Development And Application To Mevalonate Synthesis Of Synthetic Small Regulatory RNAs In Pseudomonas

Pseudomonas is common gram-negative bacteria which has plentiful metabolic pathways and high tolerance for tough environments.As a cellular chassis Pseudomonas can apply multiple regulatory tools for metabolic engineering purposes,including transposons systems,integrative plasmids,recombineering,CRISPR/Cas9,CRISPRi,and FENIX systems.However,the regulatory tools that have been developed so far still suffer from complex operational steps and inefficient regulation,and are still far from capable application in metabolic engineering on a large scale.These defects hinder the further development of Pseudomonas as a cellular chassis in synthetic biology.In recent years,small regulatory RNAs(s RNA)have been gradually applied to gene micro-regulation for metabolic engineering for their advantages of small size,low metabolic burden and capacity for large-scale target gene screening.The s RNA system has been applied extensively in several model bacterial chassis,but it has been rarely investigated in Pseudomonas and the regulatory efficiency is not ideal.This project first developed a regulatory small RNA system in Pseudomonas putida KT2440.Using exogenous green fluorescent protein as a characterization,the s RNA system based on Mic C and Sgr S scaffolds(derived from Escherichia coli)were developed in P.putida KT2440,respectively.The fluorescence results showed that the inhibition efficiency of the Mic C and Sgr S based scaffolds was30% and 38% respectively.The results significantly affect the expression of fluorescent proteins,but not enough to support subsequent experiments.In the project,the s RNA system based on the Prr F1-2scaffold(derived from Pseudomonas aeruginosa)was established,and the inhibition efficiency of the system was tested to achieve 57%.Compared with the inhibition efficiency(20%-40%)of the published Mic C scaffold,the s RNA system containing Prr F1-2 scaffold is able to decrease the expression of fluorescent proteins significantly.The ability to regulate the endogenous genes of P.putida KT2440 was further tested.Based on the antibiotic tolerance of P.putida KT2440,the endogenous gene(ttg A/mex E)related to antibiotic tolerance was selected as the target gene and weakened the antibiotic tolerance of the strain by regulating Prr F1-2 s RNA system.The experimental results revealed that the growth of the recombinant strain with ttg A as the target gene was hindered under antibiotic.The experiments verify the ability of the Prr F1-2 s RNA system to regulate the endogenous gene of P.putida KT2440 and indicate that the system can be used as a gene regulation tool for subsequent metabolic engineering modification.On the basis of the existing experiments,P.putida KT2440 was applied as a biological chassis cell to construct the mevalonate synthesis pathway by introducing the upper mevalonate pathway(mva E and mva S genes)of Enterococcus faecalis.The promoter strength of the exogenous genes and the fermentation carbon source were sequentially optimized,and the higher initiation activity of the J23102 promoter to express mva E and mva S was finally selected.The production of mevalonate was also measured in a shake flask fermentation experiment using 2,3-butanediol,glucose,and glycerol as carbon sources.The production of mevalonate was the highest when 2,3-butanediol was used as the carbon source,reaching 11.3 mmol/L.The Prr F1-2 s RNA system was further utilized in metabolic engineering of P.putida KT2440 for synthesis,and an s RNA library targeting 12 mevalonate branched genes was established via analysis of the 2,3-butanediol metabolic pathway.The large-scale screening was performed using the s RNA library to verify whether the target genes affect the biosynthesis of mevalonate.The experimental results showed that the fad B,acc D,Fab H,leu A,and ato B genes in the s RNA library enhanced mevalonate production,and the hbd,acc A,acc B,acc C,glc B,ace F,and aco C genes reduced mevalonate production.It was also found that overexpression of endogenous genes fad A and acs in recombinant strains could have a positive effect on mevalonate production.In summary,this paper has developed the first Prr F1-2 regulatory small RNA system in P.putida KT2440.This system extends the gene editing regulation platform of P.putida KT2440 and provides an effective gene regulation tool for metabolic engineering of P.putida KT2440.Meanwhile,P.putida KT2440 through metabolic engineering and gene screening using the Prr F1-2 s RNA system.The experimental results provide the necessary theoretical basis for the synthesis of mevalonate in P.putida KT2440.The results will facilitate the further modification of P.putida KT2440 using synthetic biology strategies to obtain a biosynthetic strain with high mevalonate production.