Unravelling metabolic cross‐feeding in a yeast–bacteria community using ¹³C‐based proteomics

Abstract Cross‐feeding is fundamental to the diversity and function of microbial communities. However, identification of cross‐fed metabolites is often challenging due to the universality of metabolic and biosynthetic intermediates. Here, we use 13 C isotope tracing in peptides to elucidate cross‐fed metabolites in co‐cultures of Saccharomyces cerevisiae and Lactococcus lactis . The community was grown on lactose as the main carbon source with either glucose or galactose fraction of the molecule labelled with 13 C. Data analysis allowing for the possible mass‐shifts yielded hundreds of peptides for which we could assign both species identity and labelling degree. The labelling pattern showed that the yeast utilized galactose and, to a lesser extent, lactic acid shared by L. lactis as carbon sources. While the yeast provided essential amino acids to the bacterium as expected, the data also uncovered a complex pattern of amino acid exchange. The identity of the cross‐fed metabolites was further supported by metabolite labelling in the co‐culture supernatant, and by diminished fitness of a galactose‐negative yeast mutant in the community. Together, our results demonstrate the utility of 13 C‐based proteomics for uncovering microbial interactions.