title: Engineering microbial biofuel tolerance and export using efflux pumps.
abstract: Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes.
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Dunlop
authors.forename: Zain Y
authors.initials: ZY
authors.lastname: Dossani
authors.forename: Heather L
authors.initials: HL
authors.lastname: Szmidt
authors.forename: Hou Cheng
authors.initials: HC
authors.lastname: Chu
authors.forename: Taek Soon
authors.initials: TS
authors.lastname: Lee
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.forename: Masood Z
authors.initials: MZ
authors.lastname: Hadi
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
mesh_terms: D020001:1-Butanol / Q000378:metabolism / Q000633:toxicity; D056804:Biofuels / Q000633:toxicity; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D046228:Microarray Analysis; D000439:Pentanols / Q000378:metabolism / Q000633:toxicity
chemical_list: D056804:Biofuels; D026901:Membrane Transport Proteins; D000439:Pentanols; D020001:1-Butanol; C029683:isopentyl alcohol