Design And Construction Of Far-red Light-controlled Transgene Expression Devices In Prokaryotes

The rapid development of synthetic biology makes it possible to design and assemble efficient microbial chassis cells.According to its theory and method,engineered microorganisms are widely applied in industrial production,environmental monitoring,and biomedical fields.Light is abundant in nature and there are many elements that sense light in biological cells.Researchers have developed optical systems that respond to blue,green,and red/near-infrared light in prokaryotes through rational assembly of optical components.These light-controlled systems play pivotal roles in regulating prokaryotic gene expression and specific biological functions.The primary goal of metabolic engineering is to maximize the yield of productions,but the expression level of metabolic enzyme directly affects the synthesis efficiency of metabolites.Therefore,precise regulation of metabolic enzyme expression is the primary task.Traditional constitutive expression leads to overexpression of enzyme genes,generating survival pressure of the hosts.Subsequent developments of chemical regulation methods,including IPTG,metal ions,etc.partially solve the problems of overexpression,but chemical substances have side effects and cannot be spatially regulated.Compared to chemical regulation,light-based optogenetic devices enable remote and trace-free regulation.Optogenetic device represented by blue light is used in metabolic engineering to increase the metabolites production.However,blue light is toxic to cells and has weak penetrating power.Hence,it is of great value to develop a light-conducting system with strong penetrability and low toxicity for metabolic engineering.Based on previous studies,we design and construct a far-infrared light-regulated prokaryotic gene expression device with synthetic biology strategies.First,we construct the V1 version,which includes the light-sensitive elements Bph S and Bph O,the transcriptional element Bld D-Activator,the regulatory element Yhj H,and the reporting system.The light-sensitive element Bph S catalyzes the synthesis of c-di GMP by GTP under the irradiation of far-infrared light(730 nm).The c-di GMP binds to the transcription element Bld D-Activator,recruiting RNA polymerase and driving the inducible promoter P_FRLto initiate expression of reporter gene.The regulatory element Yhj H degrades excess c-di GMP to maintain dynamic balance.The V1 device utilizing three artificially designed hybrid transcriptional activators Bld D-CAP,Bld D-Fis,and Bld D-Sox S as transcriptional elements can not regulate expression of the reporter gene in E.coli BL21(DE3).Optimizing inducible promoter structures of reporter system and the linker of recombinant protein still cannot improve the regulation efficiency of the V1 version.So we choose Mrk H,which is naturally existed in Klebsiella pneumoniae,as the transcription component and assemble the V2 version.After optimizing the RBS intensity of reporter system,the expression of transcriptional elements and regulatory elements,V2 device shows higher regulation efficiency in E.coli BL21(DE3)and the background leakage is less.Under the illumination of far-infrared light,the expression level of reporter gene in light group is 6.3 times higher than that in dark group.Quorum sensing is a system that can be regulated by population density.Microbial populations with a density exceeding the threshold can simultaneously initiate the expression of genes.We combine quorum sensing with far-infrared light control devices,which use the quorum-sensing promoter to drive the four components expression of the far-infrared light-regulating device,and construct the V3 device.V3device has higher regulation efficiency after promoter and Yhj H expression optimization.In E.coli BL21(DE3),the V3 device has a 12-fold induction efficiency in the illumination group compared to the dark group.We also test the dynamics of V3 device with illumination time and illumination intensity,and the results show that it has good far-infrared dependence.In addition,the V3 device has good spatial control ability with far-infrared light which has no effect on the growth of E.coli BL21(DE3).Finally,we test the efficiency of V3 device in different strains.The V3device also has some regulation efficiency in E.coli MG1655,E.coli Top10 and probiotic E.coli Nissle 1917,but the background leakage is high.So more in-depth optimization is needed.In summary,we design and develop far-red light-controlled transgene expression devices in prokaryotes.The microbial optogenetic device has good induction efficiency,adjustability,efficient spatiotemporal specificity and biosafety in E.coli BL21(DE3).It also has some regulatory efficiency in other strains.The far-infrared light-regulated transgene expression devices broaden the function of prokaryotes.They provide a new method for metabolic engineering to improve product yield and provide new ideas for the application of disease treatment.