title: Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli.
abstract: Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: George
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Mitchell G
authors.initials: MG
authors.lastname: Thompson
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Aram
authors.initials: A
authors.lastname: Kang
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Baidoo
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Leanne Jade G
authors.initials: LJ
authors.lastname: Chan
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA [3] Department of Bioengineering, University of California, Berkeley, CA 94720, USA [4] Department of Chemical &Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: 1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Taek Soon
authors.initials: TS
authors.lastname: Lee
mesh_terms: D000438:Alcohols / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D000439:Pentanols / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism
chemical_list: D000438:Alcohols; D029968:Escherichia coli Proteins; D000439:Pentanols; D011993:Recombinant Fusion Proteins; D013729:Terpenes; C029683:isopentyl alcohol; C546903:NudB protein, E coli; D011755:Pyrophosphatases