CN113249347B - Mutants of pyruvate dehydrogenase and methods for producing L-amino acids using the same - Google Patents
Mutants of pyruvate dehydrogenase and methods for producing L-amino acids using the same Download PDFInfo
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- C12Y102/01051—Pyruvate dehydrogenase (NADP+) (1.2.1.51)
Abstract
The pyruvate dehydrogenase mutant provided by the invention is characterized in that on the basis that the amino acid sequence is shown as SEQ ID NO. 1, the 217 th position of the pyruvate dehydrogenase mutant is mutated into any one of alanine, aspartic acid, glutamic acid, leucine and proline. The mutant can improve the yield and the conversion rate of the L-amino acid of the strain, does not inhibit the growth of the strain while improving the yield, provides a new way for producing the L-amino acid on a large scale, and has higher application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mutant of pyruvate dehydrogenase, a polynucleotide for encoding the mutant, a host cell containing the mutant, and a method for producing L-amino acid by using the mutant.
Background
Pyruvate dehydrogenase multienzyme complex (PDHC) is a group of rate-limiting enzymes, and in corynebacterium glutamicum PDHC catalyzes the irreversible oxidative decarboxylation of pyruvate produced by the glycolysis process to acetyl-coa, linking the aerobic oxidation of sugars to the tricarboxylic acid cycle and oxidative phosphorylation, is a key enzyme of the tricarboxylic acid cycle. PDHC consists of pyruvate dehydrogenase (E1 p), dihydrolipoamide acetyltransferase (E2 p) and dihydrolipoamide dehydrogenase (E3 p). Enzymes have two forms, inactive (phosphorylating) and active (dephosphorylating), which are different from phosphorylases. There are a number of allosteric modulators that regulate the conversion of both enzyme forms and are influenced by activin activity.
Wherein the E1p enzyme consists ofaceEThe gene encodes. Attenuation of the expression of AceE has been reported to increase the production of L-amino acids in coryneform bacteria. CN106715687A also reports that mutation of any one of the amino acids at positions 205 and 440 of AceE 190 can increase the production of L-lysine in Corynebacterium glutamicum. At present, no new mutation site has been reported.
Disclosure of Invention
According to the result of the AceE structure prediction, the 217 th site is subjected to saturation mutation, and the partial mutants can show the yield of the host cell L-lysine, so that the invention is completed.
The present invention aims at providing one new kind of pyruvate dehydrogenase mutant, host cell containing the mutant and L-lysine producing process with the host cell containing the mutant.
In a first aspect, the present invention provides a novel pyruvate dehydrogenase mutant, which is:
1) the amino acid sequence is shown as SEQ ID NO. 1, and the 217 th position of the amino acid sequence is any one of alanine, aspartic acid, glutamic acid, leucine and proline.
2) A polypeptide having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with SEQ ID NO. 1 and any one of alanine, aspartic acid, glutamic acid, leucine, proline at position 217 corresponding to SEQ ID NO. 1.
3) One or more bases are added or deleted at two ends of the polypeptide shown in SEQ ID NO. 1, and any one of alanine, aspartic acid, glutamic acid, leucine and proline is added or deleted at position 217 corresponding to SEQ ID NO. 1. Preferably, 1, 2, 3, 4, 5 and 6 bases are added and deleted at two ends of the polypeptide shown in SEQ ID NO. 1.
In a second aspect, the present invention provides a coding polynucleotide encoding a pyruvate dehydrogenase mutant.
In a third aspect, the present invention provides an expression cassette comprising the nucleotide encoding the pyruvate dehydrogenase mutant. The coding nucleotide is operably linked to a promoter.
In a fourth aspect, the present invention provides vectors, particularly expression vectors, comprising nucleotides encoding pyruvate dehydrogenase mutants.
In a fifth aspect, the invention provides a host cell comprising a nucleotide encoding a pyruvate dehydrogenase mutant.
Further, the host cell includes, but is not limited to, microorganisms of the genera Escherichia (genus Escherichia), Erwinia (genus Erwinia), Serratia (genus Serratia), Provesia (genus Providencia), Enterobacter (genus Enterobacteria), Salmonella (genus Salmonella), Streptomyces (genus Streptomyces), Pseudomonas (genus Pseudomonas), Brevibacterium (genus Brevibacterium), Corynebacterium (genus Corynebacterium). Optionally, the host cell is corynebacterium glutamicum, escherichia coli. Preferably, the host cells include, but are not limited to, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC 14067, and strains derived therefrom.
In a sixth aspect, the invention provides the use of said pyruvate dehydrogenase mutant or a nucleotide encoding the same for the production of an L-amino acid. Preferably, the L-amino acid is selected from L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine or L-alanine.
In a seventh aspect, the present invention provides a method for producing an L-amino acid, which comprises culturing the host cell of the fifth aspect to produce the L-amino acid, and further comprises the step of isolating and extracting or recovering the L-amino acid from the culture medium.
Further, the L-amino acids include, but are 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 invention can improve the yield and the conversion rate of L-amino acid of the strain, does not inhibit the growth of the strain while improving the yield, and provides a new strategy for producing the L-amino acid on a large scale.
Detailed Description
Definition of terms
The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.
As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.
Although the present disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless it is expressly stated that only an alternative or mutual exclusion between alternatives is present.
When used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed within the middle of the end points of the range with respect to the aforementioned end points of values.
The term "pyruvate dehydrogenase" according to the present invention refers to one of the enzymes constituting the pyruvate dehydrogenase multienzyme complex (PDHC), which participates in the conversion of pyruvate into acetyl-CoA. As used herein, pyruvate dehydrogenase is not particularly limited as long as it has a corresponding activity, and it may be pyruvate dehydrogenase derived from a microorganism of corynebacterium, particularly, corynebacterium glutamicum, but is not limited thereto. For example, the pyruvate dehydrogenase can be the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO. 1. The E1p protein having the amino acid sequence of SEQ ID NO. 1 may be composed of the polynucleotide sequence of SEQ ID NO. 2aceEThe gene encodes, but is not limited thereto. In addition, if the amino acid sequence has homology with the above-mentioned sequence and has substantially the same or corresponding biological activity as the protein of SEQ ID NO. 1, it is apparent that the amino acid sequence having deletion, modification, substitution or addition should also fall within the scope of the present disclosure. In the present disclosure, any polynucleotide sequence encoding pyruvate dehydrogenase may be within the scope of the present disclosure. For example, the polynucleotide sequence can be at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or alternatively, it can be at least 75%, specifically at least 80%, and even more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or alternatively, it can be at least the same as the polynucleotide sequence of SEQ ID NO. 2A polynucleotide sequence having 99% or greater homology. In addition, a polynucleotide sequence encoding a protein may have various variants on a coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, based on codon degeneracy or considering codons preferred by an organism to express the protein.
The pyruvate dehydrogenase mutant of the invention can comprise polypeptide which has an amino acid sequence shown as SEQ ID NO. 1 and is selected from any one of alanine, aspartic acid, glutamic acid, leucine and proline at the amino acid position 217 corresponding to the SEQ ID NO. 1.
Further, the pyruvate dehydrogenase mutants of the present invention may include not only the polypeptide having the amino acid sequence of SEQ ID NO. 1 but also pyruvate dehydrogenase mutants having homology of 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%, and 99% or more with the polypeptide having the amino acid sequence shown in SEQ ID NO. 1, as long as they are selected from any one of alanine, aspartic acid, glutamic acid, leucine, proline at the amino acid position 217 corresponding to EQ ID NO. 1 and their activities are greatly attenuated as compared with the wild-type pyruvate dehydrogenase activity. It is obvious that an amino acid sequence having substantially the same or corresponding biological activity as the polypeptide having the amino acid sequence of SEQ ID NO. 1 also falls within the scope of the present invention, and those skilled in the art know that substitution, deletion, addition and substitution of 1 to several bases based on the amino acid sequence shown in SEQ ID NO. 1 can give an amino acid sequence having substantially the same or corresponding biological activity as the polypeptide having the amino acid sequence of SEQ ID NO. 1.
The term "polynucleotide" of the present invention refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments, or may be a component of a larger nucleotide sequence structure, derived from nucleotide sequences that have been isolated at least once in quantity or concentration, and which are capable of being recognized, manipulated, and recovered in sequence, and their component nucleotide sequences, by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (either individual fragments or whole fragments), or may be an integral part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.
Specifically, the encoding polynucleotide of the pyruvate dehydrogenase mutant comprises the polynucleotide shown in SEQ ID NO. 2 and the polynucleotide mutated at position 649-651 thereof. In addition, the polynucleotide of the present invention also includes any polynucleotide having homology of 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%, and 99% or more with the polynucleotide represented by SEQ ID NO. 2.
The term "homology" in the context of the present invention refers to the percentage of identity between two polynucleotide or polypeptide parts. Homology between the sequences of one portion and another portion 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. In addition, the homology between polynucleotides can be determined by: polynucleotides are hybridized under conditions that form a stable double strand between homologous regions, cleaved with a single strand specific nuclease, and the cleaved fragments are then sized.
The term "wild-type" of the present invention refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, can be isolated from a source in nature, and is not intentionally modified by man in the laboratory, is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms. In some embodiments, wild-type pyruvate dehydrogenase in the present disclosure refers to a pyruvate dehydrogenase having an amino acid sequence as set forth in SEQ ID NO: 1.
The term "mutant" of the present invention refers to a polynucleotide that comprises alterations (i.e., substitutions, insertions, and/or deletions) at one or more (e.g., several) positions relative to a "wild-type", or "comparable" polynucleotide or polypeptide, wherein a substitution refers to the substitution of a nucleotide occupying a position with a different nucleotide.
In some embodiments, a "mutation" of the present invention is a "substitution", which is a mutation caused by the substitution of a base in one or more nucleotides with another different base, also referred to as a base substitution mutation (mutation) or a point mutation (point mutation).
The term "expression" of the present invention includes any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "vector" of the present invention refers to a DNA construct comprising a DNA sequence operably linked to suitable control sequences for the expression of 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 comprising i) genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector is 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. 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, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies.
The term "host cell" of the present invention refers to any cell type transformed, transfected, transduced, or the like, comprising a pyruvate dehydrogenase mutant or expression vector of the present invention. The term "recombinant host cell" encompasses host cells which differ from the parent cell after introduction of a transcription initiation element or a recombinant expression vector, which is effected in particular by transformation.
The term "transformation" in the present invention has the meaning commonly understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing a nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate precipitation, calcium chloride (CaCl)2) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.
The host cell of the present invention may be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of containing the pyruvate dehydrogenase mutant of the present invention. In some embodiments, the host cell refers to a prokaryotic cell, in particular, the host cell is derived from a microorganism suitable for fermentative production of amino acids, organic acids, biobased materials or pharmaceutical compounds, and may include Escherichia (R) ((R))Escherichia) Erwinia genus (a)Erwinia) Serratia (A), (B) and (C)Serratia) Provedasius genus (A), (B), (C)Providencia) Genus Enterobacter (a)Enterobacteria) Salmonella genus (A), (B)Salmonella) Streptomyces (I), (II)Streptomyces) Pseudomonas (a)Pseudomonas) Brevibacterium (Brevibacterium) (II)Brevibacterium) Corynebacterium genus (A), (B), (C)Corynebacterium) Etc., but are not limited thereto. Alternatively, it may be Corynebacterium glutamicum. Preferably, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC 14067, and derived strains thereof, and the like can be used. Illustratively, the derivative strain may be any strain as long as the strain has an ability to produce an L-amino acid.
Illustratively, the host cell is a lysine-producing host cell. In some embodiments, for lysine-producing host cells, can be in the Corynebacterium glutamicum ATCC13032 based on the expression of feedback inhibition of aspartate kinase derivative strain. In addition, lysine-producing host cells may also be other kinds of strains having lysine-producing ability.
In some embodiments, the lysine producing host cell may further include, but is not limited to, attenuated or reduced expression of one or more genes selected from the group consisting of:
a. encoding alcohol dehydrogenaseadhEA gene;
b. encoding acetate kinaseackAA gene;
c. encoding a phosphate acetyltransferaseptaA gene;
d. encoding lactate dehydrogenaseldhAA gene;
e. encoding formate transportersfocAA gene;
f. encoding pyruvate formate lyasepflBA gene;
g. process for coding pyruvate oxidasepoxBA gene;
h. encoding an aspartokinase I/homoserine dehydrogenase I bifunctional enzymethrAA gene;
i. encoding homoserine kinasethrBA gene;
j. encoding lysine decarboxylaseldcCA gene;
h. encoding lysine decarboxylasecadAA gene.
In some embodiments, one or more genes selected from the group consisting of, but not limited to:
a. encoding dihydrodipyridine synthetase for relieving lysine feedback inhibitiondapAA gene;
b. encoding dihydrodipicolinate reductasedapBA gene;
c. encoding diaminopimelate dehydrogenaseddhA gene;
d. encoding Tetrahydropyridine dicarboxylic acid succinylOf enzymesdapDAnd encoding succinyldiaminopimelate deacylasedapE；
e. Encoding aspartate-semialdehyde dehydrogenaseasdA gene;
f. encoding phosphoenolpyruvate carboxylaseppcA gene;
g. encoding nicotinamide adenine dinucleotide transhydrogenasepntABA gene;
i. transport proteins encoding lysinelysEA gene.
Illustratively, the host cell is a threonine producing host cell. In some embodiments, the threonine producing host cell is a strain that expresses the feedback-released aspartate kinase LysC on the basis of corynebacterium glutamicum ATCC 13032. In other embodiments, the threonine-producing host cell can also be other species of strain that have threonine-producing ability.
In some embodiments, one or more genes selected from the group consisting of:
a. encoding the threonine operonthrABCA gene;
b. encoding feedback inhibition-relieved homoserine dehydrogenasehomA gene;
c. encoding glyceraldehyde-3-phosphate dehydrogenasegapA gene;
d. encoding pyruvate carboxylasepycA gene;
e. encoding malic acid: process for preparing quinone oxidoreductasesmqoA gene;
f. encoding transketolasetktA gene;
g. encoding 6-phosphogluconate dehydrogenasegndA gene;
h. encoding threonine exportthrEA gene;
i. encoding enolaseenoA gene.
Illustratively, the host cell is a host cell that produces isoleucine. In some embodiments, the host cell that produces isoleucine is produced by substituting alanine for L-threonine dehydrataseilvAAmino acid 323 of geneAnd L-isoleucine-producing strains. In other embodiments, the host cell producing isoleucine may also be another species of strain having isoleucine-producing ability.
Illustratively, the host cell is a host cell producing O-acetylhomoserine. In some embodiments, the host cell producing O-acetylhomoserine is a strain that produces O-acetylhomoserine by inactivation of O-acetylhomoserine (thiol) -lyase. In other embodiments, the host cell producing O-acetylhomoserine can also be other kinds of strains having O-acetylhomoserine producing ability.
Illustratively, the host cell is a host cell that produces methionine. In some embodiments, the methionine producing host cell is a strain that produces methionine by inactivating the transcriptional regulators of methionine and cysteine. In other embodiments, the methionine-producing host cell may be a strain of another species having methionine-producing ability.
The term "L-amino acid" in the present invention refers to all L-amino acids that can be produced from different carbon sources by pyruvate. More specifically, the L-amino acid may include L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine, or L-alanine, and even more specifically, L-lysine or L-valine.
The term "culture" in the present invention may be carried out according to a conventional method in the art, including, but not limited to, a well plate culture, a shake flask culture, a batch culture, a continuous culture, a fed-batch culture, and the like, and various culture conditions such as temperature, time, pH of a medium, and the like may be appropriately adjusted according to actual circumstances.
Unless defined otherwise herein or clearly indicated by the background, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Examples
Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The experimental techniques and experimental procedures used in this example are, unless otherwise specified, conventional techniques, e.g., those in the following examples, in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.
Example 1 editing of the plasmid pCas9gRNA-aceE217 construction
The AceE enzyme is deeply analyzed, wherein the 217 th site is predicted to be a site which influences the activity of the AceE enzyme, so that the site is subjected to subsequent research on mutation. Subsequently, the cloning method of base amplification was performed according to the Goldinge cloning method reported in the literature (WANG, Yu, et alCorynebacterium glutamicumBiotechnology and bioengineering, 2019, 116: 3016-3029) construction of targetingaceEThe target DNA binding region of the sgRNA in the pCas9gRNA plasmid having the codon at amino acid residue 217 of the gene is CCAACTGTGTCCATGGGTCT. The specific method comprises the following steps: the 217-F/217-R is denatured and annealed to obtain a DNA double-stranded product with a sticky end, and then the DNA double-stranded product is mixed with pCas9gRNA-ccdBCloning of GoldenGate (NEB Golden Gate Assembly kit, # E1601) was performed on the plasmid (refer to CN 112111469B) to obtain pCas9gRNA-aceE217 plasmid expressing Cas9 protein and sgrnas targeting site-directed mutation regions. The primers used for the above plasmid construction are shown in Table 1.
The primers used in the examples of the disclosure are shown in table 1 below:
example 2 construction of Corynebacterium glutamicumaceEGene 217 th amino acid codon mutant
Since wild type Corynebacterium glutamicum can not produce lysine, but is introduced into Corynebacterium glutamicumlysC、pycAndhomlysine can be produced after the point mutation of (1). In this example, starting with ATCC13032, a lysine-producing strain was first constructed, i.e., the aspartokinase gene in Corynebacterium glutamicum ATCC13032lysCIntroduced T311I point mutation (to remove feedback inhibition of enzyme) in pyruvate carboxylase genepycIntroducing P458S point mutation (removing feedback inhibition of enzyme) in homoserine dehydrogenase genehomA V59A point mutation (weakening the activity of enzyme) is introduced to obtain the lysine high-producing strain AHP-3.
Subsequently, using the CRISPR/Cas9 genome editing system based on single-strand recombination (basic plasmid construction process refer to patent CN 112111469A), pyruvate dehydrogenase of Corynebacterium glutamicum (aceEGene coding) 217 site for saturation mutation. Firstly, preparing competent cells of a lysine high-producing strain AHP-3, and electrically transforming a recombinant helper plasmid pRecT plasmid into a Corynebacterium glutamicum L-lysine producing strain AHP-3 to obtain the AHP-3-pRecT strain. AHP-3-pRecT strain adopts a method reported in the literature to prepare competent cells (Ruan Y, Zhu L, Li Q. Improviding the electro-transformation efficiency ofCorynebacterium glutamicumby themselves, harvesting cell walls and engineering the cytoplastic membrane flow, BiotechnolLett. 2015;37: 2445-52.) to obtain AHP-3-pRecT competent cells.
To pairaceEAmino acid position 217 of the geneThe acid codon was subjected to 19 kinds of mutations other than wild type, and single-stranded DNAs of S217A, S217C, S217D, S217E, S217F, S217G, S217H, S217I, S217K, S217L, S217M, S217N, S217P, S217Q, S217R, S217T, S217V, S217W, and S217Y were designed (table 1), and recombinant templates for mutant construction were used. 1. mu.g of pCas9gRNA-aceE217 plasmid and 10. mu.g of single-stranded DNA were added to 1 mL of TSB medium preheated at 46 ℃ and incubated at 46 ℃ for 6 min and at 30 ℃ for 3h, and TSB plates supplemented with 5. mu.g/mL chloramphenicol, 15. mu.g/mL kanamycin and 0.05 mM IPTG were spread and cultured for 2 days to obtain clones. The TSB culture medium comprises the following components (g/L): glucose, 5 g/L; 5 g/L of yeast powder; soybean peptone, 9 g/L; 3 g/L of urea; succinic acid, 0.5 g/L; k2HPO4 & 3H2O, 1 g/L; MgSO4 & 7H2O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L. The single clones obtained above were subjected to colony PCR identification using specific primers S217-jd-F/aceE-jd-R (Table 1), and correct clones were sequenced and confirmed to finally obtain S217A, S217C, S217D, S217E, S217G, S217L, S217P, S217T, and S217V 9 mutants, while the other 10 mutants were not obtained.
Obtained as described aboveaceEpRecT and pCas9gRNA in 217 th amino acid codon mutant strain of geneaceE217 plasmid was lost, as follows: the monoclonals are cultured in a nonresistant TSB liquid culture medium at 37 ℃ overnight, then the monoclonals are streaked on a nonresistant TSB solid culture medium plate, then the grown monoclonals are streaked on 3 solid plates (TSB +5 mu g/mL chloramphenicol, TSB +15 mu g/mL kanamycin and TSB) respectively, and the monoclonals are cultured for 24 hours at 30 ℃ to obtain chloramphenicol-resistant plates and kanamycin-resistant plates which are not long, but the TSB plates can be long strains, namely mutant strains losing two plasmids, which are named as SCgL46 to SCgL54 respectively, and the corresponding mutants are S217A, S217C, S217D, S217E, S217G, S217L, S217P, S217T and S217V respectively.
Example 3 Corynebacterium glutamicumaceEEffect of the 217 th mutation in the Gene on L-lysine Synthesis
To test Corynebacterium glutamicumaceE217 th amino acid mutation on lysineThe influence of the high-producing strains on the production of L-lysine was determined by fermentation testing of the SCgL46 to SCgL54 strains, respectively. The wild AHP-3 strain is used as a reference, and the fermentation medium comprises the following components: glucose, 80 g/L; ammonium sulfate, 20 g/L; 5 g/L of urea; KH (Perkin Elmer)2PO4，1 g/L；K2HPO4·3H2O，1.3 g/L；MOPS，42 g/L；CaCl2，0.01 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；NiCl·6H2O，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; initial pH 7.2. The strain was first inoculated into TSB broth for 8 h, and the culture was inoculated as a seed into a 24-well plate containing 800. mu.L of fermentation medium per well, starting OD600Controlling the temperature to be about 0.1, culturing for 21 h at 30 ℃, controlling the rotation speed of a pore plate shaker to be 800 rpm, paralleling 3 strains each, detecting the yield of the L-lysine and the consumption of the glucose after the fermentation is finished, and calculating the saccharic acid conversion rate from the glucose to the L-lysine.
As a result, as shown in Table 2, the mutants S217C, S217G, S217T and S217V all had lower L-lysine productivity than the wild-type control strain; the mutants S217A, S217D, S217E, S217L and S217P all had higher L-lysine productivity than the wild-type control strain. Therefore, the amino acid site mutation has better application prospect in the production of L-lysine and derivatives thereof.
TABLE 2
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> pyruvate dehydrogenase mutant and method for producing L-amino acid using the same
<130> 2021-06-13
<160> 44
<170> PatentIn version 3.3
<210> 1
<211> 922
<212> PRT
<213> Corynebacterium glutamicum.
<400> 1
MADQAKLGGK PSDDSNFAMI RDGVASYLND SDPEETNEWM DSLDGLLQES SPERARYLML 60
RLLERASAKR VSLPPMTSTD YVNTIPTSME PEFPGDEEME KRYRRWIRWN AAIMVHRAQR 120
PGIGVGGHIS TYAGAAPLYE VGFNHFFRGK DHPGGGDQIF FQGHASPGMY ARAFMEGRLS 180
EDDLDGFRQE VSREQGGIPS YPHPHGMKDF WEFPTVSMGL GPMDAIYQAR FNRYLENRGI 240
KDTSDQHVWA FLGDGEMDEP ESRGLIQQAA LNNLDNLTFV VNCNLQRLDG PVRGNTKIIQ 300
ELESFFRGAG WSVIKVVWGR EWDELLEKDQ DGALVEIMNN TSDGDYQTFK ANDGAYVREH 360
FFGRDPRTAK LVENMTDEEI WKLPRGGHDY RKVYAAYKRA LETKDRPTVI LAHTIKGYGL 420
GHNFEGRNAT HQMKKLTLDD LKLFRDKQGI PITDEQLEKD PYLPPYYHPG EDAPEIKYMK 480
ERRAALGGYL PERRENYDPI QVPPLDKLRS VRKGSGKQQI ATTMATVRTF KELMRDKGLA 540
DRLVPIIPDE ARTFGLDSWF PTLKIYNPHG QNYVPVDHDL MLSYREAPEG QILHEGINEA 600
GSVASFIAAG TSYATHGKAM IPLYIFYSMF GFQRTGDSIW AAADQMARGF LLGATAGRTT 660
LTGEGLQHMD GHSPVLASTN EGVETYDPSF AYEIAHLVHR GIDRMYGPGK GEDVIYYITI 720
YNEPTPQPAE PEGLDVEGLH KGIYLYSRGE GTGHEANILA SGVGMQWALK AASILEADYG 780
VRANIYSATS WVNLARDGAA RNKAQLRNPG ADAGEAFVTT QLKQTSGPYV AVSDFSTDLP 840
NQIREWVPGD YTVLGADGFG FSDTRPAARR FFNIDAESIV VAVLNSLARE GKIDVSVAAQ 900
AAEKFKLDDP TSVSVDPNAP EE 922
<210> 2
<211> 2769
<212> DNA
<213> Corynebacterium glutamicum.
<400> 2
atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60
cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120
gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180
cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240
tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300
aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360
ccaggcatcg gcgtcggcgg acacatttcc acttacgcag gcgcagcccc tctgtacgaa 420
gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480
ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540
gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg cattccgtcc 600
taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660
ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720
aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780
gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840
gttaactgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900
gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960
gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020
acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080
ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140
tggaagcttc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200
cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260
ggccacaact tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320
ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380
ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440
gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500
caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560
gctaccacca tggcgactgt tcgtaccttc aaggaactga tgcgcgataa gggcttggct 1620
gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680
ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740
atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800
ggttccgtgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860
attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920
gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980
ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040
gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100
ggcatcgacc gcatgtacgg cccaggcaag ggtgaagatg ttatctacta catcaccatc 2160
tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220
aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280
tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340
gttcgtgcca acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400
cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460
cagctgaagc agacctccgg cccatacgtt gcagtgtctg acttctccac tgatctgcca 2520
aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580
ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640
gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700
gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760
gaggaataa 2769
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence
<400> 3
ttcaccaact gtgtccatgg gtct 24
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence
<400> 4
aaacagaccc atggacacag ttgg 24
<210> 5
<211> 83
<212> DNA
<213> Artificial sequence
<400> 5
cccacacggt atgaaggact tctgggagtt cccaactgtg gcaatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 6
<211> 83
<212> DNA
<213> Artificial sequence
<400> 6
cccacacggt atgaaggact tctgggagtt cccaactgtg tgcatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 7
<211> 83
<212> DNA
<213> Artificial sequence
<400> 7
cccacacggt atgaaggact tctgggagtt cccaactgtg gatatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 8
<211> 83
<212> DNA
<213> Artificial sequence
<400> 8
cccacacggt atgaaggact tctgggagtt cccaactgtg gaaatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 9
<211> 83
<212> DNA
<213> Artificial sequence
<400> 9
cccacacggt atgaaggact tctgggagtt cccaactgtg ttcatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 10
<211> 83
<212> DNA
<213> Artificial sequence
<400> 10
cccacacggt atgaaggact tctgggagtt cccaactgtg ggcatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 11
<211> 83
<212> DNA
<213> Artificial sequence
<400> 11
cccacacggt atgaaggact tctgggagtt cccaactgtg cacatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 12
<211> 83
<212> DNA
<213> Artificial sequence
<400> 12
cccacacggt atgaaggact tctgggagtt cccaactgtg atcatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 13
<211> 83
<212> DNA
<213> Artificial sequence
<400> 13
cccacacggt atgaaggact tctgggagtt cccaactgtg aagatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 14
<211> 83
<212> DNA
<213> Artificial sequence
<400> 14
cccacacggt atgaaggact tctgggagtt cccaactgtg ctgatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 15
<211> 83
<212> DNA
<213> Artificial sequence
<400> 15
cccacacggt atgaaggact tctgggagtt cccaactgtg atgatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 16
<211> 83
<212> DNA
<213> Artificial sequence
<400> 16
cccacacggt atgaaggact tctgggagtt cccaactgtg aacatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 17
<211> 83
<212> DNA
<213> Artificial sequence
<400> 17
cccacacggt atgaaggact tctgggagtt cccaactgtg ccaatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 18
<211> 83
<212> DNA
<213> Artificial sequence
<400> 18
cccacacggt atgaaggact tctgggagtt cccaactgtg cagatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 19
<211> 83
<212> DNA
<213> Artificial sequence
<400> 19
cccacacggt atgaaggact tctgggagtt cccaactgtg cgcatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 20
<211> 83
<212> DNA
<213> Artificial sequence
<400> 20
cccacacggt atgaaggact tctgggagtt cccaactgtg accatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 21
<211> 83
<212> DNA
<213> Artificial sequence
<400> 21
cccacacggt atgaaggact tctgggagtt cccaactgtg gtgatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 22
<211> 83
<212> DNA
<213> Artificial sequence
<400> 22
cccacacggt atgaaggact tctgggagtt cccaactgtg tggatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 23
<211> 83
<212> DNA
<213> Artificial sequence
<400> 23
cccacacggt atgaaggact tctgggagtt cccaactgtg tacatgggtc ttggcccaat 60
ggatgccatt taccaggcac gtt 83
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence
<400> 24
gggagttccc aactgtggca 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence
<400> 25
gggagttccc aactgtgtgc 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence
<400> 26
gggagttccc aactgtggat 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence
<400> 27
gggagttccc aactgtggaa 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence
<400> 28
gggagttccc aactgtgttc 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence
<400> 29
gggagttccc aactgtgggc 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence
<400> 30
gggagttccc aactgtgcac 20
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence
<400> 31
gggagttccc aactgtgatc 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence
<400> 32
gggagttccc aactgtgaag 20
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence
<400> 33
gggagttccc aactgtgctg 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<400> 34
gggagttccc aactgtgatg 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence
<400> 35
gggagttccc aactgtgaac 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence
<400> 36
gggagttccc aactgtgcca 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence
<400> 37
gggagttccc aactgtgcag 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence
<400> 38
gggagttccc aactgtgcgc 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence
<400> 39
gggagttccc aactgtgacc 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence
<400> 40
gggagttccc aactgtggtg 20
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence
<400> 41
gggagttccc aactgtgtgg 20
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence
<400> 42
gggagttccc aactgtgtac 20
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence
<400> 43
caaggcgatc agccaagc 18
<210> 44
<211> 25
<212> DNA
<213> Artificial sequence
<400> 44
ccaacctcta tggaacctga attcc 25
Claims (9)
1. The pyruvate dehydrogenase mutant is characterized in that the 217 th site of the amino acid sequence of the mutant is mutated into any one of alanine, aspartic acid, glutamic acid, leucine and proline on the basis of SEQ ID NO. 1.
2. The nucleotide sequence encoding the pyruvate dehydrogenase mutant according to claim 1.
3. An expression cassette comprising the nucleotide sequence encoding the pyruvate dehydrogenase mutant according to claim 2.
4. A vector comprising the nucleotide sequence encoding the pyruvate dehydrogenase mutant according to claim 2.
5. The vector of claim 4, which is an expression vector.
6. A host cell comprising the nucleotide sequence encoding a pyruvate dehydrogenase mutant according to claim 2, wherein the host cell is a microorganism of Corynebacterium glutamicum.
7. The host cell of claim 6, wherein the starting strain of the host cell is Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, or Corynebacterium glutamicum ATCC 14067.
8. Use of a pyruvate dehydrogenase mutant according to claim 1 or a nucleotide encoding the same for the production of L-amino acids; wherein the L-amino acid is selected from L-lysine.
9. A method for producing an L-amino acid, which comprises culturing the host cell of claim 6 or 7 to produce an L-amino acid, and further comprises the step of isolating and extracting or recovering the L-amino acid from the culture medium; wherein the L-amino acid is selected from L-lysine.
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| CN109022381A (en) * | 2018-08-17 | 2018-12-18 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutant and its application |
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| CN109022381A (en) * | 2018-08-17 | 2018-12-18 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutant and its application |
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