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
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
Metabolic biosensors are increasingly used in metabolic engineering and synthetic biology.In this study,using Saccha-romyces cerevisiae as a model system,we developed a methodology to identify promoter elements that a...Metabolic biosensors are increasingly used in metabolic engineering and synthetic biology.In this study,using Saccha-romyces cerevisiae as a model system,we developed a methodology to identify promoter elements that are responsive to glucaric acid.Through transcriptome analysis,it was found that multiple genes were upregulated when cells were exposed to high concentrations of glucaric acid.From the promoters of these candidate genes,the YCR012W promoter(PYCR012W)was observed to specifically respond to glucaric acid in a dose-dependent manner.To gain further insight into the binding site of glucaric acid-responsive activators,we truncated the promoter and revealed that the-564 to-464 bp regions of PYCR012W was essential for glucaric acid-responsive expression.To investigate the glucaric acid-responsive transcription factors,we predicted the transcription factor binding sites in the-564 to-464 bp region of PYCR012W and found that two transcription factors,Ash1p and Cbf1p,might be linked to glucaric acid responses.The strategies used in this study outline a method for the identification and development of metabolic biosensors.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[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.
基金This work was supported by the National Key R&D Program of China(2019YFA0905502)the National Natural Science Foundation of China(21877053)+1 种基金the Natural Science Foundation of Jiangsu Province(BK20181345)the Open Foundation of Jiangsu Key Laboratory of Industrial Biotechnology(KLIB-KF201807).
文摘Metabolic biosensors are increasingly used in metabolic engineering and synthetic biology.In this study,using Saccha-romyces cerevisiae as a model system,we developed a methodology to identify promoter elements that are responsive to glucaric acid.Through transcriptome analysis,it was found that multiple genes were upregulated when cells were exposed to high concentrations of glucaric acid.From the promoters of these candidate genes,the YCR012W promoter(PYCR012W)was observed to specifically respond to glucaric acid in a dose-dependent manner.To gain further insight into the binding site of glucaric acid-responsive activators,we truncated the promoter and revealed that the-564 to-464 bp regions of PYCR012W was essential for glucaric acid-responsive expression.To investigate the glucaric acid-responsive transcription factors,we predicted the transcription factor binding sites in the-564 to-464 bp region of PYCR012W and found that two transcription factors,Ash1p and Cbf1p,might be linked to glucaric acid responses.The strategies used in this study outline a method for the identification and development of metabolic biosensors.
