Enhanced Medium-Chain Fatty Acid Production in Megasphaera elsdenii via Adaptive Laboratory Evolution and Genomic Insights

The transition to a sustainable bioeconomy has intensified research into microbial production of medium-chain fatty acids (MCFAs) such as butyric acid (BA) and hexanoic acid (HA), which are high-value precursors for biofuels and specialty chemicals. This study investigates the metabolic and genomic differences between Megasphaera elsdenii strains, JCM 1772 (ATCC 25940) and JCM 35779, focusing on their capacity for MCFA production. Preliminary analyses of JCM 1772 and JCM 35779, conducted using adaptive laboratory evolution (ALE) with ethyl methanesulfonate (EMS) mutagenesis, revealed notable differences between the two strains. JCM 1772 showed a capacity for adaptive evolution, leading to the emergence of mutants with increased HA production. In contrast, EMS was found to be toxic to JCM 35779 and was less effective in yielding mutants. Notwithstanding, JCM 35779 demonstrates a significant enhancement over JCM 1772 and its mutants, achieving up to 4.7 to 2.8-fold improvement in HA production while generally exhibiting lower BA production. Genomic analysis of these strains revealed the absence of a BA-specific CoA transferase in JCM 35779, which may explain its metabolic preference for HA over BA synthesis. This hypothesis was further supported by perturbation experiments involving acetate supplementation. These findings highlight the connections between genomic variation, metabolic pathways, and environmental perturbations in shaping MCFA output. By integrating genomic and metabolic insights, this work identifies targets for strain engineering to enhance MCFA production, advancing the potential of M. elsdenii as a sustainable bioproduction platform.