Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries.Adenosylcobalamin is an essential coenzyme that is required in ...Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries.Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries.High-throughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level.In this study,we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin,the inverter part was an orthogonal transcriptional repressor to obtain signal inversion,and the output part was a fluorescent protein to be easily detected.The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo.This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization.An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control.Then,using such a biosensor combined with fluorescence-activated cell sorting,we established a high-throughput screening platform for screening adenosylcobalamin overproducers.This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites.展开更多
基金supported by the National Key R&D Program of China[Grant number 2021YFC2100700]the National Natural Science Foundation of China[Grant numbers 22178372,32300069]+3 种基金TIB-VIB Joint Center of Synthetic Biology[Grant number TSBICIP-IJCP-002]the National Science Fund for Distinguished Young Scholars[Grant number 22325807]the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project[Grant numbers TSBICIP-KJGG-011,TSBICIP-CXRC-055]the Yellow River Delta Industry Leading Talents[Grant number DYRC20190212].
文摘Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries.Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries.High-throughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level.In this study,we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin,the inverter part was an orthogonal transcriptional repressor to obtain signal inversion,and the output part was a fluorescent protein to be easily detected.The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo.This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization.An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control.Then,using such a biosensor combined with fluorescence-activated cell sorting,we established a high-throughput screening platform for screening adenosylcobalamin overproducers.This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites.
