CN114990138B - Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield - Google Patents

Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield Download PDF

Info

Publication number
CN114990138B
CN114990138B CN202210681569.3A CN202210681569A CN114990138B CN 114990138 B CN114990138 B CN 114990138B CN 202210681569 A CN202210681569 A CN 202210681569A CN 114990138 B CN114990138 B CN 114990138B
Authority
CN
China
Prior art keywords
codon sequence
codon
gene
sequence
pacgapdh
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210681569.3A
Other languages
Chinese (zh)
Other versions
CN114990138A (en
Inventor
梁恒宇
韩超
幸志伟
李雪亮
陈民良
周鹏
张敏
郭慧萍
杨梦园
侯剑桥
李喆
张皓
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan Jiankangyuan Biomedical Research Institute Co ltd
Original Assignee
Henan Jiankangyuan Biomedical Research Institute Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan Jiankangyuan Biomedical Research Institute Co ltd filed Critical Henan Jiankangyuan Biomedical Research Institute Co ltd
Priority to CN202210681569.3A priority Critical patent/CN114990138B/en
Publication of CN114990138A publication Critical patent/CN114990138A/en
Application granted granted Critical
Publication of CN114990138B publication Critical patent/CN114990138B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/06—Cephalosporin C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03011—L-Glutamate oxidase (1.4.3.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a gene, a vector, a strain and application of over-expression exogenous L-glutamate hydrogenase, wherein the gene is named as KclgoX, and the nucleotide sequence is shown as SEQ ID NO. 3; an expression vector for over-expressing exogenous L-glutamate hydrogenase gene, wherein the expression vector is pBARGPE 1-PAcGapdh-KclgoX; the expression vector is transformed into the Acremonium chrysogenum to form a strain, and the strain is applied to the cephalosporin C with high yield, so that the yield of the cephalosporin C is improved.

Description

Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a gene, an expression vector, a strain and application thereof in improving the yield of cephalosporin C.
Background
Currently, several decades of research have passed to essentially elucidate the enzyme-catalyzed reaction pathways in which the key genes and their encoded products are involved in the biosynthetic pathway of β -lactam antibiotics such as penicillin, cephalosporin C, and cephalosporin C. Especially in recent years, the completion of the whole genome sequence sequencing of the beta-lactam antibiotic-producing strain patulin (Penicillium chrysogenum), streptomyces clavuligerus (Streptomyces clavuligerus) and acremonium chrysogenum (Acremonium chrysogenum) and the completion of the systematic biological work of transcriptome, proteome, metabolome and the like of the beta-lactam antibiotic-producing strain greatly promote people to realize the biosynthesis and regulation of the beta-lactam antibiotic. Penicillin, cephalosporin C and cephalosporin C are typically non-ribosomal peptide compounds, and precursors involved in their synthesis include L-alpha-aminoadipic acid (L-alpha-AAA), L-cysteine and L-valine. The 3 precursor amino acids are condensed catalytically by ACV Synthase (ACVs) in the presence of ATP to form delta- (L-aminoadipyl) -L-cysteinyl-D-valine (LLD-ACV) tripeptide, which is then cyclized to form isopenicillin N (IPN) by cyclase.
The synthesis of β -lactam antibiotics such as penicillin, cephalosporin C and cephalosporin C is related to the supply of precursor substances. The yield of beta-lactam antibiotics such as cephalosporin C, cephalosporin C and the like can be remarkably increased by increasing the content of L-alpha-AAA, L-cysteine and L-valine in cells. Studies have shown that the concentration of L-alpha-AAA in A. Chrysogenum cells is directly related to the production of cephalosporin C, and that increasing the supply of L-alpha-AAA precursor increases the rate of cephalosporin C synthesis.
Unlike bacteria, the L-alpha-AAA in fungi is formed by condensing alpha-ketoglutarate with acetyl-CoA and through the homocitric, homoaconitic, homoisocitric, ketoadipic acid pathways, and is an intermediate metabolite for the formation of L-lysine. As early as the sixties of the last century, scientists have demonstrated that α -ketoglutarate is a precursor material for the formation of L- α -aminoadipic acid. It is further proved that alpha-ketoglutarate is also an important cofactor when a further important enzyme in the CPC biosynthesis pathway, namely desacetoxycephalosporin C synthetase (expandase), catalyzes five-membered thiazole ring to expand into six-membered thiazine ring. Studies have shown that the addition of alpha-ketoglutarate can effectively increase the production of cephalosporin C and cephalosporin C.
Alpha-ketoglutarate (alpha-KG) is a dicarboxylic acid which is important in the tricarboxylic acid cycle (TCA) and in the metabolism of amino acids. In the cell, isocitrate is oxidized and decarboxylated by isocitrate dehydrogenase and oxidized to succinyl-CoA by alpha-ketoglutarate dehydrogenase. Alpha-ketoglutarate can also deaminate L-glutamic acid by glutamate dehydrogenase, is a precursor of glutamic acid, succinic acid and heterocyclic compounds, and plays an important role in metabolism of biological organisms.
From the 70 s of the 20 th century, jurtchuk et al, in the study of the activity of azotobacter vinelandii, found an enzyme capable of oxidizing the substrate glutamate, designated L-glutamate oxidase (L-glutamate oxidase, GLOD). L-glutamate oxidase is a flavoproteinase with flavin adenine dinucleotide (flavin adenine dinucleotide, FAD) as prosthetic group, and can specifically oxidize L-glutamate to generate hydrogen peroxide, ammonia and alpha-ketoglutarate under the condition of no exogenous cofactor. L-glutamate oxidase mainly exists in streptomycete, has high specificity and high affinity to a reaction substrate, and has mild reaction conditions and high catalytic efficiency, so that the L-glutamate oxidase has wide application in food, industrial fermentation and pharmaceutical industries. The enzyme is discovered in the 80 s of the 20 th century, is a research hotspot at home and abroad, and is one of potential tool enzymes. However, L-glutamate oxidase is used as a new practical tool enzyme, so that the research on the yield of secondary metabolites such as beta-lactam antibiotics by heterologous expression of the enzyme has not been reported at present, and in addition, the research on the action mechanism and application of the enzyme in production strains such as cephalosporin C and the like is also rarely reported.
The proper addition of alpha-ketoglutarate to the fermentation medium of penicillin, cephalosporin C and cephalosporin C species can significantly enhance the yield of the above-mentioned beta-lactam antibiotics, however, in practical industrial production, the additional addition of alpha-ketoglutarate to the fermentation medium is quite uneconomical. Therefore, there is a need to develop a strain engineering strategy that enables the production of alpha-ketoglutarate as a precursor substance by the production strain autonomously during fermentation. By over-expressing heterologous L-glutamate oxidase into the cephalosporin C production strain, the yield of the cephalosporin C of the acremonium chrysogenum can be effectively improved.
Therefore, how to provide a gene, vector and strain that can overexpress heterologous L-glutamate oxidase is a problem that those skilled in the art are urgent to solve.
Disclosure of Invention
In view of the above, the present invention provides a gene, vector and strain for over-expressing heterologous L-glutamate oxidase, and the fermentation of the constructed strain to produce cephalosporin C, which improves the yield of cephalosporin C.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a gene for over-expressing exogenous L-glutamate hydrogenase, named KclgoX, is characterized in that the nucleotide sequence is shown in SEQ ID NO.3.
According to the technical scheme, the gene is obtained by codon optimization of a wild type gene, wherein the nucleotide sequence of the wild type gene is shown as SEQ ID NO. 1; the protein sequence is shown as SEQ ID NO.2.
As a preferred embodiment of the above-described method, the codon usage is optimized to be TTC for alanine codon usage, TGC for cysteine codon usage, GAC for aspartic acid codon usage, GAG for glutamic acid codon usage, TTC for phenylalanine codon usage, GGC for glycine codon usage, CAC for histidine codon usage, ATC for isoleucine codon usage, AAG for lysine codon usage, CTC for leucine codon usage, AAC for asparagine codon usage, CCC for proline codon usage, CAG for glutamine codon usage, CGC for arginine codon usage, AGC for serine codon usage, ACC for threonine codon usage, GTC for valine codon usage, TAA for tyrosine codon usage.
As the same inventive concept as the above technical scheme, the invention also claims an expression vector for over-expressing exogenous L-glutamate hydrogenase gene, wherein the expression vector is pBARGPE 1-PAcGapdh-KclgoX.
As a preferred technical scheme, a pBARGPE1-PAcGapdh plasmid is formed by connecting a PAcGapdh promoter and a pBARGPE1-hygro linear fragment, and then a KclgoX gene and the pBARGPE1-PAcGapdh linear fragment are connected to obtain the pBARGPE 1-PAcGapdh-KclgoX.
As the invention concept same as the technical scheme, the invention also claims the application of the expression vector in constructing the high-yield cephalosporin C strain.
As the same inventive concept as the above technical scheme, the invention also claims a strain with high cephalosporin C production, which is characterized in that the strain is obtained by converting pBARGPE1-PAcGapdh-KclgoX into Acremonium chrysogenum.
As the same invention conception as the technical scheme, the invention also claims the application of the strain in fermenting the cephalosporin C with high yield.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the electrophoresis of BamHI-BglII double digested fragments of pBARGPE1-hygro plasmid, pBARGPE1-hygro circular plasmid and PAcGapdh promoter; m: d15000 A Marker; line1: the pBARGPE1-hygro plasmid is digested with BamHI-BglII; line2: pBARGPE1-hygro circular plasmid; line3: PCR amplified fragment of PAcGapdh promoter
FIG. 2 is a diagram showing the KclgoX gene; linear plasmid after single digestion of pBARGPE1-PAcGapdh BamHI, pBARGPE1-PAcGapdh circular plasmid before single digestion, pBARGPE1-hygro circular plasmid electrophoretogram; m: d15000 A Marker; line1: kclgoX gene; line2: a linear plasmid after single cleavage of pBARGPE1-PAcGapdh BamHI; line3: pBARGPE1-PAcGapdh circular plasmid before single enzyme cutting; line4: pBARGPE1-hygro circular plasmid;
FIG. 3 is a schematic representation of the pBARGPE1-PAcGapdh-KclgoX circular plasmid, pBARGPE1-PacGapdh circular plasmid electrophoresis; wherein M: d15000 A Marker; l1 to L20: pBARGPE1-PAcGapdh-KclgoX circular plasmid; l21: pBARGPE1-PacGapdh circular plasmid;
FIG. 4 is a complete program diagram of pBARGPE1-PAcGapdh-KclgoX plasmid construction;
FIG. 5 is a graph showing the standard curve of cephalosporin C (CPC) by HPLC;
FIG. 6 is a graph showing the comparison of typical HPLC chromatograms of CGMCC3.3795-pBARGPE1-KclgoX, CGMCC3.3795-pBARGPE1 and the wild strain CGMCC3.3795 cephalosporin C (CPC).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Samples and sources used in the examples:
strains and plasmids: coli DH 5. Alpha. From Japanese Takara; the strain for over-expressing glutamate oxidase gene is Acremonium chrysogenum (Acremonium chrysogenum) CGMCC3.3795, and is collected in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC). Plasmid pBARGPE1-Hygro is a universal filamentous fungus integrated gene overexpression plasmid, purchased from vast Prod plasmid platform (cat# P0381). Construction procedures for plasmids pBARGPE1-PAcGapdh and pBARGPE1-PAcGapdh-KclgoX are described in the literature (Zhangyan et al, cloning and application of the Acremonium chrysogenum pcb AB-pcbC bidirectional promoter region [ J ], microbiology report, 2004, 44 (2): 255-257; song Jia. Preliminary investigation of Aspergillus niger ras gene function [ D ]. Harbin university of Industrial, 2018).
Enzymes and reagents: restriction enzymes BamHI (cleavage site: 5' -G ∈ GATCC-3 '), restriction enzymes Bgl II (cleavage site: 5' -A ∈ GATCT-3 '), primeSTAR Hizikiase, mighty Mix ligase, cloning vector pET-30a (+), DL 2000DNA Marker, primeScript RT Reagent Kit, SYBRPremix Ex Taq, etc. were all purchased from Takara, UNIQ-10 column Trizol total RNA extraction kit, DNase I, RNase Inhibitor, etc. from biological engineering (Shanghai) Co., lysing Enzymes from Trichoderma harzianum from Sigma, a large number of plasmid extraction kits were purchased from MN, D7010M Seamless Cloning Kit seamless cloning kit was purchased from Shanghai Biyun biotechnology Co., fungal genome DNA extraction kit was purchased from Beijing Soy technology Co., CPC standard (98% purity) was purchased from Job's health care biological Co., ltd.
LB medium: tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH=7.4.
PDA medium: 200g/L of potato, 20g/L of glucose, 15g/L of agar and 30min of 121 ℃.
CPC seed medium: glucose 5g/L, sucrose 35g/L, corn steep liquor 10mL/L, ammonium sulfate 8g/L, DL-methionine 0.5g/L, calcium carbonate 5g/L, soybean oil 5mL/L.
CPC fermentation medium: 30g/L of starch, 60g/L of dextrin, 0.2g/L of alpha-amylase, 10mL/L of corn steep liquor, 6g/L of DL-methionine, 2g/L of urea, 11g/L of ammonium sulfate and magnesium sulfate (MgSO) 4 ·7H 2 O) 3g/L, dipotassium hydrogen phosphate 9g/L, calcium carbonate 5g/L and soybean oil 5mL/L.
EXAMPLE 1 extraction and sequencing of genomic DNA
The total genome DNA of Cephalosporium chrysogenum AC-J-001 (the above microorganism and genome sample are all provided by Coke health element biological products Co., ltd.) is extracted by using a D2300-50 fungus genome DNA extraction kit produced by Beijing Soxhaust technology Co., ltd. Taking about 100mg of mycelia of Cephalosporium chrysogenum AC-J-001 cultured to logarithmic growth phase, adding liquid nitrogen into a glass grinder to grind the dispersed mycelia appropriately, adding 200 mu L of solution A provided in a genome DNA extraction kit, adding 20 mu L of RNaseA prepared by the kit, adding 100mg of crystallized sterile glass beads, oscillating for about 30min on a high-speed oscillator, and extracting the total genome DNA of the microorganisms according to the steps provided by the kit.
The extracted genomic DNA meeting the sequencing standard is sequenced and analyzed by Guangdong Meiger Gene technology Co., ltd, and about 50ng of genomic sample is left as a template for subsequent target gene amplification. The result of the genome sequencing analysis shows that the genome of the Cephalosporium chrysogenum AC-J-001 is about 28.95Mbp.
Example 2 obtaining exogenous L-glutamic acid oxidase Gene
BLAST comparison analysis was performed on the Ji Yuanshan Northlining spore (Kitasatospora cheerisanensis) KCTC 2395 genome annotation file and the protein prediction file by literature investigation and NCBI database download and analysis, and the genome was determined to contain the L-glutamate oxidase gene, which was designated as KclgoX. The wild type DNA of the gene has the length of 1731bp (genome position: JNBY01000141.1: 48966-50696), the sequence of SEQ ID NO.1, and the gene synthesizes an L-glutamic acid oxidase (protein sequence number: KDN 81488.1) protein sequence SEQ ID NO.2 consisting of 576 amino acid residues.
In order to express the gene sequence in the A.chrysogenum CGMCC3.3795 more efficiently, the inventor carries out codon optimization on wild DNA in the streptomyces. In this example, alanine codon sequence is optimized to ttc, cysteine codon sequence is optimized to tgc, aspartic acid codon sequence is optimized to gac, glutamic acid codon sequence is optimized to gag, phenylalanine codon sequence is optimized to ttc, glycine codon sequence is optimized to ggc, histidine codon sequence is optimized to cac, isoleucine codon sequence is optimized to atc, lysine codon sequence is optimized to aag, leucine codon sequence is optimized to ctc, asparagine codon sequence is optimized to aac, proline codon sequence is optimized to ccc, glutamine codon sequence is optimized to cag, arginine codon sequence is optimized to cgc, serine codon sequence is optimized to agc, threonine codon sequence is optimized to acc, valine codon sequence is optimized to gtc, tyrosine codon sequence is optimized to taa. The optimized gene sequence is named as KclgoX and SEQ ID NO.3.
EXAMPLE 3pBARGPE1-hygro plasmid promoter engineering
In order to improve the expression efficiency of the exogenous gene on the genome of Acremonium chrysogenum, the inventor specially modifies the gpdA promoter on the pBARGPE1-hygro plasmid into the PAcGapdh promoter with higher homology with host bacteria. The inventors designed primers AcGU (SEQ ID NO. 4)/AcGD (SEQ ID NO. 5) to amplify the PAcGapdh promoter fragment using the Acremonium chrysogenum AC-J-001 genome as a template. The promoter sequence is SEQ ID NO.6, and has 2068bp total. The PCR reaction system comprises: 2×Max Master Mix,25 μL; primer AcGU, 0.1. Mu.L; primer AcGD,0.1 μl; AC-J-001gDNA, 1. Mu.L; ddH 2 O, up to 50. Mu.L. The PCR reaction conditions were: pre-denaturation at 98 ℃ for 3min for 1 cycle; denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 2min,34 cycles; the final extension was 72℃X 5min,1 cycle. The PCR amplified product PAcGapdh is subjected to agarose gel electrophoresis (as shown in figure 1), and the amplified fragment is recovered for later use.
The pBARGPE1-hygro plasmid obtained by purification is subjected to BamHI and Bgl II double enzyme digestion to prepare a linearization plasmid vector, and the reaction system is as follows: 80. Mu.L of pBARGPE1-hygro circular plasmid; bamHI, 8. Mu.L; bgl II,8 μl;10 XBuffer, 20. Mu.L; ddH 2 O, up to 200. Mu.L. The enzyme digestion system is placed in a water bath at 37 ℃ for one night, agarose gel electrophoresis is carried out, and then gel digestion recovery is carried out on an electrophoresis strip.
The thus recovered PacGapdh promoter and pBARGPE1-hygro linear fragment were ligated between BamHI and BglII cleavage sites using a recombinase system to form a novel circular plasmid designated pBARGPE1-PacGapdh (see FIG. 1). The recombinase ligation system includes: 10 ng to 100ng of PAcGapdh promoter sequence; linearizing plasmid vector, 50-100 ng;2X Seamless Cloning Mix,10. Mu.L; ddH 2 O，up to 200Mu L; the molar ratio of each insert to vector = 3:1. After the recombinase connection system is incubated in a water bath at 50 ℃ for 15min, heat shock is carried out for 90s in the water bath at 42 ℃ to be converted into competent cells of escherichia coli DH5 alpha, and the competent cells are rapidly placed on ice for cooling for 3-5 min after heat shock. 1mL of LB liquid medium (without Amp) was added to the tube, and after mixing, the mixture was cultured at 37℃for 1 hour. Shaking the bacterial liquid uniformly, taking 100 mu L of the bacterial liquid, coating the bacterial liquid on a resistance screening plate containing 50 mu g/mL Amp, standing the bacterial liquid for 30min in the front side, inverting a culture dish after the bacterial liquid is completely absorbed by a culture medium, and culturing the bacterial liquid at 37 ℃ for 16-24 h. After the plate culture is finished, selecting single colony, transferring 5mL of LB liquid culture medium containing 50 mug/mL Amp resistance, carrying out shaking culture at 200rpm overnight at 37 ℃, selecting single amplified culture plasmid, verifying, extracting plasmid after the connected circular plasmid is transformed into escherichia coli DH5 alpha, and carrying out sequencing verification.
EXAMPLE 4 construction of L-glutamate oxidase Gene overexpression plasmid vector pBARGPE1-PAcGapdh-KclgoX
This example entrusts the complete gene synthesis of plasmid DNA by Changzhou-based biotechnology Co., ltd, inserts the codon-optimized coding gene KclgoX of L-glutamate oxidase (L-GOX) shown in SEQ ID NO.3 into the plasmid obtained between NdeI/XhoI sites in E.coli pET-30a (+) vector, which was named pET30a-KclgoX, for subsequent manipulation as a DNA template for amplifying the L-glutamate oxidase protein gene.
A pair of primers KcGOU (SEQ ID NO. 7)/KcGOD (SEQ ID NO. 8) is designed, and a pET30a-KclgoX plasmid is used as a template to PCR amplify the gene fragment encoding the L-glutamate oxidase. The PCR reaction system comprises:max Master Mix,25 μL; primer KcGOU, 0.1. Mu.L; primer KcGOD, 0.1. Mu.L; pET30a-KcLGOX plasmid DNA, 1. Mu.L; ddH 2 O, up to 50. Mu.L. The PCR reaction conditions were: pre-denaturation at 98 ℃ for 3min for 1 cycle; denaturation at 98℃for 10s, annealing at 57℃for 15s, extension at 72℃for 2min,34 cycles; the final extension was 72℃X 5min,1 cycle. After the completion of the PCR reaction, the reaction product was recovered (as shown in FIG. 2).
The recovered KclgoX gene anda linear fragment of pBARGPE1-PAcGapdh (shown in figure 2) is connected to a BamHI site of a pBARGPE1-PAcGapdh vector by utilizing a recombinase to construct a gene integration type over-expression plasmid of an exogenous L-glutamate oxidase (LGOX) encoding gene KclgoX of Acremonium chrysogenum, which is named as pBARGPE 1-PacGapdh-KclgoX. The recombinase ligation system includes: kclgoX gene fragment, 10-100 ng; linearizing pBARGPE1-PAcGapdh plasmid vector, 50-100 ng;2X Seamless Cloning Mix,10. Mu.L; ddH 2 O, up to 200. Mu.L; the molar ratio of each insert to vector = 3:1.
After the recombinase connection system is incubated in a water bath at 50 ℃ for 15min, heat shock is carried out for 90s in the water bath at 42 ℃ to be converted into competent cells of escherichia coli DH5 alpha, and the competent cells are rapidly placed on ice for cooling for 3-5 min after heat shock. 1mL of LB liquid medium (without Amp) was added to the tube, and after mixing, the mixture was cultured at 37℃for 1 hour. Shaking the bacterial liquid uniformly, taking 100 mu L of the bacterial liquid, coating the bacterial liquid on a resistance screening plate containing 50 mu g/mL Amp, standing the bacterial liquid for 30min in the front side, inverting a culture dish after the bacterial liquid is completely absorbed by a culture medium, and culturing the bacterial liquid at 37 ℃ for 16-24 h. After the plate culture is finished, selecting single colony, transferring 5mL of LB liquid culture medium containing 50 mug/mL Amp resistance, carrying out shaking culture at 200rpm overnight at 37 ℃, selecting single amplified culture plasmid, verifying, extracting plasmid after the connected circular plasmid is transformed into escherichia coli DH5 alpha, and carrying out sequencing verification. After verification, the plasmid was identified as pBARGPE1-PAcGapdh-KclgoX (shown in FIG. 3), and the complete procedure for plasmid construction is shown in FIG. 4.
Example 5 construction of a Acremonium chrysogenum strain overexpressing the heterologous L-glutamate oxidase Gene lgoX culture and protoplast preparation of Acremonium chrysogenum CGMCC3.3795 mycelium:
(1) Scraping appropriate amount of Acremonium chrysogenum CGMCC3.3795 spores from the culture inclined plane, respectively inoculating into 100mL YPS liquid culture medium (glucose 20g/L, yeast extract 5g/L, polypeptone 10g/L, mgSO) 4 ·7H 2 O 1g/L，K 2 HPO 4 ·3H 2 O1.3 g/L, ph=7.0), at 28 ℃, shaking culture at 230rpm for 4-5 d;
(2) After the cultivation is finished, the mycelia are collected by centrifugation at 8000rpm for 15min, and the supernatant is removed by centrifugation and washed once by sterile water;
(3) 50mL of dithiothreitol (DTT, 5 mmol/L) solution is filtered by a sterile filter membrane with the thickness of 0.22 mu m, and the solution is incubated for 40 to 60 minutes at 30 ℃ under shaking at 150 rpm;
(4) Centrifuging at 8000rpm for 5min after incubation, and adding P Buffer (KCl 44.7g/L, mgCl) 2 ·6H 2 O 2.03g/L，CaCl 2 2.78 g/L) for 2 times at normal temperature;
(5) 60mL of lying enzymolysis liquid (prepared by P Buffer and 10 mg/mL) filtered by a sterile filter membrane with the thickness of 0.22 mu m is added, and the mixture is incubated for 3 to 4 hours at the temperature of 30 ℃ and the shaking speed of 150 rpm;
(6) Microscopic examination is carried out on the protoplast cell suspension after enzymolysis, after most mycelia in the visual field release protoplasts, 4 times of P Buffer is added, and the residual mycelia are removed by filtration through a sterilized needle cylinder filled with absorbent cotton;
(7) Centrifuging at 3000rpm for 5min, washing with P Buffer for 2 times, suspending protoplast in appropriate amount of PBbuffer to make protoplast concentration>10 8 CFU/mL；
(8) The protoplast of the Acremonium chrysogenum CGMCC3.3795 is subpackaged in 1.5mL centrifuge tubes with 100 mu L each.
EXAMPLE 6 plasmid transformation of pBARGPE1-PAcGapdh-KclgoX Acremonium chrysogenum protoplasts and identification
PEG-CaCl is adopted 2 The plasmid pBARGPE1-PAcGapdh-KclgoX and the empty plasmid vector pBARGPE1-PAcGapdh are respectively transformed into the prepared Cephalosporium chrysogenum CGMCC3.3795 protoplast by a mediated protoplast transformation method. The experimental procedure was as follows:
(1) 10 mug of plasmid DNA is added, gently mixed and ice-bathed for 30min;
(2) Add 900. Mu.L 30% PEG4000/CaCl 2 The solution was incubated at 25℃for 15min.
(3) 6000rpm x 5min, sucking out PEG4000 solution as much as possible, washing with P Buffer for 1 time;
(4) The protoplast obtained in the third embodiment is resuspended in 100 mu L of P Buffer respectively, added into the upper soft agar medium with temperature kept at 45 ℃, gently mixed by shaking on a vortex oscillator, poured onto a regeneration plate, and rapidly rotated to make the soft agar cover the surface of the lower medium uniformly; culturing at 28deg.C for 36h, covering with soft NaCl agar containing hygromycin to make final hygromycin concentration in the plate 5 μg/mL, solidifying the soft agar, and culturing at 28deg.C;
(5) And after 7d of culture, respectively picking up two strains of bleomycin resistance transformants for slant culture, and after 7d, extracting genome DNA for PCR verification.
The mutant strain after PCR verification is named CGMCC3.3795-PAcGapdh-KclgoX and negative control strain CGMCC 3.3795-PAcGapdh.
Example 7 fermentation of Acremonium chrysogenum and detection of the fermentation product CPC
The triangular flasks in this example were 500mL in size, with a spring ring at the bottom and three shake flask replicates per experimental strain.
A proper amount of acremonium chrysogenum spores are scraped from the inclined plane of the culture for 10d, and the strain containing the transformant is an experimental group by taking the wild type initial strain CGMCC3.3795 as a control. Wherein the transformant 1 is a Acremonium chrysogenum strain CGMCC3.3795-PAcGapdh transformed with an empty plasmid pBARGPE1-PAcGapdh, the transformant 2 is a Acremonium chrysogenum strain CGMCC3.3795-pBARGPE1-KclgoX transformed with an exogenous L-glutamate oxidase gene overexpression plasmid pBARGPE1-PAcGapdh-KclgoX, and the strain is respectively inoculated into 500mL shake flasks filled with 30mL of seed culture medium, and the strain is cultured for 3d in a rotary shaking table at the rotating speed of 230rpmn and the temperature of 28 ℃. Then, the inoculated cells were transferred to a 250mL shaking flask containing 30mL of fermentation medium at 25℃and 230rpm, and cultured for 7 days.
CPC standard preparation and standard curve determination: (1) precisely weighing 20mg of CPC standard substance with purity of 98%, transferring to a 100mL volumetric flask, adding ultrapure water for ultrasonic dissolution, and fixing the volume to a scale; (2) preparing standard sample solutions of 2, 4, 6, 8 and 10mg/mL respectively; (3) and (3) performing HPLC detection on the concentration standard substance solutions respectively, sampling each concentration for 3 times, and drawing a standard curve for the area mean value of the chromatographic peak. The standard solutions in this example need to be prepared on-the-fly, and the standard curve for CPC assay is shown in fig. 5.
Sample pretreatment method: (1) the broth was filtered using medium speed quantitative filter paper into a 50mL clean small beaker; (2) adding 990. Mu.L deionized water into a 2mL centrifuge tube; (3) sucking 10 mu L of filtrate into the centrifuge tube; (4) vortex mixing; (5) the sample was filtered into a sample bottle using a 0.22 μm filter and left to stand at 4℃for detection (detection completed within 24 hours).
The high performance liquid chromatography detection method comprises the following steps: the analytical time is 10min, the column temperature is 40 ℃, the mobile phase is 10mmol/L sodium acetate solution, acetonitrile=99:1, the sample injection amount is 10 μl, the flow rate is 1.0mL/min, the upper pressure limit is 280bar, the analytical time is 10min, the column temperature is 30 ℃, the detection wavelength is 254nm, and the analytical time is 9min. And calculating the CPC content in the sample according to the peak area and the effective content of the standard substance.
CPC sample concentration calculation method:
FIG. 6 is a comparison of HPLC determination of CPC yield after fermentation of wild-type strain CGMCC3.3795, negative control CGMCC3.3795-pBARGPE1 and mutant strain CGMCC3.3795-pBARGPE 1-KclgoX; as a result of the detection, the CPC yield of the wild-type starting control strain CGMCC3.3795 was 3196.3 + -77.8 mg/L, the CPC yield of the mutant control strain CGMCC3.3795-pBARGPE1-PAcGapdh transformed with the empty plasmid pBARGPE1-PAcGapdh transformant was 3008+ -214.3 mg/mL, and the CPC yield of the mutant strain CGMCC3.3795-pBARGPE1-KclgoX transformed with the exogenous L-glutamate oxidase gene overexpression plasmid pBARGPE1-PAcGapdh-KclgoX transformant was 4182.7 + -238.9 mg/mL. After statistical analysis of the data, the wild strain CGMCC3.3795 has no significant difference with the CPC yield of the negative control strain CGMCC3.3795-pBARGPE1-PAcGapdh transformed with the empty plasmid pBARGPE1-PAcGapdh, but has significant difference with the experimental mutant strain CGMCC3.3795-PAcGapdh-KclgoX transformed with the L-glutamate oxidase gene overexpression plasmid pBARGPE1-PAcGapdh-KclgoX (p=0.012); there was also a significant difference (p=0.024) between the control strain CGMCC3.3795-pBARGPE1-PAcGapdh transformed with the empty plasmid pBARGPE1-PAcGapdh and the experimental mutant strain CGMCC3.3795-pBARGPE1-KclgoX transformed with the L-glutamate oxidase gene overexpression plasmid pBARGPE 1-PAcGapdh-KclgoX. The yield of acremonium chrysogenum CPC over-expressing the L-glutamate oxidase KclgoX was increased by 30.8% compared to the wild type.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. A gene over-expressing exogenous L-glutamate hydrogenase, designated KclgoX, characterized in that KclgoX is obtained from wild-type gene by codon optimization; the nucleotide sequence of the wild-type gene is JNBY01000141.1:48966-50696; the codon is optimized for alanine codon sequence to TTC, cysteine codon sequence to TGC, aspartic acid codon sequence to GAC, glutamic acid codon sequence to GAG, phenylalanine codon sequence to TTC, glycine codon sequence to GGC, histidine codon sequence to CAC, isoleucine codon sequence to ATC, lysine codon sequence to AAG, leucine codon sequence to CTC, asparagine codon sequence to AAC, proline codon sequence to CCC, glutamine codon sequence to CAG, arginine codon sequence to CGC, serine codon sequence to AGC, threonine codon sequence to ACC, valine codon sequence to GTC, tyrosine codon sequence to TAA.
2. The use of the gene according to claim 1 for constructing a high-yield cephalosporin C expression vector.
3. Use of the gene according to claim 1 for the construction of a high-yielding cephalosporin C strain.
4. An expression vector for over-expressing exogenous L-glutamate hydrogenase gene, which is characterized in that the expression vector is pBARGPE1-PAcGapdh-KclgoX, and the KclgoX is obtained by codon optimization of wild type gene; the nucleotide sequence of the wild-type gene is JNBY01000141.1:48966-50696; the codon is optimized for alanine codon sequence to TTC, cysteine codon sequence to TGC, aspartic acid codon sequence to GAC, glutamic acid codon sequence to GAG, phenylalanine codon sequence to TTC, glycine codon sequence to GGC, histidine codon sequence to CAC, isoleucine codon sequence to ATC, lysine codon sequence to AAG, leucine codon sequence to CTC, asparagine codon sequence to AAC, proline codon sequence to CCC, glutamine codon sequence to CAG, arginine codon sequence to CGC, serine codon sequence to AGC, threonine codon sequence to ACC, valine codon sequence to GTC, tyrosine codon sequence to TAA.
5. The expression vector of claim 4, wherein the plasmid pBARGPE1-PAcGapdh is formed by ligating the PAcGapdh promoter and the linear fragment pBARGPE1-hygro, and wherein the gene KclgoX is ligated to the linear fragment pBARGPE1-PAcGapdh to obtain the gene pBARGPE 1-PAcGapdh-KclgoX.
6. Use of the expression vector according to claim 5 for constructing a high-yielding cephalosporin C strain.
7. A strain with high cephalosporin C production, characterized in that it is obtained by transformation of the expression vector pBARGPE1-PAcGapdh-KclgoX according to claim 4 into acremonium chrysogenum.
8. Use of the strain of claim 7 for fermenting high-yielding cephalosporin C.
CN202210681569.3A 2022-06-15 2022-06-15 Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield Active CN114990138B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210681569.3A CN114990138B (en) 2022-06-15 2022-06-15 Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210681569.3A CN114990138B (en) 2022-06-15 2022-06-15 Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield

Publications (2)

Publication Number Publication Date
CN114990138A CN114990138A (en) 2022-09-02
CN114990138B true CN114990138B (en) 2023-11-14

Family

ID=83035903

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210681569.3A Active CN114990138B (en) 2022-06-15 2022-06-15 Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield

Country Status (1)

Country Link
CN (1) CN114990138B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174420A (en) * 2011-01-28 2011-09-07 中国科学院微生物研究所 Genetic engineering bacteria for producing high-purity cephalosporin C and application thereof
CN109971660A (en) * 2017-12-28 2019-07-05 上海医药工业研究院 A kind of preparation method of cephalosporin and its genetic engineering bacterium used
CN113106028A (en) * 2021-05-11 2021-07-13 河南省健康元生物医药研究院有限公司 Construction method and application of genetically engineered bacterium for high yield of cephalosporin C

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174420A (en) * 2011-01-28 2011-09-07 中国科学院微生物研究所 Genetic engineering bacteria for producing high-purity cephalosporin C and application thereof
CN109971660A (en) * 2017-12-28 2019-07-05 上海医药工业研究院 A kind of preparation method of cephalosporin and its genetic engineering bacterium used
CN113106028A (en) * 2021-05-11 2021-07-13 河南省健康元生物医药研究院有限公司 Construction method and application of genetically engineered bacterium for high yield of cephalosporin C

Also Published As

Publication number Publication date
CN114990138A (en) 2022-09-02

Similar Documents

Publication Publication Date Title
JPH07501931A (en) 7-New biochemical production method of ADCA
JP2021506316A (en) Microorganisms capable of producing nepetalactor oxidoreductase, nepetalactor synthase, and nepetalactone
CN113564142A (en) Burkholderia ester synthetase, coding gene and application thereof
CN114990138B (en) Gene, expression vector, bacterial strain and application thereof in improving cephalosporin C yield
CN110129305B (en) Cephalosporin C acylase mutant for preparing 7-ACA
CN113913399B (en) Ketopantolactone reductase from Candida maltosa Xu316
CN114085820B (en) Ketopantolactone reductase from Candida viswanathii
CN115261342A (en) Burkholderia BJQ 0011-derived ester synthetase JFN _18195, encoding gene and application thereof
CN111254181B (en) Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemical enzyme method
CN112410353B (en) fkbS gene, genetic engineering bacterium containing fkbS gene, and preparation method and application of fkbS gene
CN111254180B (en) Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution
CN107287256A (en) The method that whole-cell catalytic synthesizes L-2- piperidine carboxylic acids
CN113174378A (en) Glutamic dehydrogenase mutant, encoding gene thereof, genetically engineered bacterium and application of genetically engineered bacterium in preparation of L-2-aminobutyric acid
CN109722455B (en) Method for producing glutacoside by microbial fermentation, engineering bacteria and application
CN114540397B (en) Method for enhancing regulatory protein expression to increase glutamine transaminase fermentation level
CN114196642B (en) Glutamate dehydrogenase variants and their use in the preparation of L-amino acids
CN116948999B (en) Ketone reductase mutant, composition, biological material and application thereof
CN113493745B (en) Genetically engineered bacterium for producing cephalosporin C and construction method thereof
JP4729919B2 (en) Microbial culture method and optically active carboxylic acid production method
CN114317631B (en) Application of monoamine oxidase in preparation of topiroxone
CN112442523B (en) Method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution
CN114958877A (en) Deacetoxycephalosporin C synthetase mutant and coding gene and application thereof
CN114540335B (en) Immobilized modified threonine transaldolase and application thereof
CN109370997B (en) Phenylalanine aminomutase mutant
WO2020125531A1 (en) Spiramycin-producing strain, kelimycin-producing strain, construction method therefor, use thereof and method for increasing product yield thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant