CN114686388A - Method for efficiently screening high-yield glutamine transaminase production strains by using kanamycin and application - Google Patents

Method for efficiently screening high-yield glutamine transaminase production strains by using kanamycin and application Download PDF

Info

Publication number
CN114686388A
CN114686388A CN202011565693.0A CN202011565693A CN114686388A CN 114686388 A CN114686388 A CN 114686388A CN 202011565693 A CN202011565693 A CN 202011565693A CN 114686388 A CN114686388 A CN 114686388A
Authority
CN
China
Prior art keywords
ipio
strain
glutamine transaminase
promoter
gene
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.)
Granted
Application number
CN202011565693.0A
Other languages
Chinese (zh)
Other versions
CN114686388B (en
Inventor
步国建
步绪亮
白林泉
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.)
Taixing Dongsheng Bio Tech Co ltd
Original Assignee
Jiangsu Donghui Biotechnology 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 Jiangsu Donghui Biotechnology Co ltd filed Critical Jiangsu Donghui Biotechnology Co ltd
Priority to CN202011565693.0A priority Critical patent/CN114686388B/en
Publication of CN114686388A publication Critical patent/CN114686388A/en
Application granted granted Critical
Publication of CN114686388B publication Critical patent/CN114686388B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Landscapes

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

Abstract

The invention provides a method for efficiently screening high-yield glutamine transaminase production strains by using kanamycin, which comprises the steps of controlling the expression of a resistance gene neo by using a promoter of a glutamine transaminase gene in Streptomyces mobaraensis IPIO to obtain a mutagenized starting strain IPIO-BXL 1; the mutant strain after screening and mutagenesis is carried out by improving the concentration of antibiotics in a solid culture medium, so that the high-yield glutamine transaminase production strain is obtained. The invention also provides an initial strain IPIO-BXL1 and a construction method thereof. The invention obviously improves the efficiency of screening and obtaining the high-yield glutamine transaminase bacterial strain, greatly saves manpower and material resources for screening the bacterial strain, and has wide industrial application prospect.

Description

Method for efficiently screening high-yield glutamine transaminase production strains by using kanamycin and application
Technical Field
The invention relates to the field of bioengineering, in particular to a method for efficiently screening a bacterial strain for producing high-yield transglutaminase by using kanamycin and application of the bacterial strain.
Background
Glutamine transaminase (TGase) is a single subunit protein produced by Streptomyces mobaraensis (Streptomyces mobaraensis) and is capable of catalyzing transamidation reactions between the γ -amide group of glutamine residues and the epsilon-amino group of lysine in proteins to form the heterotypic peptide bond of epsilon- (γ -glutamine) -lysine, thereby altering the functional properties of the protein. TGase is an exocrine protein, is in a pre-pro-MTGase initial form in a cell, passes through a cell membrane to become inactive zymogen pro-TGase, is cut into signal peptide by metalloprotease TAMEP to become FRAP-TGase, and is cut into final mature TGase by serine protease SM-TAP. TGase is used as a protein cross-linking agent, is widely applied due to the advantages of good stability, safe use and the like, small pieces of meat can be combined into large pieces to improve the aesthetic degree of food in the field of food by cross-linking glutamine residues and lysine residues, the nutrition is increased by integrating amino acid, and TGase is biosynthesized to prepare degradable plastic packages; in the medical field, the method can be used for crosslinking antibodies and drug molecules to produce antibody coupling drugs, catalyzing gelatin and collagen to form a scaffold to be implanted into a human body to regenerate organs and the like. At present, most of strains for obtaining high-yield TGase in industrial production are screened after mutagenesis, but the traditional screening is time-consuming and labor-consuming, and the efficiency for obtaining high-yield TGase strains is low.
Disclosure of Invention
The invention overcomes the problems in the prior art, and provides a method for efficiently screening high-yield transglutaminase production strains by using kanamycin.
The invention provides a method for efficiently screening a bacterial strain for producing high-yield glutamine transaminase. The mutant strain is based on an original strain which utilizes a promoter containing a glutamine transaminase gene to control the expression of a resistance gene neo, and the screening efficiency of a high-yield glutamine transaminase producing strain is improved through high-concentration resistance screening.
The invention provides a strain Streptomyces mobaraensis IPIO, which is classified as Streptomyces mobaraensis IPIO and is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation number is CCTCC NO: m2020196, the preservation date is 2020, 6 and 10.
The strain Streptomyces mobaraensis IPIO is obtained by strain mutagenesis of Taixing east Sheng Biotech limited company.
The invention also provides a starting strain, namely IPIO-BXL1, which is obtained by integrating a promoter containing a glutamine transaminase gene in the Streptomyces mobaraensis IPIO to control the plasmid expression of a resistance gene neo.
The starting strain IPIO-BXL1 can be used for kanamycin screening.
The starting strain IPIO-BXL1 contains an expression cassette of a resistance gene neo controlled by a promoter of a glutamine transaminase gene.
Furthermore, the strain is obtained by integrating a plasmid containing an expression cassette of a resistance gene neo controlled by a promoter of a glutamine transaminase gene into Streptomyces mobaraensis IPIO.
Wherein the sequence of the promoter of the glutamine transaminase gene is shown as SEQ ID NO. 1.
The present invention also provides an expression cassette comprising: the promoter of glutamine transaminase gene, and the resistance gene neo.
The invention also provides application of the expression cassette in efficient screening of strains producing high-yield transglutaminase.
The invention also provides a screening method for improving the high-yield glutamine transaminase bacterial strain by utilizing the kanamycin high-efficiency screening, which comprises the following steps:
step (1): controlling the expression of a resistance gene neo by using a promoter of a glutamine transaminase gene in Streptomyces mobaraensis IPIO to obtain a mutagenized starting strain IPIO-BXL 1;
step (2): the high-yield glutamine transaminase producing strain with improved screening efficiency is obtained by increasing the concentration of antibiotics in a solid culture medium for screening.
The step (1) specifically comprises the following steps:
(1.1): designing and constructing an integrative plasmid vector pTDS001 of which the resistance gene neo is controlled by a promoter of a glutamine transaminase gene for resistance screening;
wherein, the construction method of the integrative plasmid vector pTDS001 is that 1008bp of promoter sequence of glutamine transaminase gene and 795bp of PCR fragment of resistance gene neo are obtained by PCR amplification and are connected into XbaI/EcoRI site of the integrative plasmid pSET152 by a one-step assembly method.
(1.2): the integrated plasmid vector pTDS001 (phi C31 integration site, derived from pSET 152) constructed in the first step is introduced into a recipient streptomyces mobaraensis IPIO through conjugative transfer, a promoter of a glutamine transaminase gene and a resistance gene neo controlled by the promoter are inserted into a chromosome, and a mutagenized starting strain IPIO-BXL1 is obtained through resistance and PCR verification screening.
The step (2) specifically comprises the following steps:
(2.1): coating the starting strain IPIO-BXL1 on solid culture media of different kanamycin to determine the resistance of the starting strain IPIO-BXL 1;
(2.2): the spores of the original strain IPIO-BXL1 are mutagenized by ARTP or NTG to obtain mutagenized spores.
(2.3): the mutagenized spores were plated on solid medium with higher concentration of kanamycin for selection.
In step (2.1), the concentration of kanamycin is 0. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL, 250. mu.g/mL, 300. mu.g/mL, 350. mu.g/mL and 400. mu.g/mL.
In the step (2.2), the mutagenesis condition of the ARTP is that the working gas flow is set to be 10SLM, helium is used as the working gas, the power supply power and the processing time are 100W and 10 s; the specific conditions for mutagenesis with NTG were to treat spores of IPIO-BXL1 with 50mM Tris-malate buffer containing 0.1mg/mL and 0.2mg/mL nitrosoguanidine, incubate at 30 ℃ and 200rpm for 30min, and then mix with 1% sodium thiosulfate to terminate the reaction.
In step (2.3), the higher concentrations refer to 300. mu.g/mL and 400. mu.g/mL.
The invention also provides a construction method of the original strain IPIO-BXL1, wherein in Streptomyces mobaraensis IPIO, the expression of the resistance gene neo is controlled by using the promoter of the glutamine transaminase gene to obtain the mutagenized original strain IPIO-BXL 1.
The preparation method of the starting strain comprises the following steps:
the first step is as follows: designing and constructing an integrative plasmid vector pTDS001 of which the resistance gene neo is controlled by a promoter of a glutamine transaminase gene for resistance screening;
the second step is that: the integrated plasmid vector pTDS001 obtained by the first step is transferred into a recipient strain Streptomyces mobaraensis IPIO by conjugation, a promoter of a glutamine transaminase gene and a resistance gene neo controlled by the promoter are inserted into a chromosome, and a mutagenized starting strain IPIO-BXL1 is obtained by verifying and screening resistance and PCR.
Wherein, the construction method of the integrative plasmid vector pTDS001 obtains a promoter sequence of a glutamine transaminase gene with 1008bp and a PCR fragment of a resistance gene neo with 795bp through PCR amplification, and the PCR fragment is connected into an XbaI/EcoRI site of the integrative plasmid pSET152 through a one-step assembly method.
The invention also provides a high-yield glutamine transaminase producing strain obtained by screening by the method.
The invention also provides application of the high-yield glutamine transaminase producing strain in improving the fermentation level of glutamine transaminase.
The invention also provides a method for efficiently screening high-yield glutamine transaminase production strains to improve the fermentation level of glutamine transaminase by using kanamycin, which comprises the following steps: inoculating activated spores of a strain capable of growing on a high-concentration kanamycin culture medium into a seed culture medium, culturing for 24h at 30 ℃ and 200rpm, transferring the activated spores into a fermentation culture medium according to the inoculation amount of 10%, fermenting for 28h at 30 ℃ and 200rpm, collecting fermentation liquor, and performing enzyme activity detection.
The seed culture medium comprises: 1-3 w/v% of glycerol, 0.5-0.8 w/v% of yeast extract, 2-4 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.3w/v％，K2HPO4·3H2O 0.2-0.4w/v％；
The fermentation medium comprises: 1-3 w/v% of glycerol, 0.5-0.8 w/v% of yeast extract, 2-4 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.3w/v％，K2HPO4·3H20.2-0.4 w/v% of O and 0.1% w/v of fermentation accelerator.
In Streptomyces mobaraensis IPIO, an integrated vector pTDS001 is utilized, an expression cassette of a resistance gene neo controlled by a promoter containing a glutamine transaminage gene is inserted on a chromosome, so as to obtain a starting strain IPIO-BXL1, and after mutagenesis, 31 strains grow in a high-concentration kanamycin solid medium, wherein the glutamine transaminage yield of 27 strains is higher than that of the starting strain IPIO-BXL 1; among 27 strains, 26 strains showed a 110% higher glutamine transaminase yield than IPIO-BXL, 12 strains showed a higher glutamine transaminase yield than IPIO-BXL, and 1 strain showed a higher glutamine transaminase yield than IPIO-BXL in 20% and 30% respectively. The method can obviously improve the efficiency of screening and obtaining the high-yield glutamine transaminase bacterial strain, thereby greatly saving manpower and material resources for screening the bacterial strain.
The invention also provides a gene sequence for expressing the resistance gene neo, wherein the gene sequence is a nucleotide sequence of a promoter of a glutamine transaminase gene or a nucleotide sequence with homology of more than 90 percent with the promoter, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
The invention also provides a construction method of the plasmid vector pTDS001, wherein a 1008bp promoter sequence of a glutamine transaminase gene and a 795bp PCR fragment of a resistance gene neo are obtained by PCR amplification, and are connected into the XbaI/EcoRI site of the integrative plasmid pSET152 by a one-step assembly method to obtain the plasmid vector pTDS 001.
The plasmids pSET152 and pDR3-K according to the invention have been described in SCI database documents "Xinjuan Ning, Xinran Wang, quantifying Wu, Qianjin Kang and Linquan Bai:identificationand Engineering of Post-PKS Modification Bottlenecks for analysis of P-3Titer Improvement in expression of ATCC 31280 Biotechnology Journal 2017,12, 1700484.
The invention relates to a method for improving the screening efficiency of a high-yield glutamine transaminase producing strain by resistance screening by using a starting strain which controls the expression of a resistance gene neo by using a promoter containing a glutamine transaminase gene. The expression of a resistance gene neo is controlled by using a promoter of a glutamine transaminase gene in Streptomyces mobaraensis IPIO to obtain a mutagenized starting strain IPIO-BXL 1. After mutagenesis, most of the mutant strains grow on a culture medium with high kanamycin concentration, and the strains are strains with high glutamine transaminase gene yield, so that the screening efficiency of the strains producing high-yield glutamine transaminase is obviously improved.
The invention innovatively provides a starting strain for controlling the expression of a resistance gene neo by using a promoter containing a glutamine transaminase gene, and the strain subjected to screening mutagenesis is obtained by increasing the concentration of antibiotics in a solid culture medium, so that the strain with high expression of the glutamine transaminase gene is obtained, and the screening efficiency of a high-yield glutamine transaminase production strain is improved. The expression of a resistance gene neo is controlled by using a promoter of a glutamine transaminase gene in Streptomyces mobaraensis IPIO to obtain a mutagenized development strain IPIO-BXL 1. After mutagenesis, most of the mutant strains grow on a culture medium with high kanamycin concentration, and the strains are strains with high glutamine transaminase gene yield, so that the screening efficiency of the strains producing high-yield glutamine transaminase is obviously improved. The 31 mutant strains obtained by the invention have 27 strains with higher glutamine transaminase yield than the original strain IPIO-BXL1, and 26 strains, 12 strains and 1 strain respectively have the glutamine transaminase yield of 10 percent, 20 percent and 30 percent of 27 strains with higher glutamine transaminase yield than the IPIO-BXL.
Drawings
FIG. 1 is a schematic diagram of the construction of an integrative plasmid pTDS001 containing a resistance gene neo controlled by a promoter of a glutamine transaminase gene.
FIG. 2 is a graph showing kanamycin resistance of the Streptomyces mobaraensis IPIO-BXL1 strain itself.
FIG. 3 is a graph showing the relative fermentation yields of transglutaminase from strains grown on high-concentration kanamycin-resistant plates and control strains.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1
The embodiment is a specific process for preparing an IPIO-BXL1 starting strain of a neo expression cassette of a promoter control resistance gene containing a glutamine transaminase gene, and specifically comprises the following steps:
the first step is as follows: construction of plasmid pTDS 001: a promoter fragment (1008bp) of the glutamine transaminase gene was obtained by PCR amplification using the genomic DNA of Streptomyces mobaraensis IPIO as a template and the primer PrTG-F/PrTG-R. Resistance gene neo fragment (795bp) was amplified by PCR using primers neo-F/neo-R in plasmid pDR3-K as template. The promoter fragment of the glutamine transaminase gene obtained by amplification and the neo fragment of the resistance gene were introduced into the XbaI/EcoRI site of pSET152 by a one-step cloning method to obtain pTDS 001.
The endonuclease recognition sites (restriction sites) involved in the first step are as follows:
XbaI recognition site: an EcoRI recognition site:
5'...T^CTAGA...3' 5'...G^AATTC...3'
3'...AGATC^T...5' 3'...CTTAA^G...5'
the primer sequences used in the first step were:
Figure RE-GDA0003151785540000051
PCR system and conditions used for gene fragment preparation in the first step:
and (3) PCR reaction system: PrimeSTAR Max Premix (2X) 25. mu.L, primers 15pmol each, DMSO 2. mu.L, DNA template 50ng, and purified water to make up to 30. mu.L;
PCR conditions were as follows: 10min at 95 ℃; 30s at 95 ℃; 30s at 60 ℃; 15s at 72 ℃; circulating for 30 times; 10min at 72 ℃.
The second step is that: the integrated plasmid vector pTDS001 obtained in the first step is transferred into a recipient strain Streptomyces mobaraensis IPIO by conjugation for site-specific recombination, and a correct binder is verified by resistance screening and PCR, so that the mutagenized starting strain IPIO-BXL1 is obtained. The method specifically comprises the following steps:
the plasmid pTDS001 which has been constructed is transformed into E.coli host ET12567(pUZ8002), and E.coli is cultured overnight at 37 ℃ after addition of the corresponding antibiotic for use. The corresponding antibiotic was added to 3mL of fresh LB solution, transferred to 300. mu.L of E.coli overnight culture, and cultured at 37 ℃ until OD reached between 0.4 and 0.6 for about 3.5 h. Centrifuging Streptomyces mobaraensis spore suspension at high speed, discarding supernatant, resuspending with 1mL fresh TES buffer solution, resuspending with 500 μ L, heat shocking in 50 deg.C water bath for 10min, rapidly cooling with flowing water to room temperature, adding 500 μ L spore pre-germination solution and 1 μ L5 mol/L CaCl2The solution was incubated at 37 ℃ for 2 h. Taking 1mL of escherichia coli liquid, centrifuging at high speed, removing supernatant, washing with a fresh LB culture medium without antibiotic, ensuring no antibiotic residue, and finally re-suspending with 400 mu L of fresh LB culture medium. Meanwhile, taking the spore suspension subjected to the pre-culture, centrifuging at a high speed, removing the supernatant, washing once with a fresh LB culture medium to thoroughly wash away the residues of the TES-free buffer solution, and finally resuspending with 400 mu L of a fresh nonreactive LB culture medium.
The LB culture medium comprises the following components: tryptone 1 w/v%, yeast extract 0.5 w/v%, NaCl 1 w/v%;
mixing the above suspensions, blowing and sucking with a gun head, uniformly coating on ISP4 (MgCl)220mM final concentration, 5% YT) and dried by air, and cultured in an inverted state at 30 ℃ for 16 h. Adding appropriate amount of antibiotic, nalidixic acid and 2mL of sterilized water, uniformly covering, completely drying the plate, and culturing at 30 deg.C for 5-7 days to observe zygote. The mutant strain is transferred to ISP4MYM solid culture medium containing apramycin and nalidixic acid for amplification culture, and colony PCR verification screening is carried out to obtain the mutant strain containing a promoter control resistance gene neo expression cassette containing glutamine transaminase gene.
The ISP4 solid medium comprises the following components: soluble starch 1 w/v%, MgSO4·7H2O 0.1w/v％、(NH4)2SO4 0.2w/v％、FeSO4·7H2O 0.0001w/v％、K2HPO4 0.1w/v％、NaCl 0.1w/v％、CaCO30.2w/v％、 MnCl2·4H2O 0.0001w/v％、ZnSO4·7H2O0.0001 w/v%, agar 2 w/v%, pH 7.0-7.4.
In the second step, PCR system and conditions used in screening the mutant strains were verified by PCR:
and (3) PCR system: 10-100 ng of DNA template, 10pmol of primer, 1 mu L of DMSO, 10 mu L of 2 xMix buffer solution and 20 mu L of pure water; the 2 xmix buffer comprises the following components: taq DNA polymerase, dNTP mixture, MgCl2(ii) a (2 XMix is a commercial enzyme System)
PCR conditions were as follows: 10min at 95 ℃; 30s at 95 ℃; 30s at 60 ℃; 60s at 72 ℃; circulating for 30 times; 10min at 72 ℃.
Example 2
This example is a screening process using kanamycin for mutant strains mutagenized from IPIO-BXL1 as the starting strain. The method comprises the following specific steps:
in the first step, the kanamycin resistance concentration of the strain IPIO-BXL1 itself is determined: the IPIO-BXL1 was cultured on solid Gow's medium to produce spores, and after 7 days, a spore suspension was collected. The spore suspension was streaked onto solid high-grade medium containing kanamycin at different concentrations, and after 4 days of growth, it was observed whether it could grow, with streptomyces mobaraensis IPIO as a control strain.
In the second step, random mutagenesis and resistance screening are carried out on the strain IPIO-BXL 1: sucking 10 mu L of spore suspension of IPIO-BXL1, uniformly coating the spore suspension on a sterile metal sheet with the diameter of 6mm, moving the sterile metal sheet to an ARTP mutagenic instrument preheated in advance, setting the working gas flow rate to be 10SLM, using helium as working gas, setting the power supply power and the processing time to be 100W and 10s, putting the processed metal gasket into a 2mL centrifuge tube, adding 1000 mu L of sterile water, shaking for 1min, coating the processed spore liquid on a Gao-plate containing 300 mu g/mL kanamycin, and culturing for 5d in a 30-DEG constant-temperature incubator. In addition, spores of IPIO-BXL1 were treated with 50mM Tris-malate buffer containing 0.1mg/mL and 0.2mg/mL nitrosoguanidine, incubated at 30 ℃ for 30min at 200rpm, and then mixed with 1% sodium thiosulfate to terminate the reaction. Centrifuged at 8000rpm, the supernatant was discarded, and the suspension was resuspended in sterile water and plated on a Gao plate containing 300. mu.g/mL kanamycin and incubated at 30 ℃ for 5 days in a constant temperature incubator.
The preparation of the Gauss solid culture medium comprises the following steps: gao's first agar medium: weighing 100g of soluble starch, putting the soluble starch into a 5L beaker, adding 200mL of distilled water, uniformly stirring by using a glass rod, adding 1L of boiling water, stirring by using the glass rod while adding until the starch is changed into a transparent viscous state, adding 3L of distilled water, stirring by using a magnetic stirrer, and accurately weighing 2.5g of MgSO 24·7H2O、2.5g K2HPO4·3H2O、2.5g NaCl、0.05g FeSO4·7H2O、5g KNO3Adding into starch solution, dissolving, measuring pH with pH meter (generally 7.5-7.6), adjusting pH, and sequentially metering to 5L with 2L measuring cylinder. Weighing 2% agar strips in each 400mL conical flask, adding 400mL culture medium, sealing with bottle membrane and kraft paper, sterilizing at 121 deg.C for 20min, and pouring into flat plate.
FIG. 2 is a schematic diagram of the determination of kanamycin resistance concentration of the strain IPIO-BXL1 itself. The results show that the tolerated concentration of IPIO-BXL1 was 250. mu.g/mL.
Example 3
This example is a process of producing TGase by fermentation using a mutant strain grown on a solid culture with high concentration of kanamycin. The method comprises the following specific steps: coating the bacterial strain growing on the solid culture medium with high kanamycin on the solid culture medium for activation, culturing for 5-7 days at 30 ℃, scraping a flat spore, inoculating the flat spore into a seed culture medium, culturing for 24h at 30 ℃ and 200rpm, transferring the flat spore to a fermentation culture medium according to 10 percent of inoculum size, fermenting for 28h at 30 ℃ and 200rpm, collecting the fermentation liquid, and carrying out enzyme activity detection and observation.
Example 4
This example is a method for detecting the enzyme activity of TGase by colorimetric method. The method specifically comprises the following steps: 200 mu L of the supernatant of the 20-fold diluted fermentation liquid is placed in two test tubes, 200 mu L of water is added into one tube as a control, 2mL of solution A preheated at 37 ℃ is added into the other tube, and after the reaction is carried out for 10min at 37 ℃, 2mLB solution is added to stop the reaction. The absorbance of the fermentation broth was measured at 525nm of a spectrophotometer using a 1cm quartz cuvette. Finally will OD525And substituting the formula obtained by conversion of the standard curve to calculate the enzyme activity of the TGase.
The solution preparation method comprises the following steps:
solution A: 9.688g of tris (hydroxymethyl) aminomethane, 2.780g of hydroxylamine hydrochloride, 1.229g of reduced glutathione and 4.048 g of a substrate Na-CBZ-GLN-GLY were weighed into a beaker, 350mL of water was added, the pH was adjusted to 6.0, and the volume was adjusted to 400mL by adding water.
And B, liquid B: 3mol/L hydrochloric acid, 12% trichloroacetic acid and 5% FeCl3Dissolved in 0.1mol/LHCl and the three solutions are mixed homogeneously in equal amounts.
FIG. 3 is a graph showing the relative fermentation yields of transglutaminase from strains grown on high-concentration kanamycin-resistant plates and control strains. The result shows that 31 strains grow on a high-concentration kanamycin solid culture medium at a laboratory shake flask level, wherein the glutamine transaminase yield of 27 strains is higher than that of the starting bacterium IPIO-BXL 1; among 27 strains, 26 strains showed a glutamine transaminase production of 110%, 12 strains showed a glutamine transaminase production of 20% or more, and 1 strain showed a glutamine transaminase production of 30% or more.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art are intended to be included within the present invention without departing from the spirit and scope of the inventive concept and are intended to be protected by the following claims.
SEQUENCE LISTING
<110> Jiangsu Donghui Biotech Co., Ltd
<120> method for efficiently screening high-yield glutamine transaminase producing strains by using kanamycin and application
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1008
<212> DNA
<213> Artificial sequence
<400> 1
tcgtcgtcct cctggcgatg atggcggcgg agggcacgtc cgtactgcgc aacgtgtacg 60
tgatcaaccg gggttacgag gacctggcgg accggctgaa ctcgatcggc gcccagatcg 120
agatcttccg ggacatctga cggacggatt ccgccgcacc ccgtctgaat tgcttctcgc 180
ggtcgaagag ttgaagaagg ggtgcggcgg cgtctctggg acttctccgg gaccccgtgg 240
ccctcggcag gccacgtccc aggggcactt ccggcgccga tcggccgtgg ctacgcggag 300
atcgcgtcga tggcggcgcc gcctcgggta ccggcgcggg gcgggaagcg ggagttcctc 360
cgcgaagtcg aagtcctcca aggcgagtcc aagcgccttc ccttgcccga ggccagcgcc 420
gaccctgctg cgtcgatgac ggacgcaggc gcaccgagtc ccgcggtctc gctcgcccgg 480
aggggatgcg gcggtgtccg gcgcccagcc ggattccgct cctgtgacgg agtggccggt 540
tttggagccg tggtgttgcc ggggagttaa ctgggagaca taatcacttc tcgtagcgac 600
ccgatcactc gtccgggagt cgagaagtgt tacgccgaac cccattccgc accatcaccc 660
ctgccgccgt gaccgcggcc ggcagtctgc ctctcgccga gagagccacc cggagaaccg 720
cccggacggg gtccgcttca ccgctccggt gacggcttcg acgtaacacg accgcgccgt 780
caccggccgt atccggtacg caccgcatcc ccattccgcc gtgcggccgc ggcctcttcc 840
tcaccgccgt taccggcgcg gcaccgcagg acgggcaccg cccgacgtta tgcgcggcca 900
ctcgccgcaa cctccacccc ccgcgtcgca ctctggcatg ccctcgttcc gcgaggttcg 960
ccagattcag ccctttcgtc acgttcgcca aaggagttgt tgttcttc 1008
<210> 2
<211> 56
<212> DNA
<213> Artificial sequence
<400> 2
tgccaagctt gggctgcagg tcgactctag atcgtcgtcc tcctggcgat gatggc 56
<210> 3
<211> 50
<212> DNA
<213> Artificial sequence
<400> 3
gcgtgcaatc catcttgttc aatcatgaag aacaacaact cctttggcga 50
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence
<400> 4
tgattgaaca agatggattg cacgc 25
<210> 5
<211> 56
<212> DNA
<213> Artificial sequence
<400> 5
aacagctatg acatgattac gaattactag ttcagaagaa ctcgtcaaga aggcga 56

Claims (17)

1. The strain Streptomyces mobaraensis IPIO is characterized in that the classification name of the strain is Streptomyces mobaraensis IPIO, the strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020196, the preservation date is 2020, 6 and 10.
2. A strain IPIO-BXL1, characterized in that the strain IPIO-BXL1 is obtained by integrating a promoter comprising a glutamine transaminase gene in the Streptomyces mobaraensis IPIO according to claim 1 to control the expression of a plasmid for the resistance gene neo.
3. The strain IPIO-BXL1 of claim 2, which is strain IPIO-BXL1 that can be screened with kanamycin; and/or the sequence of the promoter of the glutamine transaminase gene is shown as SEQ ID NO. 1; and/or the starting strain IPIO-BXL1 contains an expression cassette of a resistance gene neo controlled by a promoter of a glutamine transaminase gene.
4. The strain IPIO-BXL1 of claim 3, wherein the expression cassette comprises: the promoter of glutamine transaminase gene, and the resistance gene neo.
5. A method for efficiently screening a bacterial strain for producing high-yield glutamine transaminase by using kanamycin is characterized by comprising the following steps of:
step (1): obtaining a mutagenized starting species IPIO-BXL1 by controlling the expression of the resistance gene neo in Streptomyces mobaraensis IPIO according to claim 1 using the promoter of the glutamine transaminase gene;
step (2): the mutant strain after mutagenesis is screened by improving the concentration of antibiotics in a solid culture medium, so that the high-yield glutamine transaminase production strain with improved screening efficiency is obtained.
6. The method of claim 5, wherein step (1) comprises the steps of:
(1.1) designing and constructing an integrative plasmid vector pTDS001 for controlling a resistance gene neo by the promoter of the glutamine transaminase gene for resistance screening;
(1.2) introducing the integrated plasmid vector pTDS001 obtained by the first step into a recipient strain Streptomyces mobaraensis IPIO through conjugative transfer, inserting a promoter of a glutamine transaminase gene and a resistance gene neo controlled by the promoter on a chromosome, and obtaining the mutagenized starting strain IPIO-BXL1 through resistance and PCR verification screening.
7. The method of claim 5, wherein step (2) comprises the steps of:
(2.1) coating the starting strain IPIO-BXL1 on solid culture media of different kanamycin to determine the resistance of the starting strain IPIO-BXL 1;
(2.2) carrying out mutagenesis on the spores of the starting strain IPIO-BXL1 through ARTP or NTG to obtain mutagenized spores;
(2.3) the mutagenized spores were plated on a solid medium with a higher concentration of kanamycin for selection.
8. The method of claim 6, wherein the integrative plasmid vector pTDS001 is obtained by PCR amplification of 1008bp of the promoter sequence of glutamine transaminase gene and 795bp of the PCR fragment of resistance gene neo, and is connected into the XbaI/EcoRI site of the integrative plasmid pSET152 by a one-step assembly method.
9. The construction method of the strain IPIO-BXL1 is characterized in that in Streptomyces mobaraensis IPIO, a promoter of a glutamine transaminase gene is used for controlling the expression of a resistance gene neo to obtain a mutagenized starting strain IPIO-BXL 1; the method comprises the following steps:
the first step is as follows: designing and constructing an integrative plasmid vector pTDS001 of which the resistance gene neo is controlled by a promoter of a glutamine transaminase gene for resistance screening;
the second step is that: the integrated plasmid vector pTDS001 obtained by the first step is transferred into a recipient strain Streptomyces mobaraensis IPIO by conjugation, a promoter of a glutamine transaminase gene and a resistance gene neo controlled by the promoter are inserted into a chromosome, and the mutagenized starting strain IPIO-BXL1 is obtained by verification and screening of resistance and PCR.
10. The method of claim 9, wherein the integrative plasmid vector pTDS001 is a PCR fragment obtained by PCR amplification of 1008bp of the promoter sequence of glutamine transaminase gene and 795bp of resistance gene neo, and is ligated into the XbaI/EcoRI site of the integrative plasmid pSET152 by a one-step assembly method.
11. A method for efficiently screening high-yield glutamine transaminase producing strains to improve the fermentation level of glutamine transaminase by using kanamycin is characterized by comprising the following steps of inoculating spores of strains growing on a high-kanamycin solid culture medium into a seed culture medium, culturing for 24 hours at 30 ℃ and 200rpm, transferring the spores into a fermentation culture medium according to 10% inoculation amount, fermenting for 28 hours at 30 ℃ and 200rpm, collecting fermentation liquor and carrying out enzyme activity detection;
wherein the strain is selected according to the method of any one of claims 5 to 8.
12. The method of claim 11,
the seed culture medium comprises: 1-3 w/v% of glycerol, 0.5-0.8 w/v% of yeast extract, 2-4 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.3w/v％，K2HPO4·3H2O 0.2-0.4w/v％；
The fermentation medium comprises: 1-3 w/v% of glycerol, 0.5-0.8 w/v% of yeast extract, 2-4 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.3w/v％，K2HPO4·3H20.2-0.4 w/v% of O and 0.1% w/v of fermentation accelerator.
13. Use of the strain Streptomyces mobaraensis IPIO according to claim 1 and the strain IPIO-BXL1 according to claim 2 for efficiently screening strains producing high-yield transglutaminase.
14. Use of a high-producing transglutaminase producing strain selected by the method according to any one of claims 5 to 10 for increasing the fermentation level of transglutaminase.
15. A gene sequence for expressing a resistance gene neo is characterized in that the gene sequence is a nucleotide sequence of a promoter of a glutamine transaminase gene or a nucleotide sequence with homology of more than 90% with the promoter, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
16. An expression cassette comprising: the promoter of glutamine transaminase gene, and the resistance gene neo.
17. Use of the expression cassette of claim 16 for efficient screening of high-producing transglutaminase producing strains.
CN202011565693.0A 2020-12-25 2020-12-25 Method for efficiently screening high-yield glutamine transaminase production strain by using kanamycin and application Active CN114686388B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011565693.0A CN114686388B (en) 2020-12-25 2020-12-25 Method for efficiently screening high-yield glutamine transaminase production strain by using kanamycin and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011565693.0A CN114686388B (en) 2020-12-25 2020-12-25 Method for efficiently screening high-yield glutamine transaminase production strain by using kanamycin and application

Publications (2)

Publication Number Publication Date
CN114686388A true CN114686388A (en) 2022-07-01
CN114686388B CN114686388B (en) 2024-05-14

Family

ID=82130909

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011565693.0A Active CN114686388B (en) 2020-12-25 2020-12-25 Method for efficiently screening high-yield glutamine transaminase production strain by using kanamycin and application

Country Status (1)

Country Link
CN (1) CN114686388B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05199883A (en) * 1990-10-19 1993-08-10 Amano Pharmaceut Co Ltd Recombinant transglutaminase
US20020187525A1 (en) * 1999-10-18 2002-12-12 Ajinomoto Co., Inc. Method of producing microbial transglutaminase
US20060035366A1 (en) * 2002-09-10 2006-02-16 Kensuke Yuuki Transglutaminase-producing strain
CN102517242A (en) * 2011-12-30 2012-06-27 齐河百多安生物医药科技有限公司 Method for improving transglutaminase protein expression index of streptoverticillium mobaraense and dedicated engineering bacterium thereof
CN107384820A (en) * 2017-07-25 2017-11-24 华东理工大学 One plant of glutamine transaminage high yield mutagenic strain and its application
CN110358708A (en) * 2019-07-15 2019-10-22 泰兴市东圣生物科技有限公司 A kind of high-yield glutamine transaminase bacterial strain and its application
CN111690570A (en) * 2020-07-13 2020-09-22 江苏东汇生物科技有限公司 Glutamine transaminase producing strain

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05199883A (en) * 1990-10-19 1993-08-10 Amano Pharmaceut Co Ltd Recombinant transglutaminase
US20020187525A1 (en) * 1999-10-18 2002-12-12 Ajinomoto Co., Inc. Method of producing microbial transglutaminase
US20060035366A1 (en) * 2002-09-10 2006-02-16 Kensuke Yuuki Transglutaminase-producing strain
CN102517242A (en) * 2011-12-30 2012-06-27 齐河百多安生物医药科技有限公司 Method for improving transglutaminase protein expression index of streptoverticillium mobaraense and dedicated engineering bacterium thereof
CN107384820A (en) * 2017-07-25 2017-11-24 华东理工大学 One plant of glutamine transaminage high yield mutagenic strain and its application
CN110358708A (en) * 2019-07-15 2019-10-22 泰兴市东圣生物科技有限公司 A kind of high-yield glutamine transaminase bacterial strain and its application
CN111690570A (en) * 2020-07-13 2020-09-22 江苏东汇生物科技有限公司 Glutamine transaminase producing strain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KEIICHI YOKOYAMA ET AL: "Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis", 《APPL MICROBIOL BIOTECHNOL》, vol. 87, no. 6, pages 2087 - 2096, XP019841654 *
严文君: "产谷氨酰胺转氨酶的茂源链霉菌高产菌株高产机制的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》, pages 006 - 448 *

Also Published As

Publication number Publication date
CN114686388B (en) 2024-05-14

Similar Documents

Publication Publication Date Title
CN113699128B (en) Method for producing nicotinamide phosphoribosyl transferase by fermentation
CN110904174B (en) Application of bacillus licheniformis with deletion of leucine dehydrogenase gene in production of heterologous protein
CN112574977A (en) Enzyme special for galactooligosaccharide production and preparation and application thereof
CN112961845A (en) Method for improving fermentation level of glutamine transaminase by knocking out cslA gene
CN112980759B (en) Method for improving TG enzyme fermentation level by enhancing transcription level of Subtilisin gene
CN113005071B (en) Application of SsgA coding gene SMDS _1018, recombinant strain and construction method of recombinant strain
CN114196556A (en) Metarhizium anisopliae strain with high locust toxicity and construction method thereof
CN113817762A (en) Recombinant escherichia coli for producing pentamethylene diamine and application thereof
CN112126613B (en) Recombinant Streptomyces mobaraensis and application thereof in production of glutamine transaminase
CN111117942B (en) Genetic engineering bacterium for producing lincomycin and construction method and application thereof
CN112094792A (en) Brucella Rev.1 eryA gene deletion strain, and construction method and application thereof
CN114686388B (en) Method for efficiently screening high-yield glutamine transaminase production strain by using kanamycin and application
CN110878293B (en) Application of bacillus licheniformis with deletion of yceD gene in production of heterologous protein
CN114149934B (en) Protein glutaminase production strain, screening and characteristic analysis method
CN109593701B (en) Acid-resistant recombinant lactic acid bacteria and construction method thereof
CN107475140B (en) Recombinant pichia pastoris mutant with high pullulanase yield and improved fermentation speed under acidic condition
CN114686409B (en) Method for enhancing expression of superoxide dismutase gene and improving glutamine transaminase yield
CN108148853B (en) Genome editing vector, genome editing system composed of same and application of genome editing system
CN114686389B (en) Glutamine transaminase high-yield strain for enhancing transcription level of vgbS gene and preparation and fermentation methods thereof
CN114686408B (en) Method for enhancing VOC family protein gene expression and improving glutamine transaminase yield
CN114540397B (en) Method for enhancing regulatory protein expression to increase glutamine transaminase fermentation level
CN114790436B (en) Gene afsR_4 mutation and application thereof
CN116286575B (en) Method for efficiently expressing raw starch alpha-amylase by using bacillus subtilis
CN110643647B (en) Application of bacillus licheniformis glutamate dehydrogenase mutant S277W in poly-gamma-glutamic acid synthesis
CN113801831B (en) Bacillus subtilis capable of producing neutral protease with high yield and application 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
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20240411

Address after: No. 91-92 Junmin Road, Huangqiao Town, Taixing City, Taizhou City, Jiangsu Province, 225411

Applicant after: TAIXING DONGSHENG BIO-TECH CO.,LTD.

Country or region after: China

Address before: No.91 Junmin Road, Huangqiao Town, Taixing City, Taizhou City, Jiangsu Province 225411

Applicant before: Jiangsu Donghui Biotechnology Co.,Ltd.

Country or region before: China

GR01 Patent grant
GR01 Patent grant