Model-driven exploration of lactose and galactose metabolism via an oxidoreductive pathway in Sungouiella intermedia for cell factory applications

Converting industrial side streams into value-added chemicals using microbial cell factories is of increasing interest, as such processes offer sustainable solutions to reduce waste and production costs. However, developing new, efficient non-model cell factories for precision fermentation remains challenging due to limited knowledge about their metabolic capabilities. Here, we investigate the lactose and galactose metabolism of the understudied yeast Sungouiella intermedia (formerly Candida intermedia), using knowledge-matching of high-quality genome-scale metabolic model (GEM) with extensive experimental analysis, and determine its potential as a future cell factory on lactose-rich industrial side-streams. We show that this yeast possesses the conserved Leloir pathway as well as an oxidoreductive route for galactose catabolism. Model simulations and experimental data from continuous and batch bioreactors, transcriptomics, and metabolite analysis indicate that while the Leloir pathway dominates galactose metabolism in S. intermedia, the oxidoreductive pathway is employed in a condition-dependent manner. The yeast produces galactitol as a carbon overflow metabolite, facilitating redox cofactor balance during both lactose and galactose growth. Furthermore, the new metabolic insights facilitated the development of an improved bioprocess design, where an engineered S. intermedia strain could achieve galactitol yields of > 90% of the theoretical maximum using the industrial side-stream cheese whey permeate as feedstock. Additional strain engineering resulted in galactitol-to-tagatose conversion, proving the versatility of the future production host. Overall, this work sheds new light on the intrinsic interplay between parallel metabolic pathways that shape the lactose and galactose catabolism in S. intermedia. It also demonstrates how a GEM combined with experimental analysis can work in synergy to fast-forward metabolic characterization and development of new, non-model yeast cell factories.