WHAT DID THE 2011 EARLY CAREER AWARD ALLOW YOU TO DO?

Replacements for gasoline, diesel, and jet fuel can be derived directly from living organisms. However, a major technical challenge in engineering microbial biofuel synthesis is that cells can only tolerate limited concentrations of the  fuels that they are synthesizing -- the fuel that they produce is itself toxic. Thus, there is an upper limit to the quantity of biofuels that microorganisms can produce. Cells must trade off biofuel production against survival.

Support from my Early Career Research Award enabled my lab to find and optimize novel mechanisms for improving microbes’ tolerance of biofuels. We focused on microbes that thrive in hydrocarbon-rich environments, such as around natural oil seepages in the ocean and in the soil. Using genomic DNA from hydrocarbon-tolerant microbes, we identified several promising biofuel tolerance mechanisms. Some examples of these mechanisms include approaches that change the composition of the cell membrane and those that export biofuel from the cell.

We next focused on optimizing biofuel export using efflux pumps, which are proteins that work to transport biofuels out of the cells and thereby improve tolerance. However, efflux pumps require energy and therefore place an additional burden on the cell.

We developed a mathematical model to describe these opposing effects. The model predicts that there is an optimal number of pumps that can balance the benefits of biofuel export against the energy demands associated with the pumps.  We tested this prediction experimentally and demonstrated that cell growth is indeed maximized at intermediate pump levels. We constructed a synthetic feedback loop in E. coli using a sensor for biofuel stress coupled with an efflux pump that can export biofuel.

These multi-disciplinary approaches have allowed us to improve biofuel tolerance within microbes, setting the stage for future strain engineering efforts to increase production.

ABOUT:

Mary Dunlop is an associate professor in the College of Engineering at Boston University.

SUPPORTING THE DOE SC MISSION:

The Early Career Research Program provides financial support that is foundational to early career investigators, enabling them to define and direct independent research in areas important to DOE missions. The development of outstanding scientists and research leaders is of paramount importance to the Department of Energy Office of Science. By investing in the next generation of researchers, the Office of Science champions lifelong careers in discovery science.

For more information, please go to the Early Career Research Program.

THE 2011 PROJECT ABSTRACT:

Engineering Robust Hosts for Microbial Biofuel Production

Microbes contain a vast diversity of metabolic pathways that can be subtly tweaked and redesigned for the conversion of biomass to biofuels compounds. Next‐generation biofuels such as short‐chain hydrocarbons are particularly attractive target molecules since they would be compatible with existing engines and infrastructure. However, high levels of these compounds are often toxic to the microbes synthesizing them, limiting the potential rate and yield of industrial biofuel production.

The objective of this research is to understand hydrocarbon tolerance mechanisms used by microbes inhabiting natural hydrocarbon seeps or oil‐contaminated sites, searching genome sequences of these organisms for efflux pumps and other molecular machines that microbes use to separate toxic hydrocarbons from their delicate biological systems. 

Promising candidates will be introduced into biofuel synthesizing strains of E. coli and tuned for optimal gene expression to determine if it is possible to engineer strains with enhanced tolerance to hydrocarbons and improved efficiency of overall synthesis.

RESOURCES:

Y. Siu, J. Fenno, J. Lindle, and M.J. Dunlop, "Design and selection of a synthetic feedback loop for optimizing biofuel tolerance." ACS Synthetic Biology 7, 16 (2017). [DOI: 10.1021/acssynbio.7b00260]

T. Tomko and M.J. Dunlop, "Expression of heterologous sigma factor expands the searchable space for biofuel tolerance mechanisms." ACS Synthetic Biology 6, 1343 (2017). [DOI: 10.1021/acssynbio.6b00375]

T. Tomko and M.J. Dunlop, "Engineering improved bio-jet fuel tolerance in Escherichia coli using a transgenic library from the hydrocarbon-degrader Marinobacter aquaeolei." Biotechnology for Biofuels 8, 165 (2015). [DOI: 10.1186/s13068-015-0347-3]

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Additional profiles of the Early Career Research Program award recipients can be found at /science/listings/early-career-program

The Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. For more information, please visit www.energy.gov/science.

Sandra Allen McLean is a Communications Specialist in the Office of Science, Office of Communications and Public Affairs [email protected].

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