Adaptive laboratory evolution (ALE) as a tool to ameliorate xylose consumption in Rhodosporidium toruloides-1588

Rhodosporidium toruloides, an oleaginous yeast, is extensively studied for biofuel production due to its ability to accumulate lipids. However, its industrial application is constrained by its inefficiency in xylose uptake and utilization, particularly when cultivated on lignocellulosic hydrolysates, where xylose is the second most abundant sugar. To address this challenge, R. toruloides-1588 was evolved using Adaptive Laboratory Evolution (ALE) as a tool in minimal media with xylose as the sole carbon source to enhance its utilization. The evolved strain achieved 99 % xylose consumption by the 13th generation, with a 70 % increase in xylose uptake within 16 h compared to the native strain. When cultivated in yeast malt synthetic media, the adapted strain showed a 15 % increase in xylose consumption and a 23.40 % increase in lipid accumulation with a two-fold increase in oleic acid content (52.42 %). Further, in bench-scale-up studies (1L) using xylose-wood hydrolysate (C5-wood hydrolysate) as a culture media, the evolved strain showed a maximum lipid accumulation of 23.42 g/L at 156 h, representing an 18.35 % increase compared to the native strain (19.79 g/L) under the same conditions. These results highlighted the effectiveness of adaptive laboratory evolution in enhancing xylose uptake by R. toruloides-1588. This led to an improved growth, sugar co-utilization, and lipid accumulation on complex substrates for sustainable biofuel production.