Figure 3.
Efflux pumps can improve tolerance and production yields for selected biofuels. (A) Plasmid levels for each of the efflux pumps in the library after 72 h of competition. The pool culture was grown without an inhibitor. For all others, biofuel was added at an inhibitory level (Materials and methods). Data from three biological replicates and their average are shown. (B) Inhibition experiments with the top-performing pump (shown with an arrow in (A)) showed that for n-butanol and isopentanol the pump provides no growth advantage over the negative control. Taken together, (A) and (B) demonstrate that none of the efflux pumps we tested improved tolerance to n-butanol or isopentanol. (C) Competition results for biofuels where efflux pumps provided a growth advantage. (D) Top-performing pumps (shown with an arrow in (C)) showed improvements over the negative control strain. Measurements shown in (B) and (D) were taken in triplicate and averaged; error bars show standard deviation. Note that all efflux pumps that survived the α-pinene competition showed good performance when grown individually with α-pinene (Supplementary Figures S3, S4). (E) Limonene yield in a production strain expressing an efflux pump compared with an identical strain without the pump. Error bars show standard deviation between biological replicates.
