Design And Construction Of A Two-component Biosensor For Gamma-aminobutyric Acid

Gamma-aminobutyric acid(GABA)as a natural non-protein amino acid is widely used in medicine,food,plastics and other fields.At present,there is still a bottleneck in the production of GABA by microbial fermentation,so it is necessary to develop suitable biosensors for high-throughput screening.PctC,a chemotactic receptor in Pseudomonas aeruginosa,naturally responds to GABA.In this study,the extracellular sensory domain of PctC was fused with the intracellular domain of Sensor Kinase(SK)in the endogenous TCSs of Escherichia coli to construct a chimeric TCSs responsive to GABA.In order to better apply the biosensor in the field of metabolic engineering,the performance of the biosensor was modified as follows:(1)FACS-based PctC/PhoQ chimeric library screening biosensors with high dynamic range.In order to improve the dynamic range of the biosensor,different PctC and PhoQ were intercepted for fusion expression to construct a chimeric library.Later,several chimeric TCSs with improved dynamic range were obtained by combining FACS screening,one of which had a response rate of 2.02 times to GABA.(2)Create a biosensor of orthogonal GABA by reducing other TCSs crosstalk.Biosensors based on chimeric TCSs are susceptible to crosstalk,such as endogenous PhoQP and other TCSs in E.coli.In order to eliminate these crosstalk,phoQ gene was knocked out,and a mutant pair PhoQ*P*was constructed,which successfully reduced the basic leakage level of biosensors and increased the dynamic range to 3.50 times.(3)Functional regulation of biosensors using different PhoP binding regulated DNA sequences.PhoP can bind to different DBS to control the expression of different genes.The binding affinity of PhoP and its different DBS was verified in vitro.The final screening results showed that PhoP and OyrbL had the highest binding affinity.The expression of sfGFP was controlled by PhoP and OyrbL,and the dynamic range of the biosensor was increased to 3.97 times.(4)Combined promoter,ribosome binding site engineering and degradation enzyme control system to improve the dynamic range of biosensors.By replacing the promoter regulating sfGFP expression with a lower intensity,the dynamic range of the biosensor was finally increased to 8.11 times.The dynamic range of the biosensor was further improved to 15.81-fold by using a protein-reporting control system based on TEV protease.Base leak level reduced from 4222(GFP/OD600)to 615(GFP/OD600).Finally.the constructed and optimized biosensor combined with ARTP technology was applied to the screening of GABA-producing strains.The engineered GABAproducing strains were first mutated by ARTP,and then cultured by fermentation.Finally,the fermentation supernatant was used to induce chimeric TCSs.Finally,four mutant strains with higher GABA yield than the control were screened.The GABA yield of these strains was positively correlated with the expression level of sfGFP.In conclusion,this study constructed a low leakage and orthogonal GABA chimeric TCS in E.coli and successfully realized its application in the screening of GABa-producing bacteria,opening the door for the creation of customized gene-coded biosensors by fusion of chemotactic receptors and histidine kinases,and setting an example for the creation of low base leakage and orthogonal TCSs for application in synthetic biology.