Adaptive laboratory evolution of Lachancea thermotolerans for enhanced production of 2-Phenylethanol and 2-Phenylethyl acetate in wine

2-Phenylethyl alcohol (2-PE) and 2-phenylethyl acetate (2-PEA), responsible for “rose,” “fruity” and “floral” aromas in wine, are derived from L -phenylalanine (L-Phe). L. thermotolerans , a common yeast used in wine fermentation, has a limited ability to utilize L-Phe, restricting 2-PE and 2-PEA production. This study aimed to evolve an L. thermotolerans A38 strain with enhanced L-Phe utilization through ALE, in order to increase the production of the wine aroma compounds 2-PE and 2-PEA. After 200 generations of adaptive laboratory evolution (ALE), four mutants were selected, exhibiting an 82.8 % increase in 2-PE (from 15.06 to 28.22 mg/L) and a 2.07-fold increase in 2-PEA (from 75.73 to 157.27 μg/L) in chemically defined grape juice medium (CDGJM). The evolved strains also showed improved tolerance to high sugar concentrations, SO 2 , ethanol, and pH. In wine micro-vinification, they produced 14.02-fold (from 47.63 to 667.96 mg/L) more 2-PEA and 4.10-fold more 2-PE than the original strain, surpassing the commercial yeast CT10. This study demonstrates ALE as an effective strategy for boosting 2-PE and 2-PEA production in wine. • ALE as a promising strategy for enhancing the production capacity of 2-PE and 2-PEA. • Evolved L. thermotolerans increased 2-PEA by up to 14 times and 2-PE by 4.10 times in wine. • ALE also improved the strains tolerance in high sugar concentration, SO 2 , ethanol, and pH levels. • Evolved strains boosted ethyl ester and terpene compounds in wines, enhancing floral and fruity aromas.