CRISPR-based gene editing technology and its application in microbial engineering

Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype.Recent advances in clustered regularly interspaced short palindromic repeats(CRISPR)-Cas-mediated technolo-gies have provided an efficient tool for genetic engineering of cells and organisms.Here,we review the three emerging gene editing tools(ZFNs,TALENs,and CRISPR-Cas)and briefly introduce the principle,classification,and mechanisms of the CRISPR-Cas systems.Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems,as well as the novel base editor(BE),prime editor(PE),and CRISPR-associated transposase(CAST)technologies,are described in detail.In addition,we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications.Finally,the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.

Construction and application of high-quality genome-scale metabolic model of Zymomonas mobilis to guide rational design of microbial cell factories

High-quality genome-scale metabolic models(GEMs)could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies.Despite of the constant establishment and update of GEMs for model microorganisms such as Escherichia coli and Saccharomyces cerevisiae,high-quality GEMs for non-model industrial microorganisms are still scarce.Zymomonas mobilis subsp.mobilis ZM4 is a non-model ethanologenic microorganism with many excellent industrial characteristics that has been developing as microbial cell factories for biochemical production.Although five GEMs of Z.mobilis have been constructed,these models are either generating ATP incorrectly,or lacking information of plasmid genes,or not providing standard format file.In this study,a high-quality GEM iZM516 of Z.mobilis ZM4 was constructed.The information from the improved genome annotation,literature,datasets of Biolog Phenotype Microarray studies,and recently updated Gene-Protein-Reaction information was combined for the curation of iZM516.Finally,516 genes,1389 reactions,1437 metabolites,and 3 cell compartments are included in iZM516,which also had the highest MEMOTE score of 91%among all published GEMs of Z.mobilis.Cell growth was then predicted by iZM516,which had 79.4%agreement with the experimental results of the substrate utilization.In addition,the potential endogenous succinate synthesis pathway of Z.mobilis ZM4 was proposed through simulation and analysis using iZM516.Furthermore,metabolic engineering strategies to produce succinate and 1,4-butanediol(1,4-BDO)were designed and then simulated under anaerobic condition using iZM516.The results indicated that 1.68 mol/mol succinate and 1.07 mol/mol 1,4-BDO can be achieved through combinational metabolic engineering strategies,which was comparable to that of the model species E.coli.Our study thus not only established a high-quality GEM iZM516 to help understand and design microbial cell factories for economic biochemical production using Z.mobilis as the chassis,but also provided guidance on building accurate GEMs for other non-model industrial microorganisms.