Research efforts are underway at JBEI and elsewhere to engineer microorganisms, such as E. coli, to produce advanced biofuels in a cost-effective manner. These fuels, which encompass short-to-medium carbon-chain alcohols, such as butanol, isopentanol and geraniol, can replace gasoline on a gallon-for-gallon basis and be used in today’s infrastructures and engines, unlike ethanol.

Biofuels made from branched carbon-chain compounds, such as geranyl acetate and farnesyl hexanoate, would also be superior to today’s biodiesel, which is made from esters of linear fatty acids. Cyclic alkenes, such as limonene and pinene, could serve as precursors to jet fuel. Although biosynthetic pathways to the production of these carbon compounds in microbes have been identified, product toxicity to microbes is a common problem in strain engineering for biofuels and other biotechnology applications.

Working with all available microbial genome sequence data, we generated a library of largely uncharacterized genes and were able to devise a simple but highly effective strategy to identify efflux pumps that could alleviate biofuel toxicity in E. coli and, as a consequence, help improve biofuel production.

—Aindrila Mukhopadhyay, chemist with JBEI’s Fuels Synthesis Division, and corresponding author

Mukhopadhyay, who also holds an appointment with the Lawrence Berkeley National Laboratory (Berkeley Lab)’s Physical Biosciences Division, is the corresponding author on a paper published in the journal Molecular Systems Biology, titled “Engineering Microbial Biofuel Tolerance and Export Using Efflux Pumps.” Molecular Systems Biology is an open-access journal published by European Molecular Biology Organization and Nature Publishing Group.

Co-authoring the paper with Mukhopadhyay were Mary Dunlop, Zain Dossani, Heather Szmidt, Hou-Cheng Chu, Taek Soon Lee, Jay Keasling and Masood Hadi.

Since all known solvent-resistant efflux pumps in Gram-negative bacteria fall into the hydrophobe/amphiphile efflux (HAE1) family, Mukhopadhyay and her colleagues constructed a database of all HAE1 pumps from sequenced bacterial genomes. They then performed a bioinformatics screen to compare regions predicted to be responsible for substrate specificity to those of TtgB, a well-characterized solvent-resistant efflux pump.

This metric allowed them to rank the complete set of pumps and select a subset that represented a uniform distribution of candidate genes, says Mukhopadhyay. To construct their library of 43 pumps, they amplified efflux pump operons from the genomic DNA of the selected bacteria, cloned them into a vector, and transformed the vector into an E. coli host strain.

...we asked whether competing the cultures in the presence of different biofuels would identify unique sets of resistant efflux pumps for each biofuel. With respect to the compounds tested, our results fall broadly into two classes: (i) biofuels that are toxic, but where pumps do not reduce toxicity and (ii) biofuels where the pumps do reduce toxicity. Both n-butanol and isopentanol fall into the first class of fuels.

This could be because none of the pumps in the library export these compounds or, alternatively, the rate of export may not be sufficient to counteract intracellular accumulation. Pumps improved tolerance for the second class of fuels. We saw a distinct separation between the competition winners and those pumps that were not beneficial. The competition survivors and their relative abundances in the culture were biofuel-specific.

—Dunlop et al.

The two microbial efflux pumps that performed best in the survival competitions were the native E. coli pump AcrAB and a previously uncharacterized pump from a marine microbe Alcanivorax borkumensis.

We focused on the A. borkumensis pump and tested it in a strain of host microbe engineered to produce the limonene jet fuel precursor. Microbes expressing the pump produced significantly more limonene than those with no pump, providing an important proof of principle demonstration that efflux pumps that increase tolerance to exogenous biofuel can also improve the yield of a production host.

—Aindrila Mukhopadhyay

Mukhopadhyay and her JBEI colleagues have begun evaluating microbial efflux pumps for other important compounds as well as inhibitors present in the carbon source from lignocellulose. They are also looking to improve the A. borkumensis pump and other high performers in their current library, and to optimize the systems by which pump genes are expressed in engineered biofuel-producing microbial strains.

Resources

• Mary J Dunlop, Zain Y Dossani, Heather L Szmidt, Hou Cheng Chu, Taek Soon Lee, Jay D Keasling, Masood Z Hadi, & Aindrila Mukhopadhyay (2011) Engineering microbial biofuel tolerance and export using efflux pumps. Molecular Systems Biology 7 Article number: 487 doi: 10.1038/msb.2011.21