Construction And Application Of Multiple Quorum Sensing Systems In Escherichia Coli

The quorum sensing system is a ubiquitous tool for information exchange in single celled organisms.In recent years,quorum sensing systems have been widely used in synthetic biology and metabolic engineering.In this study,three independent quorum sensing systems were constructed and applied to the construction of different systems in synthetic biology.Firstly,we constructed a self-induced Sal quorum sensing system orthogonal to the classical Lux system and applied it to the dynamic regulation of salicylic acid synthesis.Sal system is an artificially constructed population sensing system with few synthase genes in the system,and the structure of signal molecules differs greatly from that of natural population sensing system.Therefore,Sal system has good application potential.However,there are no reports of constructing a self-induced Sal system in single cells.We first constructed a self-induced Sal system on the high copy plasmid skeleton of Escherichia coli.Then,precise expression regulation of key genes is performed to construct a system with high sensitivity and wide induction range.Subsequently,on the basis of this system,a self-induced Sal system was successfully constructed in the same cell for the first time by adding a sal synthase gene.Finally,it was applied to the production of salicylic acid,and the yield of salicylic acid from the engineered strain increased by 1 51%compared to the control strain.In the second part,combining the Sal system with the Las system and the Tra*system,a three bacteria microbial ecosystem was initially constructed.In the research of synthetic ecosystems,more attention has been paid to the study of dual strains,while the study of multiple strains is still lacking.We chose the Las system,Tra*system,and Sal system to combine positive and negative feedback to build a three-strain synthetic microbial ecosystem.The quorum sensing system serves as a communication module,and the toxin antitoxin system serves as an effect module to design and construct three different strains in the ecosystem.By regulating the expression of key genes in the system,two systems in each bacterium can work normally.Next,select suitable co cultured strains and further optimize the co culture inoculation ratio.Finally,the dominant population in the system is changed by adding different combinations of inducers.This part of work has certain application potential for constructing a synthetic biological coculture artificial bacterial population system for efficient production of valuable products.Finally,the optimization group sensing system Las system Tra*system,and stable phase specific promoter subsystem were constructed and applied to the threelevel dynamic regulation system of metabolic engineering.The Las system,Tra*system,and stable phase specific promoter system with different startup times obtained through screening and optimization were constructed into a three-layer regulatory system with time difference.In this system,the first to start is the Las system.followed by the stable startup specific dynamic subsystem,and finally the Tra*system.After the construction of the three-layer regulatory system,it was applied to the production of 3-hydroxypropionic acid through the malonyl coenzyme A pathway.Combining a three-layer regulatory system with an artificially encoded trans-microRNA suppression system,the dynamic growth inhibitory genes gltA and fabD are dynamically expressed,and acc and mcr are dynamically expressed to efficiently synthesize 3hydroxypropionic acid.Compared to static regulation,single layer dynamic regulation.and double layer dynamic regulation,the final yield of 3-hydroxypropionic acid is the highest by using a three-layer dynamic regulation system,with a shake flask fermentation rate of 7.0 g/L.The results demonstrate the necessity and great application potential of three-layer dynamic regulation.