Construction And Application Of Quorum Sensing-Based Dual-Function Switches And Synthetic Oscillators

Quorum sensing(QS)enables bacteria to communicate through QS signals to synchronize individuals,which is an effective tool for coordinating and inducing group behavior.Currently,synthetic circuits based on the QS module mainly rely on the selfinduced activation function caused by LuxR and its homologous proteins when binding QS signals.The regulation of timely up-regulation and down-regulation of different genes is a common problem in synthetic biology research and metabolic engineering.Therefore.the development of multi-functional QS regulatory elements is of great significance.In addition,synthetic oscillators that simulate pomplex biological clocks have become a research hotspot of synthetic biology..The construction and stable operation of oscillators in large-scale non-microfluidic environments are important and challenging.Developing new regulation elements and updating the circuit structure are possible solutions.In this study,we designed a series of dual-function regulatory switches,which could synchronize the inhibition and activation of specific genes at different periods of bacterial growth in Escherichia coli.Then,the potential application of switches in metabolic engineering is explored through two cases of regulating biosynthesis.Based on the dual-function switch,this study further integrated the cascade element and protease regulatory components to optimize the circuit,and constructed a synthetic oscillation circuit that could oscillate autonomously in a larger culture system.Finally,the possibility of synthetic oscillator in regulating cell morphology and microbial metabolism was explored.1.The dual-function switch based on quorum sensingIn this paper,the Esa QS system from Pantoea stewartii ssp.Stewartii is selected as the starting point for the construction of dual-function,regulation switch by analyzing the regulation mechanisms of different QS systems and related synthetic components.In the Esa QS system,EsaI is the AHL synthase,EsaR is the transcriptional repressor of promoter PesaR and the transcriptional activator of promoter PesaS,We first verified the expression status of promoter PesaS,PesaR,PesaR-P and PesaR-C by exogenous addition of signal molecules.The synthetic promoters of different regulatory modes were combined and transformed into strains L19,L24 and L31 with different AHL production rates.The results of fluorescence characterization showed that the promoter PesaS and PesaR-C have similar sensitivity to AHL during the process of self-induction of QS.The combination of L24SR-C,L19SR-R,L19SR-C and L31SR-C synchronously up-regulated and down-regulated specific genes at different stages of microbial growth.respectively.We tested the effect of the QS switches on regulating the production of poly-βhydroxybutyrate(PHB)and 5-aminolevulinic acid(ALA)in E.coli.Acetyl-coA is a precursor to PHB biosynthesis as well as to the tricarboxylic acid(TCA)cycle,which leads to competition between cell growth and PHB production.The QS switches which switched at different times were used to dynamically redirect metabolic flux from the TCA cycle to PHB production.The results showed that the PHB yield of the experimental strains were higher than that of the control strains.In addition,the experiment showed that regulation in stationary phase was more beneficial to PHB production.In the downstream pathway of ALA,hemB is an essential gene for microbial growth,and the ALA dehydratase encoded by it is a key link related to the accumulation of ALA.Direct modification of hemB will not have a positive effect on the yield but will affect the growth.In this case,we used QS switches for dual dynamic regulation of ALA production,that is,down-regulation of hemB expression and upregulation of ALA production.The ALA yield of dual-regulated strain L19B1-R was nearly twice that of single-regulated strain LI 9BI-R.The use of dual-function switch improved the ALA production ’more effectively than single-regulation.The above cases demonstrated that the QS switch could be used to solve two key problems in metabolic engineering:the redirection of metabolic flux and the dynamic regulation of essential gene knockout.2.The synthetic population-level oscillator based on dual-function switchIn this part,we designed a synthetic population-level oscillation circuit based on the dual-function QS switch.Delayed negative feedback can cause oscillations.Esa QS is able to couple the down-regulation of PesaS-controlled genes at the population level,which means that when the signaling synthase EsaI is set as the regulatory object of PesaS,the delayed negative feedback at the population level is formed.In the design of our circuit,PesaS regulated the expression of EsaI,and the accumulated AHL combined with EsaR to down-regulate the expression of esaI,forming a negative feedback at the transcriptional level to reduce AHL synthesis and prevent signal overload.In addition,the expression of the signal degrading enzyme Aii A regulated by PesaR was up-regulated.AiiA directly degraded AHL to enhance the negative feedback effect and accelerate signal resetting.The cascade element was integrated into the circuit to increase the delay of negative feedback,and the protease regulatory elements were further introduced to add regulation at post-translational level to ensure timely turnover of AiiA to complete multiple signal reset.We tested the circuit in 24-well plates,shake flasks,and quadruple tanks to demonstrate that the oscillator could achieve oscillation behavior in non-microfluidic environments without the addition of inducers or frequent dilution.Next,we constructed the circuit QPK-H to facilitate subsequent operations.Visualization characterization and flow cytometry were performed to further demonstrate the oscillations at the population level.Finally,we tested some potential applications for synthetic oscillators.In the case of regulating cell morphology,strains that periodically expressed genes related to cell division showed strip-rod-sphere-rodstrip morphological changes during culture,which proved that our synthetic oscillator could be an effective regulatory tool for synthetic morphology.In the case of regulating cell metabolism,the production of succinate and mevalonate increased in strains with periodically expressed ptsG.and the synthesis rates of products showed periodic changes.The results suggested that synthetic oscillator,could improve the production by regulating cell metabolism.In summary,we constructed a set of QS based dual-function regulation switches to synchronously up-regulate and down-regulate specific genes at different periods of bacterial growth.The QS switches were used to regulate the production of PHB and ALA,which proved its application potential in metabolic engineering.Based on the QS switch,we optimized the control elements and circuitstructure,and constructed a synthetic population-level oscillator.The synthetic oscillator could oscillate stably in non-microfluidic environments and was an effective tool for regulating cell morphology and cell metabolism.This study extended the toolbox of synthetic biology research,provided a useful element for dynamic regulation in metabolic engineering,and provided useful information for the design,testing and application of large-scale synthetic clocks.