Genome-scale metabolic modeling of Pichia kudriavzevii reveals citric acid utilization mechanisms and engineering strategies for improved wine deacidification

• High-quality genome sequencing of a citrate-utilizing P. kudriavzevii strain (GS1-1) and development of a curated genome-scale metabolic model ( i QS924) encompassing 924 genes and 1199 reactions. • Integrated omics–model analysis revealed distinct metabolic adaptations under glucose vs. citrate conditions, including enhanced TCA cycle flux and altered energy distribution. • In silico simulations identified genetic targets, such as SCT1, to relieve glucose repression and enhance citrate utilization, offering a blueprint for yeast strain optimization in winemaking. High acidity in fruit wines can adversely affect flavor balance and consumer acceptance. Pichia kudriavzevii (PK) is a non-Saccharomyces yeast with strong citrate metabolism potential, making it a promising strain for wine deacidification. In this study, we combined genomic sequencing with genome-scale metabolic modeling to elucidate the metabolic basis of citrate utilization and to identify strategies for enhancing its efficiency during wine fermentation. The high-quality model i QS924, encompassing 924 genes, 1,431 metabolites, and 1,199 reactions, accurately reproduced 90% of experimentally validated growth phenotypes across 20 carbon sources. Comparative analyses under glucose and citrate conditions revealed distinct growth dynamics, essential gene/reaction profiles, and large-scale flux redistributions, with citrate assimilation characterized by enhanced tricarboxylic acid cycle activity, gluconeogenesis, and redox balancing. Integration of transcriptomic data and in silico simulations identified multiple genetic intervention strategies; notably, upregulation of SCT1 alone increased the predicted citrate-to-glucose uptake ratio from 0.01 to 0.93 in mixed-carbon environments. These findings provide mechanistic insights into PK’s organic acid metabolism and propose targeted engineering strategies to improve its deacidification efficiency in wine fermentation.