Butyl Butyrate Production by Meyerozyma guilliermondii YS34 and Its Contribution to Flavour Ester Biosynthesis

Butyl butyrate is a high‐value natural flavour ester with a characteristic pineapple‐like aroma that is widely used in food flavourings, beverages, fragrances, cosmetics and personal care products. The increasing demand for clean‐label aroma ingredients has stimulated interest in sustainable microbial production as an environmentally friendly alternative to conventional chemical synthesis. This study investigated butyl butyrate biosynthesis by Meyerozyma guilliermondii YS34 through an integrated approach combining fermentation optimization, targeted and untargeted volatile metabolite profile and whole‐genome analysis. Fermentation was performed using glucose as the carbon source with butanol supplementation at 3, 5 and 7 g/L. Supplementation with 5 g/L butanol yielded the highest butyl butyrate concentration (10.47 g/L after 24 h), representing a 2.13‐fold increase over the non‐optimized condition. Untargeted metabolomic profiling identified 32 volatile metabolites, with butyl butyrate as the predominant ester accompanied by coordinated changes in alcohols, aldehydes and short‐chain fatty acids associated with ester biosynthesis. Whole‐genome analysis identified genes encoding key enzymes involved in β‐oxidation, alcohol metabolism and acyl transfer reactions, providing genomic evidence supporting the proposed butyl butyrate biosynthetic pathway. To our knowledge, this is the first study integrating fermentation optimization, metabolomic profiling and genome‐based pathway analysis to elucidate the molecular basis of butyl butyrate biosynthesis in M. guilliermondii . These findings establish M. guilliermondii YS34 as a promising microbial cell factory for the sustainable production of natural butyl butyrate and provide a molecular framework for future flavour biotechnology and industrial bioprocess development.