title: Engineering Escherichia coli to produce branched-chain fatty acids in high percentages.
abstract: Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.
authors.affiliation: Division of Biological and Biomedical Sciences, Washington University in St. Louis, Saint Louis, MO 63130, USA.
authors.forename: Gayle J
authors.initials: GJ
authors.lastname: Bentley
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA; Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Linda P
authors.initials: LP
authors.lastname: Guamán
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Division of Biological and Biomedical Sciences, Washington University in St. Louis, Saint Louis, MO 63130, USA; Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA; Institute of Materials Science and Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA. Electronic address: fzhang@seas.wustl.edu.
authors.forename: Fuzhong
authors.initials: F
authors.lastname: Zhang
mesh_terms: D053898:Biosynthetic Pathways / Q000502:physiology; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis* / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D050155:Lipogenesis / Q000502:physiology; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005227:Fatty Acids; D010088:Oxidoreductases; D005947:Glucose
keywords: Biofuels; Branched-chain fatty acid; Cold flow; Protein lipoylation