Endogenous pathway dysregulation in Kluyveromyces marxianus under nitrogen limitation drives 2-phenylethyl acetate and 2-phenylethanol overproduction

The aromatic compounds 2-phenylethanol (2-PE) and 2-phenylethyl acetate (2-PEA) are of high industrial relevance due to their distinctive sensory properties and extensive use in food, fragrance, and wine industries. However, their natural production is costly, motivating interest in sustainable microbial alternatives. This study examined the metabolic regulation of Kluyveromyces marxianus ITD0090, a non-conventional yeast, under nitrogen-limited conditions for enhanced 2-PE and 2-PEA production. Combined genomic and transcriptomic analyses revealed key regulatory points associated with aromatic biosynthesis. K. marxianus ITD0090 showed remarkable metabolic flexibility, favoring 2-PEA over 2-PE. ARO9 overexpression appeared to compensate for ARO8 inactivation, while the shikimate pathway remained active despite external L-phenylalanine, supporting de novo phenylalanine synthesis. The preferential accumulation of 2-PEA may represent a cellular detoxification strategy. These results emphasize the potential of K. marxianus ITD0090 as a robust, non-engineered platform for cost-effective aroma production without genetic modification or expensive supplementation. This work provides novel insights into alternative metabolic strategies in non-conventional yeasts and opens perspectives for optimizing nitrogen- and carbon-regulated pathways to improve the sustainable production of natural flavor compounds.