阅读说明：本技术 一种提高木糖利用率的基因、重组载体、重组细胞及应用方法 (A kind of gene, recombinant vector, recombinant cell and application method improving xylose utilization rate ) 是由 王志强 吕鑫 李刚 罗明刚 郑英敏 杨秋霞 吴泽华 孙敬善 于 2019-08-14 设计创作，主要内容包括：本发明涉及分子生物学领域,具体涉及一种提高木糖利用率的基因、重组载体、重组细胞及应用方法。一种提高木糖利用率基因,所述基因的核苷酸序列如SEQ ID NO.1所示,通过扩增技术对该基因进行扩增,构建了含有该基因的重组载体,并通过热激法转化至大肠杆菌感受态细胞中获得大肠杆菌工程菌,增强了大肠杆菌中木糖异构酶的表达水平,提高了大肠杆菌代谢木糖的能力,能够显著提高产物中赖氨酸的浓度,提高糖酸转化率。(The present invention relates to molecular biology fields, and in particular to a kind of gene, recombinant vector, recombinant cell and application method for improving xylose utilization rate.A kind of raising xylose utilization rate gene, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, the gene is expanded by amplification technique, construct the recombinant vector containing the gene, and converted by heat shock method and obtain colibacillus engineering into competent escherichia coli cell, the expression of xylose isomerase in Escherichia coli is enhanced, the ability of Metabolism of E. coli xylose is improved, the concentration of lysine in product can be significantly improved, saccharic acid conversion ratio is improved.)

1. a kind of raising xylose utilization rate gene, which is characterized in that the nucleotide sequence of the gene such as SEQ ID NO.1 institute Show.

2. containing recombinant vector, the recombinant cell described in claim 1 for improving xylose utilization rate gene.

3. the xylose utilization rate gene described in claim 1 that improves is in the Escherichia coli work using stalk hydrolyzate production lysine Application in journey bacterium.

4. a kind of preparation method for improving xylose utilization rate gene, which is characterized in that specifically includes the following steps:

Obtain bacillus subtilis 168 and bacillus coli gene；And according to 168 P43 of bacillus subtilis and Escherichia coli xylose Isomerase gene sequence design amplimer；

P43 gene is expanded using 168 gene of bacillus subtilis as template, obtains P43 promoter；

Using bacillus coli gene gene as template, Escherichia coli xylose isomerase gene is expanded, obtains Escherichia coli wood Sugared isomerase gene segment；

Using the P43 promoter and Escherichia coli xylose isomerase gene segment as template, improved using overlapping amplification Xylose utilization rate gene.

5. the preparation method according to claim 4 for improving xylose utilization rate gene, which is characterized in that the acquisition P43 The gene order of the amplimer of promoter is as shown in SEQ ID NO.2 and SEQ ID NO.3, amplification condition are as follows:

Initial denaturation: 95 DEG C, 5min；Denaturation: 95 DEG C, 1min, renaturation: 55 DEG C, 30s, extend: 72 DEG C, 30s, 30 circulations；After prolong It stretches: 72 DEG C, 10min.

6. the preparation method according to claim 4 for improving xylose utilization rate gene, which is characterized in that the acquisition large intestine The gene order of the amplimer of bacillus xylose isomerase gene segment is as shown in SEQ ID NO.4 and SEQ ID NO.5, amplification Condition are as follows:

Initial denaturation: 95 DEG C, 5min；Denaturation: 95 DEG C, 1min；Renaturation: 57 DEG C, 30s；Extend: 72 DEG C, 1min；30 circulations；After prolong It stretches: 72 DEG C, 10min.

7. the preparation method according to claim 4 for improving xylose utilization rate gene, which is characterized in that the overlapping amplification Amplimer is amplification condition shown in SEQ ID NO.2 and SEQ ID NO.5 in method are as follows:

Initial denaturation: 95 DEG C, 5min；Denaturation: 95 DEG C, 1min；Renaturation: 56 DEG C, 30s；Extend: 72 DEG C, 2min；30 circulations；After prolong It stretches: 72 DEG C, 10min.

8. the xylose utilization rate gene according to claim 3 that improves is in the large intestine bar using stalk hydrolyzate production lysine Application in bacterium engineering bacteria, which is characterized in that the step of application is as follows:

The raising xylose utilization rate gene and plasmid vector are obtained into recombinant vector by digestion, connection reaction；

The recombinant vector is converted by heat shock method into competent escherichia coli cell, and carries out positive-selecting and obtains large intestine Bacillus engineering bacteria.

9. applying step according to claim 8, which is characterized in that the digestion system during the digestion are as follows:

Restriction enzyme EcoRI and HindIII, inscribe enzyme buffer solution, distilled water.

10. applying step according to claim 8, which is characterized in that the positive-selecting specifically includes the following steps:

The permissive cell is coated on the plating medium containing 25 μ g/mL streptomysins and is cultivated, single colonie note is obtained For containing the colibacillus engineering for being improved xylose utilization rate gene.

Technical field

The present invention relates to molecular biology fields, and in particular to a kind of to improve the gene of xylose utilization rate, recombinant vector, again Group cell and application method.

Background technique

Stalk is that the remainder after seed is harvested in existing agricultural, wherein containing a large amount of carbohydrate and various beneficial members How element is recycled the nutritional ingredient in stalk, and obtaining reproducible living resources is the emphasis studied now.

Summary of the invention

Weaker using xylose ability for Escherichia coli existing in the prior art, metabolic efficiency is low, is unable to reach conversion It is required that the technical issues of, the technical solution provided by the present invention is:

A kind of raising xylose utilization rate gene, the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

Recombinant vector, recombinant cell containing the raising xylose utilization rate gene.

The xylose utilization rate gene that improves is in the colibacillus engineering using stalk hydrolyzate production lysine Using.

A kind of preparation method improving xylose utilization rate gene, specifically includes the following steps:

Obtain bacillus subtilis 168 and bacillus coli gene；And according to bacillus subtilis 168P43 and Escherichia coli Xylose isomerase gene sequence design amplimer；

P43 gene is expanded using 168 gene of bacillus subtilis as template, obtains P43 promoter；

Using bacillus coli gene gene as template, Escherichia coli xylose isomerase gene is expanded, obtains large intestine bar Bacterium xylose isomerase gene segment；

Using the P43 promoter and Escherichia coli xylose isomerase gene segment as template, obtained using overlapping amplification Improve xylose utilization rate gene.

Described improves xylose utilization rate gene in the colibacillus engineering using stalk hydrolyzate production lysine Application, the step of application is as follows:

The raising xylose utilization rate gene and plasmid vector are obtained into recombinant vector by digestion, connection reaction；

The recombinant vector is converted by heat shock method into competent escherichia coli cell, and carries out positive-selecting acquisition Colibacillus engineering.

The utility model has the advantages that

The application provides a kind of raising xylose utilization rate gene for the first time, and is expanded by amplification technique the gene Increase, constructs the recombinant vector containing the gene, and convert by heat shock method and obtain large intestine into competent escherichia coli cell Bacillus engineering bacteria enhances the expression of xylose isomerase in Escherichia coli, improves the ability of Metabolism of E. coli xylose, The concentration of lysine in product can be significantly improved, saccharic acid conversion ratio is improved.

Specific embodiment

In order to more fairly set out technology contents of the invention, it is described in detail in conjunction with specific embodiments herein, it is clear that Cited embodiment is the preferred embodiment of the technical program, and those skilled in the art can be according to disclosed skill The other technologies scheme that art content is apparent from still falls within protection scope of the present invention.

The embodiment of the invention provides a kind of raising xylose utilization rate gene, the nucleotide sequence of the gene such as SEQ Shown in IDNO.1.

The embodiment of the invention also provides recombinant vector, recombinant cells containing the raising xylose utilization rate gene.

The embodiment of the invention also provides the raising xylose utilization rate genes to produce bad ammonia using stalk hydrolyzate Application in the colibacillus engineering of acid.

The embodiment of the invention provides a kind of preparation methods for improving xylose utilization rate gene, specifically includes the following steps:

Obtain bacillus subtilis 168 and bacillus coli gene；And according to bacillus subtilis 168P43 and Escherichia coli Xylose isomerase gene sequence design amplimer；

P43 gene is expanded using 168 gene of bacillus subtilis as template, obtains P43 promoter；

Using bacillus coli gene gene as template, Escherichia coli xylose isomerase gene is expanded, obtains large intestine bar Bacterium xylose isomerase gene segment；

Using the P43 promoter and Escherichia coli xylose isomerase gene segment as template, obtained using overlapping amplification Improve xylose utilization rate gene.

The gene order of the amplimer of P43 promoter is wherein obtained as shown in SEQ ID NO.2 and SEQ ID NO.3, Amplification condition is preferred are as follows:

Initial denaturation: 95 DEG C, 5min；Denaturation: 95 DEG C, 1min, renaturation: 55 DEG C, 30s, extend: 72 DEG C, 30s, 30 circulations；Afterwards Extend: 72 DEG C, 10min.

Wherein obtain Escherichia coli xylose isomerase gene segment amplimer gene order such as SEQ ID NO.4 and Shown in SEQ ID NO.5, amplification condition is preferred are as follows:

Initial denaturation: 95 DEG C, 5min；Denaturation: 95 DEG C, 1min；Renaturation: 57 DEG C, 30s；Extend: 72 DEG C, 1min；30 circulations； Extend afterwards: 72 DEG C, 10min.

Wherein amplimer is amplification condition shown in SEQ ID NO.2 and SEQ ID NO.5 in the overlapping amplification Are as follows:

Initial denaturation: 95 DEG C, 5min；Denaturation: 95 DEG C, 1min；Renaturation: 56 DEG C, 30s；Extend: 72 DEG C, 2min；30 circulations； Extend afterwards: 72 DEG C, 10min.

The embodiment of the invention provides the raising xylose utilization rate genes to produce lysine using stalk hydrolyzate Colibacillus engineering in application, the step of application is as follows:

The raising xylose utilization rate gene and plasmid vector are obtained into recombinant vector by digestion, connection reaction；

The recombinant vector is converted by heat shock method into competent escherichia coli cell, and carries out positive-selecting acquisition Colibacillus engineering.

The wherein digestion system during the digestion are as follows: restriction enzyme EcoRI and HindIII, inscribe enzyme buffer Solution, distilled water.

Wherein the positive-selecting is specifically includes the following steps: being coated on the permissive cell containing 25 μ g/mL strepto-s It is cultivated on the plating medium of element, obtaining single colonie is to contain the engineered E. coli for being improved xylose utilization rate gene Bacterium.