Fermentation is a major type of metabolism, which is carried out by both prokaryotes and eukaryotes. Organisms carrying out this metabolism live in many habitats, including the rumen of cows, gastrointestinal tracts of other animals, aquatic environments, and anaerobic digesters. These organisms ferment organic compounds and produce small molecular metabolites. Three important metabolites formed during fermentation are acetate, propionate, and butyrate. The production of these short-chain fatty acids during fermentation has broad importance for foods, agriculture, industry, and human health. For example, up to 70% of energy metabolized in a cow is from fermentation products formed in the rumen. Despite the importance of fermentation acids, we still lack a full understanding of how microbes form these fermentation acids. While explanations of fermentation acid formation have existed in textbooks for decades, genomic studies suggested that some bacteria lack pathways described in textbooks and instead they may possess unrecognized alternate pathways. For example, some bacteria lack genes for enzymes that form acetate. Instead, they have genes for a previously unrecognized pathway involving the enzymes succinyl-CoA:acetate CoA-transferase (SCACT) and succinyl-CoA synthetase (SCS). Bacteria may use the SCACT/SCS pathway to form acetate. Likewise, some bacteria lack a step for forming propionate; however, they have genes for Rnf (Rhodobacter nitrogen fixation), an enzyme that is able to fill the missing step. Though the genomic evidence for these pathways is strong, biochemical evidence is still needed.
The first aim was to test if bacteria can use the SCACT/SCS pathway to form acetate. We found that the bacterium Cutibacterium granulosum formed acetate during fermentation of glucose. Using genomics, proteomics, and enzymatic assays, we demonstrated this bacterium used the SCACT/SCS pathway, rather than the typical pathway found in nearly all acetate-forming bacteria. Among nearly 600 genomes of bacteria known to form acetate, we found 36 genomes of bacteria encoded homologs with SCACT and SCS activity. These species belong to 5 different phyla, suggesting the SCACT/SCS pathway is important for acetate formation in many branches of the tree of life.
The second aim was to test if the enzyme Rnf fills in a missing step to complete the pathway for forming propionate during fermentation in bacteria. This missing step is for reducing oxidized NAD and oxidizing reduced ferredoxin, which are both redox cofactors. We confirmed that two rumen bacteria (Prevotella ruminicola and Prevotella brevis) formed propionate (or its precursor, succinate) during fermentation of glucose. Genes for Rnf were identified and their expression in the cell were confirmed with shotgun proteomics. Enzyme assays confirmed these bacteria had the ferredoxin:NAD+ oxidoreductase activity characteristic of Rnf. Other redox related enzymes were identified using the same methods. We searched for Rnf genes in the genomes of bacteria that form propionate/succinate, and found 44 type strains from many habitats encode it. This suggests that Rnf is important to propionate/succinate formation in bacteria from many habitats.
In sum, our work identified the SCACT/SCS pathway for forming acetate and also identified an ion pump Rnf involved in forming propionate during fermentation in bacteria. These pathways can be used by bacteria living in various environments. Our work not only filled the knowledge gap in understanding biochemical pathways for forming fermentation acids in bacteria, but also provide insights for modifying fermentation. Enzymes in these pathways could be targets for modifying acetate and propionate production during fermentation, such as fermentation in ruminants.
