A method for improving 1-deoxynojirimycin biological production strain, ybbA gene and application

Abstract

The present invention belongs to the field of industrial biotechnology, and discloses a method for improving 1-deoxynojirimycin bioproduction strains, ybbA gene and application, the method comprising introducing the ybbA gene, an expression cassette comprising the ybbA gene or a vector comprising the ybbA gene into Bacillus amyloliquefaciens. The present invention uses precise gene knockout and backfilling technology to conduct a comprehensive functional analysis of the ybbA gene in Bacillus amyloliquefaciens. It aims to clarify the exact role of the ybbA gene in DNJ biosynthesis and provide a scientific basis and theoretical support for the development of novel and efficient DNJ production strategies. The research results of the present invention are expected to break through the bottleneck of DNJ yield improvement and open up new paths and methods for related research in the field of industrial biotechnology.

Claims

1. A kind of utilization ybbA A method for genetically improving a 1-deoxynojirimycin bioproduction strain, characterized by: The method comprises the ybbA Gene, comprising said ybbA The gene expression cassette or the ybbA The gene vector is introduced into Bacillus amyloliquefaciens; The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, classification name is: Bacillus amyloliquefaciens , the deposit number is: CGMCC No.29550; described ybbA The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A ybbA A recombinant strain of Bacillus amyloliquefaciens for the bioproduction of 1-deoxynojirimycin by a modified gene; ybbA The nucleotide sequence of the gene is shown in SEQ ID No. 1; The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, classification name is: Bacillus amyloliquefaciens , the deposit number is: CGMCC No.29550.

3. The method for constructing a recombinant strain according to claim 2, wherein: The steps include: First, electroporation ybbA The gene overexpression plasmid pLH2388 was introduced into Bacillus amyloliquefaciens LH-2 competent cells, and preliminary transformants were obtained by screening using LB medium containing 5 μg / mL chloramphenicol. The transformants were passaged at 42°C for three times, with transfer every 12 hours, to induce single exchange between the plasmid and the genome, and integration was confirmed by PCR. Subsequently, further high-temperature passage was performed to promote double exchange recombination, and double exchange strains were screened by applying LB medium containing 25 μg / mL 5-fluorouracil, and PCR was used to confirm the integration. ybbA Stable integration of gene overexpression structure; finally, sensitive strains were screened by chloramphenicol sensitivity test, residual plasmids were excluded, and the accuracy of gene editing was verified by sequencing to obtain genetically stable ybbA Overexpression recombinant strains.

4. Use of the recombinant strain according to claim 3 in the fermentation production of 1-deoxynojirimycin.

5. A method for producing 1-deoxynojirimycin by fermentation using the recombinant strain according to claim 3, characterized in that: The steps include: The recombinant strain was removed from the ultra-low temperature freezer and streaked onto LB solid medium. The culture was incubated at 37°C for 12 hours to grow a single strain. The single colony was transferred to 5 mL of fresh LB liquid medium and incubated at 37°C and 200 rpm for 12 hours to prepare a seed culture. The seed solution of the cultured bacteria was inoculated into the fermentation medium at an inoculum rate of 4% and incubated at 37°C and 100 rpm for 72 hours to ferment 1-deoxynojirimycin. The formula of the fermentation medium used is: (NH4)2SO4: 4 g / L, lactose: 25 g / L, K2HPO4: 14 g / L, KH2PO4: 6 g / L, MgSO4·7H2O: 0.2 g / L, CaCl2: 0.15 g / L, MnSO4·H2O: 1.9 mg / L, FeCl3·6H2O: 37.8 mg / L, ZnCl2: 7 mg / L, the solvent is water, pH 7.0, and sterilized at 121 ℃ for 20 min.

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