Functional Characterization of CRISPR-Cas System in the Ethanologenic Bacterium Zymomonas mobilis ZM4

CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into the bacterial cells. Zymomonas mobilis is an ethanologenic bacterium, which encodes a subtype I-F CRISPR-Cas system containing three CRISPR loci and a far distant cas gene cluster. Reverse transcription (RT)-PCR analysis revealed that the CRISPR loci were transcribed on both strands. The Cas proteins were suggested to be expressed based on the previous transcriptomic analysis. Challenging with the invader plasmids containing the artificial protospacer with the protospacer adjacent motif (PAM) of NGG or GG exhibited immune interference activity. However, PAM motif of GG seems more effective than NGG in interference activity. Further, the artificial CRISPR arrays with the spacer sequences targeting to the specific genome sites could also lead to strong immune activity, resulting in almost no transformant grown on the agar plates. It was suggested that bacteria like Z. mobilis ZM4 are lack of the rejoining function to heal the double breakage of genomic DNA made by the CRISPR system. Conclusively, the Type I-F CRISPR-Cas system in Z. mobilis ZM4 is active to functionally defense the invading DNA elements.

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.

Prospects for engineering Ralstonia eutropha and Zymomonas mobilis for the autotrophic production of 2,3-butanediol from CO_(2)and H_(2)

The decarbonization of the chemical industry and a shift toward circular economies because of high global CO_(2) emissions make CO_(2) an attractive feedstock for manufacturing chemicals.Moreover,H_(2) is a low-cost and carbon-free reductant because technologies such as solar-driven electrolysis and supercritical water(scH_(2)O) gasification enable sustainable production of molecular hydrogen(H_(2)).We review the recent advances in engineering Ralsto-nia eutropha,the representative species of“Knallgas”bacteria,for utilizing CO_(2) and H_(2) to autotrophically produce 2,3-butanediol(2,3-BDO).This assessment is focused on state-of-the-art approaches for splitting H_(2) to supply en-ergy in the form of ATP and NADH to power cellular reactions and employing the Calvin-Benson-Bassham cycle for CO_(2) fixation.Major challenges and opportunities for application and future perspectives are discussed in the context of developing other promising CO_(2) and H_(2)-utilizing microorganisms,exemplified by Zymomonas mobilis.

CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements(MGEs)through uptake of invader-derived spacers.De novo adaptation samples spacers from both invaders and hosts,whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis.Self-derived spacers will lead to CRISPR self-interference.However,our in vivo study demonstrated that this species used the microhomology-mediated end joining(MMEJ)pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers.MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers.Importantly,CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA.Moreover,CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway,probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA in Z.mobilis.Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element(MGE)DNA,and maintains genome stability post CRISPR immunity in Z.mobilis.