Striking the right balance: Researchers counteract biofuel toxicity in microbes

May 11, 2011
Microbial production of biofuels from cellulose can be enhanced with the use of efflux pumps to export toxic fuels from the microbes. Credit: Image courtesy of Aindrila Mukhopadhyay

Advanced biofuels – liquid transportation fuels derived from the cellulosic biomass of perennial grasses and other non-food plants, as well as from agricultural waste – are highly touted as potential replacements for gasoline, diesel and jet fuels. Equally touted is the synthesis of these fuels through the use of microbes. However, many of the best candidate compounds for advanced biofuels are toxic to microbes, which presents a "production versus survival" conundrum. Researchers at the U.S. Department of Energy (DOE)'s Joint BioEnergy Institute (JBEI) have provided a solution to this problem by developing a library of microbial efflux pumps that were shown to significantly reduce the toxicity of seven representative biofuels in engineered strains of Escherichia coli.

"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," says Aindrila Mukhopadhyay, a chemist with JBEI's Fuels Synthesis Division, who led this research.

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

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 have been identified, product toxicity to microbes is a common problem in strain engineering for biofuels and other biotechnology applications.

"In order for microbial biofuel production to be cost effective, yields must exceed native microbial tolerance levels, necessitating the development of stress-tolerant microbe strains," Mukhopadhyay says. "It is crucial that we improve tolerance in parallel with the development of metabolic pathways for the production of next-generation biofuels."

Microbes employ various strategies for addressing cell toxicity but perhaps the most effective are efflux pumps, proteins in the cytoplasmic membrane of cells whose function is to transport toxic substances out of the cell. This is done actively, using proton motive force. However, to date very few of these have been characterized for efficacy against biofuel like compounds.

"Sequenced bacterial genomes include many efflux pumps but remain a largely unexplored resource for use in engineering fuel tolerance," Mukhopadhyay says. "We took a systematic approach to screen a library of primarily uncharacterized heterologous pumps for engineering biofuel tolerant host strains. We were then able to demonstrate that expression of a heterologous pump can increase the yield of a biofuel in the production strain."

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 us to rank the complete set of pumps and select a subset that represented a uniform distribution of candidate genes," says Mukhopadhyay. "To construct the library, we 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."

In a series of survival competitions, the two microbial efflux pumps that performed best 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," Mukhopadhyay says. "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."

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.

"We believe our bioprospecting strategy for biofuel tolerance mechanisms is going to be a valuable and widely applicable tool in the biotechnology field for engineering new microbial production strains," Mukhopadhyay says.

Explore further: Scientists tap trees' evolutionary databanks to discover environment adaptation strategies

Related Stories

Barren deserts can host complex ecosystems in their soils

Dec 22, 2014

"Biological soil crusts" don't look like much. In fact, people often trample right over these dark, or green-tinted, sometimes raised patches in the desert soil. But these scruffy stretches can house delicate ...

Robots may receive urine-powered artificial 'hearts'

Nov 27, 2013

(Phys.org) —It's a first: researchers have built the first artificial-heart-like pump that is powered by microbial fuel cells fed on human urine. But instead of being used as a prosthetic device for human ...

Recommended for you

Keep dogs and cats safe during winter

Dec 27, 2014

(HealthDay)—Winter can be tough on dogs and cats, but there are a number of safe and effective ways you can help them get through the cold season, an expert says.

Scientists target mess from Christmas tree needles

Dec 26, 2014

The presents are unwrapped. The children's shrieks of delight are just a memory. Now it's time for another Yuletide tradition: cleaning up the needles that are falling off your Christmas tree.

Top Japan lab dismisses ground-breaking stem cell study

Dec 26, 2014

Japan's top research institute on Friday hammered the final nail in the coffin of what was once billed as a ground-breaking stem cell study, dismissing it as flawed and saying the work could have been fabricated.

User comments : 0

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.