 and the control process shown in Table 2. The experiment with each strain was repeated three times, and the results are shown in Table 7.

The results are shown in Table 7. Overexpression of the NCgl1575 gene in Corynebacterium glutamicum, or the NCgl1575 ^A1775T point mutation and overexpression of the coding region of the NCgl1575 gene promoted an increase in L-lysine production, while attenuation or knockout of the gene did not promote L-lysine accumulation.

Example 14: Constructing a Transformation Vector , containing the promoter region of the gene gene with point mutation

Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were designed and synthesized to amplify the sequences of the promoter region of the gene , and a point mutation was introduced into the promoter region of the lysC gene (SEQ ID NO: 57) based on the YP97158 strain through allelic substitution. G at position -45 bp of the nucleotide sequence of the promoter region of the lysC gene was replaced with A, and G at position -47 bp was replaced with T (SEQ ID NO: 58).

The primers were designed as follows (synthesized by Shanghai mvitrogen Company):

Construction method: Corynebacterium glutamicum ATCC13032 was used as a template for PCR amplification with primers P1'' and P2'', P3'' and P4'', respectively. PCR system: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl. PCR amplification was performed as follows: pre-denaturation for 5 min at 94°C, denaturation for 30 sec at 94°C, annealing for 30 sec at 52°C, elongation for 40 sec at 72°C (30 cycles), and extension for 10 min at 72°C. Two DNA fragments with a point mutation of 729 bp and 760 bp, respectively (promoter fragments) were obtained. (upstream and downstream fragments). After separation and purification of the above two fragments by agarose gel electrophoresis, the two purified DNA fragments were used as templates, and P1'' and P4'' were used as primers to amplify a fragment of approximately 1459 bp (upstream fragment) by overlapping primer PCR. PCR system: 10×Ex Taq buffer 5 μl, dNTP mix (2.5 mM each) 4 μl, Mg2 ⁺ (25 mM) 4 μl, primers (10 pM) 2 μl each, Ex Taq (5 U/μl) 0.25 μl, total volume 50 μl. PCR amplification was performed as follows: pre-denaturation for 5 minutes at 94°C, denaturation for 30 seconds at 94°C, annealing for 30 seconds at 52°C, elongation for 90 seconds at 72°C (30 cycles), and extension for 10 minutes at 72°C. The above-described upstream fragment was isolated and purified by agarose gel electrophoresis, and the fragment contained the promoter region of the gene and its upstream and downstream sequences and both ends of the fragment containing digestion sites for the enzymes EcoRI and SphI, respectively. This DNA fragment causes a guanine (G) nucleotide substitution at position -45 bp in the promoter region of the YP97158 gene to adenine (A), and the nucleotide guanine (G) at position -47 bp to thymine (T). The fragment was purified and isolated after dual enzyme digestion (EcoR I/Sph I) and was fused to the shuttle plasmid pK18mobsacB (purchased from Addgene) after dual digestion with the same enzymes (EcoR I/Sph I) to yield plasmid with an allelic substitution containing a kanamycin resistance marker Vector was sent to a sequencing company for sequencing and identification, and the vector containing the correct point mutation was retained for use.

Example 15: Construction of engineered strains containing with point mutation

Plasmid with allelic substitution were transformed into L-lysine-producing strain YP97158 by electroshock. A single colony obtained by culture was identified using primer P1'' and universal primer M13F, respectively, and the strains that could amplify a band of 1500 bp were positive strains. The positive strains were cultured in a medium containing 15% sucrose, and a single colony obtained by culture was grown in a medium containing kanamycin and a medium without kanamycin, respectively; the strains that grew on the medium without kanamycin but did not grow on the medium containing kanamycin were then identified by PCR using the following primers (synthesized by Shanghai Invitrogen Company):

The above PCR amplification product was denatured at high temperature and subjected to ice bath, followed by sscp electrophoresis (amplified plasmid fragment (used as a positive control, the amplified fragment of YP97158 was used as a negative control, and water was used as a blank control). Due to the different fragment structures and electrophoresis positions, the strains whose electrophoresis positions were different from those of the negative control fragments and the same as those of the positive control fragments were the strains with successful allelic substitutions. The target fragment of the positive strain was PCR amplified again and fused into the PMD19-T vector for sequencing. Through sequence alignment, the sequence in which the base sequence was mutated confirmed that the allelic substitution of the strain was successful, and the strain was named YPL-4-009.

Example 16: L-Lysine fermentation experiment

The fermentation experiment of the strain YPL-4-009 constructed in Example 15 and the original strain YP97158 was performed in a BLBIO-5GC-4-H fermentation tank (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the culture medium shown in Table 1 and the control process shown in Table 2. The experiment with each strain was repeated three times, and the results are shown in Table 8.

The above conversion rate = total lysine/total glucose intake * 100%

The results are shown in Table 8. Point mutation gene promoter in Corynebacterium glutamicum promotes increased production of L-lysine.

The embodiment of the invention is described above. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made in accordance with the essence and principle of the invention are included in the scope of legal protection of this invention.

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SEQUENCE LIST

<110> INNER MONGOLIA EPPEN BIOTECH CO., LTD.

<120> RECOMBINANT STRAIN FOR PRODUCTION

L-AMINO ACIDS, THE METHOD OF ITS CONSTRUCTION AND ITS APPLICATION

<130> CPWO20411699

<160> 64

<170> PatentIn version 3.3

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<211> 1083

<212> DNA

<213> Corynebacterium glutamicum

<400> 1

gtgtcacaca ccgcgtccac accgacgcca gaggaatact ccgcgcagca acccagcacc 60

cagggcactc gcgttgagtt ccgcggcata accaaagtct ttagcaacaa taaatctgct 120

aaaaccaccg cgcttgataa tgtcactctc accgtagaac ccggtgaggt aatcggcatc 180

atcggttact ctggcgccgg caagtccact cttgtccgcc tcatcaatgg ccttgactcc 240

cccacgagcg gttcgttgct gctcaacggc accgacatcg tcggaatgcc cgagtctaag 300

ctgcgtaaac tgcgcagtaa tatcggcatg attttccagc agttcaacct gttccagtcg 360

cgtactgcgg ctggaaatgt ggagtacccg ctggaagttg ccaagatgga caaggcagct 420

cgtaaagctc gcgtgcaaga aatgctcgag ttcgtcggcc tgggcgacaa aggcaaaaac 480

taccccgagc agctgtcggg cggccagaag cagcgcgtcg gcattgcccg tgcactggcc 540

accaatccaa cgcttttgct tgccgacgaa gccacctccg ctttggaccc agaaaccacc 600

catgaagttc tggagctgct gcgcaaggta aaccgcgaac tgggcatcac catcgttgtg 660

atcacccacg aaatggaagt tgtgcgttcc atcgcagaca aggttgctgt gatggaatcc 720

ggcaaagttg tggaatacgg cagcgtctac gaggtgttct ccaatccaca aacacaggtt 780

gctcaaaagt tcgtggccac cgcgctgcgt aacaccccag accaagtgga atcggaagat 840

ctgcttagcc atgagggacg tctgttcacc attgatctga ctgaaacgtc cggcttcttt 900

gcagcaaccg ctcgtgctgc cgaacaaggt gcttttgtca acatcgttca cggtggcgtg 960

accaccttgc aacgccaatc atttggcaaa atgactgttc gactcaccgg caacaccgct 1020

gcgattgaag agttctatca aaccttgacc aagaccacga ccatcaagga gatcacccga 1080

tga 1083

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gtgtcacaca ccgcgtccac accgacgcca gaggaatact ccgcgcagca acccagcacc 60

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aaaaccaccg cgcttgataa tgtcactctc accgtagaac ccggtgaggt aatcggcatc 180

atcggttact ctggcgccgg caagtccact cttgtccgcc tcatcaatgg ccttgactcc 240

cccacgagcg gttcgttgct gctcaacggc accgacatcg tcggaatgcc cgagtctaag 300

ctgcgtaaac tgcgcagtaa tatcggcatg attttccagc agttcaacct gttccagtcg 360

cgtactgcgg ctggaaatgt ggagtacccg ctggaagttg ccaagatgga caaggcagct 420

cgtaaagctc gcgtgcaaga aatgctcgag ttcgtcggcc tgggcgacaa aggcaaaaac 480

taccccgagc agctgtcggg cggccagaag cagcgcgtcg gcattgcccg tgcactggcc 540

accaatccaa cgcttttgct tgccgacgaa gccacctccg ctttggaccc agaaaccacc 600

catgaagttc tggagctgct gcgcaaggta aaccgcgaac tgggcatcac catcgttgtg 660

atcacccacg aaatggaagt tgtgcgttcc atcgcagaca aggttgctgt gatggaatcc 720

ggcaaagttg tggaatacgg cagcgtctac gaggtgttct ccaatccaca aacacaggtt 780

gctcaaaagt tcgtggccac cgcgctgcgt aacaccccag accaagtgga atcggaagat 840

ctgcttagcc atgagggacg tctgttcacc attgatctga ctgaaacgtc cggcttcttt 900

gcagcaaccg ctcgtgctgc cgaacaaggt gcttttgtca acatcgttca cggtggcgtg 960

accaccttgc aacgccaatc atttggcaaa atgactgttt gactcaccgg caacaccgct 1020

gcgattgaag agttctatca aaccttgacc aagaccacga ccatcaagga gatcacccga 1080

tga 1083

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<211> 360

<212> PRT

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Met Ser His Thr Ala Ser Thr Pro Thr Pro Glu Glu Tyr Ser Ala Gln

1 5 10 15

Gln Pro Ser Thr Gln Gly Thr Arg Val Glu Phe Arg Gly Ile Thr Lys

20 25 30

Val Phe Ser Asn Asn Lys Ser Ala Lys Thr Thr Ala Leu Asp Asn Val

35 40 45

Thr Leu Thr Val Glu Pro Gly Glu Val Ile Gly Ile Ile Gly Tyr Ser

50 55 60

Gly Ala Gly Lys Ser Thr Leu Val Arg Leu Ile Asn Gly Leu Asp Ser

65 70 75 80

Pro Thr Ser Gly Ser Leu Leu Leu Asn Gly Thr Asp Ile Val Gly Met

85 90 95

Pro Glu Ser Lys Leu Arg Lys Leu Arg Ser Asn Ile Gly Met Ile Phe

100 105 110

Gln Gln Phe Asn Leu Phe Gln Ser Arg Thr Ala Ala Gly Asn Val Glu

115 120 125

Tyr Pro Leu Glu Val Ala Lys Met Asp Lys Ala Ala Arg Lys Ala Arg

130 135 140

Val Gln Glu Met Leu Glu Phe Val Gly Leu Gly Asp Lys Gly Lys Asn

145 150 155 160

Tyr Pro Glu Gln Leu Ser Gly Gly Gln Lys Gln Arg Val Gly Ile Ala

165 170 175

Arg Ala Leu Ala Thr Asn Pro Thr Leu Leu Leu Ala Asp Glu Ala Thr

180 185 190

Ser Ala Leu Asp Pro Glu Thr Thr His Glu Val Leu Glu Leu Leu Arg

195 200 205

Lys Val Asn Arg Glu Leu Gly Ile Thr Ile Val Val Ile Thr His Glu

210 215 220

Met Glu Val Val Arg Ser Ile Ala Asp Lys Val Ala Val Met Glu Ser

225 230 235 240

Gly Lys Val Val Glu Tyr Gly Ser Val Tyr Glu Val Phe Ser Asn Pro

245 250 255

Gln Thr Gln Val Ala Gln Lys Phe Val Ala Thr Ala Leu Arg Asn Thr

260 265 270

Pro Asp Gln Val Glu Ser Glu Asp Leu Leu Ser His Glu Gly Arg Leu

275 280 285

Phe Thr Ile Asp Leu Thr Glu Thr Ser Gly Phe Phe Ala Ala Thr Ala

290 295 300

Arg Ala Ala Glu Gln Gly Ala Phe Val Asn Ile Val His Gly Gly Val

305 310 315 320

Thr Thr Leu Gln Arg Gln Ser Phe Gly Lys Met Thr Val Arg Leu Thr

325 330 335

Gly Asn Thr Ala Ala Ile Glu Glu Phe Tyr Gln Thr Leu Thr Lys Thr

340 345 350

Thr Thr Ile Lys Glu Ile Thr Arg

355 360

<210> 4

<211> 333

<212> PRT

<213> artificial sequence

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Met Ser His Thr Ala Ser Thr Pro Thr Pro Glu Glu Tyr Ser Ala Gln

1 5 10 15

Gln Pro Ser Thr Gln Gly Thr Arg Val Glu Phe Arg Gly Ile Thr Lys

20 25 30

Val Phe Ser Asn Asn Lys Ser Ala Lys Thr Thr Ala Leu Asp Asn Val

35 40 45

Thr Leu Thr Val Glu Pro Gly Glu Val Ile Gly Ile Ile Gly Tyr Ser

50 55 60

Gly Ala Gly Lys Ser Thr Leu Val Arg Leu Ile Asn Gly Leu Asp Ser

65 70 75 80

Pro Thr Ser Gly Ser Leu Leu Leu Asn Gly Thr Asp Ile Val Gly Met

85 90 95

Pro Glu Ser Lys Leu Arg Lys Leu Arg Ser Asn Ile Gly Met Ile Phe

100 105 110

Gln Gln Phe Asn Leu Phe Gln Ser Arg Thr Ala Ala Gly Asn Val Glu

115 120 125

Tyr Pro Leu Glu Val Ala Lys Met Asp Lys Ala Ala Arg Lys Ala Arg

130 135 140

Val Gln Glu Met Leu Glu Phe Val Gly Leu Gly Asp Lys Gly Lys Asn

145 150 155 160

Tyr Pro Glu Gln Leu Ser Gly Gly Gln Lys Gln Arg Val Gly Ile Ala

165 170 175

Arg Ala Leu Ala Thr Asn Pro Thr Leu Leu Leu Ala Asp Glu Ala Thr

180 185 190

Ser Ala Leu Asp Pro Glu Thr Thr His Glu Val Leu Glu Leu Leu Arg

195 200 205

Lys Val Asn Arg Glu Leu Gly Ile Thr Ile Val Val Ile Thr His Glu

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Met Glu Val Val Arg Ser Ile Ala Asp Lys Val Ala Val Met Glu Ser

225 230 235 240

Gly Lys Val Val Glu Tyr Gly Ser Val Tyr Glu Val Phe Ser Asn Pro

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Gln Thr Gln Val Ala Gln Lys Phe Val Ala Thr Ala Leu Arg Asn Thr

260 265 270

Pro Asp Gln Val Glu Ser Glu Asp Leu Leu Ser His Glu Gly Arg Leu

275 280 285

Phe Thr Ile Asp Leu Thr Glu Thr Ser Gly Phe Phe Ala Ala Thr Ala

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Arg Ala Ala Glu Gln Gly Ala Phe Val Asn Ile Val His Gly Gly Val

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Thr Thr Leu Gln Arg Gln Ser Phe Gly Lys Met Thr Val

325 330

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<212> DNA

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gttgccggtg agtcaaacag tcattttgc 29

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<212> DNA

<213> artificial sequence

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gcaaaatgac tgtttgactc accggcaac 29

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<212> DNA

<213> artificial sequence

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ctagccggtt ccagtcag 18

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ggacgtctgt tcaccattg 19

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<212> DNA

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<212> DNA

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<212> DNA

<213> artificial sequence

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catctgttct cggtgcacca gctgcgagga tcatctc 37

<210> 14

<211> 46

<212> DNA

<213> artificial sequence

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gatttaattg cgccatctga ttctggcaac aactccttcc ttgacc 46

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ggtcaaggaa ggagttgttg ccagaatcag atggcgcaat taaatcaag 49

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<212> DNA

<213> artificial sequence

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<212> DNA

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<212> DNA

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<212> DNA

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<212> DNA

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cgttggaatc ttgcgttg 18

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<211> 55

<212> DNA

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atcaggctga aaatcttctc tcatccgcca aaaccaacaa ctccttcctt gacc 54

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<212> DNA

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cagtgccaag cttgcatgcc tgcaggtcga ctctagaatg aatgggatgg gtcg 54

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<212> DNA

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catcatcggt tactctggcc gaaatggaag ttgtgcg 37

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<211> 37

<212> DNA

<213> artificial sequence

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cgcacaactt ccatttcggc cagagtaacc gatgatg 37

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<211> 58

<212> DNA

<213> artificial sequence

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<210> 27

<211> 18

<212> DNA

<213> artificial sequence

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aatgaatggg atgggtcg 18

<210> 28

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<212> DNA

<213> artificial sequence

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caacaactcc ttccttgacc 20

<210> 29

<211> 2679

<212> DNA

<213> Corynebacterium glutamicum

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atggcagaat caaacgctat ggaccgggca caaatctctg cactgctaga tagagcacag 60

cacacaatca accttgccga acaagcaaac aacgtgctcc gactgttgaa aacacccgga 120

acggccacag taggggacaa cgggacactc ggcaccgata cctatctgat cccacccgc 180

aacatcacct ggcctgacaa cctgtatgtc aacgtctttc tagacggcat gaatgcagaa 240

gccaccctta ccgattacgt cgcatcagtc gcttcgatcc cacgcctatg ccagatcatc 300

aacgagggcc aaggcggcat gttccgcaga ctattcaacc ccaccaaggt ccaagccggc 360

gaccaagctg tcttcgacct catggtcaaa ctcgacgaga tttcatctac cacccacgaa 420

gtctcccgca tgctcgaggg cgtccacgct gcccgcaccc gccaacaaca aggcgttgca 480

cttttcccag gtattcatgg agtgggagag cgctacatcg aacgcgcaca acaggtactc 540

gcctcagccc tcggtatcgc tggattcggt gccgaaccct gggacggaca tacccttgcc 600

caagcgcgcc gggtagtcca acgctacgcc caagatccta actccgaata ccggctgaaa 660

agcgaagccg agaaacacct cacatccatc aacgagctcc gcgtacagat actcctcgaa 720

caactccccg ttgatgccct acgcatggct accgaccacc gcctgcgctt tggatccctc 780

gattccatcc acgtcgcaac cgtcgccgac gtcctaaaaa cacacacctc catcctcacc 840

accgtgcaag gtatcggcgc ccaaaccgcg gggcggatga aagccgcagc agaaacactc 900

aaacaagaag cactacgccg ccaaaacacc tccatcggcg acgaacctac ccaacccgcc 960

atgcgtctaa tcaacgtgct ggcccgcttc gaccaaaccg aaaccatcac gcccgaagaa 1020

cgcgcccgcc gcacccgcgt catcgactac gtagaacaca tacccccaag cctcgacccc 1080

tacatcgtca tcaacccagc aacgcctgag ttcaacaact tcaccgacga cctccgctgg 1140

atcgacgcaa accccaacct cttccaccca caaacaatca ccaccccacc cgccgacatc 1200

tgggacgact acatctcccg tcccgctcac taccaaggcc tgctagccac gctgctcggc 1260

cgcgacatcg aaggcgcaga cgaactcctc gacgccacca ccctccaaaa aatcagagac 1320

ctcaccctcg acaaaactca tctcaccgac ctccacctcc gcggatacca atcattcggc 1380

gcccgcttcg ccatcatcca aaagaaaacc ctcctcggcg acgacatggg actcggcaaa 1440

acagtccaag ccctctccgc agctgcacac cttgccgcca ccgaaaaaga cttccgcacc 1500

ctcgtcgtcg tacccgcatc cgtcattgtt aactggaccc gcgaatgcaa acgcttcctc 1560

aacctccccg tattcatcgc ccacggagac aacaaacaag acgccatcaa cgcctggtct 1620

aacaccaacg gaatcgcaat ctgcacctac gacggcgtcc gcaccatgga catccccgcg 1680

ccgggtctgg tcattgccga tgaagcccac ctgatcaaaa acccctccac caaacgcacc 1740

caagcactgc gcaaacttat cgacgccgcc ccatacaccc ttctgatgac cggcacacca 1800

ctagaaaaca aagtggaaga gtttgtaaat ctcgtgcgct acatccaacc ggagctgatc 1860

acccgtggca tgtccaaaat gcaggccgag aatttccgcg agcgcatcgc accagcctat 1920

ctgcgcagaa atcaagctga tgtgcttgac gaactcccag agcgcaccga ctccatcgac 1980

tggatcgacc tcaccccaga agaccgcagc gcctacgacg accaagtccg ccaaggcagc 2040

tggatgggca tgcgccgctc cgccatgctc tcaccaacac cacgcctaac ttccgcaaaa 2100

atgcaacgca tcctagaact cttcgaagaa gcagaagaac acggccgcaa agccctcatc 2160

ttcacctact tcctcgacgt cctcgacgaa ctggaaaagc atctaggcga gcgcgtcatc 2220

ggccgcattt ccggcgacgt gccagccacc aagcgccaat tgcttgtcga cgccctgtcc 2280

cactccaaac ccggatccgc cctcattgcc caaatcaccg ccgggggagt aggcctaaac 2340

atccaatccg cgagcctatg cattatttgt gaacctcaag taaagccaac catcgaacag 2400

caggccgtcg cccgagtcca ccgcatgggc caaaccgcca ccgtccaagt ccaccgactc 2460

atcggcgacg aaaccgcaga cgaacgcatg ctagaaatcc tggcaggcaa aactcacgtc 2520

ttcgacgtct acgcccggct atctgaaacc gcagagattc cagatgctgt ggatatcact 2580

gaatcacagc tggcagcacg ggttattgat gaggagcgtg cacggttagg gcttactgaa 2640

tccactggcc ctaaagatga agaaacggcc ttaagctag 2679

<210> 30

<211> 2679

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 30

atggcagaat caaacgctat ggaccgggca caaatctctg cactgctaga tagagcacag 60

cacacaatca accttgccga acaagcaaac aacgtgctcc gactgttgaa aacacccgga 120

acggccacag taggggacaa cgggacactc ggcaccgata cctatctgat cccacccgc 180

aacatcacct ggcctgacaa cctgtatgtc aacgtctttc tagacggcat gaatgcagaa 240

gccaccctta ccgattacgt cgcatcagtc gcttcgatcc cacgcctatg ccagatcatc 300

aacgagggcc aaggcggcat gttccgcaga ctattcaacc ccaccaaggt ccaagccggc 360

gaccaagctg tcttcgacct catggtcaaa ctcgacgaga tttcatctac cacccacgaa 420

gtctcccgca tgctcgaggg cgtccacgct gcccgcaccc gccaacaaca aggcgttgca 480

cttttcccag gtattcatgg agtgggagag cgctacatcg aacgcgcaca acaggtactc 540

gcctcagccc tcggtatcgc tggattcggt gccgaaccct gggacggaca tacccttgcc 600

caagcgcgcc gggtagtcca acgctacgcc caagatccta actccgaata ccggctgaaa 660

agcgaagccg agaaacacct cacatccatc aacgagctcc gcgtacagat actcctcgaa 720

caactccccg ttgatgccct acgcatggct accgaccacc gcctgcgctt tggatccctc 780

gattccatcc acgtcgcaac cgtcgccgac gtcctaaaaa cacacacctc catcctcacc 840

accgtgcaag gtatcggcgc ccaaaccgcg gggcggatga aagccgcagc agaaacactc 900

aaacaagaag cactacgccg ccaaaacacc tccatcggcg acgaacctac ccaacccgcc 960

atgcgtctaa tcaacgtgct ggcccgcttc gaccaaaccg aaaccatcac gcccgaagaa 1020

cgcgcccgcc gcacccgcgt catcgactac gtagaacaca tacccccaag cctcgacccc 1080

tacatcgtca tcaacccagc aacgcctgag ttcaacaact tcaccgacga cctccgctgg 1140

atcgacgcaa accccaacct cttccaccca caaacaatca ccaccccacc cgccgacatc 1200

tgggacgact acatctcccg tcccgctcac taccaaggcc tgctagccac gctgctcggc 1260

cgcgacatcg aaggcgcaga cgaactcctc gacgccacca ccctccaaaa aatcagagac 1320

ctcaccctcg acaaaactca tctcaccgac ctccacctcc gcggatacca atcattcggc 1380

gcccgcttcg ccatcatcca aaagaaaacc ctcctcggcg acgacatggg actcggcaaa 1440

acagtccaag ccctctccgc agctgcacac cttgccgcca ccgaaaaaga cttccgcacc 1500

ctcgtcgtcg tacccgcatc cgtcattgtt aactggaccc gcgaatgcaa acgcttcctc 1560

aacctccccg tattcatcgc ccacggagac aacaaacaag acgccatcaa cgcctggtct 1620

aacaccaacg gaatcgcaat ctgcacctac gacggcgtcc gcaccatgga catccccgcg 1680

ccgggtctgg tcattgccga tgaagcccac ctgatcaaaa acccctccac caaacgcacc 1740

caagcactgc gcaaacttat cgacgccgcc ccattcaccc ttctgatgac cggcacacca 1800

ctagaaaaca aagtggaaga gtttgtaaat ctcgtgcgct acatccaacc ggagctgatc 1860

acccgtggca tgtccaaaat gcaggccgag aatttccgcg agcgcatcgc accagcctat 1920

ctgcgcagaa atcaagctga tgtgcttgac gaactcccag agcgcaccga ctccatcgac 1980

tggatcgacc tcaccccaga agaccgcagc gcctacgacg accaagtccg ccaaggcagc 2040

tggatgggca tgcgccgctc cgccatgctc tcaccaacac cacgcctaac ttccgcaaaa 2100

atgcaacgca tcctagaact cttcgaagaa gcagaagaac acggccgcaa agccctcatc 2160

ttcacctact tcctcgacgt cctcgacgaa ctggaaaagc atctaggcga gcgcgtcatc 2220

ggccgcattt ccggcgacgt gccagccacc aagcgccaat tgcttgtcga cgccctgtcc 2280

cactccaaac ccggatccgc cctcattgcc caaatcaccg ccgggggagt aggcctaaac 2340

atccaatccg cgagcctatg cattatttgt gaacctcaag taaagccaac catcgaacag 2400

caggccgtcg cccgagtcca ccgcatgggc caaaccgcca ccgtccaagt ccaccgactc 2460

atcggcgacg aaaccgcaga cgaacgcatg ctagaaatcc tggcaggcaa aactcacgtc 2520

ttcgacgtct acgcccggct atctgaaacc gcagagattc cagatgctgt ggatatcact 2580

gaatcacagc tggcagcacg ggttattgat gaggagcgtg cacggttagg gcttactgaa 2640

tccactggcc ctaaagatga agaaacggcc ttaagctag 2679

<210> 31

<211> 892

<212> PRT

<213> Corynebacterium glutamicum

<400> 31

Met Ala Glu Ser Asn Ala Met Asp Arg Ala Gln Ile Ser Ala Leu Leu

1 5 10 15

Asp Arg Ala Gln His Thr Ile Asn Leu Ala Glu Gln Ala Asn Asn Val

20 25 30

Leu Arg Leu Leu Lys Thr Pro Gly Thr Ala Thr Val Gly Asp Asn Gly

35 40 45

Thr Leu Gly Thr Asp Thr Tyr Leu Ile Pro Ser Arg Asn Ile Thr Trp

50 55 60

Pro Asp Asn Leu Tyr Val Asn Val Phe Leu Asp Gly Met Asn Ala Glu

65 70 75 80

Ala Thr Leu Thr Asp Tyr Val Ala Ser Val Ala Ser Ile Pro Arg Leu

85 90 95

Cys Gln Ile Ile Asn Glu Gly Gln Gly Gly Met Phe Arg Arg Leu Phe

100 105 110

Asn Pro Thr Lys Val Gln Ala Gly Asp Gln Ala Val Phe Asp Leu Met

115 120 125

Val Lys Leu Asp Glu Ile Ser Ser Thr Thr His Glu Val Ser Arg Met

130 135 140

Leu Glu Gly Val His Ala Ala Arg Thr Arg Gln Gln Gln Gly Val Ala

145 150 155 160

Leu Phe Pro Gly Ile His Gly Val Gly Glu Arg Tyr Ile Glu Arg Ala

165 170 175

Gln Gln Val Leu Ala Ser Ala Leu Gly Ile Ala Gly Phe Gly Ala Glu

180 185 190

Pro Trp Asp Gly His Thr Leu Ala Gln Ala Arg Arg Val Val Gln Arg

195 200 205

Tyr Ala Gln Asp Pro Asn Ser Glu Tyr Arg Leu Lys Ser Glu Ala Glu

210 215 220

Lys His Leu Thr Ser Ile Asn Glu Leu Arg Val Gln Ile Leu Leu Glu

225 230 235 240

Gln Leu Pro Val Asp Ala Leu Arg Met Ala Thr Asp His Arg Leu Arg

245 250 255

Phe Gly Ser Leu Asp Ser Ile His Val Ala Thr Val Ala Asp Val Leu

260 265 270

Lys Thr His Thr Ser Ile Leu Thr Thr Val Gln Gly Ile Gly Ala Gln

275 280 285

Thr Ala Gly Arg Met Lys Ala Ala Ala Glu Thr Leu Lys Gln Glu Ala

290 295 300

Leu Arg Arg Gln Asn Thr Ser Ile Gly Asp Glu Pro Thr Gln Pro Ala

305 310 315 320

Met Arg Leu Ile Asn Val Leu Ala Arg Phe Asp Gln Thr Glu Thr Ile

325 330 335

Thr Pro Glu Glu Arg Ala Arg Arg Thr Arg Val Ile Asp Tyr Val Glu

340 345 350

His Ile Pro Pro Ser Leu Asp Pro Tyr Ile Val Ile Asn Pro Ala Thr

355 360 365

Pro Glu Phe Asn Asn Phe Thr Asp Asp Leu Arg Trp Ile Asp Ala Asn

370 375 380

Pro Asn Leu Phe His Pro Gln Thr Ile Thr Thr Pro Pro Ala Asp Ile

385 390 395 400

Trp Asp Asp Tyr Ile Ser Arg Pro Ala His Tyr Gln Gly Leu Leu Ala

405 410 415

Thr Leu Leu Gly Arg Asp Ile Glu Gly Ala Asp Glu Leu Leu Asp Ala

420 425 430

Thr Thr Leu Gln Lys Ile Arg Asp Leu Thr Leu Asp Lys Thr His Leu

435 440 445

Thr Asp Leu His Leu Arg Gly Tyr Gln Ser Phe Gly Ala Arg Phe Ala

450 455 460

Ile Ile Gln Lys Lys Thr Leu Leu Gly Asp Asp Met Gly Leu Gly Lys

465 470 475 480

Thr Val Gln Ala Leu Ser Ala Ala Ala His Leu Ala Ala Thr Glu Lys

485 490 495

Asp Phe Arg Thr Leu Val Val Val Pro Ala Ser Val Ile Val Asn Trp

500 505 510

Thr Arg Glu Cys Lys Arg Phe Leu Asn Leu Pro Val Phe Ile Ala His

515 520 525

Gly Asp Asn Lys Gln Asp Ala Ile Asn Ala Trp Ser Asn Thr Asn Gly

530 535 540

Ile Ala Ile Cys Thr Tyr Asp Gly Val Arg Thr Met Asp Ile Pro Ala

545 550 555 560

Pro Gly Leu Val Ile Ala Asp Glu Ala His Leu Ile Lys Asn Pro Ser

565 570 575

Thr Lys Arg Thr Gln Ala Leu Arg Lys Leu Ile Asp Ala Ala Pro Tyr

580 585 590

Thr Leu Leu Met Thr Gly Thr Pro Leu Glu Asn Lys Val Glu Glu Phe

595 600 605

Val Asn Leu Val Arg Tyr Ile Gln Pro Glu Leu Ile Thr Arg Gly Met

610 615 620

Ser Lys Met Gln Ala Glu Asn Phe Arg Glu Arg Ile Ala Pro Ala Tyr

625 630 635 640

Leu Arg Arg Asn Gln Ala Asp Val Leu Asp Glu Leu Pro Glu Arg Thr

645 650 655

Asp Ser Ile Asp Trp Ile Asp Leu Thr Pro Glu Asp Arg Ser Ala Tyr

660 665 670

Asp Asp Gln Val Arg Gln Gly Ser Trp Met Gly Met Arg Arg Ser Ala

675 680 685

Met Leu Ser Pro Thr Pro Arg Leu Thr Ser Ala Lys Met Gln Arg Ile

690 695 700

Leu Glu Leu Phe Glu Glu Ala Glu Glu His Gly Arg Lys Ala Leu Ile

705 710 715 720

Phe Thr Tyr Phe Leu Asp Val Leu Asp Glu Leu Glu Lys His Leu Gly

725 730 735

Glu Arg Val Ile Gly Arg Ile Ser Gly Asp Val Pro Ala Thr Lys Arg

740 745 750

Gln Leu Leu Val Asp Ala Leu Ser His Ser Lys Pro Gly Ser Ala Leu

755 760 765

Ile Ala Gln Ile Thr Ala Gly Gly Val Gly Leu Asn Ile Gln Ser Ala

770 775 780

Ser Leu Cys Ile Ile Cys Glu Pro Gln Val Lys Pro Thr Ile Glu Gln

785 790 795 800

Gln Ala Val Ala Arg Val His Arg Met Gly Gln Thr Ala Thr Val Gln

805 810 815

Val His Arg Leu Ile Gly Asp Glu Thr Ala Asp Glu Arg Met Leu Glu

820 825 830

Ile Leu Ala Gly Lys Thr His Val Phe Asp Val Tyr Ala Arg Leu Ser

835 840 845

Glu Thr Ala Glu Ile Pro Asp Ala Val Asp Ile Thr Glu Ser Gln Leu

850 855 860

Ala Ala Arg Val Ile Asp Glu Glu Arg Ala Arg Leu Gly Leu Thr Glu

865 870 875 880

Ser Thr Gly Pro Lys Asp Glu Glu Thr Ala Leu Ser

885 890

<210> 32

<211> 892

<212> PRT

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 32

Met Ala Glu Ser Asn Ala Met Asp Arg Ala Gln Ile Ser Ala Leu Leu

1 5 10 15

Asp Arg Ala Gln His Thr Ile Asn Leu Ala Glu Gln Ala Asn Asn Val

20 25 30

Leu Arg Leu Leu Lys Thr Pro Gly Thr Ala Thr Val Gly Asp Asn Gly

35 40 45

Thr Leu Gly Thr Asp Thr Tyr Leu Ile Pro Ser Arg Asn Ile Thr Trp

50 55 60

Pro Asp Asn Leu Tyr Val Asn Val Phe Leu Asp Gly Met Asn Ala Glu

65 70 75 80

Ala Thr Leu Thr Asp Tyr Val Ala Ser Val Ala Ser Ile Pro Arg Leu

85 90 95

Cys Gln Ile Ile Asn Glu Gly Gln Gly Gly Met Phe Arg Arg Leu Phe

100 105 110

Asn Pro Thr Lys Val Gln Ala Gly Asp Gln Ala Val Phe Asp Leu Met

115 120 125

Val Lys Leu Asp Glu Ile Ser Ser Thr Thr His Glu Val Ser Arg Met

130 135 140

Leu Glu Gly Val His Ala Ala Arg Thr Arg Gln Gln Gln Gly Val Ala

145 150 155 160

Leu Phe Pro Gly Ile His Gly Val Gly Glu Arg Tyr Ile Glu Arg Ala

165 170 175

Gln Gln Val Leu Ala Ser Ala Leu Gly Ile Ala Gly Phe Gly Ala Glu

180 185 190

Pro Trp Asp Gly His Thr Leu Ala Gln Ala Arg Arg Val Val Gln Arg

195 200 205

Tyr Ala Gln Asp Pro Asn Ser Glu Tyr Arg Leu Lys Ser Glu Ala Glu

210 215 220

Lys His Leu Thr Ser Ile Asn Glu Leu Arg Val Gln Ile Leu Leu Glu

225 230 235 240

Gln Leu Pro Val Asp Ala Leu Arg Met Ala Thr Asp His Arg Leu Arg

245 250 255

Phe Gly Ser Leu Asp Ser Ile His Val Ala Thr Val Ala Asp Val Leu

260 265 270

Lys Thr His Thr Ser Ile Leu Thr Thr Val Gln Gly Ile Gly Ala Gln

275 280 285

Thr Ala Gly Arg Met Lys Ala Ala Ala Glu Thr Leu Lys Gln Glu Ala

290 295 300

Leu Arg Arg Gln Asn Thr Ser Ile Gly Asp Glu Pro Thr Gln Pro Ala

305 310 315 320

Met Arg Leu Ile Asn Val Leu Ala Arg Phe Asp Gln Thr Glu Thr Ile

325 330 335

Thr Pro Glu Glu Arg Ala Arg Arg Thr Arg Val Ile Asp Tyr Val Glu

340 345 350

His Ile Pro Pro Ser Leu Asp Pro Tyr Ile Val Ile Asn Pro Ala Thr

355 360 365

Pro Glu Phe Asn Asn Phe Thr Asp Asp Leu Arg Trp Ile Asp Ala Asn

370 375 380

Pro Asn Leu Phe His Pro Gln Thr Ile Thr Thr Pro Pro Ala Asp Ile

385 390 395 400

Trp Asp Asp Tyr Ile Ser Arg Pro Ala His Tyr Gln Gly Leu Leu Ala

405 410 415

Thr Leu Leu Gly Arg Asp Ile Glu Gly Ala Asp Glu Leu Leu Asp Ala

420 425 430

Thr Thr Leu Gln Lys Ile Arg Asp Leu Thr Leu Asp Lys Thr His Leu

435 440 445

Thr Asp Leu His Leu Arg Gly Tyr Gln Ser Phe Gly Ala Arg Phe Ala

450 455 460

Ile Ile Gln Lys Lys Thr Leu Leu Gly Asp Asp Met Gly Leu Gly Lys

465 470 475 480

Thr Val Gln Ala Leu Ser Ala Ala Ala His Leu Ala Ala Thr Glu Lys

485 490 495

Asp Phe Arg Thr Leu Val Val Val Pro Ala Ser Val Ile Val Asn Trp

500 505 510

Thr Arg Glu Cys Lys Arg Phe Leu Asn Leu Pro Val Phe Ile Ala His

515 520 525

Gly Asp Asn Lys Gln Asp Ala Ile Asn Ala Trp Ser Asn Thr Asn Gly

530 535 540

Ile Ala Ile Cys Thr Tyr Asp Gly Val Arg Thr Met Asp Ile Pro Ala

545 550 555 560

Pro Gly Leu Val Ile Ala Asp Glu Ala His Leu Ile Lys Asn Pro Ser

565 570 575

Thr Lys Arg Thr Gln Ala Leu Arg Lys Leu Ile Asp Ala Ala Pro Phe

580 585 590

Thr Leu Leu Met Thr Gly Thr Pro Leu Glu Asn Lys Val Glu Glu Phe

595 600 605

Val Asn Leu Val Arg Tyr Ile Gln Pro Glu Leu Ile Thr Arg Gly Met

610 615 620

Ser Lys Met Gln Ala Glu Asn Phe Arg Glu Arg Ile Ala Pro Ala Tyr

625 630 635 640

Leu Arg Arg Asn Gln Ala Asp Val Leu Asp Glu Leu Pro Glu Arg Thr

645 650 655

Asp Ser Ile Asp Trp Ile Asp Leu Thr Pro Glu Asp Arg Ser Ala Tyr

660 665 670

Asp Asp Gln Val Arg Gln Gly Ser Trp Met Gly Met Arg Arg Ser Ala

675 680 685

Met Leu Ser Pro Thr Pro Arg Leu Thr Ser Ala Lys Met Gln Arg Ile

690 695 700

Leu Glu Leu Phe Glu Glu Ala Glu Glu His Gly Arg Lys Ala Leu Ile

705 710 715 720

Phe Thr Tyr Phe Leu Asp Val Leu Asp Glu Leu Glu Lys His Leu Gly

725 730 735

Glu Arg Val Ile Gly Arg Ile Ser Gly Asp Val Pro Ala Thr Lys Arg

740 745 750

Gln Leu Leu Val Asp Ala Leu Ser His Ser Lys Pro Gly Ser Ala Leu

755 760 765

Ile Ala Gln Ile Thr Ala Gly Gly Val Gly Leu Asn Ile Gln Ser Ala

770 775 780

Ser Leu Cys Ile Ile Cys Glu Pro Gln Val Lys Pro Thr Ile Glu Gln

785 790 795 800

Gln Ala Val Ala Arg Val His Arg Met Gly Gln Thr Ala Thr Val Gln

805 810 815

Val His Arg Leu Ile Gly Asp Glu Thr Ala Asp Glu Arg Met Leu Glu

820 825 830

Ile Leu Ala Gly Lys Thr His Val Phe Asp Val Tyr Ala Arg Leu Ser

835 840 845

Glu Thr Ala Glu Ile Pro Asp Ala Val Asp Ile Thr Glu Ser Gln Leu

850 855 860

Ala Ala Arg Val Ile Asp Glu Glu Arg Ala Arg Leu Gly Leu Thr Glu

865 870 875 880

Ser Thr Gly Pro Lys Asp Glu Glu Thr Ala Leu Ser

885 890

<210> 33

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 33

cagtgccaag cttgcatgcc tgcaggtcga ctctagtgcg ttcgtctgcg gtttcg 56

<210> 34

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 34

atcgacgccg ccccattcac ccttctgatg 30

<210> 35

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 35

catcagaagg gtgaatgggg cggcgtcgat 30

<210> 36

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 36

cagctatgac catgattacg aattcgagct cggtacccaa gcctcgaccc ctacatc 57

<210> 37

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 37

cacatcagct tgatttctgc 20

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 38

ggtcattgcc gatgaagccc 20

<210> 39

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 39

cagtgccaag cttgcatgcc tgcaggtcga ctctagaatg cgttctggac tgagg 55

<210> 40

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 40

gaaacggcct taagctaggt gcaccgagaa cagatg 36

<210> 41

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 41

catctgttct cggtgcacct agcttaaggc cgtttc 36

<210> 42

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 42

cttgatttaa ttgcgccatc aagcttttcc cgcccggtt 39

<210> 43

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 43

aaccgggcgg gaaaagcttg atggcgcaat taaatcaag 39

<210> 44

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 44

cagctatgac catgattacg aattcgagct cggtacccgc tatgacacct tcaacggatc 60

<210> 45

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 45

tccaaggaag atacacgcc 19

<210> 46

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 46

cttctgatga ccggcacacc 20

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 47

tagtcgatga cgcgggtgcg 20

<210> 48

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 48

cgttggaatc ttgcgttg 18

<210> 49

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 49

gcttgcatgc ctgcaggtcg actctagagg atccccctag cttaaggccg tttc 54

<210> 50

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 50

atcaggctga aaatcttctc tcatccgcca aaacaagctt ttcccgcccg gtt 53

<210> 51

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 51

cagtgccaag cttgcatgcc tgcaggtcga ctctagaccg gcgcagatgc caacgc 56

<210> 52

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 52

cccagaactg aaggtctaat tgcctaaggc cggaatt 37

<210> 53

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 53

aattccggcc ttaggcaatt agaccttcag ttctggg 37

<210> 54

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 54

cagctatgac catgattacg aattcgagct cggtacccgc ttgatgaagg ctccag 56

<210> 55

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 55

accggcgcag atgccaacgc 20

<210> 56

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 56

gcttgatgaa ggctccag 18

<210> 57

<211> 242

<212> DNA

<213> Corynebacterium glutamicum

<400> 57

ttcagggtag ttgactaaag agttgctcgc gaagtagcac ctgtcacttt tgtctcaaat 60

attaaatcga atatcaatat atggtctgtt tattggaacg cgtcccagtg gctgagacgc 120

atccgctaaa gccccaggaa ccctgtgcag aaagaaaaca ctcctctggc taggtagaca 180

cagtttataa aggtagagtt gagcgggtaa ctgtcagcac gtagatcgaa aggtgcacaa 240

ag 242

<210> 58

<211> 242

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 58

ttcagggtag ttgactaaag agttgctcgc gaagtagcac ctgtcacttt tgtctcaaat 60

attaaatcga atatcaatat atggtctgtt tattggaacg cgtcccagtg gctgagacgc 120

atccgctaaa gccccaggaa ccctgtgcag aaagaaaaca ctcctctggc taggtagaca 180

cagtttataa aggtataatt gagcgggtaa ctgtcagcac gtagatcgaa aggtgcacaa 240

ag 242

<210> 59

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 59

ccggaattcg accaaggatg agggctttg 29

<210> 60

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 60

agttacccgc tcaattatac ctttataaac 30

<210> 61

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 61

gtttataaag gtataattga gcgggtaact 30

<210> 62

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 62

acatgcatgc gcgtacgcga agtggcacat 30

<210> 63

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 63

atcaatatat ggtctgttta 20

<210> 64

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 64

cttggtggca acgatccgtt 20

<---

Claims (44)

1. A bacterium producing an L-amino acid selected from L-lysine and L-glutamic acid, wherein said bacterium has improved expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3, in which arginine at position 334 is replaced by a terminator. 2. A bacterium producing L-lysine, wherein said bacterium has improved expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3, in which arginine at position 334 is replaced by a terminator, or improved expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31, in which tyrosine at position 592 is replaced by other amino acids, or mutations of bases at positions -45 bp and -47 bp in the promoter region shown in SEQ ID NO: 57. 3. The bacterium according to claim 1 or 2, characterized in that the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 contains the nucleotide sequence of SEQ ID NO: 1; or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 comprises the nucleotide sequence of SEQ ID NO: 29. 4. The bacterium of any one of claims 1 to 3, characterized in that the polynucleotide sequence with point mutations encoding the amino acid sequence of SEQ ID NO: 3 is formed by mutating the 1000th base of the polynucleotide sequence shown in SEQ ID NO: 1; or the mutation comprises mutating the 1000th base of the polynucleotide sequence shown in SEQ ID NO: 1 from cytosine (C) to thymine (T); or the polynucleotide sequence with point mutations comprises the polynucleotide sequence shown in SEQ ID NO: 2; or a polynucleotide sequence with point mutations encoding the amino acid sequence of SEQ ID NO: 31 is formed by mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29; or the mutation comprises mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29 from adenine (A) to thymine (T); or the polynucleotide sequence with point mutations comprises the polynucleotide sequence shown in SEQ ID NO: 30. 5. The bacterium according to any one of claims 2 to 4, characterized in that the base mutations at positions -45 bp and -47 bp in the promoter region shown in SEQ ID NO: 57 are selected from a substitution of guanine (G) at position -45 bp with adenine (A) and a substitution of guanine (G) at position -47 bp with thymine (T) in the promoter region shown in SEQ ID NO: 57; or the nucleotide sequence of the promoter contains: (a) the nucleotide sequence shown in SEQ ID NO: 58; or (b) a nucleotide sequence having a sequence identity of more than 90%, preferably more than 95%, 98% with the nucleotide sequence shown in SEQ ID NO: 58 and retaining an increased promoter activity of (a), while retaining an adenine (A) at position -45 bp and a thymine (T) at position -47 bp. 6. The bacterium according to any one of paragraphs 1-5, characterized in that said bacterium is Corynebacterium glutamicum. 7. The bacterium of claim 6, wherein said bacterium is YP97158 or ATCC 13869. 8. A recombinant bacterial strain for producing L-lysine, comprising any of (I) and (IV), (II) and (IV), (III) and (IV), (V), (VI) or (VII) listed below: (I) a polynucleotide sequence characterized in that said polynucleotide sequence comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 3, and the arginine at position 334 is replaced by a terminator; (II) a polynucleotide sequence characterized in that said polynucleotide sequence comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 31, wherein tyrosine at position 592 is replaced by other amino acids; (III) a recombinant vector, characterized in that said recombinant vector comprises a polynucleotide sequence as defined in (I) or (II); (IV) a promoter nucleotide sequence comprising a nucleotide sequence formed by mutation of bases at positions -45 bp and -47 bp of the promoter region as shown in SEQ ID NO: 57; (V) an expression cassette comprising a promoter nucleotide sequence as defined in (IV) and a coding sequence operably linked downstream of the promoter nucleotide sequence; (VI) a recombinant vector containing a nucleotide sequence of a promoter as defined in (IV); And (VII) a nucleotide sequence of a promoter as defined in (IV) or a recombinant vector as defined in (VI). 9. A recombinant bacterial strain according to claim 8, characterized in that said polynucleotide sequence (I) comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 4; said polynucleotide sequence (I) is formed by mutation of the 1000th base of the polynucleotide sequence shown in SEQ ID NO: 1; said polynucleotide sequence (I) comprises a mutation of the 485th base of the polynucleotide sequence shown in SEQ ID NO: 1 from cytosine (C) to thymine (T); or said polynucleotide sequence (I) comprises the polynucleotide sequence shown in SEQ ID NO: 2. 10. A recombinant bacterial strain according to claim 8, characterized in that said polynucleotide sequence (II) comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 32; said polynucleotide sequence (II) is formed by mutation of the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29; said polynucleotide sequence (II) comprises a mutation at the 1775th base of the polynucleotide sequence shown in SEQ ID NO: 29 from adenine (A) to thymine (T); and said polynucleotide sequence (II) comprises the polynucleotide sequence shown in SEQ ID NO: 30. 11. A recombinant bacterial strain according to claim 8, characterized in that the nucleotide guanine (G) at position -45 bp in the promoter region is mutated to adenine (A) and the nucleotide guanine (G) at position -47 bp is mutated to thymine (T) in the promoter region shown in SEQ ID NO: 57. 12. A recombinant bacterial strain according to claim 8, characterized in that said nucleotide sequence of promoter (IV) additionally comprises (a) or (b): (a) the nucleotide sequence shown in SEQ ID NO:58; or (b) a nucleotide sequence having a sequence identity of more than 90% to the nucleotide sequence shown in SEQ ID NO: 58 and retaining an enhanced promoter activity of (a), while retaining an adenine (A) at position -45 bp and a thymine (T) at position -47 bp. 13. A recombinant bacterial strain according to claim 8, characterized in that the coding sequence is the coding sequence of the lysC gene. 14. A recombinant bacterial strain according to claim 8, characterized in that said nucleotide sequence of the promoter (VI) is linked to a shuttle plasmid for constructing a recombinant vector; and said shuttle plasmid is the pK18mobsacB plasmid. 15. A recombinant bacterial strain according to claim 8, characterized in that said nucleotide sequence (VII) is the nucleotide sequence shown in SEQ ID NO: 58; or said nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 58, linked to the coding sequence of the lysC gene. 16. A recombinant strain of bacteria according to claim 8, characterized in that tyrosine at position 592 is replaced by phenylalanine. 17. A recombinant bacterial strain according to claim 12, characterized in that said nucleotide sequence of the promoter (IV) additionally contains a nucleotide sequence having a sequence identity of more than 95% to the nucleotide sequence shown in SEQ ID NO: 58. 18. A recombinant bacterial strain according to claim 12, characterized in that said nucleotide sequence of the promoter (IV) additionally contains a nucleotide sequence having a sequence identity of more than 98% to the nucleotide sequence shown in SEQ ID NO: 58. 19. A method for producing an L-amino acid, comprising culturing a bacterium according to any one of claims 1-5 or a recombinant strain of a bacterium according to claim 8 and isolating the L-amino acid from the culture, wherein the L-amino acid is L-lysine.