阅读说明：本技术 提高枯草芽孢杆菌CRISPR-Cas9n系统基因组多位点编辑效率的方法 (Method for improving genome multi-site editing efficiency of bacillus subtilis CRISPR-Cas9n system ) 是由 王智文 刘丁玉 黄灿 辛国省 陈涛 于 2019-09-19 设计创作，主要内容包括：本发明公开了提高枯草芽孢杆菌CRISPR-Cas9n系统基因组多位点编辑效率的方法,包括如下步骤：根据编辑位点,构建枯草芽孢杆菌CRISPR-Cas9n基因组编辑系统；(2)敲除待编辑枯草芽孢杆菌菌株的ligD基因,所述ligD基因的核苷酸序列如SEQ ID NO.90所示；(3)将步骤(1)获得的枯草芽孢杆菌CRISPR-Cas9n基因组编辑系统导入步骤(2)获得的重组菌中进行基因组多位点编辑。本发明的方法可以提高枯草芽孢杆菌CRISPR-Cas9n系统基因组多位点编辑效率。利用枯草芽孢杆菌CRISPR-Cas9n基因组编辑系统,提升枯草芽孢杆菌BS89的核黄素生产能力。(The invention discloses a method for improving genome multi-site editing efficiency of a bacillus subtilis CRISPR-Cas9n system, which comprises the following steps: constructing a genome editing system of the bacillus subtilis CRISPR-Cas9n according to the editing sites; (2) knocking out a ligaD gene of a bacillus subtilis strain to be edited, wherein the nucleotide sequence of the ligaD gene is shown as SEQ ID No. 90; (3) and (3) introducing the genome editing system of the bacillus subtilis CRISPR-Cas9n obtained in the step (1) into the recombinant bacteria obtained in the step (2) for genome multi-site editing. The method can improve the genome multi-site editing efficiency of the bacillus subtilis CRISPR-Cas9n system. The riboflavin production capacity of the bacillus subtilis BS89 is improved by utilizing a bacillus subtilis CRISPR-Cas9n genome editing system.)

1. The method for improving the multi-site editing efficiency of the genome editing system of the bacillus subtilis CRISPR-Cas9n is characterized by comprising the following steps of:

(1) Constructing a genome editing system of the bacillus subtilis CRISPR-Cas9n according to the editing sites;

(2) Knocking out a ligaD gene of a bacillus subtilis strain to be edited, wherein the nucleotide sequence of the ligaD gene is shown as SEQ ID No. 90;

(3) And (3) introducing the genome editing system of the bacillus subtilis CRISPR-Cas9n obtained in the step (1) into the recombinant bacteria obtained in the step (2) for genome multi-site editing.

2. The method for improving the riboflavin production capacity of the bacillus subtilis BS89 by utilizing a bacillus subtilis CRISPR-Cas9n genome editing system is characterized by comprising the following steps:

(1) simultaneously modifying RBS regions of three genes of ribA, ribB and ribH on a riboflavin operon of the bacillus subtilis BS89, and constructing a bacillus subtilis CRISPR-Cas9n genome editing system according to three modification sites, wherein the nucleotide sequence of the ribA gene is shown as SEQ ID NO. 47; the nucleotide sequence of the ribB gene is shown as SEQ ID NO. 48; the nucleotide sequence of the ribH gene is shown as SEQ ID NO. 49;

(2) Knocking out a ligaD gene of a bacillus subtilis BS89 strain, wherein the nucleotide sequence of the ligaD gene is shown as SEQ ID No. 90;

(3) And (3) introducing the CRISPR-Cas9n genome editing system obtained in the step (1) into the recombinant bacteria obtained in the step (2), carrying out random mutation on RBS regions of three genes of ribA, ribB and ribH on a riboflavin operon to obtain strains with different mutation combinations, and screening the strains with significantly improved riboflavin production capacity.

Technical Field

The invention belongs to the field of genome editing technology and metabolic engineering, and particularly relates to a method for improving the genome multi-site editing efficiency of a bacillus subtilis CRISPR-Cas9n system and application of the method in metabolic engineering.

Background

Efficient genome editing tools are key to metabolic network regulation. With the development of system biology and the need to develop microbial cell factories, optimal regulation of metabolic networks has gradually evolved from regulation of a single gene or pathway to simultaneous modification of multiple targets and pathways. Therefore, the development of multiple genome editing tools is an important aspect of the field of gene editing technology.

The CRISPR-Cas9n genome editing technology based on the Cas9n with the nickase activity overcomes the defects that the growth pressure of a Cas9 system on host cells is large, double-strand nicks are not beneficial to repair and the off-target rate is high. Meanwhile, when the genome is edited at multiple sites, the single-stranded nicks do not excessively damage the integrity of the genome, so that the editing efficiency is higher.

At present, in the traditional genome editing technology of bacillus subtilis, the efficient gene editing of more than 2 sites cannot be realized. In addition, with the wide application of the CIRSPR technology in the field of gene editing in recent years, the gene editing efficiency of the bacillus subtilis is remarkably improved. Currently, the multi-site editing efficiency of the Bacillus subtilis CRISPR-Cas9n system is low.

disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for improving the genome multi-site editing efficiency of a bacillus subtilis CRISPR-Cas9n system.

The second purpose of the invention is to provide a method for improving the riboflavin production capacity of bacillus subtilis BS89 by utilizing a bacillus subtilis CRISPR-Cas9n genome editing system.

The technical scheme of the invention is summarized as follows:

The method for improving the multi-site editing efficiency of the genome editing system of the bacillus subtilis CRISPR-Cas9n comprises the following steps:

(1) Constructing a genome editing system of the bacillus subtilis CRISPR-Cas9n according to the editing sites;

(2) Knocking out a ligaD gene of a bacillus subtilis strain to be edited, wherein the nucleotide sequence of the ligaD gene is shown as SEQ ID No. 90;

(3) And (3) introducing the genome editing system of the bacillus subtilis CRISPR-Cas9n obtained in the step (1) into the recombinant bacteria obtained in the step (2) for genome multi-site editing.

The method for improving the riboflavin production capacity of the bacillus subtilis BS89 by utilizing the bacillus subtilis CRISPR-Cas9n genome editing system comprises the following steps:

(1) Simultaneously modifying RBS regions of three genes of ribA, ribB and ribH on a riboflavin operon of the bacillus subtilis BS89, and constructing a bacillus subtilis CRISPR-Cas9n genome editing system according to three modification sites, wherein the nucleotide sequence of the ribA gene is shown as SEQ ID NO. 47; the nucleotide sequence of the ribB gene is shown as SEQ ID NO. 48; the nucleotide sequence of the ribH gene is shown as SEQ ID NO. 49;

(2) Knocking out a ligaD gene of a bacillus subtilis BS89 strain, wherein the nucleotide sequence of the ligaD gene is shown as SEQ ID No. 90;

(3) And (3) introducing the CRISPR-Cas9n genome editing system obtained in the step (1) into the recombinant bacteria obtained in the step (2), carrying out random mutation on RBS regions of three genes of ribA, ribB and ribH on a riboflavin operon to obtain strains with different mutation combinations, and screening the strains with significantly improved riboflavin production capacity.

The invention has the advantages that:

The method can improve the genome multi-site editing efficiency of the bacillus subtilis CRISPR-Cas9n system.

The riboflavin production capacity of the bacillus subtilis BS89 is improved by utilizing a bacillus subtilis CRISPR-Cas9n genome editing system.

Drawings

Fig. 1 is a comparison of efficiency for three-site editing.

FIG. 2 shows the results of fermentation in 96-well plates of riboflavin operon-optimized strains.

FIG. 3 shows the results of shake flask rescreening of the riboflavin operon-optimized strain.

Detailed Description

the present invention is further illustrated by the following examples, which are provided to enable those skilled in the art to better understand the present invention and are not intended to limit the present invention in any way.

The host strain Bacillus subtilis 168 used in the invention is derived from BGSC (Bacillus Genetic Stock Center, http:// www.bgsc.org /). Bacillus subtilis BS89 is derived from references ShiT, Wang Y, Wang Z, Wang G, Liu D, Fu J, Chen T, ZHao X. definition of purpurathway in Bacillus subtilis and its use in riboflavin biosyntheses. Microbialcell industries.2014, 13(1):101.

The original plasmids pAX01 and pHP13 used in the present invention were derived from BGSC (Bacillus Genetic Stock Center, http:// www.bgsc.org /).

the plasmid pEBS-cop1 was derived from the Bacillus subtilis genome traceless modification method (application publication No.: CN 103451224A).

Plasmid pUC18 and plasmid pCas9cur were derived from addgene (http:// www.addgene.org).

pGRB plasmids are derived from the reference Li et al, Metabolic engineering of Escherichia coli using CRISPR-Cas9 media encoding. Metab Eng.2015,31:13-21.

Molecular biological reagents used, such as restriction enzymes, dephosphorylates, DNA ligases, etc., were purchased from thermo (http:// www.thermoscientificbio.com/fermentas), and other biochemical reagents used were purchased from Biotechnology (Shanghai) GmbH (http:// www.sangon.com /).