title: Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis.
abstract: Advanced high-throughput screening methods for small molecules may have important applications in the metabolic engineering of the biosynthetic pathways of these molecules. Ectoine is an excellent osmoprotectant that has been widely used in cosmetics. In this study, the Escherichia coli regulatory protein AraC was engineered to recognize ectoine as its non-natural effector and to activate transcription upon ectoine binding. As an endogenous reporter of ectoine, the mutated AraC protein was successfully incorporated into high-throughput screening of ectoine hyper-producing strains. The ectoine biosynthetic cluster from Halomonas elongata was cloned into E. coli. By engineering the rate-limiting enzyme L-2,4-diaminobutyric acid (DABA) aminotransferase (EctB), ectoine production and the specific activity of the EctB mutant were increased. Thus, these results demonstrated the effectiveness of engineering regulatory proteins into sensitive and rapid screening tools for small molecules and highlighted the importance and efficacy of directed evolution strategies applied to the engineering of genetic components for yield improvement in the biosynthesis of small molecules.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Peixia
authors.initials: P
authors.lastname: Jiang
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: Jun
authors.initials: J
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yongzhi
authors.initials: Y
authors.lastname: He
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Lincai
authors.initials: L
authors.lastname: Chen
authors.affiliation: College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China.
authors.forename: Xiwu
authors.initials: X
authors.lastname: Gui
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: dongzy@im.ac.cn.
authors.forename: Zhiyang
authors.initials: Z
authors.lastname: Dong
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: D000599:Amino Acids, Diamino / Q000096:biosynthesis* / Q000235:genetics; D051777:AraC Transcription Factor / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D009154:Mutation
chemical_list: D000599:Amino Acids, Diamino; D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; C045628:ectoine
keywords: Directed evolution; Ectoine; High-throughput screening; Metabolic engineering; Regulatory protein