Engineering microbial biofuel tolerance and export using efflux pumps.

Molecular systems biology (2011-05-11)
Mary J Dunlop, Zain Y Dossani, Heather L Szmidt, Hou Cheng Chu, Taek Soon Lee, Jay D Keasling, Masood Z Hadi, Aindrila Mukhopadhyay
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3-Methyl-1-butanol, anhydrous, ≥99%
Sigma-Aldrich
Isoamyl alcohol, ≥98%, FG
Sigma-Aldrich
3-Methylbutanol, BioReagent, for molecular biology, ≥98.5%
Sigma-Aldrich
3-Methyl-1-butanol, reagent grade, 98%
Sigma-Aldrich
Isoamyl alcohol, natural, ≥98%, FG
Sigma-Aldrich
3-Methylbutanol, puriss. p.a., ACS reagent, ≥98.5% (GC)
Sigma-Aldrich
3-Methyl-1-butanol, ACS reagent, ≥98.5%
Sigma-Aldrich
3-Methylbutanol, BioUltra, for molecular biology, ≥99.0% (GC)
Supelco
3-Methylbutanol, analytical standard

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