The oxidoreductase activity of Rnf balances redox cofactors during fermentation of glucose to propionate in Prevotella

Abstract

Propionate is a microbial metabolite formed in the gastrointestinal tract, and it affects host physiology as a source of energy and signaling molecule. Despite the importance of propionate, the biochemical pathways responsible for its formation are not clear in all microbes. For the succinate pathway used during fermentation, a key enzyme appears to be missing—one that oxidizes ferredoxin and reduces NAD. Here we show that Rnf [ferredoxin—NAD+ oxidoreductase (Na+-transporting)] is this key enzyme in two abundant bacteria of the rumen (Prevotella brevis and Prevotella ruminicola). We found these bacteria form propionate, succinate, and acetate with the classic succinate pathway. Without ferredoxin:NAD+ oxidoreductase, redox cofactors would be unbalanced; it would produce almost equal excess amounts of reduced ferredoxin and oxidized NAD. By combining growth experiments, genomics, proteomics, and enzyme assays, we point to the possibility that these bacteria solve this problem by oxidizing ferredoxin and reducing NAD with Rnf [ferredoxin—NAD+ oxidoreductase (Na+-transporting)]. Genomic and phenotypic data suggest many bacteria may use Rnf similarly. This work shows the ferredoxin:NAD+ oxidoreductase activity of Rnf is important to propionate formation in Prevotella species and other bacteria from the environment, and it provides fundamental knowledge for manipulating fermentative propionate production.

Propionate production by Bacteroidia gut bacteria and its dependence on substrate concentrations differs among species

“…Since increased laboratory safety measures increases costs for biotechnological processes, we solely focused on species that belong to risk group 1 according to the German technical rule for biological agencies 466 (TRBA). Unfortunately, a well-studied propionate producer, Xylanibacter ruminicola [ 29 ] (formerly Prevotella ruminicola [ 30 ]) belongs to risk group 2. The species were selected either because they utilized a wide spectrum of polymeric substrates, for example B. cellulosilyticus , which had been described to degrade crystalline cellulose [ 31 ], or because comparably high propionate concentrations were reported.…”

“…Genes for the energy metabolisms identified were an Na + -gradient generating NADH-quinone reductase (Nqr), the Rnf complex and the cytochrome bd ubiquinol oxidase, which have been described previously for Bacteroidia [ 22 , 29 , 42 ]. In addition, B. graminisolvens harbored the genes for the NADH-quinone oxidoreductase without the subunits E, F and G, similar to the headless variant of this enzyme, that has been described before in Bacteroidia [ 20 ].…”

The vast landscape of carbohydrate fermentation in prokaryotes

“…5A ), three unrecognized enzymes have been found in two studies (Schleicher et al 2021 , Zhang et al 2023 ). The first study (Zhang et al 2023 ) found that Rnf is present, and as in P. ruminis , this enzyme transfers electrons away from reduced ferredoxin. This is important because a second enzyme was found that forms reduced ferredoxin.…”