title: Improving olefin tolerance and production in E. coli using native and evolved AcrB.
abstract: Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha-olefins; 1-hexene, 1-octene, and 1-nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB-TolC, in enhancing tolerance towards these olefin compounds. AcrAB-TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1-hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for production in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB-TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1-hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha-olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergistic manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production.
authors.affiliation: Total New Energies Inc., Emeryville, California, 94608.
authors.forename: Florence
authors.initials: F
authors.lastname: Mingardon
authors.forename: Camille
authors.initials: C
authors.lastname: Clement
authors.forename: Kathleen
authors.initials: K
authors.lastname: Hirano
authors.forename: Melissa
authors.initials: M
authors.lastname: Nhan
authors.forename: Eric G
authors.initials: EG
authors.lastname: Luning
authors.forename: Angelique
authors.initials: A
authors.lastname: Chanal
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
mesh_terms: D000475:Alkenes / Q000378:metabolism* / Q000633:toxicity; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007218:Industrial Microbiology; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary
chemical_list: D000475:Alkenes; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli; C117224:1-hexene; C037690:1-octene
keywords: directed evolution; host engineering; olefin production; solvent tolerance