WO2021143727A1 - 生产赖氨酸的微生物以及赖氨酸的生产方法 - Google Patents

生产赖氨酸的微生物以及赖氨酸的生产方法 Download PDF

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WO2021143727A1
WO2021143727A1 PCT/CN2021/071517 CN2021071517W WO2021143727A1 WO 2021143727 A1 WO2021143727 A1 WO 2021143727A1 CN 2021071517 W CN2021071517 W CN 2021071517W WO 2021143727 A1 WO2021143727 A1 WO 2021143727A1
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lysine
gene encoding
polypeptide
strain
seq
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French (fr)
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孙际宾
陈久洲
郑平
周文娟
郭轩
石拓
刘娇
马延和
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中国科学院天津工业生物技术研究所
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Priority to EP21741763.3A priority patent/EP4092040A4/en
Publication of WO2021143727A1 publication Critical patent/WO2021143727A1/zh

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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0091—Oxidoreductases (1.) oxidizing metal ions (1.16)
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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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    • C12N15/09—Recombinant DNA-technology
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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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)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01016—Diaminopimelate dehydrogenase (1.4.1.16)
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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)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the present disclosure relates to the field of biotechnology. Specifically, the present disclosure relates to L-lysine high-yielding strains, construction methods and applications of the high-yielding strains.
  • L-Lysine lysine for short, is the most important essential amino acid in human and animal nutrition. It is widely used in industries such as medicine, health, food, animal feed, and cosmetics. Lysine is mainly produced by microbial fermentation. At present, the main production strains include microorganisms such as Escherichia coli and Corynebacterium glutamicum. Due to the physiological superiority of Corynebacterium glutamicum, Corynebacterium glutamicum has become an industrial The most important production strain. With the continuous development of biotechnology, in recent years, methods for metabolic engineering of Corynebacterium glutamicum to increase its lysine production have also emerged, including the transformation of lysine synthesis pathways, and lysine synthesis competition pathways. Weakening and so on, the acid production capacity of the current industrial strains of lysine has reached a high level.
  • the purpose of the present disclosure is to provide a new lysine-producing strain, a method for constructing the strain, and a method for producing lysine using the constructed lysine-producing strain.
  • the present disclosure provides a L-lysine production strain, which has the following (i)-(iii) compared with a wild-type strain containing an endogenous polypeptide At least one of the characteristics shown:
  • the endogenous polypeptide is a polypeptide of the amino acid sequence shown in SEQ ID NO:1, or has at least 90% of the amino acid sequence shown in SEQ ID NO:1, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably a polypeptide encoded by a sequence with at least 99% sequence identity;
  • the target polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2, or at least 90% of the nucleotide sequence shown in SEQ ID NO: 2, optionally at least 95%, preferably at least A polynucleotide encoded by a sequence of 97%, more preferably at least 98%, and most preferably at least 99% sequence identity.
  • the strain has a homology of more than 98% with the polypeptide shown in SEQ ID NO:1 and a polypeptide with a FhuF domain at the C-terminal is inactivated.
  • the strain is Corynebacterium glutamicum.
  • polypeptide shown in SEQ ID NO:1 in the strain is inactivated.
  • the polypeptide inactivation means that the transcription and expression of the gene encoding the polypeptide are reduced by at least 30%, preferably at least 40%, more preferably at least 50%, compared with the endogenous polypeptide, for example At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or the gene encoding the polypeptide is removed; or,
  • the activity of the polypeptide is reduced by at least 30%, preferably at least 40%, more preferably at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, At least 98% or at least 99%.
  • the L-lysine production of the L-lysine producing strain is increased by at least 5%, preferably at least 15%, More preferably at least 20%, more preferably at least 25%, more preferably at least 30%, most preferably at least 35%.
  • the glucose conversion rate of the L-lysine producing strain during the production of L-lysine is increased by at least 10% , Preferably 20%, more preferably 30%.
  • the inactivation of the endogenous polypeptide can be achieved by one or a combination of the following methods: partial knockout or complete knockout of the polypeptide coding gene; coding gene mutation; changing the promoter of the coding gene, translation Codons in the regulatory region or coding region weaken its transcription or translation; change the coding gene sequence to weaken the stability of the mRNA or the structure of the encoded protein; or any other modification of the gene coding region and its adjacent upstream and downstream regions to make it Ways of inactivation, etc.
  • frameshift mutations, missense mutations, deletions, start codon changes, etc. occur in the coding sequence of the gene.
  • one or more genes selected from the following in the lysine-producing strain are enhanced or overexpressed:
  • ddh The ddh gene encoding diaminopimelate dehydratase
  • ppc gene encoding phosphoenolpyruvate carboxylase
  • h 1 The pntAB gene encoding niacinamine adenine dinucleotide transhydrogenase.
  • one or more genes selected from the following in the strain are attenuated or reduced in expression:
  • thrA gene encoding the bifunctional enzyme of aspartate kinase I/homoserine dehydratase I;
  • the cadA gene encoding lysine decarboxylase.
  • the present disclosure provides a method for constructing an L-lysine producing strain, which includes the following steps:
  • the wild-type strain containing the endogenous polypeptide is modified to reduce or disappear the polypeptide activity of the endogenous polypeptide of the wild-type strain; or,
  • the wild-type strain containing the endogenous polypeptide is modified so that the expression level of the gene encoding the endogenous polypeptide of the wild-type strain is reduced or disappeared; or,
  • the endogenous polypeptide is a polypeptide of the amino acid sequence shown in SEQ ID NO:1, or has at least 90% of the amino acid sequence shown in SEQ ID NO:1, optionally at least 95%, preferably at least 97%, and more Preferably, a polypeptide encoded by a sequence with at least 98%, and most preferably at least 99% sequence identity;
  • the target polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2, or at least 90% of the nucleotide sequence shown in SEQ ID NO: 2, optionally at least 95%, preferably at least A polynucleotide encoded by a sequence of 97%, more preferably at least 98%, and most preferably at least 99% sequence identity.
  • the strain is modified in which a polypeptide with a homology to the polypeptide shown in SEQ ID NO:1 is higher than 98% and a polypeptide with a FhuF domain at the C-terminus is inactivated.
  • the strain is Corynebacterium glutamicum.
  • the strain is modified in which the polypeptide shown in SEQ ID NO:1 is inactivated.
  • the polypeptide inactivation means that the transcription and expression of the gene encoding the polypeptide are reduced by at least 30%, preferably at least 40%, more preferably at least 50%, compared with the endogenous polypeptide, for example At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or the gene encoding the polypeptide is removed; or, The activity of the polypeptide is reduced by at least 30%, preferably at least 40%, more preferably at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 % Or at least 99%.
  • the L-lysine production of the L-lysine producing strain is increased by at least 5%, preferably at least 15%, More preferably at least 20%, more preferably at least 25%, more preferably at least 30%, most preferably at least 35%.
  • the glucose conversion rate of the L-lysine producing strain during the production of L-lysine is increased by at least 10% , Preferably 20%, more preferably 30%.
  • the inactivation of the endogenous polypeptide can be achieved by one or a combination of the following methods: partial knockout or complete knockout of the coding gene; coding gene mutation; changing the promoter and translation control region of the coding gene Or the coding region codons weaken its transcription or translation; change the coding gene sequence to reduce the stability of the mRNA or the structure of the encoded protein; or any other modification of the gene coding region and its adjacent upstream and downstream regions in the cell to make it Ways of weakening or inactivation, etc.
  • frameshift mutations, missense mutations, deletions, start codon changes, etc. occur in the coding sequence of the polypeptide gene.
  • the method further includes enhancing or overexpressing one or more genes selected from the following in the strain:
  • ddh The ddh gene encoding diaminopimelate dehydratase
  • ppc gene encoding phosphoenolpyruvate carboxylase
  • h 1 The pntAB gene encoding niacinamine adenine dinucleotide transhydrogenase.
  • the method further comprises attenuating one or more genes selected from the following in the strain or reducing the expression of one or more genes selected from the following in the strain:
  • thrA gene encoding the bifunctional enzyme of aspartate kinase I/homoserine dehydratase I;
  • the cadA gene encoding lysine decarboxylase.
  • the present disclosure provides a method for preparing L-lysine, the method comprising:
  • the present disclosure provides the use of the L-lysine producing strain described in the first aspect or the L-lysine producing strain prepared by the method described in the second aspect in the production of L-lysine.
  • SEQ ID NO: 1 MSIWKRLLVQYPRFADTLTAGQPITLEELATPEVILEAVAKGQEIFGIEQPKHAAQLWFHSLCTAIVGPAVTAMVEFDVIPSLDIRRGQLHNIDGYWFGFRPEEMLVDASLHLSGTQFGESIRVVIDALCAATDLRPAPLWAVASDALGIAASGAGVEAFEEEHAREVAEALIEGMNSVNSVPSPRFNDDDYFIRAGCCMIFHSPRADFCTSCPQKR
  • SEQ ID NO: 2 shown polypeptide derived from Corynebacterium glutamicum, which encodes the nucleotide sequence SEQ ID NO: 2 as shown in (ATGAGCATCTGGAAACGTCTGTTAGTGCAGTACCCGCGCTTCGCCGACACCCTCACAGCCGGCCAACCCATCACGCTCGAGGAATT), because it contains the C-terminus of a FhuF
  • the domain is also annotated as (2Fe-2S) binding protein.
  • Polynucleotides generally refer to polyribonucleotides and polydeoxyribonucleotides, which can be unmodified RNA or DNA, or modified RNA or DNA.
  • the nucleotide sequence of the present disclosure may be the nucleotide sequence described in SEQ ID NO: 2, or a nucleotide sequence that hybridizes with the nucleotide sequence described in SEQ ID NO: 2 under stringent conditions. It may be a nucleotide sequence that hybridizes with the probe prepared by SEQ ID NO: 2.
  • stringent conditions referred to here refer to conditions under which specific hybridization can occur but not specific hybridization does not occur.
  • probe length can be selected according to hybridization conditions, usually 100bp to 1kb.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).
  • expression includes any step involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • sequence identity and “percent identity” refer to the percentage of nucleotides or amino acids that are identical (ie identical) between two or more polynucleotides or polypeptides.
  • sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: aligning the nucleotide or amino acid sequence of the polynucleotides or polypeptides and aligning the aligned polynucleotides or polypeptides The number of positions containing the same nucleotide or amino acid residue is scored and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotide or polypeptide.
  • Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides).
  • Polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides).
  • Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
  • wild-type/endogenous refers to the unmodified state of the polypeptide or polynucleotide in the microorganism, that is, the natural state.
  • a polypeptide, polynucleotide sequence, or microorganism that can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory is naturally occurring.
  • naturally occurring and “wild type” are synonymous.
  • the wild-type strain of the present disclosure refers to a strain containing an endogenous polypeptide
  • the endogenous polypeptide is a polypeptide with an amino acid sequence as shown in SEQ ID NO:1, or with a sequence shown in SEQ ID NO:1.
  • the sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% sequence identity.
  • the wild-type strain is Corynebacterium glutamicum containing an endogenous polypeptide.
  • the present disclosure performs genetic modification on wild-type strains to obtain recombinant strains.
  • the recombinant strains have at least one of the following characteristics (i)-(iii):
  • the endogenous polypeptide is a polypeptide of the amino acid sequence shown in SEQ ID NO:1, or has at least 90% of the amino acid sequence shown in SEQ ID NO:1, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably a polypeptide encoded by a sequence with at least 99% sequence identity;
  • the polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2, or at least 90% of the nucleotide sequence shown in SEQ ID NO: 2, optionally at least 95%, preferably at least 97 %, more preferably at least 98%, most preferably at least 99% sequence identity of the polynucleotide encoded by the sequence.
  • the present disclosure improves the lysine production of the strain by inactivating the polypeptide shown in SEQ ID NO: 1 of Corynebacterium glutamicum. Therefore, the "inactivation of the polypeptide shown in SEQ ID NO:1" as described herein means that compared with the wild-type strain, the expression of the polypeptide shown in SEQ ID NO:1 is reduced, attenuated or even completely disappeared, or it means that the expression of the polypeptide shown in SEQ ID NO: 1 The gene, or although expressed, the expression product has no activity or has reduced activity.
  • the transcription, expression of the gene or the activity of the encoded protein after the mutation is reduced by at least 30%, preferably at least 40%, more preferably at least 50%, such as at least 60% , At least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or the gene encoding the wild-type or endogenous polypeptide is deleted.
  • modification in the present disclosure refers to any genetic manipulation performed on a wild-type strain or a parent strain, including but not limited to various molecular biological methods.
  • modification can be replaced with each other.
  • activation in the present disclosure can be achieved by modification, including but not limited to deleting part or all of the coding gene, mutation of the gene reading frame, weakening the transcription or translation strength, or using the corresponding enzyme or code with lower activity.
  • the gene or allele of the protein, or the corresponding gene or enzyme is inactivated, and these methods are optionally used in combination.
  • the use of appropriate culture methods or genetic modification (mutation) of the signal structure of gene expression can reduce gene expression.
  • the signal structure of gene expression is a repressor gene, active gene, operator gene, promoter, attenuator, and ribosome binding site. Point, start codon and terminator.
  • lysine can be increased by inactivating the gene encoding the polypeptide shown in SEQ ID NO:1, or making the polypeptide shown in SEQ ID NO:1 unable to function normally in cells.
  • the output Those skilled in the art can achieve the above-mentioned objects through technical means known in the art. For example, it can be achieved by making the enzyme-encoding gene defect on the chromosome, or by modifying an expression control sequence such as a promoter or SD sequence; it can also be achieved by introducing amino acid substitutions (missense mutations) and stop codons (nonsense mutations) into the coding region.
  • glucose conversion rate refers to the molar ratio of conversion of the substrate glucose to the product lysine.
  • the "lysine-producing strain” mentioned herein means that when the bacteria are cultured in the medium, they can produce lysine and can accumulate lysine, or can secrete lysine into the medium, that is, they can get Extracellular free lysine strain.
  • it may be a naturally-occurring lysine-producing strain, or it may be a lysine-producing engineered strain obtained through genetic modification. These modifications include, but are not limited to, that one or more genes selected from the following are enhanced in the strain Or express more:
  • ddh The ddh gene encoding diaminopimelate dehydratase
  • h 1 The pntAB gene encoding niacinamine adenine dinucleotide transhydrogenase.
  • genes selected from the following including but not limited to the strain are weakened or their expression is reduced:
  • thrA gene encoding the bifunctional enzyme of aspartate kinase I/homoserine dehydratase I;
  • the cadA gene encoding lysine decarboxylase.
  • the reduced or disappeared protein activity, the expression level of the reduced or disappeared protein-encoding gene, the reduced or disappeared enzyme activity, the reduced or disappeared enzyme-encoding gene in the genetically modified lysine production engineering strain The expression level of the protein, including the recombinant strain obtained by the following genetic engineering method: introducing a weak promoter, a weak ribosome binding site into the strain, knocking out or knocking down the coding genes of proteins and enzymes, Inserting random fragments into the encoding gene of the enzyme causes the protein and enzyme activity to be lost.
  • the enhanced protein activity, enhanced expression level of protein encoding genes, enhanced enzyme activity, and enhanced expression level of enzyme encoding genes in the genetically modified lysine production engineering strains include the following genes:
  • the engineering method is used for transformation: introducing a strong promoter and a strong ribosome binding site into the strain, introducing a non-integrated protein and enzyme recombinant expression vector, and introducing a chromosomal integrated protein and enzyme recombinant expression vector.
  • vector refers to a DNA construct that contains a DNA sequence operably linked to a suitable control sequence to express the gene of interest in a suitable host.
  • "Recombinant expression vector” refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide.
  • Recombinant expression vectors may include, for example, i) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) structures or coding sequences that are transcribed into mRNA and translated into proteins; and iii) appropriate transcription And the transcription subunits of translation initiation and termination sequences.
  • the recombinant expression vector is constructed in any suitable manner.
  • vectors include plasmids, viruses, phages, and transposons.
  • Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, such as vaccinia, adenovirus, chicken DNA of viruses such as pox, baculovirus, SV40 and pseudorabies.
  • the "recombinant expression vector" of the present disclosure refers to a mutant vector.
  • transformation, transfection, transduction in the present disclosure have the meaning generally understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host.
  • the methods of transformation, transfection, and transduction include any method of introducing nucleic acid into cells. These methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, and microinjection. , Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.
  • the culture of the strains of the present disclosure can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and each can be adjusted appropriately according to actual conditions.
  • Kind of culture conditions such as temperature, time and pH value of the culture medium.
  • the present disclosure provides a new method for constructing lysine-producing strains, thereby opening up new ideas for the construction of L-lysine-producing strains;
  • the upstream and downstream homology arms for lysC mutation were obtained by PCR amplification Fragment (wherein the 932th base C of the lysC gene is mutated to T). After the above PCR fragments were recovered, they were recombined and ligated with the pK18mobsacB vector digested with EcoRI and BamHI to obtain the mutant vector pK18-lysC of the lysC gene.
  • Competent cells of Corynebacterium glutamicum ATCC13032 were prepared, the pK18-lysC plasmid constructed above was transformed into this strain, and the LBHIS solid medium containing 25 ⁇ g/mL kanamycin (yeast powder 2.5g/L, peptone 5g/ L, sodium chloride 5g/L, brain heart infusion 18.5g/L, sorbitol 91g/L) were cultured at 30°C to obtain the first recombined transformant.
  • the correct transformants were transferred to LB medium containing 5g/L glucose for overnight culture, and then transferred to LB medium containing 100g/L sucrose, cultured at 30°C for 6 hours, and then coated with LB medium supplemented with 100g/L sucrose for selection.
  • lysC mutant strain SCgL30 were prepared, the pK18-lysC plasmid constructed above was transformed into this strain, and the LBHIS solid medium containing 25 ⁇ g/mL kanamycin (yeast powder 2.5
  • Example 3 Construction of a knockout vector for the polypeptide encoding gene shown in Corynebacterium glutamicum SEQ ID NO:1
  • primers SEQ ID NO:1-F1/R1 and SEQ ID NO:1-F2/R2 were designed respectively, and SEQ ID NO: was obtained by PCR amplification using the ATCC13032 genome as a template.
  • After the above PCR fragments were recovered, they were recombined with the pK18mobsacB vector digested with EcoRI and BamHI to obtain the mutant vector pK18-SEQ ID NO:1 of the polypeptide gene shown in SEQ ID NO:1.
  • Example 5 The effect of deletion of the polypeptide shown in SEQ ID NO: 1 on the synthesis of lysine by Corynebacterium glutamicum
  • the strain was inoculated into LB medium containing 10g/L glucose for overnight culture, and the culture was inoculated as seeds into a 24-well plate containing 600 ⁇ l of fermentation medium per well.
  • the initial OD was controlled to 0.5, and the plate was cultured at 30 degrees for 29 hours.
  • the rotating speed of the shaker was 800 rpm, and each strain had 3 parallels.
  • the lysine production and glucose consumption were measured. The results are shown in Table 1. After the polypeptide shown in SEQ ID NO: 1 is knocked out, the Lys production and glucose conversion rate are significantly increased.
  • Example 6 Construction of a weakened polypeptide strain based on SEQ ID NO:1 of dCas9
  • the Cas9 gene of pCas9 (LIU, Jiao, et al. Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microbial cell factories, 2017, 16.1: 205) is mutated with D10A and H840A, and the plasmid skeleton The BsaI restriction site was removed to obtain the pdCas9 plasmid; pnCas9(D10A)-AID-gRNA-ccdBTS(WANG,Yu,etal.Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum.Biotechnology and bioengineering, 2019, 116: 3016-3029)
  • the gRNA-ccdB expression cassette amplified on the plasmid was cloned into the same position of pdCas9, and the CRISPRi plasmid pdCas9gRNA-ccdB that can be
  • the weakening vector of the polypeptide shown in SEQ ID NO:1 and its control vector were constructed.
  • the primers dCas-F/R were designed respectively, and the two primers were subjected to denaturation and annealing procedures to obtain complementary region fragments.
  • the above fragment and the pdCas9-ccdB plasmid digested with BsaI were ligated through Goldengate to construct a weakened vector pdCas-SEQ ID NO:1 with the polypeptide gRNA shown in SEQ ID NO:1.
  • the primers Cas-1, Cas-2, Cas-3 and Cas-4 were designed according to the sequence of pdCas9gRNA-ccdB, and two plasmid fragments were obtained by PCR amplification, and the control vector pdCas9 with gRNA without any complementary region was obtained by recombination with Vazyme recombinase.
  • the aforementioned recombinant vectors pdCas-SEQ ID NO:1 and pdCas9 were respectively transformed into the SCgL30 strain to obtain the attenuated strain SCgL30/pdCas-SEQ ID NO:1 of the polypeptide shown in SEQ ID NO:1 and the control strain SCgL30/pdCas9.
  • Example 7 The effect of weakening of the polypeptide shown in SEQ ID NO: 1 on the synthesis of lysine by Corynebacterium glutamicum
  • the fermentation medium Ingredients (g/L): glucose, 80; yeast powder 8; urea, 9; K 2 HPO 4 , 1.5; MnSO 4 , 0.01; MgSO 4 , 0.6; FeSO 4 , 0.01; MOPS, 42.
  • the strain was inoculated into LB medium containing 10g/L glucose for overnight culture, and the culture was inoculated as seeds into a 24-well plate containing 600 ⁇ l of fermentation medium per well.
  • the initial OD was controlled to 0.5, and the plate was cultured at 30 degrees for 29 hours.
  • the rotating speed of the shaker was 800 rpm, and each strain was 3 in parallel.
  • the lysine production and glucose consumption were measured. The results are shown in Table 2. After the weakening of the polypeptide shown in SEQ ID NO: 1, the yield of Lys and the glucose conversion rate were significantly increased. RT-PCR detection results showed that the transcription level of the polypeptide shown in SEQ ID NO:1 in the SCgL30/pdCas-SEQ ID NO:1 strain was reduced by 31% compared with the control strain.
  • Example 8 Application of the deletion of the polypeptide shown in SEQ ID NO: 1 in the lysine high-producing strain
  • the threonine at position 311 of the aspartate kinase (encoded by the lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 was mutated to isoleucine, and the pyruvate carboxylase (encoded by the pyc gene) was mutated to isoleucine.
  • the 279th to 317th core region sequence of the gene promoter is changed from CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG to CGGCCTTTGATTGTAAGATAAGACATTTAGTATAATTAG, and the diaminopimelate dehydrogenase (ddh gene encoding) gene promoter's 292th to 300th core region sequence is changed from ATGCATCTC was mutated to CCTTGTTAT, and a lysine high-producing strain SCgL40 was constructed.
  • fermentation tests were performed on the SCgL45 and SCgL40 strains.
  • the fermentation medium components were: glucose, 80g/L; yeast powder, 1g/ L; Soy Peptone, 1g/L; NaCl, 1g/L; Ammonium sulfate, 1g/L; Urea, 10g/L; K 2 HPO 4 ⁇ 3H 2 O, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.45g /L; FeSO 4 ⁇ 7H 2 O, 0.05 g/L; Biotin, 0.4 mg/L; Vitamin B1, 0.1 mg/L; MOPS, 40 g/L; initial pH 7.2.
  • the strain was inoculated into TSB liquid medium and cultured for 8 hours, and the culture was inoculated as seeds into a 24-well plate containing 800 ⁇ l of fermentation medium per well.
  • the initial OD 600 was controlled to be about 0.1, cultivated at 30°C for 21 hours, and the plate shaker speed At 800 rpm, each strain is 3 in parallel.
  • the L-lysine production and glucose consumption are detected, and the sugar acid conversion rate from glucose to L-lysine is calculated.
  • TSB medium ingredients (g/L): glucose, 5g/L; yeast powder, 5g/L; soy peptone, 9g/L; urea, 3g/L; succinic acid, 0.5g/L; K 2 HPO 4 ⁇ 3H 2 O, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.1g/L; Biotin, 0.01mg/L; Vitamin B1, 0.1mg/L; MOPS, 20g/L.
  • the results are shown in Table 3. The lysine production and sugar acid conversion rate of the strain after the deletion of the polypeptide shown in SEQ ID NO:1 were significantly improved.
  • the above two strains were subjected to fermentation tests in a 5L fermentor.
  • the components of the fermentation medium are: glucose, 60g/L; molasses: 15g/L; corn steep liquor, 1.5g/L; KCl, 0.5g/L; ammonium sulfate, 20g/L; phosphoric acid, 0.5g/L; ferrous sulfate , 150mg/L; Manganese sulfate, 150mg/L; MgSO 4 ⁇ 7H 2 O, 1g/L; Biotin, 1mg/L; Vitamin B1, 5mg/L; Add glucose mother liquor and ammonium sulfate mother liquor.
  • the strain was activated with TSB solid medium, and then inoculated into TSB liquid medium for 12 hours, 200ml of the above culture was inoculated as seeds into a 5L fermentor equipped with 1.8L fermentation medium, and then fermented at 37°C for 24 hours.
  • the production of L-lysine was detected during the fermentation process.
  • the fermentation results showed that the initial strain SCgL40 had a 24h lysine yield of 30g/L, while the strain SCgL45 lacking the polypeptide shown in SEQ ID NO:1 reached a lysine yield of 48g/L, and the improvement effect was very significant.

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Abstract

提供了一种生产L-赖氨酸的谷氨酸棒杆菌、该L-赖氨酸生产菌株的构建方法和利用所述菌株制备L-赖氨酸的方法，所述L-赖氨酸生产菌株的赖氨酸产量和葡萄糖转化率提高，从而降低生产成本。

Description

生产赖氨酸的微生物以及赖氨酸的生产方法 技术领域
本公开涉及生物技术领域。具体地说，本公开涉及L-赖氨酸高产菌株、所述高产菌株的构建方法和应用。
背景技术
L-赖氨酸，简称赖氨酸，是人类和动物营养中最重要的必需氨基酸，被广泛应用于医药、健康、食品、动物饲料和化妆品等行业中。赖氨酸主要采用微生物发酵法来生产，目前，主要的生产菌株包括大肠杆菌、谷氨酸棒杆菌等的微生物，而由于谷氨酸棒杆菌的生理优越性，谷氨酸棒杆菌已成为工业中最重要的生产菌株。随着生物技术的不断发展，近年来，对谷氨酸棒杆菌进行代谢工程改造以提高其赖氨酸产量的方法也不断出现，包括赖氨酸合成途径的改造，赖氨酸合成竞争途径的弱化等等，当前赖氨酸工业生产菌株的产酸能力已达到较高的水平。
然而，工业上仍然需要生产能力(产量及转化率)更高的赖氨酸生产菌株，以便进一步降低生产成本。因此，本领域急需新的赖氨酸生产菌株的构建方法，以便进一步提高赖氨酸的产量。
发明内容
本公开的目的在于提供一种新的赖氨酸生产菌株，该菌株的构建方法以及利用所构建的赖氨酸生产菌株生产赖氨酸的方法。
在第一方面，本公开提供一种L-赖氨酸的生产菌株，所述L-赖氨酸生产菌株与包含内源性多肽的野生型菌株相比，具有如下(i)-(iii)至少一项所示的特性：
(i)降低或消失的内源性多肽的多肽活性；
(ii)降低或消失的内源性多肽的编码基因的表达水平；
(iii)降低或消失的目标多核苷酸的表达水平；
所述内源性多肽为如SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示氨基酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列编码的多肽；
所述目标多核苷酸为如SEQ ID NO:2所示核苷酸序列的多核苷酸，或与SEQ  ID NO:2所示核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列编码的多核苷酸。
在具体的实施方式中，所述菌株中与SEQ ID NO:1所示多肽同源性为98％以上且C末端具有FhuF结构域的多肽失活。
在具体的实施方式中，所述菌株是谷氨酸棒杆菌。
在具体的实施方式中，所述菌株中SEQ ID NO:1所示多肽失活。
在具体的实施方式中，所述多肽失活是指，与内源性多肽相比，所述多肽的编码基因的转录、表达降低至少30％，优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％，或所述多肽的编码基因被去除；或者，
所述多肽的活性降低至少30％、优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％。
在优选的实施方式中，与包含内源性多肽的L-赖氨酸生产菌株相比，所述L-赖氨酸生产菌株的L-赖氨酸产量提高至少5％、优选至少15％、更优选至少20％、更优选至少25％、更优选至少30％、最优选至少35％。
在优选的实施方式中，与包含内源性多肽的L-赖氨酸生产菌株相比，所述L-赖氨酸生产菌株在生产L-赖氨酸过程中的葡萄糖转化率提高至少10％，优选20％，更优选30％。
在优选的实施方式中，所述内源性多肽的失活可以通过以下方法之一或组合实现：部分敲除或完全敲除多肽的编码基因；编码基因突变；改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译弱化；改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定；或其他任何通过修饰基因编码区及其临近的上下游区域使其失活的方式等。
在优选的实施方式中，使得基因编码序列发生移码突变、错义突变、缺失、起始密码子改变等。
在优选的实施方式中，所述赖氨酸生产菌株中选自以下的一个或几个基因被增强或过表达：
a 1.编码解除赖氨酸反馈抑制的天冬氨酸激酶lysC基因；
b 1.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
c 1.编码二氢二吡啶二羧酸还原酶的dapB基因；
d 1.编码二氨基庚二酸脱水酶的ddh基因；
e 1.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
f 1.编码天冬氨酸-半醛脱水酶的asd基因；
g 1.编码磷酸烯醇丙酮酸羧化酶的ppc基因；或
h 1.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因。
在优选的实施方式中，所述菌株中选自以下的一个或几个基因被弱化或表达降低：
a 2.编码乙醇脱水酶的adhE基因；
b 2.编码乙酸激酶的ackA基因；
c 2.编码磷酸乙酰转移酶的pta基因；
d 2.编码乳酸脱水酶的ldhA基因；
e 2.编码甲酸转运蛋白的focA基因；
f 2.编码丙酮酸甲酸裂解酶的pflB基因；
g 2.编码丙酮酸氧化酶的poxB基因；
h 2.编码天冬氨酸激酶I/高丝氨酸脱水酶I双功能酶的thrA基因；
i 2.编码高丝氨酸激酶的thrB基因；
j 2.编码赖氨酸脱羧酶的ldcC基因；和
h 2.编码赖氨酸脱羧酶的cadA基因。
在第二方面，本公开提供一种L-赖氨酸生产菌株的构建方法，包括以下步骤：
对包含内源性多肽的野生型菌株进行修饰，使所述野生型菌株的内源性多肽的多肽活性降低或消失；或者，
对包含内源性多肽的野生型菌株进行修饰，使所述野生型菌株的内源性多肽的编码基因的表达水平降低或消失；或者，
对野生型菌株进行修饰，使所述野生型菌株内目标多核苷酸的表达水平降低或消失；
其中，所述内源性多肽为如SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示氨基酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列编码的多肽；
所述目标多核苷酸为如SEQ ID NO:2所示核苷酸序列的多核苷酸，或与SEQ ID NO:2所示核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列编码的多核苷酸。
在具体的实施方式中，所述菌株经修饰，其中与SEQ ID NO:1所示多肽同源性高于98％以上且C末端具有FhuF结构域的多肽失活。
在具体的实施方式中，所述菌株是谷氨酸棒杆菌。
在具体的实施方式中，所述菌株经修饰，其中SEQ ID NO:1所示多肽失活。
在具体的实施方式中，所述多肽失活是指，与内源性多肽相比，所述多肽的编码基因的转录、表达降低至少30％，优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％，或所述多肽的编码基因被去除；或者，所述多肽的活性降低至少30％、优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％。
在优选的实施方式中，与包含内源性多肽的L-赖氨酸生产菌株相比，所述L-赖氨酸生产菌株的L-赖氨酸产量提高至少5％、优选至少15％、更优选至少20％、更优选至少25％、更优选至少30％、最优选至少35％。
在优选的实施方式中，与包含内源性多肽的L-赖氨酸生产菌株相比，所述L-赖氨酸生产菌株在生产L-赖氨酸过程中的葡萄糖转化率提高至少10％，优选20％，更优选30％。
在优选的实施方式中，所述内源性多肽的失活可以通过以下方法之一或组合实现：部分敲除或完全敲除编码基因；编码基因突变；改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译弱化；改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定；或其他任何通过修饰细胞中基因编码区及其临近的上下游区域使其弱化或失活的方式等。
在优选的实施方式中，使得多肽基因编码序列发生移码突变、错义突变、缺失、起始密码子改变等。
在优选的实施方式中，所述方法还包括增强或过表达菌株中选自以下的一个或几个基因：
a 1.编码解除赖氨酸反馈抑制的天冬氨酸激酶lysC基因；
b 1.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
c 1.编码二氢二吡啶二羧酸还原酶的dapB基因；
d 1.编码二氨基庚二酸脱水酶的ddh基因；
e 1.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
f 1.编码天冬氨酸-半醛脱水酶的asd基因；
g 1.编码磷酸烯醇丙酮酸羧化酶的ppc基因；或
h 1.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因。
在优选的实施方式中，所述方法还包括弱化菌株中选自以下的一个或几个基因或使菌株中选自以下的一个或几个基因的表达降低：
a 2.编码乙醇脱水酶的adhE基因；
b 2.编码乙酸激酶的ackA基因；
c 2.编码磷酸乙酰转移酶的pta基因；
d 2.编码乳酸脱水酶的ldhA基因；
e 2.编码甲酸转运蛋白的focA基因；
f 2.编码丙酮酸甲酸裂解酶的pflB基因；
g 2.编码丙酮酸氧化酶的poxB基因；
h 2.编码天冬氨酸激酶I/高丝氨酸脱水酶I双功能酶的thrA基因；
i 2.编码高丝氨酸激酶的thrB基因；
j 2.编码赖氨酸脱羧酶的ldcC基因；和
h 2.编码赖氨酸脱羧酶的cadA基因。
在第三方面，本公开提供一种制备L-赖氨酸的方法，所述方法包括：
1)培养第一方面所述的L-赖氨酸生产菌株或第二方面所述方法构建的L-赖氨酸生产菌株，使之产生L-赖氨酸；和
2)任选地从步骤1)所得到的培养液中分离L-赖氨酸。
在第四方面，本公开提供第一方面所述的L-赖氨酸生产菌株或第二方面所述的方法制备的L-赖氨酸生产菌株在生产L-赖氨酸中的应用。
应理解，在本公开范围内中，本公开的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本公开经过广泛而深入的研究，出乎意料地发现一种编码假定蛋白的基因，使得该基因失活能够显著提高赖氨酸的产量，从而获得赖氨酸的高产菌株。在此基础上完成了本公开。
SEQ ID NO:1所示多肽
本公开所述的SEQ ID NO:1(MSIWKRLLVQYPRFADTLTAGQPITLEELATPEVILEAVAKGQEIFGIEQPKHAAQLWFHSLCTAIVGPAVTAMVEFDVIPSLDIRRGQLHNIDGYWFGFRPEEMLVDASLHLSGTQFGESIRVVIDALCAATDLRPAPLWAVASDALGIAASGAGVEAFEEEHAREVAEALIEGMNSVNSVPSPRFNDDDYFIRAGCCMIFHSPRADFCTSCPQKR)所示多肽来源于谷氨酸棒杆菌，其编码核苷酸序列如SEQ ID NO:2所示(ATGAGCATCTGGAAACGTCTGTTAGTGCAGTACCCGCGCTTCGCCGACACCCTCACAGCCGGCCAACCCATCACGCTCGAGGAATT AGCAACCCCGGAAGTGATCTTGGAAGCTGTTGCCAAAGGCCAAGAAATTTTCGGCATTGAGCAGCCAAAACATGCAGCACAACTCTGGTTTCACTCCCTGTGCACCGCAATTGTCGGCCCCGCCGTCACCGCCATGGTGGAATTCGATGTCATCCCCAGCCTCGACATACGTCGAGGTCAGCTGCATAACATCGACGGTTACTGGTTCGGCTTCAGGCCGGAGGAGATGCTTGTCGACGCCTCCCTCCACCTGTCGGGCACCCAATTCGGCGAGAGTATCCGCGTGGTGATTGATGCATTATGCGCTGCCACGGATCTGCGACCGGCACCCCTGTGGGCGGTTGCCTCAGATGCGTTGGGAATCGCAGCTAGCGGCGCAGGTGTCGAGGCCTTTGAAGAAGAACATGCCCGCGAGGTGGCGGAAGCCCTCATTGAAGGAATGAATAGTGTGAACTCAGTTCCATCGCCGCGGTTTAACGACGACGATTATTTCATTCGAGCTGGATGCTGCATGATTTTCCACTCACCACGAGCTGATTTTTGCACGTCGTGCCCACAGAAGAGGTGA)，因其C末端含有一个FhuF的结构域，也被注释为(2Fe-2S)结合蛋白。通过BLAST分析发现，该蛋白多肽在不同谷氨酸棒杆菌中保守性极高，且C末端都存在FhuF的结构域。因此，其在不同谷氨酸棒杆菌中具有相同的功能。
术语定义
多核苷酸，一般是指多核糖核苷酸和多脱氧核糖核苷酸，其可以是非修饰的RNA或DNA，或修饰的RNA或DNA。本公开的核苷酸序列可以是SEQ ID NO:2所述的核苷酸序列，也可以是在严格条件下与SEQ ID NO:2所述的核苷酸序列杂交的核苷酸序列，也可以是与由SEQ ID NO:2制备的探针杂交的核苷酸序列。这里所说的“严格条件”是指特异性杂交可发生而非特异性杂交不发生的条件。如，在一定浓度的盐溶液中洗涤，洗涤1次，优选洗涤2次或3次，相应地浓度为1*SSC、0.1％SDS，优选60℃下0.1*SSC、0.1％SDS，更优选68℃下0.1*SSC、0.1％SDS，探针长度可根据杂交条件选择，通常为100bp到1kb。
术语“多肽”、“肽”和“蛋白质”在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。
术语“表达”包括涉及多肽产生的任何步骤，包括但不限于：转录、转录后修饰、翻译、翻译后修饰、和分泌。
术语“序列同一性”和“同一性百分比”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定：将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分，且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即 一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸插入或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言，可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
本文所用的术语“野生型/内源性”指的是在微生物中多肽或多核苷酸处于未修饰状态，即自然状态。例如，一种可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽、多核苷酸序列或微生物是天然存在的。如本公开所用的，“天然存在”和“野生型”是同义词。
在一些实施方式中，本公开的野生型菌株是指包含内源性多肽的菌株，内源性多肽为氨基酸序列如SEQ ID NO:1所示序列的多肽，或者与SEQ ID NO:1所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性的序列编码的多肽。
在具体的实施方式中，野生型菌株为包含内源性多肽的谷氨酸棒杆菌。
在一些实施方式中，本公开对野生型菌株进行基因改造，得到重组菌株，重组菌株与野生型菌株相比，具有如下(i)-(iii)至少一项所示的特性：
(i)降低或消失的内源性多肽的多肽活性；
(ii)降低或消失的内源性多肽的编码基因的表达水平；
(iii)降低或消失的多核苷酸的表达水平；
所述内源性多肽为如SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示氨基酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列编码的多肽；
所述多核苷酸为如SEQ ID NO:2所示核苷酸序列的多核苷酸，或与SEQ ID NO:2所示核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列编码的多核苷酸。
基于本公开的教导，本领域技术人员应该理解，本公开通过将谷氨酸棒杆菌的SEQ ID NO:1所示多肽失活来提高菌株的赖氨酸产量。因此，本文所述的“SEQ ID NO:1所示多肽失活”表示，与野生型菌株相比，SEQ ID NO:1所示多肽的表达被降低、减弱甚至完全消失，或指产生不表达的基因，或尽管表达但表达产物不具有活性或者具有降低的活性。
在具体实施方式中，相比于野生型或内源性多肽，突变后基因的转录、表达或编码的蛋白的活性降低至少30％，优选至少40％，更优选至少50％，例如至少60％、 至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％，或者该野生型或内源性多肽的编码基因被去除。
本公开所述“修饰”是指任何对野生型菌株或亲本菌株进行的遗传操作，包括但不限于各种分子生物学手段。本公开中的术语“修饰”、“基因编辑”、“基因改造”可相互替换。
本公开中术语“失活”可以通过修饰来实现，包括但不限于通过删除部分或全部编码基因、基因阅读框移码突变、弱化转录或翻译强度、或使用编码具有较低活性的相应酶或蛋白质的基因或等位基因，或使对应基因或酶失去活性，及任选地组合使用这些方法。采用合适的培养方法或基因表达的信号结构的遗传修饰(突变)可以实现基因表达的降低，例如基因表达的信号结构是阻遏基因，活性基因，操纵基因，启动子，弱化子，核糖体结合位点，起始密码子和终止子。
基于本公开的教导，本领域技术人员知晓，可以通过失活SEQ ID NO:1所示多肽的编码基因，或者使得SEQ ID NO:1所示多肽在细胞中不能正常发挥功能来提高赖氨酸的产量。本领域技术人员可以通过本领域已知的技术手段实现上述目的。例如，可以通过使染色体上的酶编码基因缺陷，或者通过修饰一个表达控制序列如启动子或SD序列来实现；也可以通过向编码区域引入氨基酸取代(错义突变)、终止密码子(无义突变)，或向编码区引入一个或两个碱基的插入或缺失(移码突变)或缺失部分基因来实现(Journal of Biological Chemistry 1997，272:8611-8617)，包括但不限于上述方法。
本文所述的“葡萄糖转化率”是指底物葡萄糖转化为产物赖氨酸的摩尔比例。
本文所说的“赖氨酸生产菌株”是指，当细菌在培养基中培养时可以生产赖氨酸并且能够积累赖氨酸，或者能够将赖氨酸分泌到培养基中，也就是能够得到胞外的游离赖氨酸的菌株。例如，可以是天然存在的赖氨酸生产菌株，也可以是经过遗传改造获得的赖氨酸生产工程菌株，这些改造包括但不限于所述菌株中的选自以下的一个或几个基因被增强或多表达：
a 1.编码解除赖氨酸反馈抑制的天冬氨酸激酶lysC基因；
b 1.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
c 1.编码二氢二吡啶二羧酸还原酶的dapB基因；
d 1.编码二氨基庚二酸脱水酶的ddh基因；
e 1.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
f 1.编码天冬氨酸-半醛脱水酶的asd基因；
g 1.编码磷酸烯醇丙酮酸羧化酶的ppc基因；
h 1.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因。
此外，包含但不限于所述菌株中选自以下的一个或几个基因被弱化或表达降低：
a 2.编码乙醇脱水酶的adhE基因；
b 2.编码乙酸激酶的ackA基因；
c 2.编码磷酸乙酰转移酶的pta基因；
d 2.编码乳酸脱水酶的ldhA基因；
e 2.编码甲酸转运蛋白的focA基因；
f 2.编码丙酮酸甲酸裂解酶的pflB基因；
g 2.编码丙酮酸氧化酶的poxB基因；
h 2.编码天冬氨酸激酶I/高丝氨酸脱水酶I双功能酶的thrA基因；
i 2.编码高丝氨酸激酶的thrB基因；
j 2.编码赖氨酸脱羧酶的ldcC基因；
h 2.编码赖氨酸脱羧酶的cadA基因。
在一些实施方式中，经过遗传改造获得的赖氨酸生产工程菌株内降低或消失的蛋白活性、降低或消失的蛋白编码基因的表达水平、降低或消失的酶活性、降低或消失的酶编码基因的表达水平，包括以下述基因工程的方法改造得到的重组菌株：向菌株中引入弱启动子、弱核糖体结合位点，对蛋白、酶的编码基因进行基因敲除或敲减，向蛋白、酶的编码基因中插入随机片段使蛋白、酶的活性丧失。
在一些实施方式中，经过遗传改造获得的赖氨酸生产工程菌株内增强的蛋白活性、增强的蛋白编码基因的表达水平、增强的酶活性、增强的酶编码基因的表达水平，包括以下述基因工程的方法进行改造：向菌株中引入强启动子、强核糖体结合位点，引入非整合型的蛋白、酶的重组表达载体，引入染色体整合型的蛋白、酶的重组表达载体。
术语“载体”指的是DNA构建体，其含有与合适的控制序列可操作地连接的DNA序列，从而在合适的宿主中表达目的基因。“重组表达载体”指用于表达例如编码所需多肽的多核苷酸的DNA结构。重组表达载体可包括，例如包含i)对基因表达具有调控作用的遗传元素的集合，例如启动子和增强子；ii)转录成mRNA并翻译成蛋白质的结构或编码序列；以及iii)适当的转录和翻译起始和终止序列的转录亚单位。重组表达载体以任何合适的方式构建。载体的性质并不重要，并可以使用任何载体，包括质粒、病毒、噬菌体和转座子。用于本公开的可能载体包括但不限于染色体、非染色体和合成DNA序列，例如细菌质粒、噬菌体DNA、酵母质粒以及从质粒和噬菌体DNA的组合中衍生的载体，来自如牛痘、腺病毒、鸡痘、杆状病毒、SV40和伪狂犬病等病毒的DNA。在一些实施方式中，本公开的“重组表达载体”是指突变载体。
本公开中的术语“转化、转染、转导”具有本领域技术人员普遍理解的意思，即将外源性的DNA导入宿主的过程。所述转化、转染、转导的方法包括任何将核酸导入细胞的方法，这些方法包括但不限于电穿孔法、磷酸钙(CaPO4)沉淀法、氯化钙(CaCl2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
本公开的菌株的培养可以根据本领域的常规方法进行，包括但不限于孔板培养、摇瓶培养、批次培养、连续培养和分批补料培养等，并可以根据实际情况适当地调整各种培养条件如温度、时间和培养基的pH值等。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
本公开的优点：
1.本公开提供了一种构建赖氨酸生产菌株的新方法，从而为L-赖氨酸高产菌株的构建开辟了新的思路；
2.本公开构建的赖氨酸生产菌株的赖氨酸产量以及葡萄糖转化率均有所提高；和
3.通过本公开方法生产赖氨酸的成本有所降低。
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
除非另有定义，本文所用的所有技术和科学术语与本公开所属领域普通技术人员通常理解的意义相同。虽然可利用与本文所述相似或等价的任何方法和材料来实施或检验本公开，但优选本文所述的方法和材料。
实施例1.谷氨酸棒杆菌lysC基因突变载体构建
由于关键基因存在反馈调控作用，野生型谷氨酸棒杆菌ATCC13032不能积累赖氨酸。文献报道解除天冬氨酸激酶(lysC基因编码)反馈抑制的菌株可以积累赖氨酸，因此发明人首先通过突变天冬氨酸激酶(第311位Thr突变为Ile)构建一株具有一定赖氨酸合成能力的菌株。根据已公开的谷氨酸棒杆菌ATCC13032基因组序列，分别设计引物lysC-F1/R1和lysC-F2/R2，以ATCC13032基因组为模板，通过PCR扩增获得用于lysC突变的上游和下游同源臂片段(其中lysC基因的第932位碱基C突 变为T)。上述PCR片段回收后，与EcoRI和BamHI酶切处理的pK18mobsacB载体进行重组连接，获得lysC基因的突变载体pK18-lysC。
实施例2.谷氨酸棒杆菌lysC基因突变菌株构建
制备谷氨酸棒杆菌ATCC13032的感受态细胞，将上述构建的pK18-lysC质粒转化该菌株，涂布含有25μg/mL卡那霉素的LBHIS固体培养基(酵母粉2.5g/L，蛋白胨5g/L，氯化钠5g/L，脑心浸液18.5g/L，山梨醇91g/L)上，30℃培养获得第一次重组的转化子。正确转化子转接含有5g/L葡萄糖的LB培养基过夜培养，然后转接含有100g/L蔗糖的LB培养基，30℃培养6h后涂布添加100g/L蔗糖的LB培养基进行筛选，获得lysC突变的菌株SCgL30。
实施例3.谷氨酸棒杆菌SEQ ID NO:1所示多肽编码基因敲除载体构建
根据已报道的谷氨酸棒杆菌ATCC13032基因组序列，分别设计引物SEQ ID NO:1-F1/R1和SEQ ID NO:1-F2/R2，以ATCC13032基因组为模板通过PCR扩增获得SEQ ID NO:1所示多肽基因的上下游同源臂。上述PCR片段回收后，与EcoRI和BamHI酶切处理的pK18mobsacB载体进行重组连接，获得SEQ ID NO:1所示多肽基因的突变载体pK18-SEQ ID NO:1。
实施例4.谷氨酸棒杆菌SEQ ID NO:1所示多肽缺失菌株构建
制备SCgL30菌株的感受态细胞，将上述构建的pK18-SEQ ID NO:1质粒转化该菌株，涂布含有5g/L葡萄糖和25μg/mL卡那霉素的LBHIS固体培养基上，30℃培养获得第一次重组的转化子。正确转化子转接含有5g/L葡萄糖的LB培养基过夜培养，然后转接含有100g/L蔗糖的LB培养基，30℃培养6h后涂布添加100g/L蔗糖的LB培养基进行筛选，获得SEQ ID NO:1所示多肽缺失的菌株SCgL31。
实施例5.SEQ ID NO:1所示多肽缺失对谷氨酸棒杆菌赖氨酸合成的影响
为了测试谷氨酸棒杆菌中SEQ ID NO:1所示多肽基因敲除对菌株产赖氨酸的影响，分别对SCgL30和SCgL31进行发酵测试，发酵培养基为CGXII培养基，主要成份为(g/L)：(NH 4) 2SO 4，20；尿素，5；KH 2PO 4，1；K 2HPO 4·3H 2O，1.3；3-(N-吗啉基)丙磺酸(MOPS)，42；CaCl 2，0.01；FeSO 4·7H 2O，0.01、MnSO 4·H 2O，0.01、ZnSO 4·7H 2O，0.001；CuSO 4，0.0002；NiCl·6H 2O，0.00002；MgSO 4·7H 2O，0.25；原儿茶酸，0.03；维生素B 1，0.0001；生物素，0.0002；葡萄糖，80。首先将菌株接种到含有10g/L葡萄糖的LB培养基中过夜培养，培养物作为种子接种到每孔含有600μl发酵培养基的24孔板中，初始OD控制为0.5，30度培养29h，孔板摇床转速为 800rpm，每个菌株3个平行，发酵结束后检测赖氨酸产量和葡萄糖消耗量。结果如表1所示，SEQ ID NO:1所示多肽敲除后Lys产量及葡萄糖转化率均显著提升。
表1.
Figure PCTCN2021071517-appb-000001
实施例6.基于dCas9的SEQ ID NO:1所示多肽弱化菌株构建
首先将pCas9(LIU,Jiao,et al.Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.Microbial cell factories,2017,16.1:205)质粒的Cas9基因进行D10A和H840A突变，同时将质粒骨架中的BsaI酶切位点去除，获得pdCas9质粒；再从pnCas9(D10A)‐AID‐gRNA‐ccdBTS(WANG,Yu,et al.Expanding targeting scope,editing window,and base transition capability of base editing in Corynebacterium glutamicum.Biotechnology and bioengineering,2019，116：3016-3029)质粒上扩增gRNA‐ccdB表达盒克隆至pdCas9的相同位置，获得可以高效构建的CRISPRi质粒pdCas9gRNA-ccdB。
利用上述基于dCas9的弱化系统，构建SEQ ID NO:1所示多肽的弱化载体及其对照载体。根据已报道的谷氨酸棒杆菌ATCC13032基因组序列，分别设计引物dCas-F/R，两条引物通过变性和退火程序，获得互补区片段。上述片段与BsaI酶切处理的pdCas9-ccdB质粒通过Goldengate连接，构建获得带有SEQ ID NO:1所示多肽gRNA的弱化载体pdCas-SEQ ID NO:1。根据pdCas9gRNA-ccdB序列设计引物Cas-1、Cas-2、Cas-3和Cas-4，通过PCR扩增获得两个质粒片段，通过Vazyme重组酶重组获得gRNA不含任何互补区的对照载体pdCas9。将上述重组载体pdCas-SEQ ID NO:1和pdCas9分别转化SCgL30菌株，获得SEQ ID NO:1所示多肽的弱化菌株SCgL30/pdCas-SEQ ID NO:1及其对照菌株SCgL30/pdCas9。
实施例7.SEQ ID NO:1所示多肽弱化对谷氨酸棒杆菌赖氨酸合成的影响
为了测试谷氨酸棒杆菌中SEQ ID NO:1所示多肽基因表达弱化对菌株产赖氨酸的影响，分别对SCgL30/pdCas9和SCgL30/pdCas-SEQ ID NO:1进行发酵测试，发酵培养基成份为(g/L)：葡萄糖，80；酵母粉8；尿素，9；K 2HPO 4，1.5；MnSO 4，0.01；MgSO 4，0.6；FeSO 4，0.01；MOPS，42。首先将菌株接种到含有10g/L葡萄糖的LB培养基中过夜培养，培养物作为种子接种到每孔含有600μl发酵培养基的24孔板中，初始OD控制为0.5，30度培养29h，孔板摇床转速为800rpm，每个菌株3 个平行，发酵结束后检测赖氨酸产量和葡萄糖消耗量。结果如表2所示，SEQ ID NO:1所示多肽弱化后，Lys产量及葡萄糖转化率均显著提升。RT-PCR检测结果显示SCgL30/pdCas-SEQ ID NO:1菌株中SEQ ID NO:1所示多肽的转录水平与对照菌株相比下降31％。
表2.
Figure PCTCN2021071517-appb-000002
实施例8.SEQ ID NO:1所示多肽缺失在赖氨酸高产菌株中的应用
(1)赖氨酸高产菌株中SEQ ID NO:1所示多肽缺失菌株构建
利用基于pK18mobsacB的同源重组技术将谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸，将丙酮酸羧化酶(pyc基因编码)基因启动子的第279位至第317位核心区序列由CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG突变为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG，将二氨基庚二酸脱氢酶(ddh基因编码)基因启动子的第292位至第300位核心区序列由ATGCATCTC突变为CCTTGTTAT，构建获得一株赖氨酸高产菌株SCgL40。制备SCgL40菌株的感受态细胞，将上述构建的pK18-SEQ ID NO:1质粒转化该菌株，涂布含有5g/L葡萄糖和25μg/mL卡那的LBHIS培养基，30℃培养6h后涂布添加100g/L蔗糖的LB培养基进行筛选，获得SEQ ID NO:1所示多肽缺失的菌株SCgL45。
(2)SEQ ID NO:1所示多肽缺失对高产菌株赖氨酸合成的影响
为了测试SEQ ID NO:1所示多肽缺失对高产菌株产L-赖氨酸的影响，分别对SCgL45和SCgL40菌株进行发酵测试，发酵培养基成份为：葡萄糖，80g/L；酵母粉，1g/L；大豆蛋白胨，1g/L；NaCl，1g/L；硫酸铵，1g/L；尿素，10g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.45g/L；FeSO 4·7H 2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。首先将菌株接种到TSB液体培养基中培养8h，培养物作为种子接种到每孔含有800μl发酵培养基的24孔板中，初始OD 600控制约为0.1，30℃培养21h，孔板摇床转速为800rpm，每个菌株3个平行，发酵结束后检测L-赖氨酸产量和葡萄糖消耗量，并计算从葡萄糖到L-赖氨酸的糖酸转化率。TSB培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，5g/L；大豆蛋白胨，9g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。 结果如表3所示，SEQ ID NO:1所示多肽缺失后菌株的赖氨酸产量和糖酸转化率均有显著的提高。
表3.
Figure PCTCN2021071517-appb-000003
为了进一步确定高产菌株中SEQ ID NO:1所示多肽缺失的效果，将上述两菌株在5L发酵罐中进行发酵测试。发酵培养基成份为：葡萄糖，60g/L；糖蜜：15g/L；玉米浆，1.5g/L；KCl，0.5g/L；硫酸铵，20g/L；磷酸，0.5g/L；硫酸亚铁，150mg/L；硫酸锰，150mg/L；MgSO 4·7H 2O，1g/L；生物素，1mg/L；维生素B1，5mg/L；流加葡萄糖母液和硫酸铵母液。首先将菌株利用TSB固体培养基活化，然后接种到TSB液体培养基中培养12h，200ml上述培养物作为种子接种到装有1.8L发酵培养基的5L发酵罐，37℃发酵24h。发酵过程中检测L-赖氨酸产量。发酵结果显示出发菌株SCgL40发酵24h赖氨酸产量为30g/L，而SEQ ID NO:1所示多肽缺失的菌株SCgL45赖氨酸产量达到了48g/L，提升效果非常显著。
本公开实施例中所用的引物见下表：
Figure PCTCN2021071517-appb-000004
Figure PCTCN2021071517-appb-000005
实施例9.SEQ ID NO:1所示多肽在不同谷氨酸棒杆菌中同源性的比较
利用NCBI数据库对SEQ ID NO:1所示多肽进行序列比对分析，结果显示该蛋白多肽在不同谷氨酸棒杆菌中DNA和氨基酸序列的一致性分别达到98.01％和98.56％以上，表明该蛋白多肽在谷氨酸棒杆菌中的保守性非常高。此外，通过序列分析发现该蛋白的C末端含有一个FhuF(大肠杆菌细胞质中的一种含有2Fe-2S的铁还原酶)的C-末端结构域，且该结构域在上述谷氨酸棒杆菌的同源序列中均存在。因此，在不同谷氨酸棒杆菌中上述同源基因的缺失或弱化都有利于赖氨酸的合成。
在本公开提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本公开的上述讲授内容之后，本领域技术人员可以对本公开作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种L-赖氨酸生产菌株，其中，所述L-赖氨酸生产菌株与包含内源性多肽的野生型菌株相比，具有如下(i)-(iii)至少一项所示的特性：
    (i)降低或消失的内源性多肽的多肽活性；
    (ii)降低或消失的内源性多肽的编码基因的表达水平；
    (iii)降低或消失的目标多核苷酸的表达水平；
    所述内源性多肽为如SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示氨基酸序列具有至少90％的序列同一性的序列编码的多肽；
    所述目标多核苷酸为如SEQ ID NO:2所示核苷酸序列的多核苷酸，或与SEQ ID NO:2所示核苷酸序列具有至少90％的序列同一性的序列编码的多核苷酸。
  2. 根据权利要求1所述的L-赖氨酸生产菌株，其中，所述L-赖氨酸生产菌株为产L-赖氨酸的谷氨酸棒杆菌。
  3. 根据权利要求2所述的L-赖氨酸生产菌株，其中，所述菌株中与SEQ ID NO:1所示多肽同源性为98％以上且C末端具有FhuF结构域的多肽失活。
  4. 根据权利要求2或3所述的L-赖氨酸生产菌株，其中，所述菌株中SEQ ID NO:1所示多肽失活。
  5. 根据权利要求3或4所述的L-赖氨酸生产菌株，其中，所述多肽失活是指，与内源性多肽相比，所述多肽的编码基因的转录、表达降低至少30％，或所述多肽的编码基因被去除；或者，所述多肽的活性降低至少30％。
  6. 根据权利要求1-5任一项所述的L-赖氨酸生产菌株，其中，与包含内源性多肽的L-赖氨酸生产菌株相比，所述L-赖氨酸生产菌株的L-赖氨酸产量提高至少5％；
    优选地，与包含内源性多肽的L-赖氨酸生产菌株相比，所述L-赖氨酸生产菌株在生产L-赖氨酸过程中的葡萄糖转化率提高至少10％。
  7. 根据权利要求1-6任一项所述的L-赖氨酸生产菌株，其中，所述的L-赖氨酸生产菌株中选自以下的一个或几个基因被增强或过表达：
    a 1.编码解除赖氨酸反馈抑制的天冬氨酸激酶lysC基因；
    b 1.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
    c 1.编码二氢二吡啶二羧酸还原酶的dapB基因；
    d 1.编码二氨基庚二酸脱水酶的ddh基因；
    e 1.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
    f 1.编码天冬氨酸-半醛脱水酶的asd基因；
    g 1.编码磷酸烯醇丙酮酸羧化酶的ppc基因；
    h 1.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因；
    可选地，所述L-赖氨酸生产菌株中选自以下的一个或几个基因被弱化或表达降低：
    a 2.编码乙醇脱水酶的adhE基因；
    b 2.编码乙酸激酶的ackA基因；
    c 2.编码磷酸乙酰转移酶的pta基因；
    d 2.编码乳酸脱水酶的ldhA基因；
    e 2.编码甲酸转运蛋白的focA基因；
    f 2.编码丙酮酸甲酸裂解酶的pflB基因；
    g 2.编码丙酮酸氧化酶的poxB基因；
    h 2.编码天冬氨酸激酶I/高丝氨酸脱水酶I双功能酶的thrA基因；
    i 2.编码高丝氨酸激酶的thrB基因；
    j 2.编码赖氨酸脱羧酶的ldcC基因；
    h 2.编码赖氨酸脱羧酶的cadA基因。
  8. 一种L-赖氨酸生产菌株的构建方法，其中，包括以下步骤：
    对包含内源性多肽的野生型菌株进行修饰，使所述野生型菌株的内源性多肽的多肽活性降低或消失；或者，
    对包含内源性多肽的野生型菌株进行修饰，使所述野生型菌株的内源性多肽的编码基因的表达水平降低或消失；或者，
    对野生型菌株进行修饰，使所述野生型菌株内目标多核苷酸的表达水平降低或消失；
    其中，所述内源性多肽为如SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示氨基酸序列具有至少90％的序列同一性的序列编码的多肽；
    所述目标多核苷酸为如SEQ ID NO:2所示核苷酸序列的多核苷酸，或与SEQ ID NO:2所示核苷酸序列具有至少90％的序列同一性的序列编码的多核苷酸。
  9. 根据权利要求8所述的构建方法，其中，所述L-赖氨酸生产菌株为产L-赖氨酸的谷氨酸棒杆菌。
  10. 根据权利要求9所述的构建方法，其中，所述菌株经修饰，其中与SEQ ID NO:1所示多肽同源性高于98％以上且C末端具有FhuF结构域的多肽失活。
  11. 根据权利要求9或10所述的方法，其中，所述菌株经修饰，其中SEQ ID NO:1所示多肽失活。
  12. 根据权利要求10或11所述的方法，其中，所述多肽失活是指，与内源性多肽相比，所述多肽的编码基因的转录、表达降低至少30％，或所述多肽的编码基因被去除；或者，
    所述多肽的活性降低或消失是指，所述多肽的活性降低至少30％。
  13. 根据权利要求8-12任一项所述的方法，其中，所述构建方法还包括增强或过表达菌株中选自以下的一个或几个基因：
    a 1.编码解除赖氨酸反馈抑制的天冬氨酸激酶lysC基因；
    b 1.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
    c 1.编码二氢二吡啶二羧酸还原酶的dapB基因；
    d 1.编码二氨基庚二酸脱水酶的ddh基因；
    e 1.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
    f 1.编码天冬氨酸-半醛脱水酶的asd基因；
    g 1.编码磷酸烯醇丙酮酸羧化酶的ppc基因；
    h 1.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因；
    可选地，所述构建方法还包括弱化菌株中选自以下的一个或几个基因或使菌株中选自以下的一个或几个基因的表达降低：
    a 2.编码乙醇脱水酶的adhE基因；
    b 2.编码乙酸激酶的ackA基因；
    c 2.编码磷酸乙酰转移酶的pta基因；
    d 2.编码乳酸脱水酶的ldhA基因；
    e 2.编码甲酸转运蛋白的focA基因；
    f 2.编码丙酮酸甲酸裂解酶的pflB基因；
    g 2.编码丙酮酸氧化酶的poxB基因；
    h 2.编码天冬氨酸激酶I/高丝氨酸脱水酶I双功能酶的thrA基因；
    i 2.编码高丝氨酸激酶的thrB基因；
    j 2.编码赖氨酸脱羧酶的ldcC基因；
    h 2.编码赖氨酸脱羧酶的cadA基因。
  14. 一种制备L-赖氨酸的方法，其中，所述方法包括：
    1)培养权利要求1-7中任一项所述的L-赖氨酸生产菌株，或者权利要求8-13中任一项所述方法构建的L-赖氨酸生产菌株，使之产生L-赖氨酸；和
    2)任选地从步骤1)所得到的培养液中分离L-赖氨酸。
  15. 权利要求1-7中任一项所述的L-赖氨酸生产菌株或权利要求8-13中任一项所述的方法制备的L-赖氨酸生产菌株在生产L-赖氨酸中的应用。
PCT/CN2021/071517 2020-01-15 2021-01-13 生产赖氨酸的微生物以及赖氨酸的生产方法 WO2021143727A1 (zh)

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