CN114457103A

CN114457103A - Method for improving TG enzyme yield by using CRISPR/dCas9 to knock down and regulate protein expression

Info

Publication number: CN114457103A
Application number: CN202210224071.4A
Authority: CN
Inventors: 白林泉; 刘先; 步建国
Original assignee: Taixing Dongsheng Bio Tech Co ltd; Shanghai Jiaotong University
Current assignee: Taixing Dongsheng Bio Tech Co ltd; Shanghai Jiaotong University
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-05-10
Anticipated expiration: 2042-03-07
Also published as: CN114457103B

Abstract

The invention discloses a method for improving the yield of TG enzyme by knocking down the expression of regulatory protein by using CRISPR/dCas 9; the mutant strains with improved TG enzyme yield are obtained by simultaneously knocking down encoding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 of regulatory proteins in a Streptomyces mobaraensis C2 genome through a CRISPR/dCas9 technology. And 4 regulatory protein coding genes are knocked down simultaneously, so that the negative regulation of the genes on TG enzyme is reduced, and the yield of the TG enzyme is improved. The TG enzyme fermentation final yield of the engineering strain obtained by the invention is increased by 74.3% compared with a control strain transferred into an empty carrier. The invention can obviously improve the fermentation yield of the TG enzyme and greatly reduce the fermentation cost.

Description

Method for improving TG enzyme yield by using CRISPR/dCas9 to knock down and regulate protein expression

Technical Field

The invention belongs to the field of bioengineering, and relates to a method for improving TG enzyme yield by using CRISPR/dCas9 to knock down and regulate protein expression; in particular to a method for improving the fermentation level of glutamine Transaminase (TG) by combining and knocking down regulatory proteins SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 by using CRISPR/dCas9 technology.

Background

Transglutaminase (TG enzyme) is a single subunit protein produced by Streptomyces mobaraensis (Streptomyces mobaraensis) and is capable of catalyzing transamidation between the γ -amide group of glutamine residues and the ∈ -amino group of lysine in proteins to form an allotypic peptide bond of ∈ - (γ -glutamine) -lysine, thereby changing the functional properties of the protein. TG enzyme is an exocrine protein, is in a pre-pro-MTGase initial form in a cell, penetrates 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 finally mature TG enzyme by serine protease SM-TAP. TG enzyme is used as a protein cross-linking agent, and 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 in the field of food by cross-linking glutamine residues and lysine residues, the attractiveness of the food is improved, nutrition is increased by integrating amino acid, and the degradable plastic package is manufactured by biosynthesis of TG enzyme; 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. The regulatory pathways of TG enzyme are not clear, and are key factors for limiting the improvement of TG enzyme yield in industry. In the invention, through genomics information and over-expression and knock-down experiment results, negative regulatory proteins SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 of TG enzyme coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 are found. Meanwhile, 4 negative regulation protein coding genes are knocked down, so that negative regulation on TG enzyme synthesis can be reduced, and the TG enzyme yield can be obviously improved finally.

Disclosure of Invention

The invention aims to provide a method for knocking down the expression of regulatory protein by using CRISPR/dCas9 to improve the yield of TG enzyme; by simultaneously knocking down regulatory protein coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 in the streptomyces mobaraensis C2 genome, the negative regulation of TG enzyme synthesis can be reduced, and the TG enzyme yield can be obviously improved finally.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention relates to a method for improving TG enzyme yield by knocking down regulatory protein expression, which simultaneously knockdown coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 of regulatory proteins SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 in a Streptomyces mobaraensis C2 genome (by using CRISPR/dCas9 technology) so as to improve TG enzyme fermentation level. By simultaneously knocking down regulatory proteins SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 in the genome of Streptomyces mobaraensis C2, coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 are reduced, the negative regulation and control on the synthesis of TG enzyme are reduced, and finally, the yield of TG enzyme can be obviously improved.

As one embodiment, the sequences of the genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 coded by the regulatory proteins are shown as SEQ ID NO.1-4 in sequence.

As one embodiment, the method comprises the steps of:

s1, constructing CRISPR/dCas9 plasmid vector I for simultaneously knocking down SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _ 720;

s2, introducing the constructed CRISPR/dCas9 plasmid vector I into a receptor strain streptomyces mobaraensis C2 through conjugative transfer to perform specific inhibition;

s3, screening by apramycin resistance and PCR verification, and obtaining mutant strains with reduced SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _ 720.

As one embodiment, the construction of plasmid vector I comprises the following steps:

a1, designing 20nt sequences of sgRNAs targeting and inhibiting SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720, respectively obtaining sgRNA fragments containing target 20nt through PCR amplification, and assembling integrated plasmids pSET-dCas9 subjected to double enzyme digestion by Gibson assembly and SpeI/EcoRI to obtain 4 plasmids inhibiting 4 genes respectively;

a2, designing a universal plasmid pGGA vector, 4nt protruding ends of 4 sgRNA fragments and primers, carrying out PCR amplification to respectively obtain 4 fragments, and assembling the 4 sgRNA fragments and the vector together; 4 sgRNA fragments were amplified together by PCR, and assembled with pSET-dCas9 digested simultaneously with EcoRI/EcoRV by Gibson assembly, to obtain plasmid vector I which inhibits 4 genes simultaneously.

As an embodiment, in step a1, the 20nt sequences of sgrnas that suppress SMDS _4150, SMDS _1792, SMDS _3072, and SMDS _720 are shown in sequence as SEQ ID nos. 5 to 8.

As one embodiment, in step a1, the primers for PCR amplification include: 4150-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO.9/10, 1792-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO.11/10, 3072-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO.12/10 and 720-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO. 13/10.

As one embodiment, in step A2, the primer comprises part1-F/R shown in SEQ ID NO.14/15, part2-F/R shown in SEQ ID NO.16/17, part3-F/R shown in SEQ ID NO.18/19, and part4-F/R shown in SEQ ID NO. 20/21.

In one embodiment, in step A2, the 4 sgRNA fragments are amplified together using the primer GGA-Gibson-F/R with the sequence shown in SEQ ID NO. 24/25.

As one embodiment, in step A2, 4 sgRNA fragments and a vector are assembled together to form a plasmid, and the correctness of the plasmid is verified by PCR, PstI/NotI double digestion and sequencing by using a primer pGGA-F/R with the sequence shown in SEQ ID NO. 22/23.

As an embodiment, in step A2, the correctness of the plasmid vector I is verified by PCR, EcoRI/EcoRV double digestion and sequencing with the primer GGA-Gibson-F/R having the sequence shown in SEQ ID NO. 24/25.

As one embodiment, the method further comprises a method of producing TG enzymes by fermenting the obtained mutant strain in which the genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 are knocked down.

As an embodiment, the fermentation comprises the steps of: inoculating the activated gene-knocked-down mutant strain LX-60 spores of the regulatory protein in a seed culture medium, culturing for 24h at 30 ℃ and 200rpm, transferring the cultured strain into a fermentation culture medium according to 10% of the inoculum size, fermenting for 30h at 30 ℃ and 200rpm, collecting fermentation liquor, and performing enzyme activity detection.

As one embodiment, the seed culture medium comprises 2 w/v% of glycerol, 0.6 w/v% of yeast extract, 2.5 w/v% of fish meal peptone, MgSO 2₄·7H₂O 0.2w/v％，K₂HPO₄·3H₂O 0.2w/v％；

As an embodiment, the fermentation medium comprises 2 w/v% glycerol, 0.6 w/v% yeast extract, 2.5 w/v% fish meal peptone, MgSO 2₄·7H₂O 0.2w/v％，K₂HPO₄·3H₂O0.2 w/v%, fermentation accelerator 0.1 w/v%.

The invention also relates to a genetically engineered bacterium for high-yield glutamine transaminase production, and simultaneously, regulatory protein coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 in Streptomyces mobaraensis C2 are knocked down to obtain the genetically engineered bacterium.

The strain Streptomyces mobaraensis C2 is obtained by strain mutagenesis from Jiangsu Donghui Biotechnology limited company, and is preserved in China Center for Type Culture Collection (CCTCC) with a preservation address of Wuhan university in Wuhan, China with a preservation number of M2020194 and a preservation date of 2020.6.10.

The invention increases zymogen synthesis from the source from the perspective of upstream intracellular product synthesis regulation, thereby realizing high yield. Compared with the prior art, the invention has the following beneficial effects:

in Streptomyces mobaraensis C2, CRISPR/dCas9 technology is used for inhibiting the transcription of regulatory protein genes, so that regulatory protein coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 are knocked down.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram of the construction of genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 for simultaneous knocking of low plasmid I;

FIG. 2 is a graph showing the comparison of the TG enzyme fermentation yields of the knock-down mutants of genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 with those of the empty vector-transferred strain.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention. In the following examples, the experimental methods without specifying the specific conditions were carried out under the conventional conditions or the conditions recommended by the manufacturers.

The plasmid pSET-dCas9 according to the present invention has been described in SCI database literature "He Huang, Guosong Zheng, Weihong Jiang, Haifeng Hu, and Yinhua Lu. one-step high-efficiency CRISPR/dCas9-mediated genome editing in Streptomyces. acta Biochimica Et Biophysica Sinica (4), 231-43".

Example 1

This example is a specific process for preparing a mutant with a knock-down gene encoding a regulatory protein (LX-60. the process specifically includes the following steps:

the method comprises the following steps: plasmid I was constructed as shown in FIG. 1. The specific operation is as follows:

firstly, a 20nt sequence of sgRNA of targeted inhibition SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 (sequences are shown as SEQ ID NO. 1-4) is designed on line by using website crispy.second microorganisms.org, and is shown as SEQ ID NO. 5-8; using pSET-dCas9 as a template, respectively using primers 4150-gRNA-F/dCas9-gRNA-R, 1792-gRNA-F/dCas9-gRNA-R, 3072-gRNA-F/dCas9-gRNA-R and 720-gRNA-FdCas9-gRNA-R to obtain 252bp sgRNA fragment containing target 20nt through PCR amplification, assembling the sgRNA fragment and an integrated plasmid pSET-dCas9 subjected to double digestion by Gibson assobly and SpeI/EcoRI to obtain 4 plasmids II, III, IV and V which respectively inhibit 4 genes, and verifying the correctness of the plasmids through double digestion by SpeI/EcoRI and sequencing; designing a universal plasmid pGGA-select vector and 4nt protruding ends of 4 sgRNA fragments and a primer part1-F/R, part2-F/R, part3-F/R, part4-F/R on line by utilizing a website Golden gate. neb. com, respectively using plasmids II, III, IV and V as templates, amplifying by PCR to obtain 4 sgRNA fragments of 256bp, assembling the 4 sgRNA fragments and the vector pGGA-select into a plasmid VI by Golden gate assombly, and verifying the correctness of the plasmid by utilizing the primer pGGA-F/R through PCR, PstI/NotI double enzyme digestion and sequencing; VI is used as a template, 4 sgRNA fragments are amplified together by PCR (polymerase chain reaction) by utilizing a primer GGA-Gibson-F/R, plasmid I capable of inhibiting 4 genes simultaneously is obtained by assembling Gibson assembly and pSET-dCas9 subjected to double enzyme digestion by EcoRI/EcoRV, and the correctness of the plasmid is verified by PCR, EcoRI/EcoRV double enzyme digestion and sequencing by utilizing the primer GGA-Gibson-F/R.

The plasmid pGGA-select related to the present invention has been described in SCI database literature "Potapov, V.et. al." Comprehensive Profiling of Four Base elevation Ligation Fidelity by T4 DNA library and Application to DNA Assembly ACS Synth. biol.,2018,7,1, 2665-fold 2674 ".

Step two: plasmid I for CRISPR/dCas9 was introduced into a highly productive strain Streptomyces mobaraensis C2, and SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 knockdown mutants were selected.

FIG. 1 illustrates the process of knocking down the genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _ 720. The specific operation is as follows:

the CRISPR/dCas9 plasmid vector I was transformed into the host ET12567(pUZ 8002). Corresponding ET12567(pUZ8002) was inoculated into LB containing three antibiotics 1 ‰ Apr, Kan and Chl, cultured at 37 ℃ for 20 hours, and then the cells were rinsed with fresh LB solution to remove the antibiotics from the culture. Meanwhile, scraping fresh spores (about 7d culture) of Streptomyces mobaraensis C2 into a2 XYT solution, thermally shocking for 10min at 50 ℃, adding a spore pre-germination solution, pre-germinating for 2h at 37 ℃, rinsing for 2-3 times by using the 2 XYT solution, uniformly mixing the spores with a previously prepared host bacterium ET12567(pUZ8002) (the ratio of acceptor bacterium cells to donor bacterium is about 1: 10), spreading the mixture on an ISP4MYM solid medium containing 10mM magnesium ions, and carrying out inverted culture in a 37 ℃ culture box. After 16h, taking out the plate, respectively adding the two antibiotics of apramycin (with the final concentration of 50 mu g/mL) and nalidixic acid (with the final concentration of 50 mu g/mL) into 1mL of sterile water, uniformly mixing, covering the mixture on an ISP4MYM solid culture medium, airing the solid culture medium, and transferring the solid culture medium to a 30 ℃ incubator for inverted culture. After 3-5 days, the joint grows out on the visible flat plate, and the visible flat plate is transferred to an ISP4MYM solid culture medium containing 1 per mill of apramycin and nalidixic acid for amplification culture to obtain a single colony. Single colonies were screened and verified by mycelium PCR using GGA-Gibson-F/R primers to obtain mutants with simultaneous knockdown of SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _ 720.

ISP4MYM medium configuration method is as follows:

ISP4(Difco)37g, mannitol 1g/L, yeast extract 1g/L, malt extract 2.5g/L, adding distilled water to volume of 1L, and sterilizing at 121 deg.C for 20 min.

The endonuclease recognition sites (restriction enzyme sites) involved in the first step are as follows:

the primer sequences used in the first and second steps are shown in table 1:

TABLE 1 primer sequence Listing

The PCR system and conditions used for preparing the gene fragment in the first step:

and (3) PCR reaction system: 30ng of DNA template, 20pmol of primer, 5 mu L of 50% DMSO, 10 nmol of dNTP, 25 mu L of buffer solution and 1 unit of Taq DNA polymerase, and adding pure water to make up to 50 mu L;

PCR conditions were as follows: 5min at 95 ℃; 15s at 95 ℃; 15s at 60 ℃; 30s-2min at 72 ℃; circulating for 30 times; 10min at 72 ℃.

And step two, verifying PCR systems and conditions adopted in mutant strain screening through PCR:

and (3) PCR system: 10-100 ng of DNA template, 10pmol of primer, 2 mu L of 50% DMSO and 10 mu L of 2 xMix buffer solution, and adding pure water to make up to 20 mu L;

PCR conditions were as follows: 10min at 95 ℃; 30s at 95 ℃; 30s at 60 ℃; 30s-2min at 72 ℃; circulating for 30 times; 10min at 72 ℃.

Example 2

This example is a process for producing TG enzyme by fermentation using a mutant strain LX-60 in which the gene encoding a regulatory protein is knocked down. The method comprises the following specific steps: coating the regulatory protein coding gene knock-down mutant strain LX-60 on a solid ISP4MYM 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 24 hours at 30 ℃ and 200rpm, transferring to a fermentation culture medium according to 10% of inoculum size, fermenting for 30 hours at 30 ℃ and 200rpm, and collecting fermentation liquor for enzyme activity detection.

TABLE 2 composition of seed Medium and fermentation Medium

Example 3

This example is a method for detecting the enzymatic activity of TG enzyme by a colorimetric method. The method specifically comprises the following steps: 100 mu L of fermentation broth supernatant is taken and put into a test tube, 100 mu L of water is added into one tube as a control, 1mL of solution A preheated at 37 ℃ is added, after reaction is carried out for 10min at 37 ℃, 1mL of solution B is added to stop the reaction. The absorbance of the reaction solution was measured at 525nm in an ultraviolet spectrophotometer using a 1cm quartz cuvette. Finally will OD₅₂₅Substituting into a formula obtained by conversion of a standard curve, and calculating the enzyme activity of the TG enzyme.

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 4.048g 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% FeCl₃Dissolve in 0.1mol/L HCl, mix three solutions equally well.

FIG. 2 is a graph showing the TG enzyme fermentation yields of the gene SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 knocked-down mutants and a control strain transferred into the empty vector pSET-dCas 9. The results show that the yield of the mutant strain is increased by 74.3% compared with the control strain at the laboratory shake flask level.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> Shanghai university of transportation

Tech east sage Biotech Ltd

<120> method for improving TG enzyme production by using CRISPR/dCas9 to knock down regulatory protein expression

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tcgcactggt tccaggacca ggagacccgc gagcgggcgc cggagccggc ggccgagccc 3240

gaggcgcccc gcggtctgcc gcgccgggcc ccgcgcgccg aggcccccgc tgcccccacc 3300

gcccccgacg tcacggccca gaccccgctg ccggactggc gcgagtcccc caacgacgag 3360

cgctggcgcc gggccgagca ggtccgggtg cccgccgcgg acggaaccac ctcgtcgggc 3420

ctgccccgcc gggtcccccg ggccaacctc gtcgagggca cggcccagca gcaggccctg 3480

ccggagggtc cgcaggtctc gcgcgcgccc gacgacgtgc gcggccggct gaccaacctc 3540

cgccggggca ttcagcaagg acgtcaggcc ggcaccagcc cggccaccgg tagccaccac 3600

ggtggcccca cctaccagca ggagcgttag 3630

<210> 3

<211> 4527

<212> DNA

<213> Streptomyces mobaraensis C2

<400> 3

atgcgcctgc ccgaggaccg gacgaacggc gggcgcacca agccgacgcg catgatcgcg 60

cggcggacgg acggcacgca gttccccgtc gaggtcacca gcgccaacct gcccgacagc 120

cgcaccccgt acgccgaaca gggtccgtac accggcgacg agctgctgat gctcgtcgtc 180

cgcgacctga ccggcacgct cgacaccgag gccgaactcg cccgccagca gcgccagacg 240

gagatgatcc tgcgcgcggc ggccgagggc gtggtcggcg tggacacggc gggcaaggtc 300

gtcctcgtca acccctccgc cgcgcagatc ctcggctacc gcgccagcga cctgggcggg 360

cgcgaactcc acccgttgat ccaccactcg cgcgcggacg gcacgccgtt cccgttcgag 420

gactcgccga tcgccgacac cctgcgttcc ggtcgcaaac accgggtccg tgggcaggtg 480

ctgtgggcga aggacgggcg cgcggtgccc gtcgacctga cgacggcgcc ggtgcgcgac 540

ggcgaccagc tggtcggcgc ggtgatgacg ttcaccgacc ggcgtccgta cgacgcgctg 600

gccgcgcggc acgcccagtt ggtggctgtg ctcgatggct cgctgcacgg gccgctggag 660

cggctgcggg gcgagctcgg cacgctggcc gccgatccgg ccgggcagtt gtggcccgag 720

gccaaccaga tcctgcacca cctcgccgcc ggctacgggc ggatggcccg gctggtcgag 780

agtgtgctcg cctaccagcg actggaggag ggcgaggggc ggctggagcg ggtggcgacc 840

gccctggacg aggtcgtcgc cgccggcgtc gagggggccg tcgagctgat cggccccggg 900

cgtgcgcagt tcgcggtgca cgcgccggcc atagaggccg agatcgacgc ggagtggttc 960

gcccgggcgc tcacccatct gatcgcggac gtcgcgggcg tggacgcgac gggcgagacc 1020

gccgccggca cggcgccgtc cggcgactcg acgatcgtcg tcgcggccgc gaagagggac 1080

acggccgtcc ggatcgaggt ccgcggcccg caccccggcg gcgacccggt ccacgggccc 1140

atcgtgcggg gcatcgtccg gcggcacggc ggcgtcctcc agacccacga cgtgccgggc 1200

agcgggggcg gcaaggcgta cgtcctggag gtgccggtct cggcggaggc cgggcccgtc 1260

atcccgtccg accggcccga ctccccggcc agcatcggca gcggtacgac gatcatgccg 1320

atgccggggc agcggtcggg agagacggcg gcggctcggg ggacggtcgg ggccgacggg 1380

agttctgttt ctgctgtttc tgcagcttct tctggtgcca cgggagttgg cggggccggg 1440

ggcccggcgg gctctccggg ttccggaggt cccgcggctt ccggtggtgt tgtcggggct 1500

gggggagctc ggggttccgg ggactccggc ggtggggtgg ggccgtcggg ttccgccgga 1560

gcggcggttc ccgccgcggc cgccgggagc gccggggacg gcgggggttc cgcgggcacc 1620

gaagtcaccg ggaatggcgg cggtgcgacg tccggcgcac cggccgctgc gcatgcgccg 1680

aactccgcgg cacggccgat tgccccaccc catcagaacc agcaggcgaa gcccggccat 1740

ccggctcccc cgccgcatgc ttctccttcg cctcatccgt ctcagccggt cgccgtggct 1800

catccggcct accccgttcc gcaggcgcgg caagggagtt cgcccgccag ggacgtcccc 1860

ccggtcaccg gtgcctctgg tgcctctggt gcccccgcgc agcccggcgt tccgaacgcc 1920

gcggtacctc tcggtcccac gccatccgcc ggtgacgggc cggggggtgc ggcgccgcac 1980

cccggttccg tacccggcgt gcccgtgccc gcccagccga ccgggcgccg gcgcggtccg 2040

gcccgtcccg acgaggagga ggcggccgcg gccgagcggg tacgggccaa ccggcacggc 2100

cgcggcgccg accaggcccc cgcaccgggt gccgggctcg tgccgccgca ggtggccgcg 2160

cccgcgcagc cgaccggacg ccgcgcccgg cgggaggccg ccgcggccga accggcggag 2220

ccggcgggcg cggacgcccg gcgcgcggcg ttcgcgctgc cgcccgccga ggcggaccgc 2280

ggacccggcc cggccgccga ggccgccgcc gcgcccaccg ggcgccgcgc ccggcgcgct 2340

ctcaccgagg cgcccgagcg gcccatcccg ggccaggccg acgccgaacc cgacattccg 2400

cgcgccgcgt tcgcgctgcc gccggccgaa gccgaccgca agccggcgcc cggcccgggt 2460

gccgaacccg tcccgggccg tccggtgccg gcccagggcg ggccgctcga cggcgggccc 2520

gcggagggcc ggccgacgga cccggcggtg gccgggcatg gtggccccgg tcacgggggt 2580

cctggtagtc acggcagtcc cggtggccac ggcggtcccg gtggtcacag cggtcccggt 2640

catggcaacg cggtagacgg cgcagggggc ggggtccggc ccgacgcttc gaccggctcc 2700

gacggctccg tgccgctgcc ggccgggcag tccggtgcgc ccggcaccac acccgtcacc 2760

gcgcccaacg ccgcgcccgt caccgcgcgg cctgcggacg ccgccgggcc gatgggcccc 2820

gcccggcccg tcgggcccgc cgcctcgccc gccgcgccgg ggcactccgg cccggccgag 2880

gccggtgcgg ggcccgcggg tgcgggacat gccggggccg gggtaggggc cggctctgtc 2940

ggtcatgccg ggcaccctgg acagcccgga catcccgccc acgccggccc caccggcccc 3000

accgggggcg ccgaccgcac cgacgtcgcg catcccgaag caggccggac ggccatcccc 3060

gtcgaccccg tcggccccgt cggtcccgtc gaccctgtcg gccccgctgg tcaccacgcc 3120

cccgtcggct ccacaggtcc cgagagtccc gtcagcccgg cgggtcagcc cggttctccg 3180

gcgtctccgc cgccggccgc gcccggggtc gccggtcccg ccgcgccggc cggtcaggac 3240

gttctccacg cccgtacgga tctcgacggt ccggctgagg cgccgcccac cgggcgccgg 3300

cgtgcgcgcc gggcgctcgc cgaggaaccg ggcgcgacgg cgctgccggg cgccggcgac 3360

gtcgcccgcg ccatcagcgt gcgcacgctc gggcagggcg cgccctacgc ccagcagggc 3420

ggggagaccc cgcccggcgg caccccgaac gcgtcgggag ccggttccgg gcggcggcgc 3480

aaactgggca atccggccga caacgccggt gagcgggaag cccaggcccg ggcgcaggcg 3540

cccgccgccg cgccgcgccc cggcccgacg ggcggcccga tcggtcccgg cgccggcacc 3600

agcaccggtc ccggaaccgg caccggccac ccgggtcctc tcggcctccg cccgcccgcc 3660

gccgcgccgg agggccgcgc cttcgccatt ggggcgccgg acgagggcgc cgagggcccg 3720

gagccgctgg acggccccaa cggcgccgtg gagatcaacc ccgctgcgtc cgtaccgccg 3780

cccgtggacg acgagttgcc gcccgagccg ctcgacaacc cgcgccgcct cctcgtctgg 3840

ccggccccgg acacgtccac ccggcaggcc ctgaccgaac gcggctaccg cccggtgatc 3900

gtgcactcgc gcgaggaggt cgacgcgcag atggcggcgt atccggccgc gctgttcgtc 3960

gatccgctca ccgggccgat cacccgtacc gccctccagg cgctgcgtca ggcggcggtg 4020

gccgccggcg ttcccgtcct ggtgacggcc ggtctcgggc aggccacgcg ggaggcggcg 4080

tacggcgccg acccggccgt cctcctcaag gcgctggcgc cgcgcgacag cgagatgcac 4140

ccggcccgcg tgctgctcgt cgaggagcac gaaccgatcg ccgacgcgct cacggcgacg 4200

ctggagcggc gcggcgtgca ggtggcctgc gcggccacgg acgcggaggc cgtggcgctg 4260

gcccagcaga tccgtcccaa cctggtggtg atggacctga tgcaggtacg gcgccgccgg 4320

gccgggatcc tggactggct gcgggcgcag gggctcctga accggacgcc cctggtcgtc 4380

tacacctcgg ccgggatcga cccggcccag ctcccccggc tggcctccgg cgagaccgtt 4440

ctcttcctcg ccgagcgttc gaccagcgcc gaggtgcaga gccggatcgt cgacctcctc 4500

acgaagatcg gcaccagccc ggggtga 4527

<210> 4

<211> 4308

<212> DNA

<213> Streptomyces mobaraensis C2

<400> 4

atgggcgagt ccacggccgg cgcggcggcg accgcggtac cgggccgcag gcgggacgac 60

ggccaaggtc ccgacgtcgg cgaggcggag ctgcggcagc tgctcgccgg gctgacggcg 120

gtgcgcgacg gcgacttcgg catccggctg ccggaggacg ccgacgggct gctcggcgaa 180

atagcgaccg tcttcaacgg gatgacggac cagctgtccc tgttcacctc cgaggtgacc 240

cgggtcgccc gcgaggtggg cagcgagggc cggctcggcg ggcaggcgcg ggtgccgggg 300

gtctcgggca cctggaagga cctcaccgac tcggtgaacg cgatggcggg gaacctcacc 360

acgcaggtcc gcgacatcgc gcaggtggcc accgcggtgg ccaagggcga cctgtcgcag 420

aagatcgacg tggcggcgca gggcgagatc ctggagctga agaacaccgt caacacgatg 480

gtcgaccagc tctccgcctt cgccgacgaa gtcacccgcg tcgcccgcga ggtgggcagc 540

gagggccggc tcggcgggca ggcgcaggtg cccggcgtgg gcggggtgtg gcgggatctg 600

accgattcgg tcaacttcat ggccggcaac ctcaccgccc aggtccgcaa catcgcccag 660

gtcaccacgg ccgtggccaa gggcgacctc tcgcagaaga tcacggtcga cgcgcgcggc 720

gagatcctcg ccctcaagaa caccatcaac gccatggtcg accagctctc ggccttcgcc 780

gacgaggtca cccgcgtcgc ccgcgaggtg ggcacggagg ggcggctcgg cgggcaggcc 840

gacgtcgagg gcatctccgg gacctggaag aacctcaccg agtcggtcaa cgtgatggcc 900

gacaacctca cggcgcaggt gcggtcgatc gcgcaggtca ccacggcggt ggccaagggc 960

gacctctcgc agaagatcaa cgtcggggcg cgcggggaga tccaggagct caaggagacc 1020

atcaacacga tggtcgacca gctctcctcg ttcgccgacg aggtgacccg cgtcgcccgc 1080

gaggtgggca ccgaggggaa cctcggcggc caggcgaccg tgcgcggggt ctcgggcacg 1140

tggaaggacc tgaccgacaa cgtcaacgtg atggcgtcca acctcaccgg gcaggtccgt 1200

tcgatcgccc aggtggcggc ggcggtggcg cgcggcgacc tgtcgcagaa gatcacggtg 1260

gaggcgaagg gcgaggtcgc cgcgctcgcc gacgtgatca accggatggt cgacacgctg 1320

tccgccttcg ccgacgaagt gacccgggtg gcccgcgagg tgggcaccga ggggatgctc 1380

ggcggccagg cgcgggtgcc caacgtcgcg ggcacctgga aggacctcac cgacaacgtc 1440

aactcgatgg ccaacaacct caccgggcag gtccgcaaca tcgcgcaggt caccacggcc 1500

gtcgccaacg gcgacctgac ccgcaagatc gacgtggacg cccgcggcga gatcctcgaa 1560

ctcaagacca ccatcaacac gatggtcgac cagctctcgt cgttcgccgc cgaggtcacc 1620

cgggtggccc gcgaggtcgg cagcgagggc cggctggggg gccaggccga ggtcgagggc 1680

gtctcgggca cctggaagcg gctcaccgag aacgtcaacg agctggccgg gaacctcacc 1740

cgccaggtgc gggcgatcgc cgaggtcacc agcgccgtcg cggagggcga cctgacccgg 1800

tcgatcaccg tcgaggcgtc cggcgaggtc gccgatctca aggacaacat caacgcgatg 1860

gtccgctccc tgcgcgagac cacccgcgcc aaccaggagc aggactggct gaaatccaat 1920

ctggcccgca cctccgggat gatgcagggc caccgcgacc tcaccctcgt cgcccggctg 1980

atcatggagg agctggcccc gctcgtcggc gcccagtacg gcggcttcta cctcgccgag 2040

gagacggagg ccggcccgtc gctgcgcatg atcggctcgt acgggcgccc cgagggcggc 2100

gagggggccg acgcgccccg gttctcgttc gggcagtcgc tcgtcggaca ggcggcctct 2160

gggcgccgga cgatcgcggt ggacgacctg cccgccggat cggtcaccgt gccctccggg 2220

ttcgggttca tcgagccgtc gcacctggtg gtcctgccga tcgtggtcga ggaccaggtg 2280

ctcggcgtca tcgagctggc ctccgtccac cgtttcacgc ccgtgcagcg ggacttcctg 2340

gagcagctgc gggagaccat cggcgtcaac gtcaacacca tcatcgccaa cgctcgtacg 2400

gatgaactgc tggatgagtc gcagcggctg accgcggagt tgcaggcccg ctccgaggaa 2460

ctccaggtcg gtcaggagga gttgcggcgc tccaacgccg agctggagga gaaggcggcg 2520

ctgctcgccc ggcagaaccg cgacatcgag accaagaacc tggagatcga acaggcccgc 2580

caggagctgg agacccgcgc gcaggagctc gcgctcgcct ccaagtacaa gtcggagttc 2640

ctggccaaca tgagccacga gctgcgcacc ccgctcaaca gcctgctgat cctggcgcag 2700

ctgctgtccc agaacccgag cggcaacctc accggcaagc aggtcgagta cgccgagatc 2760

atccactcgg ccggctccga cctgctccag ctcatcaacg acatcctcga cctgtcgaag 2820

gtcgaggcgg ggaagatgga tatctccccc gagtgggtgc cgctgcaccg gctgctcgcc 2880

tacgtggagt cgaccttccg gccgatgacc ggccagaagg ggctcggctt cgacgtggtg 2940

accgagccgg gcgtgcccgt cgccctgctg accgacgact cgcgcctccg ccaggtgctg 3000

cgcaacctgc tgtccaacgc ggtgaagttc accgagaccg ggcacgtgga gctgagcatc 3060

gagcccgtca ccggcgcgga actgccgccc gccgtacggc ggcacggcgc ggcgctcgcc 3120

ttccgggtac gggacaccgg catcggcatc gccgagcacc agctggaggc catcttcggc 3180

gcgttccagc aggcggacgg caccaccagc cgcaagtacg gcgggaccgg gctcggcctg 3240

tcgatcagcc gggagatcgc gtacctgctc ggcggctcga tcaccgcgca cagcacaccg 3300

ggcgagggca gcacgttcac cctctacctg ccggtggccc ggccggactt ccacgagcag 3360

acggcaccct ccgccgaggg cgagcccgag ctcgagtccg cggacaccga tcggcggtcc 3420

gcaccggccg taccccggca gcggcgcctc ctcgtcatcg aacagcagcc gcgcgggctg 3480

ctgtcgatgg tcgccgagag cgcccgcgcc ggcctcgcgc ccgtcgccgg cgcgacggcg 3540

cccgacacgg tcgacatcat cagcgcggtc ggcgcgcagg aggcggcgac cgtgctcgcc 3600

accgaggtct gccactgcgt cgtcctcgac ctcgacatgc ccgacgagga ggtgctgcgg 3660

ttcgtcgagg cgatgggcgc cgacccggcg ctgcgcacca tgcccgtcct ggcgcacaac 3720

agccggagcg tcggcacccc gcgcgagcgg gagatccggg agcggttcgc cggccggccg 3780

ctggacctgc tgtccagcct cgacgagctg cggcagcgga tcgcgctgca cctgtcggcc 3840

gagcggcccg gcgacgtccc gccgccgctg cccgcggccc gggaggcgcg cccggccgcc 3900

gcgtcggcgc cgggacggga cctggatccg gtgctggccg ggcggacggc cctcgtcgtc 3960

gacgacgacg cccgcaacct ctacgcgctc accggcatgc tcgaactcca gggcatgacc 4020

gtgctgcacg ccgagaacgg gcgggccggg atcgagacac tcaccgggca ccccgaggtc 4080

gacatcgtgc tgatggacgt gatgatgccg gagatggacg ggtatacggc aacggcggcc 4140

atccgggcca tgcccgcgta cgcggacctg ccgatcatcg cggtgacggc gaaggcgatg 4200

ccgggagacg aggagaagac catggcgtcg ggggcgagcg actacgtcac gaagccggtc 4260

gacgccgacg acctgatcgg ccgcatccgc cgcaggctgg caccgtga 4308

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

tggcgctaag cccccctcgg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 6

gcagcttcat gtggtcgagc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

gcgagtcgaa cgccggcagc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

cgcgccaagg ggcggcacac 20

<210> 9

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 9

cagctagctc agtcctaggt ataatactag ttggcgctaa gcccccctcg ggttttagag 60

ctagaaatag caagtt 76

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 10

caggaaacag ctatgacatg attac 25

<210> 11

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 11

cagctagctc agtcctaggt ataatactag tgcagcttca tgtggtcgag cgttttagag 60

ctagaaatag caagtt 76

<210> 12

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 12

cagctagctc agtcctaggt ataatactag tgcgagtcga acgccggcag cgttttagag 60

ctagaaatag caagtt 76

<210> 13

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 13

cagctagctc agtcctaggt ataatactag tcgcgccaag gggcggcaca cgttttagag 60

ctagaaatag caagtt 76

<210> 14

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 14

ggctacggtc tccggaggga tccttgacag ctagctca 38

<210> 15

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 15

ggctacggtc tcatagctcc agtaatgacc tcagaact 38

<210> 16

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 16

ggctacggtc tcagctagga tccttgacag ctagctca 38

<210> 17

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 17

ggctacggtc tcgtccatcc agtaatgacc tcagaact 38

<210> 18

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 18

ggctacggtc tcgtggagga tccttgacag ctagctca 38

<210> 19

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 19

ggctacggtc tccagtatcc agtaatgacc tcagaact 38

<210> 20

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 20

ggctacggtc tcctactgga tccttgacag ctagctca 38

<210> 21

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 21

ggctacggtc tccatggtcc agtaatgacc tcagaact 38

<210> 22

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 22

ctgcaggaag gtttaaacgc atttagg 27

<210> 23

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 23

taatacgact cactataggg agacgc 26

<210> 24

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 24

tttttcacgt tgaaaatctc gatatctcaa gggtaccgtg tgtatcgctc gagggatc 58

<210> 25

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 25

aacagctatg acatgattac gaattcgggt gtacagcctc gagaattctg acgtct 56

Claims

1. A method for knocking down expression of regulatory protein to improve TG enzyme yield is characterized in that genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 encoding the regulatory protein in a streptomyces mobaraensis C2 genome are knocked down simultaneously.

2. The method of claim 1, wherein the regulatory protein-encoding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 have the sequences shown in SEQ ID No.1, No.2, No.3 and No.4, respectively.

3. The method of claim 1, wherein the method comprises the steps of:

4. The method of claim 3, wherein the construction of plasmid vector I comprises the steps of:

5. The method of claim 4, wherein the 20nt sequences of sgRNAs inhibiting SMDS _4150, SMDS _1792, SMDS _3072, and SMDS _720 in step A1 are set forth in sequence as SEQ ID nos. 5-8.

6. The method of claim 4, wherein in step A1, the primers for PCR amplification comprise: 4150-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO.9/10, 1792-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO.11/10, 3072-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO.12/10 and 720-gRNA-F/dCas9-gRNA-R with the sequence shown in SEQ ID NO. 13/10.

7. The method of claim 4, wherein in step A2, the primers comprise part1-F/R having the sequence shown in SEQ ID No.14/15, part2-F/R having the sequence shown in SEQ ID No.16/17, part3-F/R having the sequence shown in SEQ ID No.18/19, and part4-F/R having the sequence shown in SEQ ID No. 20/21.

8. The method of claim 4, wherein in step A2, the 4 sgRNA fragments are amplified together using a primer GGA-Gibson-F/R having the sequence shown in SEQ ID NO. 24/25.

9. The method of claim 4, wherein in step A2, 4 sgRNA fragments and the vector are assembled together to form a plasmid, and the correctness of the plasmid is verified by PCR, PstI/NotI double digestion and sequencing by using a primer pGGA-F/R with a sequence shown in SEQ ID No. 22/23; the correctness of the plasmid vector I is verified by PCR, EcoRI/EcoRV double digestion and sequencing through a primer GGA-Gibson-F/R with the sequence shown as SEQ ID NO. 24/25.

10. The genetically engineered bacterium for high-yield glutamine transaminase production is characterized in that regulatory protein coding genes SMDS _4150, SMDS _1792, SMDS _3072 and SMDS _720 in Streptomyces mobaraensis C2 are simultaneously knocked down to obtain the genetically engineered bacterium.