title: Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor.
abstract: Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Xiong
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Shikun
authors.initials: S
authors.lastname: Lu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jieyuan
authors.initials: J
authors.lastname: Wu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Chaoning
authors.initials: C
authors.lastname: Liang
authors.affiliation: School of Chemistry and Biotechnology, Yunnan Minzu University, Kunming 650500, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Wenzhao
authors.initials: W
authors.lastname: Wang
authors.affiliation: Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing 100048, China. Electronic address: jinjianming@btbu.edu.cn.
authors.forename: Jian-Ming
authors.initials: JM
authors.lastname: Jin
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: tangsy@im.ac.cn.
authors.forename: Shuang-Yan
authors.initials: SY
authors.lastname: Tang
mesh_terms: D015374:Biosensing Techniques; D053898:Biosynthetic Pathways / Q000235:genetics*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000502:physiology*; D044950:Flavanones / Q000302:isolation & purification / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D020005:Propanols / Q000378:metabolism*; D000077185:Resveratrol; D013267:Stilbenes / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: D003373:Coumaric Acids; D044950:Flavanones; D020005:Propanols; D013267:Stilbenes; C439395:1-phenylpropanol; D003066:Coenzyme A Ligases; C005273:naringenin; D000077185:Resveratrol
keywords: Biosensor; High-throughput screening; Phenylpropanoids pathway; Protein engineering; Resveratrol; p-Coumarate:coenzyme A ligase