WO2022133917A1 - Modified phosphoenolpyruvate carboxylase and application thereof in increasing yield of amino acids of corynebacterium glutamicum - Google Patents
Modified phosphoenolpyruvate carboxylase and application thereof in increasing yield of amino acids of corynebacterium glutamicum Download PDFInfo
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- WO2022133917A1 WO2022133917A1 PCT/CN2020/139053 CN2020139053W WO2022133917A1 WO 2022133917 A1 WO2022133917 A1 WO 2022133917A1 CN 2020139053 W CN2020139053 W CN 2020139053W WO 2022133917 A1 WO2022133917 A1 WO 2022133917A1
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- Prior art keywords
- phosphoenolpyruvate carboxylase
- amino acid
- amino acids
- wild
- modified
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- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 0.000 title claims abstract description 165
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Definitions
- the present invention relates to the field of biology. Specifically, the present invention relates to the modified phosphoenolpyruvate carboxylase and its application in improving the amino acid production of Corynebacterium glutamicum.
- Amino acids are the basic building blocks of proteins, and these nutritionally critical compounds are widely used in the preparation of feed additives, food ingredients, nutraceuticals and pharmaceuticals.
- aspartic amino acids including L-lysine and L-threonine
- Corynebacterium glutamicum or Escherichia coli Fermentation has occupied an increasing share of the amino acid market.
- oxaloacetate (OAA)-derived amino acids such as L-lysine or L-threonine mainly depends on the carbon flux through the complementation pathway.
- Corynebacterium glutamicum has phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (Pyruvate carboxylase, PYC), which are used to supplement the consumed oxaloacetate.
- PEPC phosphoenolpyruvate carboxylase
- PYC pyruvate carboxylase
- Phosphoenolpyruvate carboxylase is an enzyme found in most bacteria and all plants.
- Phosphoenolpyruvate (PEP) is an intermediate product of glycolysis. After PEP is carboxylated to oxaloacetate by PEPC, it is quickly converted to malate by Malate Dehydrogenase. ), thus supplementing the tricarboxylic acid cycle (TCA cycle) with intermediates.
- TCA cycle tricarboxylic acid cycle
- Many intermediate products in the TCA cycle are substrates required for amino acid synthesis, so in most organisms, the main role of PEPC is to shunt glycolysis to provide raw materials for amino acid synthesis.
- Some intermediate products in the TCA cycle are the precursors for the synthesis of many important organic compounds, such as OAA is the carbon skeleton for the synthesis of aspartic amino acids.
- Alpha-ketoglutarate is the carbon skeleton for the synthesis of glutamate amino acids.
- Phosphoenolpyruvate carboxylase catalyzes the reaction of phosphoenolpyruvate with CO2 to form oxaloacetate.
- the enhanced activity of phosphoenolpyruvate carboxylase helps to increase the accumulation of oxaloacetate.
- Phosphoenolpyruvate carboxylase was first discovered in spinach leaves. In vivo it exists in the form of homotetramers with a monomer size of 100-110 kDa. PEP carboxylase also undergoes various modulations in its active expression. Studies have reported the isolation and purification of PEPC enzymes from many different bacteria, and found that their activities are regulated by many metabolites. In Escherichia coli, PEPC enzyme activity can be activated by acetyl-CoA and fructose 1,6-bisphosphate, and can produce synergistic activation, but is severely inhibited by aspartate and malate.
- PEPC enzymatic activity is inhibited by aspartate and some TCA cycle metabolites, especially succinate.
- the regulation of PEPC is different from that of E. coli PEPC. Its enzymatic activity cannot be activated by acetyl-CoA, but it can also be strongly inhibited by aspartic acid.
- glutamate inhibited the enzyme activity of Corynebacterium glutamicum PEPC more obviously.
- the present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent.
- the present invention proposes modified phosphoenolpyruvate carboxylase, nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vector, genetically engineered bacteria, test kit, and obtained modified phosphoenolacetone
- the method for acid carboxylase and its application, the method for improving the yield of amino acid of Corynebacterium glutamicum and the method for producing amino acid, by carrying out codon optimization and specific amino acid site mutation of wild-type phosphoenolpyruvate carboxylase, the The obtained modified phosphoenolpyruvate carboxylase has a great influence on the carbon flux of amino acids, and the encoding gene of the modified phosphoenolpyruvate carboxylase is expressed in Corynebacterium glutamicum, which can effectively improve the amino acid carbon flux. Yield, the application prospect is good.
- the present invention provides an engineered phosphoenolpyruvate carboxylase.
- the amino acid sequence of the engineered phosphoenolpyruvate carboxylase is at position 771 and/or position 931 and/or the amino acid at position 943 is substituted;
- the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or has the amino acid sequence shown in SEQ ID NO: 1 with at least 80 % homology to the amino acid sequence.
- the above-mentioned modified phosphoenolpyruvate carboxylase may also have the following additional technical features:
- the wild-type phosphoenolpyruvate carboxylase is derived from a plant.
- the wild-type phosphoenolpyruvate carboxylase is derived from sorghum.
- the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and/or lysine at position 931 by glutamine and/or Or aspartic acid at position 943 is replaced by asparagine.
- the 931st lysine of the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by glutamine and the 943rd aspartic acid is substituted by asparagine.
- the present invention provides a nucleic acid encoding an engineered phosphoenolpyruvate carboxylase.
- the nucleotide sequence encoding the modified phosphoenolpyruvate carboxylase is codon-optimized in advance , and the optimized nucleotide sequence has a mutation at the 2312th base, the 2791st base and/or the 2827th base, the nucleotide sequence encoding the wild-type phosphoenolpyruvate carboxylase Has the nucleotide sequence shown in SEQ ID NO:2 or has a nucleotide sequence with at least 80% homology to the nucleotide sequence shown in SEQ ID NO:2.
- the nucleic acid according to the embodiment of the present invention expresses phosphoenolpyruvate carboxylase in Coryne
- the optimized nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 3 source nucleotide sequence.
- the optimized nucleotide sequence has mutations of c.2312C>A, c.2791C>A and/or c.2827G>A.
- the optimized nucleotide sequence has c.2791C>A and c.2827G>A mutations.
- a 6 ⁇ HIS tag can be attached to the C-terminus of the protein consisting of the amino acid residue sequence shown in SEQ ID NO: 1.
- the present invention provides a recombinant expression vector.
- the recombinant expression vector is selected from expression vectors containing the aforementioned nucleic acids.
- the recombinant expression vector according to the embodiment of the present invention expresses phosphoenolpyruvate carboxylase in Corynebacterium glutamicum, which helps to improve the yield of amino acids.
- the expression vector is selected from Escherichia coli-Corynebacterium glutamicum shuttle expression vector.
- the present invention provides a genetically engineered bacteria.
- the genetically engineered bacteria are obtained by transforming the aforementioned recombinant expression vector into the recipient bacteria.
- the genetically engineered bacteria according to the embodiments of the present invention can produce high amino acids.
- the recipient bacteria are selected from Corynebacterium glutamicum.
- the present invention provides a kit.
- the kit includes the aforementioned recombinant expression vector or the aforementioned genetically engineered bacteria.
- the purpose of high-yield amino acid can be achieved by using the kit according to the embodiment of the present invention.
- the present invention provides a method for obtaining an engineered phosphoenolpyruvate carboxylase.
- the method comprises: mutating amino acids at positions 771 and/or 931 and/or 943 in the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase in the amino acid sequence to obtain the modified phosphoenolpyruvate carboxylase, and the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 Amino acid sequences are amino acid sequences that have at least 80% homology.
- the wild-type phosphoenolpyruvate carboxylase is derived from plants, preferably sorghum.
- the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and/or lysine at position 931 by glutamine and/or Or aspartic acid at position 943 is replaced by asparagine.
- the present invention proposes the aforementioned modified phosphoenolpyruvate carboxylase, the nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vectors, and genetically engineered bacteria in Applications for improving amino acid production.
- the modified phosphoenolpyruvate carboxylase, the nucleic acid encoding it, the recombinant expression vector, and the genetically engineered bacteria according to the embodiments of the present invention help to improve the amino acid yield fermented by Corynebacterium glutamicum, and have good application prospects.
- the present invention provides a method for improving the amino acid production of Corynebacterium glutamicum.
- the method includes: transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum, and fermenting and culturing the transformed genetically engineered bacteria. Therefore, expressing phosphoenolpyruvate carboxylase during the fermentation process of genetically engineered bacteria is helpful to improve the amino acid production of Corynebacterium glutamicum.
- the amino acids include aspartic amino acids and/or glutamic amino acids.
- the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine;
- the glutamic acid amino acids include glutamic acid amino acid, glutamine, proline and/or arginine.
- the present invention provides a method for producing amino acids.
- the method includes: fermenting and culturing the aforementioned genetically engineered bacteria.
- the genetically engineered bacteria have high amino acid yields, so the purpose of high amino acid production can be achieved by fermenting and culturing the genetically engineered bacteria.
- the amino acids include aspartic amino acids and/or glutamic amino acids.
- the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine;
- the glutamic acid amino acids include glutamic acid amino acid, glutamine and/or arginine.
- Figure 1 shows a plasmid map according to one embodiment of the present invention.
- the present invention provides modified phosphoenolpyruvate carboxylase, nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vector, genetically engineered bacteria, kit, and obtained modified phosphoenolpyruvate carboxylation
- the method and application of the enzyme, the method for improving the amino acid production of Corynebacterium glutamicum and the method for producing the amino acid will be described in detail below respectively.
- the present invention provides an engineered phosphoenolpyruvate carboxylase.
- the amino acid sequence of the modified phosphoenolpyruvate carboxylase has optimized codons, and has an enzyme with optimized codons
- the amino acid sequence 771 and/or 931 and/or 943 of the amino acid sequence is substituted.
- codon optimization refers to redesigning the gene sequence according to the preference of codons expressed in different species protein systems, so as to achieve high-level protein expression.
- the present invention uses phosphoenolacetone derived from sorghum
- the gene encoding acid carboxylase is optimized for expression in Corynebacterium glutamicum.
- Site-directed mutagenesis is a conventional technical means in the field, and site-directed mutagenesis refers to the introduction of desired changes (usually favorable for characterization) into the target DNA fragment (which may be a genome or a plasmid) by methods such as polymerase chain reaction (PCR). Changes in direction), including base additions, deletions, point mutations, etc. Site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful method in genetic research.
- PCR polymerase chain reaction
- the wild-type phosphoenolpyruvate carboxylase is derived from a plant.
- the wild-type phosphoenolpyruvate carboxylase is derived from sorghum.
- Sorghum belongs to C4 plants.
- PEPC of C4 plants has a higher affinity for its substrate HCO3 - .
- HCO3 - In the photosynthetic pathway, it catalyzes the initial fixation of CO 2 and concentrates CO 2 , making it have a higher photosynthetic rate. ability to function.
- the inventor analyzed the primary structure of PEPC through Uniprot and ExPASy, found its binding sites with aspartic acid and malic acid, and simulated the Ser-771 Ser by Pymol software to Tyr, and found the conformation of the protein.
- the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 Sexual amino acid sequence.
- the amino acid sequence shown in SEQ ID NO: 1 is derived from sorghum phosphoenolpyruvate carboxylase, and the enzyme and the enzyme with at least 80% homology with it can be expressed in microorganisms to improve amino acid production.
- the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted with tyrosine at position 771 of serine.
- lysine at position 931 of the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted with glutamine.
- the aspartic acid at position 943 of the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted with asparagine.
- the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and lysine at position 931 by glutamine.
- the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and aspartic acid at position 943 by asparagine.
- the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase has 931 lysine substituted with glutamine and 943 aspartic acid substituted with asparagine.
- the affinity for phosphoenolpyruvate carboxylase is increased while the sensitivity to glycine is decreased, and the enzyme activity is improved.
- the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771, lysine at position 931 by glutamine and aspartic acid at position 943 amino acid is replaced by asparagine.
- the present invention provides a nucleic acid encoding an engineered phosphoenolpyruvate carboxylase.
- the nucleotide sequence encoding the modified phosphoenolpyruvate carboxylase is directed against glutamate rods Bacillus is codon-optimized, and the optimized nucleotide sequence has mutations at base 2312, base 2791 and/or base 2827.
- the production of amino acids by microbial fermentation has occupied an increasing proportion.
- the plant-derived phosphoenolpyruvate carboxylase was preferentially selected as the research object. Since plant-derived enzymes are difficult to achieve heterologous expression in microorganisms, according to the characteristics of the target microorganisms, the nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase was codon-optimized in advance to make it more efficient. Heterologous expression is well achieved.
- the activity of Corynebacterium glutamicum further affects the amino acid production of Corynebacterium glutamicum, and further, through site-directed mutation of the bases of these three sites, it is helpful to improve the amino acid production of Corynebacterium glutamicum, and the application prospect is good.
- the nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase has the nucleotide sequence as shown in SEQ ID NO: 2 or has the nucleotide sequence as shown in SEQ ID NO: 2
- Acid sequences are nucleotide sequences with at least 80% homology.
- the optimized nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 3 nucleotide sequence.
- the nucleotide sequence can be heterologously expressed in microorganisms with high efficiency. Further, by performing at least one of the above-mentioned three nucleotide positions, the enzyme encoded by the nucleotide can increase the yield of amino acids.
- the optimized nucleotide sequence has mutations of c.2312C>A, c.2791C>A and/or c.2827G>A.
- the 2312th C base is mutated to an A base
- the 2791st C base is mutated to an A base
- the 2827th G base is mutated to an A base.
- At least one mutation can improve the enzyme activity and further improve the amino acid production.
- the 2791st C base was mutated to A base
- the 2827th G base was mutated to A base.
- the codon-optimized nucleotide sequence is shown in SEQ ID NO: 2 or has at least 80% homology with SEQ ID NO: 2.
- the codon-optimized enzyme can be highly expressed in microorganisms, especially Corynebacterium glutamicum.
- the present invention provides a recombinant expression vector.
- the recombinant expression vector is selected from expression vectors containing the aforementioned nucleic acids.
- the recombinant expression vector according to the embodiment of the present invention expresses phosphoenolpyruvate carboxylase in Corynebacterium glutamicum, which helps to improve the yield of amino acids.
- the expression vector is selected from the Escherichia coli-Corynebacterium glutamicum shuttle expression vector.
- the Escherichia coli-Corynebacterium glutamicum shuttle expression vector means that it can replicate and exist stably in Escherichia coli and Corynebacterium, and contains a promoter with transcription initiation function and a terminator with transcription termination function in both Escherichia coli and Corynebacterium.
- Constitutive expression vectors are vectors that add expression elements (such as promoters, terminators, etc.) to the basic skeleton of the cloning vector, so that the target gene can be expressed.
- a promoter is a specific DNA sequence that RNA polymerase binds to and initiates transcription
- a terminator is a DNA sequence that gives RNA polymerase a transcription termination signal.
- Inducible expression vector is to realize the secreted expression of exogenous protein, and the most direct method is to insert the gene behind the expression element (promoter and secretion signal signal peptide sequence) of the host secreted protein.
- the introduction of a tightly regulated strong promoter into the expression vector enables the expression of the target gene under the condition of low inducer concentration.
- the induction methods are usually chemical induction and environmental induction.
- the Escherichia coli-Corynebacterium glutamicum shuttle expression vector is a constitutive expression vector PVcaseG, and the constitutive expression vector includes gapa derived from Corynebacterium glutamicum itself as a promoter and a constitutive expression vector derived from Escherichia coli - rrnB terminator of the C. glutamicum shuttle vector PXMJ19.
- the nucleic acid sequence (referred to as pepcTB) of the phosphoenolpyruvate carboxylase encoding the aforementioned transformation was inserted into the PVcaseG carrier, and the PVcaseG-pepcTB recombinant expression vector was constructed (see Figure 1 for the plasmid map), and the 931-position lysine was added simultaneously.
- the acid was mutated to glutamine, the 943rd aspartic acid was mutated to asparagine, and electrotransferred into Corynebacterium glutamicum, and the expression was induced by IPTG in Corynebacterium glutamicum, which can improve the carboxylation of phosphoenolpyruvate enzyme activity.
- the present invention provides a genetically engineered bacteria.
- the genetically engineered bacteria are obtained by transforming the aforementioned recombinant expression vector into the recipient bacteria.
- the genetically engineered bacteria according to the embodiments of the present invention can highly produce amino acids.
- the recipient bacteria are selected from Corynebacterium glutamicum.
- the present invention provides a kit.
- the kit includes the aforementioned recombinant expression vector or the aforementioned genetically engineered bacteria.
- the purpose of high-yield amino acid can be achieved by using the kit according to the embodiment of the present invention.
- the present invention provides a method for obtaining an engineered phosphoenolpyruvate carboxylase.
- the method comprises: mutating amino acids at positions 771, 931 and/or 943 in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase to obtain the modified Phosphoenolpyruvate carboxylase.
- the wild-type phosphoenolpyruvate carboxylase is derived from a plant, preferably sorghum.
- the modified phosphoenolpyruvate carboxylase helps to improve the carbon flux of amino acids fermented by Corynebacterium glutamicum and achieve high yield of amino acids.
- the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 Amino acid sequence; amino acid sequence of wild-type phosphoenolpyruvate carboxylase with substitution of serine 771 by tyrosine, lysine 931 by glutamine and/or aspartic acid 943 replaced by asparagine.
- the modified phosphoenolpyruvate carboxylase helps to improve the carbon flux of amino acids fermented by Corynebacterium glutamicum and achieve high yield of amino acids.
- the present invention proposes the aforementioned modified phosphoenolpyruvate carboxylase, the nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vectors, and genetically engineered bacteria in Applications for improving amino acid production.
- the modified phosphoenolpyruvate carboxylase, the nucleic acid encoding it, the recombinant expression vector, and the genetically engineered bacteria according to the embodiments of the present invention help to improve the amino acid yield fermented by Corynebacterium glutamicum, and have good application prospects.
- the present invention provides a method for improving the amino acid production of Corynebacterium glutamicum.
- the method includes: transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum, and fermenting and culturing the transformed genetically engineered bacteria. Therefore, the recombinant expression vector expresses phosphoenolpyruvate carboxylase in the fermentation process of Corynebacterium glutamicum, and the enzyme activity is high, which helps to improve the yield of amino acids.
- the amino acids include aspartic amino acids and glutamic amino acids.
- the inventors found that the production of aspartate amino acids and glutamic acid amino acids can be significantly improved by transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum.
- the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine;
- the glutamic acid amino acids include glutamic acid, pro amino acid, glutamine, arginine.
- the present invention provides a method for producing amino acids.
- the method includes: fermenting and culturing the aforementioned genetically engineered bacteria. Therefore, the recombinant expression vector expresses phosphoenolpyruvate carboxylase in the fermentation process of Corynebacterium glutamicum, and the enzyme activity is high, which helps to improve the yield of amino acids.
- the amino acids include aspartic amino acids and glutamic amino acids.
- the inventors found that the production of aspartate amino acids and glutamic acid amino acids can be significantly improved by using the aforementioned genetically engineered bacteria.
- the aspartic amino acids include aspartic acid, lysine, lysine, threonine, methionine and/or isoleucine;
- the glutamic acid family of amino acids includes glutamic acid Amino acid, proline, glutamine, arginine.
- Plasmid pk18mobsacB was purchased from Wuhan Miaoling Biotechnology Co., Ltd., shuttle plasmid PXMJ19 was purchased from Protin Biotechnology (Beijing) Co., Ltd., Escherichia coli DH5 ⁇ was routinely used in the field; DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.; restriction endonucleases (EcoRI, SalI, EcoRV), DNAmarker, plasmid extraction kit, DNA gel recovery and purification kit, all purchased from Takara Bioengineering (Dalian) Co., Ltd.; bacterial genomic DNA extraction kit Purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Onestep cloning and recombination kit was purchased from NEB Beijing Company; kanamycin sulfate was purchased from Biosharp Company; other chemical reagents such as sucrose were of analytical grade from Sinopharm.
- the amino acid content in the fermentation broth was detected by an amino acid analyzer (Hitachi 8800).
- the enzyme activity catalyzes phosphoenolpyruvate and carbon dioxide to generate oxaloacetate and hydrogen phosphate ions through PEPC
- malate dehydrogenase further catalyzes oxaloacetate and NADH to generate malate and NAD+
- the reduction of NADH is measured at 340nm rate to calculate PEPC activity.
- the consumption of 1 nmol NADH per mg tissue protein per minute was defined as one unit of enzyme activity.
- the method for determining the protein content adopted in the present invention is the BCA kit detection method.
- the formula of isoleucine seed medium is: glucose 3.0%, ammonium sulfate 2.5%, corn steep liquor 3.5%, yeast extract 0.3%, silk peptide powder 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, calcium carbonate 4% %, the balance is deionized water, pH is 7; The % is the weight volume (g/mL) percentage.
- the formula of isoleucine fermentation medium is: glucose 16.0%, ammonium sulfate 0.8%, corn steep liquor 3.5%, yeast extract 0.3%, silk peptide powder 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and the balance is Deionized water, pH 7; the % are weight volume (g/mL) percentages.
- arginine seed medium glucose 3.0%, dipotassium hydrogen phosphate 0.2%, urea 0.12%, ammonium sulfate 0.5%, yeast extract 0.5%, corn steep liquor 5.5%, magnesium sulfate 0.04%, biotin 50ug/L, Silk peptide powder 1.0%, corn oil 10 drops/L, light calcium carbonate 1%.
- arginine fermentation medium glucose 12.5%, dipotassium hydrogen phosphate 0.15%, urea 0.1%, ammonium sulfate 4.5%, yeast extract 0.5%, corn steep liquor 5.5%, magnesium sulfate 0.05%, biotin 0.05%, vitamins B1 0.1%, corn oil 10 drops/L, molasses 4.0%, silk peptide powder 0.3%, light calcium carbonate 0.5%, the balance is deionized water, pH is 7; the % is weight volume (g/mL) percentage.
- the phosphoenolpyruvate carboxylase (SEQ ID NO: 2) encoding sorghum was codon optimized to obtain the nucleotide sequence shown in SEQ ID NO: 3.
- the following primers were used to amplify the mutated phosphoenolpyruvate carboxylase-encoding gene PEPC by inverse PCR, and a 6 ⁇ HIS tag was added to the C-terminal of the gene for the convenience of subsequent purification.
- the enzyme used was the Q5 high-fidelity enzyme.
- the primers are as follows:
- Lysine 931 was mutated to glutamine, and the primers were as follows:
- the 943rd aspartic acid was mutated to asparagine, and the primers were as follows:
- PEPC amplification primers
- High-fidelity Q5 High-Fidelity DNA Polymerase PCR amplifies the nucleotide fragment pepc of about 2.9kb.
- the PCR reaction system (50 ⁇ l) was: 5 ⁇ Q5 reaction buffer 10 ⁇ l, 10 mM dNTP 1 ⁇ l, primers 2.5 ⁇ l each, template depending on the sample concentration, Q5 enzyme 0.5 ⁇ l, and water to 50 ⁇ l.
- the reaction conditions were: 98°C for 30s, 98°C for 10s, 55°C-72°C for 30s, 72°C for 1.5 min, 33 cycles; 72°C for 2 min. 1% agarose gel electrophoresis detection and DNA gel recovery kit purification and recovery of 4 PCR products of 2.9kb.
- the PEPC gene vector mutated at position 931 is used as a template, and the 771-mutated primer is used for reverse PCR at position 771.
- the resulting product is detected by 1% agarose gel electrophoresis and DNA gel recovery kit After purification and recovery, the phosphoenolpyruvate carboxylase gene mutated at positions 771 and 931 at the same time was amplified with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
- the PEPC gene vector mutated at position 771 is used as a template, and the 943-mutated primer is used for reverse PCR at position 943.
- the resulting product is detected by 1% agarose gel electrophoresis and DNA gel recovery kit After purification and recovery, the phosphoenolpyruvate carboxylase gene mutated at positions 771 and 943 was amplified with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
- the PEPC gene vector mutated at position 931 is used as the template, and the 943-position mutated primer is used for reverse PCR at position 943.
- the resulting product is detected by 1% agarose gel electrophoresis and DNA gel recovery kit After purification and recovery, the phosphoenolpyruvate carboxylase gene mutated at positions 931 and 943 at the same time was amplified with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
- the PEPC gene vector mutated at the 771st and 931st positions is used as the template, and the 943th mutated primer is used for reverse PCR at the 943rd position, and the resulting product is electrophoresed on a 1% agarose gel Detection, purification and recovery of DNA gel recovery kit, and amplification of the phosphoenolpyruvate carboxylase gene simultaneously mutated at positions 771, 931 and 943 with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
- the plasmid pet28a was digested with XbaI/BamHI double enzyme (plasmid pet28a was purchased from Wuhan Miaoling Biotechnology Co., Ltd.), and the reaction system was: plasmid 1 ⁇ g, 10 ⁇ buffer 5 ⁇ l, XbaI 1 ⁇ l, BamHI 1 ⁇ l, and water to 50 ⁇ l. Digestion at 37°C for 2h. 1% agarose gel electrophoresis detection and DNA gel recovery and purification kit to recover 5.3kb nucleotide fragments.
- Onestep cloning and recombination kit recombines the above double-enzyme digestion product (i.e. XbaI/BamHI double-enzyme digestion plasmid pet28a) and the mutated pepc fragment.
- the recombinant product is transformed into Escherichia coli BL21 and spread on a plate containing kanamycin sulfate. After 37 After culturing overnight at °C, the transformants were selected for sequencing and verification.
- the transformants with correct sequencing contained the recombinant plasmid pet28a-pepcTB, which were named pet28a-pepcTB1 (serine 771 was mutated to tyrosine), pet28a-pepcTB2 (lysine 931 was mutated).
- the recombinant vectors pet28a-pepcTB1, pet28a-pepcTB2, pet28a-pepcTB3, pet28a-pepcTB12, pet28a-pepcTB13, pet28a-pepcTB23, pet28a-pepcTB123 were transformed into E. coli strain BL21(DE3) CodonPlus RIPL.
- the recombinant E. coli strains generated from the transformation experiments were designated: E.coli pepc1, E.coli pepc2, E.coli pepc3, E.coli pepc12, E.coli pepc13, E.coli pepc23, E.coli pepc123.
- the above recombinant E. coli strains were cultured in LB medium containing 50 mg/L kanamycin at 37°C until OD600 reached 0.4-1.0. Then, 0.1-0.5Mm/L IPTG was added to the medium, and the bacteria were cultured at 30°C for another 4-16 hours. The cells were collected by centrifugation, washed with buffer (20 mM Tris-HCl, Ph6.8) and suspended. Ultrasonic crushing, the conditions are 100W, 30min, 4°C, 12 000r/min centrifugation for 2min, the obtained supernatant is the crude enzyme liquid.
- the above crude enzyme solution was separated by SDS-PAGE electrophoresis, the size of the target protein was about 102KDa, and the protein content was detected by BCA protein detection kit.
- the target protein was purified by nickel column affinity chromatography. The principle is: polyhistidine can bind to various transition metals and transition metal chelates, so the protein with exposed 6X His-tag can bind to the immobilized Ni2+ resin, thereby distinguishing his-tag histidine-tagged fusion proteins from other proteins. When we eluted with a high concentration of imidazole solution, imidazole competed with the imidazole ring of the fusion protein his-tag for binding, and finally the fusion protein was eluted. The purified protein was detected by SDS-PAGE electrophoresis again.
- the enzyme activity was determined by phosphoenolpyruvate carboxylase enzyme activity detection kit.
- the principle is that PEPC catalyzes phosphoenolpyruvate and carbon dioxide to generate oxaloacetate and hydrogen phosphate ions, and malate dehydrogenase further catalyzes oxalyl Acetic acid and NADH generate malic acid and NAD+, and the change in absorbance at 340 nm is measured with a UV spectrophotometer. The consumption of 1 nmol NADH per mg protein per minute was defined as one unit of enzyme activity.
- the 931st lysine was mutated to glutamine, which improved the affinity for phosphoenolpyruvate, and finally the enzyme activity was increased by 3.79 times on the original basis;
- the 943rd aspartic acid was mutated to asparagine amide, reducing the sensitivity to glycine, and finally increasing the enzyme activity by 6.3 times on the original basis;
- the phosphorylation site 771 was mutated from serine to tyrosine, and the 931 lysine was mutated to glutamine.
- the activity of the enzyme was increased by 5.44 times on the original basis; the phosphorylation site 771 was mutated from serine to tyrosine, and the 943rd aspartic acid was mutated to asparagine, which finally made the enzyme live on the original basis.
- the enzyme activity was increased by 3.41 times; the 931st lysine was mutated to glutamine, and the 943rd aspartic acid was mutated to asparagine, which finally increased the enzyme activity by 12.05 times on the original basis; the 771st phosphate
- the serine was mutated to tyrosine, the 931st lysine was mutated to glutamine, and the 943rd aspartate was mutated to asparagine, which finally increased the enzyme activity by 6.41 times.
- the constitutive expression vector PVcaseG with gapa as the promoter and rrnB as the termination on the basic plasmid PVcase (the plasmid map is shown in Figure 1).
- the amplification of the promoter and terminator is based on the existing strain m13 genome in the laboratory as the template, and the primers used are as follows:
- the plasmid PVcase was digested with KpnI/SalI, and the constitutive expression vector PVcaseG was constructed by the one-step cloning method as described above.
- the target product obtained by inverse PCR amplification is a linear vector after gel recovery, and the amplification primers are as follows:
- amplification primers are as follows:
- Onestep cloning recombination kit recombines the above linear vector and the mutated pepc fragment.
- the recombinant product is transformed into Escherichia coli DH5 ⁇ and spread on a plate containing kanamycin sulfate. After overnight incubation at 37°C, the selected transformants are sequenced to verify that the sequencing is correct.
- the transformants contained the recombinant plasmid PVcaseG-pepcTB, named PVcaseG-pepcTB1, PVcaseG-pepcTB2, PVcaseG-pepcTB3, PVcaseG-pepcTB12, PVcaseG-pepcTB13, PVcaseG-pepcTB23, PVcaseG-pepcTB123.
- control strains C.glutamicum H5 and C.glutamicum H1 and the above-mentioned recombinant Corynebacterium glutamicum were inoculated into isoleucine and arginine fermentation medium respectively, and after culturing at 30 °C for 36 h, the fermentation broth was taken, and the fermentation broth was taken at 4 °C for 10 000 r. Cells were collected by centrifugation at /min for 2 min, washed twice with 0.1 mol HCL, washed 3 times with pH 7.5, 0.1 mol/L phosphate buffer, resuspended and sonicated at 300 W for 1 h.
- C.glutamicum H5 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on November 1, 2016.
- the strain name is Corynebacterium glutamicum, the strain number is H5, and the deposit number is CCTCC NO: M2016609.
- C.glutamicum H1 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on October 26, 2020.
- the strain name is Corynebacterium glutamicum H1
- the classification number is Corynebacterium glutamicum H1
- the deposit number is CCTCC NO: M2020644.
- the recombinant strains H5-Vpepc1, H5-Vpepc2, H5-Vpepc3, H5-Vpepc12, H5-Vpepc13, H5-Vpepc23, H5-Vpepc123 were put into a 5L fermenter for cultivation. Take 1ml of solid-state activated medium of glycerol strains of control bacteria and recombinant bacteria to streak, and cultivate at 30°C for 16h; pick an inoculated loop from the activated medium and transfer it to, for example, 100ml of seed medium, at 30°C, 200rpm Shake culture for 12-16h.
- the seeds were inoculated into a 5L fermentation tank containing 3L fermentation medium at a volume ratio of 10%, the temperature was controlled at 30°C, the pH of the ammonia water was controlled at 7.0, and the dissolved oxygen was maintained at 30% by adjusting the rotational speed and ventilation rate.
- the residual sugar content was lower than At 1.5%, 80% glucose was added in flow to maintain the residual sugar content at 1.5-2.5%, and the fermentation time was more than 55h, and the fermentation time in this example was 65h.
- the fermentation broth was centrifuged to take the supernatant for derivatization, and HPLC was used to analyze the main amino acids. The statistics of the experimental results are shown in Table 3.
- the recombinant strains H1-Vpepc1, H1-Vpepc2, H1-Vpepc3, H1-Vpepc12, H1-Vpepc13, H1-Vpepc23, H1-Vpepc123 were put into a 5L fermenter for cultivation.
- In the feeding bottle connect the feeding pipeline, inoculate the seed culture liquid into the fermentation medium, and start the fermentation culture.
- the yields of aspartate amino acids and glutamic acid amino acids of the recombinant strains were improved to varying degrees, and the recombinant strain H1-Vpepc23 had the most obvious effect.
- g/L increased to 33.89g/L, an increase of 61.23%
- glutamic acid group amino acids increased from 98.61g/L to 127.94g/L, an increase of 29.74%, especially arginine
- the yield increased from the previous 98.61g/L to 127.94g/L 66.83g/L increased to 83.67g/L, an increase of 25.20%.
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Abstract
A modified Phosphoenolpyruvate carboxylase. Compared to an amino acid sequence of a wild-type Phosphoenolpyruvate carboxylase, amino acids at bit 771 and/or bit 931 and/or bit 943 of an amino acid sequence of the modified Phosphoenolpyruvate carboxylase are substituted. By performing specific site mutation on the amino acid sequence of the wild-type Phosphoenolpyruvate carboxylase, the modified Phosphoenolpyruvate carboxylase obtained has a great influence on metabolism of an amino acid carbon flow; and expressing a coding gene of the modified Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum can effectively increase the yield of amino acids to achieve good application prospect.
Description
本发明涉及生物领域。具体地，本发明涉及改造的磷酸烯醇丙酮酸羧化酶及其在提高谷氨酸棒杆菌氨基酸产量中的应用。The present invention relates to the field of biology. Specifically, the present invention relates to the modified phosphoenolpyruvate carboxylase and its application in improving the amino acid production of Corynebacterium glutamicum.
氨基酸是蛋白质的基本组成部分，这些营养关键化合物广泛用于制备饲料添加剂、食品成分、营养品和药物。近年来世界氨基酸产量稳步增长。特别地，天冬氨酸族氨基酸(包括L-赖氨酸和L-苏氨酸)已经在世界范围内广泛应用，并且通过使用谷氨酸棒杆菌(Corynebacterium glutamicum)或者大肠杆菌(Escherichia coli)进行发酵，在氨基酸市场中已经占据了越来越大的份额。Amino acids are the basic building blocks of proteins, and these nutritionally critical compounds are widely used in the preparation of feed additives, food ingredients, nutraceuticals and pharmaceuticals. In recent years, the world's amino acid production has grown steadily. In particular, aspartic amino acids (including L-lysine and L-threonine) have been widely used worldwide, and by using Corynebacterium glutamicum or Escherichia coli Fermentation has occupied an increasing share of the amino acid market.
先前的研究表明，草酰乙酸(Oxaloacetate,OAA)衍生的氨基酸如L-赖氨酸或L-苏氨酸的产率主要取决于通过补缺途径的碳通量。谷氨酸棒杆菌具有磷酸烯醇丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC)和丙酮酸羧化酶(Pyruvate carboxylase,PYC)，用于补充消耗的草酰乙酸。Previous studies have shown that the yield of oxaloacetate (OAA)-derived amino acids such as L-lysine or L-threonine mainly depends on the carbon flux through the complementation pathway. Corynebacterium glutamicum has phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (Pyruvate carboxylase, PYC), which are used to supplement the consumed oxaloacetate.
磷酸烯醇丙酮酸羧化酶是一种见于大部分细菌和所有植物的酶。磷酸烯醇丙酮酸(Phosphoenolpyruvate,PEP)是糖酵解的中间产物，在PEP被PEPC羧化为草酰乙酸后，又会很快被苹果酸脱氢酶(Malate Dehydrogenase)转化为苹果酸(Malate)，因此为三羧酸循环(TCA循环)补充了中间产物。而TCA循环过程中的许多中间产物都是氨基酸合成所需的底物，所以在大多数生物中，PEPC的主要作用是分流糖酵解，为氨基酸的合成提供原料。TCA循环中某些中间产物是合成许多重要有机物的前体，如OAA是天冬氨酸族氨基酸合成的碳架。α-酮戊二酸是谷氨酸族氨基酸合成的碳架。磷酸烯醇式丙酮酸羧化酶催化磷酸烯醇式丙酮酸和CO2反应生成草酰乙酸。反应方程式如下：PEP+CO2＝OAA+Pi。磷酸烯醇式丙酮酸羧化酶的活性增强，有助于增加草酰乙酸的积累，作为天冬氨酸族氨基酸和谷氨酸族氨基酸合成途径中重要前体，草酰乙酸供给充足才能保证相关氨基酸的高效合成，因此，增强前体物草酰乙酸供给对高产天冬氨酸族氨基酸和谷氨酸族氨基酸也是十分重要的。Phosphoenolpyruvate carboxylase is an enzyme found in most bacteria and all plants. Phosphoenolpyruvate (PEP) is an intermediate product of glycolysis. After PEP is carboxylated to oxaloacetate by PEPC, it is quickly converted to malate by Malate Dehydrogenase. ), thus supplementing the tricarboxylic acid cycle (TCA cycle) with intermediates. Many intermediate products in the TCA cycle are substrates required for amino acid synthesis, so in most organisms, the main role of PEPC is to shunt glycolysis to provide raw materials for amino acid synthesis. Some intermediate products in the TCA cycle are the precursors for the synthesis of many important organic compounds, such as OAA is the carbon skeleton for the synthesis of aspartic amino acids. Alpha-ketoglutarate is the carbon skeleton for the synthesis of glutamate amino acids. Phosphoenolpyruvate carboxylase catalyzes the reaction of phosphoenolpyruvate with CO2 to form oxaloacetate. The reaction equation is as follows: PEP+CO2=OAA+Pi. The enhanced activity of phosphoenolpyruvate carboxylase helps to increase the accumulation of oxaloacetate. As an important precursor in the synthesis pathway of aspartate amino acids and glutamate amino acids, sufficient supply of oxaloacetate can ensure Therefore, enhancing the supply of the precursor oxaloacetate is also very important for the high yield of aspartic and glutamic amino acids.
磷酸烯醇式丙酮酸羧化酶首先在菠菜叶片中发现的。在体内它是以同源四聚体的形式 存在的，单体的大小为100-1l0kDa。PEP羧化酶在其活性表达方面也经历各种调节。已有研究报道了从许多不同细菌中分离纯化了PEPC酶，发现他们的活性都受到许多代谢物的调控。在大肠杆菌中，PEPC酶活性可被乙酰辅酶A和果糖1，6-二磷酸激活，并可产生协同激活作用，但受到天冬氨酸和苹果酸的严重抑制。在醋酸杆菌(Acetobacter aceti)中，PEPC酶活受天冬氨酸和一些TCA循环代谢物抑制，尤其是琥珀酸。在谷氨酸棒状杆菌中，PEPC的调控情况与大肠杆菌PEPC不同，其酶活不能被乙酰辅酶A所激活，但同样可被天冬氨酸所强烈抑制。此外，与黄短杆菌相比，谷氨酸对谷氨酸棒状杆菌PEPC的酶活抑制更明显。PEPC酶的这些精细的调控表明PEPC是从碳源转变为氨基酸的代谢流中的重要调控节点，这使其成为氨基酸生产菌株改造的重要靶点。Phosphoenolpyruvate carboxylase was first discovered in spinach leaves. In vivo it exists in the form of homotetramers with a monomer size of 100-110 kDa. PEP carboxylase also undergoes various modulations in its active expression. Studies have reported the isolation and purification of PEPC enzymes from many different bacteria, and found that their activities are regulated by many metabolites. In Escherichia coli, PEPC enzyme activity can be activated by acetyl-CoA and fructose 1,6-bisphosphate, and can produce synergistic activation, but is severely inhibited by aspartate and malate. In Acetobacter aceti, PEPC enzymatic activity is inhibited by aspartate and some TCA cycle metabolites, especially succinate. In Corynebacterium glutamicum, the regulation of PEPC is different from that of E. coli PEPC. Its enzymatic activity cannot be activated by acetyl-CoA, but it can also be strongly inhibited by aspartic acid. In addition, compared with Brevibacterium flavus, glutamate inhibited the enzyme activity of Corynebacterium glutamicum PEPC more obviously. These fine regulation of PEPC enzymes suggest that PEPC is an important regulatory node in the metabolic flow from carbon source to amino acid, which makes it an important target for amino acid-producing strain engineering.
在现有技术中，开发了多种技术以在氨基酸发酵中进行有效生产，其中以对磷酸烯醇丙酮酸羧化酶进行修饰为主。但是，目前对磷酸烯醇丙酮酸羧化酶的修饰一般是通过过表达细菌本身的磷酸烯醇式丙酮酸羧化酶编码基因或者是对其进行突变修饰，从而提高草酰乙酸的积累量，而且产酸提高效果并不显著，难以满足工业需求，而且迄今为止，没有专利报道异源表达植物中的磷酸烯醇丙酮酸羧化酶编码基因来提高谷氨酸棒状杆菌的CO2固定能力，从而提高草酰乙酸的积累量，进而提高产酸。In the prior art, a variety of techniques have been developed for efficient production in amino acid fermentation, mainly the modification of phosphoenolpyruvate carboxylase. However, at present, the modification of phosphoenolpyruvate carboxylase is generally by overexpressing the phosphoenolpyruvate carboxylase encoding gene of bacteria itself or mutating it, thereby increasing the accumulation of oxaloacetate. Moreover, the effect of improving acid production is not significant, and it is difficult to meet industrial needs. And so far, there is no patent report on heterologous expression of the gene encoding phosphoenolpyruvate carboxylase in plants to improve the CO2 fixation capacity of Corynebacterium glutamicum, thereby Increase the accumulation of oxaloacetic acid, thereby increasing acid production.
因此，目前通过改造磷酸烯醇丙酮酸羧化酶以获得高产氨基酸菌株仍有待研究。Therefore, it remains to be studied to obtain high-producing amino acid strains by modifying phosphoenolpyruvate carboxylase.
发明内容SUMMARY OF THE INVENTION
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提出了改造的磷酸烯醇丙酮酸羧化酶、编码改造的磷酸烯醇丙酮酸羧化酶的核酸、重组表达载体、基因工程菌、试剂盒、获得改造的磷酸烯醇丙酮酸羧化酶的方法及其应用、提高谷氨酸棒杆菌氨基酸产量的方法和生产氨基酸的方法，通过将野生型磷酸烯醇丙酮酸羧化酶进行密码子优化以及特定氨基酸位点突变，所得到的改造的磷酸烯醇丙酮酸羧化酶对氨基酸碳通量有很大影响，利用该改造的磷酸烯醇丙酮酸羧化酶的编码基因在谷氨酸棒杆菌中表达，可以高效提高氨基酸产量，应用前景好。The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. To this end, the present invention proposes modified phosphoenolpyruvate carboxylase, nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vector, genetically engineered bacteria, test kit, and obtained modified phosphoenolacetone The method for acid carboxylase and its application, the method for improving the yield of amino acid of Corynebacterium glutamicum and the method for producing amino acid, by carrying out codon optimization and specific amino acid site mutation of wild-type phosphoenolpyruvate carboxylase, the The obtained modified phosphoenolpyruvate carboxylase has a great influence on the carbon flux of amino acids, and the encoding gene of the modified phosphoenolpyruvate carboxylase is expressed in Corynebacterium glutamicum, which can effectively improve the amino acid carbon flux. Yield, the application prospect is good.
在本发明的一个方面，本发明提出了一种改造的磷酸烯醇丙酮酸羧化酶。根据本发明的实施例，与野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列相比，所述改造的磷酸烯醇丙酮酸羧化酶的氨基酸序列中的第771位和/或第931位和/或第943位氨基酸被取代；所述野生型磷酸烯醇丙酮酸羧化酶具有如SEQ ID NO：1所示的氨基酸序列或者具有与SEQ ID NO：1所示的氨基酸序列具有至少80％同源性的氨基酸序列。通过将野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列上的3个氨基酸残基进行突变会显著影响磷酸烯醇丙酮酸羧化酶的活 性，进一步增强谷氨酸棒杆菌氨基酸代谢途径中的碳流量，进而，有助于提高谷氨酸棒杆菌的氨基酸产量，应用前景好。In one aspect of the present invention, the present invention provides an engineered phosphoenolpyruvate carboxylase. According to an embodiment of the present invention, compared with the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase, the amino acid sequence of the engineered phosphoenolpyruvate carboxylase is at position 771 and/or position 931 and/or the amino acid at position 943 is substituted; the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or has the amino acid sequence shown in SEQ ID NO: 1 with at least 80 % homology to the amino acid sequence. By mutating 3 amino acid residues in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase, the activity of phosphoenolpyruvate carboxylase will be significantly affected, and the amino acid metabolism pathway of C. glutamicum will be further enhanced. The carbon flux, in turn, helps to improve the amino acid production of Corynebacterium glutamicum, and the application prospect is good.
根据本发明的实施例，上述改造的磷酸烯醇丙酮酸羧化酶还可以具有下列附加技术特征：According to an embodiment of the present invention, the above-mentioned modified phosphoenolpyruvate carboxylase may also have the following additional technical features:
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶来源于植物。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase is derived from a plant.
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶来源于高粱。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase is derived from sorghum.
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代和/或第931位赖氨酸被谷氨酰胺所取代和/或第943位天冬氨酸被天冬酰胺所取代。According to an embodiment of the present invention, the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and/or lysine at position 931 by glutamine and/or Or aspartic acid at position 943 is replaced by asparagine.
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第931位赖氨酸被谷氨酰胺所取代和第943位天冬氨酸被天冬酰胺所取代。According to an embodiment of the present invention, the 931st lysine of the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by glutamine and the 943rd aspartic acid is substituted by asparagine.
在本发明的另一方面，本发明提出了一种编码改造的磷酸烯醇丙酮酸羧化酶的核酸。根据本发明的实施例，与编码野生型磷酸烯醇丙酮酸羧化酶的核苷酸序列相比，所述编码改造的磷酸烯醇丙酮酸羧化酶的核苷酸序列预先经过密码子优化，且优化后的核苷酸序列的第2312位碱基、第2791位碱基和/或第2827位碱基具有突变，所述编码野生型磷酸烯醇丙酮酸羧化酶的核苷酸序列具有如SEQ ID NO：2所示的核苷酸序列或者具有与SEQ ID NO：2所示的核苷酸序列具有至少80％同源性的核苷酸序列。由此，根据本发明实施例的核酸在谷氨酸棒杆菌中表达磷酸烯醇丙酮酸羧化酶，有助于提高氨基酸产量。In another aspect of the present invention, the present invention provides a nucleic acid encoding an engineered phosphoenolpyruvate carboxylase. According to an embodiment of the present invention, compared with the nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase, the nucleotide sequence encoding the modified phosphoenolpyruvate carboxylase is codon-optimized in advance , and the optimized nucleotide sequence has a mutation at the 2312th base, the 2791st base and/or the 2827th base, the nucleotide sequence encoding the wild-type phosphoenolpyruvate carboxylase Has the nucleotide sequence shown in SEQ ID NO:2 or has a nucleotide sequence with at least 80% homology to the nucleotide sequence shown in SEQ ID NO:2. Thus, the nucleic acid according to the embodiment of the present invention expresses phosphoenolpyruvate carboxylase in Corynebacterium glutamicum, which helps to improve the yield of amino acids.
根据本发明的实施例，所述优化后的核苷酸序列具有如SEQ ID NO：3所示的核苷酸序列或者具有与SEQ ID NO：3所示的核苷酸序列具有至少80％同源性的核苷酸序列。According to an embodiment of the present invention, the optimized nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 3 source nucleotide sequence.
根据本发明的实施例，所述优化后的核苷酸序列具有c.2312C＞A、c.2791C＞A和/或c.2827G＞A的突变。According to an embodiment of the present invention, the optimized nucleotide sequence has mutations of c.2312C>A, c.2791C>A and/or c.2827G>A.
根据本发明的实施例，所述优化后的核苷酸序列具有c.2791C＞A和c.2827G＞A突变。According to an embodiment of the present invention, the optimized nucleotide sequence has c.2791C>A and c.2827G>A mutations.
为了使PEPC便于纯化，可在由SEQ ID NO：1所示氨基酸残基序列组成的蛋白质C端连接上6×HIS标签。In order to facilitate the purification of PEPC, a 6×HIS tag can be attached to the C-terminus of the protein consisting of the amino acid residue sequence shown in SEQ ID NO: 1.
在本发明的又一方面，本发明提出了一种重组表达载体。根据本发明的实施例，所述重组表达载体选自含有前面所述核酸的表达载体。由此，根据本发明实施例的重组表达载体在谷氨酸棒杆菌中表达磷酸烯醇丙酮酸羧化酶，有助于提高氨基酸产量。In yet another aspect of the present invention, the present invention provides a recombinant expression vector. According to an embodiment of the present invention, the recombinant expression vector is selected from expression vectors containing the aforementioned nucleic acids. Thus, the recombinant expression vector according to the embodiment of the present invention expresses phosphoenolpyruvate carboxylase in Corynebacterium glutamicum, which helps to improve the yield of amino acids.
根据本发明的实施例，所述表达载体选自大肠杆菌-谷氨酸棒杆菌穿梭表达载体。According to an embodiment of the present invention, the expression vector is selected from Escherichia coli-Corynebacterium glutamicum shuttle expression vector.
在本发明的又一方面，本发明提出了一种基因工程菌。根据本发明的实施例，所述基因工程菌是通过将前面所述重组表达载体转化到受体菌内所获得的。由此，根据本发明实 施例的基因工程菌可以高产氨基酸。In yet another aspect of the present invention, the present invention provides a genetically engineered bacteria. According to an embodiment of the present invention, the genetically engineered bacteria are obtained by transforming the aforementioned recombinant expression vector into the recipient bacteria. Thus, the genetically engineered bacteria according to the embodiments of the present invention can produce high amino acids.
根据本发明的实施例，所述受体菌选自谷氨酸棒杆菌。According to an embodiment of the present invention, the recipient bacteria are selected from Corynebacterium glutamicum.
本发明的又一方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括前面所述重组表达载体或前面所述基因工程菌。由此，利用根据本发明实施例的试剂盒可以实现高产氨基酸的目的。In yet another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned recombinant expression vector or the aforementioned genetically engineered bacteria. Thus, the purpose of high-yield amino acid can be achieved by using the kit according to the embodiment of the present invention.
本发明的又一方面，本发明提出了一种获得改造的磷酸烯醇丙酮酸羧化酶的方法。根据本发明的实施例，所述方法包括：将野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列中的氨基酸序列中的第771位和/或第931位和/或第943位氨基酸进行突变，得到所述改造的磷酸烯醇丙酮酸羧化酶，所述野生型磷酸烯醇丙酮酸羧化酶具有如SEQ ID NO：1所示的氨基酸序列或者具有与SEQ ID NO：1所示的氨基酸序列具有至少80％同源性的氨基酸序列。通过将野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列上的三个氨基酸残基进行突变，会显著影响磷酸烯醇丙酮酸羧化酶的活性，进一步影响谷氨酸棒杆菌的氨基酸产量，进而，通过对这三个位点进行氨基酸取代，有助于提高谷氨酸棒杆菌的氨基酸产量，应用前景好。In yet another aspect of the present invention, the present invention provides a method for obtaining an engineered phosphoenolpyruvate carboxylase. According to an embodiment of the present invention, the method comprises: mutating amino acids at positions 771 and/or 931 and/or 943 in the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase in the amino acid sequence to obtain the modified phosphoenolpyruvate carboxylase, and the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 Amino acid sequences are amino acid sequences that have at least 80% homology. By mutating three amino acid residues in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase, the activity of phosphoenolpyruvate carboxylase will be significantly affected, and the amino acid production of Corynebacterium glutamicum will be further affected. Furthermore, by performing amino acid substitutions on these three sites, it is helpful to improve the amino acid yield of Corynebacterium glutamicum, and the application prospect is good.
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶来源于植物，优选高粱。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase is derived from plants, preferably sorghum.
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代和/或第931位赖氨酸被谷氨酰胺所取代和/或第943位天冬氨酸被天冬酰胺所取代。According to an embodiment of the present invention, the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and/or lysine at position 931 by glutamine and/or Or aspartic acid at position 943 is replaced by asparagine.
在本发明的又一方面，本发明提出了前面所述改造的磷酸烯醇丙酮酸羧化酶、所述编码改造的磷酸烯醇丙酮酸羧化酶的核酸、重组表达载体、基因工程菌在提高氨基酸产量中的应用。由此，根据本发明实施例的改造的磷酸烯醇丙酮酸羧化酶、编码其的核酸、重组表达载体、基因工程菌有助于提高谷氨酸棒杆菌发酵的氨基酸产量，应用前景好。In another aspect of the present invention, the present invention proposes the aforementioned modified phosphoenolpyruvate carboxylase, the nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vectors, and genetically engineered bacteria in Applications for improving amino acid production. Thus, the modified phosphoenolpyruvate carboxylase, the nucleic acid encoding it, the recombinant expression vector, and the genetically engineered bacteria according to the embodiments of the present invention help to improve the amino acid yield fermented by Corynebacterium glutamicum, and have good application prospects.
在本发明的又一方面，本发明提出了一种提高谷氨酸棒杆菌氨基酸产量的方法。根据本发明的实施例，所述方法包括：将前面所述重组表达载体转化到谷氨酸棒杆菌中，并对转化后的基因工程菌进行发酵培养。由此，在基因工程菌发酵过程中表达磷酸烯醇丙酮酸羧化酶，有助于提高谷氨酸棒杆菌氨基酸产量。In yet another aspect of the present invention, the present invention provides a method for improving the amino acid production of Corynebacterium glutamicum. According to an embodiment of the present invention, the method includes: transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum, and fermenting and culturing the transformed genetically engineered bacteria. Therefore, expressing phosphoenolpyruvate carboxylase during the fermentation process of genetically engineered bacteria is helpful to improve the amino acid production of Corynebacterium glutamicum.
根据本发明的实施例，所述氨基酸包括天冬氨酸族氨基酸和/或谷氨酸族氨基酸。According to an embodiment of the present invention, the amino acids include aspartic amino acids and/or glutamic amino acids.
根据本发明的实施例，所述天冬氨酸族氨基酸包括天冬氨酸、赖氨酸、苏氨酸、甲硫氨酸和/或异亮氨酸；所述谷氨酸族氨基酸包括谷氨酸、谷氨酰胺、脯氨酸和/或精氨酸。According to an embodiment of the present invention, the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine; the glutamic acid amino acids include glutamic acid amino acid, glutamine, proline and/or arginine.
在本发明的又一方面，本发明提出了一种生产氨基酸的方法。根据本发明的实施例，所述方法包括：将前面所述基因工程菌进行发酵培养。如前所述，该基因工程菌的氨基酸产量高，由此，将该基因工程菌进行发酵培养，可以实现高产氨基酸的目的。In yet another aspect of the present invention, the present invention provides a method for producing amino acids. According to an embodiment of the present invention, the method includes: fermenting and culturing the aforementioned genetically engineered bacteria. As mentioned above, the genetically engineered bacteria have high amino acid yields, so the purpose of high amino acid production can be achieved by fermenting and culturing the genetically engineered bacteria.
根据本发明的实施例，所述氨基酸包括天冬氨酸族氨基酸和/或谷氨酸族氨基酸。According to an embodiment of the present invention, the amino acids include aspartic amino acids and/or glutamic amino acids.
根据本发明的实施例，所述天冬氨酸族氨基酸包括天冬氨酸、赖氨酸、苏氨酸、甲硫氨酸和/或异亮氨酸；所述谷氨酸族氨基酸包括谷氨酸、谷氨酰胺和/或精氨酸。According to an embodiment of the present invention, the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine; the glutamic acid amino acids include glutamic acid amino acid, glutamine and/or arginine.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein:
图1显示了根据本发明一个实施例的质粒图谱。Figure 1 shows a plasmid map according to one embodiment of the present invention.
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below. The embodiments described below are exemplary, only for explaining the present invention, and should not be construed as limiting the present invention.
本发明提出了改造的磷酸烯醇丙酮酸羧化酶、编码改造的磷酸烯醇丙酮酸羧化酶的核酸、重组表达载体、基因工程菌、试剂盒、获得改造的磷酸烯醇丙酮酸羧化酶的方法、用途、提高谷氨酸棒杆菌氨基酸产量的方法和生产氨基酸的方法，下面将分别对其进行详细描述。The present invention provides modified phosphoenolpyruvate carboxylase, nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vector, genetically engineered bacteria, kit, and obtained modified phosphoenolpyruvate carboxylation The method and application of the enzyme, the method for improving the amino acid production of Corynebacterium glutamicum and the method for producing the amino acid will be described in detail below respectively.
改造的磷酸烯醇丙酮酸羧化酶Engineered phosphoenolpyruvate carboxylase
在本发明的一个方面，本发明提出了一种改造的磷酸烯醇丙酮酸羧化酶。根据本发明的实施例，与野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列相比，该改造的磷酸烯醇丙酮酸羧化酶的氨基酸序列具有优化密码子，且具有优化密码子的酶的氨基酸序列的第771位和/或第931位和/或第943位氨基酸被取代。In one aspect of the present invention, the present invention provides an engineered phosphoenolpyruvate carboxylase. According to an embodiment of the present invention, compared with the amino acid sequence of wild-type phosphoenolpyruvate carboxylase, the amino acid sequence of the modified phosphoenolpyruvate carboxylase has optimized codons, and has an enzyme with optimized codons The amino acid sequence 771 and/or 931 and/or 943 of the amino acid sequence is substituted.
发明人发现，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列上的第771位、第931位、第943位氨基酸残基会显著影响磷酸烯醇丙酮酸羧化酶的活性，进一步影响谷氨酸棒杆菌的氨基酸产量，进而，通过对这三个位点的氨基酸进行定点突变，有助于提高谷氨酸棒杆菌的氨基酸产量，应用前景好。The inventors found that the amino acid residues at positions 771, 931 and 943 in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase can significantly affect the activity of phosphoenolpyruvate carboxylase, and further affect the The amino acid yield of Corynebacterium glutamicum, and further, by site-directed mutation of the amino acids at these three positions, is helpful to improve the amino acid yield of Corynebacterium glutamicum, and the application prospect is good.
本发明术语中，密码子优化指根据不同物种蛋白系统表达对密码子的偏好性，进行基因序列的重新设计，从而实现蛋白的高水平表达，本发明通过将来源于高粱的磷酸烯醇式丙酮酸羧化酶编码基因进行优化，使其适合在谷氨酸棒状杆菌中表达。In the terms of the present invention, codon optimization refers to redesigning the gene sequence according to the preference of codons expressed in different species protein systems, so as to achieve high-level protein expression. The present invention uses phosphoenolacetone derived from sorghum The gene encoding acid carboxylase is optimized for expression in Corynebacterium glutamicum.
定点突变为本领域常规技术手段，其为定点突变是指通过聚合酶链式反应(PCR)等 方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。Site-directed mutagenesis is a conventional technical means in the field, and site-directed mutagenesis refers to the introduction of desired changes (usually favorable for characterization) into the target DNA fragment (which may be a genome or a plasmid) by methods such as polymerase chain reaction (PCR). Changes in direction), including base additions, deletions, point mutations, etc. Site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful method in genetic research.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶来源于植物。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase is derived from a plant.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶来源于高粱。高粱属于C4植物，与其他C3植物相比C4植物的PEPC对其底物HCO3
-有较高的亲合力，在光合途径中，催化CO
2原初固定，浓缩CO
2，使得其有较高的光合作用能力。发明人通过Uniprot和ExPASy对PEPC一级结构进行分析，找到其与天冬氨酸及苹果酸的结合位点，通过Pymol软件模拟，将Ser-第771位的Ser突变为Tyr，发现蛋白质的构象发生很大变化，其与苹果酸的分子间作用力减弱，推测此突变可能会在很大程度上影响磷酸烯醇式丙酮酸羧化酶的活性；同时分析Lys931可能是与PEP结合的关键位点，将Lys931突变为Gln，其与PEP的分子间作用力增强，且整体能量变低，推测此突变能够增强与PEP的亲和力，蛋白结构更加稳定；第943位的天冬氨酸突变成天冬酰胺，其与甘氨酸结合的活性空腔变小，推测可以降低对甘氨酸的敏感性。
According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase is derived from sorghum. Sorghum belongs to C4 plants. Compared with other C3 plants, PEPC of C4 plants has a higher affinity for its substrate HCO3 - . In the photosynthetic pathway, it catalyzes the initial fixation of CO 2 and concentrates CO 2 , making it have a higher photosynthetic rate. ability to function. The inventor analyzed the primary structure of PEPC through Uniprot and ExPASy, found its binding sites with aspartic acid and malic acid, and simulated the Ser-771 Ser by Pymol software to Tyr, and found the conformation of the protein. There is a great change, and the intermolecular force between it and malic acid is weakened. It is speculated that this mutation may affect the activity of phosphoenolpyruvate carboxylase to a large extent. At the same time, Lys931 may be the key site for binding to PEP. At this point, by mutating Lys931 to Gln, its intermolecular force with PEP is enhanced, and the overall energy becomes lower. It is speculated that this mutation can enhance the affinity with PEP and the protein structure is more stable; the aspartic acid at position 943 is mutated to aspartic acid The amide, which binds to glycine in a smaller active cavity, presumably reduces sensitivity to glycine.
根据本发明的实施例，所述野生型磷酸烯醇丙酮酸羧化酶具有如SEQ ID NO：1所示的氨基酸序列或者具有与SEQ ID NO：1所示的氨基酸序列具有至少80％同源性的氨基酸序列。具有如SEQ ID NO：1所示的氨基酸序列来源于高粱磷酸烯醇丙酮酸羧化酶，该酶以及与其具有至少80％同源性的酶均可以在微生物中表达，提高氨基酸产量。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 Sexual amino acid sequence. The amino acid sequence shown in SEQ ID NO: 1 is derived from sorghum phosphoenolpyruvate carboxylase, and the enzyme and the enzyme with at least 80% homology with it can be expressed in microorganisms to improve amino acid production.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代。由此，有效缓解苹果酸的抑制作用，提高酶活力。According to an embodiment of the present invention, the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted with tyrosine at position 771 of serine. As a result, the inhibitory effect of malic acid is effectively relieved and the enzyme activity is improved.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第931位赖氨酸被谷氨酰胺所取代。由此，以便提高对磷酸烯醇丙酮酸羧化酶的亲和力，提高酶活力。According to an embodiment of the present invention, lysine at position 931 of the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted with glutamine. Thus, in order to improve the affinity for phosphoenolpyruvate carboxylase and improve the enzyme activity.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第943位天冬氨酸被天冬酰胺所取代。由此，降低对甘氨酸的敏感性，提高酶活力。According to an embodiment of the present invention, the aspartic acid at position 943 of the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted with asparagine. Thereby, the sensitivity to glycine is reduced, and the enzyme activity is improved.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代和第931位赖氨酸被谷氨酰胺所取代。由此，缓解苹果酸的抑制作用同时提高对磷酸烯醇式丙酮酸羧化酶的亲和力，提高酶活力。According to an embodiment of the present invention, the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and lysine at position 931 by glutamine. Thereby, the inhibitory effect of malic acid is relieved, and the affinity for phosphoenolpyruvate carboxylase is improved, and the enzyme activity is improved.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代和第943位天冬氨酸被天冬酰胺所取代。由此，缓解苹果酸的抑制作用同时降低对甘氨酸的敏感性，提高酶活力。According to an embodiment of the present invention, the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and aspartic acid at position 943 by asparagine. Thereby, the inhibitory effect of malic acid is relieved while the sensitivity to glycine is reduced, and the enzyme activity is improved.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第931位赖氨酸被谷氨酰胺所取代和第943位天冬氨酸被天冬酰胺所取代。由此，提高对磷酸烯醇式丙酮酸羧化酶的亲和力同时降低对甘氨酸的敏感性，提高酶活力。According to an embodiment of the present invention, the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase has 931 lysine substituted with glutamine and 943 aspartic acid substituted with asparagine. Thus, the affinity for phosphoenolpyruvate carboxylase is increased while the sensitivity to glycine is decreased, and the enzyme activity is improved.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代和第931位赖氨酸被谷氨酰胺所取代和第943位天冬氨酸被天冬酰胺所取代。According to an embodiment of the present invention, the amino acid sequence of wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771, lysine at position 931 by glutamine and aspartic acid at position 943 amino acid is replaced by asparagine.
发明人发现，在上述3个突变位点中，第931位赖氨酸被谷氨酰胺所取代和第943位天冬氨酸被天冬酰胺所取代这两个突变共同作用下，效果较佳，氨基酸产量更高。The inventors found that in the above three mutation sites, the 931st lysine is replaced by glutamine and the 943rd aspartic acid is replaced by asparagine, the effect is better under the combined action of the two mutations. , the amino acid yield is higher.
编码改造的磷酸烯醇丙酮酸羧化酶的核酸Nucleic acid encoding an engineered phosphoenolpyruvate carboxylase
在本发明的另一方面，本发明提出了一种编码改造的磷酸烯醇丙酮酸羧化酶的核酸。根据本发明的实施例，与编码野生型磷酸烯醇丙酮酸羧化酶的核苷酸序列相比，所述编码改造的磷酸烯醇丙酮酸羧化酶的核苷酸序列针对谷氨酸棒杆菌进行了密码子优化，且优化后的核苷酸序列的第2312位碱基、第2791位碱基和/或第2827位碱基具有突变。In another aspect of the present invention, the present invention provides a nucleic acid encoding an engineered phosphoenolpyruvate carboxylase. According to an embodiment of the present invention, compared with the nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase, the nucleotide sequence encoding the modified phosphoenolpyruvate carboxylase is directed against glutamate rods Bacillus is codon-optimized, and the optimized nucleotide sequence has mutations at base 2312, base 2791 and/or base 2827.
目前，采用微生物发酵(如谷氨酸棒杆菌或大肠杆菌)生产氨基酸已经是占据了越来 越高的比重。由于植物的固碳能力较强，所以优先选择植物来源的磷酸烯醇丙酮酸羧化酶作为研究对象。由于植物源的酶难于在微生物中实现异源表达，所以，依据目标微生物的特性，预先对编码野生型的磷酸烯醇丙酮酸羧化酶的核苷酸序列进行密码子优化，使其能够更好地实现异源表达。进一步地，发明人发现，经密码子优化所得的核苷酸序列上的第第2312位碱基、第2791位碱基和/或第2827位碱基会显著影响磷酸烯醇丙酮酸羧化酶的活性，进一步影响谷氨酸棒杆菌的氨基酸产量，进而，通过对这3个位点的碱基进行定点突变，有助于提高谷氨酸棒杆菌的氨基酸产量，应用前景好。At present, the production of amino acids by microbial fermentation (such as Corynebacterium glutamicum or Escherichia coli) has occupied an increasing proportion. Due to the strong carbon fixation ability of plants, the plant-derived phosphoenolpyruvate carboxylase was preferentially selected as the research object. Since plant-derived enzymes are difficult to achieve heterologous expression in microorganisms, according to the characteristics of the target microorganisms, the nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase was codon-optimized in advance to make it more efficient. Heterologous expression is well achieved. Further, the inventors found that the 2312th base, the 2791st base and/or the 2827th base on the nucleotide sequence obtained by codon optimization can significantly affect phosphoenolpyruvate carboxylase The activity of Corynebacterium glutamicum further affects the amino acid production of Corynebacterium glutamicum, and further, through site-directed mutation of the bases of these three sites, it is helpful to improve the amino acid production of Corynebacterium glutamicum, and the application prospect is good.
根据本发明的实施例，编码野生型磷酸烯醇丙酮酸羧化酶的核苷酸序列具有如SEQ ID NO：2所示的核苷酸序列或者具有与SEQ ID NO：2所示的核苷酸序列具有至少80％同源性的核苷酸序列。According to an embodiment of the present invention, the nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase has the nucleotide sequence as shown in SEQ ID NO: 2 or has the nucleotide sequence as shown in SEQ ID NO: 2 Acid sequences are nucleotide sequences with at least 80% homology.
根据本发明的实施例，优化后的核苷酸序列具有如SEQ ID NO：3所示的核苷酸序列或者具有与SEQ ID NO：3所示的核苷酸序列具有至少80％同源性的核苷酸序列。该核苷酸序列可以在微生物中高效异源表达。进一步地，通过对上述3个核苷酸位点至少之一进行，使得该核苷酸编码的酶可以提高氨基酸产量。According to an embodiment of the present invention, the optimized nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 3 nucleotide sequence. The nucleotide sequence can be heterologously expressed in microorganisms with high efficiency. Further, by performing at least one of the above-mentioned three nucleotide positions, the enzyme encoded by the nucleotide can increase the yield of amino acids.
根据本发明的实施例，优化后的核苷酸序列具有c.2312C＞A、c.2791C＞A和/或c.2827G＞A的突变。具有优化密码子的核苷酸序列的第2312位C碱基突变为A碱基、第2791位C碱基突变为A碱基、第2827位G碱基突变为A碱基这三个中的至少一个发生突 变，均可以使得酶活性提高，进一步提高氨基酸产量。其中第2791位C碱基突变为A碱基、第2827位G碱基突变为A碱基这两种突变同时存在，氨基酸产量较高。According to an embodiment of the present invention, the optimized nucleotide sequence has mutations of c.2312C>A, c.2791C>A and/or c.2827G>A. In the nucleotide sequence with optimized codons, the 2312th C base is mutated to an A base, the 2791st C base is mutated to an A base, and the 2827th G base is mutated to an A base. At least one mutation can improve the enzyme activity and further improve the amino acid production. Among them, the 2791st C base was mutated to A base, and the 2827th G base was mutated to A base. These two mutations coexisted, and the amino acid yield was higher.
根据本发明的实施例，密码子优化后的核苷酸序列如SEQ ID NO：2所示或者与SEQ ID NO：2具有至少80％同源性。由此，经密码子优化后的酶可以在微生物中高效表达，尤其是谷氨酸棒杆菌。According to an embodiment of the present invention, the codon-optimized nucleotide sequence is shown in SEQ ID NO: 2 or has at least 80% homology with SEQ ID NO: 2. Thus, the codon-optimized enzyme can be highly expressed in microorganisms, especially Corynebacterium glutamicum.
重组表达载体recombinant expression vector
在本发明的又一方面，本发明提出了一种重组表达载体。根据本发明的实施例，该重组表达载体选自含有前面所述核酸的表达载体。由此，根据本发明实施例的重组表达载体在谷氨酸棒杆菌中表达磷酸烯醇丙酮酸羧化酶，有助于提高氨基酸产量。In yet another aspect of the present invention, the present invention provides a recombinant expression vector. According to an embodiment of the present invention, the recombinant expression vector is selected from expression vectors containing the aforementioned nucleic acids. Thus, the recombinant expression vector according to the embodiment of the present invention expresses phosphoenolpyruvate carboxylase in Corynebacterium glutamicum, which helps to improve the yield of amino acids.
根据本发明的实施例，表达载体选自大肠杆菌-谷氨酸棒杆菌穿梭表达载体。According to an embodiment of the present invention, the expression vector is selected from the Escherichia coli-Corynebacterium glutamicum shuttle expression vector.
大肠杆菌-谷氨酸棒杆菌穿梭表达载体指能在大肠杆菌和棒状杆菌中复制并稳定存在,含有一个在大肠杆菌和棒状杆菌中皆具有转录启动功能的启动子以及转录终止功能的终止子。组成型表达载体是在克隆载体基本骨架的基础上增加表达元件(如启动子、终止子等)，使目的基因能够表达的载体。其中，启动子(promoter)是RNA聚合酶结合并启动转录的特异DNA序列，终止子(terminator)是给予RNA聚合酶转录终止信号的DNA序列。诱导型表达载体是为实现外源蛋白分泌表达，最直接的方法就是将该基因插人到宿主分泌蛋白的表达元件(启动子和分泌信号信号肽序列)的后面。在表达载体中引入严格调控的强启动子，能够使目的基因在低诱导物浓度条件下仍能表达。诱导方式通常为化学诱导和环境诱导。The Escherichia coli-Corynebacterium glutamicum shuttle expression vector means that it can replicate and exist stably in Escherichia coli and Corynebacterium, and contains a promoter with transcription initiation function and a terminator with transcription termination function in both Escherichia coli and Corynebacterium. Constitutive expression vectors are vectors that add expression elements (such as promoters, terminators, etc.) to the basic skeleton of the cloning vector, so that the target gene can be expressed. Among them, a promoter is a specific DNA sequence that RNA polymerase binds to and initiates transcription, and a terminator is a DNA sequence that gives RNA polymerase a transcription termination signal. Inducible expression vector is to realize the secreted expression of exogenous protein, and the most direct method is to insert the gene behind the expression element (promoter and secretion signal signal peptide sequence) of the host secreted protein. The introduction of a tightly regulated strong promoter into the expression vector enables the expression of the target gene under the condition of low inducer concentration. The induction methods are usually chemical induction and environmental induction.
根据本发明的实施例，大肠杆菌-谷氨酸棒杆菌穿梭表达载体为组成型表达载体PVcaseG，组成型表达载体为包括来源于谷氨酸棒杆菌本身的gapa为启动子和为来源于大肠杆菌-谷氨酸棒杆菌穿梭载体PXMJ19的rrnB终止子。将前面所述编码改造的磷酸烯醇丙酮酸羧化酶的核酸序列(简称pepcTB)插入到PVcaseG载体上，构建成PVcaseG-pepcTB重组表达载体(质粒图参见图1)，同时将931位赖氨酸突变为谷氨酰胺，第943位天冬氨酸突变为天冬酰胺，电转到谷氨酸棒状杆菌中，在谷氨酸棒杆菌中通过IPTG诱导表达，可以提高磷酸烯醇丙酮酸羧化酶的活性。According to an embodiment of the present invention, the Escherichia coli-Corynebacterium glutamicum shuttle expression vector is a constitutive expression vector PVcaseG, and the constitutive expression vector includes gapa derived from Corynebacterium glutamicum itself as a promoter and a constitutive expression vector derived from Escherichia coli - rrnB terminator of the C. glutamicum shuttle vector PXMJ19. The nucleic acid sequence (referred to as pepcTB) of the phosphoenolpyruvate carboxylase encoding the aforementioned transformation was inserted into the PVcaseG carrier, and the PVcaseG-pepcTB recombinant expression vector was constructed (see Figure 1 for the plasmid map), and the 931-position lysine was added simultaneously. The acid was mutated to glutamine, the 943rd aspartic acid was mutated to asparagine, and electrotransferred into Corynebacterium glutamicum, and the expression was induced by IPTG in Corynebacterium glutamicum, which can improve the carboxylation of phosphoenolpyruvate enzyme activity.
本领域技术人员能够理解的是，前面针对编码改造的磷酸烯醇丙酮酸羧化酶的核酸所描述的特征和优点，同样适用于该重组表达载体，在此不再赘述。Those skilled in the art can understand that the features and advantages described above with respect to the nucleic acid encoding the modified phosphoenolpyruvate carboxylase are also applicable to the recombinant expression vector, and will not be repeated here.
基因工程菌Genetically engineered bacteria
在本发明的又一方面，本发明提出了一种基因工程菌。根据本发明的实施例，所述基因工程菌是通过将前面所述重组表达载体转化到受体菌内所获得的。由此，根据本发明实施例的基因工程菌可以高产氨基酸。In yet another aspect of the present invention, the present invention provides a genetically engineered bacteria. According to an embodiment of the present invention, the genetically engineered bacteria are obtained by transforming the aforementioned recombinant expression vector into the recipient bacteria. Thus, the genetically engineered bacteria according to the embodiments of the present invention can highly produce amino acids.
根据本发明的实施例，受体菌选自谷氨酸棒杆菌。According to an embodiment of the present invention, the recipient bacteria are selected from Corynebacterium glutamicum.
本领域技术人员能够理解的是，前面针对重组表达载体所描述的特征和优点，同样适用于该基因工程菌，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the recombinant expression vector are also applicable to the genetically engineered bacteria, and are not repeated here.
试剂盒Reagent test kit
本发明的又一方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括前面所述重组表达载体或前面所述基因工程菌。由此，利用根据本发明实施例的试剂盒可以实现高产氨基酸的目的。In yet another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned recombinant expression vector or the aforementioned genetically engineered bacteria. Thus, the purpose of high-yield amino acid can be achieved by using the kit according to the embodiment of the present invention.
本领域技术人员能够理解的是，前面针对重组表达载体和基因工程菌所描述的特征和优点，同样适用于该试剂盒，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the recombinant expression vector and the genetically engineered bacteria are also applicable to the kit, which will not be repeated here.
获得改造的磷酸烯醇丙酮酸羧化酶的方法Method for obtaining engineered phosphoenolpyruvate carboxylase
本发明的又一方面，本发明提出了一种获得改造的磷酸烯醇丙酮酸羧化酶的方法。根据本发明的实施例，所述方法包括：将野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列中的第771位、第931位和/或第943位氨基酸进行突变，得到所述改造的磷酸烯醇丙酮酸羧化酶。通过将野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列上的3个氨基酸残基进行突变会显著影响磷酸烯醇丙酮酸羧化酶的活性，进一步影响谷氨酸棒杆菌发酵的氨基酸产量。进而，通过对这三个位点至少之一的氨基酸取代，有助于提高谷氨酸棒杆菌发酵的氨基酸产量，应用前景好。In yet another aspect of the present invention, the present invention provides a method for obtaining an engineered phosphoenolpyruvate carboxylase. According to an embodiment of the present invention, the method comprises: mutating amino acids at positions 771, 931 and/or 943 in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase to obtain the modified Phosphoenolpyruvate carboxylase. By mutating three amino acid residues in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase, the activity of phosphoenolpyruvate carboxylase was significantly affected, and the amino acid yield of C. glutamicum fermentation was further affected. Furthermore, by substituting amino acids for at least one of the three sites, it is helpful to improve the yield of amino acids fermented by Corynebacterium glutamicum, and the application prospect is good.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶来源于植物，优选高粱。由此，改造的磷酸烯醇丙酮酸羧化酶有助于提高谷氨酸棒杆菌发酵的氨基酸碳通量，实现高产氨基酸。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase is derived from a plant, preferably sorghum. Thus, the modified phosphoenolpyruvate carboxylase helps to improve the carbon flux of amino acids fermented by Corynebacterium glutamicum and achieve high yield of amino acids.
根据本发明的实施例，野生型磷酸烯醇丙酮酸羧化酶具有如SEQ ID NO：1所示的氨基酸序列或者具有与SEQ ID NO：1所示的氨基酸序列具有至少80％同源性的氨基酸序列；野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代、第931位赖氨酸被谷氨酰胺所取代和/或第943位天冬氨酸被天冬酰胺所取代。由此，改造的磷酸烯醇丙酮酸羧化酶有助于提高谷氨酸棒杆菌发酵的氨基酸碳通量，实现高产氨基酸。According to an embodiment of the present invention, the wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 Amino acid sequence; amino acid sequence of wild-type phosphoenolpyruvate carboxylase with substitution of serine 771 by tyrosine, lysine 931 by glutamine and/or aspartic acid 943 replaced by asparagine. Thus, the modified phosphoenolpyruvate carboxylase helps to improve the carbon flux of amino acids fermented by Corynebacterium glutamicum and achieve high yield of amino acids.
本领域技术人员能够理解的是，前面针对磷酸烯醇丙酮酸羧化酶所描述的特征和优点， 同样适用于该获得改造的磷酸烯醇丙酮酸羧化酶的方法，在此不再赘述。It can be understood by those skilled in the art that the features and advantages described above for the phosphoenolpyruvate carboxylase are also applicable to the method for obtaining the modified phosphoenolpyruvate carboxylase, and will not be repeated here.
用途use
在本发明的又一方面，本发明提出了前面所述改造的磷酸烯醇丙酮酸羧化酶、所述编码改造的磷酸烯醇丙酮酸羧化酶的核酸、重组表达载体、基因工程菌在提高氨基酸产量中的应用。由此，根据本发明实施例的改造的磷酸烯醇丙酮酸羧化酶、编码其的核酸、重组表达载体、基因工程菌有助于提高谷氨酸棒杆菌发酵的氨基酸产量，应用前景好。In another aspect of the present invention, the present invention proposes the aforementioned modified phosphoenolpyruvate carboxylase, the nucleic acid encoding the modified phosphoenolpyruvate carboxylase, recombinant expression vectors, and genetically engineered bacteria in Applications for improving amino acid production. Thus, the modified phosphoenolpyruvate carboxylase, the nucleic acid encoding it, the recombinant expression vector, and the genetically engineered bacteria according to the embodiments of the present invention help to improve the amino acid yield fermented by Corynebacterium glutamicum, and have good application prospects.
本领域技术人员能够理解的是，前面针对改造的磷酸烯醇丙酮酸羧化酶、所述编码改造的磷酸烯醇丙酮酸羧化酶的核酸、重组表达载体、基因工程菌所描述的特征和优点，同样适用于该用途，在此不再赘述。It can be understood by those skilled in the art that the features and characteristics described above for the modified phosphoenolpyruvate carboxylase, the nucleic acid encoding the modified phosphoenolpyruvate carboxylase, the recombinant expression vector, the genetically engineered bacteria and the The advantages are also applicable to this purpose, and will not be repeated here.
提高谷氨酸棒杆菌氨基酸产量的方法Method for improving amino acid yield of Corynebacterium glutamicum
在本发明的又一方面，本发明提出了一种提高谷氨酸棒杆菌氨基酸产量的方法。根据本发明的实施例，所述方法包括：对前面所述重组表达载体转化到谷氨酸棒杆菌中，并对转化后的基因工程菌进行发酵培养。由此，重组表达载体在谷氨酸棒杆菌发酵过程中表达磷酸烯醇丙酮酸羧化酶，酶活性高，有助于提高氨基酸产量。In yet another aspect of the present invention, the present invention provides a method for improving the amino acid production of Corynebacterium glutamicum. According to an embodiment of the present invention, the method includes: transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum, and fermenting and culturing the transformed genetically engineered bacteria. Therefore, the recombinant expression vector expresses phosphoenolpyruvate carboxylase in the fermentation process of Corynebacterium glutamicum, and the enzyme activity is high, which helps to improve the yield of amino acids.
根据本发明的实施例，氨基酸包括天冬氨酸族氨基酸和谷氨酸族氨基酸。发明人发现，采用前述重组表达载体转化到谷氨酸棒杆菌中，可以显著提升天冬氨酸族氨基酸和谷氨酸族氨基酸产量。According to an embodiment of the present invention, the amino acids include aspartic amino acids and glutamic amino acids. The inventors found that the production of aspartate amino acids and glutamic acid amino acids can be significantly improved by transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum.
根据本发明的实施例，天冬氨酸族氨基酸包括天冬氨酸、赖氨酸、苏氨酸、甲硫氨酸和/或异亮氨酸；谷氨酸族氨基酸包括谷氨酸、脯氨酸、谷氨酰胺、精氨酸。发明人发现，采用前述重组表达载体转化到谷氨酸棒杆菌中，可以显著提升上述天冬氨酸族氨基酸和谷氨酸族氨基酸产量。According to an embodiment of the present invention, the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine; the glutamic acid amino acids include glutamic acid, pro amino acid, glutamine, arginine. The inventors found that by transforming the aforementioned recombinant expression vector into Corynebacterium glutamicum, the yields of the above-mentioned aspartic amino acids and glutamic acid amino acids can be significantly increased.
本领域技术人员能够理解的是，前面针对重组表达载体所描述的特征和优点，同样适用于该提高谷氨酸棒杆菌氨基酸产量的方法，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the recombinant expression vector are also applicable to the method for improving the amino acid production of Corynebacterium glutamicum, which will not be repeated here.
生产氨基酸的方法Method for producing amino acids
在本发明的又一方面，本发明提出了一种生产氨基酸的方法。根据本发明的实施例，所述方法包括：将前面所述基因工程菌进行发酵培养。由此，重组表达载体在谷氨酸棒杆菌发酵过程中表达磷酸烯醇丙酮酸羧化酶，酶活性高，有助于提高氨基酸产量。In yet another aspect of the present invention, the present invention provides a method for producing amino acids. According to an embodiment of the present invention, the method includes: fermenting and culturing the aforementioned genetically engineered bacteria. Therefore, the recombinant expression vector expresses phosphoenolpyruvate carboxylase in the fermentation process of Corynebacterium glutamicum, and the enzyme activity is high, which helps to improve the yield of amino acids.
根据本发明的实施例，氨基酸包括天冬氨酸族氨基酸和谷氨酸族氨基酸。发明人发现， 采用前述基因工程菌可以显著提升天冬氨酸族氨基酸和谷氨酸族氨基酸产量。According to an embodiment of the present invention, the amino acids include aspartic amino acids and glutamic amino acids. The inventors found that the production of aspartate amino acids and glutamic acid amino acids can be significantly improved by using the aforementioned genetically engineered bacteria.
根据本发明的实施例，天冬氨酸族氨基酸包括天冬氨酸、赖氨酸、赖氨酸、苏氨酸、甲硫氨酸和/或异亮氨酸；谷氨酸族氨基酸包括谷氨酸、脯氨酸、谷氨酰胺、精氨酸。发明人发现，采用前述基因工程菌可以显著提升上述天冬氨酸族氨基酸和谷氨酸族氨基酸产量。According to an embodiment of the present invention, the aspartic amino acids include aspartic acid, lysine, lysine, threonine, methionine and/or isoleucine; the glutamic acid family of amino acids includes glutamic acid Amino acid, proline, glutamine, arginine. The inventors found that the use of the aforementioned genetically engineered bacteria can significantly increase the yields of the above-mentioned aspartate amino acids and glutamic acid amino acids.
本领域技术人员能够理解的是，前面针对基因工程菌所描述的特征和优点，同样适用于该生产氨基酸的方法，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the genetically engineered bacteria are also applicable to the method for producing amino acids, and details are not repeated here.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
质粒pk18mobsacB购自武汉淼灵生物科技有限公司、穿梭质粒PXMJ19购自普如汀生物技术(北京)有限公司、大肠杆菌DH5α为本领域常规使用；DNA聚合酶(Q5 High-Fidelity DNA Polymerase)购自基因有限公司；限制性内切酶(EcoRI、SalI、EcoRV)、DNAmarker、质粒提取试剂盒、DNA胶回收纯化试剂盒，均购自Takara宝生物工程(大连)有限公司；细菌基因组DNA提取试剂盒购自天根生化科技(北京)有限公司、Onestep clonning克隆重组试剂盒购自NEB北京公司；硫酸卡那霉素购自Biosharp公司；蔗糖等其余化学药品试剂均为国药分析纯。质粒提取操作步骤参照质粒小提取试剂盒说明书；DNA胶回收操作步骤参照DNA胶回收试剂盒说明书；谷氨酸棒杆菌基因组提取操作步骤参照细菌基因组DNA提取试剂盒说明书；DNA片段重组连接操作步骤参照Onestep clonning克隆重组试剂盒明书；谷氨酸棒杆菌感受态的制备及转化方法参照vander Rest等的方法(M.E.vander Rest,C.Lange,D.Molenaar.A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA.Appl.Microbiol.Biotechnol.1999,52:541-545)。通过氨基酸分析仪(日立8800)进行发酵液中的氨基酸含量检测。Plasmid pk18mobsacB was purchased from Wuhan Miaoling Biotechnology Co., Ltd., shuttle plasmid PXMJ19 was purchased from Protin Biotechnology (Beijing) Co., Ltd., Escherichia coli DH5α was routinely used in the field; DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.; restriction endonucleases (EcoRI, SalI, EcoRV), DNAmarker, plasmid extraction kit, DNA gel recovery and purification kit, all purchased from Takara Bioengineering (Dalian) Co., Ltd.; bacterial genomic DNA extraction kit Purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Onestep cloning and recombination kit was purchased from NEB Beijing Company; kanamycin sulfate was purchased from Biosharp Company; other chemical reagents such as sucrose were of analytical grade from Sinopharm. The operation steps of plasmid extraction refer to the instructions of the plasmid mini-extraction kit; the operation steps of DNA gel recovery refer to the instructions of DNA gel recovery kit; the operation steps of Corynebacterium glutamicum genome extraction refer to the instructions of bacterial genome DNA extraction kit; the operation steps of DNA fragment recombination and ligation refer to Onestep cloning cloning and recombination kit instructions; the preparation and transformation method of the competent Corynebacterium glutamicum refer to the method of vander Rest, etc. (M.E.vander Rest, C. Lange, D. Molenaar. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasma DNA. Appl. Microbiol. Biotechnol. 1999, 52:541-545). The amino acid content in the fermentation broth was detected by an amino acid analyzer (Hitachi 8800).
下述实施例中酶活通过PEPC催化磷酸烯醇式丙酮酸和二氧化碳生成草酰乙酸和磷酸氢离子，苹果酸脱氢酶进一步催化草酰乙酸和NADH生成苹果酸和NAD+，在340nm测定NADH减少速率，从而计算PEPC活性。每mg组织蛋白每分钟消耗1nmol NADH定义为一个酶活力单位。本发明中所采用的的测定蛋白质含量的方法为BCA试剂盒检测法。In the following examples, the enzyme activity catalyzes phosphoenolpyruvate and carbon dioxide to generate oxaloacetate and hydrogen phosphate ions through PEPC, and malate dehydrogenase further catalyzes oxaloacetate and NADH to generate malate and NAD+, and the reduction of NADH is measured at 340nm rate to calculate PEPC activity. The consumption of 1 nmol NADH per mg tissue protein per minute was defined as one unit of enzyme activity. The method for determining the protein content adopted in the present invention is the BCA kit detection method.
异亮氨酸种子培养基配方为：葡萄糖3.0％、硫酸铵2.5％、玉米浆3.5％、酵母膏0.3％、丝肽粉0.3％、磷酸氢二钾0.1％、硫酸镁0.05％、碳酸钙4％，余量为去离子水，pH为7； 所述％为重量体积(g/mL)百分比。The formula of isoleucine seed medium is: glucose 3.0%, ammonium sulfate 2.5%, corn steep liquor 3.5%, yeast extract 0.3%, silk peptide powder 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, calcium carbonate 4% %, the balance is deionized water, pH is 7; The % is the weight volume (g/mL) percentage.
异亮氨酸发酵培养基配方为：葡萄糖16.0％、硫酸铵0.8％、玉米浆3.5％、酵母膏0.3％、丝肽粉0.3％、磷酸氢二钾0.1％、硫酸镁0.05％，余量为去离子水，pH为7；所述％为重量体积(g/mL)百分比。The formula of isoleucine fermentation medium is: glucose 16.0%, ammonium sulfate 0.8%, corn steep liquor 3.5%, yeast extract 0.3%, silk peptide powder 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and the balance is Deionized water, pH 7; the % are weight volume (g/mL) percentages.
精氨酸种子培养基配方为：葡萄糖3.0％，磷酸氢二钾0.2％，尿素0.12％，硫酸铵0.5％，酵母膏0.5％，玉米浆5.5％，硫酸镁0.04％，生物素50ug/L，丝肽粉1.0％，玉米油10滴/L，轻质碳酸钙1％。The formula of arginine seed medium is: glucose 3.0%, dipotassium hydrogen phosphate 0.2%, urea 0.12%, ammonium sulfate 0.5%, yeast extract 0.5%, corn steep liquor 5.5%, magnesium sulfate 0.04%, biotin 50ug/L, Silk peptide powder 1.0%, corn oil 10 drops/L, light calcium carbonate 1%.
精氨酸发酵培养基配方为：葡萄糖12.5％，磷酸氢二钾0.15％，尿素0.1％，硫酸铵4.5％，酵母膏0.5％，玉米浆5.5％，硫酸镁0.05％，生物素0.05％，维生素B1 0.1％，玉米油10滴/L，糖蜜4.0％，丝肽粉0.3％，轻质碳酸钙0.5％，余量为去离子水，pH为7；所述％为重量体积(g/mL)百分比。The formula of arginine fermentation medium is: glucose 12.5%, dipotassium hydrogen phosphate 0.15%, urea 0.1%, ammonium sulfate 4.5%, yeast extract 0.5%, corn steep liquor 5.5%, magnesium sulfate 0.05%, biotin 0.05%, vitamins B1 0.1%, corn oil 10 drops/L, molasses 4.0%, silk peptide powder 0.3%, light calcium carbonate 0.5%, the balance is deionized water, pH is 7; the % is weight volume (g/mL) percentage.
实施例1Example 1
基于谷氨酸棒杆菌的特性，对编码高粱的磷酸烯醇丙酮酸羧化酶(SEQ ID NO：2)进行密码子优化，获得SEQ ID NO：3所示的核苷酸序列。Based on the characteristics of Corynebacterium glutamicum, the phosphoenolpyruvate carboxylase (SEQ ID NO: 2) encoding sorghum was codon optimized to obtain the nucleotide sequence shown in SEQ ID NO: 3.
实施例2Example 2
对磷酸烯醇丙酮酸羧化酶基因(PEPC)的定点诱变及构建能在大肠杆菌中表达的重组载体pet28a-pepcTBSite-directed mutagenesis of phosphoenolpyruvate carboxylase gene (PEPC) and construction of recombinant vector pet28a-pepcTB expressing in Escherichia coli
以合成的PEPC载体为模板，使用以下引物通过反向PCR扩增突变的磷酸烯醇丙酮酸羧化酶的编码基因PEPC，同时为了便于后续纯化在基因C端加上6×HIS标签。所用酶为Q5高保真酶。Using the synthetic PEPC vector as a template, the following primers were used to amplify the mutated phosphoenolpyruvate carboxylase-encoding gene PEPC by inverse PCR, and a 6×HIS tag was added to the C-terminal of the gene for the convenience of subsequent purification. The enzyme used was the Q5 high-fidelity enzyme.
对N端第771位磷酸化位点丝氨酸突变为酪氨酸，引物如下：To mutate serine to tyrosine at the 771st phosphorylation site at the N-terminal, the primers are as follows:
5’-CTTCTACTGGACCCAGACCCGCTTCCACCTGCCAGTTTGGCTGGG-3’(SEQ ID NO：4)5'-CTTCTACTGGACCCAGACCCGCTTCCACCTGCCAGTTTGGCTGGG-3' (SEQ ID NO: 4)
3’-TGGGTCCAGTAGAAGATCCATGGGATAGCGCGCAGGGTGGTGATG-5’(SEQ ID NO：5)3'-TGGGTCCAGTAGAAGATCCATGGGATAGCGCGCAGGGTGGTGATG-5' (SEQ ID NO: 5)
第931位赖氨酸突变为谷氨酰胺，引物如下：Lysine 931 was mutated to glutamine, and the primers were as follows:
5’-CCTGGTTCAGCTGAACGGCGAGCGCGTTCCACCAGGCCTGGAGAA-3’(SEQ ID NO：6)5'-CCTGGTTCAGCTGAACGGCGAGCGCGTTCCACCAGGCCTGGAGAA-3' (SEQ ID NO: 6)
3’-TTCAGCTGAACCAGGCCAGCTGGCTTGTTCTCGTCAGCGAACTCC-5’(SEQ ID NO：7)3'-TTCAGCTGAACCAGGCCAGCTGGCTTGTTCTCGTCAGCGAACTCC-5' (SEQ ID NO: 7)
第943位天冬氨酸突变为天冬酰胺，引物如下：The 943rd aspartic acid was mutated to asparagine, and the primers were as follows:
5’-CCTGGAGAACACCCTGATCCTGACCATGAAGGGCATCGCTGCTGG-3’(SEQ ID NO：8)5'-CCTGGAGAACACCCTGATCCTGACCATGAAGGGCATCGCTGCTGG-3' (SEQ ID NO: 8)
3’-AGGGTGTTCTCCAGGCCTGGTGGAACGCGCTCGCCGTTCAGCTGA-5’(SEQ ID NO：9)3'-AGGGTGTTCTCCAGGCCTGGTGGAACGCGCTCGCCGTTCAGCTGA-5' (SEQ ID NO: 9)
PEPC扩增引物：PEPC amplification primers:
5’-CAATTCCCCTCTAGAATGGCTTCCGAGCGCCACCAC-3’(SEQ ID NO：10)5'-CAATTCCCCTCTAGAATGGCTTCCGAGCGCCACCAC-3' (SEQ ID NO: 10)
5’-TTAGTGGTGGTGGTGGTGGTG-3’(SEQ ID NO：11)5'-TTAGTGGTGGTGGTGGTGGTG-3' (SEQ ID NO: 11)
高保真Q5 High-Fidelity DNA Polymerase聚合酶PCR扩增核苷酸片段pepc约2.9kb。PCR反应体系(50μl)为：5×Q5 reaction buffer 10μl、10mM dNTP 1μl、引物各2.5μl、模板视样品浓度而定、Q5酶0.5μl、加水至50μl。反应条件为：98℃30s，98℃10s，55℃-72℃30s，72℃1.5min，33个循环；72℃2min。1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收2.9kb的4个PCR产物。High-fidelity Q5 High-Fidelity DNA Polymerase PCR amplifies the nucleotide fragment pepc of about 2.9kb. The PCR reaction system (50 μl) was: 5×Q5 reaction buffer 10 μl, 10 mM dNTP 1 μl, primers 2.5 μl each, template depending on the sample concentration, Q5 enzyme 0.5 μl, and water to 50 μl. The reaction conditions were: 98°C for 30s, 98°C for 10s, 55°C-72°C for 30s, 72°C for 1.5 min, 33 cycles; 72°C for 2 min. 1% agarose gel electrophoresis detection and DNA gel recovery kit purification and recovery of 4 PCR products of 2.9kb.
第771位和931位同时突变时，以931位突变的PEPC基因载体为模板，在771位使用771位突变引物进行反向pcr，所得产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收，再以Pepc扩增引物扩增第771位和931位同时突变的磷酸烯醇式丙酮酸羧化酶基因。最终产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收。When positions 771 and 931 are mutated at the same time, the PEPC gene vector mutated at position 931 is used as a template, and the 771-mutated primer is used for reverse PCR at position 771. The resulting product is detected by 1% agarose gel electrophoresis and DNA gel recovery kit After purification and recovery, the phosphoenolpyruvate carboxylase gene mutated at positions 771 and 931 at the same time was amplified with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
第771位和943位同时突变时，以771位突变的PEPC基因载体为模板，在943位使用943位突变引物进行反向pcr，所得产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收，再以Pepc扩增引物扩增第771位和943位同时突变的磷酸烯醇式丙酮酸羧化酶基因。最终产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收。When positions 771 and 943 are mutated at the same time, the PEPC gene vector mutated at position 771 is used as a template, and the 943-mutated primer is used for reverse PCR at position 943. The resulting product is detected by 1% agarose gel electrophoresis and DNA gel recovery kit After purification and recovery, the phosphoenolpyruvate carboxylase gene mutated at positions 771 and 943 was amplified with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
第931位和943位同时突变时，以931位突变的PEPC基因载体为模板，在943位使用943位突变引物进行反向pcr，所得产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收，再以Pepc扩增引物扩增第931位和943位同时突变的磷酸烯醇式丙酮酸羧化酶基因。最终产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收。When the 931st and 943th positions are mutated at the same time, the PEPC gene vector mutated at position 931 is used as the template, and the 943-position mutated primer is used for reverse PCR at position 943. The resulting product is detected by 1% agarose gel electrophoresis and DNA gel recovery kit After purification and recovery, the phosphoenolpyruvate carboxylase gene mutated at positions 931 and 943 at the same time was amplified with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
第771位、931位和943位同时突变时，以771位和931位同时突变的PEPC基因载体为模板，在943位使用943位突变引物进行反向pcr，所得产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收，再以Pepc扩增引物扩增第771位、931位和943位同时突变的磷酸烯醇式丙酮酸羧化酶基因。最终产物1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收。When the 771st, 931st and 943th positions are mutated at the same time, the PEPC gene vector mutated at the 771st and 931st positions is used as the template, and the 943th mutated primer is used for reverse PCR at the 943rd position, and the resulting product is electrophoresed on a 1% agarose gel Detection, purification and recovery of DNA gel recovery kit, and amplification of the phosphoenolpyruvate carboxylase gene simultaneously mutated at positions 771, 931 and 943 with Pepc amplification primers. The final product was detected by 1% agarose gel electrophoresis and purified and recovered by DNA gel recovery kit.
利用XbaI/BamHI双酶切质粒pet28a(质粒pet28a购自武汉淼灵生物科技有限公司)，反应体系为：质粒1μg、10×buffer 5μl、XbaI 1μl、BamHI 1μl、补水至50μl。37℃酶切2h。1％ 琼脂糖凝胶电泳检测并DNA胶回收纯化试剂盒回收5.3kb核苷酸片段。The plasmid pet28a was digested with XbaI/BamHI double enzyme (plasmid pet28a was purchased from Wuhan Miaoling Biotechnology Co., Ltd.), and the reaction system was: plasmid 1 μg, 10×buffer 5 μl, XbaI 1 μl, BamHI 1 μl, and water to 50 μl. Digestion at 37°C for 2h. 1% agarose gel electrophoresis detection and DNA gel recovery and purification kit to recover 5.3kb nucleotide fragments.
Onestep clonning克隆重组试剂盒重组上述双酶切产物(即XbaI/BamHI双酶切质粒pet28a)及突变的pepc片段，重组产物转化大肠杆菌BL21，涂布于含硫酸卡那霉素的平板，经过37℃过夜培养后，挑选转化子测序验证，测序正确的转化子即含有重组质粒pet28a-pepcTB，分别命名为pet28a-pepcTB1(771位丝氨酸突变为酪氨酸)、pet28a-pepcTB2(931位赖氨酸突变为谷氨酰胺)、pet28a-pepcTB3(943位天冬氨酸突变为天冬酰胺)、pet28a-pepcTB12(771位丝氨酸突变为酪氨酸，同时931位赖氨酸突变为谷氨酰胺)、pet28a-pepcTB13(771位丝氨酸突变为酪氨酸，同时943位天冬氨酸突变为天冬酰胺)pet28a-pepcTB23(931位赖氨酸突变为谷氨酰胺，同时943位天冬氨酸突变为天冬酰胺)、pet28a-pepcTB123(771位丝氨酸突变为酪氨酸，931位赖氨酸突变为谷氨酰胺，同时943位天冬氨酸突变为天冬酰胺)。Onestep cloning and recombination kit recombines the above double-enzyme digestion product (i.e. XbaI/BamHI double-enzyme digestion plasmid pet28a) and the mutated pepc fragment. The recombinant product is transformed into Escherichia coli BL21 and spread on a plate containing kanamycin sulfate. After 37 After culturing overnight at ℃, the transformants were selected for sequencing and verification. The transformants with correct sequencing contained the recombinant plasmid pet28a-pepcTB, which were named pet28a-pepcTB1 (serine 771 was mutated to tyrosine), pet28a-pepcTB2 (lysine 931 was mutated). Mutation to glutamine), pet28a-pepcTB3 (mutation of aspartic acid at position 943 to asparagine), pet28a-pepcTB12 (mutation of serine 771 to tyrosine, and mutation of lysine 931 to glutamine), pet28a-pepcTB13 (serine 771 is mutated to tyrosine and aspartic acid 943 is mutated to asparagine) pet28a-pepcTB23 (lysine 931 is mutated to glutamine, and aspartic acid 943 is mutated to asparagine), pet28a-pepcTB123 (serine 771 was mutated to tyrosine, lysine 931 was mutated to glutamine, and aspartic acid 943 was mutated to asparagine).
实施例3Example 3
根据常规方案，将重组载体pet28a-pepcTB1、pet28a-pepcTB2、pet28a-pepcTB3、pet28a-pepcTB12、pet28a-pepcTB13、pet28a-pepcTB23、pet28a-pepcTB123均转化到大肠杆菌菌株BL21(DE3)CodonPlus RIPL中。由转化实验产生的重组大肠杆菌菌株被指定为：E.coli pepc1、E.coli pepc2、E.coli pepc3、E.coli pepc12、E.coli pepc13、E.coli pepc23、E.coli pepc123。According to conventional protocols, the recombinant vectors pet28a-pepcTB1, pet28a-pepcTB2, pet28a-pepcTB3, pet28a-pepcTB12, pet28a-pepcTB13, pet28a-pepcTB23, pet28a-pepcTB123 were transformed into E. coli strain BL21(DE3) CodonPlus RIPL. The recombinant E. coli strains generated from the transformation experiments were designated: E.coli pepc1, E.coli pepc2, E.coli pepc3, E.coli pepc12, E.coli pepc13, E.coli pepc23, E.coli pepc123.
将上述重组大肠杆菌菌株在37℃下在含有50mg/L卡那霉素的LB培养基中培养，直至OD600达到0.4-1.0。然后，将0.1-0.5Mm/L IPTG加入到培养基中，并将细菌在30℃再培养4-16小时。离心收集菌体用缓冲液(20mM Tris-HCl，Ph6.8)洗涤并悬浮。超声破碎，条件为100W，30min，4℃、12 000r/min离心2min，所得到的上清液即为粗酶液。The above recombinant E. coli strains were cultured in LB medium containing 50 mg/L kanamycin at 37°C until OD600 reached 0.4-1.0. Then, 0.1-0.5Mm/L IPTG was added to the medium, and the bacteria were cultured at 30°C for another 4-16 hours. The cells were collected by centrifugation, washed with buffer (20 mM Tris-HCl, Ph6.8) and suspended. Ultrasonic crushing, the conditions are 100W, 30min, 4℃, 12 000r/min centrifugation for 2min, the obtained supernatant is the crude enzyme liquid.
实施例4Example 4
大肠杆菌突变的磷酸烯醇丙酮酸羧化酶酶学性质分析：Analysis of the enzymatic properties of Escherichia coli mutant phosphoenolpyruvate carboxylase:
根据常规方案，将上述粗酶液进行SDS-PAGE电泳分离，目的蛋白大小约102KDa，通过BCA蛋白检测试剂盒进行蛋白含量检测。通过镍柱亲和层析法纯化目的蛋白，其原理为：多聚组氨酸能与多种过渡金属和过渡金属螯合物结合，因此带暴露的6 X His-tag的蛋白质能结合于固化Ni2+树脂，从而将带有his-tag组氨酸标签的融合蛋白与其它蛋白区分开来。当我们用高浓度的咪唑(imidazole)溶液洗脱的时侯，咪唑便与融合蛋白his-tag的咪唑环竞争结合，最终将融合蛋白洗脱下来。纯化后的蛋白再次进行SDS-PAGE电泳检测。According to the conventional protocol, the above crude enzyme solution was separated by SDS-PAGE electrophoresis, the size of the target protein was about 102KDa, and the protein content was detected by BCA protein detection kit. The target protein was purified by nickel column affinity chromatography. The principle is: polyhistidine can bind to various transition metals and transition metal chelates, so the protein with exposed 6X His-tag can bind to the immobilized Ni2+ resin, thereby distinguishing his-tag histidine-tagged fusion proteins from other proteins. When we eluted with a high concentration of imidazole solution, imidazole competed with the imidazole ring of the fusion protein his-tag for binding, and finally the fusion protein was eluted. The purified protein was detected by SDS-PAGE electrophoresis again.
通过磷酸烯醇式丙酮酸羧化酶酶活检测试剂盒测定酶活，其原理为PEPC催化磷酸烯醇式丙酮酸和二氧化碳生成草酰乙酸和磷酸氢离子，苹果酸脱氢酶进一步催化草酰乙酸和NADH生成苹果酸和NAD+，并用紫外分光光度计测定340nm处吸光度的变化。每mg蛋白每分钟消耗1nmol NADH定义为一个酶活力单位。The enzyme activity was determined by phosphoenolpyruvate carboxylase enzyme activity detection kit. The principle is that PEPC catalyzes phosphoenolpyruvate and carbon dioxide to generate oxaloacetate and hydrogen phosphate ions, and malate dehydrogenase further catalyzes oxalyl Acetic acid and NADH generate malic acid and NAD+, and the change in absorbance at 340 nm is measured with a UV spectrophotometer. The consumption of 1 nmol NADH per mg protein per minute was defined as one unit of enzyme activity.
不同位点的突变对磷酸烯醇式丙酮酸羧化酶酶活的影响不一样，酶活测定结果如表1所示。可以看出，突变后磷酸烯醇式丙酮酸羧化酶酶活均有不同程度的变化，第771位磷酸化位点丝氨酸突变为酪氨酸，最终使酶活在原来的基础上提高了1.96倍；第931位赖氨酸突变为谷氨酰胺，提高对磷酸烯醇式丙酮酸的亲和力，最终使酶活在原来的基础上提高了3.79倍；第943位天冬氨酸突变为天冬酰胺，降低对甘氨酸的敏感性，最终使酶活在原来的基础上提高了6.3倍；第771位磷酸化位点丝氨酸突变为酪氨酸，同时第931位赖氨酸突变为谷氨酰胺，最终使酶活在原来的基础上提高了5.44倍；第771位磷酸化位点丝氨酸突变为酪氨酸，同时第943位天冬氨酸突变为天冬酰胺，最终使酶活在原来的基础上提高了3.41倍；第931位赖氨酸突变为谷氨酰胺，同时第943位天冬氨酸突变为天冬酰胺，最终使酶活在原来的基础上提高了12.05倍；第771位磷酸化位点丝氨酸突变为酪氨酸，同时第931位赖氨酸突变为谷氨酰胺，第943位天冬氨酸突变为天冬酰胺，最终使酶活在原来的基础上提高了6.41倍。Mutations at different sites have different effects on the enzymatic activity of phosphoenolpyruvate carboxylase. The results of the enzymatic activity assay are shown in Table 1. It can be seen that the enzymatic activity of phosphoenolpyruvate carboxylase changes to varying degrees after the mutation. The serine at the 771st phosphorylation site is mutated to tyrosine, which finally increases the enzymatic activity by 1.96% on the original basis. The 931st lysine was mutated to glutamine, which improved the affinity for phosphoenolpyruvate, and finally the enzyme activity was increased by 3.79 times on the original basis; the 943rd aspartic acid was mutated to asparagine amide, reducing the sensitivity to glycine, and finally increasing the enzyme activity by 6.3 times on the original basis; the phosphorylation site 771 was mutated from serine to tyrosine, and the 931 lysine was mutated to glutamine. Finally, the activity of the enzyme was increased by 5.44 times on the original basis; the phosphorylation site 771 was mutated from serine to tyrosine, and the 943rd aspartic acid was mutated to asparagine, which finally made the enzyme live on the original basis. The enzyme activity was increased by 3.41 times; the 931st lysine was mutated to glutamine, and the 943rd aspartic acid was mutated to asparagine, which finally increased the enzyme activity by 12.05 times on the original basis; the 771st phosphate The serine was mutated to tyrosine, the 931st lysine was mutated to glutamine, and the 943rd aspartate was mutated to asparagine, which finally increased the enzyme activity by 6.41 times.
表1大肠杆菌突变菌株酶活测定Table 1 Determination of enzyme activity of Escherichia coli mutant strains
实施例5Example 5
构建单点突变和组合突变PEPC基因的谷氨酸棒杆菌表达载体Construction of single point mutation and combined mutation PEPC gene expression vector of Corynebacterium glutamicum
首先在基础质粒PVcase(质粒图谱如图1所示)上构建以gapa为启动子，rrnB为终止之的组成型表达载体PVcaseG。启动子与终止子的扩增都以实验室现有菌株m13基因组为模板，所用引物如下：First, construct the constitutive expression vector PVcaseG with gapa as the promoter and rrnB as the termination on the basic plasmid PVcase (the plasmid map is shown in Figure 1). The amplification of the promoter and terminator is based on the existing strain m13 genome in the laboratory as the template, and the primers used are as follows:
Gapa扩增Gapa amplification
5’-ATTCGAGCTCGGTACCCCGAAGATCTGAAGATTCCTG-3’(SEQ ID NO：12)5'-ATTCGAGCTCGGTACCCCGAAGATCTGAAGATTCCTG-3' (SEQ ID NO: 12)
5’-ACAGCCATGGAGATCTGGTGTGTCTCCTCTAAAGATTG-3’(SEQ ID NO：13)5'-ACAGCCATGGAGATCTGGTGTGTCTCCTCTAAAGATTG-3' (SEQ ID NO: 13)
RrnB扩增RrnB amplification
5’-TTAGAGGAGACACACCAGATCTCCATGGCTGTTTTG-3’(SEQ ID NO：14)5'-TTAGAGGAGACACACCAGATCTCCATGGCTGTTTTG-3' (SEQ ID NO: 14)
5’-TGCCTGCAGGTCGACATGAAAGAGTTTGTAGAAACGC-3’(SEQ ID NO：15)5'-TGCCTGCAGGTCGACATGAAAGAGTTTGTAGAAACGC-3' (SEQ ID NO: 15)
利用KpnI/SalI双酶切质粒PVcase，通过如上所述一步克隆的方法构建组成型表达载体PVcaseG。The plasmid PVcase was digested with KpnI/SalI, and the constitutive expression vector PVcaseG was constructed by the one-step cloning method as described above.
以质粒PVcaseG为模板，反向PCR扩增所得目的产物经胶回收即为线性载体，扩增引物如下：Using plasmid PVcaseG as a template, the target product obtained by inverse PCR amplification is a linear vector after gel recovery, and the amplification primers are as follows:
5’-CACCACCACCACTAAAGATCTCCATGGCTGTTTTG-3’(SEQ ID NO：16)5'-CACCACCACCACTAAAGATCTCCATGGCTGTTTTG-3' (SEQ ID NO: 16)
5’-GCGCTCGGAAGCCATGGTGTGTCTCCTCTAAAGATTG-3’(SEQ ID NO：17)5'-GCGCTCGGAAGCCATGGTGTGTCTCCTCTAAAGATTG-3' (SEQ ID NO: 17)
分别以质粒pet28a-pepcTB1、pet28a-pepcTB2、pet28a-pepcTB3、pet28a-pepcTB12、pet28a-pepcTB13、pet28a-pepcTB23、pet28a-pepcTB123为模板，扩增单点突变及组合突变PEPC基因，扩增引物如下：Using plasmids pet28a-pepcTB1, pet28a-pepcTB2, pet28a-pepcTB3, pet28a-pepcTB12, pet28a-pepcTB13, pet28a-pepcTB23, pet28a-pepcTB123 as templates, amplify single-point mutation and combined mutation PEPC gene, amplification primers are as follows:
5’-TAGAGGAGACACACCATGGCTTCCGAGCGCCACCAC-3’(SEQ ID NO：18)5'-TAGAGGAGACACACCATGGCTTCCGAGCGCCACCAC-3' (SEQ ID NO: 18)
5’CAGCCATGGAGATCTTTAGTGGTGGTGGTGGTGGTGGCCGGTGTTCTGCATGCCAG-3’(SEQ ID NO：19)5' CAGCCATGGAGATCTTTAGTGGTGGTGGTGGTGGTGGCCGGTGTTCTGCATGCCAG-3' (SEQ ID NO: 19)
Onestep clonning克隆重组试剂盒重组上述线性载体及突变的pepc片段，重组产物转化大肠杆菌DH5α，涂布于含硫酸卡那霉素的平板，经过37℃过夜培养后，挑选转化子测序验证，测序正确的转化子即含有重组质粒PVcaseG-pepcTB，分别命名为PVcaseG-pepcTB1、PVcaseG-pepcTB2、PVcaseG-pepcTB3、PVcaseG-pepcTB12、PVcaseG-pepcTB13、PVcaseG-pepcTB23、PVcaseG-pepcTB123。Onestep cloning recombination kit recombines the above linear vector and the mutated pepc fragment. The recombinant product is transformed into Escherichia coli DH5α and spread on a plate containing kanamycin sulfate. After overnight incubation at 37°C, the selected transformants are sequenced to verify that the sequencing is correct. The transformants contained the recombinant plasmid PVcaseG-pepcTB, named PVcaseG-pepcTB1, PVcaseG-pepcTB2, PVcaseG-pepcTB3, PVcaseG-pepcTB12, PVcaseG-pepcTB13, PVcaseG-pepcTB23, PVcaseG-pepcTB123.
根据常规方案，将不同的PVcaseG-pepcTB载体同时电转到谷氨酸棒状杆菌C.glutamicum H5和C.glutamicum H1中，由转化实验产生的重组谷氨酸棒状杆菌被命名为：H5-Vpepc1、H5-Vpepc2、H5-Vpepc3、H5-Vpepc12、H5-Vpepc13、H5-Vpepc23、H5-Vpepc123；H1-Vpepc1、H1-Vpepc2、H1-Vpepc3、H1-Vpepc12、H1-Vpepc13、H1-Vpepc23、H1-Vpepc123。According to the conventional protocol, different PVcaseG-pepcTB vectors were electroporated into Corynebacterium glutamicum C.glutamicum H5 and C.glutamicum H1 at the same time, and the recombinant Corynebacterium glutamicum produced by the transformation experiment was named: H5-Vpepc1, H5 -Vpepc2, H5-Vpepc3, H5-Vpepc12, H5-Vpepc13, H5-Vpepc23, H5-Vpepc123; H1-Vpepc1, H1-Vpepc2, H1-Vpepc3, H1-Vpepc12, H1-Vpepc13, H1-Vpepc23, H1-Vpepc123 .
实施例6Example 6
重组菌株磷酸烯醇丙酮酸羧化酶的表达与酶活测定Expression and activity assay of phosphoenolpyruvate carboxylase from recombinant strains
将对照菌株C.glutamicum H5和C.glutamicum H1以及上述重组谷氨酸棒杆菌分别接种于异亮氨酸和精氨酸发酵培养基，30℃培养36h后，取发酵液，4℃、10 000r/min离 心2min收集菌体，用0.1mol HCL洗两次，再用pH 7.5，0.1mol/L磷酸盐缓冲液洗涤3次，重悬后超声破碎，破碎条件为300W，1h。4℃、10 000r/min离心2min取上清液进行酶活测定，通过磷酸烯醇式丙酮酸羧化酶酶活检测试剂盒测定酶活，结果如表1所示。与对照菌株C.glutamicum H5相比，重组菌株H5-Vpepc1、H5-Vpepc2、H5-Vpepc3、H5-Vpepc12、H5-Vpepc13、H5-Vpepc23、H5-Vpepc123酶活均有不同程度的提高，尤其是H5-Vpepc23最终酶活提高了7.68倍。与对照菌株C.glutamicum H1相比，重组菌株H1-Vpepc1、H1-Vpepc2、H1-Vpepc3、H1-Vpepc12、H1-Vpepc13、H1-Vpepc23、H1-Vpepc123酶活也均有不同程度的提高，H1-Vpepc23酶活提高效果最为显著，最终酶活提高了7.49倍。The control strains C.glutamicum H5 and C.glutamicum H1 and the above-mentioned recombinant Corynebacterium glutamicum were inoculated into isoleucine and arginine fermentation medium respectively, and after culturing at 30 °C for 36 h, the fermentation broth was taken, and the fermentation broth was taken at 4 °C for 10 000 r. Cells were collected by centrifugation at /min for 2 min, washed twice with 0.1 mol HCL, washed 3 times with pH 7.5, 0.1 mol/L phosphate buffer, resuspended and sonicated at 300 W for 1 h. Centrifuge at 4°C and 10 000 r/min for 2 min to take the supernatant for enzyme activity assay, and measure the enzyme activity by phosphoenolpyruvate carboxylase enzyme activity detection kit. Compared with the control strain C.glutamicum H5, the enzyme activities of recombinant strains H5-Vpepc1, H5-Vpepc2, H5-Vpepc3, H5-Vpepc12, H5-Vpepc13, H5-Vpepc23, H5-Vpepc123 were improved to varying degrees, especially The final enzyme activity of H5-Vpepc23 was increased by 7.68 times. Compared with the control strain C.glutamicum H1, the enzyme activities of recombinant strains H1-Vpepc1, H1-Vpepc2, H1-Vpepc3, H1-Vpepc12, H1-Vpepc13, H1-Vpepc23, H1-Vpepc123 were also improved to varying degrees, and H1 - The enzyme activity of Vpepc23 has the most significant improvement effect, and the final enzyme activity is increased by 7.49 times.
表2谷氨酸棒状杆菌突变菌株酶活测定Table 2 Determination of enzyme activity of mutant strains of Corynebacterium glutamicum
注：C.glutamicum H5于2016年11月1日保藏在中国典型培养物保藏中心(地址：中国 武汉 武汉大学)，菌种名称为谷氨酸棒杆菌Corynebacterium glutamicum，株号为H5，保藏编号为CCTCC NO：M2016609。Note: C.glutamicum H5 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on November 1, 2016. The strain name is Corynebacterium glutamicum, the strain number is H5, and the deposit number is CCTCC NO: M2016609.
C.glutamicum H1于2020年10月26日保藏在中国典型培养物保藏中心(地址：中国 武汉 武汉大 学)，菌种名称为谷氨酸棒杆菌H1，分类号为Corynebacterium glutamicum H1，保藏编号为CCTCC NO：M 2020644。C.glutamicum H1 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on October 26, 2020. The strain name is Corynebacterium glutamicum H1, the classification number is Corynebacterium glutamicum H1, and the deposit number is CCTCC NO: M2020644.
实施例7Example 7
以菌株C.glutamicum H5为对照，将重组菌H5-Vpepc1、H5-Vpepc2、H5-Vpepc3、H5-Vpepc12、H5-Vpepc13、H5-Vpepc23、H5-Vpepc123装入5L发酵罐进行培养。分别取对照菌和重组菌的甘油菌种1ml固态活化培养基上划线，30℃培养16h；从活化培养基上挑一接种环菌苔转接到例如100ml种子培养基中，30℃、200rpm振荡培养12～16h。按10％的体积比接种种子至含有3L发酵培养基的5L发酵罐中，控温30℃，氨水控制pH在7.0，通过调整转速及通气量维持溶氧在30％，当残糖含量低于1.5％时，流加80％的葡萄糖，维持残糖含量在1.5～2.5％，发酵时间为55h以上，本实施例发酵时间65h。发酵结束后，发酵液离心取上清衍生化后，使用HPLC对各主要氨基酸进行分析其实验结果统计如表3所示，结果表明增强PEPC酶活性可显著改变天冬氨酸族氨基酸和谷氨酸族氨基酸产量。与对照菌株相比，重组菌的天冬氨酸族氨基酸和谷氨酸族氨基酸产量都有不同程度的提高，重组菌H5-Vpepc23效果最为明显，天冬氨酸族氨基酸总产量由之前的46.5g/L提高到54.94g/L，提高了18.15％，特别是异亮氨酸，产量由之前的38.83g/L提高到42.95g/L，提高了10.61％；谷氨酸族氨基酸由之前的3.81g/L提高到9.48g/L，提高了1.49倍。Taking the strain C.glutamicum H5 as a control, the recombinant strains H5-Vpepc1, H5-Vpepc2, H5-Vpepc3, H5-Vpepc12, H5-Vpepc13, H5-Vpepc23, H5-Vpepc123 were put into a 5L fermenter for cultivation. Take 1ml of solid-state activated medium of glycerol strains of control bacteria and recombinant bacteria to streak, and cultivate at 30°C for 16h; pick an inoculated loop from the activated medium and transfer it to, for example, 100ml of seed medium, at 30°C, 200rpm Shake culture for 12-16h. The seeds were inoculated into a 5L fermentation tank containing 3L fermentation medium at a volume ratio of 10%, the temperature was controlled at 30°C, the pH of the ammonia water was controlled at 7.0, and the dissolved oxygen was maintained at 30% by adjusting the rotational speed and ventilation rate. When the residual sugar content was lower than At 1.5%, 80% glucose was added in flow to maintain the residual sugar content at 1.5-2.5%, and the fermentation time was more than 55h, and the fermentation time in this example was 65h. After the fermentation, the fermentation broth was centrifuged to take the supernatant for derivatization, and HPLC was used to analyze the main amino acids. The statistics of the experimental results are shown in Table 3. The results show that enhancing the activity of PEPC can significantly change the aspartic amino acids and glutamine. Acid group amino acid production. Compared with the control strain, the yields of aspartate amino acids and glutamic acid amino acids of the recombinant strains were improved to varying degrees, and the recombinant strain H5-Vpepc23 had the most obvious effect. g/L increased to 54.94g/L, an increase of 18.15%, especially for isoleucine, the yield increased from 38.83g/L to 42.95g/L, an increase of 10.61%; 3.81g/L increased to 9.48g/L, an increase of 1.49 times.
表3发酵液中的氨基酸分析Amino acid analysis in table 3 fermentation broth
实施例8Example 8
以菌株C.glutamicum H1为对照，将重组菌H1-Vpepc1、H1-Vpepc2、H1-Vpepc3、H1-Vpepc12、H1-Vpepc13、H1-Vpepc23、H1-Vpepc123装入5L发酵罐进行培养。从平板上挑取单菌落接种至种子培养基中，于温度为32℃、转速为200r/min的条件下摇床培养12h，得到种子液；按照5％接种量，将种子培养液进行合并至补料瓶中，连接补料管道，将种子培养液接种至发酵培养基中，开始发酵培养。控温30℃，罐压：0.03～0.08Mpa，氨水控制pH在7.0，通过调整转速及通气量维持溶氧在30％，当残糖含量低于3.0％时，流加80％的葡萄糖，维持残糖含量在1.5～3.0％，发酵时间为72h以上。发酵结束后，发酵液离心取上清衍生化后，使用HPLC对各主要氨基酸进行分析，其实验结果统计如表4所示，结果表明增强PEPC酶活性可显著改变天冬氨酸族氨基酸和谷氨酸族氨基酸的碳通量。与对照菌株相比，重组菌的天冬氨酸族氨基酸和谷氨酸族氨基酸产量都有不同程度的提高，重组菌H1-Vpepc23效果最为明显，天冬氨酸族氨基酸总产量由之前的21.02g/L提高到33.89g/L，提高了61.23％，；谷氨酸族氨基酸由之前的98.61g/L提高到127.94g/L，提高了29.74％，特别是精氨酸，产量由之前的66.83g/L提高到83.67g/L，提高了25.20％。Taking the strain C.glutamicum H1 as the control, the recombinant strains H1-Vpepc1, H1-Vpepc2, H1-Vpepc3, H1-Vpepc12, H1-Vpepc13, H1-Vpepc23, H1-Vpepc123 were put into a 5L fermenter for cultivation. Pick a single colony from the plate and inoculate it into the seed medium, and inoculate it with a shaker for 12 hours at a temperature of 32 °C and a rotation speed of 200 r/min to obtain seed liquid; In the feeding bottle, connect the feeding pipeline, inoculate the seed culture liquid into the fermentation medium, and start the fermentation culture. Temperature control 30℃, tank pressure: 0.03～0.08Mpa, ammonia water is controlled at pH 7.0, dissolved oxygen is maintained at 30% by adjusting rotational speed and ventilation volume, when the residual sugar content is lower than 3.0%, 80% glucose is added to maintain The residual sugar content is 1.5-3.0%, and the fermentation time is more than 72h. After the fermentation, the fermentation broth was centrifuged to obtain the supernatant for derivatization, and HPLC was used to analyze the main amino acids. The statistics of the experimental results are shown in Table 4. The results show that enhancing the activity of PEPC can significantly change the aspartic amino acids and glutamic acid. Carbon fluxes of amino acids in the amino acid family. Compared with the control strain, the yields of aspartate amino acids and glutamic acid amino acids of the recombinant strains were improved to varying degrees, and the recombinant strain H1-Vpepc23 had the most obvious effect. g/L increased to 33.89g/L, an increase of 61.23%; glutamic acid group amino acids increased from 98.61g/L to 127.94g/L, an increase of 29.74%, especially arginine, the yield increased from the previous 98.61g/L to 127.94g/L 66.83g/L increased to 83.67g/L, an increase of 25.20%.
表4氨基酸产量Table 4 Amino acid production
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一 个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Embodiments are subject to variations, modifications, substitutions and variations.
Claims (25)
- 一种改造的磷酸烯醇丙酮酸羧化酶，其特征在于，与野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列相比，所述改造的磷酸烯醇丙酮酸羧化酶的氨基酸序列中的第771位和/或第931位和/或第943位氨基酸被取代；A modified phosphoenolpyruvate carboxylase, characterized in that, compared with the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase, in the amino acid sequence of the modified phosphoenolpyruvate carboxylase amino acids at positions 771 and/or 931 and/or 943 are substituted;所述野生型磷酸烯醇丙酮酸羧化酶具有如SEQ ID NO：1所示的氨基酸序列或者具有与SEQ ID NO：1所示的氨基酸序列具有至少80％同源性的氨基酸序列。The wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1.
- 根据权利要求1所述的改造的磷酸烯醇丙酮酸羧化酶，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶来源于植物。The modified phosphoenolpyruvate carboxylase according to claim 1, wherein the wild-type phosphoenolpyruvate carboxylase is derived from a plant.
- 根据权利要求1所述的改造的磷酸烯醇丙酮酸羧化酶，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶来源于高粱。The modified phosphoenolpyruvate carboxylase according to claim 1, wherein the wild-type phosphoenolpyruvate carboxylase is derived from sorghum.
- 根据权利要求1所述的改造的磷酸烯醇丙酮酸羧化酶，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代和/或第931位赖氨酸被谷氨酰胺所取代和/或第943位天冬氨酸被天冬酰胺所取代。The modified phosphoenolpyruvate carboxylase according to claim 1, wherein the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and/or Either lysine at position 931 is replaced by glutamine and/or aspartic acid at position 943 is replaced by asparagine.
- 根据权利要求1所述的改造的磷酸烯醇丙酮酸羧化酶，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第931位赖氨酸被谷氨酰胺所取代和第943位天冬氨酸被天冬酰胺所取代。The modified phosphoenolpyruvate carboxylase according to claim 1, wherein the 931st lysine in the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is replaced by glutamine and aspartic acid at position 943 was replaced by asparagine.
- 一种编码改造的磷酸烯醇丙酮酸羧化酶的核酸，其特征在于，与编码野生型磷酸烯醇丙酮酸羧化酶的核苷酸序列相比，所述编码改造的磷酸烯醇丙酮酸羧化酶的核苷酸序列预先经过密码子优化，且优化后的核苷酸序列的第2312位碱基、第2791位碱基和/或第2827位碱基具有突变；A nucleic acid encoding a modified phosphoenolpyruvate carboxylase, characterized in that, compared with a nucleotide sequence encoding a wild-type phosphoenolpyruvate carboxylase, the modified phosphoenolpyruvate encoding The nucleotide sequence of the carboxylase is codon-optimized in advance, and the optimized nucleotide sequence has mutations at the 2312th base, the 2791st base and/or the 2827th base;所述编码野生型磷酸烯醇丙酮酸羧化酶的核苷酸序列具有如SEQ ID NO：2所示的核苷酸序列或者具有与SEQ ID NO：2所示的核苷酸序列具有至少80％同源性的核苷酸序列。The nucleotide sequence encoding wild-type phosphoenolpyruvate carboxylase has the nucleotide sequence shown in SEQ ID NO: 2 or has at least 80 nucleotides with the nucleotide sequence shown in SEQ ID NO: 2 % Homology of Nucleotide Sequences.
- 根据权利要求6所述的核酸，其特征在于，所述优化后的核苷酸序列具有如SEQ ID NO：3所示的核苷酸序列或者具有与SEQ ID NO：3所示的核苷酸序列具有至少80％同源性的核苷酸序列。The nucleic acid according to claim 6, wherein the optimized nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence shown in SEQ ID NO: 3 Sequences have at least 80% homology to nucleotide sequences.
- 根据权利要求6所述的核酸，其特征在于，所述优化后的核苷酸序列具有c.2312C＞A、c.2791C＞A和/或c.2827G＞A的突变。The nucleic acid according to claim 6, wherein the optimized nucleotide sequence has mutations of c.2312C>A, c.2791C>A and/or c.2827G>A.
- 根据权利要求6所述的核酸，其特征在于，所述优化后的核苷酸序列具有c.2791C＞A和c.2827G＞A突变。The nucleic acid according to claim 6, wherein the optimized nucleotide sequence has mutations c.2791C>A and c.2827G>A.
- 一种重组表达载体，其特征在于，所述重组表达载体选自含有权利要求6～9任一 项所述核酸的表达载体。A recombinant expression vector, characterized in that the recombinant expression vector is selected from the expression vector containing the nucleic acid of any one of claims 6-9.
- 根据权利要求10所述的重组表达载体，其特征在于，所述表达载体选自大肠杆菌-谷氨酸棒杆菌穿梭表达载体。The recombinant expression vector according to claim 10, wherein the expression vector is selected from Escherichia coli-Corynebacterium glutamicum shuttle expression vector.
- 一种基因工程菌，其特征在于，所述基因工程菌是通过将权利要求10或11所述重组表达载体转化到受体菌内所获得的。A genetically engineered bacterium, characterized in that, the genetically engineered bacterium is obtained by transforming the recombinant expression vector of claim 10 or 11 into a recipient bacterium.
- 根据权利要求12所述的基因工程菌，其特征在于，所述受体菌选自谷氨酸棒杆菌。The genetically engineered bacterium according to claim 12, wherein the recipient bacterium is selected from Corynebacterium glutamicum.
- 一种试剂盒，其特征在于，包括权利要求10或11所述重组表达载体或权利要求12或13所述基因工程菌。A kit, characterized by comprising the recombinant expression vector of claim 10 or 11 or the genetically engineered bacteria of claim 12 or 13.
- 一种获得改造的磷酸烯醇丙酮酸羧化酶的方法，其特征在于，包括：A method for obtaining a modified phosphoenolpyruvate carboxylase, comprising:将野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列中的第771位、第931位和/或第943位氨基酸进行突变，得到所述改造的磷酸烯醇丙酮酸羧化酶；Mutating amino acids at positions 771, 931 and/or 943 in the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase to obtain the modified phosphoenolpyruvate carboxylase;所述野生型磷酸烯醇丙酮酸羧化酶具有如SEQ ID NO：1所示的氨基酸序列或者具有与SEQ ID NO：1所示的氨基酸序列具有至少80％同源性的氨基酸序列。The wild-type phosphoenolpyruvate carboxylase has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1.
- 根据权利要求15所述的方法，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶来源于植物。The method of claim 15, wherein the wild-type phosphoenolpyruvate carboxylase is derived from a plant.
- 根据权利要求15所述的方法，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶来源于高粱。The method of claim 15, wherein the wild-type phosphoenolpyruvate carboxylase is derived from sorghum.
- 根据权利要求15所述的方法，其特征在于，所述野生型磷酸烯醇丙酮酸羧化酶的氨基酸序列的第771位丝氨酸被酪氨酸所取代、第931位赖氨酸被谷氨酰胺所取代和/或第943位天冬氨酸被天冬酰胺所取代。The method according to claim 15, wherein the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase is substituted by tyrosine at position 771 and lysine at position 931 by glutamine Substituted and/or aspartic acid at position 943 is replaced by asparagine.
- 权利要求1～5任一项所述改造的磷酸烯醇丙酮酸羧化酶、权利要求6～9任一项所述编码改造的磷酸烯醇丙酮酸羧化酶的核酸、权利要求10或11所述重组表达载体、权利要求12或13所述基因工程菌在提高氨基酸产量中的应用。The modified phosphoenolpyruvate carboxylase according to any one of claims 1 to 5, the nucleic acid encoding the modified phosphoenolpyruvate carboxylase according to any one of claims 6 to 9, and claims 10 or 11 Application of the recombinant expression vector and the genetically engineered bacteria of claim 12 or 13 in improving amino acid production.
- 一种提高谷氨酸棒杆菌氨基酸产量的方法，其特征在于，包括：将权利要求10或11所述重组表达载体转化到谷氨酸棒杆菌中，并对转化后的基因工程菌进行发酵培养。A method for improving the amino acid yield of Corynebacterium glutamicum, comprising: transforming the recombinant expression vector described in claim 10 or 11 into Corynebacterium glutamicum, and fermenting and culturing the transformed genetically engineered bacteria .
- 根据权利要求20所述的方法，其特征在于，所述氨基酸包括天冬氨酸族氨基酸和/或谷氨酸族氨基酸。The method of claim 20, wherein the amino acids comprise aspartic amino acids and/or glutamic amino acids.
- 根据权利要求20所述的方法，其特征在于，所述天冬氨酸族氨基酸包括天冬氨酸、赖氨酸、苏氨酸、甲硫氨酸和/或异亮氨酸；The method of claim 20, wherein the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine;所述谷氨酸族氨基酸包括谷氨酸、谷氨酰胺、脯氨酸和/或精氨酸。The glutamic amino acids include glutamic acid, glutamine, proline and/or arginine.
- 一种生产氨基酸的方法，其特征在于，包括：将权利要求12或13所述基因工程 菌进行发酵培养。A method for producing amino acids, comprising: fermenting and culturing the genetically engineered bacteria of claim 12 or 13.
- 根据权利要求23所述的方法，其特征在于，所述氨基酸包括天冬氨酸族氨基酸和/或谷氨酸族氨基酸。The method of claim 23, wherein the amino acids comprise aspartic amino acids and/or glutamic amino acids.
- 根据权利要求23所述的方法，其特征在于，所述天冬氨酸族氨基酸包括天冬氨酸、赖氨酸、苏氨酸、甲硫氨酸和/或异亮氨酸；The method of claim 23, wherein the aspartic amino acids include aspartic acid, lysine, threonine, methionine and/or isoleucine;所述谷氨酸族氨基酸包括谷氨酸、谷氨酰胺、脯氨酸和/或精氨酸。The glutamic amino acids include glutamic acid, glutamine, proline and/or arginine.
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| EP2495317A1 (en) * | 2011-03-01 | 2012-09-05 | Technische Universität Hamburg-Harburg | Modified phosphoenolpyruvate carboxylase from Corynebacterium glutamicum and uses 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 |
| CN111206011A (en) * | 2020-03-05 | 2020-05-29 | 江南大学 | Recombinant corynebacterium glutamicum and application thereof in production of L-glutamic acid |
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2020
- 2020-12-24 WO PCT/CN2020/139053 patent/WO2022133917A1/en active Application Filing
- 2020-12-24 CN CN202080105049.7A patent/CN116113699A/en active Pending
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| EP2495317A1 (en) * | 2011-03-01 | 2012-09-05 | Technische Universität Hamburg-Harburg | Modified phosphoenolpyruvate carboxylase from Corynebacterium glutamicum and uses 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 |
| CN111206011A (en) * | 2020-03-05 | 2020-05-29 | 江南大学 | Recombinant corynebacterium glutamicum and application thereof in production of L-glutamic acid |
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