title: High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production.
abstract: Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an "IPP-bypass" isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either kcator Kiof PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.
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: Corey W
authors.initials: CW
authors.lastname: Meadows
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Canu
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; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
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: D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism*; D000439:Pentanols / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D000439:Pentanols; C056744:isopentenol; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase; D008798:Mevalonic Acid
keywords: Biofuel; Enzyme screening; Isopentenol; Isoprenol; Mevalonate pathway; Phosphomevalonate decarboxylase