Construction And Metabolic Engineering Application Of Cascade Circuit Library For Dynamic Temporal Regulation

In the production of high value-added chemicals based on microbial cell factories,metabolic engineering is generally used to maximize the yield and productivity of target compounds.Among them,the dynamic regulation strategy can well modify the metabolic pathway and effectively solve the problems of low biological yield,growth retardation and metabolic imbalance.However,the current dynamic regulatory elements have the limitations of few types,narrow response threshold,limited regulation range and unstable regulation time.and are mainly used to regulate the expression of a single gene at a specific time point.Therefore,it is necessary to expand the application scope of dynamic regulation strategy and explore new general components of synthetic biology for metabolic engineering optimization.This study attempts to use the quorum sensing(QS)system,which has gradually become more valuable in synthetic biology,to modify the metabolic pathway together with the dual regulatory protein cascade.In the early stage,our laboratory has constructed a self-induced dynamic temporal regulation cascade circuit,which did not significantly improve productivity after being applied to the synthesis of poly-β-hydroxybutyric acid(PHB).Therefore,this study plans to construct a dynamic temporal regulation cascade circuit library based on this circuit in E.coli to realize the self-induced dynamic temporal expression of different genes in the metabolic pathway,in order to solve the balance between cell growth and product synthesis in two-stage fermentation or other metabolic applications.Firstly,the constructed temporal regulation circuit is characterized,and it is found that the time difference between the cascaded circuit of Tra,Las and Lux and Tra cascades could reach 200 min and 150 min,respectively.In order to preliminarily verify the effectiveness of the circuit,the PHB fermentation experiment was redesigned.The results showed that the PHB production and content of the three strains in the experimental group were increased compared with the control group,and the P3 strain had the highest production at 7.2 g/L,which was 2.2 times higher than that of the control check.The T3 strain had the highest PHB content at 64.6%,which was 1.5 times higher than that of the control check.The PHB production and content of the experimental groups P3,T3 and PTO were different,indicating that the two target genes could be expressed in three different sequences at different time periods by using this circuit.In order to provide more alternative synthetic biology universal elements for metabolic applications,the core components of the cascade circuit,LasI synthase and TraR,LasR receptor proteins,were modified by promoter engineering and ribosomebinding site(RBS)replacement.A dynamic temporal regulation cascade circuit library with a time interval ranging from 110 min to 310 min was constructed,which contained 34 different circuit modifications,and the different circuit time intervals in the tool library were verified at the protein level and the transcription level.At the same time,it is found that when the LasI synthase and TraR,LasR receptor proteins are modified,there is a rule that the higher the strength of the promoter and the RBS,the smaller the response time difference of the cascade circuit.In dynamic regulation studies,the production process of related chemicals with acetylCoA as the precursor is often selected as the regulation model of metabolic flux.However,most studies use switch devices for metabolic regulation,and do not study the regulation sequence and interval time.In this study,it is believed that the dynamic temporal of metabolic flux regulation has practical application significance for the efficient synthesis of products.Therefore,the library was applied to PHB production again to verify its effectiveness and application value.The results show that different time interval circuits in the library have different application potentials,and different expression sequences of upstream and downstream genes will also affect the production and content of PHB.The production and content of PHB in C2-max strain were the highest,reaching 6.1 g/L and 57.4%,while the control strain P3 had only 3.9 g/L and 42%.Combined with the systematic error,we can use the existing time interval library to screen out the better PHB production circuit.In addition,it is found that when the time interval library is applied to PHB production,there is a law that PHB content and production gradually increase with the increase of circuit time interval.This study firstly verified the effectiveness of the self-induced dynamic timing temporal cascade circuit,and applied it to PHB production to demonstrate the potential application value of the circuit.Then,a library of dynamic temporal control cascade circuit is constructed,and the time interval rule and dynamic control characteristics in the library are analyzed and studied.Finally,the PHB production platform is used again to illustrate that the library can realize the self-induced dynamic temporal regulation of genes,separate the growth stage and production stage of strains to effectively improve the production of target compounds,and provide a new idea for the modification of metabolic pathways in synthetic biology.