Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts

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 Rhodosporidiumtoruloides (also known as Rhodotorulatoruloides) 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. 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). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R.toruloides. Deletion of RTO4_11990 increased the 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.

EngineeringRhodosporidium toruloidesfor sustainable production of value-added punicic acid from glucose and wood residues

“…On the contrary, oleaginous R. toruloides can naturally assimilate pentose sugars, which is a great advantage in serving as a platform for converting low-value lignocellulose feedstock to high-value unusual fatty acid via metabolic engineering. Moreover, previous studies indicated that R. toruloides ’ ability to convert lignocellulosic feedstock into bioproducts could be improved through the enhancement of xylose assimilation and the adaptive evolution of the engineered strain (Coradetti et al, 2023; Díaz et al, 2018; Z. Liu et al, 2021).…”

“…R. toruloides has an unusual xylose metabolism featuring the reduction to Darabitol, oxidation to D-ribulose, and phosphorylation to ribulose 5-phosphate (Adamczyk et (which , 2023). By overexpressing a putative transcription factor (RTO4_12978, Pnt1) that acts as a major regulator of pentose metabolism, the expression of enzymes involved in xylose catabolism was increased and the specific growth rate was improved significantly in cultures on xylose (Coradetti et al, 2023). Furthermore, the adaptation of R. toruloides through evolutionary strategies has also led to the development of strains with increased tolerance to the major inhibitors present in lignocellulosic hydrolysates (Díaz et al, 2018;Z.…”

“…R. toruloides , a non-conventional yeast capable of concomitant synthesis of lipids and carotenoids, is typically regarded as a promising candidate for biofuel production due to its excellent lipid accumulation capability, which often exceeds 60% of dry cell weight (González-García et al, 2017). More interestingly, R. toruloides has the natural ability to use a wide range of substrates, including pentose sugars such as xylose (Adamczyk et al, 2023; Coradetti et al, 2023). This advantage makes R. toruloides promising for sustainable bioindustry and agriculture applications since xylose assimilation capability is essential for converting lignocellulosic biomass, the most abundant raw material derived from agriculture and forestry waste, into value-added unusual fatty acids.…”