title: Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction.
abstract: Alkanes and alkenes are ideal biofuels, due to their high energy content and ability to be safely transported. To date, fatty acid-derived pathways for alkane and alkene bioproduction have been thoroughly explored. In this study, we engineered the pathway of the iterative Type I polyketide synthase (PKS) SgcE with the cognate thioesterase (TE) SgcE10 in Escherichia coli, with the goal of overproducing pentadecaheptaene (PDH) followed by its hydrogenation to pentadecane (PD). Based on initial in vitro titration assays, we learned that PDH production is strongly dependent on the SgcE10:SgcE ratio. Thus, we engineered a high-yield E. coli strain by fine-tuning SgcE10 expression via synthetic promoters. We analyzed engineered E. coli strains using a modified multiple reactions monitoring mass spectrometry (MRM-MS)-based targeted proteomic approach, using a chimeric SgcE10 and SgcE fusion construct to gain insight into expression levels of the two proteins. Lastly, through fed-batch fermentation followed by flow chemical hydrogenation, we obtained a PD yield of nearly 140mg/L in single-alkane form. Thus, we not only employed a metabolic engineering approach to the iterative polyketide pathway, we highlighted the potential of PKS shunt products to play a role in the production of single-form and high-value chemicals.
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; J1 Biotech, Co. Ltd., Wuhan 430075, China.
authors.forename: Kaiyue
authors.initials: K
authors.lastname: Wu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.
authors.forename: Yongbo
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Lu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.
authors.forename: Erting
authors.initials: E
authors.lastname: Xiao
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China. Electronic address: liutg@whu.edu.cn.
authors.forename: Tiangang
authors.initials: T
authors.lastname: Liu
mesh_terms: D000473:Alkanes / Q000378:metabolism*; D000475:Alkenes / Q000378:metabolism*; D056804:Biofuels; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism; D061065:Polyketides / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism
chemical_list: D000473:Alkanes; D000475:Alkenes; D056804:Biofuels; D029968:Escherichia coli Proteins; D061065:Polyketides; D048630:Polyketide Synthases; D013869:Thiolester Hydrolases
keywords: Alkane; Alkene; Iterative Pks; Precise protein regulation; Targeted engineering; Thioesterases; in vitro reconstitution