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Efflux Pumps to Increase Microbial Tolerance and Biofuel Production




Aindrila Mukhopadhyay, Jay Keasling, and Mary Dunlop at the Joint BioEnergy Institute (JBEI) have developed a method for providing industrial host microbes with resistance to valuable but potentially toxic molecules, such as solvents and fuel-like compounds. Providing such tolerance is a crucial step in engineering organisms to produce desirable substances. The scientists used efflux pumps to confer resistance on E. coli and developed a library of the most effective pumps for protection against several compounds, such as geraniol, limonene, pinene, and farnesyl hexanoate. These compounds represent biogasoline, biodiesel, and biojetfuel candidates. Moreover, the method for deriving this library is applicable to determining the most effective pumps for any given host and target compound.

The scientists first selected the potential pump candidates from an extensive database of sequenced microbes, expressed these in bacterial hosts, and then grew the hosts in competitive cultures in the presence of the compounds of interest. They then analyzed the pumps that provided the greatest growth advantage. For example, monoterpenes such as limonene have antimicrobial activity and present toxicity at sub-micromolar levels. Use of efflux pumps specific to this compound rendered the microbe tolerant to this compound at levels greater than required for a biofuel production strain.

As metabolic engineering increases the biological production titers of compounds, there is a growing need to overcome limitations posed by each compound’s toxicity, inhibition of cell growth, and intracellular feedback inhibition (i.e., the slowing of production by accumulated product). Until now, these problems have been addressed primarily through combinatorial approaches, such as adaptation, genome shuffling, and random mutatgenesis. These techniques may work under certain settings but are often not transferrable to other hosts or target compounds, because they do not identify the mechanism of the resistance. On the other hand, the JBEI technology uses a known, transferrable mechanism—an efflux pump—to optimize the tolerance of various hosts to any compound of interest. In several cases where the target compound is highly water immiscible, successful export of the compound from the cell can also improve product extraction from the culture.  

The Joint BioEnergy Institute (JBEI, is a scientific partnership led by the Lawrence Berkeley National Laboratory and including the Sandia National Laboratories, the University of California campuses of Berkeley and Davis, the Carnegie Institution for Science and the Lawrence Livermore National Laboratory. JBEI’s primary scientific mission is to advance the development of the next generation of biofuels.

DEVELOPMENT STAGE: Proven concept.

STATUS: Published US Patent US20110294183 available at Available for licensing or collaborative research.


Dunlop, M. J., Dossani, Z. Y., Szmidt, H. L., Chu, H. C., Lee, T. S., Keasling, J. D., Hadi, M. Z., Mukhopadhyay, A. “Engineering microbial biofuel tolerance and export using efflux pumps,” Molecular Systems Biology 7, Article no. 487; doi: 10.1038/msb.2011.21.

Yarris, Lynn. “Striking the Right Balance: JBEI Researchers Counteract Biofuel Toxicity in Microbes,” Berkeley Lab News Center, May 11, 2011


Engineered Biosynthesis of Alternative Biodiesel Fuel in E. Coli and Yeast, EIB-2391

Novel Biosynthetic Pathway for Production of Fatty Acid Derived Molecules, IB-2386, IB-2387



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