Genomics and phenotypic potential of non-Saccharomyces cocoa yeast strains

The use of non-Saccharomyces yeast starter culture strains has caught the attention of brewers, bakers, and winemakers during the last 30 years to obtain fermented food products with a distinct flavour and/or characteristics of added value in the market. Cocoa fermentation is, however, still a spontaneous process in which starter cultures have been applied only at research level. The present study aimed at assessing the phenotypic and genomic potential of candidate yeast starter culture strains to ferment the cocoa pulp-bean mass and deliver an end-product with a distinct flavour profile. First, a phenotypic screening to test the growth and fermentation capacities of different yeast strains in media containing different carbon sources was performed. Hanseniaspora opuntiae IMDO 020003 and Pichia kudriavzevii IMDO 060005 were selected and, together with the previously used P. kudriavzevii IMDO 020508 and H. opuntiae IMDO 040108, their genome was sequenced and assembled using a hybrid approach that applied long- and short-read sequencing. This way, a telomere-to-telomere genome assembly was achieved for both P. kudriavzevii IMDO strains and the most contiguous genome assembly of an H. opuntiae was accomplished, suggesting the presence of seven nuclear chromosomes. The combination of an in silico functional analysis of the sequenced strains together with their extensive phenotypical characterization in cocoa pulp simulation medium revealed a distinct enzyme machinery to metabolize organic acids and produce volatile organic compounds. Finally, the sequenced strains were compared to other publicly available P. kudriavzevii and Hanseniaspora spp., which highlighted a gene loss in the H. opuntiae genomes regarding metabolic activities of relevance for the cocoa fermentation process.