Fungal nitrogen overflow-driven cross-feeding alleviates bacterial growth limitations and promotes γ-aminobutyric acid and ethyl ester biosynthesis

D. hansenii D1 nitrogen overflow drives cross-kingdom bacterial growth. • Amino acid dependencies reveal niche differentiation in bacterial partners. • Cross-feeding supplies precursors but substrates limit GABA and ester synthesis. • Co-supplementation of amino acids and α-keto acids enhances metabolite production. • Nitrogen availability and TORC1 signaling regulate fungal overflow and cross-feeding. Microbial interactions are important for the biosynthesis of flavor and functional metabolites in fermented ecosystems, yet how inter-kingdom nitrogen flux is allocated and contributes to these processes remains poorly understood. In this study, a defined microecosystem comprising the core starter yeast Debaryomyces hansenii D1 and two bacteria, Lactiplantibacillus plantarum LP-2 and Staphylococcus xylosus JH-3, was established to investigate this metabolic network using gas chromatography coupled with tandem mass spectrometry and high-performance liquid chromatography. Results revealed distinct metabolic niches: L. plantarum heavily relied on exogenous amino acids, whereas S. xylosus demonstrated autonomous synthesis. The release of diffusible fungal nitrogen metabolites, including cysteine and glutamine, was associated with nitrogen overflow and enhanced bacterial growth. This cross-feeding enhanced γ-aminobutyric acid (GABA) and ethyl ester biosynthesis. Moreover, co-supplementation with amino acids and α-keto acids increased the accumulation of these key metabolites. Fungal amino acid release appeared to involve regulated efflux rather than passive leakage and was associated with inorganic nitrogen assimilation and the Target of Rapamycin Complex 1 (TORC1) signaling pathway. These findings provide insights into cross-kingdom interactions in a defined three-species community and may inform the future development of synthetic microbial starters. Download: Download high-res image (429KB) Download: Download full-size image