Screening Of Caffeic Acid-responsive Promoter And Regulatory Protein Modification

Caffeic acid is an important branch of many long-term metabolic pathways in E.coli and can rapidly cross cell membranes.In order to solve the problem of caffeic acid accumulation and cytotoxicity in the process of co-culture,to achieve dynamic regulation and balance the whole metabolic pathway,this paper studied a caffeic acid sensor and RNAi-based dual-function dynamic control network,which can simultaneously provide up-regulation and down-regulation of cells metabolites.The conclusions reached are as follows:Based on the similarity of structure,this paper first constructed a DmpR transcription factor-based biosensor that responds to phenol and a NahR biosensor that responds to salicylic acid to explore the caffeic acid sensing system.The fluorescence intensity was measured by a microplate reader to determine the induction effect of different concentration inducers on gene transcription.The results show that the DmpR sensor responds to caffeic acid,and as the concentration of caffeic acid increases,the fluorescence intensity also increases,which is transcriptionally activated.It showed a maximum dynamic range of about 12.5(a.u.)at 12 h after the addition of 2 mM caffeic acid.while NahR showed a decreasing trend with increasing caffeic acid concentration,so further research on NahR-based sensors was abandoned.To further increase the sensitivity,dynamic range and stability of the DmpR sensor to caffeic acid response,the ability to bind is then altered by random mutations and site-directed mutagenesis.Finally,the highest dynamic range of the two mutant strains F42V and C137A was exceeded,and the dynamic range was stable within 48 hours.In order to determine whether it can be applied to the designed silybin synthesis pathway,the responsiveness of the synthetic caffeic acid precursor analog L-dopa,to coumaric acid and tyrosine was tested..The results indicate that tyrosine is unresponsive.L-dopa showed an induction rate of about 1.8 to 2.3 times,and dynamic range of about 1.97?2.8 times for coumaric acid.However,in contrast to caffeic acid,the responsiveness of caffeic acid is significantly higher than that of the precursor,providing a basis for further application.Finally,under the control of the caffeic acid-responsive promoter Po,the expression of eGFP was down-regulated by RNAi,which interfered with 0-100 bp,-50-50 bp,-100-0 bp,and-21-79 bp,respectively.Among them,interference 0-100 bp,compared with no caffeic acid,the relative fluorescence intensity decreased by 14%,25%,30%,37%within 48 h,interference-21-79 bp,relative fluorescence intensity within 48 h Reduced by 7%,16%,40%,46%,respectively.It is shown that the integration of RNAi and Po promoter regulatory factors provides a timely dynamic down-regulation response by sensing different thickness of caffeic acid.