title: CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production.
abstract: Methods for simple and efficient regulation of metabolic pathway genes are essential for maximizing product titers and conversion yields, and for minimizing the metabolic burden caused by heterologous expression of multiple genes often in the operon context. Clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) is emerging as a promising tool for transcriptional modulation. In this study, we developed a regulatable CRISPRi system for fine-tuning biosynthetic pathways and thus directing carbon flux toward target product synthesis. By exploiting engineered Escherichia coli harboring a biosynthetic mevalonate (MVA) pathway and plant-derived terpenoid synthases, the CRISPRi system successfully modulated the expression of all the MVA pathway genes in the context of operon and blocked the transcription of the acetoacetyl-CoA thiolase enzyme that catalyzes the first step in the MVA pathway. This CRISPRi-guided balancing of expression of MVA pathway genes led to enhanced production of (-)-α-bisabolol (C15) and lycopene (C40) and alleviation of cell growth inhibition that may be caused by expression of multiple enzymes or production of toxic intermediate metabolites in the MVA pathway. Coupling CRISPRi to cell growth by regulating an endogenous essential gene (ispA) increased isoprene (C5) production. The regulatable CRISPRi system proved to be a robust platform for systematic modulation of biosynthetic and endogenous gene expression, and can be used to tune biosynthetic metabolic pathways. Its application can enable the development of microbial 'smart cell' factories that can produce other industrially valuable products in the future.
authors: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
authors: Seong Keun
authors: SK
authors: Kim
authors: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
authors: Gui Hwan
authors: GH
authors: Han
authors: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
authors: Wonjae
authors: W
authors: Seong
authors: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
authors: Haseong
authors: H
authors: Kim
authors: Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 52828, Republic of Korea.
authors: Seon-Won
authors: SW
authors: Kim
authors: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea. Electronic address: dhlee@kribb.re.kr.
authors: Dae-Hee
authors: DH
authors: Lee
authors: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea. Electronic address: sglee@kribb.re.kr.
authors: Seung-Goo
authors: SG
authors: Lee
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D053898:Biosynthetic Pathways / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008798:Mevalonic Acid / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism
chemical_list: D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D008798:Mevalonic Acid
keywords: CRISPR interference; Escherichia coli; Mevalonate pathway; Terpenoid