Construction And Applications Of CRISPR/Cas9n System In Bacillus Licheniformis DW2

Bacillus licheniformis is an important industrial microorganism which can produce a variety of biological products.However,low transformation efficiency and lack of effective genome editing tools hamper its application.In recent years,the CRISPR/Cas9 system has been developed and widely applied to various cells due to its advantages of high efficiency,ease of operations and short operation cycles.However,the genome editing strategy of Cas9 endonuclease equipped with NHEJ repair pathway still has some problems in prokaryotic microorganisms,such as the off-target effect and lethal effects.In order to solve these problems,Cas9 n mutated from Cas9,a single-incision endonuclease is found and often used in the genome editing technique with homologous recombination repair pathways.In this study,an improved CRISPR/Cas9 n system was built to develop systematic genome editing toolkits for improving the genome editing efficiency in B.licheniformis.On the basis of a shuttle vector pHY300,the Cas9 n expression cassette was added,and DW2/pCas9 n overexpression engineering strain was constructed.The SDS-PAGE experiment showed that Cas9 n protein could express in B.licheniformis DW2.In order to reduce load capacity of this vector,Cas9 n expression cassete driven by P43 promoter was firstly integrated into the genome of the B.licheniformis,and the Cas9 n integrated strain was named DWC9 n.Based on DWC9 n strains,a series of genome editing strategies were constucted,including single gene knockout,two-gene knockout,large DNA fragment knockout and single genes integtation,which greatly improves the editing efficiency of B.licheniformis,and provides strategies for engineering microorganisms.In this study,the pulcherrimin synthetase gene yvmC was chosen as the target gene,and a specific gRNA sequence was built to develop single gene knockout vector.Then,a series of different sizes of homology arms were designed(0.1 kb + 0.1 kb,0.2kb + 0.2 kb,0.3 kb + 0.3 kb,0.5 kb + 0.5 kb,0.7kb + 0.7 kb,1.0kb + 1.0kb)to optimize the knockout vector.The experimental results showed that CRISPR/Cas9 n system could be well applied to single gene deletion even with shortest homology arm(0.1kb),and the editing efficiency increased with increasing homology arm size.However,given the load capacity of vector and the strategy development,0.3 kb and0.5kb homology arms may be the best choice,and the editing efficiency up to80.6%.At the same time,pHY300-?epr?wprA was constructed for a simultaneous twogene disruption,and the efficiency was 11.6%.Based on the pHY300 vector,a large DNA fragment knockout strategy based on CRISPR/Cas9 n was constructed to delete a 42.7 kb DNA fragment with an efficiency of 79.0%.In order to establish the systematic CRISPR/Cas9 n genome editing toolkits,nattokinase expression cassette was integrated into the genome of the B.licheniformis DWC9 n by CRISPR/Cas9 n system with an efficiency of 76.5%.Finally,in order to test the applicability of CRISPR/Cas9 n system established in DWC9 n,a suitable host strain was constructed for heterologous protein expression by knocking out some intracellular and extracellular protease genes with this system.In this section,the genes epr,vpr,mpr,bprA,aprE,clpP,clpE,clpYQ,lonB,andbacABC were continuously knocked out to construct the engineering strain DWC9n?10.Then,DWC9 n and DWC9n?10 were transformed with pPSNT-SsacC to test the secretion ability of nattokinase.The fermentation results showed that the engineered strain DWC9n?10 was more suitable for nattokinase expression,and the nattokinase activity reached 75.0 FU/mL,an increase of 30.4% compared to that of the control strain.This study has described a systematic and efficient genome editing method in B.licheniformis that can be applied to the development of Bacillus strains in the future.