ES2976595B2 - PYRUVATE DEHYDROGENASE MUTANT AND METHOD FOR PRODUCING L-AMINO ACID USING THE SAME - Google Patents

Description

[0001] DESCRIPTION

[0004] PYRUVATE DEHYDROGENASE MUTANT AND METHOD FOR PRODUCING

[0006] L-AMINO ACID USING THE SAME

[0008] TECHNICAL FIELD

[0009] The present description relates to the field of biotechnology, and specifically relates to a pyruvate dehydrogenase mutant, a polynucleotide encoding the mutant, a host cell containing the mutant, and a method for producing L-amino acids using the mutant.

[0011] BACKGROUND

[0012] The pyruvate dehydrogenase multienzyme complex (PDHC) is a group of rate-limiting enzymes. In Corynebacterium glutamicum, PDHC catalyzes the irreversible oxidative decarboxylation of pyruvate generated during glycolysis to acetyl-CoA, linking the aerobic oxidation of sugars to the tricarboxylic acid cycle and oxidative phosphorylation, and is a key enzyme for the tricarboxylic acid cycle. PDHC is composed of pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p), and dihydrolipoamide dehydrogenase (E3p). The enzyme differs from phosphorylase in that it has both inactive (phosphorylated) and active (dephosphorylated) forms. There are a plurality of allosteric modulators that regulate the conversion of the two forms of the enzyme and are affected by hormonal activity.

[0013] In particular, the E1p enzyme is encoded by the AceE gene. It has been observed that weakening AceE expression can improve L-amino acid yield in Corynebacterium. Document CN106715687A also indicates that any amino acid mutation at positions 190 to 205 or 415 to 440 of AceE can improve L-lysine yield by Corynebacterium glutamicum. No new mutation sites have been indicated at this time.

[0015] BRIEF DESCRIPTION

[0016] Based on the results of the AceE structure prediction, in the present disclosure a saturation mutagenesis at position 217 has been carried out and it was observed that some of the mutants can significantly increase L-lysine yield in host cells, thus completing the present disclosure.

[0017] One objective of the present description 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.

[0018] In the first aspect, the present disclosure provides a novel pyruvate dehydrogenase mutant, wherein the mutant is as follows:

[0019] 1) a mutant in which an amino acid sequence is exposed at SEQ ID NO:1 and position 217 of SEQ ID NO:1 is any of alanine, aspartic acid, glutamic acid, leucine, and proline;

[0020] 2) a polypeptide having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO:1 and position 217 corresponding to SEQ ID NO:1 is any of alanine, aspartic acid, glutamic acid, leucine, and proline; or 3) a mutant in which one or more amino acids are added or deleted at both ends of a polypeptide exposed in SEQ ID NO:1 and position 217 corresponding to SEQ ID NO:1 is any of alanine, aspartic acid, glutamic acid, leucine, and proline; preferably, 1, 2, 3, 4, 5, or 6 amino acids are added or deleted at both ends of the polypeptide exposed in SEQ ID NO:1.

[0021] In the second aspect, the present description provides a coding polynucleotide that encodes the pyruvate dehydrogenase mutant.

[0022] In the third aspect, the present description provides an expression cassette containing the coding nucleotide of the pyruvate dehydrogenase mutant. The coding nucleotide is operationally linked to a promoter.

[0023] In the fourth aspect, the present description provides a vector containing the nucleotide encoding the pyruvate dehydrogenase mutant, in particular, an expression vector.

[0024] In the fifth aspect, the present description provides a host cell containing the nucleotide encoding the pyruvate dehydrogenase mutant.

[0025] The host cell also includes, but is not limited to, microorganisms of the genus Escherichia, genus Erwinia, genus Serratia, genus Providencia, genus Enterobacteriaceae, genus Salmonella, genus Streptomyces, genus Pseudomonas, genus Brevibacterium, and the genus Corynebacterium. Optionally, the host cell is Corynebacterium glutamicum or Escherichia coli. Preferably, the host cell includes, but is not limited to, ATCC13032 of Corynebacterium glutamicum, ATCC13869 of Corynebacterium glutamicum, ATCC 14067 of Corynebacterium glutamicum, and strains derived from these.

[0026] In the sixth aspect, the present disclosure provides the use of the pyruvate dehydrogenase mutant or its coding nucleotide in the production of an L-amino acid.

[0027] Preferably, the L-amino acid is selected from the group consisting of L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine and L-alanine.

[0028] In the seventh aspect, the present disclosure provides a method for producing an L-amino acid, wherein the method comprises culturing the host cell according to the fifth aspect to produce an L-amino acid, and further comprises a step of isolating and extracting or recovering the L-amino acid from a medium.

[0029] In addition, the L-amino acid includes, but is not limited to, L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine, and L-alanine; most preferably, the L-amino acid is L-lysine.

[0030] The pyruvate dehydrogenase mutant provided in the present description can improve the yield and conversion rate of L-amino acids in strains, and does not inhibit the growth of the strains while improving the yield, thus providing a new strategy for large-scale L-amino acid production.

[0032] DETAILED DESCRIPTION

[0033] Definitions of terms

[0034] When used in combination with the term "including" in the claims and/or in the descriptive memorandum, the term "a" may refer to "one", or to "one or more", "at least one" and "one or more than one".

[0035] As used in the claims and in the descriptive memorandum, the term "including", "features", "comprising", or "containing" is intended to be inclusive or open-ended and does not exclude additional or unmentioned elements, methods, or steps.

[0036] Throughout the application document, the term "approximately" means that a value includes the standard deviation of the error caused by the device or method used to measure the value.

[0037] It is applicable to the content described herein that the term "or" is defined only as alternatives and "and/or", but the term "or" used in the claims refers to "and/or" unless expressly stated otherwise as either alternatives only or mutual exclusion between alternatives.

[0038] The selected/optional/preferred "numerical range", when used in the claims or in the descriptive memory, includes not only the numerical endpoints at both ends of the range, but also all natural numbers covered between the above numerical endpoints with respect to these numerical endpoints.

[0039] The term “pyruvate dehydrogenase” as used herein refers to one of the enzymes comprising the pyruvate dehydrogenase multienzyme complex (PDHC), and participates in the conversion of pyruvate to acetyl-CoA. As used herein, pyruvate dehydrogenase is not specifically limited to any one enzyme, provided it has corresponding activity, and may be, but is not limited to, a pyruvate dehydrogenase derived from a microorganism of the genus Corynebacterium, specifically Corynebacterium glutamicum. For example, pyruvate dehydrogenase may be an amino acid sequence from SEQ ID NO:1 or an amino acid sequence that exhibits at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence of SEQ ID NO:1. The E1p protein, which exhibits an amino acid sequence from SEQ ID NO:1, may be encoded by the gene that exhibits a polynucleotide sequence from SEQ ID NO:2, but is not limited to it. Furthermore, if an amino acid sequence has homology with the above sequence and has substantially the same or a corresponding activity as the protein in SEQ ID NO:1, it is evident that an amino acid sequence with a deletion, modification, substitution, or addition should fall within the scope of this disclosure. In this description, any polynucleotide sequence encoding pyruvate dehydrogenase may fall within the scope of this disclosure. For example, the polynucleotide sequence may be one that exhibits at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the polynucleotide sequence of SEQ ID NO:2. Furthermore, depending on codon degeneracy or considering the codons preferred by organisms for protein expression, the polynucleotide sequence encoding the protein may exhibit various variants in the coding region, to the point of not altering the amino acid sequence of the protein expressed from the coding region.

[0040] The pyruvate dehydrogenase mutant of the present description may include polypeptides having an amino acid sequence set out in SEQ ID NO:1, and the amino acid corresponding to position 217 of SEQ ID NO:1 being any selected from the group consisting of alanine, aspartic acid, glutamic acid, leucine, and proline.

[0041] Furthermore, the pyruvate dehydrogenase mutant of the present description may include not only a polypeptide having the amino acid sequence of SEQ ID NO:1, but also a pyruvate dehydrogenase mutant having 75% or more, specifically 80% or more, more specifically 85% or more, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the polypeptide having the amino acid sequence of SEQ ID NO:1, provided that its amino acids corresponding to position 217 of SEQ ID NO:1 are any selected from the group consisting of alanine, aspartic acid, glutamic acid, leucine, and proline, and its activities are significantly weakened compared to the activity of wild-type pyruvate dehydrogenase. It is evident that amino acid sequences exhibiting substantially the same or corresponding biological activity as the polypeptide in SEQ ID NO:1 should also fall within the scope of this disclosure. All experts in the field know that one or more substitutions, deletions, additions, and replacements of amino acids in the amino acid sequence in SEQ ID NO:1 result in amino acid sequences exhibiting substantially the same or corresponding biological activity as the polypeptide in SEQ ID NO:1.

[0042] The term “polynucleotide” as used herein refers to a polymer composed of nucleotides. The polynucleotide may be in the form of an individual fragment, or it may be a constituent part of a larger nucleotide sequence structure, which is derived from a nucleotide sequence that has been isolated at least once in quantity or concentration, and could be recognized, manipulated, and recovered from the sequence and its component nucleotide sequence by a standard molecular biology method (e.g., using a cloning vector). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C), where “U” replaces “T”. In other words, “polynucleotide” refers to a polymer of nucleotides removed from other nucleotides (an individual fragment or a complete fragment), or it may be a constituent part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include sequences of DNA, RNA, and cDNA.

[0043] Specifically, the polynucleotide encoding the pyruvate dehydrogenase mutant of the present description includes a polynucleotide disclosed in SEQ ID NO:2, and polynucleotides having mutations at positions 649 to 651. Also, the polynucleotide of the present disclosure further includes any polynucleotide having 75% or more, specifically 80% or more, more specifically 85% or more, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the polynucleotide disclosed in SEQ ID NO:2.

[0044] The term “homology” as used herein refers to the percentage of identity between two polynucleotide or polypeptide portions. Homology between sequences of one fraction and the other fraction can be determined using a known technique. For example, homology can be determined by directly aligning the sequence information of two polynucleotide molecules or two polypeptide molecules using an accessible computer program. Examples of such programs include BLAST (NCBI), CLC Main Workbench (CLC bio), and MegAlign™ (DNASTAR Inc.), etc. Furthermore, homology between polynucleotides can be determined by the following steps: hybridizing the polynucleotides to form a stable double strand between homologous regions, disassembling with a specific single-strand nuclease, and then performing size determination on the disassembled fragments.

[0045] The term "wild-type," as used herein, refers to an object that can be found in nature. For example, a sequence of polypeptides or polynucleotides present in an organism that can be isolated from a source in nature and that has not been intentionally modified by humans in the laboratory is of natural origin. As used herein, "of natural origin" and "wild-type" are synonymous. In some embodiments, the wild-type pyruvate dehydrogenase described herein refers to a polypeptide with an amino acid sequence set out in SEQ ID NO:1.

[0046] The term "mutant" as used herein refers to a polynucleotide or polypeptide that, with respect to the "wild-type" or "comparative" polynucleotide or polypeptide, contains alteration(s) (i.e., substitution, insertion, and/or deletion) of polynucleotide(s) at one or more (e.g., multiple) positions, where substitution refers to the replacement of a different nucleotide by a nucleotide occupying a position; deletion refers to the removal of a nucleotide occupying a certain position; and insertion refers to the addition of a nucleotide after the adjacent nucleotide and immediately after the occupied position.

[0047] In some embodiments, the "mutation" employed herein is a "substitution" caused by the replacement of one or more bases in one or more nucleotides by a different base, also called a base replacement mutation or point mutation.

[0048] The term "expression" as used herein includes any stage involving the production of RNA and proteins, including, but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0049] The term “vector” as used herein refers to a DNA construct containing DNA sequences operationally linked to appropriate control sequences for the purpose of expressing a 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. The recombinant expression vector may include, for example: (i) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; (ii) a coding structure or sequence that is transcribed into mRNA and translated into a protein; and (iii) transcriptional subunits that appropriately transcribe and translate the initiation and termination sequences. Recombinant expression vectors may be constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmids, viruses, phages, and transposons. The potential vectors used in this document 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, and viral DNA such as vaccinia, adenovirus, avipoxvirus, baculovirus, SV40, and pseudorabies.

[0050] The term "host cell" as used herein refers to any cell type resulting from transformation, transfection, transduction, or the like, of the pyruvate dehydrogenase mutant or expression vector described herein. The term "recombinant host cell" encompasses a host cell that is distinct from the parent cell following the introduction of a transcription initiation element or recombinant expression vector. The recombinant host cell is specifically obtained by transformation.

[0051] The term "transformation" as used herein has the meaning generally understood by a person skilled in the art, namely, a process of introducing exogenous DNA into a host. Methods for transformation include any method of introducing a nucleic acid into a cell. These methods include, but are not limited to, electroporation, calcium phosphate precipitation, calcium chloride precipitation, microinjection, a polyethylene glycol (PEG) method, a DEAE-dextran method, a cationic liposome method, and a lithium acetate-DMSO method.

[0052] The host cell of the present description may be a prokaryotic or eukaryotic cell, provided that the cells contain the pyruvate dehydrogenase mutant of the present disclosure. In some embodiments, the host cell refers to a prokaryotic cell. Specifically, the host cell is derived from microorganisms suitable for the fermentative production of amino acids, organic acids, bio-based materials, or pharmaceutical compounds, which may include, but are not limited to, strains such as Escherichia, Erwinia, Serratia, Providencia, Enterobacteriaceae, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, and Corynebacterium. Optionally, the host cell can be Corynebacterium glutamicum. Preferably, the host cell can be ATCC 13032 of Corynebacterium glutamicum, ATCC 13869 of Corynebacterium glutamicum, ATCC 14067 of Corynebacterium glutamicum, and strains derived from these1. By way of example, the derived strain can be any strain, provided that the strain is capable of producing L-amino acids.

[0053] By way of example, the host cell is a lysine-producing host cell. In some embodiments, the lysine-producing host cell may be a derived strain expressing aspartate kinase that relieves feedback inhibition based on ATCC 13032 from Corynebacterium glutamicum. In addition, the lysine-producing host cell may also be other types of strains that exhibit the ability to produce lysine.

[0054] In some embodiments, the host cell that produces lysine may also include, but is not limited to, one or more genes selected from the following genes that have attenuated or reduced expression:

[0055] a. genadhEque encodes ethanol dehydrogenase;

[0056] b. genackAque encodes acetate kinase;

[0057] c. genptaque encodes phosphate acetyltransferase;

[0058] d. genldhAque encodes lactate dehydrogenase;

[0059] e. genfocAque encodes formate transporter;

[0060] f. genpflBque encodes pyruvate formate lyase;

[0061] g. genpoxB which encodes pyruvate oxidase;

[0062] h. genthrAque encodes aspartate kinase I/homoserine dehydrogenase I as a bifunctional enzyme;

[0063] i. genthrBque codes for homoserine kinase;

[0064] j. genldcC which encodes lysine decarboxylase; and

[0065] k. gencadAque codes lysine decarboxylase.

[0066] In some embodiments, the host cells that produce lysine may also include, but are not limited to, one or more genes selected from the following genes that are enhanced or overexpressed:

[0067] a. gendapAque encodes dihydrodipyridine synthase, which relieves feedback inhibition of lysine;

[0068] b. gendapB which encodes dihydrodipicolinate reductase;

[0069] c. genddhque codes for diaminopimelate dehydrogenase;

[0070] d.dapD which encodes tetrahydrodipicolinate succinylase and dapE which encodes succinyldiaminopimelate deacylase;

[0071] e. genasdque encodes aspartate-semialdehyde dehydrogenase;

[0072] f. genppcwhich encodes phosphoenolpyruvate carboxylase;

[0073] g. genpntAB which encodes niacinamide adenine dinucleotide transhydrogenase; and h. genlysE which encodes the lysine transport protein.

[0074] By way of example, the host cells are host cells that produce threonine. In some embodiments, the threonine-producing host cells may be strains expressing the LysC aspartate kinase that relieves the ATCC 13032-based feedback inhibition of Corynebacterium glutamicum. In other embodiments, the threonine-producing host cells may also be other types of strains that have the ability to produce threonine.

[0075] In some embodiments, one or more genes selected from the following genes are enhanced or overexpressed in the host cells that produce threonine:

[0076] a. genthrABCque encodes the threonine operon;

[0077] b. genhomque encodes homoserine dehydrogenase, which relieves feedback inhibition;

[0078] c. gengapque encodes glyceraldehyde-3-phosphate dehydrogenase;

[0079] d. genpycque encodes pyruvate carboxylase;

[0080] e. genmqoque encodes malate: quinone oxidoreductase;

[0081] f. gentkt which encodes transketolase;

[0082] g. gengndwhich encodes 6-phosphogluconate dehydrogenase;

[0083] h. genthrEque encodes the threonine transporter; e

[0084] i. genenoque encodes enolase.

[0085] By way of example, the host cells are host cells that produce isoleucine. In some embodiments, the isoleucine-producing host cells are strains that produce L-isoleucine by substituting the amino acid at position 323 of the genilvA-encoded L-threonine dehydratase with alanine. In other embodiments, the isoleucine-producing host cells may also be other types of strains that have the ability to produce isoleucine.

[0086] By way of example, the host cells are host cells that produce O-acetylhomoserine. In some embodiments, the host cells that produce O-acetylhomoserine are strains that produce O-acetylhomoserine by inactivating O-acetylhomoserine(thiol)-lyase. In other embodiments, the host cells that produce O-acetylhomoserine may also be other types of strains that have the ability to produce O-acetylhomoserine.

[0087] By way of example, the host cells are host cells that produce methionine. In some embodiments, the host cells that produce methionine are strains that produce methionine by inactivating the transcriptional regulators of methionine and cysteine. In other embodiments, the host cells that produce methionine may also be other types of strains that have the ability to produce methionine.

[0088] The term “L-amino acid” used herein refers to all L-amino acids that can be produced from different carbon sources via pyruvate. More specifically, L-amino acids may include L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine, or L-alanine, and even more specifically, L-lysine and L-valine.

[0089] The term "culture" as used herein may refer to cultivation carried out using conventional methods in the art, including, but not limited to, plate culture, shake culture, batch culture, continuous culture, semi-continuous culture, etc. In addition, various culture conditions, such as temperature, time, and pH value of the medium, may be appropriately adjusted to suit actual situations.

[0090] Unless otherwise defined in the invention or clearly indicated by the context, all technical and scientific terms used herein have the same meanings as are normally understood by a person skilled in the art to which this disclosure pertains.

[0092] Examples

[0093] Other objectives, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be appreciated that the detailed description and specific examples (although they indicate the specific examples in this description) are provided for illustrative purposes only, since, after reading the detailed description, various alterations and modifications made within the spirit and scope of this disclosure will be evident to a person skilled in the art.

[0094] All experimental techniques and methods used in the examples, unless otherwise specified, are conventional techniques and methods. For example, experimental methods for which no specific conditions are given in the following examples are generally performed under conventional conditions, such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturers. All materials, reagents, etc., used in the examples, unless otherwise specified, are commercially available.

[0096] Example 1. Construction of the pCas9gRNA-aceE217 editing plasmid

[0097] After intensive analysis of the AceE enzyme, the present description predicted that position 217 might be the site affecting its activity, so this site was mutated for further study. Subsequently, according to the documented cloning method of Goldengate (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), a pCas9gRNA plasmid was constructed targeting the codon of the amino acid residue at position 217 in the AceE gene, and the target DNA-binding region of the sgRNA was CCAACTGTGTCCATGGGTCT. The specific method was as follows: 217-F/217-R was denatured and hybridized to obtain a double-stranded DNA product with sticky ends, which was then subjected to Goldengate cloning (NEB® Golden Gate assembly kit, #E1601) with the pCas9gRNA-cco® plasmid (reference CN112111469B) to obtain the pCas9gRNA-aceE217 plasmid. This plasmid expressed the Cas9 protein and the sgRNA targeting the mutation site region. The primers used to construct the above plasmid are listed in Table 1.

[0098] The primers used in the examples in this disclosure are listed in Table 1 below.

[0101]

[0102]

[0103]

[0106] Example 2. Construction of mutant with an amino acid codon mutation at position 217 in the aceE gene of Corynebacterium glutamicum

[0107] Wild-type Corynebacterium glutamicum was unable to produce lysine, but Corynebacterium glutamicum was able to produce lysine after the introduction of point mutations in lysC, pycyhom. In the present Example, ATCC13032 was used as the original strain. First, a lysine-producing strain was constructed. That is, in the ATCC13032 strain of Corynebacterium glutamicum, a T311I point mutation (which releases feedback inhibition of the enzyme) was introduced in the gene lysC for aspartate kinase, a P458S point mutation (which releases feedback inhibition of the enzyme) was introduced in the gene pyc for pyruvate carboxykinase, and a V59A point mutation (which weakens enzyme activity) was introduced in the gene ho for homoserine dehydrogenase in order to obtain the high lysine-producing AHP-3 strain.

[0108] Subsequently, a CRISPR/Cas9 genome editing system based on single-strand recombination (referring to patent CN112111469A for the basic plasmid construction process) was used to perform the saturation mutation at site 217 of pyruvate dehydrogenase (encoded by genaceE) in Corynebacterium glutamicum. Competent cells of the high-lysine-producing AHP-3 strain were first prepared, and the pRecT plasmid as an auxiliary plasmid for recombination was transformed by electroporation into an L-lysine-producing AHP-3 strain of Corynebacterium glutamicum to obtain the AHP-3-pRecT strain. Competent cells were prepared from the AHP-3-pRecT strain using the documented method (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 competent AHP-3-pRecT cells.

[0109] To perform 19 different wild-type mutations in the amino acid codon at position 217 in the genaceE, single-stranded DNAs of S217A, S217C, S217D, S217E, S217F, S217G, S217H, S217I, S217K, S217L, S217M, S217N, S217P, S217Q, S217R, S217T, S217V, S217W and S217Y (Table 1) were designed as recombinant templates for mutant construction. One pg of the pCas9gRNA-aceE 217 plasmid and 10 pg of single-stranded DNA were transformed by electroporation into previously prepared AHP-3-pRecT competent cells, added to 1 ml of TSB medium preheated to 46 °C, incubated at 46 °C for 6 min, incubated at 30 °C for 3 h, coated onto a TSB plate containing 5 pg/ml chloramphenicol, 15 pg/ml kanamycin, and 0.05 mM IPTG, and cultured for 2 days to obtain clones. The ingredients (g/l) of the TSB medium were as follows: glucose, 5 g/l; yeast powder, 5 g/l; soy peptone, 9 g/l; urea, 3 g/l; succinic acid, 0.5 g/l; K2HPO4AH2O, 1 g/L; MgSO4-7H2O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; and MOPS, 20 g/L. The monoclones obtained above were identified by colony PCR using a specific primer S217-jd-F/aceE-jd-R (Table 1), respectively. The correct clones were confirmed by sequencing, ultimately yielding nine mutants of S217A, S217C, S217D, S217E, S217G, S217L, S217P, S217T, and S217V, while the other ten mutants were not obtained.

[0110] The pRecT and pCas9gRNA-aceE217 plasmids were lost from the above resulting strains with the amino acid codon mutant at position 217 in the genaceE. The specific process was as follows: monoclones were cultured overnight at 37 °C in TSB liquid medium without the resistance; then, the monoclones were streaked onto a plate of TSB solid medium without the resistance; Subsequently, the cultured monoclonal bacteria were streaked onto three solid plates (TSB 5 pg/ml chloramphenicol, TSB 15 pg/ml kanamycin, and TSB) respectively, and cultured at 30 °C for 24 h to obtain strains that did not grow on chloramphenicol and kanamycin plates, but were able to grow on TSB plates, which were mutant strains from which two plasmids were lost and were named SCgL46 to SCgL54, respectively, and the corresponding mutants were S217A, S217C, S217D, S217E, S217G, S217L, S217P, S217T and S217V, respectively.

[0112] Example 3. Effect of the mutation at position 217 of the aceE gene in Corynebacterium glutamicum on L-lysine synthesis

[0113] To analyze how the amino acid mutation at position 217 of the gene in Corynebacterium glutamicum affected L-lysine production by high-lysine production staining, fermentation tests were performed on strains SCgL46 to SCgL54, respectively. The wild-type strain AHP-3 was used as a control. The fermentation medium ingredients were as follows: glucose 80 g/l; ammonium sulfate, 20 g/l; urea, 5 g/l; KH₂PO₄, 1 g/l; K₂HPO₄·AH₂O, 1.3 g/l; MOPS, 42 g/l; CaC₆H₅, 0.01 g/l; FeSO₄·7H₂O, 0.01 g/l; MnSO₄·H₂O, 0.01 g/l; ZnSO₄·7H₂O, 0.001 g/L; CuSO₄, 0.0002 g/L; NiClA₂O, 0.00002 g/L; MgSO₄·7H₂O, 0.25 g/L; protocatechuic acid, 0.03 g/L; biotin, 0.0002 g/L; vitamin B1, 0.0001 g/L; and an initial pH of 7.2. First, the strains were inoculated into TSB liquid medium and cultured for 8 h. The cultures were then inoculated as seeds into a 24-well plate containing 800 lp of fermentation medium in each well with an initial OD₆ < 600 controlled to approximately 0.1, and cultured at 30 °C for 21 h. The plate shaker rotation speed was 800 rpm. Three samples were fixed for each strain. After fermentation, L-lysine yield and glucose consumption were detected, and the sugar-acid conversion rate of glucose to L-lysine was calculated.

[0114] The results were those shown in Table 2. All L-lysine yields from mutants S217C, S217G, S217T, and S217V were lower than those from the wild-type control strain, while all L-lysine yields from mutants S217A, S217D, S217E, S217L, and S217P were higher than those from the wild-type control strain. Therefore, the mutations at these amino acid sites showed greater potential for applications in the production of L-lysine and its derivatives.

[0115] Table 2

[0117]

[0119] AceE(SEQ ID NO:1):

[0120]

[0121]

[0123] All technical features described in this specification can be combined in any way. Each of the technical features described herein can be substituted by other features that have the same, equivalent, or similar effects. Therefore, unless specifically stated, each of the features described herein is only one example of a series of equivalent or similar features.

[0124] Furthermore, with the foregoing descriptions in this disclosure, the key features of this description are evident to a person skilled in the art. Many modifications to the invention may be made without departing from the spirit and scope of this disclosure, in order to adapt the invention to various purposes and conditions of application. Accordingly, it is also intended that such modifications fall within the scope of the appended claims.

Claims (12)

1. CLAIMS 1. A pyruvate dehydrogenase mutant, wherein the mutant is any selected from the group consisting of: 1) a mutant in which an amino acid sequence is exposed at SEQ ID NO:1 and position 217 of SEQ ID NO:1 has mutated to any of alanine, aspartic acid, glutamic acid, leucine, and proline; and 2) a mutant in which 1, 2, 3, 4, 5 or 6 amino acids are added or deleted from both ends of a polypeptide exposed at SEQ ID NO:1 and position 217 corresponding to SEQ ID NO:1 is any of alanine, aspartic acid, glutamic acid, leucine and proline. 2. A nucleotide encoding the pyruvate dehydrogenase mutant according to claim 1. 3. An expression cassette containing the nucleotide encoding the pyruvate dehydrogenase mutant according to claim 2. 4. A vector containing the nucleotide encoding the pyruvate dehydrogenase mutant according to claim 2. 5. The vector according to claim 4, wherein the vector is an expression vector. 6. A host cell containing the nucleotide encoding the pyruvate dehydrogenase mutant according to claim 2. 7. A host cell containing the coding nucleotide according to claim 2 or the vector according to claim 4, wherein the host cell is a microorganism of Escherichia, Erwinia, Serratia, Providencia, Enterobacteriaceae, Salmonella, Streptomyces, Pseudomonas, Brevibacterium or Corynebacterium. 8. The host cell according to claim 7, wherein the host cell is an ATCC13032 strain of Corynebacterium glutamicum, an ATCC13869 strain of Corynebacterium glutamicum, or an ATCC 14067 strain of Corynebacterium glutamicum. 9. Use of the pyruvate dehydrogenase mutant according to claim 1 or a nucleotide encoding the same in the production of an L-amino acid. 10. The use according to claim 9, wherein the L-amino acid is selected from the group consisting of L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine and L-alanine. 11. A method for producing an L-amino acid, comprising culturing the host cell according to claim 7 or 8 so that the host cell produces an L-amino acid, and further comprising a step of isolating and extracting or recovering the L-amino acid from a medium. 12. The method according to claim 11, wherein the L-amino acid is selected from the group consisting of L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine and L-alanine.