Engineering an asymmetric cross-feeding consortium in Ogataea polymorpha driven by sulfur metabolism.

Microbial cell factories offer a sustainable route for chemical production, yet reconstructing complex pathways in a single host often leads to metabolic burden and imbalanced flux. Here we establish an auxotrophy-based cross-feeding strategy in the non-conventional yeast Ogataea polymorpha. Through systematic screening of pairwise auxotrophic mutants, we identify several complementary combinations, among which the met10Δ-str3Δ pair exhibits robust and reproducible growth restoration under both glucose and methanol conditions. Mechanistically, this pair displays asymmetric metabolic dependency. met10Δ can utilize multiple sulfur-containing intermediates, whereas str3Δ relies exclusively on homocysteine. Population dynamics further reveal that the initial inoculation ratio critically determines community establishment and final biomass, with all co-cultures converging toward met10Δ enrichment over time. This work provides an experimental foundation for designing stable synthetic consortia in non-conventional yeasts.