Adaptive Laboratory Evolution of the Non-Conventional Yeast Pichia ethanolica for Improved Molasses Utilization Reveals Multilevel Adaptive Remodeling.

Molasses is an abundant and low-cost fermentation feedstock, but its direct utilization is often constrained by its complex composition and inhibitory components. In this study, a molasses-utilizing yeast strain, M0, was isolated from tea rhizosphere soil and identified as Pichia ethanolica. M0 grew directly in untreated molasses diluted only with water, and adaptive laboratory evolution (ALE) was performed under stepwise increasing molasses concentrations. After 70 transfers, the evolved population reached an OD560 of 26.41, representing a 73.1% increase relative to the parental strain, and a superior isolate was designated M70. M70 showed lower residual sucrose after 48 h in 100 g/L molasses medium (16.02 vs. 21.11 g/L) and improved growth under high-molasses, -temperature, and -NaCl conditions, with additional growth advantages under selected ethanol and oxidative-stress conditions. Genome resequencing identified five small candidate variants and one interchromosomal duplicative insertion, including alterations involving RIB1, SOL3, and a CET1-like/REX2-like region. Transcriptomic and metabolomic analyses identified 179 differentially expressed genes and 169 differentially accumulated metabolites. Integrated analysis highlighted coordinated changes in D-amino acid, arginine and proline, beta-alanine, and glyoxylate and dicarboxylate metabolism. These findings indicate multilevel genomic, transcriptional, and metabolic remodeling associated with improved molasses adaptation in P. ethanolica.