Enhancing the Signature Rose Aroma of Kluyveromyces marxianus-Fermented Milk Beer via Adaptive Laboratory Evolution

Milk beer, a modern Chinese dairy beverage, is usually fermented by the co-culture of lactic acid bacteria (LAB) and Kluyveromyces marxianus (K. marxianus), with the latter known for its ability to produce aroma compounds. However, the accumulation of lactic acid produced by LAB can inhibit the growth of K. marxianus, which inevitably hinders the diversity and intensity of flavor compounds in milk beer. In this study, adaptive laboratory evolution (ALE) was applied to the parental strain Kluyveromyces marxianus CICC1953 (Km-P) under different concentrations of lactic acid to obtain an evolved strain Km-ALE-X20 with enhanced acid tolerance and increased titer of phenylethyl alcohol, which has a floral, rose-like aroma. Km-ALE-X20 demonstrated a 16-fold increase in OD₆₀₀ and a 28-fold increase in phenylethyl alcohol production compared with Km-P in chemically defined medium (CDM) containing 20 g/L lactic acid. Comparative genomics analysis suggested that mutated genes CTA1, TSL1, ERG2 were related to enhanced acid tolerance, while ARO8, ARO9, FKS2 were related to increased production of aroma compounds. Furthermore, Km-ALE-X20-fermented milk beer showed 33.87% and 32.43% higher production in alcohol and ester compounds than that of Km-P-fermented milk beer. Interestingly, sensory analysis showed that while Km-ALE-X20-fermented milk beer had higher sensory scores for rose and fruity aroma attributes, Km-P-fermented milk beer possessed a more balanced aroma profile. This paper highlights the first application of ALE to enhance the signature rose aroma of K. marxianus-fermented milk beer and provides an efficient framework for ALE-based breeding of aroma-producing food microorganisms.