Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Abstract

R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l-arabinose and d-xylose metabolism proceeding through d-arabitol and d-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d-xylose to d-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

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

“…Liu et al, 2021). R. toruloides has an unusual xylose metabolism featuring the reduction to D-arabitol, oxidation to D-ribulose, and phosphorylation to ribulose 5-phosphate (Adamczyk et al, 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).…”

“…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.…”