Zymomonas mobilis chassis cells and their construction method and application

Abstract

The present invention belongs to the field of genetic engineering technology, and more particularly to a chassis cell of Zymomonas mobilis and its construction method and application. The chassis cell is a recombinant strain of Zymomonas mobilis with the Cas12a gene integrated into the genome as a starting strain, and the RecET gene is integrated into the genome of the recombinant strain of Zymomonas mobilis to obtain a chassis cell with improved efficiency of large-fragment gene editing. The present invention provides an efficient editing method for the directional editing of large fragments of the Zymomonas mobilis genome, which is suitable for the construction of chassis cells that need to be frequently gene-edited, and is conducive to greatly saving the time cost of chassis cell gene editing; moreover, the chassis cell genome is greatly streamlined, which is conducive to improving the biosynthesis efficiency of biological elements and pathways, and has good industrial application value.

Claims

1. A method for constructing chassis cells of Zymomonas mobilis, characterized in that: The chassis cell is a ZMO0038 site of the genome of Zymomonas mobilis ZMNP-Cas12a with Cas12a gene integrated as the starting strain, the RecET gene is integrated into the ZMO0028 site of the Zymomonas mobilis ZMNP-Cas12a genome, and the upstream of the RecET gene is connected to P tet Promoter, the Cas12a gene upstream is connected to P BAD Promoter; wherein the nucleotide sequence of the Cas12a gene is shown in SEQ ID NO.1, the nucleotide sequence of the RecET gene is shown in SEQ ID NO.2, and the P tet The nucleotide sequence of the promoter is shown in SEQ ID NO.

3. BAD The nucleotide sequence of the promoter is shown in SEQ ID NO.4; The method for constructing the chassis cells comprises the following steps: Obtain the upstream homology arm 0038US and downstream homology arm 0038DS sequences of the genomic ZMO0038 site, and convert P BAD The promoter and Cas12a gene were connected in series between the 0038US and 0038DS sequences to construct the first homologous recombination plasmid; Obtain the upstream homology arm 0028US and downstream homology arm 0028DS of the genomic ZMO0028 site, and convert P tet The promoter and RecET gene were tandemly inserted between the 0028US and 0028DS sequences to construct a second homologous recombination plasmid; The first homologous recombination plasmid and the second homologous recombination plasmid were transformed into Zymomonas mobilis ZMNP-Cas12a respectively, and P BAD The promoter and Cas12a gene were integrated into the ZMO0038 site and the P tet The promoter and RecET gene were integrated into the ZMO0028 site, and Zymomonas mobilis chassis cells were screened.

2. The method for constructing Zymomonas mobilis chassis cells according to claim 1, characterized in that: The first homologous recombination plasmid and the second homologous recombination plasmid carry different resistance marker genes.

3. The method for constructing Zymomonas mobilis chassis cells according to claim 1, characterized in that: The first homologous recombination plasmid and the second homologous recombination plasmid further include an Ori sequence, which is derived from a pUC57 vector.

4. The method for constructing Zymomonas mobilis chassis cells according to claim 3, characterized in that: The construction of the first homologous recombination plasmid includes the following steps: obtaining the upstream homology arm 0038US and the downstream homology arm 0038DS sequence of the ZMO0038 site by PCR amplification, and obtaining the P BAD Promoter, Cas12a gene, spectinomycin gene and Ori sequence of pUC57 vector, the amplified 0038US sequence, Cas12a gene, P BAD The promoter, spectinomycin gene and 0038DS sequence were connected with the Ori sequence by Gibson assembly to obtain the first homologous recombination plasmid; The construction of the second homologous recombination plasmid includes the following steps: obtaining the upstream homology arm 0028US and the downstream homology arm 0028DS sequence of the ZMO0028 site by PCR amplification, and obtaining the P tet promoter, RecET gene, chloramphenicol gene and Ori sequence of pUC57 vector, the amplified 0028US sequence, RecET gene, P tet The promoter, chloramphenicol gene and 0028DS sequence were connected with the Ori sequence by Gibson assembly to obtain a second homologous recombination plasmid; Among them, the nucleotide sequence of the 0038US sequence amplification primer pair is shown in SEQ ID NO.20-21, the nucleotide sequence of the 0038DS sequence amplification primer pair is shown in SEQ ID NO.22-23, the nucleotide sequence of the 0028US sequence amplification primer pair is shown in SEQ ID NO.10-11, the nucleotide sequence of the 0028DS sequence amplification primer pair is shown in SEQ ID NO.12-13, the nucleotide sequence of the Cas12a gene amplification primer pair is shown in SEQ ID NO.18-19, the nucleotide sequence of the RecET gene amplification primer pair is shown in SEQ ID NO.5-6, P BAD The nucleotide sequences of the promoter amplification primer pairs are shown in SEQ ID NOs. 7 and 9. tet The nucleotide sequences of the promoter amplification primer pair are shown in SEQ ID NOs. 7-8, the nucleotide sequences of the spectinomycin gene amplification primer pair are shown in SEQ ID NOs. 24-25, the nucleotide sequences of the chloramphenicol gene amplification primer pair are shown in SEQ ID NOs. 14-15, and the nucleotide sequences of the Ori sequence amplification primer pair are shown in SEQ ID NOs. 16-17.

5. Use of the Zymomonas mobilis chassis cells constructed by the method for constructing Zymomonas mobilis chassis cells according to any one of claims 1 to 4 in large-fragment gene editing, characterized in that: The sequence length of the gene ranges from 9 to 25 kb.

6. The use of the Zymomonas mobilis chassis cells in large-fragment gene editing according to claim 5, characterized in that: The gene editing is gene knockout, gene replacement and / or point mutation.

7. A gene editing method for chassis cells of Zymomonas mobilis, characterized in that: The following steps are involved: Designing guide RNA and donor DNA according to the target sequence to be edited, wherein the donor DNA includes upstream homology arm and downstream homology arm sequences of the guide RNA targeting sequence, and connecting the guide RNA and donor DNA to an expression vector to obtain an editing plasmid; The editing plasmid is transformed into the Zymomonas mobilis chassis cells constructed by the method for constructing the Zymomonas mobilis chassis cells according to any one of claims 1 to 4, and the positive chassis cells transformed with the editing plasmid are cultured.

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