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 <i>Kluyveromyces marxianus</i> (<i>K. marxianus</i>), 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 <i>K. marxianus</i>, 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 <i>Kluyveromyces marxianus</i> 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 <i>CTA1</i>, <i>TSL1</i>, <i>ERG2</i> were related to enhanced acid tolerance, while <i>ARO8</i>, <i>ARO9</i>, <i>FKS2</i> 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 <i>K. marxianus</i>-fermented milk beer and provides an efficient framework for ALE-based breeding of aroma-producing food microorganisms.