Characterization Of Mmej Pathway In Zymomonas Mobilis And Its Application In Genome Engineering

Zymomonas mobilis has a unique ED metabolic pathway and outstanding ethanol production capacity,making it a main chassis cell for the study of cellulosic biomass biorefining.Zymomonas mobilis could used the microhomology-mediated end joining(MMEJ)pathway to repair CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers.However,the mechanism and rules of prokaryotic MMEJ is not clear,which restricts its applicationThis study revealed the occurrence regularity of MMEJ,including:the length of microhomologies are mostly 6 bp and 12 bp,and the sequence of microhomologies are conservative to 5’-(+1)GNANAA(+6)-3’,and the length of MMEJ-mediated DNA deletion fragments are mostly 40-70 bp and 300-500 bp,and the location of the deletion fragments is biased to the 5 ’end of the protospacersIn this study,we developed a novel gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the microhomology-mediated end j oining(MMEJ)pathway.This tool only requires a self-interference plasmid carrying the mini-CRISPR(Repeat-Spacer-Repeat)expression cassette,where the spacer matches the target DNA Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA,leaving deletions in predicted sizesUsing this tool,eight genes have been successfully deleted.Among them,a σ28 deletion strain was generated and its function in regulation of motility was characterized Then,15.7 kb large fragment deletions were achieved by transformation of the selfinterference plasmids carrying two spacers cloned in tandem.Moreover,the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette,that reduced spacer loss probably via recombination,and enhanced CRISPR-Cas interferenceIn summary,based on endogenous CRISPR-Cas system and MMEJ repair system,this study constructed a gene editing tool for Zymomonas mobilis,which can efficiently and quickly complete the work of single gene editing,gene function identification and large fragment knockout,etc.,which will help to fully analyze the biological components and provide a solid foundation for modification and optimization of Zymomonas mobilis.