Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis

• Published in: Metabolic engineering 2015-07, Vol.30, p.149-155
• Main Authors: Chen, Wei, Zhang, Shan, Jiang, Peixia, Yao, Jun, He, Yongzhi, Chen, Lincai, Gui, Xiwu, Dong, Zhiyang, Tang, Shuang-Yan
• Format: Article
• Language: English
• Subjects: Amino Acids, Diamino - biosynthesis; Amino Acids, Diamino - genetics; AraC Transcription Factor - genetics; AraC Transcription Factor - metabolism; Directed evolution; Directed Molecular Evolution - methods; Ectoine; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; High-throughput screening; Metabolic engineering; Metabolic Engineering - methods; Mutation; Regulatory protein
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2015.05.004

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. [Display omitted] •AraC was engineered to recognize ectoine as its non-natural effector.•Mutated AraC protein was incorporated into high-throughput screening of ectoine.•Ectoine hyper-producing strains were successfully selected.•Specific activity of the key enzyme of ectoine biosynthetic pathway was improved.•Regulatory proteins could be engineered into small molecule screening tools.