Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production

Abstract

Background Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. Results Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. Conclusions The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates. Electronic supplementary material The online version of this article (10.1186/s13068-019-1478-8) contains supplementary material, which is available to authorized users.

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery

“…This response has been previously reported under nitrogen limitation for R. toruloides grown in both glucose and xylose (Zhu et al, 2012;Tiukova et al, 2019b). Additionally, activation of autophagy process has been described as a direct response via TOR activation to recycle nitrogenous compounds (Zhu et al, 2012;Tiukova et al, 2019b). Although initially expressed at a low level, upregulation of autophagy-related proteins (RHTO_05541, RHTO_06526) was detected.…”

“…In response to nitrogen limitation, proteins involved in central nitrogen metabolism, such as glutamate dehydrogenase (GDH, RHTO_04650, RHTO_07718) and glutamine synthetase (GLN, RHTO_00673, RHTO_00401) were upregulated ( Figure 3A). This response has been previously reported under nitrogen limitation for R. toruloides grown in both glucose and xylose (Zhu et al, 2012;Tiukova et al, 2019b). Additionally, activation of autophagy process has been described as a direct response via TOR activation to recycle nitrogenous compounds (Zhu et al, 2012;Tiukova et al, 2019b).…”

“…Downregulation was predominantly noted in amino acid biosynthesis pathways, while NADPH consumption in lipid metabolism and the glutamate production pathway showed upregulation. Upregulation of enzymes in glutamate biosynthesis in response to nitrogen starvation have been demonstrated previously (Zhu et al, 2012;Tiukova et al, 2019b). Additionally, metabolites related to lipid synthesis (CoA, acetyl-CoA, acetate, and pyruvate) showed increased protein allocation during nitrogen limitation, while xylitol related proteins were downregulated (in all cases adj.…”

“…However, xylose metabolism in R. toruloides is still not completely understood. Only few studies have focussed on the metabolism of xylose assimilation in this oleaginous yeast (Jagtap and Rao, 2018;Tiukova et al, 2019b;Lopes et al, 2020a). Therefore, in our study, detailed physiology characterisation was combined with genome-scale modelling and absolute proteomics with the goal to investigate xylose metabolism in R. toruloides and use oxidative stress as a strategy to improve the production of lipids and carotenoids.…”

“…Complete genome sequences are available for several R. toruloides strains (Kumar et al, 2012;Zhu et al, 2012;Morin et al, 2014;Hu and Ji, 2016;Sambles et al, 2017;Coradetti et al, 2018). The lipid formation process during different growth phases of cultivation on glucose has been investigated through proteomic analysis (Liu et al, 2009) and compared with cells grown on xylose (Tiukova et al, 2019b). Multi-omics analyses have identified metabolism modification under nitrogen (Zhu et al, 2012) and phosphate limitation (Wang et al, 2018b).…”