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CN114686410B - Glutamine transaminase high-yield strain for enhancing transcription level of 1-phosphofructokinase gene and preparation and fermentation methods thereof - Google Patents

Glutamine transaminase high-yield strain for enhancing transcription level of 1-phosphofructokinase gene and preparation and fermentation methods thereof Download PDF

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CN114686410B
CN114686410B CN202011567728.4A CN202011567728A CN114686410B CN 114686410 B CN114686410 B CN 114686410B CN 202011567728 A CN202011567728 A CN 202011567728A CN 114686410 B CN114686410 B CN 114686410B
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streptomyces mobaraensis
phosphofructokinase
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CN114686410A (en
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步国建
武立清
白林泉
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Taixing Dongsheng Bio Tech Co ltd
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Abstract

The invention discloses a method for improving the yield of glutamine transaminase, which improves the yield of the glutamine transaminase by enhancing the transcription level of a 1-phosphofructokinase (1-phosphofructokinase) gene SMDS_3792. The mutant strain TGS104 with high yield of glutamine transaminase is obtained by using an artificial strong promoter kasOp to excessively express an endogenous 1-phosphofructokinase gene SMDS_3792 in Streptomyces mobaraensis C2, and the yield of the glutamine transaminase is obviously improved by enhancing the transcription level of the 1-phosphofructokinase gene SMDS_3792.

Description

Glutamine transaminase high-yield strain for enhancing transcription level of 1-phosphofructokinase gene and preparation and fermentation methods thereof
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a glutamine transaminase high-yield strain for enhancing the transcription level of a 1-phosphofructokinase (1-phosphofructokinase) gene SMDS_3792, and a preparation and fermentation method thereof.
Background
Transglutaminase (EC 2.3.2.13, tgase), also known as Transglutaminase, is produced by Streptomyces mobaraensis (Streptomyces mobaraensis). TGase mainly catalyzes an acyl transfer reaction between a gamma-hydroxylamine group of a glutamine residue in a protein and a primary amine compound (acyl acceptor), so that covalent crosslinking of the protein occurs, and the functional properties of the protein are changed through three ways of amine introduction, crosslinking and deamination. TGase mediated molecular crosslinking can improve the heat stability, water holding capacity and other characteristics of the protein, and is favorable for forming powerful gel and improving the quality of the protein. TGase is an exocrine protein that exists intracellularly as pre-pro-TGase, recognizes signal peptides by transmembrane transport, and is transported extracellularly as a precursor protein of pro-TGase. The extracellular pro-TGase is subjected to incision metal protease TAMEP to cut a polypeptide sequence from the N end to obtain FRAP-TGase, and the enzyme has a certain catalytic capability and can catalyze protein to crosslink into a polymer; then cutting off the N-terminal tetrapeptide Phe-Arg-Ala-Pro (namely FRAP) by cutting off serine proteinase TAP to form the final mature TGase. The molecular weight of the enzyme is 37.9kD, and the enzyme consists of 331 amino acids.
Since TGase has a very specific structure and strong crosslinking ability, it is currently widely used as a novel food additive in the food industry worldwide. The texture and the structure of the meat product after mechanical treatment can be improved, the gel property is enhanced through the crosslinking effect, the breaking degree is reduced, and the product quality and the water holding capacity are improved; in dairy products, the quality of the yoghurt is improved, the emulsification property of the milk protein is improved, the heat stability of the milk protein is improved, and the like; are widely used in the field of food packaging and films to improve the mechanical and barrier properties of films of biopolymers, such as gelatin, egg white proteins, whey proteins and their combinations with pectin, chitosan and the like. Meanwhile, the method has great application prospect in the processing processes of flour products, baked foods, soy protein products and the like.
Disclosure of Invention
In order to solve the disadvantages of the prior art, the present invention aims to provide a strain TGS104 which produces a high level of glutamine transaminase TGase, and a method for increasing the fermentation level of TGase by enhancing the transcription level of a fructokinase 1 (1-phosphofructokinase) gene SMDS_3792. Based on genomics information, the invention finally improves the output of glutamine transaminase by utilizing an artificial strong promoter kasOp to excessively express an endogenous 1-phosphofructokinase gene SMDS_3792 in Streptomyces mobaraensis C2.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides Streptomyces mobaraensis C2, which is obtained by strain mutagenesis of Thailand east Saint Biotech Co.Ltd, wherein the classification name of the strain is Streptomyces mobaraensis Streptomyces mobaraensis C, and the strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020194, the preservation date is 6 months and 10 days in 2020.
The present invention also provides a strain TGS104 which produces a high level of glutamine transaminase TGase, and which is obtained by enhancing the transcription level of 1-phosphofructokinase gene SMDS_3792.
Further, the strain is obtained by over-expressing a 1-phosphofructokinase gene SMDS_3792 derived from Streptomyces mobaraensis C2.
Further, the strain is obtained by overexpressing the 1-phosphofructokinase gene smds_3792 in streptomyces mobaraensis C2 using the artificial strong promoter kasOp.
The 1-phosphofructokinase gene SMDS_3792 of the strain is overexpressed.
"overexpression" refers to the insertion of a copy of the 1-phosphofructokinase gene SMDS_3792 (derived from Streptomyces mobaraensis C2) on the C2 chromosome of the recipient Streptomyces mobaraensis, enhancing the transcription level of the 1-phosphofructokinase gene SMDS_3792.
The strain contains an expression cassette for artificially strong promoter kasOp to overexpress the 1-phosphofructokinase gene SMDS_3792 derived from Streptomyces mobaraensis C2.
The expression cassette contains: the artificial strong promoter kanop, 1-phosphofructokinase gene smds_3792, transcription termination sequence.
The sequence of the 1-phosphofructokinase gene SMDS_3792 is shown in SEQ ID NO. 1.
The invention also provides an expression cassette comprising: the artificial strong promoter kanop, 1-phosphofructokinase gene smds_3792, transcription termination sequence.
The invention also provides application of the expression cassette in efficient screening of strain TGS104.
The invention also provides a method for improving the fermentation level of TGase by enhancing the transcription level of a 1-phosphofructokinase gene SMDS_3792, which comprises the following specific steps:
the first step: an integrated plasmid vector pTDS104 for overexpressing the endogenous 1-phosphofructokinase gene SMDS_3792 was designed and constructed;
and a second step of: a copy of the 1-phosphofructokinase gene SMDS_3792 was inserted into the C2 chromosome of the recipient Streptomyces mobaraensis using the integrative plasmid vector pTDS104 (ΦC31 integration site, pSET152 derivative, harboring the kasOp promoter), and the recombinant mutant TGS104 was obtained by resistance and PCR-verified selection.
And a third step of: spores of the cultured 1-phosphokinase gene SMDS_3792 overexpressing mutant strain TGS104 were inoculated into a seed medium, cultured at 25 to 35℃and 180 to 220rpm (preferably, 30℃and 200 rpm) for 20 to 24h (preferably, 24 hours), transferred into a fermentation medium at an inoculum size of 8 to 15% (preferably, 10%) and fermented at 25 to 35℃and 180 to 220rpm (preferably, 30℃and 200 rpm) for 28 to 32 hours (preferably, 28 hours), and the fermentation broth was collected and subjected to enzyme activity detection.
In the first step, the 1-phosphofructokinase gene SMDS_3792 is derived from Streptomyces mobaraensis C2.
In the first step, the construction method of the vector pTDS104 is as follows: the PCR fragment of 954bp of 1-phosphofructinase gene SMDS_3792 sequence is obtained through PCR amplification, and is connected into NdeI/EcoRI sites of an integrated plasmid pDR3-K through an enzyme cutting connection method, so that the vector pTDS104 is obtained.
In the third step, the seed culture medium comprises glycerin 1-3w/v%, yeast extract 0.5-1w/v%, fish meal peptone 2-3 w/v%, and MgSO 4 ·7H 2 O 0.1-0.5w/v％，K 2 HPO 4 ·3H 2 O0.1-0.5 w/v%, pH 7.4; preferably, it is glycerol 2w/v% of yeast extract 0.6w/v%, fish meal peptone 2.5w/v%, mgSO 4 ·7H 2 O 0.2w/v％，K 2 HPO 4 ·3H 2 O 0.2w/v％， pH 7.4。
In the third step, the fermentation medium comprises glycerol 1-3w/v%, yeast extract 0.5-1w/v%, fish meal peptone 2-3 w/v%, and MgSO 4 ·7H 2 O 0.1-0.5w/v％，K 2 HPO 4 ·3H 2 0.1-0.5w/v% of O, 0.1-0.3w/v% of fermentation promoter and pH 7.4; preferably, the fermentation medium comprises glycerol 2w/v%, yeast extract 0.6w/v%, fish meal peptone 2.5w/v%, mgSO 4 ·7H 2 O 0.2w/v％，K 2 HPO 4 ·3H 2 O0.2 w/v%, fermentation promoter 0.1w/v%, pH 7.4.
The invention also provides a preparation method of the glutamine transaminase high-producing strain TGS104, which comprises the following steps:
1) Designing and constructing an integrated plasmid vector pTDS104 for overexpressing the 1-phosphofructokinase gene SMDS_3792;
2) Using the integrative plasmid vector pTDS104 (ΦC31 integration site, pSET152 derivative, harboring the kasOp promoter), a copy of the 1-phosphofructokinase gene SMDS_3792 was inserted into the C2 chromosome of the recipient Streptomyces mobaraensis, and the recombinant mutant TGS104 was obtained by resistance and PCR-verified selection.
In step 1), the 1-phosphofructokinase gene SMDS_3792 is derived from Streptomyces mobaraensis C2.
In step 1), the construction method of the vector pTDS104 is as follows: the PCR fragment of 954bp of 1-phosphofructokinase gene SMDS_3792 sequence is obtained by PCR amplification and is connected into NdeI/EcoRI site of integrated plasmid pDR3-K by enzyme cutting connection method.
The invention also provides a 1-phosphofructokinase gene SMDS_3792, and the sequence of the 1-phosphofructokinase gene SMDS_3792 is shown as SEQ ID NO. 1.
The invention also provides a gene sequence for expressing the 1-phosphofructokinase gene SMDS_3792, wherein the gene sequence is a nucleotide sequence of an artificial strong promoter kasOp or a nucleotide sequence with more than 90% of homology with the artificial strong promoter kasOp, and the nucleotide sequence of the artificial strong promoter kasOp is shown as SEQ ID No. 2.
The invention also provides a construction method of the plasmid vector pTDS104, wherein a PCR fragment of 954bp 1-phosphofructiokinase gene SMDS_3792 sequence is obtained through PCR amplification, and the PCR fragment is connected into NdeI/EcoRI sites of an integrated plasmid pDR3-K by an enzyme digestion connection method to obtain the plasmid vector pTDS104.
The invention also provides application of the Streptomyces mobaraensis C2 in screening of a glutamine transaminase high-yield strain TGS104.
The invention also provides application of the glutamine transaminase high-producing strain TGS104 in improving the fermentation yield of glutamine transaminase.
The plasmid pDR3-K according to the present invention is described in SCI database documents "Xinjuan Ning, xinran Wang, yuanning Wu, qianjin Kang and Linquan Bai Identification and Engineering of Post-PKS Modification Bottlenecks for Ansamitocin P-3Titer Improvement in Actinosynnema pretiosum subsp.pretiosumATCC 31280.Biotechnology Journal 2017,12,1700484".
The strain Streptomyces mobaraensis C2 related by the invention is obtained by strain mutagenesis of Taixing east Sheng biotechnology limited company, and is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020194, date of preservation 2020.6.10.
The beneficial effects of the invention include: in Streptomyces mobaraensis C2, 1-phosphofructokinase gene SMDS_3792 derived from Streptomyces mobaraensis C2 is inserted in one copy on the chromosome of Streptomyces mobaraensis C2 by using the integrated vector pDR3-K, and the enzyme activity of the strain is improved by 10.43% compared with that of a control strain in a shake flask level in a laboratory. The invention can improve the fermentation yield of TGase and reduce the fermentation cost.
Drawings
FIG. 1 is a schematic diagram of construction of SMDS_3792 gene overexpression plasmid.
FIG. 2 is a diagram showing comparison of the fermentation yield of 1-phosphofructokinase gene SMDS_3792 enhanced expression mutant strain and a control strain TGase.
Detailed Description
The invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
Example 1
This example is a specific procedure for preparing a mutant strain TGS104 overexpressing the 1-phosphofructokinase gene SMDS_3792, comprising the steps of:
the first step: construction of plasmid pTDS104: the genomic DNA of Streptomyces mobaraensis C2 was used as a template, and a primer smds_3792-F/R having NdeI/EcoRI cleavage sites introduced at both ends was used to obtain a gene fragment of SMDS_3792 (954 bp) by PCR amplification. The digested amplified fragment was inserted into NdeI/EcoRI site of plasmid pDR3-K to obtain plasmid pTDS104.
* The recognition sites (cleavage sites) of the endonucleases involved in the first step are as follows:
NdeI recognition site: ecoRI recognition site:
5'...CA^TATG...3' 5'...G^AATTC...3'
3'...GTAT^AC...5' 3'...CTTAA^G...5'
* The primer sequences used in the first step are as follows:
* PCR system and conditions adopted for preparing gene fragment in the first step:
PCR reaction system: 30ng of DNA template, 20pmol of primer F/R, 5 mu L of 50% DMSO, 10nmol of dNTP, 25 mu L of buffer solution, 1 unit of Taq DNA polymerase, and adding pure water to fill up to 30 mu L;
PCR conditions: 95 ℃ for 5min; cycling for 30 times at 95 ℃ for 15s,60 ℃ for 15s and 72 ℃ for 30 s; and at 72℃for 10min.
And a second step of: by using an integrative plasmid vector pTDS104 (ΦC31 integration site, pSET152 derivative, with a kasOp promoter), a copy of the 1-phosphofructokinase gene SMDS_3792 (derived from Streptomyces mobaraensis C2) was inserted into the C2 chromosome of the acceptor Streptomyces mobaraensis, and the correct zygote was selected by resistance and PCR verification, a mutant strain overexpressing the 1-phosphofructokinase gene SMDS_3792 was obtained, which specifically comprises the following steps:
plasmid pTDS104 with over-expressed gene was transformed into host ET12567 (pUZ 8002), the corresponding ET12567 (pUZ 8002) was inoculated into LB containing three antibiotics Apr, kan and Chl, cultured for 20h at 37℃and then the cells were rinsed with fresh LB solution to remove the antibiotics in the culture; meanwhile, preparing a C2 spore pre-germination liquid, collecting spores of C2 which grow for 7 days, adding a 2 XYT culture medium after heat shock for 10min at 50 ℃ for pre-germination for 2 hours at 37 ℃, and then rinsing with a fresh LB culture medium for 2 times.
The LB culture medium comprises the following components: 1w/v% tryptone, 0.5w/v% yeast extract, 1w/v% NaCl, pH 7.0;
the 2 XYT culture medium comprises the following components: tryptone 1.6w/v%, yeast extract 1w/v%, naCl 0.5w/v%, pH 7.0.
The C2 spore pregermination solution was mixed with host bacteria ET12567 (pUZ 8002) prepared previously (the ratio of recipient bacteria cells to donor bacteria is about 1:10) uniformly, then spread on ISP4 solid medium containing 10mM magnesium ions, and cultured upside down in a 30℃incubator. After 16h, the plate is taken out, two antibiotics of apramycin (final concentration 50 mug/mL) and nalidixic acid (final concentration 25 mug/mL) are added into 1mL sterile water to be evenly mixed, covered on a joint transfer plate, and the joint transfer plate is transferred to a 30 ℃ incubator for inversion culture after blow-drying. The zygote grows out on a common plate after 3-5 days, and the zygote is transferred to a Gao's I solid culture medium containing two antibiotics of apramycin and nalidixic acid for expansion culture, and a mutant strain doubled with a 1-phosphofructinase gene SMDS_3792 is obtained through mycelium PCR verification and screening.
The ISP4 solid culture medium comprises the following components: 1w/v% of soluble starch and MgSO 4 ·7H 2 O 0.1w/v％、(NH 4 ) 2 SO 4 0.2w/v％、FeSO 4 ·7H 2 O 0.0001w/v％、K 2 HPO 4 0.1 w/v％、NaCl 0.1w/v％、CaCO 3 0.2 w/v％、 MnCl 2 ·4H 2 O 0.0001w/v％、ZnSO 4 ·7H 2 O0.0001 w/v%, agar 2w/v%, pH 7.0-7.4.
The solid culture medium of Gao's I comprises the following components: soluble starch 2w/v%, mgSO 4 ·7H 2 O 0.05w/v％、KNO 3 0.1w/v％、FeSO 4 ·7H 2 O 0.001w/v％、K 2 HPO 4 0.05w/v%, naCl 0.05w/v%, agar 2w/v%, pH 7.2-7.4.
* In the second step, the zygote mycelium was used as a DNA template, and the PCR system and conditions used in screening the mutant strain were verified by PCR using the primers smds_2075-F/R having NdeI/EcoRI cleavage sites introduced at both ends:
PCR system: 10-100 ng of DNA template, 10pmol of primer F/R, 2 mu L of 50% DMSO, 10 mu L of 2 Xmix buffer solution, and adding pure water to fill up to 20 mu L;
the 2 Xmix buffer comprises the following components: taq DNAPolymerase (recombiant) 0.05 units/. Mu. L, mgCl 2 4 mM、dNTPs(dATP、dCTP、dGTP、dTTP)0.4mM；
PCR conditions: 95 ℃ for 10min; cycling for 30 times at 95 ℃ for 15s,60 ℃ for 15s and 72 ℃ for 30 s; and at 72℃for 10min.
Example 2
This example shows the fermentation of TGS104 to produce TGase using a mutant strain overexpressed by the 1-phosphofructokinase gene SMDS_3792. The method comprises the following specific steps: the strain TGS104 with the over-expression of the 1-phosphofructokinase is respectively coated on a Gao I culture medium for activation, after 7d of culture at 30 ℃, a flat spore is scraped and inoculated into a seed culture medium, and is cultured for 24h under the conditions of 30 ℃ and 200rpm, 10% of the inoculation amount is transferred into a fermentation culture medium, and fermentation is carried out for 28h under the conditions of 30 ℃ and 200rpm, and then fermentation liquor is collected for enzyme activity measurement.
Example 3
The embodiment is a method for detecting the enzyme activity of TGase by using a colorimetric method, which specifically comprises the following steps: 200 mu L of 20-fold diluted supernatant of the fermentation broth was taken in two test tubes, one of which was filled with 200 mu LL water was used as a control, 2mL of preheated solution A at 37℃was added to the other tube, and after 10min of reaction at 37℃the reaction was terminated by adding 2mL of solution B. The absorbance of the fermentation broth was measured at 525nm using a 1cm quartz cuvette. Will eventually OD 525 And carrying out a formula obtained by conversion of a standard curve, and calculating the enzyme activity of the TGase.
The preparation method of the solution comprises the following steps:
and (3) solution A: 9.688g of tris (hydroxymethyl) aminomethane, 2.780g of hydroxylamine hydrochloride, 1.229g of reduced glutathione, 4.048 g substrate Na-CBZ-GLN-GLY are weighed into a beaker, 350mL of water is added, the pH is adjusted to 6.0, and the volume is fixed to 400mL by adding water.
And (2) liquid B: 3mol/L hydrochloric acid, 12% trichloroacetic acid, 5% FeCl 3 Dissolving in 0.1mol/LHCl, and uniformly mixing the three solutions in equal amounts.
FIG. 2 is a schematic diagram showing the relative fermentation yield of 1-phosphofructokinase gene SMDS_3792 enhanced expression mutant strain and control strain TGase. The results showed a 10.43% improvement in mutant yield over wild-type strain at laboratory shake flask level.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
SEQUENCE LISTING
<110> Jiangsu Donghui biotechnology Co., ltd
<120> a glutamine transaminase-producing strain having enhanced transcription level of fructose-1-phosphate kinase gene, and a process for producing the same and fermentation
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 954
<212> DNA
<213> artificial sequence
<400> 1
gtgatcctca ccgtcacccc caaccccagc ctcgaccgca cctacgaggt ccccgcgctg 60
cggcgcggcg ccgtcctccg cgcggcggcc gaccgcgtcg acccgggcgg caagggcgtc 120
aacgtctcgc gcgcggtcgc ggcggccggc cacgccacca cggccgtgct gccgctcggc 180
ggcgcggagg gcgccctgct ggaacgcctc ctccgtgaac agggcatcga cgtcgcgggg 240
gtccccgtct ccggcgcgac gcgggtcaac gtggccgtcg ccgaacccga cggcacgctc 300
accaaggtca acgccgccgg ccccgaactg acggccgccg aggccgaggc gctgctggac 360
agcgtggggg aacggtcggc gggcgccggc tggatcgcct gctgcggcag cctgccgcgc 420
ggactcgcgc ccgagtggta cgggcggctg gtggcccgcg cccacgccgc cggggccagg 480
atcgcgctcg acacctcggg gccggccctc accgccgccc tgcgcgaacg ccccgacgtc 540
gtcaaaccca acaccgagga actcgcccag gccgtcggcc gccccctgac gaccgtcggg 600
gacgcggccc gggcggcgga ggaactgcgc ggcctcggcg cgcggaccgt cctcgccagc 660
cttggcgcgg acggccagct cctcgtcgac ggctccggaa cgcactacgg aacggccccg 720
gtgacgcgcg tccgcagcaa cgtcggcgcg ggcgacgcct ccctggcggg cttcctcgcc 780
gccggcggcg agagcccggc cgccctcgcg gcggccctgg cccacggcgc ggcggccgtc 840
cggctgcccg gcagcgcgat gccggccccg ggcgacctcg aaccgtccac cgtgacggtc 900
acccgggacg tcccgggcga ccgcgccctg accgaaccgg cgtccggcgt ctga 954
<210> 2
<211> 97
<212> DNA
<213> artificial sequence
<400> 2
tgttcacatt cgaacggtct ctgctttgac aacatgctgt gcggtgttgt aaagtcgtgg 60
ccaggagaat acgacagcgt gcaggactgg gggagtt 97
<210> 3
<211> 30
<212> DNA
<213> artificial sequence
<400> 3
atatcatatg gtgatcctca ccgtcacccc 30
<210> 4
<211> 30
<212> DNA
<213> artificial sequence
<400> 4
atatgaattc tcagacgccg gacgccggtt 30

Claims (6)

1. A glutamine transaminase-producing strain TGS104, wherein the strain is enhanced by an enhancement method1- phosphofructokinaseGeneSMDS_3792Is obtained at the transcription level of (a);
the strain utilizes an artificial strong promoter in Streptomyces mobaraensis C2kasOp* Overexpression of C2 from Streptomyces mobaraensis1-phosphofructokinaseGeneSMDS_3792Obtaining the product; the classification name of the Streptomyces mobaraensis C2 is Streptomyces mobaraensisStreptomyces mobaraensis C2The compound is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020194, the date of preservation is 6 months and 10 days in 2020;
the strain1-phosphofructokinaseGeneSMDS_3792Over-expression; the strain contains an artificial strong promoterkasOp* Overexpression of C2 from Streptomyces mobaraensis1-phosphofructokinaseGeneSMDS_3792Is a gene expression cassette; the said1-phosphofructokinaseGeneSMDS_3792The sequence of (2) is shown as SEQ ID NO. 1.
2. The high glutamine transaminase producing strain TGS104 of claim 1, wherein the expression cassette contains an artificial strong promoterkasOp*、1-phosphofructokinaseGeneSMDS_3792Transcription termination sequences.
3. Enhancement1-phosphofructokinaseGeneSMDS_3792A method for increasing the level of glutamine transaminase fermentation by the transcription of a compound having a chain of a metallocene originUse of an artificial strong promoter in mould C2kasOp* Overexpression of C2 from Streptomyces mobaraensis1-phosphofructokinaseGeneSMDS_3792Thereby improving the output of glutamine transaminase; the classification name of the Streptomyces mobaraensis C2 is Streptomyces mobaraensisStreptomyces mobaraensis C2The compound is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020194, the date of preservation is 6 months and 10 days in 2020;
the said1-phosphofructokinaseGeneSMDS_3792Over-expression; the artificial strong promoterkasOp* Overexpression of C2 from Streptomyces mobaraensis1-phosphofructokinaseGeneSMDS_3792Is a gene expression cassette; the said1- phosphofructokinaseGeneSMDS_3792The sequence of (2) is shown as SEQ ID NO. 1;
the method comprises the following specific steps:
the first step: design and construction for overexpression1-phosphofructokinaseGeneSMDS_3792Is an integrated plasmid vector pTDS104;
and a second step of: insertion of a copy of C2 from Streptomyces mobaraensis on the recipient Streptomyces mobaraensis C2 chromosome using the integrative plasmid vector pTDS1041-phosphofructokinaseGeneSMDS_3792And obtaining a recombinant mutant strain TGS104 with gene overexpression through resistance and PCR verification screening; the construction method of the integrated plasmid vector pTDS104 comprises the following steps: amplified by PCR to give 954bp1-phosphofructokinaseGeneSMDS_3792PCR fragments of the sequences were ligated into the integrative plasmid pDR3-K by means of restriction ligationNdeI / EcoRI site to obtain the integrated plasmid vector pTDS104;
and a third step of: will be cultivated1-phosphofructokinaseGeneSMDS_3792Spores of the over-expression mutant strain TGS104 are inoculated in a seed culture medium, cultured for 20-24 h at 25-35 ℃ and 180-220rpm, transferred into a fermentation culture medium at 8-15% of inoculum size, fermented for 28-32h at 25-35 ℃ and 180-220rpm, and fermentation liquor is collected and subjected to enzyme activity detection.
4. A method as claimed in claim 3Wherein the seed culture medium comprises glycerol 1-3w/v%, yeast extract 0.5-1w/v%, fish meal peptone 2-3 w/v%, mgSO 4 •7H 2 O 0.1-0.5 w/v%，K 2 HPO 4 •3H 2 O 0.1-0.5 w/v%，pH 7.4；
The fermentation medium comprises glycerol 1-3w/v%, yeast extract 0.5-1w/v%, fish meal peptone 2-3 w/v%, and MgSO 4 •7H 2 O 0.1-0.5 w/v%，K 2 HPO 4 •3H 2 0.1-0.5w/v% of O, 0.1-0.3w/v% of fermentation promoter and pH 7.4.
5. A process for the preparation of a glutamine transaminase high producing strain TGS104, characterized in that the process comprises the following steps:
the first step: design and construction for overexpression1-phosphofructokinaseGeneSMDS_3792Is an integrated plasmid vector pTDS104; the construction method of the integrated plasmid vector pTDS104 comprises the following steps: amplified by PCR to give 954bp1- phosphofructokinaseGeneSMDS_3792PCR fragments of the sequences were ligated into the integrative plasmid pDR3-K by means of restriction ligationNdeI / EcoRI site to obtain the integrated plasmid vector pTDS104;
and a second step of: insertion of a copy of C2 from Streptomyces mobaraensis on the recipient Streptomyces mobaraensis C2 chromosome using the integrative plasmid vector pTDS1041-phosphofructokinaseGeneSMDS_3792And obtaining a recombinant mutant strain TGS104 with gene overexpression through resistance and PCR verification screening; wherein the classification name of the Streptomyces mobaraensis C2 is Streptomyces mobaraensisStreptomyces mobaraensis C2The compound is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020194, the date of preservation is 6 months and 10 days in 2020;
the said1-phosphofructokinaseGeneSMDS_3792Over-expression; the strain contains an artificial strong promoterkasOp* Overexpression of C2 from Streptomyces mobaraensis1-phosphofructokinaseGeneSMDS_3792Is a gene expression cassette; the said1-phosphofructokinaseGeneSMDS_3792The sequence of (2) is shown as SEQ ID NO. 1.
6. Use of the glutamine transaminase high producing strain TGS104 of claim 1 or 2 for increasing the fermentation yield of glutamine transaminase.
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