The Establishment And Application Of The Dual Reporter-gene System And CRISPR-cas12a Genome-editing Tool In Zymomonas Mobilis

Zymomonas mobilis is a Gram-negative facultative anaerobic bacterium.As a natural ethanologen,Z.mobilis is a chassis to be engineered for producing biofuels and biochemicals.However,the lack of quantitative toolkits to identify and characterize biological parts such as promoters and ribosomal binding sites(RBS)as well as efficient and effective genome editing tools severely impedes the development of recombinant Z.mobilis strains for those purposes.In this study,we aim to develop toolkits to address these challenges to expand biological parts and to provide genome editing tools for metabolic engineering and genome engineering practices in Z.mobilis.We developed a dual-reporter system based on flow cytometry for the quantitative characterization of biological parts in Z.mobilis.The reporter gene m Cherry driven by promoter Lac UV5 was served as the internal reference,and the candidate biological part to be tested was used to control the expression of another reporter gene EGFP.The fluorescence intensities of these two reporter genes detected by flow cytometry were quantified,and the EGFP/m Cherry ratio was calculated to represent the relative strength of the candidate biological part.This system was then applied to quantify the intensities of 38 promoters and 4 RBSs that were predicted to be of different strengths based on bioinformatics analysis of omics datasets of Z.mobilis in different conditions.The result demonstrated that the experimental results had a good correlation with the predicted strengths of these biological parts,which indicated that this system can be used to characterize biological parts such as promoters and RBSs quantitatively.These biological parts will help expand the inventory of biological parts for metabolic engineering and synthetic biology practices in Z.mobilis.In addition,we established a CRISPR-Cas genome editing system using Cas12a(Fn Cpf1)from Francisella novicida.A recombinant strain with inducible cas12a expression for genome editing was constructed in Z.mobilis ZM4,which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci.gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100%curing efficiency for three native plasmids.In addition,CRISPR/Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%.Combined with single-stranded DNA(ss DNA),CRISPR/Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity.Furthermore,the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z.mobilis with a recombinant lactate-producing strain constructed.The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z.mobilis for functional genomic research,strain improvement,as well as synthetic microbial chassis development for economic biochemical production.Moreover,efforts were initiated to further establish a transcriptional inhibition and activation(CRISPRi/a)system in Z.mobilis based on the CRISPR-dCas12a system.A single-point mutation on dcas12a resulted in two mutants of dCas12a(D917A)and dCas12a(E1006A)with similar inhibition effect on target genes,which can be used for CRISPRi applications.An efficient suppression of gene expression can be achieved when gRNA was used to target either strands of the promoter region,but only the coding strand of coding regions.In addition,the gRNA length(<15 nt)and the mismatch of gRNA with the targeting sequence greatly affected the gene interference effect of this CRISPRi system.The presence of mismatch(s)within the2^nd to 6^th base positions of gRNA sequence resulted in the loss of gene interference.In order to further establish a transcriptional activation system in Z.mobilis,the?subunit of RNA polymerase was fused to dCas12a.However,the preliminary results indicated that this CRISPRa system was not able to activate the expression of downstream genes and further modification is needed.In summary,a dual-reporter gene system was established in Z.mobilis in this work to quantify biological parts such as promoters,RBSs,and UTR,which can help expand regulatory biological parts inventory for future research in synthetic biology and metabolic engineering.In addition,a CRISPR-Cas12a-based gene editing system was established for effective nucleotide substitution,deletion,and insertion.A CRISPRi system was also established with the effect of gRNA mismatch and gRNA length on interference investigated,which provides a versatile and powerful genome-editing tool in Z.mobilis for functional genomic research,strain improvement,as well as synthetic microbial chassis development.