阅读说明：本技术 2-脱氧-3脱氧磷酸葡萄糖酸醛缩酶在控制1-脱氧野尻霉素产量中的应用 (Application of 2-deoxy-3-deoxyphosphogluconate aldolase in controlling yield of 1-deoxynojirimycin ) 是由 陈守文 蔡冬波 芦玉 冀志霞 马昕 陈建刚 李鑫 于 2020-05-11 设计创作，主要内容包括：本发明属于基因工程领域,具体涉及2-脱氧-3脱氧磷酸葡萄糖酸醛缩酶在控制1-脱氧野尻霉素产量中的应用,本发明通过基因工程的方法敲除了解淀粉芽胞杆菌LX-12的基因组内的<I>kdgA</I>基因,成功得到了缺失<I>kdgA</I>基因的解淀粉芽胞杆菌LX-12Δ<I>kdgA</I>。相对于解淀粉芽胞杆菌LX-12,本发明所构建得到的工程菌株LX-12ΔkdgA发酵后1-脱氧野尻霉素产量至少提高了21%,为提高1-脱氧野尻霉素产量提供了一种新的方法。(The invention belongs to the field of genetic engineering, and particularly relates to application of 2-deoxy-3-deoxy-phosphogluconate aldolase in controlling the yield of 1-deoxy nojirimycin kdgA Gene, successfully obtained deletion kdgA Resolution of genesBacillus amyloliquefaciens LX-12 delta kdgA . Compared with Bacillus amyloliquefaciens LX-12, the yield of the 1-deoxynojirimycin is improved by at least 21% after the constructed engineering strain LX-12 delta kdgA is fermented, and a novel method is provided for improving the yield of the 1-deoxynojirimycin.)

1. The application of knocking out, inactivating or reducing the activity of 2-deoxy-3 deoxyphosphogluconate aldolase in improving the capability of producing 1-deoxynojirimycin by fermenting bacillus amyloliquefaciens, wherein the bacillus amyloliquefaciens is a strain for producing the 1-deoxynojirimycin, and the amino acid sequence of the enzyme is shown in SEQ ID No. 2.

2. The use of claim 1, wherein the bacillus amyloliquefaciens has a preservation number of CCTCC NO: m2015234.

3. The application of claim 1, the application process comprising:

(1) the preservation number is CCTCC NO: m2015234 genome DNA of Bacillus amyloliquefaciens LX-12 is used as a template, and PCR amplification is carried outkdgAUpstream homology arms of genes andkdgAa downstream homology arm of a gene;

(2) by overlap extension PCRkdgAUpstream homology arms of genes andkdgAthe downstream homology arms of the genes are connected together to form a target gene segment;

(3) by usingSacI andXbai, carrying out double enzyme digestion on a target gene fragment by using restriction endonuclease to obtain an enzyme digestion gene fragment;

(4) preparation of plasmid T2(2) -ori and useSacI andXbacarrying out double enzyme digestion on plasmid T2(2) -ori by using I restriction enzyme to obtain a linear plasmid fragment;

(5) connecting the enzyme-digested gene fragment obtained in the step (3) with the linear plasmid fragment obtained in the step (4) by using DNA ligase to obtain a knockout plasmid T2(2) -deltakdgA；

(6) The knockout plasmid T2(2) - Δ was ligatedkdgATransferring the bacillus amyloliquefaciens into the bacillus amyloliquefaciens LX-12, and screening by taking kanamycin as a screening marker to obtain a positive transformant;

(7) carrying out colony PCR detection on the positive transformant after several times of transfer culture to obtainkdgAUpstream homology arms of genes orkdgAThe downstream homology arm of the gene and the genome DNA of the bacillus amyloliquefaciens LX-12 generate a positive single-exchange binder strain with single exchange;

(8) selectingkdgAUpstream homology arms and amylolytic buds of genesPositive single crossover binder strains producing single crossover from genomic DNA of Bacillus sp LX-12 andkdgAthe downstream homologous arm of the gene and the positive single-exchange conjugator strain which generates single exchange with the genome DNA of the bacillus amyloliquefaciens LX-12 are mixed and inoculated in a culture medium without kanamycin for a plurality of times of transfer culture, and the strain is screened by a PCR method to obtain the strain with knockoutkdgAGenetically Bacillus amyloliquefaciens (Bacillus amyloliquefaciens）LX-12ΔkdgA。

4. The use of claim 1, which comprises fermenting 1-deoxynojirimycin produced by the engineered strain of Bacillus amyloliquefaciens prepared in claim 3 in a fermentation medium of the formula:

60-100g/L of soybean meal; 50-80g/L corn starch; 0.5-1.0g/L KH₂PO₄；0.3-0.6g/L MgSO₄·7H₂O；0.1-0.3 g/LFeSO₄·7H₂O；0.01-0.05 g/L MnSO₄·H₂O and the balance of water.

Technical Field

The invention belongs to the field of microbial genetic breeding, and particularly relates to application of 2-deoxy-3-deoxyphosphogluconate aldolase in controlling the yield of 1-deoxynojirimycin.

Background

With the change and development of production life style of people, diabetes has become the third killer threatening human beings after cardiovascular and cerebrovascular diseases and tumors, and brings great threat to global health and economy, and the problem is particularly serious in Asia and particularly China. 1-deoxynojirimycin (1-deoxynojirimycin) is a polyhydroxy piperidine alkaloid and has strong alpha-glucosidase inhibition activity. Therefore, 1-deoxynojirimycin and its derivatives are widely regarded as a potent glycosidase inhibitor in drug development. In previous researches, 1-deoxynojirimycin is mostly extracted from mulberry leaves, and is high in cost and not beneficial to industrial production, so that the synthesis of 1-deoxynojirimycin by using microorganisms becomes a research hotspot at present. However, the current microbial synthesis of 1-deoxynojirimycin has low yield and cannot meet the market demand.

KdgA encodes 2-deoxy-3 deoxy-phosphogluconate aldolase, and the regulation network and specific regulation genes thereof in Bacillus amyloliquefaciens have not been reported yet. According to the bacillus amyloliquefaciens 1-deoxynojirimycin preparation, the 2-dehydrogenation-3-deoxyglucuronosyl phosphate aldolase gene kdgA in the bacillus amyloliquefaciens LX-12 is knocked out, so that the bacillus amyloliquefaciens is not expressed in the bacillus amyloliquefaciens, and the yield of the bacillus amyloliquefaciens 1-deoxynojirimycin is improved. The invention improves the yield of 1-deoxynojirimycin by knocking out the 2-dehydro-3-deoxyglucuronosyl phosphate aldolase gene kdgA for the first time, and provides a new method for the high yield of 1-deoxynojirimycin.

Disclosure of Invention

The invention aims to provide application of knocking out, inactivating or reducing activity of 2-deoxy-3-deoxyphosphogluconate aldolase in improving capability of producing 1-deoxynojirimycin by fermentation of bacillus amyloliquefaciens, wherein an amino acid sequence of the enzyme is shown in SEQ ID NO. 2.

In order to achieve the purpose, the invention adopts the following technical measures:

the application of knocking out, inactivating or reducing the activity of 2-deoxy-3-deoxyphosphogluconate aldolase in improving the capability of Bacillus amyloliquefaciens to ferment and produce 1-deoxynojirimycin comprises knocking out, mutating or silencing a kdgA gene of 2-deoxy-3-deoxyphosphogluconate aldolase edited in the Bacillus amyloliquefaciens by using the conventional technology of the invention so as to reduce or inactivate the enzyme activity of the Bacillus amyloliquefaciens, wherein the knocked-out strain improves the fermentation production yield of the Bacillus amyloliquefaciens 1-deoxynojirimycin, and the amino acid sequence of the enzyme is shown in SEQ ID No. 2;

the bacillus amyloliquefaciens is a bacterial strain for producing 1-deoxynojirimycin.

In the above application, preferably, the collection number of the bacillus amyloliquefaciens LX-12 is CCTCC NO: m2015234.

In the application, the method for knocking out the gene kdgA of the bacillus amyloliquefaciens comprises the following steps:

(1) taking the genome DNA of bacillus amyloliquefaciens LX-12(CCTCC NO: M2015234) as a template, and carrying out PCR amplification to obtain an upstream homologous arm of the kdgA gene and a downstream homologous arm of the kdgA gene;

(2) connecting an upstream homology arm of the kdgA gene and a downstream homology arm of the kdgA gene together by overlap extension PCR to form a target gene fragment;

(3) carrying out double enzyme digestion on the target gene fragment by adopting SacI and XbaI restriction endonucleases to obtain an enzyme digestion gene fragment;

(4) carrying out double enzyme digestion on plasmid T2(2) -ori by adopting SacI and XbaI restriction enzymes to obtain a linear plasmid fragment;

(5) connecting the enzyme-digested gene fragment obtained in the step (3) with the linear plasmid fragment obtained in the step (4) by using DNA ligase to obtain a knockout plasmid T2(2) -delta kdgA;

(6) transferring the knockout plasmid T2(2) -delta kdgA into Bacillus amyloliquefaciens LX-12, and screening by taking kanamycin as a screening marker to obtain a positive transformant;

(7) after the positive transformant is subjected to transfer culture for several times, colony PCR detection is carried out to obtain a positive single-exchange conjugator strain which generates single exchange between the upstream homologous arm of the kdgA gene or the downstream homologous arm of the kdgA gene and the genome DNA of the bacillus amyloliquefaciens LX-12;

(8) selecting an upstream homology arm of a kdgA gene and a positive single-exchange binder strain which generates single exchange with the genomic DNA of the Bacillus amyloliquefaciens LX-12, mixing a downstream homology arm of the kdgA gene and a positive single-exchange binder strain which generates single exchange with the genomic DNA of the Bacillus amyloliquefaciens LX-12, inoculating the mixture into a culture medium which does not contain kanamycin, performing transfer culture for a plurality of times, and screening by a PCR (polymerase chain reaction) method to obtain the Bacillus amyloliquefaciens LX-12 delta kdgA of which the kdgA gene is knocked out; in the above application, preferably, when 1-deoxynojirimycin is produced by fermentation of a bacillus amyloliquefaciens engineering strain with a kdgA gene knocked out in the application process, the formula of a fermentation medium is as follows:

60-100g/L of soybean meal; 50-80g/L corn starch; 0.5-1.0g/L KH₂PO₄；0.3-0.6g/L MgSO₄·7H₂O；0.1-0.3g/LFeSO₄·7H₂O；0.01-0.05g/L MnSO₄·H₂O and the balance of water.

Compared with the prior art, the invention has the following advantages:

compared with the Bacillus amyloliquefaciens LX-12, the yield of the 1-deoxynojirimycin of the Bacillus amyloliquefaciens LX-12 delta kdgA constructed by the invention is improved by more than 20%. The research result of the invention shows that: the kdgA gene in the genome DNA of the bacillus amyloliquefaciens LX-12 is knocked out, so that a novel method is provided for improving the yield of the 1-deoxynojirimycin.

Drawings

FIG. 1 is an agarose gel of the upstream homology arm of the kdgA gene and the downstream homology arm of the kdgA gene obtained in step (1) of example 1;

wherein, Lane M is DNA marker, Lane 1 is the upstream homology arm of the kdgA gene, and Lane 2 is the downstream homology arm of the kdgA gene.

FIG. 2 is a diagram showing confirmation of colony PCR of the knockout plasmid T2(2) - Δ kdgA obtained in step (5) of example 1;

wherein, lane M is DNA marker, lane 1 is the band of knockout plasmid T2(2) - Δ kdgA for colony PCR verification.

FIG. 3 is a test band of Bacillus amyloliquefaciens LX-12. delta. kdgA in which the kdgA gene was knocked out, obtained in step (8) of example 1.

Wherein, Lane M is DNA marker, Lane 1 is the verification band of Bacillus amyloliquefaciens LX-12. delta. kdgA, and Lane 2 is the control band of Bacillus amyloliquefaciens LX-12.

Wherein, the corresponding molecular weights of the top to bottom bands in the DNA marker lane are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100 bp.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The technical scheme of the invention is a conventional scheme in the field if not specifically stated; the reagents or materials, if not specifically mentioned, are commercially available. The invention takes the kdgA gene of knock-out Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LX-12 as an example to prove the effect of 2-deoxy-3 deoxyphosphogluconate aldolase in controlling the yield of 1-deoxynojirimycin; other means conventional in the art, such as kdgA gene siRNA, kdgA gene RNAi lentivirus, kdgA gene mutation, can increase the ability of bacillus amyloliquefaciens to produce 1-deoxynojirimycin if the enzyme activity of 2-deoxy-3 deoxyphosphogluconate aldolase is lost or decreased.

The bacillus amyloliquefaciens is a strain which can produce 1-deoxynojirimycin by a wild type.