WO2021248902A1 - 产l-氨基酸的重组菌株及其构建方法与应用 - Google Patents

产l-氨基酸的重组菌株及其构建方法与应用 Download PDF

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WO2021248902A1
WO2021248902A1 PCT/CN2020/142133 CN2020142133W WO2021248902A1 WO 2021248902 A1 WO2021248902 A1 WO 2021248902A1 CN 2020142133 W CN2020142133 W CN 2020142133W WO 2021248902 A1 WO2021248902 A1 WO 2021248902A1
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seq
sequence
amino acid
polynucleotide
mutation
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English (en)
French (fr)
Inventor
魏爱英
孟刚
周晓群
赵春光
马风勇
贾慧萍
杨立鹏
苏厚波
郭小炜
田斌
高晓航
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Inner Mongolia Eppen Biotech Co Ltd
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Inner Mongolia Eppen Biotech Co Ltd
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Priority claimed from CN202010514037.1A external-priority patent/CN111909944A/zh
Priority claimed from CN202010790887.4A external-priority patent/CN111961635B/zh
Priority claimed from CN202011105063.5A external-priority patent/CN112266891B/zh
Priority to EP20939601.9A priority Critical patent/EP4163377A4/en
Priority to KR1020227045499A priority patent/KR20230042224A/ko
Priority to JP2022574296A priority patent/JP7728802B2/ja
Priority to KR1020267006205A priority patent/KR20260066713A/ko
Application filed by Inner Mongolia Eppen Biotech Co Ltd filed Critical Inner Mongolia Eppen Biotech Co Ltd
Priority to KR1020267006206A priority patent/KR20260061169A/ko
Priority to US18/001,218 priority patent/US12584100B2/en
Publication of WO2021248902A1 publication Critical patent/WO2021248902A1/zh
Anticipated expiration legal-status Critical
Priority to US19/366,448 priority patent/US20260043000A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02004—Aspartate kinase (2.7.2.4)
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the invention belongs to the field of genetic engineering and microbial technology, and specifically relates to a recombinant strain producing L-amino acid and a construction method and application thereof.
  • L-Lysine (L-Lysine) has a wide range of applications, including medicine, food, feed and other aspects. Among them, L-Lysine used in feed additives accounts for more than 90% of the total. At present, my country is the second largest consumer market for L-lysine and the largest producer of L-lysine.
  • L-lysine is mainly produced by direct fermentation, which uses strains with a complete L-lysine biosynthetic pathway, and uses waste molasses and starch hydrolysate as substrates to produce through aerobic fermentation.
  • This method accounts for 2/3 of the L-lysine production in the world today, and the technology is very mature. This method mainly exists in yeast, bacteria, and molds, and is widespread in microorganisms.
  • the industrial production strains used for L-lysine fermentation are mainly coryneform bacteria and Brevibacterium mutant strains. With the development of metabolic engineering and genetic engineering, gene mutations can be controlled.
  • L-glutamic acid is mainly used in the production of monosodium glutamate, spices, as well as salt substitutes, nutritional supplements and biochemical reagents.
  • L-glutamic acid itself can be used as a medicine to participate in the metabolism of protein and sugar in the brain and promote the oxidation process.
  • the product combines with ammonia in the body to form non-toxic glutamine, which reduces blood ammonia and relieves symptoms of liver coma.
  • MSG was mainly carried out by the hydrolysis of wheat gluten (gluten), but now it uses the microbial fermentation method for large-scale production.
  • the purpose of the present invention is to develop a new strain with L-amino acid production capacity, thereby providing a method for efficiently producing L-amino acid.
  • the inventors of the present invention have discovered through research that the NCgl0609 gene and/or NCgl1575 gene, which are not known to have fermented amino acid production capacity in the prior art, can be equipped with high-efficiency L-amino acids by modifying the gene or improving its expression. Production capacity; In addition, the inventors of the present invention also found that mutations in a certain promoter sequence can also improve the L-amino acid production capacity of the corresponding microorganism. Based on these findings, the present invention has been completed.
  • the present invention provides bacteria that produce L-amino acids, wherein the expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 is improved, and/or the expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 is improved To improve, and/or, the -45bp and -47bp bases of the promoter region shown in SEQ ID NO: 57 are mutated.
  • the present invention also provides a method for producing L-amino acid by using the microorganism.
  • the expression improvement is that the expression of the polynucleotide is increased, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 31 has a point mutation, or the polynucleotide encoding SEQ ID NO: 3 or SEQ The polynucleotide of the amino acid sequence of ID NO: 31 has a point mutation and its expression is enhanced.
  • the first aspect of the present invention provides bacteria producing L-amino acids, wherein the expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 is improved.
  • the L-amino acid is L-lysine or L-glutamic acid.
  • amino acid sequence of SEQ ID NO: 3 is the protein encoded by the gene NCgl0609.
  • the bacteria have enhanced L-amino acid production capacity.
  • the bacteria having L-amino acid production ability may be bacteria capable of accumulating the target L-amino acid in an amount of preferably 0.5 g/L or more, more preferably 1.0 g/L or more in the medium.
  • the polynucleotide may encode the amino acid sequence of SEQ ID NO: 3 that has about 90% or higher, about 92% or higher, about 95% or higher, about 97% or higher, about 98% or higher. Amino acid sequences with high, or about 99% or higher sequence homology.
  • the polynucleotide with improved expression comprises the nucleotide sequence of SEQ ID NO:1.
  • the expression improvement means that the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 has a point mutation, so that the arginine at position 334 of the amino acid sequence of SEQ ID NO: 3 is replaced For the terminator.
  • amino acid sequence shown in SEQ ID NO: 3 wherein the amino acid sequence after the 334th arginine is replaced by a terminator is shown in SEQ ID NO: 4.
  • the polynucleotide sequence with a point mutation is formed by a mutation at the 1000th base of the polynucleotide sequence shown in SEQ ID NO:1.
  • the mutation includes the mutation of base 1000 of the polynucleotide sequence shown in SEQ ID NO:1 from cytosine (C) to thymine (T).
  • the polynucleotide sequence with point mutations includes the polynucleotide sequence shown in SEQ ID NO: 2.
  • the present invention also provides L-amino acid producing bacteria having improved expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 31.
  • the L-amino acid is L-lysine.
  • the bacteria are bacteria belonging to the genus Corynebacterium.
  • amino acid sequence of SEQ ID NO: 31 is the protein encoded by the gene NCgl1575.
  • the microorganism has enhanced L-lysine production capacity compared with wild-type or parent strains.
  • the polynucleotide may encode the amino acid sequence of SEQ ID NO: 31 that has about 90% or higher, about 92% or higher, about 95% or higher, about 97% or higher, about 98% or higher. Amino acid sequences with high, or about 99% or higher sequence homology.
  • the polynucleotide may include the nucleotide sequence of SEQ ID NO: 29.
  • the polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 has a point mutation, so that the tyrosine 592 of the amino acid sequence of SEQ ID NO: 31 is replaced by a different amino acid .
  • tyrosine at position 592 is replaced by phenylalanine.
  • amino acid sequence shown in SEQ ID NO: 31 wherein the amino acid sequence after the 592th tyrosine (Y) is replaced by phenylalanine (F) is shown in SEQ ID NO: 32.
  • the polynucleotide sequence with a point mutation is formed by a mutation at base 1775 of the polynucleotide sequence shown in SEQ ID NO:29.
  • the mutation includes the mutation of base 1775 of the polynucleotide sequence shown in SEQ ID NO: 29 from adenine (A) to thymine (T).
  • the polynucleotide sequence with point mutations includes the polynucleotide sequence shown in SEQ ID NO: 30.
  • the bacteria may be microorganisms belonging to the genus Corynebacterium, such as Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes (Corynebacterium) ammoniagenes), Corynebacterium pekinense.
  • Corynebacterium glutamicum such as Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes (Corynebacterium) ammoniagenes), Corynebacterium pekinense.
  • the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YP97158, preservation number: CGMCC No. 12856, preservation date: August 16, 2016, preservation unit: Chinese microorganism strains General Microbiology Center of the Preservation Management Committee, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355, recorded in Chinese Patent Application CN106367432A (application date September 1, 2016, publication date February 1, 2017 )middle.
  • the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC 13869.
  • polynucleotides can be enhanced by the following means: expression of regulatory sequences by substitution or mutation, introduction of mutations to polynucleotide sequences, increase in the copy number of polynucleotides introduced via chromosome insertion or vector introduction, or a combination thereof, and the like.
  • the expression regulatory sequence of the polynucleotide can be modified.
  • the expression regulatory sequence controls the expression of the polynucleotide to which it is operably linked, and may include, for example, a promoter, a terminator, an enhancer, a silencer, and the like.
  • Polynucleotides can have variations in the start codon.
  • the polynucleotide can be incorporated into a specific site on the chromosome, thereby increasing the number of copies.
  • the specific site may include, for example, a transposon site or an intergenic site.
  • the polynucleotide can be incorporated into an expression vector, and the expression vector can be introduced into a host cell, thereby increasing the copy number.
  • the number of copies is increased by incorporating polynucleotides or polynucleotides with point mutations into specific sites on microbial chromosomes.
  • a polynucleotide with a promoter sequence or a polynucleotide with a point mutation with a promoter sequence is incorporated into a specific site on a microbial chromosome to overexpress the Nucleic acid sequence.
  • a polynucleotide or a polynucleotide with a point mutation is incorporated into an expression vector, and the expression vector is introduced into a host cell, thereby increasing the copy number.
  • a polynucleotide with a promoter sequence or a polynucleotide with a point mutation with a promoter sequence is incorporated into an expression vector, and the expression vector is introduced into a host cell, Thus, the amino acid sequence is overexpressed.
  • the promoter is a promoter of a polynucleotide (NCgl0609 gene) encoding the amino acid sequence of SEQ ID NO: 3.
  • the promoter is a promoter of a polynucleotide (NCgl1575 gene) encoding the amino acid sequence of SEQ ID NO: 31.
  • the vector used is pK18mobsacB plasmid, pXMJ19 plasmid.
  • the bacteria may also have other improvements related to increasing the production of L-amino acids.
  • the second aspect of the present invention provides a polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector including the polynucleotide sequence, and a recombinant strain containing the polynucleotide sequence.
  • the polynucleotide sequence has improved expression, and the improvement includes a point mutation in the polynucleotide encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3, so that the 334th position of the amino acid sequence is refined.
  • the amino acid is replaced by a terminator.
  • amino acid sequence shown in SEQ ID NO: 3 wherein the amino acid sequence after the 334th arginine is replaced by a terminator is shown in SEQ ID NO: 4.
  • the polynucleotide sequence encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 contains the polynucleotide sequence shown in SEQ ID NO: 1.
  • the mutated polynucleotide sequence provided by the present invention is formed by a mutation at the 1000th base of the polynucleotide sequence shown in SEQ ID NO:1.
  • the mutation includes the mutation of base 1000 of the polynucleotide sequence shown in SEQ ID NO:1 from cytosine (C) to thymine (T).
  • the mutated polynucleotide sequence includes the polynucleotide sequence shown in SEQ ID NO: 2.
  • the substituted amino acid sequence includes the amino acid sequence shown in SEQ ID NO:4.
  • the polynucleotide sequence includes a polynucleotide encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 31, in which the 592th tyrosine is replaced by a different amino acid.
  • tyrosine at position 592 is replaced by phenylalanine.
  • amino acid sequence shown in SEQ ID NO: 31 wherein the amino acid sequence after the 592th tyrosine (Y) is replaced by phenylalanine (F) is shown in SEQ ID NO: 32.
  • the polynucleotide sequence encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 31 contains the polynucleotide sequence shown in SEQ ID NO: 29.
  • the polynucleotide sequence is formed by a mutation at base 1775 of the polynucleotide sequence shown in SEQ ID NO: 29.
  • the mutation includes the mutation of base 1775 of the polynucleotide sequence shown in SEQ ID NO: 29 from adenine (A) to thymine (T).
  • the polynucleotide sequence includes the polynucleotide sequence shown in SEQ ID NO: 30.
  • the amino acid sequence includes the amino acid sequence shown in SEQ ID NO:32.
  • the mutation refers to a change in the base/nucleotide of the site
  • the mutation method can be selected from at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination. kind.
  • PCR site-directed mutagenesis and/or homologous recombination are preferably used.
  • the recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid.
  • the plasmid is pK18mobsacB plasmid.
  • the plasmid is pXMJ19 plasmid.
  • the polynucleotide sequence and the plasmid can be constructed into a recombinant vector through the NEBuider recombination system.
  • the recombinant strain contains the polynucleotide sequence.
  • the starting bacteria of the recombinant strain is YP97158.
  • the starting strain of the recombinant strain is ATCC 13869.
  • the third aspect of the present invention also provides a method for constructing a recombinant strain that produces L-amino acids.
  • the construction method includes the following steps:
  • the polynucleotide sequence of wild-type NCgl0609 shown in SEQ ID NO:1 in the host strain was modified to make the 1000th base mutated to obtain a recombinant strain containing the gene encoding the mutant NCgl0609.
  • the modification includes at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to the mutation of the 1000th base in SEQ ID NO:1 from cytosine (C) to thymine (T); specifically, the polynucleoside containing the gene encoding the mutation NCgl0609
  • the acid sequence is shown in SEQ ID NO: 2.
  • construction method includes the following steps:
  • the step (1) includes: point mutation NCgl0609 gene construction: according to the genomic sequence of the unmodified strain, two pairs of primers P1 and P2, P3 and P4 for amplifying the NCgl0609 gene fragment are synthesized by PCR
  • the site-directed mutagenesis method introduces point mutations into the wild-type NCgl0609 gene SEQ ID NO:1, and obtains the nucleotide sequence SEQ ID NO:2 of the point mutation NCgl0609 gene, which is denoted as NCgl0609 C1000T .
  • the unmodified strain genome can be derived from the ATCC13032 strain, and its genome sequence can be obtained from the NCBI website.
  • the primer is:
  • the PCR amplification is performed as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30s, 52°C annealing for 30s, 72°C extension for 40s (30 cycles), 72°C over extension for 10 minutes .
  • the overlap PCR amplification is performed as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30s, 52°C annealing for 30s, and 72°C extension for 60s (30 cycles), 72°C overrun Extend for 10 minutes.
  • the step (2) includes the construction of a recombinant plasmid, including: assembling the separated and purified NCgl0609 C1000T and pK18mobsacB plasmids through the NEBuider recombination system to obtain the recombinant plasmid.
  • the step (3) includes the construction of a recombinant strain, and the recombinant plasmid is transformed into a host strain to obtain a recombinant strain.
  • the conversion of step (3) is an electroconversion method.
  • the host strain is YP97158.
  • the recombination is achieved by homologous recombination.
  • the fourth aspect of the present invention also provides a method for constructing a recombinant strain that produces L-amino acids.
  • the construction method includes the following steps:
  • NCgl0609 Amplify the upstream and downstream homologous arm fragments of the NCgl0609 gene, the coding region of the NCgl0609 gene and its promoter region sequence, or amplify the coding region of the NCgl0609 or NCgl0609 R334* gene and its promoter region sequence, and then use homologous recombination.
  • the NCgl0609 or NCgl0609 R334* gene is introduced into the genome of the host strain to obtain the strain overexpressing the NCgl0609 or NCgl0609 R334* gene.
  • the primers for amplifying upstream homology arm fragments are:
  • the primers for amplifying the downstream homology arm fragments are:
  • the primers for amplifying the sequence of the coding region of the gene and its promoter region are:
  • P10 5'GATTTAATTGCGCCATCTGATTCTGGCAACAACTCCTTCCTTGACC 3'(SEQ ID NO: 14).
  • the aforementioned P7-P12 are used as primers, and the upstream homologous fragments, downstream homologous fragments and three fragments of NCgl0609 or NCgl0609 R334* with their own promoters are mixed as templates. Amplify to obtain integrated homology arm fragments.
  • the PCR system used 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U / ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L; PCR amplification is performed as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30s, 52°C annealing for 30s, 72°C extension for 60s (30 cycles), 72°C over extension for 10 minutes.
  • the NEBuider recombination system is used to assemble the shuttle plasmid PK18mobsacB, the upper and lower homology arm fragments, the gene coding region and the promoter region fragments to obtain an integrated plasmid.
  • the integration plasmid is transfected into the host strain, and the NCgl0609 or NCgl0609 R334* gene is introduced into the genome of the host strain by homologous recombination.
  • the host strain is YP97158.
  • the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 2.
  • the fifth aspect of the present invention also provides a method for constructing a recombinant strain producing L-amino acids.
  • the construction method includes the following steps:
  • NCgl0609 gene coding region and promoter region sequence or NCgl0609 R334* gene coding region and promoter region sequence, construct an overexpression plasmid vector, and transfer the vector into the host strain to achieve the strain overexpression of NCgl0609 or NCgl0609 R334* gene.
  • the primers for amplifying the sequence of the coding region of the gene and its promoter region are:
  • the PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, a total volume of 50 ⁇ L; the PCR amplification is performed as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 52°C annealing for 30s, 72°C extension for 60s (30 cycles), 72°C over extension for 10 minutes .
  • the NEBuider recombination system is used to assemble the shuttle plasmid pXMJ19 and the NCgl0609 and NCgl0609 R334* fragments with its own promoter to obtain an overexpression plasmid.
  • the host strain is YP97158.
  • the host strain is ATCC 13869.
  • 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.
  • the construction method includes the following steps:
  • the polynucleotide sequence of the wild-type NCgl1575 shown in SEQ ID NO: 29 in the host strain is modified to mutate the 1775th base to obtain a recombinant strain of Corynebacterium containing the mutant NCgl1575 encoding gene.
  • the modification includes at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to the mutation of base 1775 in SEQ ID NO: 29 from adenine (A) to thymine (T); specifically, the polynucleoside containing the gene encoding the mutation NCgl1575
  • the acid sequence is shown in SEQ ID NO: 30.
  • construction method includes the following steps:
  • the recombinant vector is introduced into the host strain to obtain the recombinant coryneform strain containing the mutant NCgl1575 encoding gene.
  • the step (1) includes: point mutation NCgl1575 gene construction: according to the genome sequence of Corynebacterium glutamicum, two pairs of primers P1' and P2' and P3' for amplifying the NCgl1575 gene fragment are synthesized And P4', point mutations were introduced into the wild-type NCgl1575 gene SEQ ID NO: 29 by PCR site-directed mutagenesis method, and the nucleotide sequence of the point mutation NCgl1575 gene SEQ ID NO: 30 was obtained, denoted as NCgl1575 A1775T .
  • the Corynebacterium glutamicum genome can be derived from strain ATCC13032, and its genome sequence can be obtained from the NCBI website.
  • the primer is:
  • P2' 5'ATCGACGCCGCCCCATTCACCCTTCTGATG 3'(SEQ ID NO: 34);
  • the PCR amplification is performed as follows: denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 40s (30 cycles).
  • the overlap PCR amplification is performed as follows: denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 90s (30 cycles).
  • the step (2) includes the construction of a recombinant plasmid, including: assembling the isolated and purified NCgl1575 A1775T and pK18mobsacB plasmids through the NEBuider recombination system to obtain the recombinant plasmid pK18-NCgl1575 A1775T .
  • the step (3) includes the construction of a recombinant strain, and the recombinant plasmid pK18-NCgl1575 A1775T is transformed into a host strain to obtain a recombinant strain.
  • the conversion of step (3) is an electroconversion method.
  • the host strain is YP97158.
  • the recombination is achieved by homologous recombination.
  • the invention also provides a method for constructing a recombinant strain of Corynebacterium.
  • the construction method includes the following steps:
  • NCgl1575 gene the NCgl1575 gene coding region and its promoter region sequence, or the NCgl1575 A1775T gene coding region and its promoter region sequence, and introduce it into the genome of the host strain by homologous recombination NCgl1575 or NCgl1575 A1775T gene in order to achieve the strain overexpressing NCgl1575 or NCgl1575 A1775T gene.
  • the primers for amplifying upstream homology arm fragments are:
  • the primers for amplifying the downstream homology arm fragments are:
  • the primers for amplifying the sequence of the coding region of the gene and its promoter region are:
  • the aforementioned P7'/P12' is used as primers, and the upstream homologous fragments, downstream homologous fragments obtained by amplification, and the three fragments NCgl1575 or NCgl1575 A1775T with its own promoter are mixed as templates. Amplify to obtain integrated homology arm fragments.
  • the PCR system used 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U / ⁇ L) 0.25 ⁇ L, the total volume is 50 ⁇ L; PCR amplification is carried out as follows: 94°C pre-denaturation for 5min, 94°C denaturation for 30s, 52°C annealing for 30s, 72°C extension for 180s (30 cycles), 72°C over extension for 10min.
  • the NEBuider recombination system is used to assemble the shuttle plasmid PK18mobsacB and the integration homology arm fragment to obtain the integration plasmid.
  • the integration plasmid is transfected into a host strain, and the NCgl1575 or NCgl1575 A1775T gene is introduced into the genome of the host strain by homologous recombination.
  • the host strain is YP97158.
  • 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.
  • the construction method includes the following steps:
  • NCgl1575 gene coding region and promoter region sequence or NCgl1575 A1775T gene coding region and promoter region sequence, construct an overexpression plasmid vector, and transfer the vector into the host strain to achieve the strain overexpression of NCgl1575 or NCgl1575 A1775T gene.
  • the primers for amplifying the sequence of the coding region of the gene and its promoter region are:
  • the PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L; the PCR amplification is carried out as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 52°C annealing for 30s, 72°C extension for 120s (30 cycles), 72°C over extension for 10 minutes .
  • the NEBuider recombination system is used to assemble the shuttle plasmid pXMJ19 and the NCgl1575 or NCgl1575 A1775T fragment with its own promoter to obtain an overexpression plasmid.
  • the host strain is YP97158.
  • the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 30.
  • Another aspect of the present invention is to provide a promoter nucleotide sequence, which includes a nucleotide sequence formed by mutating the -45 bp and -47 bp bases of the promoter region shown in SEQ ID NO: 57.
  • the -45bp nucleotide guanine (G) of the promoter region shown in SEQ ID NO: 57 is mutated to adenine (A), and the -47 bp nucleotide guanine (G) mutation For thymine (T).
  • nucleotide sequence of the promoter is as follows:
  • nucleotide sequence that has more than 90%, preferably more than 95%, 98% identity with the nucleotide sequence shown in SEQ ID NO: 58, and retains (a) the enhancement of the promoter Active, and the -45bp position remains as adenine (A), and the -47bp position remains as thymine (T).
  • the present invention also provides an expression cassette comprising the above-mentioned promoter, which comprises the promoter and a coding sequence operably linked to the promoter.
  • the coding sequence is the coding sequence of the lysC gene.
  • the present invention also provides a recombinant vector containing the nucleotide sequence of the promoter of the present invention.
  • the promoter nucleotide sequence of the present invention is connected with a shuttle plasmid to construct the recombinant vector; as an embodiment of the present invention, the shuttle plasmid is a pK18mobsacB plasmid.
  • the present invention also provides a recombinant strain containing the above-mentioned promoter nucleotide sequence or the above-mentioned recombinant vector.
  • the recombinant strain according to the present invention includes the nucleotide sequence shown in SEQ ID NO:58.
  • the nucleotide sequence shown in SEQ ID NO: 58 is the promoter region of the lysC gene. Further, the nucleotide sequence shown in SEQ ID NO: 58 is connected to the coding sequence of the lysC gene.
  • the recombinant strain may include the above-mentioned expression cassette or recombinant vector of the present invention. Specifically, the recombinant strain of the present invention is transformed by an expression cassette or a recombinant vector.
  • the recombinant strain according to the present invention is formed by introducing the above-mentioned mutant promoter nucleotide sequence into a host strain;
  • the host strain can be selected from strains known in the art that produce L-amino acids, especially L-lysine ,
  • the Corynebacterium may be Corynebacterium glutamicum, Corynebacterium flavum, Corynebacterium crenatum, Corynebacterium Beijing; preferably Corynebacterium glutamicum.
  • the host strain is YP97158.
  • the recombinant strain according to the present invention uses the pK18mobsacB plasmid as the vector.
  • the recombinant strain according to the present invention may further include other modifications.
  • the present invention also provides a method for constructing L-lysine-producing recombinant strain, which includes the following steps:
  • the mutation refers to the mutation of the -45 bp nucleotide guanine (G) of the promoter region shown in SEQ ID NO: 57 to adenine (A), and the -47 bp nucleotide guanine Purine (G) is mutated to thymine (T).
  • G nucleotide sequence of the mutated promoter region is shown in SEQ ID NO: 58.
  • the construction method further includes the following steps:
  • the method for making the mutation includes mutagenesis, PCR site-directed mutagenesis or homologous recombination, preferably PCR site-directed mutagenesis.
  • the step (1) includes:
  • Two pairs of primers were designed to amplify the promoter region of the lysC gene, and then the nucleotide sequence of the mutant promoter region was obtained by PCR technology.
  • the primer in the step (1) is:
  • the step (1) includes: using Corynebacterium glutamicum ATCC13032 as a template, and using primers P1" and P2", P3" and P4", respectively, to perform PCR amplification to obtain two Containing DNA fragments; using the above two DNA fragments as templates, and P1" and P4" as primers, by overlapping PCR amplification (SEQ ID NO: 58) ) DNA fragments.
  • the obtained DNA fragment contains EcoR I and Sph I restriction sites at both ends of the obtained DNA fragment through overlap PCR amplification (Overlap PCR).
  • the step (2) includes: performing agarose gel electrophoresis and separation and purification on the products amplified by overlapping PCR reactions, and after double-enzyme digestion (EcoR I/Sph I), and the same double-enzyme digestion ( The shuttle plasmids after EcoR I/Sph I) are connected to obtain the allelic replacement recombinant vector.
  • the shuttle plasmid is pK18mobsacB plasmid; the constructed recombinant vector is pK18-Plys C (G(-45)A, G(-47)T) .
  • the recombinant plasmid has a kanamycin resistance marker.
  • the transformation in the step (3) is an electrotransformation method; illustratively, in the step (3), the recombinant vector is transformed into the strain YP97158.
  • the above-mentioned various recombinant strains obtained by the present invention can be used alone in the fermentation production of L-amino acid, can also be used in combination, and can also be mixed with other L-amino acid-producing bacteria to produce L-amino acid by fermentation.
  • Another aspect of the present invention provides a method of producing L-amino acids, the method comprising culturing the bacteria; and obtaining L-amino acids from the culture.
  • the culture of the bacteria can be carried out in a suitable medium under culture conditions known in the art.
  • the culture medium may contain: a carbon source, a nitrogen source, trace elements, and combinations thereof.
  • the pH of the culture can be adjusted.
  • the cultivation may include preventing the generation of bubbles, for example, by using a defoaming agent to prevent the generation of bubbles.
  • the culture may include injecting gas into the culture.
  • the gas may include any gas capable of maintaining the aerobic conditions of the culture.
  • the temperature of the culture may be 20 to 45°C.
  • the generated L-amino acid can be recovered from the culture by treating the culture with sulfuric acid or hydrochloric acid, followed by a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.
  • SEQ ID NO:1 NCgl0609 wild-type ORF sequence
  • SEQ ID NO: 3 NCgl0609 wild-type encoded protein amino acid sequence
  • SEQ ID NO: 4 NCgl0609R334* encoded protein amino acid sequence
  • SEQ ID NO: 29 NCgl1575 wild-type ORF sequence
  • SEQ ID NO: 30 NCgl1575 A1775T ORF sequence
  • SEQ ID NO: 31 NCgl1575 wild-type encoded protein sequence
  • SEQ ID NO: 32 NCgl1575 Y592F coding protein sequence
  • SEQ ID NO: 57 Wild-type promoter sequence
  • SEQ ID NO: 58 Promoter sequence after mutation
  • the term "bacteria with L-amino acid production ability" refers to the ability to produce and accumulate the desired L-amino acid in the culture medium and/or the cells of the bacteria to the following extent, so that when the bacteria are cultured in the culture medium
  • the bacteria producing L-amino acids can be collected at the time.
  • the bacteria having L-amino acid production ability may be bacteria capable of accumulating the desired L-amino acid in the culture medium and/or the cells of the bacteria in an amount larger than the amount obtainable by the unmodified strain.
  • 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-isoleuc Amino acids, L-alanine, L-valine, L-leucine, and glycine; amino acids 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-methyl Thionine; acidic amino acids, such as L-glutamic acid and L-aspartic acid; and amino acids having an amide group in the side chain, such as L-glutamine and L-asparagine.
  • basic amino acids such as L-lysine, L-or
  • 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-glutamic acid, L-lysine, L-threonine, and L-tryptophan. More specific examples of L-amino acids include L-glutamic acid, L-lysine.
  • amino acid refers to L-amino acid.
  • L-amino acid refers to L-amino acid in free form, its salt, or a mixture thereof.
  • unmodified strain refers to a control strain that has not been modified in such a way that it has specific characteristics. That is, examples of unmodified strains include wild-type strains and parent strains.
  • sequence homology refers to the percent identity between two polynucleotides or two polypeptide modules.
  • sequence homology between one type of module and another type of module can be determined by using methods known in the art. For example, such sequence homology can be determined by the BLAST algorithm.
  • operably linked refers to a functional connection between a regulatory sequence and a polynucleotide sequence, whereby the regulatory sequence controls the transcription and/or translation of the polynucleotide sequence.
  • the regulatory sequence may be a strong promoter that can increase the expression level of the polynucleotide.
  • the regulatory sequence may be a promoter derived from a microorganism belonging to the genus Corynebacterium or may be a promoter derived from another microorganism.
  • the promoter may be trc promoter, gap promoter, tac promoter, T7 promoter, lac promoter, trp promoter, araBAD promoter, or cj7 promoter.
  • vector refers to a polynucleotide construct that contains regulatory sequences and gene sequences of a gene and is configured to express a target gene in a suitable host cell.
  • a vector can also refer to a polynucleotide construct that contains sequences that can be used for homologous recombination, so that the vector introduced into the host cell can change the regulatory sequence of the endogenous gene in the genome of the host cell, or can The expressed target gene is inserted into a specific site in the host's genome.
  • the vector used in the present invention may further include a selection marker to determine the introduction of the vector into the host cell or the insertion of the vector into the chromosome of the host cell.
  • Selection markers may include markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with such selective agents, since cells expressing only the selection marker can survive or display different phenotypic traits, transformed cells can be selected.
  • the term "transformation” refers to the introduction of a polynucleotide into a host cell so that the polynucleotide can be used as an extra-genomic element or inserted into the genome of the host cell to replicate.
  • the method of transforming the vector used in the present invention may include a method of introducing nucleic acid into a cell.
  • the electric pulse method can be implemented according to the host cell.
  • the product encoded by the gene has an impact on the amino acid production capacity, and the recombinant strain is obtained by introducing point mutations in the coding sequence, or increasing the copy number or overexpression of the gene Compared with the wild-type strain, the obtained strain is conducive to the production of high-concentration amino acids.
  • a point mutation was introduced into the promoter region of the lysC gene to obtain a recombinant strain.
  • the obtained strain can also greatly increase the production of L-lysine, further improve the production efficiency, and reduce the production. Cost, easy to promote and apply.
  • composition of the basal medium used for culturing the strains is the same, and the sucrose, kanamycin, or chloramphenicol, etc., are added to the composition of the basal medium.
  • the composition of the basal medium is as follows:
  • Reagent name Ratio glucose 5.0g/L Phosphoric acid 0.38g/L Magnesium Sulfate 1.85g/L Potassium Chloride 1.6g/L Biotin 550 ⁇ g/L Vitamin B1 300 ⁇ g/L Ferrous sulfate 10mg/L Manganese sulfate 10g/dl KH 2 PO 4 2.8g/L Vitamin C 0.75mg/L Vitamin B12 2.5 ⁇ g/L P-aminobenzoic acid 0.75mg/L Defoamer 0.0015ml/dl Betaine 1.5g/L Cane molasses 7ml/L corn syrup 77ml/L Aspartic acid 1.7g/L Hair powder 2g/L
  • NCgl0609 R334* NCgl0609 R334* ).
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • P4 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCGGCTG GAAATGTGGAG3' (SEQ ID NO: 8)
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2 + (25mM) 4 ⁇ L, primers (10pM) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L, the PCR amplification is carried out as follows: 94°C pre-denaturation for 5min, (94°C denaturation for 30s, 52 Annealing at °C for 30 s, extension at 72 °C for 40 s, 30 cycles), and over-extending at 72 °C for 10 min, two DNA fragments (NCgl0609Up and NCgl0609Down) containing the coding region of NCgl0609 were obtained with sizes of 698bp and 648bp, respectively.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L, the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, (denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 60 s, 30 cycles), and over extension at 72°C for 10 min.
  • This DNA fragment causes the 1000th cytosine (C) in the coding region of YP97158NCgl0609 gene to change to thymine (T), and finally causes the 334th amino acid of the encoded protein to change from arginine (R) to a terminator.
  • the DNA fragment was purified by agarose gel electrophoresis, and the purified pK18mobsacB plasmid (purchased from Addgene, and digested with Xbal I/BamH I) with NEBuilder enzyme (purchased from NEB) After ligation at 50°C for 30 minutes, the monoclonal clones grown after the conversion of the ligation product were identified by PCR to obtain the positive vector pk18-NCgl0609 R334* , which contains the kanamycin resistance marker. Send the correct vector pk18-NCgl0609 R334* to the sequencing company for sequencing and identification, and save the vector pk18-NCgl0609 R334* containing the correct point mutation (CT) for future use.
  • CT point mutation
  • the allelic replacement plasmid pk18-NCgl0609 R334* was transformed into L-lysine production strain YP97158 by electroporation (see WO2014121669A1 for the construction method; sequencing confirmed that the wild-type NCgl0609 gene coding region is retained on the chromosome of this strain) ), the single colony produced by the culture was identified by the primer P1 and the universal primer M13R, and the strain that can amplify a band with a size of 1375 bp was a positive strain.
  • the positive strains were cultured on a medium containing 15% sucrose, and the single colonies produced by the culture were cultured on a medium containing kanamycin and a medium without kanamycin, and on a medium without kanamycin
  • the strains that did not grow on the kanamycin-containing medium were further identified by PCR using the following primers (synthesized by Shanghai Invitrogen):
  • the PCR amplified product 264bp was subjected to sscp electrophoresis after denaturation at 95°C for 10 minutes and ice bath for 5 minutes (using plasmid pk18-NCgl0609 R334* amplified fragment as a positive control, YP97158 amplified fragment as a negative control, and water as a blank control).
  • the fragment structure is different and the electrophoresis position is different. Therefore, the strain whose electrophoresis position of the fragment is inconsistent with the position of the negative control fragment and is consistent with the position of the positive control fragment is the strain with successful allelic replacement.
  • the positive strain NCgl0609 fragment was amplified by primer P5/P6 PCR again, and ligated to PMD19-T vector for sequencing. Through sequence alignment, the strain with mutation (CT) in the base sequence was the positive strain with successful allelic substitution and was named It is YPL-4-041.
  • NCBI Corynebacterium glutamicum ATCC13032 genome sequence published by NCBI
  • three pairs of primers were designed and synthesized to amplify the upstream and downstream homology arm fragments and NCgl0609 or NCgl0609 R334* gene coding region and promoter region sequence. Ways to introduce NCgl0609 or NCgl0609 R334* gene into strain YP97158.
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • Construction method use Corynebacterium glutamicum ATCC13032 or YPL-4-041 as templates, and use primers P7/P8, P9/P10, P11/P12 to carry out PCR amplification, and obtain the upstream homology arm fragment 768bp, NCgl0609 or NCgl0609 R334* gene and its promoter fragment 1626bp and downstream homology arm fragment 623bp.
  • the three amplified fragments are separately recovered by electrophoresis using a column DNA gel recovery kit.
  • the three recovered fragments were ligated with the purified pK18mobsacB plasmid (purchased from Addgene and digested with Xbal I/BamH I) with NEBuilder enzyme (purchased from NEB) at 50°C for 30 minutes, and the ligation product was transformed
  • the clones that grew later were identified by PCR to obtain a positive integrated plasmid.
  • the plasmid contains a kanamycin resistance marker, and the recombinant plasmid integrated into the genome can be obtained through kanamycin screening.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg2+(25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 60s (30 cycles), and over-extension at 72°C for 10 minutes.
  • the correctly sequenced integrated plasmid was electro-transformed into L-lysine production strain YP97158, and the single colony produced by the culture was identified by PCR with P13/P14 primers. PCR amplified a fragment with a size of 1317bp as a positive strain, and amplified Those with less than fragments are the original bacteria.
  • the positive strains were cultured on a medium containing 15% sucrose, and the single colonies produced by the culture were further identified by PCR using P15/P16 primers.
  • the bacteria with a size of 1352bp were amplified as NCgl0609 or NCgl0609 R334* gene integrated into the YP97158 genome
  • the positive strains were named YPL-4-042 (without mutation points) and YPL-4-043 (with mutation points).
  • NCgl0609 or NCgl0609 R334* gene were designed and synthesized.
  • the primers were designed as follows (synthesized by Shanghai invitrogen):
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg2+(25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq(5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 60s (30 cycles), and over-extension at 72°C for 10 minutes.
  • the correctly sequenced pXMJ19-NCgl0609 and pXMJ19-NCgl0609 R334* plasmids were electro-transformed into L-lysine production strain YP97158, and the single colonies produced by the culture were identified by PCR with primers M13F/P18, and PCR amplified a size of 1585bp.
  • the fragments are positive strains, which are named YPL-4-044 (without mutation points) and YPL-4-045 (with mutation points).
  • the recovered DNA fragment and the pK18mobsacB plasmid purified after double digestion were separately digested with Xbal I/BamH I )
  • Use NEBuilder enzyme purchased from NEB company to ligate at 50°C for 30 minutes, and the clones grown after the ligation product is transformed are identified by PCR with M13 primers to obtain the positive knockout vector pK18- ⁇ NCgl0609, and the plasmid is sent for sequencing.
  • the plasmid contains kanamycin resistance as a selection marker.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg2+(25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq(5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s (30 cycles), and over-extension at 72°C for 10 minutes.
  • sequence-correct knockout plasmid pK18- ⁇ NCgl0609 was electro-transformed into the lysine-producing strain YP97158, and the single colony produced by the culture was identified by PCR with the following primers (synthesized by Shanghai Handsome Company):
  • the above-mentioned PCR simultaneously amplified the 1334bp and 1788bp bands of the strains as positive strains, and only the 1788bp band amplified strains were the original bacteria.
  • the positive strains were screened on 15% sucrose medium and then cultured on kanamycin-containing and kanamycin-free medium. They were grown on kanamycin-free medium, and kanamycin-containing medium.
  • the strains that did not grow on the culture medium were further identified by PCR with P23/P24 primers, and the strain with a size of 1334bp amplified was the positive strain with the NCgl0609 gene coding region knocked out.
  • the fragment of the positive strain NCgl0609 was amplified by PCR with P23/P24 primers again, and ligated to the PMD19-T vector for sequencing.
  • the strain that was sequenced correctly was named YPL-4-046.
  • Example 2-5 The strain constructed in Example 2-5 and the original strain YP97158 were placed in a BLBIO-5GC-4-H fermentor (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium shown in Table 1 and shown in Table 2. The control process of the fermentation experiment was carried out. Each strain was repeated three times, and the results are shown in Table 5.
  • the glutamic acid production engineering strain YPG containing the point mutation NCgl0609 R334* was obtained.
  • the strains and original strains constructed in the examples were ATCC 13869 strains as the expression strains in the BLBIO-5GC-4-H fermentor (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium shown in Table 3 and The control process shown in Table 4 was used for fermentation experiments. Each strain was repeated three times, and the results are shown in Table 6.
  • NCgl1575 gene Based on the wild-type Corynebacterium glutamicum ATCC13032 genome sequence published by NCBI, two pairs of primers were designed and synthesized to amplify the coding region sequence of the NCgl1575 gene, and the strain YP97158 was replaced by alleles. A point mutation was introduced in the coding region of the NCgl1575 gene (SEQ ID NO: 29) in the background of the wild-type NCgl1575 gene. The amino acid sequence corresponding to the encoded protein is SEQ ID NO: 31. The nucleotide sequence of the NCgl1575 gene is the 1775th A variant.
  • T SEQ ID NO: 30: NCgl1575 A1775T
  • tyrosine at position 592 of the amino acid sequence corresponding to the encoded protein is changed to phenylalanine (SEQ ID NO: 32: NCgl1575 Y592F).
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was carried out as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 40s (30 cycles), and over-extension at 72°C for 10 minutes. Two sizes of 766bp and 766bp were obtained. 759bp, a DNA fragment containing the coding region of NCgl1575 gene (NCgl1575Up and NCgl1575Down).
  • the above two DNA fragments are separated and purified by agarose gel electrophoresis, the above two DNA fragments are used as templates and P1' and P4' are used as primers to amplify a fragment of about 1495 bp in length by overlapping PCR.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s (30 cycles), and over-extension at 72°C for 10 minutes.
  • NCgl1575 A1775T causes the 1775th adenine (A) in the coding region of the YP97158NCgl1575 gene to change to thymine (T), and finally causes the 592th amino acid of the encoded protein to change from tyrosine (Y) to phenylpropanine Acid (F).
  • NCgl1575 A1775T separated and purified by agarose gel electrophoresis and the pK18mobsacB plasmid (purchased from Addgene) digested with Xba I were assembled with the NEBuider recombination system to obtain the vector pK18-NCgl1575 A1775T , which contains kanamycin antibody. Sex mark.
  • the vector pK18-NCgl1575 A1775T was sent to a sequencing company for sequencing and identification, and the vector pK18-NCgl1575 A1775T containing the correct point mutation (AT) was stored for future use.
  • the allelic replacement plasmid pK18-NCgl1575 A1775T was transformed into L-lysine-producing strain YP97158 by electroporation, and the single colonies produced by the culture were identified by primer P1' and universal primer M13R, and the size of 1502bp could be amplified
  • the banded strain is a positive strain.
  • the positive strains were cultured on a medium containing 15% sucrose, and the single colonies produced by the culture were cultured on a medium containing kanamycin and a medium without kanamycin, and on a medium without kanamycin
  • the strains that did not grow on the kanamycin-containing medium were further identified by PCR using the following primers (synthesized by Shanghai Invitrogen):
  • the 256bp PCR amplified product was denatured at high temperature and subjected to sscp electrophoresis after ice bath (using the amplified fragment of plasmid pK18-NCgl1575 A1775T as the positive control, the amplified fragment of YP97158 as the negative control, and water as the blank control). Due to the different structure of the fragments, electrophoresis The positions are different, so the strains whose fragment electrophoresis position is inconsistent with the negative control fragment position and consistent with the positive control fragment position are the strains with successful allelic replacement.
  • NCgl1575 or NCgl1575 A1775T gene was introduced into strain YP97158.
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • Construction method Use Corynebacterium glutamicum ATCC13032 or YPL-4-023 as templates, and use primers P7'/P8', P9'/P10', P11'/P12' to carry out PCR amplification respectively to obtain the upstream homology arm Fragment 802bp, NCgl1575 gene and its promoter fragment 2737bp, or NCgl1575 A1775T gene and its promoter fragment 2737bp, downstream homology arm fragment 647bp, and then using P7'/P12' as primers, the three fragments amplified above (upstream The homology arm fragment, NCgl1575 gene and its promoter fragment, downstream homology arm fragment; or, the upstream homology arm fragment, NCgl1575 A1775T gene and its promoter fragment, the downstream homology arm fragment) are mixed as a template and amplified to obtain 4111bp of homology arm fragment is integrated.
  • the amplified product was recovered by electrophoresis, and the required 4111bp DNA fragment was recovered using the column DNA gel recovery kit (TIANGEN), and the NEBBuider recombination system and the shuttle plasmid recovered by Xba I digestion were used.
  • Connect PK18mobsacB to obtain the integrated plasmid PK18mobsacB-NCgl1575 or PK18mobsacB-NCgl1575 A1775T .
  • the plasmid contains a kanamycin resistance marker, and the recombinant plasmid integrated into the genome can be obtained by kanamycin screening.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 180s (30 cycles), and over-extension at 72°C for 10 minutes.
  • the two integrated plasmids were respectively transformed into the L-lysine production strain YP97158, and the single colony produced by the culture was identified by PCR with P13'/P14' primers.
  • the PCR amplified a fragment with a size of 1778bp as a positive strain.
  • the original bacteria cannot be amplified.
  • the positive strains were screened on 15% sucrose medium and then cultured on kanamycin-containing and kanamycin-free medium. They were grown on kanamycin-free medium, and kanamycin-containing medium.
  • strains that did not grow on the medium of the element were further identified by PCR with P15'/P16' primers, and the strains with a size of 1756 bp amplified were the strains with NCgl1575 or NCgl1575 A1775T gene integrated into the YP97158 genome, which were named YPL- 4-024 (without mutation points) and YPL-4-025 (with mutation points).
  • Construction method using ATCC13032 and YPL-4-023 as templates and primers P17'/P18' for PCR amplification to obtain NCgl1575 or NCgl1575 A1775T gene and its promoter fragment 2749bp.
  • the amplified products are recovered by electrophoresis.
  • the column DNA gel recovery kit was used to recover the required 2749bp DNA fragment, and the NEBuider recombination system was used to connect the shuttle plasmid pXMJ19 recovered by EcoRI digestion to obtain the overexpression plasmids pXMJ19-NCgl1575 and pXMJ19-NCgl1575 A1775T .
  • the plasmid contains a chloramphenicol resistance marker, and the plasmid can be transformed into the strain by chloramphenicol screening.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 120s (30 cycles), and over-extension at 72°C for 10 minutes.
  • the pXMJ19-NCgl1575 and pXMJ19-NCgl1575 A1775T plasmids were electrotransformed into the L-lysine production strain YP97158, and the single colonies produced by the culture were identified by PCR with M13(-48) and P18' primers, and the size was amplified by PCR.
  • the 2752bp fragment is the transferred strain, which is named YPL-4-026 (without mutation point) and YPL-4-027 (with mutation point).
  • PCR amplification was carried out with primers P19'/P20' and P21'/P22' to obtain an upstream homology arm fragment of 775 bp and a downstream homology arm fragment of 807 bp, and then primer P19'/ P22' performs overlapping PCR to obtain the entire homology arm fragment of 1545 bp.
  • the amplified product is recovered by electrophoresis, and the required 1545bp DNA fragment is recovered using the column DNA gel recovery kit, and the shuttle plasmid pk18mobsacB plasmid recovered by the NEBuider recombination system and Xba I digestion Connect to obtain a knockout plasmid.
  • This plasmid contains a kanamycin resistance marker.
  • the knock-out plasmid was electrotransformed into the lysine-producing strain YP97158, and the single colonies produced by the culture were identified by PCR with the following primers (synthesized by Shanghai Handsome Company):
  • the above-mentioned PCR-amplified strains with bands of 1471 bp and 4150 bp are positive strains, and strains with only 4150 bp bands are the original strains.
  • the positive strains were screened on 15% sucrose medium and then cultured on kanamycin-containing and kanamycin-free medium. They were grown on kanamycin-free medium, and kanamycin-containing medium. The strains that did not grow on the medium of the element were further identified by PCR using P23'/P24' primers.
  • the strain with a size of 1471bp amplified was a genetically engineered strain whose coding region of the NCgl1575 gene was knocked out, and it was named YPL- 4-028.
  • the strain constructed in Examples 9-12 and the original strain YP97158 were placed in a BLBIO-5GC-4-H fermentor (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium shown in Table 1 and shown in Table 2. The control process of the fermentation experiment was carried out. Each strain was repeated three times, and the results are shown in Table 7.
  • NCgl1575 gene in Corynebacterium glutamicum or the point mutation of NCgl1575 A1775T and overexpression in the coding region of NCgl1575 gene, contributes to the increase of L-lysine production, and the gene Attenuation or knockout is not conducive to the accumulation of L-lysine.
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L, the PCR amplification is carried out as follows: 94°C pre-denaturation for 5min, 94°C denaturation 30s, 52°C annealing for 30s, 72°C extension for 40s, 30 cycles, 72°C overextension for 10min, two DNA fragments (lysC promoter Up and lysC promoter Down fragments containing point mutations with lengths of 729bp and 760bp were obtained) ).
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L, the PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s, 30 cycles, and over-extension at 72°C for 10 minutes.
  • the above-mentioned Up-Down fragment is separated and purified by agarose gel electrophoresis.
  • the fragment contains the promoter region of lysC gene and its upstream and downstream sequences, and both ends of the fragment contain EcoR I and Sph I restriction sites respectively.
  • This DNA fragment causes the -45bp nucleotide guanine (G) in the promoter region of the YP97158lysC gene to change to adenine (A), and the -47bp nucleotide guanine (G) to thymine (T).
  • the fragment was double digested (EcoR I/Sph I), purified and recovered, and ligated with the shuttle plasmid pK18mobsacB (purchased from Addgene) after the same double digestion (EcoR I/Sph I) to obtain the allelic replacement plasmid pK18-PlysC (G(-45)A, G(-47)T) , this plasmid contains a kanamycin resistance marker. And the vector pK18-PlysC (G (-45) A, G (-47) T) sent sequenced Sequencing identified containing the correct point mutation vector pK18-PlysC (G (-45) A, G (-47) T) Save it for later use.
  • allelic replacement plasmid pK18-PlysC (G(-45)A, G(-47)T) was transformed into the L-lysine production strain YP97158 by electroporation, and the single colonies produced by the culture were respectively passed the primer P1” and the universal The primer M13F was used to identify the strains that can amplify a 1500bp band size as positive strains.
  • the positive strains were cultured on a medium containing 15% sucrose, and the single colonies produced by the culture were separately tested with kanamycin and without kanamycin.
  • the above PCR amplified products were denatured at high temperature and then subjected to sscp electrophoresis (using plasmid pK18-PlysC (G(-45)A, G(-47)T) amplified fragments as positive controls, and YP97158 amplified fragments as negative controls) , Water is used as a blank control), because the fragment structure is different, the electrophoresis position is different, so the electrophoresis position of the fragment is inconsistent with the position of the negative control fragment and the strain that is consistent with the position of the positive control fragment is the strain with successful allelic replacement.
  • the target fragment of the positive strain was amplified by PCR again, and ligated to PMD19-T vector for sequencing. Through sequence alignment, the sequence of the mutation in the base sequence verified that the allelic replacement of the strain was successful, and was named YPL-4-009.
  • Example 15 Put the strain YPL-4-009 constructed in Example 15 and the original strain YP97158 in a BLBIO-5GC-4-H fermentor (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium and table shown in Table 1. The control process shown in 2 is used for fermentation experiments. Each strain was repeated three times, and the results are shown in Table 8.

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Abstract

提供了一种生成L-氨基酸的细菌，该细菌具有编码SEQ ID NO:3所示的蛋白质的多核苷酸的改善的表达，编码SEQ ID NO:31所示的蛋白质的多核苷酸的改善的表达，和/或SEQ ID NO:57所示的启动子区域第-45bp位和第-47bp位碱基发生突变。还提供了一种多核苷酸，其编码的蛋白质，包含该多核苷酸序列的重组载体，重组菌株，以及生产L-氨基酸的方法，其中该多核苷酸编码含有SEQ ID NO:3所示的蛋白质且其第334位精氨酸被终止子所取代，或编码含有SEQ ID NO:31所示的蛋白质且其第592位酪氨酸被苯丙氨酸所取代，或由SEQ ID NO:57所示的启动子区域第-45bp位和第-47bp位碱基发生突变形成。

Description

产L-氨基酸的重组菌株及其构建方法与应用
本申请要求2020年10月15日向中国国家知识产权局提交的专利申请号为202011105063.5，发明名称为“一种产L-氨基酸的重组菌株及其构建方法与应用”，2020年8月7日向中国国家知识产权局提交的专利申请号为202010790887.4，发明名称为“一种产L-赖氨酸的重组菌株及其构建方法与应用”和2020年6月8日向中国国家知识产权局提交的专利申请号为202010514037.1，发明名称为“一种lysC基因改造的重组菌株及其构建方法与应用”的在先申请的优先权。所述三份在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于基因工程和微生物技术领域，具体涉及产L-氨基酸的重组菌株及其构建方法与应用。
背景技术
L-赖氨酸(L-Lysine)应用范围广泛，包括医药、食品、饲料等各方面，其中被应用于饲料添加剂的L-赖氨酸占总量的90％以上。当前，我国是L-赖氨酸第二大消费市场，第一大L-赖氨酸生产国。
目前L-赖氨酸主要采用直接发酵法生产，直接发酵法利用具备完整L-赖氨酸生物合成途径的菌株，以废糖蜜、淀粉水解液等为底物，通过好氧发酵生产。该方法占当今世界L-赖氨酸生产的2/3，工艺已十分成熟，该方法主要存在于酵母、细菌、霉菌之中，在微生物中广泛存在。目前，工业上用于L-赖氨酸发酵的生产菌种主要是棒状杆菌和短杆菌属的诱变育种突变株。随着代谢工程和基因工程的发展，使得基因突变可控，因此在利用代谢工程改造出发菌株的过程中，能够精确找到代谢过程中L-赖氨酸生产的关键酶基因，然后提高关键酶基因的表达，L-赖氨酸产量的提高才有可能实现。
L-谷氨酸主要用于生产味精、香料，以及用作代盐剂、营养增补剂和生化试剂等。L-谷氨酸本身可用作药物，参与脑内蛋白质和糖的代谢，促进氧化过程，该品在体内与氨结合成无毒的谷酰胺，使血氨下降，减轻肝昏迷症状。过去生产味精主要用小麦面筋(谷蛋白)水 解法进行，现改用微生物发酵法来进行大规模生产。
发明内容
本发明的目的是开发具有L-氨基酸生产能力的新菌株，从而提供一种有效生产L-氨基酸的方法。
为了实现上述目的，本发明的发明人通过研究发现，现有技术未知具有发酵氨基酸生产能力的NCgl0609基因、和/或NCgl1575基因，通过修饰所述基因或改善其表达，能够具备高效的L-氨基酸生产能力；此外，本发明的发明人还发现，对于某一个启动子序列进行突变，也能够提高相应微生物的L-氨基酸生产能力。基于这些发现，完成了本发明。
本发明提供了生成L-氨基酸的细菌，其中编码SEQ ID NO:3的氨基酸序列的多核苷酸的表达被改善，和/或，编码SEQ ID NO:31的氨基酸序列的多核苷酸的表达被改善，和/或，SEQ ID NO:57所示的启动子区域第-45bp位和第-47bp位碱基发生突变。本发明还提供了通过使用所述微生物生产L-氨基酸的方法。
根据本发明，所述表达改善是所述多核苷酸表达增强，或者编码SEQ ID NO:3或SEQ ID NO:31的氨基酸序列的多核苷酸具有点突变，或者编码SEQ ID NO:3或SEQ ID NO:31的氨基酸序列的多核苷酸具有点突变且表达是增强的。
本发明的第一个方面提供了生成L-氨基酸的细菌，其中编码SEQ ID NO:3的氨基酸序列的多核苷酸的表达改善。优选，所述L-氨基酸是L-赖氨酸或L-谷氨酸。
所述SEQ ID NO:3的氨基酸序列是基因NCgl0609编码的蛋白。
所述细菌具有增强的L-氨基酸生产能力。
具有L-氨基酸生产能力的细菌可以是能够在培养基中以优选0.5g/L以上，更优选1.0g/L以上的量积累目标L-氨基酸的细菌。
所述多核苷酸可以编码与SEQ ID NO:3的氨基酸序列具有约90％或更高、约92％或更高、约95％或更高、约97％或更高、约98％或更高、或约99％或更高的序列同源性的氨基酸序列。
在本发明的一个具体实施方式中，所述被改善表达的多核苷酸包含SEQ ID NO:1的核苷酸序列。
在本发明的一种实施方式中，所述表达改善是指编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变，使得SEQ ID NO:3的氨基酸序列的第334位精氨酸替换为终止子。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第334位精氨酸被终止子取代后的氨基酸序列如SEQ ID NO:4所示。
在本发明的一个实施方式中，所述具有点突变的多核苷酸序列是由SEQ ID NO:1所示的多核苷酸序列第1000位碱基发生突变而形成的。
根据本发明，所述突变包括SEQ ID NO:1所示多核苷酸序列第1000位碱基由胞嘧啶(C)突变为胸腺嘧啶(T)。
在本发明的一个实施方式中，所述具有点突变的多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
本发明还提供了生成L-氨基酸的细菌，其具有编码SEQ ID NO:31的氨基酸序列的多核苷酸的改善的表达。优选所述L-氨基酸是L-赖氨酸。优选所述细菌是属于棒杆菌属的细菌。
所述SEQ ID NO:31的氨基酸序列是基因NCgl1575编码的蛋白。
所述微生物与野生型或亲本菌株相比具有增强的L-赖氨酸生产能力。
所述多核苷酸可以编码与SEQ ID NO:31的氨基酸序列具有约90％或更高、约92％或更高、约95％或更高、约97％或更高、约98％或更高、或约99％或更高的序列同源性的氨基酸序列。
在本发明的一个具体实施方式中，所述多核苷酸可以包含SEQ ID NO:29的核苷酸序列。
在本发明的一种实施方式中，编码SEQ ID NO:31的氨基酸序列的多核苷酸的具有点突变，使得SEQ ID NO:31的氨基酸序列的第592位酪氨酸被不同的氨基酸所取代。
根据本发明，优选第592位酪氨酸被苯丙氨酸所取代。
根据本发明，SEQ ID NO:31所示的氨基酸序列，其中第592位酪氨酸(Y)被苯丙氨酸(F)所取代后的氨基酸序列如SEQ ID NO:32所示。
在本发明的一个实施方式中，所述具有点突变的多核苷酸序列是由SEQ ID NO:29所示多核苷酸序列第1775位碱基发生突变而形成的。
根据本发明，所述突变包括SEQ ID NO:29所示多核苷酸序列第1775位碱基由腺嘌呤(A)突变为胸腺嘧啶(T)。
在本发明的一个实施方式中，所述具有点突变的多核苷酸序列包括SEQ ID NO:30所示的多核苷酸序列。
根据本发明，所述细菌可以是属于棒杆菌属的微生物，例如谷氨酸棒杆菌(Corynebacterium glutamicum)、黄色短杆菌(Brevibacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)、产氨棒杆菌(Corynebacterium ammoniagenes)、北京棒杆菌(Corynebacterium pekinense)。
在本发明的一个实施方案中，所述属于棒杆菌属的微生物是谷氨酸棒杆菌YP97158，保 藏编号：CGMCC No.12856，保藏日期：2016年8月16日，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，北京市朝阳区北辰西路1号院3号，电话：010-64807355，记载于中国专利申请CN106367432A(申请日2016年9月1日，公开日2017年2月1日)中。
在本发明的一个实施方案中，所述属于棒杆菌属的微生物是谷氨酸棒杆菌ATCC 13869。
可以通过如下手段增强多核苷酸的表达：通过取代或突变表达调节序列、对多核苷酸序列引入突变、通过经由染色体插入或载体导入的多核苷酸拷贝数的增加、或其组合等。
可以修饰多核苷酸的表达调节序列。表达调节序列控制与其可操作连接的多核苷酸的表达，并且例如可以包括启动子、终止子、增强子、沉默子等。多核苷酸可以具有起始密码子的变化。可以将多核苷酸掺入染色体的特定位点中，从而增加拷贝数。在本文，特定的位点可以包括例如转座子位点或基因间位点。另外，可以将多核苷酸掺入表达载体中，将所述表达载体导入宿主细胞中，从而增加拷贝数。
在本发明的一种实施方式中，通过将多核苷酸或者具有点突变的多核苷酸掺入微生物染色体的特定位点中，从而增加拷贝数。
在本发明的一种实施方式中，通过将带有启动子序列的多核苷酸或者带有启动子序列的具有点突变的多核苷酸掺入微生物染色体的特定位点中，从而过表达所述核酸序列。
在本发明的一种实施方式中，将多核苷酸或者具有点突变的多核苷酸掺入表达载体中，将所述表达载体导入宿主细胞中，从而增加拷贝数。
在本发明的一种实施方式中，将带有启动子序列的多核苷酸或者带有启动子序列的具有点突变的多核苷酸掺入表达载体中，将所述表达载体导入宿主细胞中，从而过表达所述氨基酸序列。
在本发明的一个具体实施方式中，所述启动子是编码SEQ ID NO:3的氨基酸序列的多核苷酸(NCgl0609基因)的启动子。
在本发明的一个具体实施方式中，所述启动子是编码SEQ ID NO:31的氨基酸序列的多核苷酸(NCgl1575基因)的启动子。
在本发明的一些具体实施方式中，使用的载体是pK18mobsacB质粒，pXMJ19质粒。
根据本发明，所述细菌还可以具有与提高L-氨基酸产量有关的其他改进。
本发明的第二个方面，提供一种多核苷酸序列，由该多核苷酸序列编码的氨基酸序列，包括所述多核苷酸序列的重组载体，含有所述多核苷酸序列的重组菌株。
根据本发明，所述多核苷酸序列具有改善的表达，所述改善包括编码含有SEQ ID NO:3所示的氨基酸序列的多肽的多核苷酸发生点突变，使得所述氨基酸序列第334位精氨酸被终止 子所取代。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第334位精氨酸被终止子所取代后的氨基酸序列如SEQ ID NO:4所示。
根据本发明，所述编码含有SEQ ID NO:3所示的氨基酸序列的多肽的多核苷酸序列含有如SEQ ID NO:1所示的多核苷酸序列。
在本发明的一个实施方式中，本发明提供的突变后的多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第1000位碱基发生突变而形成的。
根据本发明，所述突变包括SEQ ID NO:1所示的多核苷酸序列第1000位碱基由胞嘧啶(C)突变为胸腺嘧啶(T)。
在本发明的一个实施方式中，所述突变后的多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
根据本发明，所述取代后的氨基酸序列包括如SEQ ID NO:4所示的氨基酸序列。
根据本发明，所述多核苷酸序列包括编码含有SEQ ID NO:31所示的氨基酸序列的多肽的多核苷酸，其中第592位酪氨酸被不同的氨基酸所取代。
根据本发明，优选第592位酪氨酸被苯丙氨酸所取代。
根据本发明，SEQ ID NO:31所示的氨基酸序列，其中第592位酪氨酸(Y)被苯丙氨酸(F)所取代后的氨基酸序列如SEQ ID NO:32所示。
根据本发明，优选所述编码含有SEQ ID NO:31所示的氨基酸序列的多肽的多核苷酸序列含有如SEQ ID NO:29所示的多核苷酸序列。
在本发明的一个实施方式中，所述多核苷酸序列是由SEQ ID NO:29所示多核苷酸序列第1775位碱基发生突变而形成的。
根据本发明，所述突变包括SEQ ID NO:29所示多核苷酸序列第1775位碱基由腺嘌呤(A)突变为胸腺嘧啶(T)。
在本发明的一个实施方式中，所述多核苷酸序列包括SEQ ID NO:30所示的多核苷酸序列。
根据本发明，所述氨基酸序列包括如SEQ ID NO:32所示的氨基酸序列。
根据本发明，所述突变是指所述位点的碱基/核苷酸发生变化，所述突变方法可以选自诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。在本发明中，优选采用PCR定点突变法和/或同源重组。
根据本发明，所述重组载体是将所述多核苷酸序列导入质粒构建而成。
在本发明的一个实施方式中，所述质粒为pK18mobsacB质粒。
在本发明的另一个实施方式中，所述质粒为pXMJ19质粒。
具体地，可以将所述多核苷酸序列和所述质粒通过NEBuider重组系统构建成重组载体。
根据本发明，所述重组菌株含有所述的多核苷酸序列。
作为本发明的一个实施方案，所述重组菌株的出发菌为YP97158。
作为本发明的一个实施方案，所述重组菌株的出发菌为ATCC 13869。
本发明的第三个方面，还提供一种生成L-氨基酸的重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
改造宿主菌株中如SEQ ID NO:1所示的野生型NCgl0609的多核苷酸序列，使其第1000位碱基发生突变，得到包含突变NCgl0609编码基因的重组菌株。
根据本发明的构建方法，所述改造包括诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明的构建方法，所述突变是指SEQ ID NO:1中第1000位碱基由胞嘧啶(C)突变为胸腺嘧啶(T)；具体地，所述包含突变NCgl0609编码基因的多核苷酸序列如SEQ ID NO:2所示。
进一步地，所述构建方法包括如下步骤：
(1)改造如SEQ ID NO:1所示的野生型NCgl0609基因的核苷酸序列，使其第1000位碱基发生突变，得到突变的NCgl0609基因多核苷酸序列；
(2)将所述突变的多核酸序列与质粒连接，构建重组载体；
(3)将所述重组载体导入宿主菌株，得到所述包含突变NCgl0609编码基因的重组菌株。
根据本发明的构建方法，所述步骤(1)包括：点突变的NCgl0609基因构建：根据未修饰菌株的基因组序列，合成两对扩增NCgl0609基因片段的引物P1和P2，P3和P4，通过PCR定点突变法在野生型NCgl0609基因SEQ ID NO:1中引入点突变，得到点突变的NCgl0609基因核苷酸序列SEQ ID NO:2，记为NCgl0609 C1000T。
在本发明的一个实施方式中，所述未修饰菌株基因组可以来源于ATCC13032菌株，其基因组序列可以从NCBI网站获取。
在本发明的一个实施方案中，所述步骤(1)中，所述引物为：
P1:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGGACGGCAAC
GTACATAAC3'(SEQ ID NO:5)
P2:5'GTTGCCGGTGAGTCAAACAGTCATTTTGC 3'(SEQ ID NO:6)
P3:5'GCAAAATGACTGTTTGACTCACCGGCAAC 3'(SEQ ID NO:7)
P4:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCGGCTG GAAATGTGGAG3'(SE Q ID NO:8)
在本发明的一个实施方案中，所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s，52℃退火30s，72℃延伸40s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方案中，所述重叠PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s，52℃退火30s，以及72℃延伸60s(30个循环)，72℃过度延伸10min。
根据本发明的构建方法，所述步骤(2)包括重组质粒的构建，包括：将分离纯化后的NCgl0609 C1000T和pK18mobsacB质粒，通过NEBuider重组系统组装，获得重组质粒。
根据本发明的构建方法，所述步骤(3)包括重组菌株的构建，将重组质粒转化至宿主菌株，得到重组菌株。
在本发明的一个实施方案中，所述步骤(3)的转化为电转化法。
在本发明的一个实施方式中，所述宿主菌株是YP97158。
在本发明的一个实施方式中，所述重组是通过同源重组实现的。
本发明的第四个方面，还提供一种生成L-氨基酸的重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
扩增NCgl0609基因的上下游同源臂片段、NCgl0609基因编码区及其启动子区序列，或者，扩增NCgl0609或NCgl0609 R334*基因编码区及其启动子区序列，再以同源重组的方式在宿主菌株的基因组中引入NCgl0609或NCgl0609 R334*基因，以获得所述菌株过表达NCgl0609或NCgl0609 R334*基因。
在本发明的一个实施方式中，扩增上游同源臂片段的引物是：
P7:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGTTCTG GACTGAGG 3'(SEQ ID NO:11)
P8:5'GAGATGATCCTCGCAGCTGGTGCACCGAGAACAGATG 3'(SEQ ID NO:12)
在本发明的一个实施方式中，扩增下游同源臂片段的引物是：
P11:5'GGTCAAGGAAGGAGTTGTTGCCAGAATCAGATGGCGCAATTA AATC AAG 3'(SEQ ID NO:15)
P12:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCTATGAC
ACCTTCAACGGATC 3'(SEQ ID NO:16)
在本发明的一个实施方式中，扩增所述基因编码区及其启动子区序列的引物是：
P9:5'CATCTGTTCTCGGTGCACCAGCTGCGAGGATCATCTC 3'(SEQ ID NO:13)
P10:5'GATTTAATTGCGCCATCTGATTCTGGCAACAACTCCTTCCTTGACC 3'(SEQ ID  NO:14)。
在本发明的一个实施方式中，以前述P7-P12为引物，以扩增获得的上游同源片段、下游同源片段和带有自身启动子的NCgl0609或NCgl0609 R334*三个片段混合为模板进行扩增，获得整合同源臂片段。
在本发明的一个实施方式中，所采用的PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL；PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸60s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方式中，采用NEBuider重组系统，将穿梭质粒PK18mobsacB和上下同源臂片段、基因编码区及启动子区片段组装，获得整合质粒。
在本发明的一个实施方式中，将整合质粒转染宿主菌株，以同源重组的方式在宿主菌株的基因组中引入NCgl0609或NCgl0609 R334*基因。
在本发明的一个实施方式中，所述宿主菌株是YP97158。
在本发明的一个实施方式中，所述宿主菌株是携带有SEQ ID NO:2所示多核苷酸序列的菌株。
本发明的第五个方面，还提供一种生产L-氨基酸的重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
扩增NCgl0609基因编码区及启动子区序列，或NCgl0609 R334*基因编码区及启动子区序列，构建过表达质粒载体，将所述载体转入宿主菌株中，以实现所述菌株过表达NCgl0609或NCgl0609 R334*基因。
在本发明的一个实施方式中，扩增所述基因编码区及其启动子区序列的引物是：
P17:5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCAGCTGCGAGG A TCATCTC 3'(SEQ ID NO:21)
P18:5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCAACAACTCCTTCCTTGACC3'(SEQ ID NO:22)
在本发明的一个实施方式中，所述PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL；所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸60s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方式中，采用NEBuider重组系统，将穿梭质粒pXMJ19和带有自身启动子的NCgl0609和NCgl0609 R334*片段组装，获得过表达质粒。
在本发明的一个实施方式中，所述宿主菌株是YP97158。
在本发明的一个实施方式中，所述宿主菌株是ATCC 13869。
在本发明的一个实施方式中，所述宿主菌株是携带有SEQ ID NO:2所示多核苷酸序列的菌株。
本发明还提供了一种棒杆菌重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
改造宿主菌株中如SEQ ID NO:29所示的野生型NCgl1575的多核苷酸序列，使其第1775位碱基发生突变，得到包含突变NCgl1575编码基因的棒杆菌重组菌株。
根据本发明的构建方法，所述改造包括诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明的构建方法，所述突变是指SEQ ID NO:29中第1775位碱基由腺嘌呤(A)突变为胸腺嘧啶(T)；具体地，所述包含突变NCgl1575编码基因的多核苷酸序列如SEQ ID NO:30所示。
进一步地，所述构建方法包括如下步骤：
(1)改造如SEQ ID NO:29所示的野生型NCgl1575基因的核苷酸序列，使其第1775位碱基发生突变，得到突变的NCgl1575基因多核苷酸序列；
(2)将所述突变的多核酸序列与质粒连接，构建重组载体；
(3)将所述重组载体导入宿主菌株，得到所述包含突变NCgl1575编码基因的棒杆菌重组菌株。
根据本发明的构建方法，所述步骤(1)包括：点突变的NCgl1575基因构建：根据谷氨酸棒杆菌的基因组序列，合成两对扩增NCgl1575基因片段的引物P1’和P2’及P3’和P4’，通过PCR定点突变法在野生型NCgl1575基因SEQ ID NO:29中引入点突变，得到点突变的NCgl1575基因核苷酸序列SEQ ID NO:30，记为NCgl1575 A1775T。
在本发明的一个实施方式中，所述谷氨酸棒杆菌基因组可以来源于ATCC13032菌株，其基因组序列可以从NCBI网站获取。
在本发明的一个实施方案中，所述步骤(1)中，所述引物为：
P1’:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTGCGTTCGTCTGCGGTTTCG 3'(SEQ ID NO:33)
P2’:5'ATCGACGCCGCCCCATTCACCCTTCTGATG 3'(SEQ ID NO:34)；
P3’:5'CATCAGAAGGGTGAATGGGGCGGCGTCGAT 3'(SEQ ID NO:35)；
P4’:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAAGCCTCGACCCCTACATC 3'(SEQ ID NO:36)
在本发明的一个实施方案中，所述PCR扩增按如下方式进行：94℃变性30s，52℃退火30s，以及72℃延伸40s(30个循环)。
在本发明的一个实施方案中，所述重叠PCR扩增按如下方式进行：94℃变性30s，52℃退火30s，以及72℃延伸90s(30个循环)。
根据本发明的构建方法，所述步骤(2)包括重组质粒的构建，包括：将分离纯化后的NCgl1575 A1775T和pK18mobsacB质粒，通过NEBuider重组系统组装，获得重组质粒pK18-NCgl1575 A1775T。
根据本发明的构建方法，所述步骤(3)包括重组菌株的构建，将重组质粒pK18-NCgl1575 A1775T转化至宿主菌株，得到重组菌株。
在本发明的一个实施方案中，所述步骤(3)的转化为电转化法。
在本发明的一个实施方式中，所述宿主菌株是YP97158。
在本发明的一个实施方式中，所述重组是通过同源重组实现的。
本发明还提供一种棒杆菌重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
扩增NCgl1575基因的上下游同源臂片段、NCgl1575基因编码区及其启动子区序列，或者，NCgl1575 A1775T基因编码区及其启动子区序列，以同源重组的方式在宿主菌株的基因组中引入NCgl1575或NCgl1575 A1775T基因，以实现所述菌株过表达NCgl1575或NCgl1575 A1775T基因。
在本发明的一个实施方式中，扩增上游同源臂片段的引物是：
P7’：5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATG
CGTTCTGGACTGAGG 3'(SEQ ID NO:39)
P8’：5'GAAACGGCCTTAAGCTAGGTGCACCGAG AACAGATG 3'(SEQ ID NO:40)。
在本发明的一个实施方式中，扩增下游同源臂片段的引物是：
P11’：5'AACCGGGCGG GAAAAGCTTGATGGCGCAATTAAATCAAG 3'(SEQ ID NO:43)
P12’：5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCTAT
GACACCTTCAACGGATC 3'(SEQ ID NO:44)。
在本发明的一个实施方式中，扩增所述基因编码区及其启动子区序列的引物是：
P9’：5'CATCTGTTCTCGGTGCAC CTAGCTTAAG GCCGTTTC 3'(SEQ ID NO:41)
P10’：5'CTTGATTTAATTGCGCCATCAAGCTTTTCC CGCCCGGTT 3'(SEQ ID NO:42)。
在本发明的一个实施方式中，以前述P7’/P12’为引物，以扩增获得的上游同源片段、下游同源片段和带有自身启动子的NCgl1575或NCgl1575 A1775T三个片段混合为模板进行扩增，获得整合同源臂片段。
在本发明的一个实施方式中，所采用的PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL；PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸180s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方式中，采用NEBuider重组系统，将穿梭质粒PK18mobsacB和整合同源臂片段组装，获得整合质粒。
在本发明的一个实施方式中，将整合质粒转染宿主菌株，以同源重组的方式在宿主菌株的基因组中引入NCgl1575或NCgl1575 A1775T基因。
在本发明的一个实施方式中，所述宿主菌株是YP97158。
在本发明的一个实施方式中，所述宿主菌株是携带有SEQ ID NO:30所示多核苷酸序列的菌株。
本发明还提供一种棒杆菌重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
扩增NCgl1575基因编码区及启动子区序列，或NCgl1575 A1775T基因编码区及启动子区序列，构建过表达质粒载体，将所述载体转入宿主菌株中，以实现所述菌株过表达NCgl1575或NCgl1575 A1775T基因。
在本发明的一个实施方式中，扩增所述基因编码区及其启动子区序列的引物是：
P17’：5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC CTAGCT
TAAG GCCGTTTC 3'(SEQ ID NO:49)
P18’：5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAAC AAGCTTT
TCC CGCCCGGTT 3'(SEQ ID NO:50)。
在本发明的一个实施方式中，所述PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL；所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸120s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方式中，采用NEBuider重组系统，将穿梭质粒pXMJ19和带有自身启动子的NCgl1575或NCgl1575 A1775T片段组装，获得过表达质粒。
在本发明的一个实施方式中，所述宿主菌株是YP97158。
在本发明的一个实施方式中，所述宿主菌株是携带有SEQ ID NO:30所示多核苷酸序列的菌株。
本发明再一个方面是提供一种启动子核苷酸序列，其包括SEQ ID NO:57所示的启动子区域第-45bp位和第-47bp位碱基发生突变形成的核苷酸序列。
根据本发明，SEQ ID NO:57所示的启动子区域的第-45bp位核苷酸鸟嘌呤(G)突变为腺嘌呤(A)、第-47bp位的核苷酸鸟嘌呤(G)突变为胸腺嘧啶(T)。
根据本发明，所述启动子核苷酸序列如下所述：
(a)SEQ ID NO:58所示的核苷酸序列；或者是，
(b)与SEQ ID NO:58所示的核苷酸序列具有90％以上，优选的具有95％、98％以上同一性的核苷酸序列，且其保留(a)所述启动子的增强活性，并且第-45bp位保持为腺嘌呤(A)、第-47bp位保持为胸腺嘧啶(T)。
本发明还提供一种包含上述启动子的表达盒，包含所述启动子和可操纵地连接在所述启动子后的编码序列。在本发明的一个实施方案中，所述编码序列为lysC基因的编码序列。
本发明还提供一种包含本发明的启动子核苷酸序列的重组载体。
根据本发明，将本发明的启动子核苷酸序列与穿梭质粒相连接来构建所述重组载体；作为本发明的一个实施方案，所述穿梭质粒为pK18mobsacB质粒。
本发明还提供一种包含上述启动子核苷酸序列或上述重组载体的重组菌株。
根据本发明的重组菌株，其包含SEQ ID NO:58所示的核苷酸序列。所述SEQ ID NO:58所示的核苷酸序列为lysC基因的启动子区域。进一步地，所述SEQ ID NO:58所示的核苷酸序列连接lysC基因编码序列。具体地，所述重组菌株可以包括本发明上述的表达盒或者重组载体。具体地，本发明的重组菌株由表达盒或者重组载体转化得到。根据本发明的重组菌株，是将上述突变的启动子核苷酸序列导入宿主菌株中重组形成；所述宿主菌株可以选自本领域已知的产L-氨基酸尤其是L-赖氨酸的菌株，例如选自棒杆菌中的至少一种，所述棒杆菌可以为谷氨酸棒杆菌、黄色棒杆菌、钝齿棒杆菌、北京棒杆菌；优选谷氨酸棒杆菌。作为本发明的一个实施方案，所述宿主菌株为YP97158。
根据本发明的重组菌株，是以pK18mobsacB质粒为载体。
根据本发明的重组菌株，还可进一步包括其他改造。
本发明还提供一种产L-赖氨酸的重组菌株的构建方法，包括如下步骤：
(1)改造如SEQ ID NO:57所示的启动子区域，使其第-45bp位和第-47bp位发生突变，得到包含突变的启动子区域的核苷酸序列。
根据本发明，所述突变是指SEQ ID NO:57所示的启动子区域的第-45bp位核苷酸鸟嘌呤(G)突变为腺嘌呤(A)、第-47bp位的核苷酸鸟嘌呤(G)突变为胸腺嘧啶(T)。具体地，所述突变后的启动子区域地核苷酸序列如SEQ ID NO:58所示。进一步地，所述构建方法还包括如下步骤：
(2)将所述突变的启动子区域核苷酸序列与质粒连接，构建重组载体；
(3)将所述重组载体导入宿主菌株，得到所述包含突变的启动子区域的产L-赖氨酸重组菌株。
根据本发明，所述步骤(1)中，使所述发生突变的方法包括诱变、PCR定点突变或同源重组，优选PCR定点突变法。
根据本发明，所述步骤(1)包括：
设计两对扩增lysC基因的启动子区域的引物，再通过PCR技术得到突变的启动子区域核苷酸序列。
在本发明的一个实施方案中，所述步骤(1)中的引物为：
P1”:5' CCGGAATTCG ACCAAGGATG AGGGCTTTG 3'(EcoR I)(SEQ ID NO:59)
P2”:5'AGTTACCCGC TCAATTATAC CTTTATAAAC 3'(SEQ ID NO:60)
P3”:5'GTTTATAAAG GTATAATTGA GCGGGTAACT 3'(SEQ ID NO:61)
P4”:5' ACATGCATGC GCGTACGCGA AGTGGCACAT 3'(Sph I)(SEQ ID NO:62)。
在本发明的一个实施方案中，所述步骤(1)包括：以谷氨酸棒杆菌ATCC13032为模板，分别以引物P1”和P2”，P3”和P4”，进行PCR扩增，获得两条含有DNA片段；以上述两条DNA片段为模板，以P1”和P4”为引物，通过重叠PCR扩增(Overlap PCR)，得到包含本发明的启动子区域核苷酸序列(SEQ ID NO:58)的DNA片段。
根据本发明，步骤(1)中，通过重叠PCR扩增(Overlap PCR)，获得的DNA片段两端分别含有EcoR I和Sph I酶切位点。
根据本发明，所述步骤(2)包括：对于重叠PCR反应扩增的产物进行琼脂糖凝胶电泳和分离纯化，将片段双酶切(EcoR I/Sph I)后，与同样双酶切(EcoR I/Sph I)后的穿梭质粒相连接，获得等位替换重组载体。
根据本发明，所述穿梭质粒为pK18mobsacB质粒；所构建的重组载体为pK18-Plys C (G(-45)A,G(-47)T)。
在本发明的一个实施方案中，所述重组质粒具有卡那霉素抗性标记。
在本发明的一个实施方案中，所述步骤(3)的转化为电转化法；示例性地，所述步骤(3)中，是将重组载体转化至菌株YP97158。
本发明获得上述各种重组菌株可以单独应用于发酵生产L-氨基酸中，也可以混合使用，也可以和其他产L-氨基酸的细菌混合发酵生产L-氨基酸。
本发明的另一个方面提供了生产L-氨基酸的方法，该方法包括培养所述细菌；并且从培养物中获得L-氨基酸。
可以在本领域中已知的培养条件下在合适的培养基中进行细菌的培养。培养基可以包含：碳源、氮源、微量元素、及其组合。在培养中，可以调节培养物的pH。此外，培养时可以包括防止气泡产生，例如通过使用消泡剂进行气泡产生的防止。此外，培养时可以包括将气体注射入培养物中。气体可以包括能够维持培养物的需氧条件的任何气体。在培养中，培养物的温度可以是20至45℃。可以从培养物回收生成的L-氨基酸，即用硫酸或氢氯酸等处理培养物，接着进行诸如阴离子交换层析、浓缩、结晶和等电点沉淀的方法的组合。
在本发明中：
SEQ ID NO:1：NCgl0609野生型ORF序列
Figure PCTCN2020142133-appb-000001
Figure PCTCN2020142133-appb-000002
SEQ ID NO:2：NCgl0609 R334*ORF序列
Figure PCTCN2020142133-appb-000003
SEQ ID NO:3：NCgl0609野生型编码蛋白氨基酸序列
Figure PCTCN2020142133-appb-000004
SEQ ID NO:4：NCgl0609R334*编码蛋白氨基酸序列
Figure PCTCN2020142133-appb-000005
Figure PCTCN2020142133-appb-000006
SEQ ID NO:29：NCgl1575野生型ORF序列
Figure PCTCN2020142133-appb-000007
Figure PCTCN2020142133-appb-000008
SEQ ID NO:30：NCgl1575 A1775T ORF序列
Figure PCTCN2020142133-appb-000009
Figure PCTCN2020142133-appb-000010
SEQ ID NO:31：NCgl1575野生型编码蛋白序列
Figure PCTCN2020142133-appb-000011
Figure PCTCN2020142133-appb-000012
SEQ ID NO:32：NCgl1575 Y592F编码蛋白序列
Figure PCTCN2020142133-appb-000013
SEQ ID NO:57：野生型启动子序列
Figure PCTCN2020142133-appb-000014
SEQ ID NO:58：突变后的启动子序列
Figure PCTCN2020142133-appb-000015
Figure PCTCN2020142133-appb-000016
术语定义：
在本发明中，术语“具有L-氨基酸生产能力的细菌”是指具有在培养基和/或细菌的细胞中以下述程度产生并累积目的L-氨基酸的能力，使得当细菌在培养基中培养时可以收集生产L-氨基酸的细菌。具有L-氨基酸生产能力的细菌可以是能够以比未修饰菌株可获得的量更大的量在培养基和/或细菌的细胞中积累目的L-氨基酸的细菌。
L-氨基酸的实例包括碱性氨基酸，如L-赖氨酸、L-鸟氨酸、L-精氨酸、L-组氨酸和L-瓜氨酸；脂肪族氨基酸，如L-异亮氨酸、L-丙氨酸、L-缬氨酸、L-亮氨酸和甘氨酸；作为羟基-单氨基羧酸的氨基酸，如L-苏氨酸和L-丝氨酸；环状氨基酸，如L-脯氨酸；芳香族氨基酸，如L-苯丙氨酸、L-酪氨酸和L-色氨酸；含硫氨基酸，如L-半胱氨酸、L-胱氨酸和L-甲硫氨酸；酸性氨基酸，如L-谷氨酸和L-天冬氨酸；和侧链具有酰胺基的氨基酸，如L-谷氨酰胺和L-天冬酰胺。L-氨基酸的具体实例包括L-谷氨酸、L-赖氨酸、L-苏氨酸、L-精氨酸、L-组氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸、L-苯丙氨酸、L-酪氨酸、L-色氨酸和L-半胱氨酸。L-氨基酸的更具体的实例包括L-谷氨酸、L-赖氨酸、L-苏氨酸和L-色氨酸。L-氨基酸的更具体实例包括L-谷氨酸、L-赖氨酸。
在本发明中，除非另有说明，术语“氨基酸”是指L-氨基酸。在本发明中，除非另有说明，术语“L-氨基酸”是指游离形式的L-氨基酸、其盐或其混合物。
术语“未修饰菌株”是指尚未以使得具有特定特征的方式进行修饰的对照菌株。即，未修饰菌株的实例包括野生型菌株和亲本菌株。
术语“同源性”指两种多核苷酸或两种多肽模块之间的百分比同一性。可以通过使用本领域中已知的方法测定一种模块和另一种模块之间的序列同源性。例如，可以通过BLAST算法测定此类序列同源性。
术语“可操作连接”指调节序列和多核苷酸序列之间的功能性连接，由此调节序列控制多核苷酸序列的转录和/或翻译。调节序列可以是能提高多核苷酸的表达水平的强启动子。调节序列可以是源自属于棒杆菌属的微生物的启动子或者可以是源自其它微生物的启动子。例如，启动子可以是trc启动子、gap启动子、tac启动子、T7启动子、lac启动子、trp启动子、araBAD启动子或cj7启动子。
术语“载体”指含有基因的调节序列和基因序列并且配置为在合适的宿主细胞中表达靶基因的多核苷酸构建体。或者，载体又可以指多核苷酸构建体，其含有可用于同源重组的序列，从而由于对宿主细胞导入的载体，可以改变宿主细胞的基因组中的内源基因的调节序列，或 者可以将可以表达的靶基因插入宿主的基因组的特定位点中。在这点上，本发明中使用的载体可以进一步包含选择标志物以确定载体对宿主细胞的导入或者载体对宿主细胞的染色体的插入。选择标志物可以包含赋予可选择表型，诸如药物抗性、营养缺陷型、针对细胞毒剂的抗性、或表面蛋白的表达的标志物。在用此类选择剂处理的环境中，由于仅表达选择标志物的细胞可以存活或者显示不同表型性状，可以选择经转化的细胞。
如本文中使用的，术语“转化”指将多核苷酸导入宿主细胞中，从而多核苷酸可以作为基因组外元件或者以插入宿主细胞的基因组中能复制。转化本发明中使用的载体的方法可以包括将核酸导入细胞的方法。另外，如相关技术中公开的，可以根据宿主细胞实施电脉冲方法。
有益效果
本发明通过对NCgl0609基因或NCgl1575基因的弱化或敲除，发现所述基因编码的产物对氨基酸生产能力产生影响，通过在编码序列引入点突变，或者增加该基因的拷贝数或过表达获得重组菌株，所获得的菌株与野生型菌株相比，有利于生产高浓度的氨基酸。而对lysC基因的启动子区域引入点突变，获得重组型菌株，所获得的菌株与未突变的菌株相比，也能大幅提高了L-赖氨酸的产量，进一步提高了生成效率，降低生成成本，便于推广应用。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备；所进行的操作都是本领域已知的，或者按照市售商品的用户手册进行。
以下实施例中培养所述菌株使用的基础培养基组成相同，在此基础培养基组成上添加相应需要的蔗糖、卡那霉素或氯霉素等，基础培养基组成如下：
成分 配方
蔗糖 10g/L
多聚蛋白胨 10g/L
牛肉膏 10g/L
酵母粉 5g/L
尿素 2g/L
氯化钠 2.5g/L
琼脂粉 20g/L
pH 7.0
培养温度 32℃
以下实施例中SSCP电泳PAGE的制备及条件为：
Figure PCTCN2020142133-appb-000017
以下实施例中，L-赖氨酸的发酵培养基和发酵工艺如下表1和2所示：
表1 L-赖氨酸发酵培养基配方
成分 配方
淀粉水解糖 30g/L
硫酸铵 12g/L
硫酸镁 0.87g/L
糖蜜 20g/L
酸化玉米浆 3mL/L
磷酸 0.4mL/L
氯化钾 0.53g/L
消泡剂(2％泡敌) 4mL/L
硫酸亚铁 120mg/L
硫酸锰 120mg/L
烟酰胺 42mg/L
泛酸钙 6.3mg/L
维生素B1 6.3mg/L
铜、锌盐溶液 0.6g/L
生物素 0.88mg/L
表2 L-赖氨酸的发酵控制工艺
Figure PCTCN2020142133-appb-000018
Figure PCTCN2020142133-appb-000019
以下实施例中，L-谷氨酸的发酵培养基和发酵工艺如下表3和4所示：
表3 L-谷氨酸发酵培养基配方
试剂名称 配比
葡萄糖 5.0g/L
磷酸 0.38g/L
硫酸镁 1.85g/L
氯化钾 1.6g/L
生物素 550μg/L
维生物素B1 300μg/L
硫酸亚铁 10mg/L
硫酸锰 10g/dl
KH 2PO 4 2.8g/L
维生素C 0.75mg/L
维生素B12 2.5μg/L
对氨基苯甲酸 0.75mg/L
消泡剂 0.0015ml/dl
甜菜碱 1.5g/L
甘蔗糖蜜 7ml/L
玉米浆 77ml/L
天冬氨酸 1.7g/L
毛发粉 2g/L
表4 L-谷氨酸发酵控制工艺
Figure PCTCN2020142133-appb-000020
实施例1构建包含点突变的NCgl0609基因编码区的转化载体pk18-NCgl0609 R334*
依据NCBI公布的谷氨酸棒杆菌ATCC13032基因组序列，设计并合成两对扩增NCgl0609基因编码区序列的引物，以等位基因置换的方式在菌株YP97158【保藏编号：CGMCC No.12856，保藏日期：2016年8月16日，保藏单位：中国科学院微生物研究所，北京市朝阳区北辰西路1号院3号，电话：010-64807355，记载于中国专利申请CN106367432A中(申请日2016年9月1日，公开日2017年2月1日)，经测序确认该菌株染色体上保留有野生型的NCgl0609基因】背景中的NCgl0609基因编码区(SEQ ID NO:1，对应编码蛋白的氨基酸序列为SEQ ID NO:3)中引入点突变，NCgl0609基因的核苷酸序列第1000位C变为T(SEQ ID NO:2)，对应编码蛋白的氨基酸序列第334位精氨酸变为终止子(SEQ ID NO:4：NCgl0609 R334*)。引物设计如下(上海invitrogen公司合成)：
P1:5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGGACGGCAAC
GTACATAAC3'(SEQ ID NO:5)
P2:5'GTTGCCGGTGAGTCAAACAGTCATTTTGC 3'(SEQ ID NO:6)
P3:5'GCAAAATGACTGTTTGACTCACCGGCAAC 3'(SEQ ID NO:7)
P4:5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCGGCTG GAAATGTGGAG3'(SEQ ID NO:8)
构建方法：以谷氨酸棒杆菌ATCC13032为模板，分别以引物P1和P2，P3和P4，进行PCR扩增，PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，(94℃变性30s、52℃退火30s、72℃延伸40s，30个循环)，72℃过度延伸10min，获得两条大小分别为698bp和648bp，含有NCgl0609基因编码区的DNA片段(NCgl0609Up和NCgl0609Down)。将上述两条DNA片段经琼脂糖凝胶电泳分离纯化后，再以上述两条DNA片段为模板，以P1和P4为引物，通过Overlap PCR扩增长为1317bp的片段。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，(94℃变性30s、52℃退火30s、72℃延伸60s，30个循环)，72℃过度延伸10min。此DNA片段导致YP97158NCgl0609基因编码区的第1000位的胞嘧啶(C)变为胸腺嘧啶(T)，最终导致编码蛋白的第334位氨基酸由精氨酸(R)变为终止子。将此DNA片段经琼脂糖凝胶电泳后进行纯化，与经过双酶切后纯化的pK18mobsacB质粒(购至Addgene公司，分别用Xbal I/BamH I双酶切)用NEBuilder酶(购至NEB公司)50℃连接30min，连接产物转化后 长出的单克隆经pcr鉴定获得阳性载体pk18-NCgl0609 R334*，该质粒上含有卡那霉素抗性标记。将酶切正确的载体pk18-NCgl0609 R334*送测序公司测序鉴定，并将含有正确点突变(C-T)的载体pk18-NCgl0609 R334*保存备用。
实施例2构建包含点突变的NCgl0609 R334*的工程菌株
构建方法：将等位替换质粒pk18-NCgl0609 R334*通过电击转化入L-赖氨酸生产菌株YP97158中(其构建方法可参见WO2014121669A1；经测序确认该菌株染色体上保留有野生型的NCgl0609基因编码区)，对培养产生的单菌落分别通过引物P1和通用引物M13R进行鉴定，能扩增出1375bp大小条带的菌株为阳性菌株。将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用如下引物(上海invitrogen公司合成)进行PCR鉴定：
P5:5'CTAGCCGGTTCCAGTCAG 3'(SEQ ID NO:9)
P6:5'GGACGTCTGTTCACCATTG 3'(SEQ ID NO:10)
上述PCR扩增产物264bp通过95℃高温变性10min、冰浴5min后进行sscp电泳(以质粒pk18-NCgl0609 R334*扩增片段为阳性对照，YP97158扩增片段为阴性对照，水作为空白对照)，由于片段结构不同，电泳位置不同，因此片段电泳位置与阴性对照片段位置不一致且与阳性对照片段位置一致的菌株为等位替换成功的菌株。再次通过引物P5/P6PCR扩增阳性菌株NCgl0609片段，并连接到PMD19-T载体进行测序，通过序列比对，碱基序列发生突变(C-T)的菌株为等位替换成功的阳性菌株，并被命名为YPL-4-041。
实施例3构建基因组上过表达NCgl0609和NCgl0609 R334*基因的工程菌株
依据NCBI公布的野生型谷氨酸棒杆菌ATCC13032基因组序列，设计并合成三对扩增上下游同源臂片段及NCgl0609或NCgl0609 R334*基因编码区及启动子区序列的引物，以同源重组的方式在菌株YP97158中引入NCgl0609或NCgl0609 R334*基因。
引物设计如下(上海invitrogen公司合成)：
P7:5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGTTCTG GACTGAGG 3'(SEQ ID NO:11)
P8:5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGTTCTG GACTGAGG 3'(SEQ ID NO:12)
P9:5'CATCTGTTCTCGGTGCACCAGCTGCGAGGATCATCTC 3'(SEQ ID NO:13)
P10:5'GATTTAATTGCGCCATCTGATTCTGGCAACAACTCCTTCCTTGACC 3'(SEQ ID NO:14)
P11:5'GGTCAAGGAAGGAGTTGTTGCCAGAATCAGATGGCGCAATTA AATC AAG 3'(SEQ ID NO:15)
P12:5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCTATGACACCTTCAACGGATC 3'(SEQ ID NO:16)
构建方法：分别以谷氨酸棒杆菌ATCC13032或YPL-4-041为模板，分别以引物P7/P8，P9/P10，P11/P12，进行PCR扩增，获得上游同源臂片段768bp，NCgl0609或NCgl0609 R334*基因及其启动子片段1626bp及下游同源臂片段623bp。PCR反应结束后，对扩增的3个片段采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3个片段与经过双酶切后纯化的pK18mobsacB质粒(购至Addgene公司，分别用Xbal I/BamH I双酶切)用NEBuilder酶(购至NEB公司)50℃连接30min，连接产物转化后长出的单克隆经pcr鉴定获得阳性整合质粒。该质粒上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得质粒整合到基因组上的重组子。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸60s(30个循环)，72℃过度延伸10min。将测序正确的整合质粒电转化入L-赖氨酸生产菌株YP97158中，对培养产生的单菌落通过P13/P14引物进行PCR鉴定，PCR扩增出含有大小1317bp的片段的为阳性菌株，扩增不到片段的为原菌。将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落进一步采用P15/P16引物进行PCR鉴定，扩增出大小为1352bp的菌为NCgl0609或NCgl0609 R334*基因整合到YP97158基因组上的阳性菌株，其被命名为YPL-4-042(不含突变点)和YPL-4-043(含突变点)。
P13:5'TCCAAGGAAGATACACGCC 3'(SEQ ID NO:17)
P14:5'CGAAATGGAAGTTGTGCG 3'(SEQ ID NO:18)
P15:5'CGATGATGCCGATTACCTC 3'(SEQ ID NO:19)
P16:5'CGTTGGAATCTTGCGTTG 3'(SEQ ID NO:20)
实施例4构建质粒上过表达NCgl0609或NCgl0609 R334*基因的工程菌株
依据NCBI公布的野生型谷氨酸棒杆菌ATCC13032基因组序列，设计并合成一对扩增NCgl0609或NCgl0609 R334*基因编码区及启动子区序列的引物，引物设计如下(上海invitrogen 公司合成)：
P17:5' GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCAGCTGCGAGG A TCATCTC 3'(SEQ ID NO:21)
P18:5' ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCAACAACTCCTTCCTTGACC3'(SEQ ID NO:22)
构建方法：分别以谷氨酸棒杆菌ATCC13032和YPL-4-041为模板，以引物P17/P18进行PCR扩增，获得NCgl0609和NCgl0609 R334*基因及其启动子片段1582bp，对扩增产物进行电泳并采用柱式DNA凝胶回收试剂盒进行纯化，回收的DNA片段与经EcoR I酶切回收的穿梭质粒pXMJ19用NEBuilder酶(购至NEB公司)50℃连接30min，连接产物转化后长出的单克隆用M13引物经pcr鉴定获得阳性过表达质粒pXMJ19-NCgl0609和pXMJ19-NCgl0609 R334*，将该质粒送测序。因质粒上含有氯霉素抗性标记，可以通过氯霉素来筛选质粒是否转化到菌株中。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸60s(30个循环)，72℃过度延伸10min。
将测序正确的pXMJ19-NCgl0609和pXMJ19-NCgl0609 R334*质粒分别电转化入L-赖氨酸生产菌株YP97158中，对培养产生的单菌落通过引物M13F/P18进行PCR鉴定，PCR扩增出含有大小1585bp片段的为阳性菌株，其被命名为YPL-4-044(不含突变点)和YPL-4-045(含突变点)。
实施例5构建基因组上缺失NCgl0609基因的工程菌株
根据NCBI公布的谷氨酸棒杆菌ATCC13032的基因组序列，合成两对扩增NCgl0609基因编码区两端片段的引物，作为上下游同源臂片段。引物设计如下(上海英俊公司合成)：
P19:5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGAA TGG GATGGGTCG 3'(SEQ ID NO:23)
P20:5'CATCATCGGTTACTCTGGCCGAAATGGAAGTTGTGCG 3'(SEQ ID NO:24)
P21:5'CGCACAACTTCCATTTCGGCCAGAGTAACCGATGATG 3'(SEQ ID NO:25)
P22:5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCAACAACTCCTTCCTTGACC3'(SEQ ID NO:26)
构建方法：以谷氨酸棒杆菌ATCC13032为模板，分别以引物P19/P20和P21/P22进行PCR 扩增，获得上游同源臂片段661bp及下游同源臂片段692bp。再用引物P19/P22进行OVERLAP PCR得到整个同源臂片段1334bp。对扩增的产物进行电泳并采用柱式DNA凝胶回收试剂盒进行纯化，回收的DNA片段与经过双酶切后纯化的pK18mobsacB质粒(购至Addgene公司，分别用Xbal I/BamH I双酶切)用NEBuilder酶(购至NEB公司)50℃连接30min，连接产物转化后长出的单克隆用M13引物经pcr鉴定获得阳性敲除载体pK18-ΔNCgl0609，将该质粒送测序。该质粒上含有卡那霉素抗性来作为筛选标记。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸90s(30个循环)，72℃过度延伸10min。
将测序正确的敲除质粒pK18-ΔNCgl0609电转化入赖氨酸生产菌株YP97158，对培养产生的单菌落通过如下引物(上海英俊公司合成)进行PCR鉴定：
P23:5'AATGAATGG GATGGGTCG 3'(SEQ ID NO:27)
P24:5'CAACAACT CCT TCCTTGACC 3'(SEQ ID NO:28)
上述PCR同时扩增出大小1334bp及1788bp的条带的菌株为阳性菌株，只扩增出1788bp条带的菌株为原菌。阳性菌株在15％蔗糖培养基上筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用P23/P24引物进行PCR鉴定，扩增出大小为1334bp条带的菌株为NCgl0609基因编码区被敲除的阳性菌株。再次通过P23/P24引物PCR扩增阳性菌株NCgl0609片段，并连接到PMD19-T载体进行测序，将测序正确的菌株命名为YPL-4-046。
实施例6 L-赖氨酸发酵实验
将实施例2-5构建的菌株和原始菌株YP97158在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表1所示的培养基和表2所示的控制工艺进行发酵实验。每个菌株重复三次，结果如表5所示。
表5 L-赖氨酸发酵实验结果
菌株 L-赖氨酸产量(g/100ml) OD(660nm)
YP97158 18.9 37.3
YPL-4-041 19.3 38.1
YPL-4-042 19.2 37.8
YPL-4-043 19.5 38.4
YPL-4-044 19.4 37.7
YPL-4-045 19.7 38.3
YPL-4-046 18.0 36.8
结果如表5所示，在谷氨酸棒杆菌中对NCgl0609基因编码区进行点突变NCgl0609 R334*及过表达，有助于L-赖氨酸产量及生长速率的提高，而对基因进行弱化或敲除，不利于L-赖氨酸的积累，同时会降低菌株的生长速率。
实施例7在谷氨酸生产菌株中引入NCgl0609基因过表达，或者对NCgl0609基因编码区进行点突变NCgl0609 R334*及过表达，并进行发酵实验
按照实施例1-5的方法，采用相同的引物和实验条件，以棒杆菌ATCC13869为出发菌，以ATCC 13869的菌为表达菌，获得包含点突变的NCgl0609 R334*的谷氨酸生产工程菌株YPG-013、基因组上过表达NCgl0609和NCgl0609 R334*基因的谷氨酸生产工程菌株YPG-014和YPG-015、质粒上过表达NCgl0609和NCgl0609 R334*基因的谷氨酸生产工程菌株YPG-016和YPG-017，和基因组上缺失NCgl0609基因的谷氨酸生产工程菌株YPG-018。
将实施例构建的菌株和原始菌株以ATCC 13869的菌为表达菌在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表3所示的培养基和表4所示的控制工艺进行发酵实验。每个菌株重复三次，结果如表6所示。
表6 L-谷氨酸发酵实验结果
菌株 L-谷氨酸产量(g/l) OD(660nm)
ATCC13869 101.0 42.3
YPG-013 103.5 43.4
YPG-014 103.9 42.8
YPG-015 103.2 43.7
YPG-016 103.6 42.6
YPG-017 103.8 43.6
YPG-018 98.5 40.5
结果如表6所示，在谷氨酸棒杆菌中对NCgl0609基因编码区进行点突变NCgl0609 R334*及过表达，有助于L-谷氨酸产量及生长速率的提高，而对基因进行弱化或敲除，不利于L谷氨酸的积累，同时会降低菌株的生长速率。
实施例8构建包含点突变的NCgl1575基因编码区的转化载体pK18-NCgl1575 A1775T
依据NCBI公布的野生型谷氨酸棒杆菌ATCC13032基因组序列，设计并合成两对扩增NCgl1575基因编码区序列的引物，以等位基因置换的方式在菌株YP97158(经测序确认该菌株染色体上保留有野生型的NCgl1575基因)背景中的NCgl1575基因编码区(SEQ ID NO:29)中 引入点突变，对应编码蛋白的氨基酸序列为SEQ ID NO:31，NCgl1575基因的核苷酸序列第1775位A变为T(SEQ ID NO:30：NCgl1575 A1775T)，对应编码蛋白的氨基酸序列第592位酪氨酸变为苯丙氨酸(SEQ ID NO:32：NCgl1575 Y592F)。
引物设计如下(上海invitrogen公司合成)：
P1’:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTGCGTTCGTCTGCGGTTTCG 3'(SEQ ID NO:33)
P2’:5'ATCGACGCCGCCCCATTCACCCTTCTGATG 3'(SEQ ID NO:34)
P3’:5'CATCAGAAGGGTGAATGGGGCGGCGTCGAT 3'(SEQ ID NO:35)
P4’:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAAGCCTCGACCCCTACATC 3'(SEQ ID NO:36)
构建方法：以谷氨酸棒杆菌ATCC13032为模板，分别以引物P1’和P2’，P3’和P4’，进行PCR扩增。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸40s(30个循环)，72℃过度延伸10min，获得两条大小分别为766bp和759bp，含有NCgl1575基因编码区的DNA片段(NCgl1575Up和NCgl1575Down)。
将上述两条DNA片段经琼脂糖凝胶电泳分离纯化后，再以上述两条DNA片段为模板，以P1’和P4’为引物，通过重叠PCR扩增长约1495bp的片段。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸90s(30个循环)，72℃过度延伸10min。
此DNA片段(NCgl1575 A1775T)导致YP97158NCgl1575基因编码区的第1775位的腺嘌呤(A)变为胸腺嘧啶(T)，最终导致编码蛋白的第592位氨基酸由酪氨酸(Y)变为苯丙氨酸(F)。
将琼脂糖凝胶电泳分离纯化后的NCgl1575 A1775T和用Xba I酶切回收的pK18mobsacB质粒(购自Addgene公司)用NEBuider重组系统组装，获得载体pK18-NCgl1575 A1775T，该质粒上含有卡那霉素抗性标记。并将载体pK18-NCgl1575 A1775T送测序公司测序鉴定，将含有正确点突变(A-T)的载体pK18-NCgl1575 A1775T保存备用。
实施例9构建包含点突变的NCgl1575 A1775T的工程菌株
构建方法：将等位替换质粒pK18-NCgl1575 A1775T通过电击转化入L-赖氨酸生产菌株YP97158中，对培养产生的单菌落分别通过引物P1’和通用引物M13R进行鉴定，能扩增出1502bp大小条带的菌株为阳性菌株。将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用如下引物(上海invitrogen公司合成)进行PCR鉴定：
P5’:5'CACATC AGCTTGATTT CTGC 3'(SEQ ID NO:37)
P6’:5'GGTCATTGCC GATGAAGCCC 3'(SEQ ID NO:38)
上述PCR扩增产物256bp通过高温变性、冰浴后进行sscp电泳(以质粒pK18-NCgl1575 A1775T扩增片段为阳性对照，YP97158扩增片段为阴性对照，水作为空白对照)，由于片段结构不同，电泳位置不同，因此片段电泳位置与阴性对照片段位置不一致且与阳性对照片段位置一致的菌株为等位替换成功的菌株。以引物P5’和P6’再次通过PCR扩增等位替换成功的菌株目的片段，并连接到PMD19-T载体测序，通过序列比对碱基序列发生突变的序列验证菌株的等位替换成功，并被命名为YPL-4-023。
实施例10构建基因组上过表达NCgl1575或NCgl1575 A1775T基因的工程菌株
依据NCBI公布的野生型谷氨酸棒杆菌ATCC13032基因组序列，设计并合成三对扩增上下游同源臂片段及NCgl1575或NCgl1575 A1775T基因编码区及启动子区序列的引物，以同源重组的方式在菌株YP97158中引入NCgl1575或NCgl1575 A1775T基因。
引物设计如下(上海invitrogen公司合成)：
P7’：5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATG
CGTTCTGGACTGAGG 3'(SEQ ID NO:39)
P8’：5'GAAACGGCCTTAAGCTAGGTGCACCGAG AACAGATG 3'(SEQ ID NO:40)
P9’：5'CATCTGTTCTCGGTGCAC CTAGCTTAAG GCCGTTTC 3'(SEQ ID NO:41)
P10’：5'CTTGATTTAATTGCGCCATCAAGCTTTTCC CGCCCGGTT 3'(SEQ ID NO:42)
P11’：5'AACCGGGCGG GAAAAGCTTGATGGCGCAATTAAATCAAG 3'(SEQ ID NO:43)
P12’：5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCTAT
GACACCTTCAACGGATC 3'(SEQ ID NO:44)
构建方法：分别以谷氨酸棒杆菌ATCC13032或YPL-4-023为模板，分别以引物P7’/P8’，P9’/P10’，P11’/P12’进行PCR扩增，获得上游同源臂片段802bp，NCgl1575基因及其启动子片 段2737bp，或NCgl1575 A1775T基因及其启动子片段2737bp，下游同源臂片段647bp，再以P7’/P12’为引物，以以上扩增的三个片段(上游同源臂片段，NCgl1575基因及其启动子片段，下游同源臂片段；或者，上游同源臂片段，NCgl1575 A1775T基因及其启动子片段，下游同源臂片段)混合为模板进行扩增，获得整合同源臂片段4111bp。
PCR反应结束后，对扩增的产物进行电泳回收，采用柱式DNA凝胶回收试剂盒(TIANGEN)进行回收所需要的4111bp的DNA片段，采用NEBBuider重组系统与经Xba I酶切回收的穿梭质粒PK18mobsacB相连接，获得整合质粒PK18mobsacB-NCgl1575或PK18mobsacB-NCgl1575 A1775T。质粒上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得质粒整合到基因组上的重组子。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸180s(30个循环)，72℃过度延伸10min。
将2个整合质粒分别电转化入L-赖氨酸生产菌株YP97158中，对培养产生的单菌落通过P13’/P14’引物进行PCR鉴定，PCR扩增出含有大小1778bp的片段的为阳性菌株，扩增不到片段的为原菌。阳性菌株在15％蔗糖培养基上筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用P15’/P16’引物进行PCR鉴定，扩增出大小为1756bp的菌为NCgl1575或NCgl1575 A1775T基因整合到YP97158基因组上的菌株，其分别被命名为YPL-4-024(不含突变点)和YPL-4-025(含突变点)。
P13’:5'TCCAAGGAAGATACACGCC 3'(SEQ ID NO:45)
P14’:5'CTTCTGATGA CCGGCACACC 3'(SEQ ID NO:46)
P15’:5'TAGTCGATGA CGCGGGTGCG 3'(SEQ ID NO:47)
P16’:5'CGTTGGAATCTTGCGTTG 3'(SEQ ID NO:48)
实施例11构建质粒上过表达NCgl1575或NCgl1575 A1775T基因的工程菌株
依据NCBI公布的野生型谷氨酸棒杆菌ATCC13032基因组序列，设计并合成一对扩增NCgl1575或NCgl1575 A1775T基因编码区及启动子区序列的引物，引物设计如下(上海invitrogen公司合成)：
P17’：5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC CTAGCT
TAAG GCCGTTTC 3'(SEQ ID NO:49)
P18’：5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAAC AAGCTTT
TCC CGCCCGGTT 3'(SEQ ID NO:50)
构建方法：分别以ATCC13032和YPL-4-023为模板，以引物P17’/P18’进行PCR扩增，获得NCgl1575或NCgl1575 A1775T基因及其启动子片段2749bp，对扩增的产物进行电泳回收，采用柱式DNA凝胶回收试剂盒进行回收所需要的2749bp的DNA片段，采用NEBuider重组系统与经EcoR I酶切回收的穿梭质粒pXMJ19相连接，获得过表达质粒pXMJ19-NCgl1575和pXMJ19-NCgl1575 A1775T。质粒上含有氯霉素抗性标记，可以通过氯霉素筛选获得质粒转化到菌株中。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸120s(30个循环)，72℃过度延伸10min。
将pXMJ19-NCgl1575和pXMJ19-NCgl1575 A1775T质粒分别电转化入L-赖氨酸生产菌株YP97158中，对培养产生的单菌落通过M13(-48)和P18’引物进行PCR鉴定，PCR扩增出含有大小2752bp的片段的为转入菌株，其被命名为YPL-4-026(不含突变点)和YPL-4-027(含突变点)。
实施例12构建基因组上缺失NCgl1575基因的工程菌株
根据NCBI公布的谷氨酸棒杆菌ATCC13032的基因组序列，合成两对扩增NCgl1575基因编码区两端片段的引物，作为上下游同源臂片段。引物设计如下(上海英俊公司合成)：
P19’：5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGACCG
GCGCAG ATGCCAACGC 3'(SEQ ID NO:51)
P20’：CCCAGAACTGAAGGTCTAATTGCCTAAGG CCGGAATT 3'(SEQ ID NO:52)
P21’：AATTCCGGCCTTAGGCAATTAGACCTTC AGTTCTGGG 3'(SEQ ID NO:53)
P22’：5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCT
TGATGAA GGCTCCAG 3'(SEQ ID NO:54)
以谷氨酸棒杆菌ATCC13032为模板，分别以引物P19’/P20’和P21’/P22’进行PCR扩增，获得上游同源臂片段775bp及下游同源臂片段807bp，再用引物P19’/P22’进行重叠PCR得到整个同源臂片段1545bp。PCR反应结束后，对扩增的产物进行电泳回收，采用柱式DNA凝胶回收试剂盒进行回收所需要的1545bp的DNA片段，并通过NEBuider重组系统与经Xba I酶切回收的穿梭质粒pk18mobsacB质粒相连接，获得敲除质粒。该质粒上含有卡那霉素抗性标记。
将敲除质粒电转化入赖氨酸生产菌株YP97158，对培养产生的单菌落分别通过如下引物(上海英俊公司合成)进行PCR鉴定：
P23’:5'ACCGGCGCAG ATGCCAACGC 3'(SEQ ID NO:55)
P24’:5'GCTTGATGAA GGCTCCAG 3'(SEQ ID NO:56)
上述PCR扩增出大小1471bp及4150bp的条带的菌株为阳性菌株，只扩增出4150bp条带的菌株为原菌。阳性菌株在15％蔗糖培养基上筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用P23’/P24’引物进行PCR鉴定，扩增出大小为1471bp条带的菌株为NCgl1575基因编码区被敲除的基因工程菌株，其被命名为YPL-4-028。
实施例13 L-赖氨酸发酵实验
将实施例9-12构建的菌株和原始菌株YP97158在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表1所示的培养基和表2所示的控制工艺进行发酵实验。每个菌株重复三次，结果如表7所示。
表7 L-赖氨酸发酵实验结果
菌株 L-赖氨酸产量(g/100ml) OD(660nm)
YP97158 18.8 37.3
YPL-4-023 19.6 36.8
YPL-4-024 19.6 37.0
YPL-4-025 19.8 35.5
YPL-4-026 19.3 36.3
YPL-4-027 19.7 37.2
YPL-4-028 18.0 36.8
结果如表7所示，在谷氨酸棒杆菌中对NCgl1575基因过表达，或者对NCgl1575基因编码区进行点突变NCgl1575 A1775T及过表达，有助于L-赖氨酸产量的提高，而对基因进行弱化或敲除，不利于L-赖氨酸的积累。
实施例14构建包含点突变的lysC基因启动子区的转化载体pK18-PlysC (G(-45)A,G(-47)T)
依据NCBI公布的谷氨酸棒杆菌ATCC13032基因组序列，设计并合成两对扩增lysC基因启动子区序列的引物，以等位基因置换的方式在菌株YP97158背景中的lysC基因启动子区(SEQ ID NO:57)中引入点突变，lysC基因启动子区核苷酸序列的-45bp位G变为A、-47bp位G变为T(SEQ ID NO:58)。
引物设计如下(上海invitrogen公司合成)：
P1”:5' CCGGAATTCG ACCAAGGATG AGGGCTTTG 3'(EcoR I)(SEQ ID NO:59)
P2”:5'AGTTACCCGC TCAATTATAC CTTTATAAAC 3'(SEQ ID NO:60)
P3”:5'GTTTATAAAG GTATAATTGAGCGGGTAACT 3'(SEQ ID NO:61)
P4”:5' ACATGCATGCGCGTACGCGAAGTGGCACAT 3'(Sph I)(SEQ ID NO:62)。
构建方法：以谷氨酸棒杆菌ATCC13032为模板，分别以引物P1”和P2”，P3”和P4”，进行PCR扩增，PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸40s，30个循环，72℃过度延伸10min，获得含有点突变的、长度分别为729bp和760bp的两条DNA片段(lysC启动子Up和lysC启动子Down片段)。将上述两条DNA片段经琼脂糖凝胶电泳分离纯化后，再以纯化后的两条DNA片段为模板，以P1”和P4”为引物，通过Overlap PCR扩增出长度约为1459bp的片段(Up-Down片段)。PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸90s，30个循环，72℃过度延伸10min。将上述Up-Down片段经琼脂糖凝胶电泳分离纯化，该片段包含lysC基因启动子区及其上下游序列，且片段两端分别含有EcoR I和Sph I酶切位点。此DNA片段导致YP97158lysC基因启动子区的-45bp位的核苷酸鸟嘌呤(G)变为腺嘌呤(A)、-47bp位的核苷酸鸟嘌呤(G)变为胸腺嘧啶(T)。将片段双酶切(EcoR I/Sph I)后纯化回收，与同样双酶切(EcoR I/Sph I)后的穿梭质粒pK18mobsacB(购自Addgene公司)相连接，获得等位替换质粒pK18-PlysC (G(-45)A,G(-47)T)，该质粒上含有卡那霉素抗性标记。并将载体pK18-PlysC (G(-45)A,G(-47)T)送测序公司测序鉴定，将含有正确点突变的载体pK18-PlysC (G(-45)A,G(-47)T)保存备用。
实施例15构建包含点突变的PlysC (G(-45)A,G(-47)T)的工程菌株
将等位替换质粒pK18-PlysC (G(-45)A,G(-47)T)通过电击转化入L-赖氨酸生产菌株YP97158中，对培养产生的单菌落分别通过引物P1”和通用引物M13F进行鉴定，能扩增出1500bp大小条带的菌株为阳性菌株。将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用如下引物(上海invitrogen公司合成)进行PCR鉴定：
P5”:5'ATCAATATATGGTCTGTTTA 3'(SEQ ID NO:63)
P6”:5'CTTGGTGGCAACGATCCGTT 3'(SEQ ID NO:64)
上述PCR扩增产物通过高温变性、冰浴后进行sscp电泳(以质粒pK18-PlysC (G(-45)A,G(-47)T)扩增片段为阳性对照，YP97158扩增片段为阴性对照，水作为空白对照)，由于片段结构不同，电泳位置不同，因此片段电泳位置与阴性对照片段位置不一致且与阳性对照片段位置一致的菌株为等位替换成功的菌株。再次通过PCR扩增阳性菌株目的片段，并连接到PMD19-T载体测序，通过序列比对，碱基序列发生突变的序列验证菌株的等位替换成功，并被命名为YPL-4-009。
实施例16 L-赖氨酸发酵实验
将实施例15构建的菌株YPL-4-009和原始菌株YP97158在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表1所示的培养基和表2所示的控制工艺进行发酵实验。每个菌株重复三次，结果如表8所示。
表8 L-赖氨酸发酵实验结果
Figure PCTCN2020142133-appb-000021
上述转化率＝赖氨酸的总质量/葡萄糖的总消耗量*100％
结果如表8所示，在谷氨酸棒杆菌中对lysC基因启动子进行点突变PlysC (G(-45)A,G(-47)T)，有助于L-赖氨酸产量的提高。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (17)

  1. 一种生成L-氨基酸的细菌，其特征在于，具有编码SEQ ID NO:3的氨基酸序列的多核苷酸的改善的表达；和/或，编码SEQ ID NO:31的氨基酸序列的多核苷酸的改善的表达，和/或，SEQ ID NO:57所示的启动子区域第-45bp位和第-47bp位碱基发生突变；
    优选，所述改善的表达是编码SEQ ID NO:3的氨基酸序列的多核苷酸的表达增强，或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变，或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变且表达是增强的；
    优选，所述改善的表达是编码SEQ ID NO:31的氨基酸序列的多核苷酸的表达增强，或者编码SEQ ID NO:31的氨基酸序列的多核苷酸具有点突变，或者编码SEQ ID NO:31的氨基酸序列的多核苷酸具有点突变且表达是增强的。
  2. 如权利要求1所述的细菌，其特征在于，编码SEQ ID NO:3的氨基酸序列的多核苷酸的点突变，使得SEQ ID NO:3的氨基酸序列的第334位精氨酸被终止子所取代；
    或者，编码SEQ ID NO:31的氨基酸序列的多核苷酸的点突变，使得SEQ ID NO:31的氨基酸序列的第592位酪氨酸被不同的氨基酸所取代；优选第592位酪氨酸被苯丙氨酸所取代。
  3. 如权利要求1-2任一项所述的细菌，其特征在于，编码SEQ ID NO:3的氨基酸序列的多核苷酸包含SEQ ID NO:1的核苷酸序列；
    或者，编码SEQ ID NO:31的氨基酸序列的多核苷酸包含SEQ ID NO:29的核苷酸序列。。
  4. 如权利要求1-3任一项所述的细菌，其特征在于，所述编码SEQ ID NO:3的氨基酸序列的具有点突变的多核苷酸序列是由SEQ ID NO:1所示的多核苷酸序列第1000位碱基发生突变而形成的；优选的，所述突变包括SEQ ID NO:1所示的多核苷酸序列第1000位碱基由胞嘧啶(C)突变为胸腺嘧啶(T)；优选，所述具有点突变的多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列；
    或者，所述编码SEQ ID NO:31的氨基酸序列的具有点突变的多核苷酸序列是由SEQ ID NO:29所示多核苷酸序列第1775位碱基发生突变而形成的；优选，所述突变包括SEQ ID NO:29所示多核苷酸序列第1775位碱基由腺嘌呤(A)突变为胸腺嘧啶(T)；优选，所述具有点突变的多核苷酸序列包括SEQ ID NO:30所示的多核苷酸序列。
  5. 如权利要求1-4所述的细菌，其特征在于，SEQ ID NO:57所示的启动子区域的第-45bp位核苷酸鸟嘌呤(G)突变为腺嘌呤(A)、第-47bp位的核苷酸鸟嘌呤(G)突变为胸腺嘧啶(T)；
    优选，所述启动子核苷酸序列包括：
    (a)SEQ ID NO:58所示的核苷酸序列；或者是，
    (b)与SEQ ID NO:58所示的核苷酸序列具有90％以上，优选的具有95％、98％以上同一性的核苷酸序列，且其保留(a)所述启动子的增强活性，并且第-45bp位保持为腺嘌呤(A)、第-47bp位保持为胸腺嘧啶(T)。
  6. 如权利要求1-5任一项所述的细菌，其特征在于，所述微生物是谷氨酸棒杆菌(Corynebacterium glutamicum)；优选为YP97158或ATCC 13869。
  7. 一种多核苷酸序列，其特征在于，包括编码含有SEQ ID NO:3所示的氨基酸序列的多核苷酸，且其第334位精氨酸被终止子所取代；
    优选所述多核苷酸序列包括编码含有SEQ ID NO:4所示的氨基酸序列的多核苷酸；
    优选所述多核苷酸序列是由包括SEQ ID NO:1所示的多核苷酸序列第1000位碱基发生突变而形成的；
    优选的，所述突变包括SEQ ID NO:1所示的多核苷酸序列第485位碱基由胞嘧啶(C)突变为胸腺嘧啶(T)。
    优选所述多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
  8. 一种多核苷酸序列，其特征在于，包括编码含有SEQ ID NO:31所示的氨基酸序列的多核苷酸，其中第592位酪氨酸被不同的氨基酸所取代；优选第592位酪氨酸被苯丙氨酸所取代；
    优选所述多核苷酸序列包括编码含有SEQ ID NO:32所示的氨基酸序列的多核苷酸；
    优选所述多核苷酸序列是由SEQ ID NO:29所示多核苷酸序列1775位碱基发生突变而形成的；优选所述突变是SEQ ID NO:29所示多核苷酸序列第1775位碱基由腺嘌呤(A)突变为胸腺嘧啶(T)；
    优选所述多核苷酸序列包括SEQ ID NO:30所示的多核苷酸序列。
  9. 一种氨基酸序列，其特征在于，所述序列如SEQ ID NO:4或SEQ ID NO:32所示。
  10. 一种重组载体，其特征在于，包含权利要求7或8所述的多核苷酸序列。
  11. 一种重组菌株，其特征在于，包含权利要求7或8所述的多核苷酸序列。
  12. 一种启动子核苷酸序列，其包括SEQ ID NO:57所示的启动子区域第-45bp位和第-47bp位碱基发生突变形成的核苷酸序列；
    优选地，SEQ ID NO:57所示的启动子区域的第-45bp位核苷酸鸟嘌呤(G)突变为腺嘌呤(A)、第-47bp位的核苷酸鸟嘌呤(G)突变为胸腺嘧啶(T)。
  13. 根据权利要求12的启动子核苷酸序列，其包括：
    (a)SEQ ID NO:58所示的核苷酸序列；或者是，
    (b)与SEQ ID NO:58所示的核苷酸序列具有90％以上，优选的具有95％、98％以上同一性的核苷酸序列，且其保留(a)所述启动子的增强活性，并且第-45bp位保持为腺嘌呤(A)、第-47bp位保持为胸腺嘧啶(T)。
  14. 一种表达盒，包含权利要求12或13所述的启动子核苷酸序列和可操纵地连接在所述启动子核苷酸序列后的编码序列；优选地，所述编码序列为lysC基因的编码序列。
  15. 一种重组载体，包含权利要求12或13所述的启动子核苷酸序列；
    优选地，所述启动子核苷酸序列与穿梭质粒相连接来构建所述重组载体；优选地，所述穿梭质粒为pK18mobsacB质粒。
  16. 一种重组菌株，包含权利要求12或13所述的启动子核苷酸序列或权利要求15所述的重组载体；优选地，其包含SEQ ID NO:58所示的核苷酸序列；优选地，所述重组菌株包含SEQ ID NO:58所示的核苷酸序列连接lysC基因编码序列。
  17. 一种生产L-氨基酸的方法，所述方法包括：培养权利要求1-6任一项所述的细菌或者权利要求11或16所述的重组菌株，并从所述培养物中回收L-氨基酸。
PCT/CN2020/142133 2020-06-08 2020-12-31 产l-氨基酸的重组菌株及其构建方法与应用 Ceased WO2021248902A1 (zh)

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EP20939601.9A EP4163377A4 (en) 2020-06-08 2020-12-31 RECOMBINANT STRAIN FOR THE PRODUCTION OF L-AMINO ACID, PRODUCTION PROCESS THEREOF AND USE THEREOF
KR1020227045499A KR20230042224A (ko) 2020-06-08 2020-12-31 L-아미노산을 생산하는 재조합 균주 및 이의 구축 방법과 응용
JP2022574296A JP7728802B2 (ja) 2020-06-08 2020-12-31 Ｌ－アミノ酸を生産する組換え菌株、並びにその構築方法及び使用
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