生物传感器已经成为实时监测特定小分子和精确控制生物系统中基因表达的强大工具。用于1,4-丁二胺生物合成的高通量传感器可以极大地提高1,4-丁二胺高产菌株的筛选效率。为研究调整生物传感器特性的策略,本研究开发了一种以转录调节因子...生物传感器已经成为实时监测特定小分子和精确控制生物系统中基因表达的强大工具。用于1,4-丁二胺生物合成的高通量传感器可以极大地提高1,4-丁二胺高产菌株的筛选效率。为研究调整生物传感器特性的策略,本研究开发了一种以转录调节因子PuuR为基础的1,4-丁二胺生物传感器,其同源的操作子puuO被组装在大肠杆菌组成型启动子PgapA中,以控制下游的高能绿色荧光蛋白(superfolder green fluorescent protein,sfGFP)作为报告蛋白表达。最终该传感器在1,4-丁二胺浓度处于0–50 mmol/L时GFP/OD600值与1,4-丁二胺浓度之间能稳定地表现出线性关系。本研究采用大肠杆菌基因组中不同强度的启动子对1,4-丁二胺生物传感器进行分子改造,探究并改进基于PuuR的1,4-丁二胺生物传感器的功能性质,为高通量筛选高产1,4-丁二胺的工程菌株奠定了基础。展开更多
L-carnitine is a quaternary amine compound essential for eukaryotic metabolism.It is mainly involved in the oxidative decomposition of medium-and long-chain fatty acids and provides energy for the body.Therefore,it is...L-carnitine is a quaternary amine compound essential for eukaryotic metabolism.It is mainly involved in the oxidative decomposition of medium-and long-chain fatty acids and provides energy for the body.Therefore,it is widely used in health care and food additives.As a pivotal transcriptional activator of L-carnitine metabolism,CaiF is notably activated by crotonobetainyl-CoA,a key intermediate product in the carnitine metabolic pathway.Capitalizing on this mechanism,a sophisticated biosensor was ingeniously developed.Nevertheless,it is worth mentioning that the biosensor currently exhibits a relatively restricted detection range,which results in some specific limitations in practical application scenarios.In this paper,we constructed a biosensor based on CaiF and developed a strategy for modifying this biosensor.The structural configuration of CaiF was formulated by computer-aided design,and the DNA binding site was simulated,which was verified by alanine scanning.Functional Diversity-Oriented Volume-Conservative Substitution Strategy of the key sites of CaiF was conducted to extend the dynamic range of the biosensor.The biosensor based on CaiFY47W/R89A,which exhibited a considerably expanded concentration response range,from 104 mM-10 mM,was obtained.The response range was 1000-fold wider and the output signal intensity was 3.3-fold higher to that of the control biosensor.These variants may have great value in improving the L-carnitine production process.展开更多
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
文摘生物传感器已经成为实时监测特定小分子和精确控制生物系统中基因表达的强大工具。用于1,4-丁二胺生物合成的高通量传感器可以极大地提高1,4-丁二胺高产菌株的筛选效率。为研究调整生物传感器特性的策略,本研究开发了一种以转录调节因子PuuR为基础的1,4-丁二胺生物传感器,其同源的操作子puuO被组装在大肠杆菌组成型启动子PgapA中,以控制下游的高能绿色荧光蛋白(superfolder green fluorescent protein,sfGFP)作为报告蛋白表达。最终该传感器在1,4-丁二胺浓度处于0–50 mmol/L时GFP/OD600值与1,4-丁二胺浓度之间能稳定地表现出线性关系。本研究采用大肠杆菌基因组中不同强度的启动子对1,4-丁二胺生物传感器进行分子改造,探究并改进基于PuuR的1,4-丁二胺生物传感器的功能性质,为高通量筛选高产1,4-丁二胺的工程菌株奠定了基础。
基金supported by the National Key R&D Program of China(2022YFC2106100)the National Science Fund for Distinguished Young Scholars(22325807)+1 种基金the National Natural Science Foundation of China(22178372,32300069,42177112)the International Partnership Program of the Chinese Academy of Sciences(306GJHZ2023019GC).
文摘L-carnitine is a quaternary amine compound essential for eukaryotic metabolism.It is mainly involved in the oxidative decomposition of medium-and long-chain fatty acids and provides energy for the body.Therefore,it is widely used in health care and food additives.As a pivotal transcriptional activator of L-carnitine metabolism,CaiF is notably activated by crotonobetainyl-CoA,a key intermediate product in the carnitine metabolic pathway.Capitalizing on this mechanism,a sophisticated biosensor was ingeniously developed.Nevertheless,it is worth mentioning that the biosensor currently exhibits a relatively restricted detection range,which results in some specific limitations in practical application scenarios.In this paper,we constructed a biosensor based on CaiF and developed a strategy for modifying this biosensor.The structural configuration of CaiF was formulated by computer-aided design,and the DNA binding site was simulated,which was verified by alanine scanning.Functional Diversity-Oriented Volume-Conservative Substitution Strategy of the key sites of CaiF was conducted to extend the dynamic range of the biosensor.The biosensor based on CaiFY47W/R89A,which exhibited a considerably expanded concentration response range,from 104 mM-10 mM,was obtained.The response range was 1000-fold wider and the output signal intensity was 3.3-fold higher to that of the control biosensor.These variants may have great value in improving the L-carnitine production process.
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.
