WO2023284419A1 - 丙酮酸脱氢酶的突变体及其用于生产l-氨基酸的方法 - Google Patents

丙酮酸脱氢酶的突变体及其用于生产l-氨基酸的方法 Download PDF

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WO2023284419A1
WO2023284419A1 PCT/CN2022/094731 CN2022094731W WO2023284419A1 WO 2023284419 A1 WO2023284419 A1 WO 2023284419A1 CN 2022094731 W CN2022094731 W CN 2022094731W WO 2023284419 A1 WO2023284419 A1 WO 2023284419A1
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amino acid
pyruvate dehydrogenase
host cell
mutant
seq
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French (fr)
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郑平
陈久洲
孙际宾
蔡柠匀
郭轩
周文娟
刘岯
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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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • 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
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01051—Pyruvate dehydrogenase (NADP+) (1.2.1.51)

Definitions

  • the disclosure belongs to the field of biotechnology, and in particular relates to a mutant of pyruvate dehydrogenase, a polynucleotide encoding the mutant, a host cell containing the mutant, and a method for producing L-amino acid using the mutant.
  • Pyruvate dehydrogenase multienzyme complex is a group of rate-limiting enzymes.
  • PDHC catalyzes the irreversible oxidative decarboxylation of pyruvate produced during glycolysis into acetyl-CoA, which converts sugar Aerobic oxidation is linked with the Krebs cycle and oxidative phosphorylation, and is a key enzyme of the Krebs cycle.
  • PDHC consists of pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3p).
  • enzymes Unlike phosphorylases, enzymes have both inactive (phosphorylated) and active (dephosphorylated) forms. There are various allosteric modulators that regulate the switch between the two enzyme forms and are influenced by hormonal activity.
  • the E1p enzyme is encoded by the aceE gene. It has been reported that attenuating the expression of AceE can increase the production of L-amino acid in Corynebacterium. CN106715687A also reported that any amino acid mutation at positions 190-205 or 415-440 of AceE can improve the output of Corynebacterium glutamicum L-lysine. So far, no new mutation sites have been reported.
  • this disclosure has carried out saturation mutation on position 217, and found that some mutants can significantly increase the production of L-lysine in host cells. On this basis, this disclosure is completed.
  • the purpose of the present disclosure is to provide a novel pyruvate dehydrogenase mutant, a host cell containing the mutant, and a method for producing L-lysine using the host cell containing the mutant.
  • the present disclosure provides a novel pyruvate dehydrogenase mutant, the mutant is:
  • amino acid sequence is as shown in SEQ ID NO: 1, and its 217th position is any one of alanine, aspartic acid, glutamic acid, leucine, and proline.
  • Polypeptides that are more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to SEQ ID NO: 1, and corresponding to The 217th position of SEQ ID NO:1 is any one in alanine, aspartic acid, glutamic acid, leucine, proline.
  • One or more amino acids are added or deleted at both ends of the polypeptide shown in SEQ ID NO: 1, and the 217th position corresponding to SEQ ID NO: 1 is alanine, aspartic acid, glutamic acid, leucine Any one of amino acid and proline.
  • 1, 2, 3, 4, 5, 6 amino acids are added or deleted at both ends of the polypeptide shown in SEQ ID NO:1.
  • the present disclosure provides an encoding polynucleotide encoding a pyruvate dehydrogenase mutant.
  • the present disclosure provides an expression cassette comprising the coding nucleotides of the pyruvate dehydrogenase mutant.
  • the encoding nucleotides are operably linked to a promoter.
  • the present disclosure provides a vector, especially an expression vector, containing a nucleotide encoding a pyruvate dehydrogenase mutant.
  • the present disclosure provides a host cell comprising a nucleotide encoding a pyruvate dehydrogenase mutant.
  • the host cells include but not limited to Escherichia (genus Escherichia), Erwinia (genus Erwinia), Serratia (genus Serratia), Providencia (genus Providencia) , genus Enterobacteria, genus Salmonella, genus Streptomyces, genus Pseudomonas, genus Brevibacterium, genus Corynebacterium of microorganisms.
  • the host cell is Corynebacterium glutamicum or Escherichia coli.
  • the host cells include, but are not limited to, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC 14067 and derivative strains thereof.
  • the present disclosure provides the use of the pyruvate dehydrogenase mutant or its encoding nucleotide in the production of L-amino acid.
  • the L-amino acid is selected from L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine or L- alanine.
  • the present disclosure provides a method for producing L-amino acids, the method comprising culturing the host cell of the fifth aspect to produce L-amino acids, further comprising the step of separating, extracting or recovering L-amino acids from the culture medium .
  • the L-amino acid includes but not limited to L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine or L-alanine; more preferably, the L-amino acid is L-lysine.
  • the pyruvate dehydrogenase mutant provided by the present disclosure can increase the yield and conversion rate of L-amino acid of the strain, and while increasing the yield, does not inhibit the growth of the strain, and provides a new strategy for large-scale production of L-amino acid.
  • the selected/optional/preferred “numeric range” not only includes the numerical endpoints at both ends of the range, but also includes all natural numbers covered in the middle of the numerical endpoints relative to the aforementioned numerical endpoints.
  • pyruvate dehydrogenase in the present disclosure refers to one of the enzymes constituting the pyruvate dehydrogenase multi-enzyme complex (PDHC), which is involved in the conversion of pyruvate into acetyl-CoA.
  • PDHC pyruvate dehydrogenase multi-enzyme complex
  • pyruvate dehydrogenase is not particularly limited as long as it has the corresponding activity, and it may be a pyruvate dehydrogenase derived from a microorganism of the genus Corynebacterium, specifically, Corynebacterium glutamicum Enzymes, but not limited thereto.
  • the pyruvate dehydrogenase may be the amino acid sequence of SEQ ID NO: 1 or have at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90% of the amino acid sequence of SEQ ID NO: 1 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homologous amino acid sequences.
  • the E1p protein having the amino acid sequence of SEQ ID NO: 1 can be encoded by the aceE gene having the polynucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
  • any polynucleotide sequence encoding a pyruvate dehydrogenase may fall within the scope of the present disclosure.
  • the polynucleotide sequence can be at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, Polynucleotide sequences that are 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homologous.
  • the polynucleotide sequence encoding the protein may have various changes in the coding region within the range of not changing the amino acid sequence of the protein expressed from the coding region. body.
  • the pyruvate dehydrogenase mutant of the present disclosure may include an amino acid sequence as shown in SEQ ID NO: 1, and the 217th amino acid corresponding to SEQ ID NO: 1 is selected from alanine, aspartic acid, glutamic acid, A polypeptide of either leucine or proline.
  • the pyruvate dehydrogenase mutants of the present disclosure may not only include polypeptides having the amino acid sequence of SEQ ID NO: 1, but also include polypeptides having amino acid sequences shown in SEQ ID NO: 1 having 75% or higher, specifically Specifically 80% or higher, more specifically 85% or higher, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% % or higher homology of pyruvate dehydrogenase mutants, as long as they are selected from the group consisting of alanine, aspartic acid, glutamic acid, leucine, Any one of the prolines, and its activity was greatly weakened compared with the wild-type pyruvate dehydrogenase activity.
  • amino acid sequence having substantially the same or corresponding biological activity as the polypeptide having the amino acid sequence of SEQ ID NO: 1 should also belong to the scope of the present disclosure, and those skilled in the art know that the amino acid sequence described in SEQ ID NO: 1 One to several amino acid substitutions, deletions, additions and substitutions are carried out on the basis of the amino acid sequence shown, and an amino acid sequence having substantially the same or corresponding biological activity as the polypeptide of the amino acid sequence of SEQ ID NO: 1 can be obtained.
  • polynucleotide in the present disclosure refers to a polymer composed of nucleotides.
  • a polynucleotide may be in the form of an individual fragment or an integral part of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in number or concentration, capable of being separated by standard Molecular biology methods (eg, using cloning vectors) identify, manipulate and restore sequences and their component nucleotide sequences.
  • a nucleotide sequence is represented by a DNA sequence (ie A, T, G, C)
  • this also includes an RNA sequence (ie A, U, G, C) where "U" replaces "T”.
  • polynucleotide refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or which may be a building block or constituent of a larger nucleotide structure, as expressed Vector or polycistronic sequence.
  • Polynucleotides include DNA, RNA and cDNA sequences.
  • the polynucleotide encoding the pyruvate dehydrogenase mutant of the present disclosure includes the polynucleotide shown in SEQ ID NO: 2, and a polynucleotide mutated at positions 649-651 thereof.
  • the polynucleotides of the present disclosure also include polynucleotides with the polynucleotides shown in SEQ ID NO: 2 having 75% or higher, specifically 80% or higher, more specifically 85% or higher, and even more specifically Any polynucleotide of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or more homology.
  • homology in this disclosure refers to the percentage of identity between two polynucleotide or polypeptide moieties. Homology between the sequences of one moiety and another can be determined by techniques known in the art. For example, homology can be determined by directly aligning the sequence information of two polynucleotide molecules or two polypeptide molecules using readily available computer programs. Examples of computer programs may include BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc.), and the like.
  • the homology between polynucleotides can be determined by hybridizing polynucleotides under the condition that a stable duplex is formed between homologous regions, dissociation with a single-strand-specific nuclease, and then analyzing the dissociated fragments Perform size determination.
  • wild-type in the present disclosure refers to an object that can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism 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 pyruvate dehydrogenase in the present disclosure refers to a polypeptide having an amino acid sequence as shown in SEQ ID NO:1.
  • mutant in the present disclosure refers to a polynucleotide or polypeptide comprising an alteration (i.e., substitution, insertion, And/or missing polynucleotides, wherein substitution refers to the replacement of a nucleotide occupying a position with a different nucleotide.
  • Deletion refers to the removal of a nucleotide occupying a position.
  • Insertion refers to a position adjacent to and immediately following Nucleotides are added after the nucleotide occupying the position.
  • the "mutation" in the present disclosure is “substitution”, which is a mutation caused by the substitution of a base in one or more nucleotides by another different base, also known as a base substitution mutation (substitution) or point mutation (point mutation).
  • RNA production and protein production includes any step involved in RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • vector in the present disclosure refers to a DNA construct containing DNA sequences operably linked to appropriate control sequences to express a gene of interest in a suitable host.
  • "Recombinant expression vector” refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide.
  • Recombinant expression vectors can include, for example, a collection of genetic elements comprising i) regulatory effects on gene expression, such as promoters and enhancers; ii) structural or coding sequences that are transcribed into mRNA and translated into protein; and iii) appropriate transcription and translation initiation and termination sequences of the transcriptional subunit.
  • Recombinant expression vectors are constructed in any suitable manner.
  • vectors are not critical, and any vector may be used, including plasmids, viruses, phage 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, from sources such as vaccinia, adenovirus, chicken DNA from viruses such as pox, baculovirus, SV40, and pseudorabies.
  • host cell refers to any cell type transformed, transfected, transduced, etc., comprising a pyruvate dehydrogenase mutant or expression vector of the present disclosure.
  • recombinant host cell encompasses a host cell that differs from the parental cell after the introduction of a transcription initiation element or a recombinant expression vector, in particular by transformation.
  • transformation in the present disclosure has the meaning commonly understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host.
  • the transformation method includes any method for introducing nucleic acid into cells, and these methods include but are not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.
  • the host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it can contain the pyruvate dehydrogenase mutant of the present disclosure.
  • the host cell refers to a prokaryotic cell.
  • the host cell is derived from a microorganism suitable for fermentative production of amino acids, organic acids, bio-based materials or pharmaceutical compounds, which may include Escherichia, Erwinia Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas (Pseudomonas), Brevibacterium (Brevibacterium), Corynebacterium (Corynebacterium) and other strains, but not limited thereto. Alternatively, it may be Corynebacterium glutamicum.
  • the derivative strain may be any strain as long as the strain has the ability to produce L-amino acid.
  • the host cell is a lysine-producing host cell.
  • the host cell for producing lysine may be a derivative strain expressed on the basis of Corynebacterium glutamicum ATCC 13032 that releases feedback inhibition of aspartokinase.
  • the lysine-producing host cells may be other types of strains capable of producing lysine.
  • the lysine-producing host cells may also include but are not limited to attenuated or reduced expression of one or more genes selected from the following:
  • the cadA gene encoding lysine decarboxylase.
  • the lysine-producing host cells may also include but are not limited to enhanced or overexpressed one or more genes selected from the following:
  • the dapA gene encoding the dihydrodipyridine synthetase that relieves lysine feedback inhibition
  • dapD encoding tetrahydrodipicolate succinylase and dapE encoding succinyldiaminopimelate deacylase
  • pntAB gene encoding nicotinamine adenine dinucleotide transhydrogenase
  • the host cell is a threonine-producing host cell.
  • the threonine-producing host cell is a strain expressing aspartokinase LysC that relieves feedback inhibition based on Corynebacterium glutamicum ATCC 13032.
  • the threonine-producing host cells may also be other types of bacterial strains capable of producing threonine.
  • one or more genes selected from the following are enhanced or overexpressed in the threonine-producing host cell:
  • the host cell is an isoleucine-producing host cell.
  • the isoleucine-producing host cell is a strain that produces L-isoleucine by substituting alanine for the amino acid at position 323 of the gene of L-threonine dehydratase ilvA.
  • the isoleucine-producing host cell can also be other kinds of strains with isoleucine production ability.
  • the host cell is a host cell that produces O-acetyl homoserine.
  • the O-acetylhomoserine-producing host cell is a strain that produces O-acetylhomoserine by inactivating O-acetylhomoserine (thiol)-lyase.
  • the host cell producing O-acetyl homoserine may also be other kinds of bacterial strains capable of producing O-acetyl homoserine.
  • the host cell is a methionine-producing host cell.
  • the methionine-producing host cell is a strain that produces methionine by inactivating transcriptional regulators of methionine and cysteine.
  • the methionine-producing host cells may also be other types of bacterial strains capable of producing methionine.
  • L-amino acid in the present disclosure refers to all L-amino acids that can be produced from different carbon sources by pyruvate. More specifically, L-amino acids may include L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine or L-alanine amino acids, and even more specifically, L-lysine or L-valine.
  • culture in the present disclosure can be carried out according to conventional methods in the art, including but not limited to orifice culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and can be appropriately adjusted according to actual conditions Various culture conditions such as temperature, time, and pH value of the medium, etc.
  • the experimental techniques and experimental methods used in this embodiment are conventional technical methods unless otherwise specified, such as the experimental methods that do not indicate specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through normal commercial channels.
  • the disclosure has carried out an in-depth analysis of the AceE enzyme, and it is predicted that position 217 may be a site that affects its activity, so the site is mutated for subsequent research. Subsequently, according to the Goldengate cloning method reported in the literature (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) to construct a targeting The pCas9gRNA plasmid of the 217th amino acid residue codon of the aceE gene, the target DNA binding region of the sgRNA is CCAACTGTGTCCATGGGTCT.
  • the specific method is as follows: 217-F/217-R is denatured and annealed to obtain a DNA double-stranded product with cohesive ends, and then the Goldengate clone is carried out with the pCas9gRNA-ccdB plasmid (referring to CN112111469B) ( Golden Gate Assembly Kit, #E1601), to obtain the pCas9gRNA-aceE217 plasmid, which expresses the Cas9 protein and the sgRNA targeting the site-directed mutation region.
  • the primers used for the above plasmid construction are listed in Table 1.
  • Embodiment 2 Construction of the mutant of the 217th amino acid codon mutation of Corynebacterium glutamicum aceE gene
  • ATCC13032 is used as the starting strain, and a lysine-producing bacterial strain is first constructed, that is, a T311I point mutation (removing the feedback inhibition of the enzyme) is introduced into the aspartokinase gene lysC of the Corynebacterium glutamicum ATCC13032 bacterial strain, and the The acid carboxylation kinase gene pyc introduced the P458S point mutation (releasing the feedback inhibition of the enzyme), and the homoserine dehydrogenase gene hom introduced the V59A point mutation (weakening the activity of the enzyme) to obtain the lysine high-yielding strain AHP-3.
  • the CRISPR/Cas9 genome editing system based on single-chain recombination (refer to patent CN112111469A for the basic plasmid construction process) was used to saturate the 217 site of pyruvate dehydrogenase (aceE gene encoding) of Corynebacterium glutamicum.
  • the competent cells of the high-lysine-producing strain AHP-3 were prepared, and the recombinant helper plasmid pRecT plasmid was electrotransformed into the L-lysine-producing strain AHP-3 of Corynebacterium glutamicum to obtain the AHP-3-pRecT strain.
  • the AHP-3-pRecT strain was prepared by the method reported in the literature (Ruan Y, Zhu L, Li Q. Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnol Lett.2015; 37:2445-52.), to obtain AHP-3-pRecT competent cells.
  • S217A, S217C, S217D, S217E, S217F, S217G, S217H, S217I, S217K, S217L, S217M, S217N, S217P, S217Q were designed , S217R, S217T, S217V, S217W, S217Y single-stranded DNA (Table 1), the recombination template used for mutant construction.
  • composition of TSB medium is (g/L): glucose, 5g/L; yeast powder, 5g/L; soybean peptone, 9g/L; urea, 3g/L; succinic acid, 0.5g/L; 1g/L; MgSO4 ⁇ 7H2O, 0.1g/L; biotin, 0.01mg/L; vitamin B1, 0.1mg/L; MOPS, 20g/L.
  • the single clones obtained above were identified by colony PCR using specific primers S217-jd-F/aceE-jd-R (Table 1), and the correct clones were confirmed by sequencing, and finally S217A, S217C, S217D, S217E, S217G, and S217L were obtained. , S217P, S217T and S217V 9 mutants, and the other 10 mutants were not obtained.
  • the pRecT and pCas9gRNA-aceE217 plasmids in the 217th amino acid codon mutant strain of the aceE gene obtained above were lost, and the specific process was as follows: the monoclonal culture was cultured overnight at 37°C in non-resistant TSB liquid medium, and then in the non-resistant TSB solid medium plate to mark single clones, and then streak the grown monoclonal bacteria on three solid plates (TSB+5 ⁇ g/mL chloramphenicol, TSB+15 ⁇ g/mL kanamycin and TSB), Cultivate at 30°C for 24 hours to obtain strains that do not grow on chloramphenicol and kanamycin-resistant plates but can grow on TSB plates, that is, mutant strains that lose the two plasmids, and are named SCgL46 to SCgL54 respectively, and the corresponding mutants are respectively For S217A, S217C, S217D, S217E, S217
  • the wild-type AHP-3 strain was used as a control, and the fermentation medium components were: glucose, 80g/L; ammonium sulfate, 20g/L; urea, 5g/L; KH 2 PO 4 , 1g/L; K 2 HPO 4 ⁇ 3H 2 O, 1.3g/L; MOPS, 42g/L; CaCl 2 , 0.01g/L; FeSO 4 ⁇ 7H 2 O, 0.01g/L; MnSO 4 ⁇ H 2 O, 0.01g/L; ZnSO 4 ⁇ 7H 2 O, 0.001g/L; CuSO 4 , 0.0002g/L; NiCl ⁇ 6H 2 O, 0.00002g/L; MgSO 4 ⁇ 7H 2 O, 0.25g/L; protocatechuic acid, 0.03g/L; Su, 0.0002g/L; vitamin B1, 0.0001g/L; initial pH7.2.
  • the strain was inoculated into TSB liquid medium and cultured for 8 hours.
  • the culture was inoculated as a seed into a 24-well plate containing 800 ⁇ L of fermentation medium per well.
  • the initial OD 600 was controlled to be about 0.1, and cultured at 30°C for 21 hours. 800rpm, 3 parallels for each strain, detect L-lysine production and glucose consumption after fermentation, and calculate the sugar-acid conversion rate from glucose to L-lysine.
  • APEEaceE gene (SEQ ID NO: 2):

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Abstract

丙酮酸脱氢酶突变体，其在氨基酸序列如SEQ ID NO:1所示的基础上，且其217位突变为丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个。该突变体可以提高菌株L-氨基酸的产量和转化率，且在提高产量的同时，没有抑制菌株的生长，为大规模生产L-氨基酸提供了新的途径。

Description

丙酮酸脱氢酶的突变体及其用于生产L-氨基酸的方法 技术领域
本公开属于生物技术领域，具体涉及一种丙酮酸脱氢酶的突变体，编码该突变体的多核苷酸，含有该突变体的宿主细胞，以及利用该突变体生产L-氨基酸的方法。
背景技术
丙酮酸脱氢酶多酶复合物(PDHC)是一组限速酶，在谷氨酸棒杆菌中，PDHC催化糖酵解过程产生的丙酮酸不可逆地氧化脱羧转化成乙酰辅酶A，将糖的有氧氧化与三羧酸循环和氧化磷酸化连接起来，是三羧酸循环的关键酶。PDHC由丙酮酸脱氢酶(E1p)、二氢硫辛酰胺乙酰转移酶(E2p)和二氢硫辛酰胺脱氢酶(E3p)组成。酶具有无活性(磷酶化)和有活性(去磷酸化)两种形式，这一点与磷酸化酶不同。有多种别构调节剂调控二种酶形式的转换，并受激素活动影响。
其中，E1p酶由aceE基因编码。已有报道弱化AceE的表达可以提高棒杆菌L-氨基酸的产量。CN106715687A也报道了AceE 190-205位或415-440位的任一氨基酸突变可提高谷氨酸棒杆菌L-赖氨酸的产量。目前，尚未有新的突变位点被报道。
发明内容
本公开根据对AceE结构预测的结果，对217位进行了饱和突变，发现部分突变体可显著宿主细胞L-赖氨酸的产量，在此基础上，完成本公开。
本公开的目的是提供一种新型的丙酮酸脱氢酶突变体，含有该突变体的宿主细胞，以及利用含有该突变体的宿主细胞生产L-赖氨酸的方法。
第一方面，本公开提供了一种新型的丙酮酸脱氢酶突变体，所述突变体为：
1)氨基酸序列如SEQ ID NO:1所示，且其217位为丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个。
2)与SEQ ID NO:1同源性高于90％、91％、92％、93％、94％、95％、96％、97％、98％、99％以上的多肽，且在对应于SEQ ID NO:1的217位为丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个。
3)在SEQ ID NO:1所示多肽的两端添加、缺失一个或多个氨基酸，且在对应于SEQ ID NO:1的217位为丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个。优选地，在SEQ ID NO:1所示多肽的两端添加、缺失1、2、3、4、5、6个氨基酸。
第二方面，本公开提供了编码丙酮酸脱氢酶突变体的编码多核苷酸。
第三方面，本公开提供含所述的丙酮酸脱氢酶突变体的编码核苷酸的表达盒。所述编码核苷酸与启动子可操作连接。
第四方面，本公开提供了含有丙酮酸脱氢酶突变体的编码核苷酸的载体，尤其是表达载体。
第五方面，本公开提供了含有丙酮酸脱氢酶突变体的编码核苷酸的宿主细胞。
进一步地，所述宿主细胞包括但不限于埃希氏杆菌属(genus Escherichia)、欧文氏菌属(genus Erwinia)、沙雷氏菌属(genus Serratia)、普罗维登斯菌属(genus Providencia)、肠道菌属(genus Enterobacteria)、沙门氏菌属(genus Salmonella)、链霉菌属(genus Streptomyces)、假单胞菌属(genus Pseudomonas)、短杆菌属(genus Brevibacterium)、棒状杆菌属(genus Corynebacterium)的微生物。可选地，所述宿主细胞为谷氨酸棒杆菌、大肠杆菌。优选地，所述宿主细胞包括但不限于谷氨酸棒杆菌ATCC13032，谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌ATCC 14067及其衍生菌株。
第六方面，本公开提供所述的丙酮酸脱氢酶突变体或其编码核苷酸在生产L-氨基酸中的应用。优选地，所述L-氨基酸选自L-赖氨酸、L-苏氨酸、L-甲硫氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸或L-丙氨酸。
第七方面，本公开提供了一种生产L-氨基酸的方法，所述方法包括培养第五方面的宿主细胞使之 生产L-氨基酸，进一步包括从培养基中分离提取或回收L-氨基酸的步骤。
进一步地，所述L-氨基酸包括但不限于L-赖氨酸、L-苏氨酸、L-甲硫氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸或L-丙氨酸；更优选地，所述L-氨基酸为L-赖氨酸。
本公开提供的丙酮酸脱氢酶突变体，可以提高菌株L-氨基酸的产量和转化率，且在提高产量的同时，没有抑制菌株的生长，为大规模生产L-氨基酸提供了新的策略。
具体实施方式
术语定义
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然本公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
当用于权利要求书或说明书时，选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
本公开的术语“丙酮酸脱氢酶”指的是组成丙酮酸脱氢酶多酶复合物(PDHC)的酶中的一种，其参与丙酮酸转变为乙酰辅酶A。如本文所使用，丙酮酸脱氢酶不被具体限制，只要其具有相应的活性，并且其可以是衍生自棒状杆菌属——具体地，谷氨酸棒状杆菌——的微生物的丙酮酸脱氢酶，但是不限于此。例如，丙酮酸脱氢酶可以是SEQ ID NO:1的氨基酸序列或者与SEQ ID NO:1的氨基酸序列具有至少75％，具体地至少80％，更具体地85％，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、或者99％或更高的同源性的氨基酸序列。具有SEQ ID NO:1的氨基酸序列的E1p蛋白可以由具有SEQ ID NO:2的多核苷酸序列的aceE基因编码，但是不限于此。另外，如果氨基酸序列与上述序列具有同源性并且与SEQ ID NO:1的蛋白质具有基本上相同或相应的生物学活性，则具有缺失、修饰、置换或添加的氨基酸序列也应当属于本公开内容的范围是显而易见的。在本公开内容中，编码丙酮酸脱氢酶的任何多核苷酸序列可以属于本公开内容的范围。例如，多核苷酸序列可以是与SEQ ID NO:2的多核苷酸序列具有至少75％，具体地至少80％，更具体地85％，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、或者99％或更高的同源性的多核苷酸序列。另外，基于密码子简并性或考虑生物体表达蛋白质优选的密码子，编码蛋白质的多核苷酸序列可以在不改变从编码区表达的蛋白质的氨基酸序列的范围内具有编码区上的各种变体。
本公开的丙酮酸脱氢酶突变体可以包括氨基酸序列如SEQ ID NO:1所示，在对应于SEQ ID NO:1的217位氨基酸选自丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个的多肽。
此外，本公开的丙酮酸脱氢酶突变体不仅可以包括具有SEQ ID NO:1的氨基酸序列的多肽，还可以包括与SEQ ID NO:1所示氨基酸序列的多肽具有75％或更高，具体地80％或更高，更具体地85％或更高，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、和99％或更高的同源性的丙酮酸脱氢酶突变体，只要它们在对应于EQ ID NO:1的217位氨基酸选自丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个，且其活性与野生型的丙酮酸脱氢酶活性相比大幅度减弱。显而易见的是，与具有SEQ ID NO:1的氨基酸序列的多肽具有基本上相同或相应生物学活性的氨基酸序列也应当属于本公开内容的范围，本领域技术人员均知道在SEQ ID NO:1所示氨基酸序列的基础上进行1个到几个氨基酸的取代、缺失、添加和置换，可以得到与SEQ ID NO:1的氨基酸序列的多肽具有基本上相同或相应生物学活性的氨基酸序列。
本公开的术语“多核苷酸”指由核苷酸组成的聚合物。多核苷酸可以是单独片段的形式，也可以是更大的核苷酸序列结构的一个组成部分，其是从至少在数量或浓度上分离一次的核苷酸序列衍生而来的，能够通过标准分子生物学方法(例如，使用克隆载体)识别、操纵以及恢复序列及其组分核苷酸 序列。当一个核苷酸序列通过一个DNA序列(即A、T、G、C)表示时，这也包括一个RNA序列(即A、U、G、C)，其中“U”取代“T”。换句话说，“多核苷酸”指从其他核苷酸(单独的片段或整个片段)中去除的核苷酸聚合物，或者可以是一个较大核苷酸结构的组成部分或成分，如表达载体或多顺反子序列。多核苷酸包括DNA、RNA和cDNA序列。
具体地，本公开的丙酮酸脱氢酶突变体的编码多核苷酸包括SEQ ID NO:2所示的多核苷酸，且在其649-651位突变的多核苷酸。此外，本公开的多核苷酸还包括与SEQ ID NO:2所示多核苷酸具有75％或更高，具体地80％或更高，更具体地85％或更高，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、和99％或更高的同源性的任何多核苷酸。
本公开的术语“同源性”指的是两个多核苷酸或多肽部分之间的同一性的百分比。一个部分与另一个部分的序列之间的同源性可以通过本领域中已知的技术测定。例如，同源性可以通过使用容易可获得的计算机程序直接排列两个多核苷酸分子或两个多肽分子的序列信息来测定。计算机程序的实例可以包括BLAST(NCBI)、CLC Main Workbench(CLC bio)、MegAlignTM(DNASTAR Inc.)等。另外，多核苷酸之间的同源性可以通过如下步骤来测定：在同源区之间形成稳定双链的条件下杂交多核苷酸，利用单链特异性核酸酶分解，然后对分解的片段进行大小测定。
本公开的术语“野生型的”指在自然界中可以找到的对象。例如，一种存在于生物体中，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或多核苷酸序列是天然存在的。如本公开所用的，“天然存在的”和“野生型的”是同义词。在一些实施方式中，本公开中野生型的丙酮酸脱氢酶是指氨基酸序列如SEQ ID NO：1所示的多肽。
本公开的术语“突变体”是指相对于“野生型”，或者“相比较的”多核苷酸或多肽，在一个或多个(例如，若干个)位置处包含改变(即，取代、插入和/或缺的多核苷酸，其中，取代是指用不同的核苷酸置换占用一个位置的核苷酸。缺失是指去除占据某一位置的核苷酸。插入是指在邻接并且紧随占据位置的核苷酸之后添加核苷酸。
在一些实施方式中，本公开的“突变”为“取代”，是由一个或多个核苷酸中的碱基被另一个不同的碱基取代所引起的突变，也称为碱基置换突变(subsititution)或点突变(point mutation)。
本公开的术语“表达”包括涉及RNA产生及蛋白产生的任何步骤，包括但不限于：转录、转录后修饰、翻译、翻译后修饰和分泌。
本公开的术语“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标基因。“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。
本公开的术语“宿主细胞”是指包含本公开的丙酮酸脱氢酶突变体或表达载体转化、转染、转导等的任何细胞类型。术语“重组宿主细胞”涵盖导入转录起始元件或重组表达载体后不同于亲本细胞的宿主细胞，重组宿主细胞具体通过转化来实现。
本公开的术语“转化”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、磷酸钙沉淀法、氯化钙(CaCl 2)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。
本公开的宿主细胞可以是原核细胞或真核细胞，只要是能够包含本公开的丙酮酸脱氢酶突变体的细胞即可。在一些实施方式中，宿主细胞指原核细胞，具体地，宿主细胞来源于适合发酵生产氨基酸、有机酸、生物基材料或药物化合物的微生物，可以包括埃希氏杆菌属(Escherichia)、欧文氏菌属(Erwinia)、沙雷氏菌属(Serratia)、普罗维登斯菌属(Providencia)、肠道菌属(Enterobacteria)、沙门氏菌 属(Salmonella)、链霉菌属(Streptomyces)、假单胞菌属(Pseudomonas)、短杆菌属(Brevibacterium)、棒状杆菌属(Corynebacterium)等的菌株，但不限于此。可选地，可以是具谷氨酸棒状杆菌。作为优选地，可以是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌ATCC 14067及其衍生菌株等。示例地，所述衍生菌株可以是任何菌株，只要该菌株具有生产L-氨基酸的能力。
示例地，宿主细胞为生产赖氨酸的宿主细胞。在一些实施方式中，对于生产赖氨酸的宿主细胞，可以是在谷氨酸棒杆菌ATCC 13032基础上表达解除反馈抑制的天冬氨酸激酶的衍生菌株。此外，生产赖氨酸的宿主细胞也可以是具有赖氨酸生产能力的其他种类的菌株。
在一些实施方式中，所述生产赖氨酸的宿主细胞中还可以包括但不限于选自以下的一个或多个基因被弱化或表达降低：
a.编码乙醇脱氢酶的adhE基因；
b.编码乙酸激酶的ackA基因；
c.编码磷酸乙酰转移酶的pta基因；
d.编码乳酸脱氢酶的ldhA基因；
e.编码甲酸转运蛋白的focA基因；
f.编码丙酮酸甲酸裂解酶的pflB基因；
g.编码丙酮酸氧化酶的poxB基因；
h.编码天冬氨酸激酶I/高丝氨酸脱氢酶I双功能酶的thrA基因；
i.编码高丝氨酸激酶的thrB基因；
j.编码赖氨酸脱羧酶的ldcC基因；和
h.编码赖氨酸脱羧酶的cadA基因。
在一些实施方式中，所述生产赖氨酸宿主细胞中还可以包括但不限于选自以下的一个或多个基因被增强或过表达：
a.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
b.编码二氢二吡啶二羧酸还原酶的dapB基因；
c.编码二氨基庚二酸脱氢酶的ddh基因；
d.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；
e.编码天冬氨酸-半醛脱氢酶的asd基因；
f.编码磷酸烯醇丙酮酸羧化酶的ppc基因；
g.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因；
i.编码赖氨酸的运输蛋白lysE基因。
示例地，宿主细胞为生产苏氨酸的宿主细胞。在一些实施方式中，生产苏氨酸的宿主细胞为在谷氨酸棒杆菌ATCC 13032基础上表达解除反馈抑制的天冬氨酸激酶LysC的菌株。在另外一些实施方式中，生产苏氨酸的宿主细胞也可以是具有苏氨酸生产能力的其他种类的菌株。
在一些实施方式中，所述生产苏氨酸的宿主细胞中选自以下的一个或多个基因被增强或过表达：
a.编码苏氨酸操纵子的thrABC基因；
b.编码解除反馈抑制的高丝氨酸脱氢酶的hom基因；
c.编码甘油醛-3-磷酸脱氢酶的gap基因；
d.编码丙酮酸羧化酶的pyc基因；
e.编码苹果酸：醌氧化还原酶的mqo基因；
f.编码转酮酶的tkt基因；
g.编码6-磷酸葡糖酸脱氢酶的gnd基因；
h.编码苏氨酸输出的thrE基因；
i.编码烯醇酶的eno基因。
示例地，宿主细胞为生产异亮氨酸的宿主细胞。在一些实施方式中，生产异亮氨酸的宿主细胞是通过用丙氨酸取代L-苏氨酸脱水酶ilvA基因第323位的氨基酸而产生L-异亮氨酸的菌株。在另外一 些实施方式中，生产异亮氨酸的宿主细胞也可以是具有异亮氨酸生产能力的其他种类的菌株。
示例地，宿主细胞为生产O-乙酰高丝氨酸的宿主细胞。在一些实施方式中，生产O-乙酰高丝氨酸的宿主细胞是通过使O-乙酰高丝氨酸(硫醇)-裂解酶失活而产生O-乙酰高丝氨酸的菌株。在另外一些实施方式中，生产O-乙酰高丝氨酸的宿主细胞也可以是具有O-乙酰高丝氨酸生产能力的其他种类的菌株。
示例地，宿主细胞为生产蛋氨酸的宿主细胞。在一些实施方式中，生产蛋氨酸的宿主细胞是通过使甲硫氨酸和半胱氨酸的转录调节因子失活而产生蛋氨酸的菌株。在另外一些实施方式中，生产蛋氨酸的宿主细胞也可以是具有蛋氨酸生产能力的其他种类的菌株。
本公开的术语“L-氨基酸”指的是可以通过丙酮酸从不同碳源生产的所有L-氨基酸。更具体地，L-氨基酸可以包括L-赖氨酸、L-苏氨酸、L-甲硫氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸或L-丙氨酸，并且甚至更具体地，L-赖氨酸或L-缬氨酸。
本公开的术语“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。
除非在发明中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。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施例)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如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。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实施例1.编辑质粒pCas9gRNA-aceE217构建
本公开通过对AceE酶进行了深入分析，其中预测217位可能是影响其活性的位点，因此对该位点进行突变进行后续研究。随后，根据文献报道的Goldengate克隆方法(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)构建靶向aceE基因第217位氨基酸残基密码子的pCas9gRNA质粒，sgRNA的靶DNA结合区为CCAACTGTGTCCATGGGTCT。具体方法如下：将217-F/217-R进行变性退火，得到带有粘性末端的DNA双链产物，再与pCas9gRNA-ccdB质粒(参考CN112111469B)进行Goldengate克隆(
Figure PCTCN2022094731-appb-000001
Golden Gate组装试剂盒，#E1601)，获得pCas9gRNA-aceE217质粒，该质粒表达Cas9蛋白和靶向定点突变区的sgRNA。上述质粒构建所用引物如表1所示。
本公开实施例中所用的引物见下表1：
Figure PCTCN2022094731-appb-000002
Figure PCTCN2022094731-appb-000003
Figure PCTCN2022094731-appb-000004
实施例2.构建谷氨酸棒杆菌aceE基因第217位氨基酸密码子突变的突变体
由于野生型谷氨酸棒杆菌不能生产赖氨酸，而在谷氨酸棒杆菌引入lysC、pyc和hom的点突变之后都可以生产赖氨酸。本实施例以ATCC13032为出发菌株，首先构建一个产赖氨酸的菌株，即在谷氨酸棒杆菌ATCC13032菌株天冬氨酸激酶基因lysC引入了T311I点突变(解除酶的反馈抑制)，在丙酮酸羧化激酶基因pyc引入P458S点突变(解除酶的反馈抑制)，在高丝氨酸脱氢酶基因hom引入V59A点突变(弱化酶的活性)，获得赖氨酸高产菌株AHP-3。
随后，利用基于单链重组的CRISPR/Cas9基因组编辑系统(基础质粒构建过程参考专利CN112111469A)，对谷氨酸棒杆菌丙酮酸脱氢酶(aceE基因编码)217位点进行饱和突变。首先制备赖氨酸高产菌株AHP-3的感受态细胞，将重组辅助质粒pRecT质粒电转化至谷氨酸棒杆菌产L-赖氨酸菌株AHP-3，获得AHP-3-pRecT菌株。AHP-3-pRecT菌株采用文献报道的方法制备感受态细胞(Ruan Y,Zhu L,Li Q.Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity.BiotechnolLett.2015；37:2445-52.)，得到AHP-3-pRecT感受态细胞。
为了对aceE基因217位的氨基酸位氨基酸密码子进行野生型以外的19种突变，设计了S217A、 S217C、S217D、S217E、S217F、S217G、S217H、S217I、S217K、S217L、S217M、S217N、S217P、S217Q、S217R、S217T、S217V、S217W、S217Y的单链DNA(表1)，用于突变体构建的重组模板。向上述制备获得的AHP-3-pRecT感受态细胞电转化1μg pCas9gRNA-aceE217质粒和10μg的单链DNA，加入1mL 46℃预热的TSB培养基，46℃孵育6min，30℃孵育3h，涂布添加5μg/mL氯霉素、15μg/mL卡那霉素和0.05mM IPTG的TSB平板，培养2天，获得克隆。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；K2HPO4·3H2O，1g/L；MgSO4·7H2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。将上述获得的单克隆分别采用特异性引物S217-jd-F/aceE-jd-R(表1)进行菌落PCR鉴定，正确克隆进行测序确认，最终获得S217A、S217C、S217D、S217E、S217G、S217L、S217P、S217T和S217V 9种突变体，另外10种突变体未获得。
将上述获得的aceE基因第217位氨基酸密码子突变体菌株中的pRecT和pCas9gRNA-aceE217质粒丢失，具体过程如下：单克隆在无抗性的TSB液体培养基37℃过夜培养，再在无抗性的TSB固体培养基平板划单克隆，然后对长出的单克隆菌分别在3种固体平板(TSB+5μg/mL氯霉素、TSB+15μg/mL卡那霉素和TSB)上划线，30℃培养24h，得到氯霉素和卡那霉素抗性平板不长，而TSB平板可以长的菌株，即为丢失两种质粒的突变菌株，分别命名为SCgL46至SCgL54，对应的突变体分别为S217A、S217C、S217D、S217E、S217G、S217L、S217P、S217T和S217V。
实施例3.谷氨酸棒杆菌aceE基因217位突变对L-赖氨酸合成的影响
为了测试谷氨酸棒杆菌aceE基因第217位氨基酸突变对赖氨酸高产菌株产L-赖氨酸的影响，分别对SCgL46至SCgL54菌株进行发酵测试。以野生型AHP-3菌株作对照，发酵培养基成份为：葡萄糖，80g/L；硫酸铵，20g/L；尿素，5g/L；KH 2PO 4，1g/L；K 2HPO 4·3H 2O，1.3g/L；MOPS，42g/L；CaCl 2，0.01g/L；FeSO 4·7H 2O，0.01g/L；MnSO 4·H 2O，0.01g/L；ZnSO 4·7H 2O，0.001g/L；CuSO 4，0.0002g/L；NiCl·6H 2O，0.00002g/L；MgSO 4·7H 2O，0.25g/L；原儿茶酸，0.03g/L；生物素，0.0002g/L；维生素B1，0.0001g/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。
结果如表2所示，突变体S217C、S217G、S217T和S217V的L-赖氨基酸产量都低于野生型对照菌株；而突变体S217A、S217D、S217E、S217L和S217P的L-赖氨基酸产量都高于野生型对照菌株。可见，这些氨基酸位点突变在L-赖氨酸及其衍生物生产中具有较好的应用前景。
表2
Figure PCTCN2022094731-appb-000005
AceE(SEQ ID NO：1):
Figure PCTCN2022094731-appb-000006
APEEaceE基因(SEQ ID NO：2):
Figure PCTCN2022094731-appb-000007
Figure PCTCN2022094731-appb-000008
本说明书公开的所有技术特征都可以任何组合方式进行组合。本说明所公开的每个特征也可以被其它具有相同、相等或相似作用的特征所替换。因此，除非特殊说明，所公开的每一特征仅仅是一系列相等或相似特征的实例。
此外，从上述描述中，本领域技术人员可从本公开中很容易清楚本公开的关键特征，在不脱离本公开的精神及范围的情况下，可对发明进行很多修改以适应各种不同的使用目的及条件，因此这类修改也旨在落入所附权利要求书的范围内。

Claims (12)

  1. 一种丙酮酸脱氢酶突变体，其特征在于，所述突变体为以下组中的任一个：
    1)氨基酸序列如SEQ ID NO:1所示的基础上，且其217位突变为丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个；
    2)在SEQ ID NO:1所示多肽的两端添加或缺失1、2、3、4、5、6个氨基酸，且在对应于SEQ ID NO:1的217位为丙氨酸、天冬氨酸、谷氨酸、亮氨酸、脯氨酸中的任一个。
  2. 如权利要求1所述丙酮酸脱氢酶突变体的编码核苷酸。
  3. 含有如权利要求2所述的丙酮酸脱氢酶突变体的编码核苷酸的表达盒。
  4. 含有如权利要求2所述的丙酮酸脱氢酶突变体的编码核苷酸的载体。
  5. 如权利要求4所述的载体，其特征在于，是表达载体。
  6. 含有如权利要求2所述的丙酮酸脱氢酶突变体的编码核苷酸的宿主细胞。
  7. 含有如权利要求2所述的编码核苷酸或如权利要求4所述的载体的宿主细胞，其特征在于，是埃希氏杆菌属(Escherichia)、欧文氏菌属(Erwinia)、沙雷氏菌属(Serratia)、普罗维登斯菌属(Providencia)、肠道菌属(Enterobacteria)、沙门氏菌属(Salmonella)、链霉菌属(Streptomyces)、假单胞菌属(Pseudomonas)、短杆菌属(Brevibacterium)、棒状杆菌属(Corynebacterium)的微生物。
  8. 如权利要求7所述的宿主细胞，其特征在于，是谷氨酸棒杆菌ATCC13032，谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌ATCC 14067菌株。
  9. 如权利要求1所述的丙酮酸脱氢酶突变体或其编码核苷酸在生产L-氨基酸中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述L-氨基酸选自L-赖氨酸、L-苏氨酸、L-甲硫氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸或L-丙氨酸。
  11. 一种生产L-氨基酸的方法，其特征在于，包括培养如权利要求7或8所述的宿主细胞使之生产L-氨基酸，进一步包括从培养基中分离提取或回收L-氨基酸的步骤。
  12. 如权利要求11所述的方法，其特征在于，所述L-氨基酸选自L-赖氨酸、L-苏氨酸、L-甲硫氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸或L-丙氨酸。
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