Construction And Optimization Of Recombinant Protein Expression Chassis Of Vibrio Natriegens

The Escherichia coli pET expression system remains one of the most popular systems for recombinant protein production.However,some heterologous genes were expressed in E.coli with weak activity,and the factories were plagued by phage contamination sometimes.Vibrio natriegens is considered the fastest growing bacterium compatible with pET series plasmids,which may be an alternative recombinant expression system.In this study,we compared the expression of 200 heterologous genes with T7 system in V natriegens and E.coli,as well as optimized the first-to-try fermentation conditions,gemome editing methods and expression chassis:V.natriegens VnDX strain was constructed to express recombinant protein using T7 system,which is a prerequisite for this work.The T7 RNA polymerase cassette was integrated into the dns locus in the genome of V.natriegens ATCC14048,resulting in a modified V.natriegens strain VnDX,which was adapted with T7 system.Then,this work evaluated the soluble expression of 196 genes of interest(GOIs)and analyzed the spectrum of highly expressed GOIs of V.natriegens.Among them,171 GOIs were biocatalytic enzymes with soluble expression in BL21(DE3),and the remaining 25 GOIs were enzymes with weak expression in BL21(DE3)from plants and fungi.196 pET plasmids with different GOIs were electroporated into VnDX.The results showed that 65%of the tested 171 well-expressed GOIs and 52%of the 25 weakly-expressed GOIs obtained soluble expression in V.natriegens,20 GOIs of which showed better expression than E.coli.The overall assessment indicates that VnDX is compatible with the commonly used T7 expression system and has a different spectrum of highly expressed GOIs from E.coli.To obtain the fermentation conditions of the expression system,a consensus "what to try first" protocol for V.natriegens was adapted.According to the first-to-try conditions for E.coli,TBv2 can be generally recommended for protein production in V.natriegens.Through the optimization experiment of induction point and temperature,the first-to-try conditions was induction by 0.3 mM IPTG at the 10th hour,at which the fermentation temperature goes from 30℃ down to 28℃.Compared with the fermentation conditions reported in the literature,the enzyme efficiency increased by 123%for GDH，82%for LkRED,76%for AbRED,128%for TvDAO,and 100%for NaRED.After the development of the V.natriegens T7 expression system,this work further optimized the expression chassis from three aspects:the regulation of T7 RNAP transcription,the dose control of heterologous genes and strain mutagenesis.(1)By modifying the+1 and-10 region of the T7 RNAP promoter placUV5 and fine-tuning the transcription of T7 RNAP,the obtained VnDX-lac1G and VnDX-lac1A strains could significantly increase the soluble:expression and enzyme activity of GDH.The GDH expressed by VnDX-lac1G was 147%higher than that of VnDX.(2)The production stability of V natriegens can be improved by integrating GOI expression cassette onto chromosome for dosage optimization.The CRISPR-associated transposases method of genome editing was established in V.natriegens to achieving site-specific integration of 0.7～5 kb cargo genes.Next,the V natriegens multicopy chromosomal integration using CRISPR-associated transposase(VnMUCICAT)was developed to obtain a various of chassis varied with 1～8 copies of GOI expression cassettes integration within two days.Taking GDH as an example,16 strains with multicopy integration were obtained through four rounds of transfer,among which the GDH activity of 3-copy integration strain was as high as 957 U/mL,and 498%higher than that of plasmid-based expression.It is concluded that multicopy chromosomal integration GOI expression cassettes in V natriegen owns great application potential.(3)This work obtained enhanced V.natriegens expression chassis and learn mechanisms of highly expressed through forward genetics.We fused C-terminal of the heterologous gene with GFP and obtained V.natriegens mutants that could increase protein yield of four enzyems by UV mutagenesis and FACS.Next-generation sequencing and strain reconstruction revealed the Y45 7 variants in the conserved site of endogenous RNA polymerase(RNAP)β’subunit rpoC was responsible for the increase of recombinant protein yield.We speculated that the mutation of rpoC Y457 should have reprogramed V.natriegens innate gene transcription,thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs.The increase of GOI expression should be at least partly due to the increase of copy number.In this paper,the construction and optimization of the T7 expression system of V．natriegens were carried out from many aspects.The three optimization strategies proposed here can help guide the optimization of other microbial hosts,and promote V natriegens to become the next generation of microbial cell factory.