Integrated metabolite, volatile, and genome annotation profiling reveals distinct functional phenotypes in cocoa-derived yeasts.

Cocoa fermentation shapes the precursor chemistry underlying chocolate flavour, but the isolate-level contributions of cocoa-associated yeasts remain incompletely resolved. This study compared six cocoa-derived yeasts with a reference Saccharomyces cerevisiae strain by profiling sugar utilisation, organic-acid turnover, volatile formation, and targeted genome annotation under controlled cocoa-relevant conditions. In cocoa pulp extract-based medium, Kluyveromyces marxianus Y1A, Meyerozyma guilliermondii Y1, and Ogataea polymorpha Y3A showed the strongest sugar depletion and organic-acid transformation, whereas the Kwoniella isolates Y4 and Y6B retained larger residual sugar pools and Lodderomyces orthopsilosis Y5 showed an intermediate phenotype. In synthetic cocoa pulp-simulating medium, volatile profiles further separated isolates across medium-chain fatty acids, alcohols, esters, and carbonyls. Y3A showed sustained fatty acid ethyl ester-associated output, Y1A displayed an acetate-ester-biased profile, and Y4 showed strong ester-associated output despite weaker primary carbon-use performance. Targeted genome annotation showed broadly conserved support for central metabolic functions, with clearer differences in sucrose- and ester-associated features. Targeted homology searches clarified key genome-phenotype gaps by revealing candidate sucrose-utilisation homologues in Y1A and candidate ester-associated homologues in Y3A and Y4 that were not fully resolved by the automated annotation panel. Overall, the integrated dataset showed clear isolate-associated differences in carbon processing, organic-acid turnover, and volatile-associated metabolism. Y1A and Y3A showed the strongest overall profiles under the conditions tested, supporting further evaluation in mixed-culture and pilot-scale cocoa fermentation studies. The genome-based taxonomic assignments, safety status, technological robustness, and regulatory suitability should be further confirmed before any starter-culture application.