(23f) Random Mutagenesis of Yarrowia Lipolytica for Enhanced Lignocellulosic Sugar Utilization

Yarrowia lipolytica represents a non-conventional yeast species exhibiting extraordinary metabolic plasticity. This oleaginous yeast demonstrates robust growth capabilities on heterogeneous carbon substrates, including n-alkanes, fatty acids, triacylglycerols, as well as conventional carbohydrates and glycerol. Y. lipolytica has garnered significant attention in industrial biotechnology due to its capacity to biosynthesize and accumulate a diverse spectrum of value-added metabolites with applications spanning biofuels, nutritional supplements, pharmaceuticals, and fine chemicals.Currently, modern bioprocesses increasingly prioritize lignocellulosic biomass as a renewable feedstock to reduce reliance on food-competing substrates like glucose. Biomass-derived hydrolysates contain mixed sugars, predominantly C6 (glucose) and C5 (xylose), which are challenging to utilize simultaneously due to catabolic repression and inefficient transport systems. While consolidated bioprocessing (CBP) strategies aim to integrate biomass hydrolysis and fermentation, native Y. lipolytica strains exhibit limited efficiency in metabolizing pentoses like xylose, often resulting in suboptimal metabolite yields. To address these limitations, this study proposes a random mutagenesis approach using ethyl methanesulfonate (EMS) to generate Y. lipolytica variants with enhanced transporter activity and simultaneous C5/C6 utilization. Following chemical mutagenesis, Y. lipolyticaTISTR 5212 wild-type strain was subjected to EMS treatment for various exposure times ranging from 0 to 8 hours. Mutant populations exhibiting survival rates between 30-40% were selected. The resulting mutants were subsequently evaluate their growth on three different selective medium: Yeast extract-Peptone-Xylose (YPX), Yeast Nitrogen Base-Xylose-Glucose (YNB-Xylose-Glucose), and Yeast Nitrogen Base-Xylose-2-deoxyglucose (YNB-Xylose-2DG).Growth profiling demonstrated that the EMS-derived mutants successfully colonized both YPX and YNB-Xylose-Glucose media, but no growth was observed on the YNB-Xylose-2DG selective medium. This indicates that the mutants had not acquired sufficient resistance to the glucose analog 2-deoxyglucose, which typically inhibits glycolysis by competing with glucose metabolism. These findings suggest that while the EMS mutagenesis successfully generated viable mutants capable of utilizing mixed carbon sources, it did not yield variants with the specific metabolic adaptations necessary to overcome 2DG inhibition while metabolizing xylose as the primary carbon source. Further optimization of mutagenesis parameters or implementation of adaptive laboratory evolution strategies may be required to obtain mutants with enhanced pentose utilization capabilities in the presence of glycolytic inhibitors.