Transaminase GabT1 mutant S106I, construction method thereof and application of transaminase GabT1 mutant S106I in biosynthesis of 1-deoxynojirimycin

Abstract

The invention belongs to the technical field of biology, and discloses a transaminase GabT1 mutant S106I, a construction method thereof and application of the transaminase GabT1 mutant S106I in biosynthesis of 1-deoxynojirimycin. According to the invention, the 106th serine near the molecular catalytic domain of the transaminase GabT1 derived from bacillus amyloliquefaciens is mutated into isoleucine in a site-directed mutagenesis manner, so that the catalytic efficiency of the transaminase GabT1 is remarkably improved, the problem that the catalytic efficiency of the transaminase GabT1 on fructose-6-phosphoric acid is not high at present is solved, and the application of the transaminase GabT1 to the fructose-6-phosphoric acid is promoted. The yield of 1-deoxynojirimycin synthesized by the mutant GabTS106I is increased by 43.87% compared with that of a wild type. The 1-deoxynojirimycin fermentation production capability of the transaminase GabT1 mutant and the engineering strain obtained by the invention is greatly improved, and the transaminase GabT1 mutant and the engineering strain have a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and protein engineering technology, specifically relating to the transaminase GabT1 mutant S106I, its construction method, and its application in the biosynthesis of 1-deoxynojirimycin. Background Technology

[0002] GabT1, a transaminase, is a key enzyme in the synthesis of 1-deoxynojirimycin, catalyzing the transamination of F-6-P to ADM. Enhancing the expression of the GabT1 gene and improving the enzyme's affinity for the substrate F-6-P and conversion efficiency are effective ways to increase the yield of 1-deoxynojirimycin. 1-Deoxynojirimycin (1-DNJ) is an important α-glucosidase inhibitor (α-GI) isolated from mulberry leaves and can be used as a raw material for treating non-addictive (type II) diabetes. Furthermore, 1-deoxynojirimycin possesses various biological activities, such as anti-obesity and antiviral activity. Summary of the Invention

[0003] This invention aims to improve enzyme-substrate affinity, which is an effective measure to enhance enzyme activity. By screening different transaminase mutants, the efficient production of 1-deoxynojirimycin is achieved. In particular, a transaminase GabT1 mutant was obtained through site-directed mutagenesis. When applied to 1-deoxynojirimycin, the yield reached 318.47 mg / L, laying the foundation for the industrial production of 1-deoxynojirimycin.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a GabT1 transaminase mutant S106I, the amino acid sequence of which is shown in SEQ ID NO.1; using the original GabT1 transaminase gene as a template, and using the mutant primer GabT1 S106I -F and GabT1 S106I -R, to obtain the GabT1 mutant gene for transaminase. gabT1 S106I mutant genes gabT1 S106I The nucleotide sequence is shown in SEQ ID NO.2; GabT1 S106I -F (SEQ ID NO.12): GTACACCCTAAAGTCAGCATCGGTTCCGGTGCAAATGAA GabT1 S106I -R (SEQ ID NO.13):TTCATTTGCACCGGAACCGATGCTGACTTTAGGGTGTAC Secondly, the present invention provides a recombinant expression plasmid vector pHY-P43U12 - gabT1 S106I -T amyl The promoter P, with a nucleotide sequence as shown in SEQ ID NO.3, is used. 43 Combined with UTR12 and the transaminase GabT1 mutant gene gabT1 S106I And the amylase gene in Bacillus licheniformis WX-02 amyL Termination sub-T amyL The target sequence P formed by concatenation 43U12 - gabT1 S106I -T amyl Using pHY300 as a vector, the recombinant expression plasmid vector pHY-P was constructed. 43U12 - gabT1 S106I -T amyl .

[0005] Thirdly, the present invention provides a recombinant genetically engineered bacterium, *Bacillus amyloliquefaciens*, which comprises the aforementioned recombinant expression plasmid vector pHY-P. 43U12 - gabT1 S106I -T amyl .

[0006] Preferably, the expression vector is pHY300PLK; Preferably, the recombinant genetically engineered bacterium Bacillus amyloliquefaciens originates from Bacillus amyloliquefaciens LX-12.

[0007] This invention also provides a method for constructing the above-mentioned recombinant genetically engineered bacterium Bacillus amyloliquefaciens, comprising the following steps: 1. The GabT1 mutant gene of transaminase was cloned using a mutant vector as a template. gabT1 S106I .

[0008] 2. Synthesize nucleotide sequences as shown in SEQ ID NO.3, P 43 Promoter and UTR12 combination P 43U12 ; 3. P 43U12 and gabT1 S106I The fragments were ligated after being digested with the same enzyme, and the ligation product was purified and cloned into the pHY300 expression vector.

[0009] 4. Recombinant genetically engineered Bacillus amyloliquefaciens was constructed by transferring it into Bacillus amyloliquefaciens LX-12.

[0010] Finally, the present invention also provides the above-mentioned transaminase GabT1 mutant and the above-mentioned recombinant expression plasmid vector pHY-P 43U12 - gabT1 S106I -T amyl Or the application of the above-mentioned recombinant genetically engineered Bacillus amyloliquefaciens in the preparation and production of 1-deoxynojirimycin.

[0011] Compared with the prior art, the present invention has the following technical advantages: The present invention uses site-directed mutagenesis to induce a mutation in bacteria derived from Bacillus amyloliquefaciens (BAM). Bacillus amyloliquefaciens The mutation of serine at position 106 near the catalytic domain of the transaminase GabT1 molecule to isoleucine significantly improves the catalytic efficiency of GabT1, solving the current problem of low catalytic efficiency of GabT1 for fructose-6-phosphate. The mutant GabT1... S106I The yield of 1-deoxynojirimycin was increased by 43.87% compared to the wild type. Specifically, a high-yielding 1-deoxynojirimycin mutant, S106I, was obtained by site-directed mutagenesis of the transaminase GabT1 gene. Subsequently, P was added... 43 Combining the promoter with UTR12 further enhances the expression of transaminase GabT1. The method is simple and easy to implement, and the highest 1-deoxynojirimycin content of the resulting recombinant transaminase GabT1 mutant strain reached 318.47 mg / L. Attached Figure Description

[0012] Figure 1 The recombinant expression vector pHY-P of the transaminase GabT1 gene 43U12 - gabT1 S106I -T amyl Atlas. Detailed Implementation

[0013] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0014] The LB medium used in the examples contained: 70 g / L peanut meal, 50 g / L corn starch, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and 0.2 g / L ferrous sulfate. The LB medium plates were made by adding agar to the above LB medium.

[0015] The method for preparing the engineered strain of Bacillus amyloliquefaciens used in the examples is as follows: (1) Take a tube of competent cells (DH5α, purchased from Wuhan Qingke Biotechnology Co., Ltd.) and add 5 μL of the following 5 recombinant plasmid DNA (50 ng / μL). Transfer the cell mixture to a pre-cooled electroporation cuvette. After electroporation, quickly add 800 μL of electroporation recovery medium (LB medium). Recover the cells at 37°C on a shaker at 110 r / min for 3 h. Spread the cells on a tetracycline-resistant plate containing 20 μg / mL and incubate at 37°C for 16-20 h. Screen for transformants.

[0016] (2) Strawberry colonies of transformants were picked and streaked onto LB agar plates containing antibiotics and incubated overnight. A suitable amount of colonies was then transferred to 1.5 mL EP tubes containing 30 μL of the culture, mixed, and incubated in a boiling water bath for 20 min. After incubation, the tubes were centrifuged at 10000 r / min for 30 s, and the supernatant was collected as a template for colony PCR. Positive clones were verified by PCR and sequencing, and the corresponding strain was named LX-12 / pHY-P. 43U12 - gabT1 -T amyl LX-12 / pHY- gabT1 Q137W LX-12 / pHY- gabT1 S106I LX-12 / pHY- gabT1 L184N LX-12 / pHY- gabT1 Y121A LX-12 / pHY- gabT1 T47N Store the glycerol tubes at -80℃ for later use, with the control strain being LX-12 / pHY-P. 43U12 - gabT1 -T amyl .

[0017] (3) The optimal engineered strain LX-12 / pHY-P was obtained through fermentation verification of different mutant strains constructed by protein engineering. 43U12 - gabT1 S106I -T amyl Store the glycerol tube at -80°C for later use.

[0018] Example 1: Recombinant plasmid pHY-P 43U12 - gabT1 -T amyl Construction GabT1-F and GabT1-R were used to isolate Bacillus amyloliquefaciens LX-12 (Bacillus cillus) Amyloliquefaciens LX-12; CCTCCNO: M2015234; A raspberry fermentation pulp and its preparation method and application CN109908060A; cloning transaminase in (amyloliquefaciens LX-12; CCTCCNO: M2015234; A raspberry fermentation pulp and its preparation method and application CN109908060A;) gabT1 Genes. Bacillus subtilis Using the 168 genome as a template, P 43U12 -F and P 43U12 -R is the primer amplification to obtain P 43 Promoter combined with UTR12; T amyL -F and T amyL -R is a primer used to amplify the amylase gene from Bacillus licheniformis WX-02. amyL Termination sub-T amyL Using P 43U12 -F and T amyL -R is a primer, which will bind P 43U12 , gabT1 T amyL Three fragments were subjected to overlap extension PCR to obtain the fusion fragment P. 43U12 - gabT1 -T amyL .

[0019] Overlap extension PCR process and system: P 43U12 , gabT1 T amyL Three fragments were obtained through primer P 43U12 -F、T amyL -R performs overlap extension PCR, P 43U12 , gabT1 T amyL The three fragments were ligated together. The reaction mixture was 50 μL, containing 45 μL of PCR high-protection enzyme and primer P. 43U12、 T amyL -R 2μL each, P 43U12 , gabT1 T amyL Take 1 μL of the mixture of the three fragments (the primer used is P). 43U12 -F, as shown in SEQ ID NO.4 and T amyL -R (as shown in SEQ ID NO. 9), constitutes the target gene fragment (2063 bp, as shown in SEQ ID NO. 3), and the sequence of this target gene fragment is: P 43U12 promoter- gabT1 Gene-T amyL Termination of contract.

[0020] Using the commonly used plasmid pHY300PLK as a template, and pHY-AmpR-GF and pHY-AmpR-GR as primers, whole-plasmid PCR amplification was performed to obtain the linearized pHY300PLK vector. After electrophoresis, the amplification products were separated by agarose gel electrophoresis from the target backbone (linearized pHY300PLK vector) and the DNA fragment (fusion fragment P). 43U12 - gabT1 -T amyL The PCR products were purified and recovered using the OMEGA Gel Extraction Kit. The fusion fragment P was then cloned using the ClonExpress II one-step cloning kit. 43U12 - gabT1 -T amyL The recombinant plasmid pHY-P was fused with the linearized vector pHY300PLK to obtain the recombinant plasmid pHY-P. 43U12 - gabT1 -T amyl (like Figure 1 (As shown).

[0021] P 43U12 -F (SEQ ID NO.4): TTTTTATAACAGGAATTCTGATAGGTGGTATGTTTTCG P 43U12 -R (SEQ ID NO.5): TATATATTCCTCCTTTTCTAATATACTTTATATTTTACATAATCGCGCGCT GabT1-F (SEQ ID NO.6): AGAAAGGAGGAATATATAGTGGTATTTGTGGGAACG GabT1-R (SEQ ID NO.7): GAAATCCGTCCTCTGCTCTTTCACTTGATTTCCTCCAA T amyL -F (SEQ ID NO.8): TTGGAGGAAATCAAGTGAAAGAGCAGAGAGGACGGAT T amyL -R (SEQ ID NO.9): GTAAACTTGGTCTGACAGCGCAATAATGCCGTCGCA pHY-AmpR-GF (SEQ ID NO. 10): CTGTCAGACCAAGTTTACTCATAT pHY-AmpR-GR (SEQ ID NO. 11): AAAGGAATATAATCATCCACTCAA The fused recombinant plasmid pHY-P 43U12 - gabT1 -T amyl Transition to receptive state E. coli DH5α was screened for positive colonies using LB agar plates containing tetracycline. After overnight incubation at 37°C on a shaker, plasmid pHY-P was extracted. 43U12 - gabT1 -T amyl And then, sequencing verification was performed.

[0022] Example 2: Construction of five GabT1 mutant transaminases With plasmid pHY-P 43U12 - gabT1 -T amyl Using the template, the designed mutant primers are used to amplify the mutant backbone.

[0023] The mutation primer sequences designed for the five mutation sites are as follows: GabT1 S106I -F (SEQ ID NO.12): GTACACCCTAAAGTCAGCATCGGTTCCGGTGCAAATGAA GabT1 S106I -R (SEQ ID NO.13): TTCATTTGCACCGGAACCGATGCTGACTTTAGGGTGTAC GabT1 Q137W -F (SEQ ID NO.14):TTTCGAAGCCACCTCGGAtggACGTATATGACTTCCGCT GabT1 Q137W R (SEQ ID NO.15):AGCGGAAGTCATATACGTccaTCCGAGGTGGCTTCGAAA G abT1 L184N -F (SEQ ID NO.16): CCTGATTCATGCGGTATGaacTGCGTAGAAAGAATT G abT1 L184N -R (SEQ ID NO.17): ATTAATTCTTTCTACGCAgttCATACCGCATGAATC G abT1 Y121A-F (SEQ ID NO.18):ATTAAAATGGCTCAGTATGCGTCCGGAAAAACCGAT G abT1 Y121A -R (SEQ ID NO.19):GGTTTTTCCGGACGCATACTGAGCCATTTTAATAGC G abT1 T47N -F (SEQ ID NO.20):ATTGATTGCGCTTCTGCCaacTTTAACTTGAATTTA G abT1 T47N -R (SEQ ID NO.21):TCCTAAATTCAAGTTAAAgttGGCAGAAGCGCAATC The designed primers for colony PCR validation are as follows: pHY-amp-F (SEQ ID NO:22):ACTCATTCCCTGATCTCGAC pHY-amp-R (SEQ ID NO. 23):TTTCGTTTTCAGAGCAAGAG The target backbone DNA was separated by agarose gel electrophoresis and purified using the OMEGA Gel Extraction Kit. A small amount of mutant backbone DNA was then added to *E. coli* DH5α competent cells, and the mutant vector was circularized using the strain's self-repair system. The bacterial cells were screened on LB agar plates containing Tet-resistant culture medium and incubated at 37°C. Colony PCR was performed to verify the transformants using pHY-amp-F and pHY-amp-R primers. If the target fragment size (1983 bp) was correct, sequencing could proceed. Analysis of the sequencing results showed that if the sequence matched the design, the mutant vector was successfully constructed.

[0024] The five successfully constructed mutant plasmids were transformed into competent cells. E. coli DH5α was screened for positive colonies using LB agar plates containing tetracycline. After overnight incubation at 37°C on a shaker, mutant plasmids were extracted and sequenced for verification. Five correctly sequenced mutant plasmids were electroporated into *Bacillus amyloliquefaciens* strain LX-12. After selecting transformants to verify correctness, the five mutant strains (LX-12 / pHY-P) were then electroporated into *Bacillus amyloliquefaciens* strain LX-12. 43U12 - gabT1 -T amyl LX-12 / pHY- gabT1 Q137W LX-12 / pHY- gabT1S106I LX-12 / pHY- gabT1 L184N LX-12 / pHY- gabT1 Y121A LX-12 / pHY- gabT1 T47N Inoculate into LB medium, incubate at 37°C for 12 hours, and store the glycerol tubes at -80°C for later use.

[0025] Example 3: Liquid fermentation of transaminase GabT1 mutant Colonies of the five mutant strains prepared in Example 2 were picked and inoculated into 5 mL of LB medium containing tetracycline (20 μg / mL), and cultured overnight at 37°C with shaking at 230 r / min. Then, 2% of the inoculum was transferred to 50 mL of fresh LB medium, and cultured until OD... 600 When the concentration was 1.0, 3% (1 mL) was inoculated into freshly prepared LB medium (70 g / L peanut meal, 50 g / L corn starch, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.2 g / L ferrous sulfate) for fermentation to produce 1-deoxynojirimycin at 37℃, 230 r / min, and for 60 h. The results are shown in Table 1, among which the mutant strain LX-12 / pHY-P 43U12 - gabT1 S106I -T amyl The highest yield of 1-deoxynojirimycin reached 318.47 mg / L, compared with the control group (LX-12 / pHY- gabT1 This represents a 43.87% increase compared to the previous year.

[0026] Table 1. Yield of 1-deoxynojirimycin in transaminase-producing GabT1 strain

[0027] SEQ ID NO.1 VVFVGTKEITNPDSLYYSVDDVVMERGEGIYLYDQEGNEYIDCASATFNNLNLGYGNKEVIDTVKEQADKLIHVTSSFQTDAVNKLAEKLVEIAPDNLTKVHPKVSIGSGANEGAIKMAQYYSGKTDVISLFRSHLGQTYMTSALSGNSFRKEPFPPQISFGLQVPDPYCSRCFYNQKPDSCGMLCVERINDFIEYASNGKIAAMIIEPISGNGGNVVPPKEYFKQLRKLCDEHDIALIFDEIQTGFGRTGKMFAADHFDVKPNMMTVAKGLGGTGFQVAATLTEDKYTGLPGYTHSFTYGSNVMAAAAACKTIDIMQRPGFLENVTTVGHYIMDRLETMKEDFAFISEVRGVGLMIGVEIVKENNEPDVELTNYIAKRAMDYGLILRTSRYGFGNVFKIRPPLTITLSEAEVLCYRLRKLLEEIK SEQ ID NO.2 SEQ ID NO.3

Claims

1. A GabT1 transaminase mutant S106I, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. The gene encoding the GabT1 mutant S106I of the transaminase as described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. Recombinant microorganisms expressing proteins as shown in SEQ ID NO.

1.

5. The recombinant microorganism according to claim 4, characterized in that, The recombinant microorganism is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciens The aforementioned Bacillus amyloliquefaciens has the ability to metabolize and produce 1-deoxynojirimycin.

6. The recombinant microorganism according to claim 5, characterized in that, The aforementioned Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LX-12.

7. The use of the mutant of claim 1, the gene of claim 2, or the recombinant microorganism of claim 4 or claim 5 in the preparation of transaminase GabT1.

8. The application according to claim 7, characterized in that, When using the gene described in claim 2 to prepare transaminase GabT1, the process involves inserting the gene shown in SEQ ID NO.2 into an expression vector and transforming it into Bacillus amyloliquefaciens for fermentation expression.

9. The use of the mutant of claim 1, the gene of claim 2, the expression vector of claim 4, or the recombinant microorganism of claim 5 in the preparation and production of 1-deoxynojirimycin.

10. The method for constructing recombinant microorganisms according to claim 5, characterized in that, Includes the following steps: 1) The GabT1 mutant gene of transaminase was cloned using a mutant vector as a template. gabT1 S106I ; 2) Synthesize nucleotide sequences as shown in SEQ ID NO.3, P 43 Promoter and UTR12 combination P 43U12 ; 3) P 43U12 and gabT1 S106I The fragments were ligated after being digested with the same enzyme, and the ligation product was purified and cloned into the pHY300 expression vector. 4) The recombinant genetically engineered bacterium Bacillus amyloliquefaciens was constructed by transferring it into Bacillus amyloliquefaciens LX-12.

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