Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient ...Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance.In this study,an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence,which could be the mutant strains with increased d-lactate production.Then,LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference(CRISPRi)library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome.Specifically,two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting(FACS),respectively.Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production,further knockout of ZMO1323 and ZMO1530 resulted in a 15%and 21%increase of d-lactate production,respectively.This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z.mobilis,but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.展开更多
基金This work was supported by the National Key Research and Development Program of China(2022YFA0911800)National Natural Science Foundation of China(CN)(21978071)+2 种基金the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)the Innovation Base for Introducing Talents of Discipline of Hubei Province(2019BJH021)We also acknowledge the support from the State Key Laboratory of Biocatalysis and Enzyme Engineering.
文摘Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance.In this study,an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence,which could be the mutant strains with increased d-lactate production.Then,LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference(CRISPRi)library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome.Specifically,two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting(FACS),respectively.Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production,further knockout of ZMO1323 and ZMO1530 resulted in a 15%and 21%increase of d-lactate production,respectively.This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z.mobilis,but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.
