The human gut microbe <i>Eubacterium limosum</i> utilizes flavodoxin over ferredoxin for lactate metabolism

ABSTRACT Eubacterium limosum is an abundant gut microbe that efficiently metabolizes lactate to produce the short-chain fatty acids, butyrate and acetate. The key oxidoreductase enzymes, formate dehydrogenase (FDH), carbon monoxide dehydrogenase (CODH) of the Wood-Ljungdahl pathway, pyruvate ferredoxin oxidoreductase (POR), and an electron-bifurcating tungsten-containing oxidoreductase (WOR) that detoxifies acetaldehyde produced as a byproduct of the POR reaction, are all utilized during lactate metabolism. Fermentative anaerobes typically utilize a single ferredoxin as an electron carrier for their primary pathways, which is replaced by flavodoxin under iron-limiting conditions. However, the E. limosum genome encodes two putative ferredoxins (Fd1 and Fd2) and three putative flavodoxins (Fld, Fld-like1, and Fld-like2). All five proteins were heterologously expressed in Escherichia coli , but the UV-visible absorption properties of purified Fld-like proteins 1 and 2 were not typical of canonical flavodoxins. Both POR and CODH reduced Fd1, Fd2, and Fld at comparable rates. Partially purified WOR and FDH had low activity using Fd1, Fd2, and Fld as electron carriers. With WOR, NAD-linked bifurcating activity could not be demonstrated with any of the electron carriers. Deletion mutants of E. limosum lacking Fd1, Fd2, or Fld exhibited similar lag phases during growth on glucose, and this increased on lactate for the Fld mutant but much less so for the Fd mutants. We conclude that E. limosum can utilize either Fd1, Fd2, or Fld as the primary redox protein, but that during growth on lactate, Fld plays a more prominent role than Fd1 or Fd2, even under iron-sufficient conditions. IMPORTANCE Eubacterium limosum is an abundant gut microbe that is beneficial to human health due to its production of short-chain fatty acids particularly during growth on lactate. It is also of interest due to its ability to metabolize H 2 /CO 2 and C1 substrates. It was assumed that E. limosum , like other fermentative anaerobes, utilizes a single ferredoxin as an electron carrier during primary carbon metabolism and only utilizes flavodoxin under iron-limited conditions. However, we show here that this organism utilizes flavodoxin as its main electron carrier. This may be a significant advantage in the gut environment where competition with the host and with other gut microbes for iron is intense. This may have both biotechnological and health implications for this organism.