title: Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol.
abstract: Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E. coli through overexpression of the cellulose binding domain (CBD) of Cellulomonas fimi exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The in vivo activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using E. coli transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. In vivo synthesis of 1-butanol using the engineered E. coli yielded 1.98g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from E. coli expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the in vitro 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of in vitro reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both in vivo and in vitro for the efficient production of desired products.
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
authors.forename: Gui Hwan
authors.initials: GH
authors.lastname: Han
authors.affiliation: 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.forename: Wonjae
authors.initials: W
authors.lastname: Seong
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
authors.forename: Yaoyao
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Yoon
authors.affiliation: 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.forename: Seong Keun
authors.initials: SK
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Yeom
authors.affiliation: 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.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.affiliation: 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.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D020001:1-Butanol / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods*; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D020001:1-Butanol
keywords: 1-Butanol; Cellulose binding domain; Enzyme cascade; Inclusion body; Leucine zipper; Metabolon