(234d) Evolutionary Engineering of Saccharomyces Cerevisiae for Improved Xylose Utilization

Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. One of the main challenges of cost-effective utilization of lignocellulosics for the ethanol production by the yeast Saccharomyces cerevisiae is efficient fermentation of D-xylose, the second most abundant sugar in lignocellulosics, as it cannot be used by natural S.cerevisiae strains. For the yeast Saccharomyces cerevisiae, evolutionary engineering has been extensively used and proved to be powerful to select for industrially relevant phenotypes such as an efficient substrate utilization, expanded substrate range, and increased stress tolerance.

In this study, a S. cerevisiae strain was metabolically engineered to obtain xylose catabolism pathway. The Piromyces XYLA, P. stipitis XYL3, TAL1, as well as the endogenous genes of the pentose phosphate pathway (RPE1, RKI1, and TKL1) were overexpressed. The constructed strain couldn't grow on xylose medium. After selection by sequential batch cultivation under aerobic and oxygen-limited conditions with xylose as the sole carbon source, the resulting strain, H131-XYLA31, is capable of fermenting xylose to ethanol under anaerobic conditions. To identify the beneficial phenotype of the evolved strain, genomic DNA libraries of the evolved strain were constructed, transformed to metabolically engineered but unevolved strain and then screened for either high growth rate on xylose medium or quick xylose utilization by a high-throughput micro-fluidic device. Both screening resulted in strains carrying tandem gene duplication of XYLA, the heterogeneous xylose isomerase and the first step of the xylose catabolism pathway.

These results illustrate the combination of metabolic engineering and evolutionary engineering as a strategy for enabling and improving recombinant pathways. Moreover, we provide significant insights on evolution engineering process, and demonstrate a applicable approach to identify the dominant beneficial genotype of an evolved strain.