S5: Application of directed evolution of xylose transport and catabolism in yeast

Metabolic engineering and directed evolution are powerful approaches for controlling and improving the production of biomolecules from biomass sugars, especially xylose.  Here we demonstrate the application of directed evolution to two key steps in biomass utilization: molecular sugar transporters and catabolic enzymes.  Specifically, metabolic flux is limited at the transport level by low pentose affinity and hexose inhibition.  Here, we present evidence that heterologous transporters may be engineered using directed evolution to improve S. cerevisiae pentose growth characteristics and ultimately biofuels production.  Likewise, xylose catabolic enzymes (including xylose isomerase) do not have sufficient activity to support high level, redox-balanced xylose utilization.  Here, we present evidence that these enzymes can be evolved for improved function in yeast.   By employing directed evolution in both of these cases, significant improvements in xylose growth rate and ethanol production are achieved.