CN-122012462-A - Method for improving expression level of bacillus subtilis recombinant protein based on low PAM dependent CRISPR regulation

Abstract

The invention discloses a method for improving the expression level of bacillus subtilis recombinant protein based on low PAM-dependent CRISPR regulation and control, belonging to the field of genetic engineering. According to the invention, the CRISPRa/i system VcaQCas-RpoZ capable of mediating the activation and inhibition regulation of gene expression in bacillus subtilis is obtained by adding the activation module RpoZ. After the system is integrated and expressed into a bacillus subtilis genome, the transcription of chaperone PrsA and penicillin binding protein PonA is activated, and the transcription level of a regulatory factor MurR is inhibited, so that the expression level of recombinant protein is obviously improved. The invention improves the enzyme activity of the ultra-high temperature amylase to 2.46 times of that of the control strain. The bacillus subtilis provides a novel gene expression regulation strategy as an exogenous protein expression host, and provides a reference for site selection of gene regulation.

Inventors

• WU JING
• ZHANG KANG
• ZHU XIONGYANG
• CHEN SHUMIN
• CHEN YINGYING

Assignees

• 江南大学

Dates

Publication Date

20260512

Application Date

20260128

Claims (10)

1. 1. The bacillus subtilis gene recombinant protein expression system is characterized by comprising a VcaQCas-RpoZ system and crRNA, wherein the VcaQCas-RpoZ system comprises TniQ, cas8, cas7, cas6 and RpoZ, and amino acid sequences of the TniQ, cas8, cas7, cas6 and RpoZ are respectively shown as SEQ ID NO. 2-6.
2. 2. The expression system of claim 1, wherein RpoZ is expressed to the C-terminus of the TniQ subunit by a Linker fusion of the amino acid sequence shown as SEQ ID No. 7.
3. 3. The expression system of claim 1, wherein TniQ and Cas7 are regulated by constitutive promoter P sunA as set forth in SEQ ID No.8, cas8 and Cas6 are regulated by constitutive promoter P groES as set forth in SEQ ID No.9, and crRNA is regulated by constitutive promoter P groES and constitutive promoter P veg as set forth in SEQ ID No. 10.
4. 4. The expression system of claim 1, wherein the VcaQCas-RpoZ system matches a CRISPR ARRAY sequence having the sequence set forth in SEQ ID No. 11.
5. 5. A method for regulating the expression level of recombinant protein of bacillus subtilis gene, which is characterized in that a VcaQCas-RpoZ system and crRNA are integrated in bacillus subtilis expressing recombinant protein gene through plasmids to regulate the transcription intensity of key genes affecting the expression level of recombinant protein, wherein the VcaQCas-RpoZ system comprises TniQ, cas8, cas7, cas6 and RpoZ, and the amino acid sequences of the TniQ, cas8, cas7, cas6 and RpoZ are respectively shown as SEQ ID NO. 2-6.
6. 6. The method of claim 5, wherein the recombinant protein is ultra-high temperature amylase Pfa.
7. 7. The method of claim 6, wherein the key genes affecting the expression level of ultra-high temperature amylase Pfa are genes prsA, ponA and murR.
8. 8. The method of claim 7, wherein the hyperthermostable amylase Pfa has an amino acid sequence as shown in SEQ ID No.13, and the n32 sequences of the targeting genes prsA, ponA and murR are shown in SEQ ID No.14 to SEQ ID No.16, respectively.
9. 9. The method of claim 5, wherein the host bacteria include, but are not limited to, bacillus subtilis WS9C.
10. 10. Use of the bacillus subtilis gene recombinant protein expression system according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9 for increasing the expression level of a bacillus subtilis recombinant protein.

Description

Method for improving expression level of bacillus subtilis recombinant protein based on low PAM dependent CRISPR regulation Technical Field The invention relates to a method for improving the expression level of bacillus subtilis recombinant protein based on low PAM-dependent CRISPR regulation and control, belonging to the technical field of biology. Background Bacillus subtilis (Bacillus subtilis) is a gram-positive strain of Bacillus. B. The subtilis is listed as a list authentication strain of American FDA (generally recognized as a safe substance) because of no toxin production and no heat-sensitive protein production, is easy to separate and culture, can ferment at high density and has strong protein secretion capability, so that the bacillus subtilis is used as an important industrial strain for producing various exogenous recombinant proteins and metabolites, and has high authentication value in excellent properties. Meanwhile, the bacillus subtilis has clear genetic background and mature molecular biology gene editing method, which is beneficial to carrying out genetic modification on the bacillus subtilis to strengthen the expression capability of the bacillus subtilis as a recombinant protein production host. How to realize accurate regulation of gene expression has become a key problem whether the biosynthesis capacity can be further improved at present. A series of effective regulation tools and strategies have been developed in bacillus subtilis, such as bacillus subtilis expression element library proposed in 2016, by randomly designing promoters with different genetic activities, RBS and protein degradation tag sequences, and performing free assembly in an expression cassette to achieve regulation of gene expression levels. In another research, a gene expression regulation tool based on a maltose operon is developed, the specificity of trans-activation after maltose induction is optimized based on promoter mutagenesis and metabolic engineering, and strict and stable gene regulation is realized in bacillus subtilis. The gene expression regulation and control based on CRISPR is to target specific genes for regulation and control by coexpression of dCAS protein and corresponding sgRNA, and the CRISPR gene expression regulation and control tool has the advantages of accurate regulation and control, programmability and the like. The CRISPR gene expression control tools commonly used in bacillus subtilis at present mainly comprise dAS 9 and dAS 12a. However, the two dCas proteins still have a certain limitation in application, and the characteristic of targeting binding by PAM recognition is a main reason that dCas9 or dCas12a systems have limited regulation efficiency in practical application, and mainly affects CRISPRa activation of the regulation process. CRISPR-dCas directed gene activation requires dCas fusion proteins to bind to appropriate sites upstream of the promoter, which is difficult to initiate when there is no recognizable PAM sequence on the genome at the site where targeted binding is desired. Therefore, developing and designing a novel CRISPR gene expression regulation tool suitable for bacillus subtilis is a current urgent problem to be solved. Disclosure of Invention In order to solve the problem of limited application of a bacillus subtilis gene expression regulation tool in the prior art, the invention provides a method for improving the expression level of bacillus subtilis gene recombinant protein based on low PAM-dependent CRISPR regulation. The first technical scheme provided by the invention is a bacillus subtilis gene recombinant protein expression system, the system comprises a VcaQCas-RpoZ system and crRNA, the VcaQCas-RpoZ system comprises TniQ, cas8, cas7, cas6 and RpoZ, the TniQ, cas8, cas7 and Cas6 in VcaQCas-RpoZ are derived from Vibrio campbelliiFXH, and RpoZ is a bacillus subtilis endogenous transcription factor. In certain embodiments, the amino acid sequences of TniQ, cas8, cas7, cas6 and RpoZ are shown as SEQ ID nos. 2 to 6, respectively. In certain embodiments, the RpoZ is expressed to the C-terminus of the TniQ subunit by Linker fusion of the amino acid sequence shown as SEQ ID No. 7. In certain embodiments, tniQ and Cas7 are regulated by constitutive promoter P sunA as shown in SEQ ID No.8, cas8 and Cas6 are regulated by constitutive promoter P groES as shown in SEQ ID No.9, and crRNA is expressed by constitutive promoter P groES and constitutive promoter P veg as shown in SEQ ID No. 10. In certain embodiments, the CRISPR ARRAY sequence to which VcaQCas-RpoZ match is identified by CRISPRCASFINDER software, which has the base sequence set forth in SEQ ID NO. 11. The second technical scheme provided by the invention is a method for regulating and controlling the expression level of recombinant protein of bacillus subtilis genes, which is to integrate and express VcaQCas-RpoZ system and crRNA in bacillus subtilis expressing recombinant protein genes through p