Developing biosensors to monitor and regulate intracellular biosynthesis pathways can significantly enhance natural product yields in microbial cell factories.This study created a novel biosensor in Saccharomyces cere...Developing biosensors to monitor and regulate intracellular biosynthesis pathways can significantly enhance natural product yields in microbial cell factories.This study created a novel biosensor in Saccharomyces cerevisiae to respond to p-coumaric acid,a critical precursor in the biosynthesis of polyphenols and flavonoids.This biosensor was constructed by expressing the BsPadR repressor from Bacillus subtilis and engineering hybrid promoters.Notably,the PBS1-CCW12 hybrid promoter exhibited tight regulation by BsPadR and enhanced activity in response to p-coumaric acid.However,excessive BsPadR expression negatively impacted yeast growth,which was mitigated by using weaker promoters,PBST1 and PERG9.Furthermore,the impact of nuclear localization signal(SV40-NLS)positioning on BsPadR functionality was explored,revealing that fusion of an SV40-NLS at the C-terminus of BsPadR enhanced the biosensor’s performance.To validate its utility,we applied this system to dynamically regulate CrtE(geranylgeranyl pyrophosphate synthase),a key enzyme in lycopene biosynthesis.By coupling p-coumaric acid production with lycopene biosynthesis,we enabled high-throughput colorimetric screening for enzyme evolution and strain selection.This novel biosensor serves as a valuable tool for future studies aimed at optimizing the production of p-coumaric acid and its derivatives in S.cerevisiae,thereby advancing the efficiency of biosynthetic processes in microbial cell factories.展开更多
基金supported by National Natural Science Foundation of China(Grant No.32371480)High-level Talent Support Program of Yangzhou University,Postgraduate Research&Practice Innovation Program of Jiangsu Province(Grant No.SJCX25_2391).
文摘Developing biosensors to monitor and regulate intracellular biosynthesis pathways can significantly enhance natural product yields in microbial cell factories.This study created a novel biosensor in Saccharomyces cerevisiae to respond to p-coumaric acid,a critical precursor in the biosynthesis of polyphenols and flavonoids.This biosensor was constructed by expressing the BsPadR repressor from Bacillus subtilis and engineering hybrid promoters.Notably,the PBS1-CCW12 hybrid promoter exhibited tight regulation by BsPadR and enhanced activity in response to p-coumaric acid.However,excessive BsPadR expression negatively impacted yeast growth,which was mitigated by using weaker promoters,PBST1 and PERG9.Furthermore,the impact of nuclear localization signal(SV40-NLS)positioning on BsPadR functionality was explored,revealing that fusion of an SV40-NLS at the C-terminus of BsPadR enhanced the biosensor’s performance.To validate its utility,we applied this system to dynamically regulate CrtE(geranylgeranyl pyrophosphate synthase),a key enzyme in lycopene biosynthesis.By coupling p-coumaric acid production with lycopene biosynthesis,we enabled high-throughput colorimetric screening for enzyme evolution and strain selection.This novel biosensor serves as a valuable tool for future studies aimed at optimizing the production of p-coumaric acid and its derivatives in S.cerevisiae,thereby advancing the efficiency of biosynthetic processes in microbial cell factories.
