Design And Construction Of Hydrogen Sulfide Based Quorum Sensing Gene Circuits In Escherichia Coli

In the bacterial quorum sensing systems,the autoinducers are usually uncommon metabolic intermediates,for instance,a specific small compound secreted by itself.Hydrogen sulfide H2S,as a small chemical produced by microorganisms,has the characteristic of freely passing cell membranes.The H2S released by Escherichia coli is mainly produced from the 3-MST pathway.We found that its production has a positive linear correlation with the cell density,and is independent of the species of E.coli,the type of culture medium and the pH of the culture medium.From the perspective of quorum sensing,H2S has the poteintial to be an autoinducer.Herein,we designed and constructed a quorum sensing gene circuits using H2S as the autoinducer.The dynamic regulation gene circuit involves several key components:signal converter,sensor and actuator.Sulfide:quinone oxidoreductase(SQR)is a sulfur oxidizing protein located on the inside of plasma membrane.It oxidizes H2S to sulfane sulfur(HSnH,n?2).Compared with H2S,the generated HSnH will be stored in the cell.Since E.coli does not have inherent SQR,we expressed PpSQR or CpSQR in it.Results showed that both of them could function normally in E.coli with different activities.A few bacterial transcriptional regulators that can specifically sense sulfane sulfu have been discovered in recent years.We tested the sensitivity of FisR,CstR and BigR to HSnH in E.coli.Results showed that only CstR specifically responded to HSnH.Therefore,we chose CstR as the signal sensor element.As for actuator,by combining different promoters and operators,we obtained a number of actuators with different transcriptional strengths.The obtained gene circuit was applied in the cell factory construction,and the automatic switch from the growth stage to the production stage of the bacteria was successfully achieved.Taking the xylose acid production as an example,the yield of 9.68g/L(lOg/L xylose)was achieved.In order to further improve the transcriptional strength of this gene circuit,we used the T7 RNA polymerase and the T7 promoter to construct a two-layered gene circuit:in the first layer,the H2S-based quorum sensing gene circuit controls the expression of T7 RNA polymerase depending on the change of the cell density;In the second layer,the T7 promoter is activated by the T7 RNA polymerase.Results showed that compared with IPTG-T7 system,the double-layered gene circuit had higher protein expression level and most importantly,self-induction.The above mentioned genetic circuits were all constructed and assembled in plasmids.Considering the plasmid capacity limitation and the influence of low conversion efficiency,we decided to integrate part of the gene circuit into E.coli genome:cstR and its promoter were integrated into E.coli BL21(DE3)genome to replace the T7 RNAP promoter in it.Subsequently,the plasmid PET30 was modified to adapt to the recombinant strain.The new protein expression system inherits the advantages of the plasmid-based two-layered gene circuits.In summary,this work explored the signaling function of H2S and constructed a series of quorum sensing gene circuits with different transcriptional strengths and different sensitivities.Application of these gene circuits in E.coli cell factory constuction realized the automatic switch from growth stage to production stage.From the perspective of the cell factory optimization,these gene circuits have obvious advantages.This study provided new tools for the research of both synthetic biology and dynamic metabolic engineering.