Table_1_Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery.XLSX

The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced β-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.

利用细胞工厂将木质纤维素生物质（lignocellulosic biomass）衍生的糖类转化为包含油脂化学品（oleochemicals）与类胡萝卜素（carotenoids）等食品添加剂在内的目标化学品，对于向可持续生产工艺转型具有关键意义。红冬孢酵母菌（Rhodotorula toruloides，简称R. toruloides）是一类可天然代谢多种底物的酵母，其底物范围涵盖木质纤维素水解液，并可将这些底物转化为油脂与类胡萝卜素。本研究以半纤维素（hemicellulose）的主要成分木糖作为红冬孢酵母菌的唯一底物，通过结合绝对蛋白质组学（absolute proteomics）与基因组规模代谢模型（genome-scale metabolic models）开展详细的生理特性表征，以解析油脂与类胡萝卜素的合成调控机制。为提升这两类产物的合成效率与产量，研究通过过氧化氢（hydrogen peroxide）与光照处理（light irradiation）诱导氧化应激（oxidative stress），并进一步通过适应性实验室进化（adaptive laboratory evolution）强化产物合成路径。基于对菌体生长与二氧化碳排放的在线监测数据，本研究在分批培养（batch cultivations）过程中鉴定出三个截然不同的生长阶段。多数胞内通量估算（intracellular flux estimations）结果与实测蛋白质水平呈现相似趋势，且显示烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的再生效率提升、磷酸酮解酶活性增强，同时β-氧化（β-oxidation）途径受到抑制，这与脂质产量的提升呈现显著正相关。与最优生长条件相比，光照处理可使类胡萝卜素含量提升70%、脂质含量提升40%。过氧化氢处理虽未对类胡萝卜素的合成产生显著影响，却实现了0.65 g/g干细胞重（dry cell weight, DCW）的最高脂质产量。经适应性实验室进化获得的菌株表现出更优的生长适应性，其类胡萝卜素含量较亲本菌株提升2.3倍。本研究系统阐明了红冬孢酵母菌将木糖转化为微生物油脂与类胡萝卜素的完整过程，为这类化学品的可再生、低成本生产提供了整体性的理论与实践参考。