Characterization and Engineering of the TPO Transporters for Biofuel Secretion in Saccharomyces cerevisiae
Concentrations of biofuel required for an industrial process at a relevant scale in microorganisms have not yet been demonstrated, because the fuels are often toxic to the host organism. Fuels are also difficult to cheaply remove from the cell. This project is introducing a small-molecule secretion "pump" engineered to efflux the fuels of interest at high yield. The insights the team gains can be applied to develop pumps and transporters that secrete other small molecules of interest, such as inhibitors from hydrolysate or toxic metabolic side products (started in 2012).
The goal of this project is to develop yeast that are more tolerant to high concentrations of alcohols. Our original approach, to apply membrane transporters, proved unsuccessful. We next set out to mimic the process of natural selection and examine what genome changes confer tolerance increases. In this way, we successfully evolved the yeast Saccharomyces cerevisiae for substantially enhanced growth in the presence of alcohols from butanol to octanol. We found that the tolerant strains contained mutations in the translation machinery, and confirmed that these confer the tolerance phenotype. We also demonstrated that alcohols significantly impair translation initiation for the wild type strain relative to the evolved strains.
The goal of this project is to develop yeast that are more tolerant to high concentrations of alcohols using two approaches: mimicking the process of natural selection, and introducing “libraries” of transporters to secrete alcohols out of the cell. Using the first approach, we successfully evolved the yeast Saccharomyces cerevisiae for substantially enhanced growth in the presence of alcohols such as pentanol and octanol. We are still evaluating these new yeast strains to determine which mutations in the genome give rise to this ability. Toward the second approach, we made thousands of separate variants of a transporter, known as the TPO antiporter, with the hope that one of these variants would have the ability to secrete these alcohols and enable growth in toxic concentrations of butanol. However, repeated attempts were unsuccessful with the TPO antiporter, and so we have expanded our search to a large set of other transporters found in the yeast membrane.
Published in 2013
Change, Exchange, and Rearrange: Protein Engineering for the Biotechnological Production of Fuels, Pharmaceuticals, and Other Chemicals, M. A. Fisher, D. Tullman-Ercek, Current Opinions in Biotechnology, 24(6), 1010-6, doi: 10.1016/j.copbio.2013.02.027, March 20, 2013.
Enhancing Tolerance to Short-Chain Alcohols by Engineering the Escherichia Coli Acrb Efflux Pump to Secrete the Non-Native Substrate N-Butanol, M. A. Fisher, S. Boyarskiy, M. R. Yamada, N. Kong, S. Bauer, D. Tullman-Ercek, ACS Synthetic Biology, 3(1), pp. 30-40, doi: 10.1021/sb400065q, September 3, 2013.
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