Development Of Novel Methods For High Efficient Expression Of Heterologous Genes And Retron-mediated Genome Editing In Escherichia Coli

Escherichia coli serves as a prominent host for contemporary protein pharmaceutical expression and a ubiquitous microbial chassis in synthetic biology research.Despite extensive studies on factors influencing heterologous protein expression in E.coli and the emergence of various genome editing technologies,including CRISPR/Cas,limitations persist.This paper introduces innovative approaches for high-efficiency expression of heterologous genes and genome editing in E.coli.Initially,diverse standardized DNA regulatory elements’ impact on heterologous gene expression in E.coli was designed and examined.Utilizing the Golden Gate cloning technique,an expression vector library was rapidly constructed and transformed into host cells.Combined with high-throughput protein expression screening using the organic arsenic dye FLAsH,a novel optimization method for heterologous protein expression was developed.Subsequently,an efficient and versatile Retron-mediated Genome Editing System(REGES)has been engineered in Escherichia coli,with the retron system serving as its foundation.By knocking out the recJ and xonA genes,REGES achieved an editing efficiency of 12%.Systematic investigations into the impact of replication,translation,and transcription levels on REGES editing,combined with optimization of editing conditions,resulted in REGES efficiently introducing substitutions,insertion,deletion,or combinatorial mutations on the genome with～100%efficiency.Building on efficient REGES,assembling different retron constructs into a retron cassette array enabled simultaneous editing at multiple sites,with editing efficiencies of 85%,69%,and 25%for double,triple,and quadruple edits,respectively.Combining SceI endonucleasemediated plasmid elimination with the multiplex genome editing system efficiently constructed a high-yield strain with five targeted mutations in the lycopene biosynthesis pathway,increasing lycopene production by 4.5 times.To further expand REGES applications,pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of genes were constructed by REGES,achieving a 208-fold range of gene expression levels.Applying this strategy to regulate the expression levels of seven global transcription factors in E.coli,a strain with reduced expression of the fumarate and nitrate reduction regulatory protein(FNR)was obtained,conferring high ethanol tolerance.Besides introducing designed mutation libraries using REGES,combining REGES with polymerase-mediated base editing and error-prone transcription further developed a continuous evolution method for in vivo protein evolution.This system introduced random mutations in the target region of the model protein SacB with an efficiency of 2.4 × 10-3,approximately 1000 times higher than spontaneous mutations.Finally,the integration of standardized DNA regulatory elements,REGES,REGES-mediated transcription factor regulation,and REGES-mediated multiplex editing were collectively applied to construct an E.coli cell factory for high biotin production.By reinforcing the endogenous biotin synthesis pathway,optimizing the biotin synthesis regulatory network,alleviating cell toxicity from key enzyme overexpression,and optimizing host energy levels,biotin production reached 7219μg/L,45 times higher than the initial strain.In conclusion,this manuscript employs case studies to elucidate the significant methodological value and multifaceted applications of the developed REGES genomic editing technology in synthetic biology research.