Biofuels Production projects
In vivo, Focused Directed Evolution of a Xylose-Specific Transporter and High-Performing Xylose Isomerase in S. cerevisiae
A continuous fermentation process in S. cerevisiae would require simultaneous import of the major monosaccharides in lignocellulosic hydrolysates, namely glucose and xylose. Currently, both sugars are imported through the hexose transporters but these have a 10 to 100-fold preference for glucose over xylose. Recent directed evolution efforts by other groups have successfully made transporters that only import xylose, but glucose still inhibits xylose transport.
Continuous fermentation of lignocellulosic feedstocks requires a yeast strain that is capable of simultaneously utilizing a mixture of sugars -- for example, glucose and xylose. Unfortunately, consumption of xylose is inhibited by the presence of glucose. It is likely that the inhibition is occurring during import of xylose into the cell, and therefore, our aim in this project, begun in 2014, is to evolve a transporter that is highly specific to xylose. Beginning with native hexose transporters, we will generate mutants using both site-directed and random mutagenesis in an attempt to alter substrate specificity. We will select for xylose-transport activity using xylose as the sole carbon source, and select against glucose import/inhibition using a non-metabolizable glucose analog, 2-deoxy-D-glucose.
Efficient conversion of biomass to fuels and commodity chemicals will require complete utilization of the various sugars present in plant cell walls. Xylose is the second most abundant sugar in plants; however, many microbes, such as the popular industrial yeast Saccharomyces cerevisiae, are unable to natively consume xylose. Many groups, including our own and others in the EBI, have enabled S. cerevisiae to grow on xylose. One remaining challenge is the ability to simultaneously consume glucose and xylose (and later, the remaining sugars), which will be essential to enable continuous fermentation processes. Expression of a cellodextrin transporter allows yeast to grow on a mixture of xylose and cellobiose without the diauxic shift observed when grown on xylose and glucose. This suggests that the primary limitation to co-consumption of xylose and glucose is competition for transport as xylose enters the cell through hexose transporters. Therefore, we are engineering a xylose-specific transporter that will not be inhibited by the presence of glucose. Using a strain that has many of its hexose transporters knocked out , growth on both sugars is dependent on complementing expression of a hexose transporter, Hxt5. We will evolve Hxt5 for xylose specificity by titrating in a non-metabolizable glucose analog, 2-deoxy-D-glucose (2DG) as a competitive inhibitor. We will generate Hxt5 mutants either by classic PCR mutagenesis and directed evolution, or using a new in vivo mutagenesis technology being developed by a collaborator at UC Irvine. After extended enrichment on xylose and 2DG, we expect to select for Hxt5 mutants that have evolved specificity for xylose while maintaining a sufficiently high rate of transport to support growth.