阅读说明：本技术 强化表达葡萄糖6-磷酸脱氢酶的地衣芽胞杆菌在异源蛋白生产中的应用 (Application of bacillus licheniformis for enhancing expression of glucose 6-phosphate dehydrogenase in production of heterologous protein ) 是由 陈守文 蔡冬波 莫非 吴晗嘉 饶忆 于 2019-12-20 设计创作，主要内容包括：本发明涉及地衣芽胞杆菌基因工程改造和蛋白高效表达领域,公开了强化表达葡萄糖6-磷酸脱氢酶Zwf的地衣芽胞杆菌在异源蛋白生产中的应用,本发明通过基因工程的方法强化表达了地衣芽胞杆菌WX-02中葡萄糖6-磷酸脱氢酶基因<I>zwf</I>,成功得到了基因<I>zwf</I>强化表达的地衣芽胞杆菌WX-02-zwf,在此基础上转入胰蛋白酶SgT游离表达质粒pHY-AprE、纳豆激酶表达载体pP43SacCNK、碱性蛋白酶游离表达载体pHY-AprE。相对于对照菌株,本发明所构建得到的工程菌株在上述蛋白酶酶活提升方面效果显著,酶活均提高了28%以上。(The invention relates to the field of Bacillus licheniformis genetic engineering modification and protein high-efficiency expression, and discloses application of Bacillus licheniformis for enhancing expression of glucose 6-phosphate dehydrogenase Zwf in heterologous protein production zwf Successfully obtain the gene zwf The enhanced expression bacillus licheniformis WX-02-zwf is transferred into a trypsin SgT free expression plasmid pHY-AprE, a nattokinase expression vector pP43SacCNK and an alkaline protease free expression vector pHY-AprE on the basis. Relative to a reference strain, the engineering strain constructed by the invention isThe protease has remarkable effect in improving the enzyme activity, and the enzyme activity is improved by more than 28%.)

1. The application of Bacillus licheniformis for enhancing expression of glucose 6-phosphate dehydrogenase in production of heterologous protein, wherein the gene of glucose 6-phosphate dehydrogenase is shown in SEQ ID NO. 1.

2. The use according to claim 1, wherein the heterologous protein is trypsin, nattokinase or alkaline protease.

3. The use of claim 1, wherein the bacillus licheniformis is bacillus licheniformis WX-02.

4. The use of claim 1, wherein the construction method of Bacillus licheniformis for enhancing expression of glucose 6-phosphate dehydrogenase comprises the following steps:

(1) using genome DNA of bacillus licheniformis as a template, carrying out PCR amplification to obtain a zwf gene, an upstream homology arm A for integrating and expressing zwf and a downstream homology arm B for integrating and expressing zwf;

(2) connecting an upstream homology arm A, Zwf gene for integrating and expressing Zwf and a downstream homology arm B for integrating and expressing Zwf together by overlap extension PCR to form a target gene segment;

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

(4) preparing plasmid T2(2) -ori, and carrying out double digestion on the plasmid T2(2) -ori by using XbaI and BamHI 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 gene integration expression plasmid T2(2) -zwf;

(6) transferring the gene integration expression plasmid T2(2) -zwf into bacillus licheniformis, 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 a plurality of times, colony PCR detection is carried out to obtain a positive single-exchange conjugant strain which is used for integrating and expressing the upstream homology arm A of zwf or the downstream homology arm B of zwf and the genome DNA of the bacillus licheniformis WX-02 to generate single exchange;

(8) selecting a positive single-exchange binder strain for generating single exchange between an upstream homologous arm A for integrating and expressing Zwf and the genomic DNA of the bacillus licheniformis WX-02, and a positive single-exchange binder strain for generating single exchange between a downstream homologous arm B for integrating and expressing Zwf and the genomic DNA of the bacillus licheniformis WX-02, mixing, inoculating the positive single-exchange binder strain and the positive single-exchange binder strain in a culture medium without kanamycin, performing transfer culture for a plurality of times, and screening by a PCR (polymerase chain reaction) method to obtain the Zwf enhanced expression bacillus licheniformis;

zwf is glucose 6-phosphate dehydrogenase gene as described above.

5. The use according to claim 2, when bacillus licheniformis expressed by glucose 6-phosphate dehydrogenase is used for enhancing the production of trypsin, or nattokinase, or alkaline protease heterologous proteins, the fermentation medium used has a formula comprising: 10-20g/L glucose, 5-13g/L soybean peptone, 8-12g/L corn steep liquor, 7-11g/L yeast powder, 8-12g/L sodium chloride, 2-5g/L K₂HPO₄And 4-8g/L (NH)₄)₂SO₄(ii) a Or 5-10g/L bone peptone, 5-10g/L soybean peptone, 8-12g/L corn steep liquor, 7-11g/L yeast powder, 8-12g/L sodium chloride, 2-5g/L K₂HPO₄And 4-8g/L (NH)₄)₂SO₄。

Technical Field

The invention relates to the field of Bacillus licheniformis gene engineering modification and protein high-efficiency expression, in particular to application of Bacillus licheniformis for enhancing expression of glucose 6-phosphate dehydrogenase in heterologous protein production.

Background

Bacillus is a good host for high-efficiency expression of heterologous proteins, and Bacillus licheniformis is a common host strain for high-efficiency industrial production of heterologous proteins, especially protease at present. The protease is an enzyme preparation product which is most widely applied at present, and is widely applied to the fields of food, medicine, chemical industry, waste treatment and the like. Heterologous protein expression is an effective strategy to increase the expression level of a protein of interest. In recent years, more and more strategies have been developed to increase the level of synthesis of proteins of interest. However, there are many genes related to the synthesis and secretion of exogenous proteins in bacillus licheniformis, and the relationship between protein yield and genes is still unknown, and further research on the way of modifying related genes to obtain high-yield protein engineering bacteria is needed.

Glucose 6-phosphate dehydrogenase (Zwf) is a key enzyme in the carbon metabolic pathway, which is the first enzyme in the pentose phosphate pathway, and the high or low expression level plays an important role in the metabolic flux distribution of the pentose phosphate pathway. However, the expression level of Zwf in relation to the expression of heterologous proteins was not resolved and was not predictable.

The invention achieves the technical effect of improving the yield of heterologous proteins by intensively expressing the Zwf in the bacillus licheniformis, and shows that the intensified expression of the Zwf is an effective strategy for improving the production level of the heterologous proteins.

Disclosure of Invention

The invention aims to provide application of Bacillus licheniformis for enhancing expression of glucose 6-phosphate dehydrogenase in production of heterologous proteins, wherein the sequence of the glucose 6-phosphate dehydrogenase is shown in SEQ ID NO. 1.

Another object of the present invention is to provide a fermentation medium of Bacillus licheniformis which expresses glucose 6-phosphate dehydrogenase.

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

the application of the bacillus licheniformis for enhancing the expression of glucose 6-phosphate dehydrogenase (or abbreviated as Zwf) in the production of heterologous proteins comprises the steps of integrating and over-expressing Zwf in the bacillus licheniformis by using the conventional method to obtain the bacillus licheniformis for enhancing the expression of Zwf, and then transferring a heterologous protein expression vector into the bacillus licheniformis for protein expression, wherein the Zwf gene is shown in SEQ ID No. 1.

In the above-mentioned application, preferably, the heterologous protein is trypsin, nattokinase or alkaline protease;

in the above application, preferably, the bacillus licheniformis is bacillus licheniformis WX-02;

in the above application, preferably, the construction of the bacillus licheniformis strain for enhancing Zwf expression comprises the following steps:

(1) using genome DNA of Bacillus licheniformis as a template, amplifying a zwf gene (containing a promoter, a target gene and a terminator) by PCR, and integrating and expressing an upstream homology arm A and a downstream homology arm B of zwf;

(2) connecting an upstream homology arm A, Zwf gene for integrating Zwf and a downstream homology arm B for integrating Zwf together by overlap extension PCR to form a target gene segment;

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

(4) preparing plasmid T2(2) -ori, and carrying out double digestion on the plasmid T2(2) -ori by using XbaI and BamHI 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 gene integration expression plasmid T2(2) -zwf;

(6) transferring the gene integration expression plasmid T2(2) -zwf into bacillus licheniformis, 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 a plurality of times, colony PCR detection is carried out to obtain a positive single-exchange conjugant strain which is used for integrating and expressing the upstream homology arm A of zwf or the downstream homology arm B of zwf and the genome DNA of the bacillus licheniformis WX-02 to generate single exchange;

(8) and selecting a positive single-exchange binder strain for integrating and expressing the upstream homology arm A of Zwf and the genomic DNA of the bacillus licheniformis WX-02 to generate single exchange, mixing the positive single-exchange binder strain with the downstream homology arm B of Zwf and the genomic DNA of the bacillus licheniformis WX-02 to generate single exchange, inoculating the mixture to a culture medium without kanamycin, performing transfer culture for a plurality of times, and screening the Zwf enhanced expression bacillus licheniformis by a PCR method.

In the above application, when the Zwf-enhanced expression of Bacillus licheniformis is used for producing heterologous proteins of trypsin, nattokinase or alkaline protease, the formula of the used fermentation medium comprises: 10-20g/L glucose, 5-13g/L soybean peptone, 8-12g/L corn steep liquor, 7-11g/L yeast powder, 8-12g/L sodium chloride, 2-5g/L K₂HPO₄And 4-8g/L (NH)₄)₂SO₄(ii) a Or 5-10g/L bone peptone, 5-10g/L soybean peptone, 8-12g/L corn steep liquor, 7-11g/L yeast powder, 8-12g/L sodium chloride, 2-5g/L K₂HPO₄And 4-8g/L (NH)₄)₂SO₄。

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

the inventor tries to integrate over-express Zwf in Bacillus licheniformis for the first time, successfully obtains a Bacillus licheniformis heterologous protein expression host strain with Zwf gene enhanced expression, and transfers trypsin, nattokinase and alkaline protease free expression vectors, and fermentation results show that the enhanced expression of Zwf obviously improves the protein expression level, and compared with corresponding control strains, the yield of the heterologous protein of the Bacillus licheniformis constructed by the invention is improved by over 28 percent. The invention provides a new strategy for the high-efficiency expression of the bacillus licheniformis protein.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

The invention takes three proteins (trypsin, nattokinase and alkaline protease) as examples to illustrate the superiority of the technical scheme of the invention; however, in practice, the method is not limited to these three proteins.