CN115806962A - Phosphoenolpyruvate carboxylase mutant and application thereof - Google Patents

Phosphoenolpyruvate carboxylase mutant and application thereof Download PDF

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CN115806962A
CN115806962A CN202111082664.3A CN202111082664A CN115806962A CN 115806962 A CN115806962 A CN 115806962A CN 202111082664 A CN202111082664 A CN 202111082664A CN 115806962 A CN115806962 A CN 115806962A
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mutant
pepc
leu
host cell
gene
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郑平
陈久洲
孙际宾
蒲伟
蔡柠匀
周文娟
刘娇
马延和
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The present invention provides a phosphoenolpyruvate carboxylase mutant, a polynucleotide encoding the mutant, an expression vector, a host cell and a method for producing a target compound using oxaloacetate as a precursor by using the mutant. The yield of lysine and glutamic acid and the glucose conversion rate of the strain expressing the PEPC mutant are obviously higher than those of the strain expressing wild PEPC, and the strain shows good application prospect.

Description

Phosphoenolpyruvate carboxylase mutant and application thereof
Technical Field
The invention belongs to the field of genetic engineering and molecular biology, and particularly relates to a phosphoenolpyruvate carboxylase mutant, an expression vector containing the mutant, a host cell, and a method for producing a target compound taking oxaloacetate as a precursor by using the mutant.
Background
The four-carbon anaplerosis pathway is a key pathway connecting glycolysis pathway and TCA cycle, while oxaloacetate is a key precursor of many amino acids and organic acids, and accumulation of target compounds that are precursors to oxaloacetate can be enhanced by enhancing the four-carbon anaplerosis pathway, such as lysine (Becker J, zelder O, hafner S, schroder H, wittmann C.Metab Eng,2011,13 (2): 159-168.), threonine (Lee KH, park JH, kimTY, kim HU, lee SY. Mol Syst Biol,2007,3 149.) and glutamic acid (Yoka, A., sawada, K.and da, M.Adv Eng Biotechnol,2017,159, 181-198.), and some organic acids such as succinic acid (Millar CS, chepo YP, liao JC, doy England Biotechnol,2017, 3262, J) (Biozoc J.8, J.).
Phosphoenolpyruvate Carboxylase (PEPC) is one of the key enzymes important in the four-carbon anaplerotic pathway,phosphoenolpyruvate and HCO can be converted 3- Conversion to oxaloacetate, but PEPC activity is tightly regulated in vivo, particularly by feedback inhibition of intracellular aspartate and malate (O' Regan M, thierbach G, bachmann B, vileval D, lepage P, viret jf. Gene,1989, 77. Researchers have previously obtained some PEPC mutants that have released feedback inhibition of aspartate and malate by evolutionary screening and rational design, and expression of these mutants helped to increase the accumulation of lysine, aspartate and glutamate (Yokota, a., sawada, k.and Wada, m.adv Biochem Eng Biotechnol,2017,159,181-198 chen, z., bommarmieddy, r.r., frank, d., rappert, s.and Zeng, a.p.appl Environ Microbiol,2014,80,1388-1393 Wada, m., sawada, k., ogura, k.shift, shift, y., hagiwarwara, t., sugimoto, m.and oni, uka.and yoka, 3763 zxft, 3763, bioeng,2016, 172, 2016. However, these mutants have a limited ability to increase the production of oxaloacetate-precursor chemicals, and some of them also have a severe effect on the growth of the cells. There is still a need in the art to develop more efficient mutants of PEPC in order to further increase the production of oxaloacetate-precursor chemicals without affecting the growth of the strain.
Disclosure of Invention
Aiming at the problems in the prior art, in order to obtain more PEPC mutants which are helpful for improving the accumulation of chemicals taking oxaloacetate as a precursor, the inventor utilizes a biosensor constructed in the early stage, constructs a random mutation library for PEPC, screens and obtains a plurality of dominant mutants, the PEPC mutants which are all helpful for improving the accumulation of lysine and glutamic acid and basically do not influence the growth, and the performances of partial mutants are superior to those of the mutants reported in the prior art. On the basis of this, the present invention has been completed.
The invention aims to provide a novel phosphoenolpyruvate carboxylase mutant, an expression vector containing the mutant, a host cell and a method for producing amino acid by using the mutant.
In a first aspect, the present invention provides a novel mutant phosphoenolpyruvate carboxylase, wherein the mutant is any one of the following group:
1, at least one of the following mutated amino acids is present in the polypeptide corresponding to the amino acid sequence shown in SEQ ID NO:
arg300His, asn806Ser, or
Lys152Glu, ser625Pro, ala674Thr, or
Ala200Val, val606Ala, ser625Pro, or
Phe356Ser, asn663Ser, or
Lys701Arg, lys702Glu, asn806Ser, or
Ala545Val, or
Ala589Val, or
Ile254Val, or
Gly17Asp, asn559Asp, or
Ile9Leu, or
His708Arg, ser757Leu, phe799Leu, or
Ala78Thr, tyr214His, his685Arg, or
Glu381Gln,Ile726Thr,Asp805Tyr。
2) 1, a polypeptide which has at least one mutation as described in 1) and has one or more bases added or deleted at both ends of the polypeptide shown in 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.
3) 1, and at least one of the mutations described in 1). Preferably, the homology is at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more.
In a specific embodiment, the present disclosure provides a preferred phosphoenolpyruvate carboxylase mutant, wherein the mutant is a polypeptide having an amino acid sequence shown in SEQ ID NO. 1 and having any one of the following mutations:
arg300His, asn806Ser, or
Lys152Glu, ser625Pro, ala674Thr, or
Ala200Val, val606Ala, ser625Pro, or
Phe356Ser, asn663Ser, or
Lys701Arg, lys702Glu, asn806Ser, or
Ala545Val, or
Ala589Val, or
Ile254Val, or
Gly17Asp, asn559Asp, or
Ile9Leu, or
His708Arg, ser757Leu, phe799Leu, or
Ala78Thr, tyr214His, his685Arg, or
Glu381Gln,Ile726Thr,Asp805Tyr。
In a second aspect, the disclosure provides polynucleotides encoding the above mutants.
In a third aspect, the present disclosure provides an expression vector comprising the above mutant.
In a fourth aspect, the present disclosure provides a host cell comprising the above mutant.
In one embodiment, the host cell is a microorganism of the genera Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providencia (Providencia), enterobacter (Enterobacteria), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), corynebacterium (Corynebacterium).
In a further embodiment, the host cell is corynebacterium glutamicum, and optionally, the starting strain of the host cell is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, corynebacterium glutamicum Z188, and derivatives thereof.
In a fifth aspect, the present disclosure provides a method of producing a compound of interest, wherein the method comprises producing the compound of interest in the presence of the mutant of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect, or culturing the host cell of the fourth aspect to produce the compound of interest. Optionally, a step of separating the target compound from the culture medium is further included.
In a further embodiment, the target compound with oxaloacetate as precursor includes but is not limited to amino acid, organic acid or derivatives thereof, preferably including aspartate family amino acid (lysine, threonine, isoleucine, methionine), glutamate family amino acid (glutamate, proline, hydroxyproline, arginine, glutamate amide), and 5-aminolevulinic acid, etc., preferably organic acid includes succinic acid, α -ketoglutaric acid, malic acid, fumaric acid, etc., and preferred derivatives include but are not limited to pentanediamine, 5-amino pentanoic acid, glutaric acid, etc.
In summary, the present disclosure provides the use of a mutant of the first aspect, a polynucleotide of the second aspect, an expression vector of the third aspect, a host cell of the fourth aspect and a production method of the fifth aspect for the production of a compound of interest, precursor to oxaloacetate.
Advantageous effects of the disclosure
1. The PEPC mutant obtained by the method can obviously improve the yield (such as lysine and glutamic acid) of the target compound taking oxaloacetate as a precursor, and basically does not affect the growth of the strain, so that the production cost of the target compound taking oxaloacetate as a precursor can be further reduced, and the PEPC mutant has better industrial application potential.
2. The PEPC mutant obtained by the method provides a new idea for constructing a strain of a target compound taking oxaloacetate as a precursor.
Drawings
FIG. 1 flow cytometry analysis of PEPC random mutation library.
FIG. 2 analysis of fluorescence intensity of PEPC mutant library 96-well rescreened mutant.
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 and "and/or," the term "or" in the claims means "and/or" unless it is explicitly stated that only alternatives or mutual exclusions between alternatives are mutually exclusive.
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 "phosphoenolpyruvate carboxylase" of the present disclosure refers to a catalytic phosphoenolpyruvate and HCO 3- An enzyme that converts to oxaloacetate. As used herein, the phosphoenolpyruvate carboxylase is not particularly limited as long as it has a corresponding activity, and may be a phosphoenolpyruvate carboxylase derived from a microorganism of the genus Corynebacterium (e.g., corynebacterium glutamicum), but is not limited thereto. For example, the phosphoenolpyruvate carboxylase can be a polypeptide represented by the amino acid sequence of SEQ ID NO. 1 or a polypeptide having an amino acid sequence with 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. In addition, amino acid sequences having deletions, modifications, substitutions or additions are also intended to be within the scope of the present disclosure if they have homology to the above sequences and have substantially the same or corresponding biological activity as the protein represented by SEQ ID NO. 1. In the present disclosure, any polynucleotide sequence encoding a phosphoenolpyruvate carboxylase can 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% identical to the polynucleotide sequence of SEQ ID NO. 2%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homologous polynucleotide sequence. 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.
Further, the phosphoenolpyruvate carboxylase mutants of the present disclosure may include not only the polypeptide having the amino acid sequence of SEQ ID NO. 1, but also phosphoenolpyruvate carboxylase 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 have any one of the mutations of the present disclosure. It is obvious that amino acid sequences having substantially the same or corresponding biological activity as the polypeptide having the amino acid sequence of SEQ ID NO. 1 shall also fall within the scope of the present disclosure. The terms "phosphoenolpyruvate carboxylase", "PEPC", "PPC" according to the invention are identical and interchangeable.
The term "polynucleotide" of the present disclosure 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 individually or as a whole) 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.
The term "homology" of the present disclosure refers to the percentage of identity between two polynucleotide or polypeptide portions. 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 disclosure 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, the wild-type PEPC in the present disclosure refers to a PEPC having an amino acid sequence as set forth in SEQ ID NO: 1.
The term "mutant" of the present disclosure 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 disclosure 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 "conservative mutation" refers to a mutation that can normally maintain the function of a protein. A representative example of conservative mutations is conservative substitutions.
As used in this disclosure, the term "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β -branches (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Examples of the substitution regarded as conservative substitution include substitution of Ala with Ser or Thr, substitution of Arg with Gln, his or Lys, substitution of Asn with Glu, gln, lys, his or Asp, substitution of Asp with Asn, glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, glu, lys, his, asp or Arg, substitution of Glu with Gly, asn, gln, lys or Asp, substitution of Gly with Pro, substitution of His with Asn, lys, gln, arg or Tyr, substitution of Ile with Leu, met, val or Phe, substitution of Leu with Ile, met, val or Phe, substitution of Lys with Asn, glu, gln, his or Arg, substitution of Met with Ile, leu, val or Phe, substitution of Phe with Trp, tyr, met, ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe, tyr, his or Phe, and substitution of Met with Met or Phe. Furthermore, conservative mutations include naturally occurring mutations due to individual differences in the origin of the gene, differences in strain, species, and the like.
The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means the position of one sequence corresponding to a specified position in the other sequence after alignment of the two sequences by homology or sequence identity. Thus, for example, with reference to "a polypeptide corresponding to SEQ ID NO:1 "if the amino acid residue at position 150 of the amino acid sequence shown in SEQ ID NO:1 with a6 × His tag at one end of the amino acid sequence shown in SEQ ID NO:1 may be at position 150 of 156.
The term "expression" of the present disclosure 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 disclosure refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences to express a gene of interest in a suitable host. "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 disclosure refers to any cell type that comprises transformation, transfection, transduction, etc., of a PEPC mutant or expression vector of the present disclosure. 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 disclosure 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 disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of containing the PEPC mutant of the present disclosure. In some embodiments, the host cell refers to a prokaryotic cell, in particular, the host cell is derived from a microorganism suitable for the fermentative production of a target compound (e.g., an L-amino acid) which is precursor to oxaloacetate, and may include strains of Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providencia (Providencia), enterobacter (enterobacter), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), corynebacterium (Corynebacterium), and the like, but is not limited thereto. Preferably, it may be Corynebacterium glutamicum. Preferably, corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, corynebacterium glutamicum Z188, and strains derived therefrom, and the like can be mentioned. Illustratively, the derivative strain may be any strain as long as the strain has the ability to produce a target compound (e.g., an L-amino acid) whose precursor is oxaloacetate.
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. the adhE gene encoding alcohol dehydrogenase;
b. the ackA gene encoding acetate kinase;
c. a pta gene encoding a phosphate acetyltransferase;
d. an ldhA gene encoding lactate dehydrogenase;
e. the focA gene encoding a formate transporter;
f. the pflB gene encoding pyruvate formate lyase;
g. a poxB gene encoding pyruvate oxidase;
h. the thrA gene of bifunctional enzyme for coding aspartokinase I/homoserine dehydrogenase I;
i. the thrB gene encoding homoserine kinase;
j. an ldcC gene encoding lysine decarboxylase; and
h. the cadA gene which codes for lysine decarboxylase.
In some embodiments, one or more genes selected from the group consisting of, but not limited to:
a. the dapA gene encoding dihydrodipicolinate synthase which relieves feedback inhibition by lysine;
b. a dapB gene encoding dihydrodipicolinate reductase;
c. a ddh gene encoding diaminopimelate dehydrogenase;
d. dapD encoding a tetrahydropyridyldicarboxylate succinylase and dapE encoding a succinyldiaminopimelate deacylase;
e. an asd gene encoding aspartate-semialdehyde dehydrogenase;
f. the ppc gene encoding phosphoenolpyruvate carboxylase;
g. the pntAB gene encoding nicotinamide adenine dinucleotide transhydrogenase;
i. the lysE gene of the transport protein which codes for lysine.
Illustratively, the host cell is a glutamate producing host cell. In some embodiments, the host cell for producing glutamic acid can be corynebacterium glutamicum Z188, or a derivative strain of corynebacterium glutamicum Z188. In some embodiments, one or more genes that may include, but are not limited to, those selected from the group consisting of:
a) Encoding the glutamate dehydrogenase gdh gene;
b) A gene encoding glutamine synthetase glnA;
c) Encoding the glutamate synthase gltBD gene;
d) Encoding an isocitrate dehydrogenase icdA gene;
e) Coding aconitate hydratase acnA/acnB gene;
f) Encoding the citrate synthase gltA gene;
g) The gene encoding pyruvate carboxylase pyc;
h) Encoding pyruvate dehydrogenase aceEF/lpdA gene;
i) The gene encoding pyruvate kinase pykA/pykF;
j) The gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase;
k) Encodes the glucose phosphate isomerase pgi gene.
In some embodiments, the host cell that produces glutamate may also include, but is not limited to, one or more genes attenuated or eliminated selected from the group consisting of:
a) Encoding the aceA gene of isocitrate lyase;
b) Encoding an alpha-ketoglutarate dehydrogenase kgd gene;
c) A gene encoding lactate dehydrogenase ldh;
d) Encoding the glutamate decarboxylase gad gene.
Illustratively, the host cell is a proline-producing host cell. In some embodiments, the proline-producing host cell may be escherichia coli or corynebacterium glutamicum, or a derivative strain thereof. In some embodiments, one or more genes that may include, but are not limited to, those selected from the group consisting of:
a) Encoding the glutamate kinase proB gene for the release of feedback inhibition;
b) The proA gene which codes for glutamate-5-semialdehyde dehydrogenase;
c) The proC gene which codes for the pyrrole-5-carboxylate dehydrogenase;
d) The gdh gene which codes for glutamate dehydrogenase;
e) The gene encoding pyruvate carboxylase pyc;
f) The gapN gene encoding glyceraldehyde-3-phosphate dehydrogenase;
g) Encoding l-proline efflux protein thrE or serE gene.
In some embodiments, the proline-producing host cell may also include, but is not limited to, attenuated or reduced expression of one or more genes selected from the group consisting of:
a) Encoding proline dehydrogenase/pyrrole-5-carboxylic acid dehydrogenase putA gene;
b) Encoding L-glutamic acid efflux protein mscCG gene;
c) Encoding the transporter proP/proY/proU gene.
Illustratively, the host cell is a host cell that produces hydroxyproline. In some embodiments, the host cell for producing hydroxyproline may be escherichia coli or corynebacterium glutamicum, or a derivative strain thereof. In some embodiments, one or more genes that may include, but are not limited to, those selected from the group consisting of:
a) A gene encoding proline hydroxylase;
b) Encoding the glutamate kinase proB gene for the release of feedback inhibition;
c) The proA gene which codes for glutamate-5-semialdehyde dehydrogenase;
d) The proC gene which codes for the pyrrole-5-carboxylate dehydrogenase;
e) Encoding the glutamate dehydrogenase gdh gene;
f) The gene encoding pyruvate carboxylase pyc;
g) The gapN gene encoding glyceraldehyde-3-phosphate dehydrogenase;
h) The gene encoding the ammonium transporter amtB;
i) Encoding the transporter proP/proY/proU gene.
In some embodiments, the hydroxyproline-producing host cell may also include, but is not limited to, attenuated or reduced expression of one or more genes selected from the group consisting of:
a) Encoding proline dehydrogenase/pyrrole-5-carboxylic acid dehydrogenase putA gene;
b) Encoding L-glutamic acid efflux protein mscCG gene;
c) Encoding L-proline efflux protein thrE or serE gene.
Illustratively, the host cell is a threonine producing host cell. In some embodiments, the threonine-producing host cell can be Escherichia coli or Corynebacterium glutamicum, or a derivative strain thereof. In some embodiments, one or more genes that may include, but are not limited to, those selected from the group consisting of:
a) Encoding aspartokinase III lysC gene;
b) Encoding aspartate semialdehyde dehydrogenase asd gene;
c) Encoding aspartokinase I thrA gene;
d) Encoding the homoserine kinase thrB gene;
e) Encoding threonine synthase thrC gene;
f) Encoding an aspartate aminotransferase aspC gene;
g) The coding 6-phosphogluconate dehydrogenase gnd gene.
In some embodiments, the threonine-producing host cell can be Escherichia coli or Corynebacterium glutamicum, or a derivative strain thereof. In some embodiments, one or more genes selected from, but not limited to, the following, may also be attenuated or eliminated in the threonine producing host cell:
a) A dapA gene encoding dihydrobipyridine synthase;
b) A dapB gene encoding dihydrodipicolinate reductase;
c) The ackA gene encoding acetate kinase;
d) A pta gene encoding a phosphate acetyltransferase;
e) Encoding the transcription regulatory factor tyrR gene.
Illustratively, the host cell is a host cell that produces 5-aminolevulinic acid. In some embodiments, the host cell for producing 5-aminolevulinic acid can be Escherichia coli or Corynebacterium glutamicum, and also can be a derivative strain thereof. In some embodiments, one or more genes in the 5-aminolevulinic acid-producing host cell, which may include but are not limited to those selected from the group consisting of:
a) A gene encoding 5-amino acid propionate synthetase hemA;
b) The gene encoding pyruvate carboxylase pyc;
c) The gene encoding pantothenate kinase coaA;
d) The rhtA gene encoding threonine/homoserine transporter;
e) Encoding cysteine/O-acetylserine transporter eamA gene;
f) The gene agt which codes for alanine, glyoxylate aminotransferase.
g) A gene encoding an antioxidant-related protein.
In some embodiments, the host cell for producing 5-amino acid propionic acid may be Escherichia coli or Corynebacterium glutamicum, or a derivative strain thereof. In some embodiments, the host cell that produces 5-amino acid propionic acid may also include, but is not limited to, one or more genes selected from the group consisting of:
a) Encoding succinyl-coa synthetase sucCD gene;
b) A gene encoding succinate dehydrogenase sdhAB;
c) The gene encoding 5-aminolevulinic acid dehydratase hemB;
d) Encoding the aceA gene of isocitrate lyase;
e) Encoding malate synthase aceB gene.
The term "oxaloacetate-precursor target compound" of the present disclosure includes, but is not limited to, amino acids including aspartate family amino acids (lysine, threonine, isoleucine, methionine), glutamate family amino acids (glutamate, proline, hydroxyproline, arginine, glutamate amide), and 5-aminolevulinic acid, etc., organic acids including succinic acid, α -ketoglutarate, malate, fumarate, etc., and derivatives thereof including, but not limited to, pentanediamine, glutarate, etc. By the method, the yield of the target compound taking oxaloacetate as a precursor of the strain can be improved.
The term "culturing" of the present disclosure 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 in the disclosure or clearly indicated by the background, all technical and scientific terms used in the disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Examples
Other objects, features and advantages of the present disclosure 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 disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure 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: conditions described in a 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 construction and engineering of lysine biosensors
Carrying out whole gene synthesis according to the reported sequence information of the eyfp gene (GeneBank No. CCD28585.1), and carrying out PCR amplification by using a primer eyfp-F/eyfp-R to obtain an eyfp gene fragment; PCR amplification was carried out using Corynebacterium glutamicum ATCC13032 genome (Gene ID: 2830649) as a template and lysGE-F/lysGE-R as a primer to obtain a fragment of lysGE Gene; the full-length plasmid was PCR-reverse-amplified using the plasmid pTRCmob (Qian Liu, et al, J Biotechnol,2007,132, 273-279) as a template and pTRCmob-Rev-F/pTRCmob-Rev-R as primers to obtain a linearized vector fragment. After the three fragments are purified and recovered, a plasmid vector, a lysGE gene fragment and an eyfp gene fragment are connected by using recombinase, and a correct recombinant vector is named as pLysGE through transformation, enzyme digestion and sequencing verification. And designing a point mutation primer LysGE-mut-F/R, and performing reverse amplification by PCR, fragment purification and transformation by taking pLysGE as a template to obtain the lysine biosensor pLysGE58 with widened response concentration range and remarkably enhanced response strength to lysine. The response principle of the lysine biosensor is that when the concentration of intracellular lysine is low, the regulatory protein LysG cannot be combined with the lysine, so that the transcription of a fluorescent protein gene cannot be started by regulating a lysE gene promoter; when the intracellular lysine reaches a certain concentration, the regulatory protein LysG is combined with the lysine, so that the conformation of the LysG is changed, and then the regulatory protein is combined with a promoter of lysE to start the expression of the downstream reporter gene yellow fluorescent protein.
Example 2 pepc knock-out Strain construction
According to a lysine strain construction method (Becker, J., et al. Metab. Eng.,2011,13,159-168) disclosed in the literature, threonine at the 311 th position of aspartokinase (lysC gene code) on a Corynebacterium glutamicum ATCC13032 genome is mutated into isoleucine by a pK18 mobsacB-based homologous recombination technology, and a strain SCgL30 with certain lysine synthesis capacity is constructed. To exclude the genomic expression of wild-type PEPC from interfering with plasmid-overexpressed PEPC mutants, we knocked out the PEPC gene of the SCgL30 genome using CRISPR-Cas 9-based gene knock-out methods reported in the literature (Liu, j., et al. Micro.cell fact.,2017,16,205). The specific experimental process is as follows:
(1) Construction of pepc Gene knockout plasmid pgRNA-delta pepc
Designing primers pepc-up-F/R and pepc-down-F/R respectively according to the disclosed genome sequence of Corynebacterium glutamicum ATCC13032, and obtaining upstream and downstream homologous arm fragments for pepc gene knockout by PCR amplification by taking the ATCC13032 genome as a template; meanwhile, pgRNA2 (Liu, J., et al. Microb. Cell fact.,2017,16,205.) is used as a template, a framework fragment 1 is amplified by using a primer pepc-pgRNA-2/pepc-pgRNA-3, and a framework fragment 2 with pepc-sgRNA (N20, 5'-CGTGCGGGATCAGCGGACGA-3') is amplified by using gRNA-pepc/pgRNA-1 as a primer. And purifying and recovering the four fragments, and recombining and connecting to obtain the pepc gene knockout plasmid pgRNA-delta pepc.
(2) Pepc Gene knockout on SCgL30 Strain chromosome
The knockout plasmid pCas9 (Liu, J., et al. Microb. Cell fact, 2017,16,205) and pgRNA- Δ pepc were co-transformed into competent cells of the strain SCgL30 by an electric pulse method, 1mL of TSB medium preheated at 46 ℃ was added, incubated at 46 ℃ for 6min, incubated at 30 ℃ for 3h, and coated with a coating containing 50 μ M IPTG; the recombinant transformants were obtained by culturing in TSB solid medium containing 25. Mu.g/mL kanamycin and 5. Mu.g/mL chloramphenicol at 30 ℃ for 2 days. Wherein the TSB culture medium comprises the following components: glucose, 5g/L; 5g/L of yeast powder; soybean peptone, 9g/L; 3g/L of urea; succinic acid, 0.5g/L; k 2 HPO 4 ·3H 2 O，1g/L；MgSO 4 ·7H 2 O,0.1g/L; biotin, 0.01mg/L; vitamin B1,0.1mg/L; MOPS,20g/L. Verifying a transformant by using a verification primer pepc-TF/TR to confirm that a target gene on a chromosome of the strain is successfully knocked out, and then discarding the pCas9 plasmid at a high temperature of 37 ℃ to obtain a pepc gene knocked-out strain named ZPCgL1. The recombinant vector pLysGE58 carrying the lysine biosensor in example 1 was transformed into ZPGGL 1 to obtain the selection host strain ZPGGL 1/pLysGE58.
Example 3 construction of PEPC and its mutant expression vectors
A primer pepc-tac-F/R is designed according to the published genome sequence of the Corynebacterium glutamicum ATCC13032, and a pepc gene fragment is obtained by PCR amplification by taking the ATCC13032 genome as a template. Designing a primer pXMJ19-Rev-2F/2R according to the sequence information of the plasmid pXMJ19, obtaining a linearized vector fragment by PCR reverse amplification by taking the plasmid pXMJ19 as a template, recovering the two fragments, and recombining and connecting to obtain the pepc over-expression vector pXMJ19-pepc.
The information on the PEPC mutant that released feedback inhibition by aspartic acid and malic acid was reported in the literature (Yokota, A., sawada, K.and W.ada, M.Adv Biochem Eng Biotechnol,2017,159,181-198; chen, Z, bommareddy, R.R., frank, D, rappert, S.and Zeng, A.P.appl Environ Microbiol,2014,80,1388-1393), designing mutation primers D299N-F/R and N917G-F/R of D299N and N917G mutant respectively, PCR amplifying plasmid fragment by taking pXMJ19-PEPC as a template, recovering and transforming E.coli DH5 alpha competent cells to obtain PEPC respectively D299N Or PEPC N917G The expression vector pXMJ19-pepc D299N And pXMJ19-pepc N917G As a positive control plasmid. Empty plasmid pXMJ19, negative control plasmid pXMJ19-pepc and positive control plasmid pXMJ19-pepc D299N And pXMJ19-pepc N917G ZPGGL 1/pLysGE58 is respectively transformed to obtain control strains for PEPC mutant screening and rescreening evaluation.
Example 4 construction of PEPC random mutation library
The recombinant vector pXMJ19-pepc containing pepc gene is used as a template, and 0.05-0.5mM MnCl is added by using a primer pepc-mut-2F/2R 2 Obtaining pepc gene segments with different mutation rates by error-prone PCR amplification under the condition of (1). Meanwhile, pXMJ19-pepc is used as a template, and a primer pPEPC-Rev-F/R is used for obtaining a linearized vector framework through PCR reverse amplification. The two fragments are recovered, recombined and connected, chemically transformed into E.coli DH5 alpha competent cells, and cultured overnight at 37 ℃. And scraping colonies from the plate, extracting mixed plasmids, and obtaining PEPC random mutation libraries with different mutation rates.
Example 5 PEPC random mutation library flow cytometry sorting
The PEPC random mutation libraries with different mutation rates obtained in example 4 were mixed in equimolar proportions, electrically pulsed into the strain ZPCGL1/pLysGE58, resuscitated at 30 ℃ for 2h, and the resuscitated solution was inoculated into TSB broth containing 25. Mu.g/mL kanamycin and 5. Mu.g/mL chloramphenicol and cultured at 30 ℃ for 16h to obtain a library of strains for screening. The negative control strains ZPGL 1/pLysGE58/pXMJ19 and ZPGCL 1/pLysGE58/pXMJ19-pepc and the mutant library strains were inoculated into a TSB broth containing 25. Mu.g/mL kanamycin and 5. Mu.g/mL chloramphenicol, cultured at 30 ℃ for 8 hours, transferred to 24-well plates containing 1mL CGXII medium (containing 25. Mu.g/mL kanamycin, 5. Mu.g/mL chloramphenicol and 25. Mu.M ITPG), and the wells were plated at 30 ℃ in 24-well platesPerforming shake culture for 6h, wherein the CGXII medium comprises the following components: 50g/L glucose,16.5g/L NH 4 Cl,5g/L urea,1g/L KH 2 PO 4 ,1g/L K 2 HPO 4 ,42g/L MOPS,0.25g/L MgSO 4 ,0.01g/LFeSO 4 ·2H 2 O,0.01g/L MnSO 4 ·H 2 O,0.001g/L ZnSO 4 ·7H 2 O,0.2mg/L CuSO 4 ,0.02mg/L NiCL·6H 2 O,0.01g/L CaCl 2 0.03g/L protocatechuic acid,0.2mg/L biotin, and 0.1mg/L vitamin B1. The cell concentration of the culture was diluted to 0.1OD with PBS buffer 600nm The fluorescence intensity of the above-mentioned strain was analyzed by a flow cytometer, and the analysis results are shown in FIG. 1. The flow cytometer sorting region (R15) of the mutant library strain was selected based on the fluorescence distribution of the strain ZPGCL 1/pLysGE58/pXMJ19, the strain ZPGCL 1/pLysGE58/pXMJ19-PEPC expressing the wild-type PEPC containing 0.02% of fluorescence enhanced cells in the R15 region, and the mutant library strain containing 0.38% of fluorescence enhanced cells in the R15 region. According to the response principle of the lysine biosensor, when the concentration of lysine is higher, the regulatory protein LysG is combined with the lysine, and further combined with the promoter of the lysE gene, so that the expression of downstream fluorescent reporter gene protein is started. Thus, the above data indicate that the mutant library contains more PEPC mutant strains that produce increased amounts of lysine, and subsequently, cells in this region are sorted and monocloned.
Example 6 re-screening of PEPC mutant libraries and Effect on lysine Synthesis
Single-cell colonies previously obtained by flow cytometry sorting were randomly picked and a portion of the single colonies was inoculated into a 96-well plate containing 200. Mu.L of TSB broth (containing 25. Mu.g/mL kanamycin and 5. Mu.g/mL chloramphenicol), together with the empty plasmid control strain ZPCGL1/pLysGE58/pXMJ19, the wild-type PEPC overexpression strain ZPCGL1/pLysGE58/pXMJ19-PEPC and the positive control strain CgPL 1/pLysGE58/pXMJ19-PEPC D299N And ZPGGL 1/pLysGE58/pXMJ19-pepc N917G Culturing at 30 deg.C and 800rpm for 8h in a well plate shaker. The seed solution was transferred at 5% (V/V) to a medium containing 200. Mu.LCGXII (containing 25. Mu.M ITPG, 25. Mu.g/mL kanamycin and 5. Mu.g/well in the blood vessel of the plant)mL chloramphenicol, 100g/L primary glucose) in a 96-well plate, incubated at 30 ℃ for 6 hours at 800rpm in a plate shaker, diluted 20-fold with PBS buffer, and then the fluorescence intensity and OD of eYFP were measured with a microplate reader (spectra max M5, molecular Devices, λ excitation =488nm, λ emission =520 nm) 600nm As shown in FIG. 2, the mutants having fluorescence values stronger than those of the wild-type PEPC-overexpressing strain ZPCGL1/pLysGE58/pXMJ19-PEPC were sequenced and the mutation sites thereof were analyzed. Meanwhile, continuously culturing the mutant strains with enhanced fluorescence values, stopping culturing after 36h, and detecting OD by using a microplate reader 600nm SBA-40D biosensing analyzer (institute of biological sciences, shandong province, academy of sciences) measures lysine concentration and residual glucose concentration. The mutant information and the lysine yield are shown in table 1, and it can be seen that the lysine yield and the glucose conversion rate of the screened PEPC-expressing mutant are obviously higher than those of wild PEPC-expressing strains, the effect is better than that of reported D299N and N917G mutants, and the application prospect is good.
TABLE 1 PEPC mutants and their lysine production
Figure BDA0003264535640000201
Example 7 PEPC mutant expression vector construction
To verify the effect of the PEPC mutants obtained in the above examples in the production of other oxaloacetate-precursor target compounds, we randomly selected the 2-50 and 5-66 mutants obtained in the above examples and tested the effect of the two mutants in the production of glutamate.
Firstly, designing a primer pepc-250-F/R according to the sequence of a pepc gene, and respectively taking strains containing 2-50 mutants and 5-66 mutants as templates to obtain corresponding gene fragments through PCR amplification. Designing a primer PEPC-pXMJ19-F/R according to the sequence information of the plasmid pXMJ19, obtaining a linearized vector fragment by PCR reverse amplification by taking the plasmid pXMJ19 as a template, recovering the two fragments, recombining and connecting to obtain the PEPC mutant expression vector pXMJ19-PEPC 2-50 And pXMJ19-pepc 5-66 。
Example 8 construction of PEPC wild type and mutant overexpression strains
Competent cells of a glutamic acid-producing strain Z188 (NCBI Reference Sequence: NZ _ AKXP 00000000.1) were prepared by a method reported in the literature (Ruan Y, et al, biotechnol. Lett.,2015, 2445-52.), 1. Mu.g of a PEPC mutant expression vector and control vectors pXMJ19 and pXMJ19-PEPC were electroporated into Z188 competent cells obtained as described above, respectively, a TSB solid medium containing 5. Mu.g/mL of chloramphenicol was spread on the cells, and the cells were cultured at 30 ℃ for 1 day to obtain recombinant strains Z188/pXMJ19, Z188/pXMJ19-PEPC, and Z188/pXMJ19-PEPC 2-50 、Z188/pXMJ19-pepc 5-66 。
Example 9 Effect of PEPC mutant expression on glutamate Synthesis
To test the effect of the overexpression of PEPC mutant by Corynebacterium glutamicum on the production of glutamic acid by the strain, fermentation tests were carried out on the recombinant strains obtained in example 8, respectively. The seed culture medium comprises the following components: glucose, 50g/L; 0.7g/L phosphoric acid; ammonium sulfate, 10g/L; mgSO (MgSO) 4 ·7H 2 O,0.8g/L; 3g/L of corn starch; 10g/L of urea; peptone, 1g/L; 0.5g/L of yeast powder; initial pH7.0. Compared with a seed culture medium, the fermentation culture medium is not added with peptone and yeast powder, and 84g/L MOPS is additionally added. The strains were first inoculated into seed medium for 14h and the cultures were inoculated as seeds into 24-well plates containing 800. Mu.L of fermentation medium per well, starting OD 600 Controlling the culture at 30 ℃ for 29h under the condition of 0.3, controlling the rotation speed of a pore plate shaker to be 800rpm, enabling 3 strains to be parallel, detecting the yield of the glutamic acid and the consumption of the glucose after the fermentation is finished, and calculating the conversion rate of the saccharic acid from the glucose to the glutamic acid. The results are shown in table-2, the glutamic acid yield of the PEPC mutant expression strain is obviously higher than that of the empty plasmid and the control strain expressing wild PEPC, and the saccharic acid conversion rate is greatly improved. Therefore, the PEPC mutant obtained by the invention has better application prospect in the production of glutamic acid and derivatives thereof.
TABLE-2 overexpression of PEPC mutant strains and their glutamic acid fermentation yields
Figure BDA0003264535640000211
TABLE 3 primers used in the examples of the present disclosure
Figure BDA0003264535640000212
Figure BDA0003264535640000221
In addition, this example only shows an example of the PEPC mutant improving the yield of lysine and glutamic acid, and as mentioned above, since oxaloacetate is a key precursor of many amino acids and organic acids, the accumulation of target compounds using oxaloacetate as a precursor, such as lysine, threonine, glutamic acid, and some organic acids, can be improved by strengthening the four-carbon anaplerosis pathway, so those skilled in the art can understand that the PEPC mutant of the present disclosure can also increase the yield of target compounds using oxaloacetate as a precursor, and the method for producing target compounds using oxaloacetate as a precursor by using the PEPC mutant of the present disclosure is within the protection scope of the present disclosure.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> phosphoenolpyruvate carboxylase mutant and application thereof
<130> CPCN21411230
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 919
<212> PRT
<213> Corynebacterium glutamicum
<400> 1
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Arg Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Gly Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Lys Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Thr Glu Val Ala
820 825 830
Glu Arg Val Tyr Ser Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915

Claims (11)

1. A phosphoenolpyruvate carboxylase mutant, wherein the mutant is any one of the group consisting of:
1) 1, at least one of the following mutated amino acids is present in the polypeptide corresponding to the amino acid sequence shown in SEQ ID NO:
arg300His, asn806Ser, or
Lys152Glu, ser625Pro, ala674Thr, or
Ala200Val, val606Ala, ser625Pro, or
Phe356Ser, asn663Ser, or
Lys701Arg, lys702Glu, asn806Ser, or
Ala545Val, or
Ala589Val, or
Ile254Val, or
Gly17Asp, asn559Asp, or
Ile9Leu, or
His708Arg, ser757Leu, phe799Leu, or
Ala78Thr, tyr214His, his685Arg, or
Glu381Gln,Ile726Thr,Asp805Tyr。
2) 1, a polypeptide which has at least one mutation as described in 1) and has one or more bases added or deleted at both ends of the polypeptide shown in 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.
3) A polypeptide having at least 90% homology with the polypeptide shown in SEQ ID NO. 1 and having at least any one of the mutations described in 1). Preferably, the homology is at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more.
2. The mutant phosphoenolpyruvate carboxylase according to claim 1, wherein the mutant is a polypeptide having an amino acid sequence as set forth in SEQ ID No. 1 and having any one of the following mutations:
arg300His, asn806Ser, or
Lys152Glu, ser625Pro, ala674Thr, or
Ala200Val, val606Ala, ser625Pro, or
Phe356Ser, asn663Ser, or
Lys701Arg, lys702Glu, asn806Ser, or
Ala545Val, or
Ala589Val, or
Ile254Val, or
Gly17Asp, asn559Asp, or
Ile9Leu, or
His708Arg, ser757Leu, phe799Leu, or
Ala78Thr, tyr214His, his685Arg, or
Glu381Gln,Ile726Thr,Asp805Tyr。
3. A polynucleotide encoding the phosphoenolpyruvate carboxylase mutant according to claim 1 to 2.
4. An expression vector comprising the phosphoenolpyruvate carboxylase mutant according to claim 1 to 2.
5. A host cell comprising the mutant phosphoenolpyruvate carboxylase of claims 1 to 2.
6. The host cell according to claim 5, characterized in that the host cell is a microorganism of the genera Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providencia (Providencia), enterobacteria (Enterobacteria), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), corynebacterium (Corynebacterium).
7. The host cell of claim 6, wherein the host cell is Corynebacterium glutamicum, and optionally wherein the starting strain of the host cell is Corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, corynebacterium glutamicum Z188, and derivatives thereof.
8. A method for producing a target compound which is a precursor to oxaloacetate, comprising producing the target compound in the presence of the mutant of any one of claims 1-2, the polynucleotide of claim 3, the expression vector of claim 4, or culturing the host cell of claims 5-7 to produce the target compound.
9. The method of claim 8, further comprising the step of separating the target compound from the culture medium.
10. Method for the production of oxaloacetate-precursor target compounds according to claims 8-9, characterized in that said oxaloacetate-precursor target compounds include but are not limited to amino acids, organic acids or their derivatives, preferably including aspartate family amino acids (lysine, threonine, isoleucine, methionine), glutamate family amino acids (glutamate, proline, hydroxyproline, arginine, glutamate amide), and 5-aminolevulinic acid, etc., preferably organic acids including succinic acid, α -ketoglutaric acid, malic acid, fumaric acid, preferably derivatives including but not limited to pentanediamine, 5-amino pentanoic acid, glutaric acid, etc.
11. Use of a phosphoenolpyruvate carboxylase mutant according to claims 1 to 2, a polynucleotide according to claim 3, an expression vector according to claim 4 and/or a host cell according to claims 5 to 7 for the production of a target compound species which is precursor for oxaloacetate.
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Publication number Priority date Publication date Assignee Title
JPH07111890A (en) * 1993-08-24 1995-05-02 Ajinomoto Co Inc Variant phosphoenolpyruvate carboxylase, its gene and production of amino acid
CN1133615A (en) * 1993-08-24 1996-10-16 味之素株式会社 Variant phosphoenolypyruvate carboxylase, gene thereof, and process for producing amino acid
CN101100661A (en) * 2007-06-25 2008-01-09 清华大学 Phosphoric acid enol type pyruvate carboxylase and coding gene thereof
CN110195087A (en) * 2019-05-16 2019-09-03 黑龙江伊品生物科技有限公司 With the method for the bacterial fermentation production L-lysine for changing ppc gene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07111890A (en) * 1993-08-24 1995-05-02 Ajinomoto Co Inc Variant phosphoenolpyruvate carboxylase, its gene and production of amino acid
CN1133615A (en) * 1993-08-24 1996-10-16 味之素株式会社 Variant phosphoenolypyruvate carboxylase, gene thereof, and process for producing amino acid
CN101100661A (en) * 2007-06-25 2008-01-09 清华大学 Phosphoric acid enol type pyruvate carboxylase and coding gene thereof
CN110195087A (en) * 2019-05-16 2019-09-03 黑龙江伊品生物科技有限公司 With the method for the bacterial fermentation production L-lysine for changing ppc gene

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