title: Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production.
abstract: Branched C5 alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C5 alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Aram
authors.initials: A
authors.lastname: Kang
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: George
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: tslee@lbl.gov.
authors.forename: Taek Soon
authors.initials: TS
authors.lastname: Lee
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D055442:Metabolome / Q000502:physiology; D008798:Mevalonic Acid / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D000439:Pentanols / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D000439:Pentanols; C056744:isopentenol; C004809:isopentenyl pyrophosphate; D008798:Mevalonic Acid
keywords: Aeration; Biofuel; IPP; Isopentenol; Isoprenol; Mevalonate pathway; Phosphomevalonate decarboxylase; Toxicity