Metabolic Flux and Growth Profiling of <scp> <i>Megasphaera cerevisiae</i> </scp> for Medium‐Chain Fatty Acid Synthesis

Megasphaera cerevisiae is a well-known beer spoilage organism, capable of producing undesirable flavours and turbidity. Although the biosynthesis of medium-chain fatty acids (MCFAs) has been extensively studied in different Megasphaera species, the metabolic behaviour of M. cerevisiae in controlled environments remains largely unexplored. This study examines the MCFAs production from diverse substrates and reports flux analyses of core metabolism for the first time using a genome-scale model. The results suggest that acetate stimulates butyrate production but not caproic acid production. Butyrate supplementation, either alone or in combination with acetate, promoted CA synthesis. Lactate supplementation primarily led to the formation of propionic acid and acetic acid. The metabolic network model was manually curated and validated against the different experimental data. The metabolic flux results showed that butyrate production facilitated via the reverse β-oxidation (RBO) pathway, with a minor contribution from the fatty acid synthesis (FAS) pathway. Conversely, CA synthesis was mainly synthesised through the FAS pathway, irrespective of the substrate used. The pathway analyses results highlighted the critical role of hydrogen production in M. cerevisiae metabolism, particularly under conditions where lactate is utilised. Collectively, these findings offer novel insights into the metabolic versatility and pathway preferences of M. cerevisiae.