An international team of bioengineers has boosted the ability of bacteria to produce isopentenol, a compound with desirable gasoline properties. Isopentenol is a five-carbon alcohol that is a highly promising candidate for biogasoline, but, like other short-chained alcohols, is toxic to E.coli at commercial levels of fuel production.

The finding, published in mBio®, the online open-access journal of the American Society for Microbiology, is a significant step toward developing a bacterial strain that can yield industrial quantities of renewable bio-gasoline.

Isopentenol Researchers Aindrila Mukhopadhyay and Heather Jansen.  Click image for the largest view.  Image Credit: Berkeley Lab.

Isopentenol Researchers Aindrila Mukhopadhyay and Heather Jansen. Click image for the largest view. Image Credit: Berkeley Lab.

Aindrila Mukhopadhyay, a chemist who directs the host engineering program for the U.S. Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) Fuels Synthesis Division, led a study in which transcriptomic data and a synthetic metabolic pathway were used to identify several genes that not only improve tolerance but also production of isopentenol in E.coli.

The metabolic engineering steps to produce short-chain alcohol solvents like isopentenol in the laboratory bacteria Escherichia coli have been worked on extensively by many research groups, explained Mukhopadhyay.

Mukhopadhyay said, “Biofuels are one tool in the array of alternative energy solutions that can be used in our infrastructure immediately.” Sustainably produced fuel compounds can be added directly into gasoline blends used today to offset reliance on fossil fuels and also lower the net carbon emissions from vehicles. “But the solvent-like compounds inhibit microbial growth and that was an aspect that we realized would come up sooner rather than later. We wanted to look at that aspect with a systems biology approach – could we engineer bacteria to also tolerate the solvent it is producing?”

Improving tolerance is key to moving production toward levels that are industrially relevant. Industrial production requires a robust strain that can stably produce for longer periods of time and withstand the accumulation of the solvent-like biofuel.

To address this challenge, the team, which also included researchers from Nanyang Technological University in Singapore, National University of Singapore, and the University of California, Berkeley, treated a non-producing E. coli strain with isopentenol by adding it to the culture. As the bacteria responded to the solvent-stressor, the team measured which genes were shifted up or down by looking at messenger RNA transcripts across the entire genome.

The team chose 40 genes that the bacteria cranked up in response to isopentenol – presumably because their actions helped mitigate the toxicity in some way. Next, they overexpressed each one in a bacterial strain actively producing isopentenol to see which ones might improve the strain’s growth.

Of the eight genes that rescued strain growth, two stood out as promising – MetR, a biosynthesis regulator, that improved isopentenol production by 55%, and MdlB, a transporter, that improved production by 12%. If the researchers bumped up the levels of the MdlB transporter protein inside the cells even further, they saw production improve by as much as 60% over the original strain.

“Finding a transporter really appealed to us because it has the potential to export the final solvent product out of the cell,” says Mukhopadhyay. “And in this case, once enough alcohol gets outside the cell, it might phase separate and not even be accessible to the organism anymore.” In other words, the biofuel would separate away to sit atop the watery broth the bacteria live in.

As an added bonus, the MdlB protein is a good candidate for directed evolution experiments that could improve the performance and specificity of the transporter for shuttling isopentenol out as quickly as possible. Combining a more efficient transporter with other genes that improve tolerance might produce a strain that can generate bio-gasoline for the gas pump in the near future.

Mukhopadhyay continued saying, “In order for microbial biofuel production to be cost effective, yields must exceed native microbial tolerance levels, necessitating the development of solvent-tolerant microbial strains. In parallel with improved tolerance it is also crucial that we improve production.”

Mukhopadhyay and her group are especially eager to further investigate the MdlB transporter, which they believe, as the first native transporter gene shown to improve production of a short-chain alcohol, will provide a valuable new avenue for host engineering in biogasoline production.

“The critical point is that you must first identify the genes that can serve as engineering targets, and then test them to find which ones work best,” Mukhopadhyay says. “Now that we have identified MdlB as a target, we are going to examine it in great depth to see how can we improve its function and optimize its use in a production microbe.”

Isopentenol, a form of the alcohol pentenol, would be an excellent gasoline extender. Nothing was said about feedstocks of other pertinent facts, but for four carbon atom alcohols and higher its good to see some action again.


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