PNNL

Abstract

Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodotorula toruloides (also known as Rhodosporidium toruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. In order to increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Over-expression of Pnt1 significantly increased rates of growth and xylose uptake when xylose was the sole carbon source, and increased fatty alcohol production 120% in an engineered strain grown on xylose. However, Pnt1 overexpression resulted in smaller improvements in growth and xylose conversion in media containing xylose and glucose. Proteomic analysis confirmed that Pnt1 is the major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.