Medium-Chain Fatty Acid Synthesis by “ Candidatus Weimeria bifida” gen. nov., sp. nov., and “ Candidatus Pseudoramibacter fermentans” sp. nov

Abstract

Chain elongation is emerging as a bioprocess to produce valuable medium-chain fatty acids (MCFA; 6 to 8 carbons in length) from organic waste streams by harnessing the metabolism of anaerobic microbiomes. Although our understanding of chain elongation physiology is still evolving, the reverse β-oxidation pathway has been identified as a key metabolic function to elongate the intermediate products of fermentation to MCFA. Here, we describe two uncultured chain-elongating microorganisms that were enriched in an anaerobic microbiome transforming the residues from a lignocellulosic biorefining process. Based on a multi-omic analysis, we describe “Candidatus Weimeria bifida” gen. nov., sp. nov., and “Candidatus Pseudoramibacter fermentans” sp. nov., both predicted to produce MCFA but using different substrates. The analysis of a time series metatranscriptomic data set suggests that “Ca. Weimeria bifida” is an effective xylose utilizer since both the pentose phosphate pathway and the bifid shunt are active. Furthermore, the metatranscriptomic data suggest that energy conservation during MCFA production in this organism is essential and occurs via the creation of an ion motive force using both the RNF complex and an energy-conserving hydrogenase. For “Ca. Pseudoramibacter fermentans,” predicted to produce MCFA from lactate, the metatranscriptomic analysis reveals the activity of an electron-confurcating lactate dehydrogenase, energy conservation via the RNF complex, H2 production for redox balance, and glycerol utilization. A thermodynamic analysis also suggests the possibility of glycerol being a substrate for MCFA production by “Ca. Pseudoramibacter fermentans.” In total, this work reveals unknown characteristics of MCFA production in two novel organisms. IMPORTANCE Chain elongation by medium-chain fatty acid (MCFA)-producing microbiomes offers an opportunity to produce valuable chemicals from organic streams that would otherwise be considered waste. However, the physiology and energetics of chain elongation are only beginning to be studied, and many of these organisms remain uncultured. We analyzed MCFA production by two uncultured organisms that were identified as the main MCFA producers in a microbial community enriched from an anaerobic digester; this characterization, which is based on meta-multi-omic analysis, complements the knowledge that has been acquired from pure-culture studies. The analysis revealed previously unreported features of the metabolism of MCFA-producing organisms.

Diagnosing and predicting mixed culture fermentations with unicellular and guild-based metabolic models

“…Pseudoramibacter fermentans. 39 This iFermGuilds789 simulation predicted ranges of ethanol, lactate, C2, and C6 that contained the experimentally observed values (Fig 6C). With these parameters, C8 and H2 production were overpredicted by iFermGuilds789, while C4 production was underpredicted.…”

“…While SEOs are predicted to use an acyl-CoA hydrolase as the terminal enzyme of reverse b-oxidation, LEOs are predicted to use a CoAT which conserves energy by activating C2 to acetyl-CoA, which would otherwise require ATP. In the previous metatranscriptomic analyses of these community members, 15,39 we found that both SEOs and LEOs expressed genes for both acyl-CoA hydrolases and CoAT. The specificity of these predicted chain elongating enzymes for different substrates, however, is not known.…”

“…3 To build and test these models we took advantage of prior metagenomic, metatranscripotomic, and metabolomic analyses of a mixed community that produces C6 and C8 fatty acids from a lignocellulosic refinery residue. 15,39 The predictions of our models generated several hypotheses that should be explored with further analyses. First, the single unit iFermCell215 predicted production of MCFAs from ethanol as a sole source of electrons and carbon ( Fig.…”

“…Weimeria bifida (SEOs), lactate-elongating Cand. Pseudoramibacter fermentans (LEOs), 39 five sugar fermenting Lactobacillus species (SFOs), and three hydrogenic sugar fermenting Coriobacteriaceae species (HSFs). 15 We constrained the abundance of each guild in iFermGuilds789 according to the relative abundance of DNA for each guild in the bioreactor.…”

Lactate-based microbial chain elongation for n-caproate and iso-butyrate production: genomic and metabolic features of three novel Clostridia isolates

“…In addition to our newly isolated strains, we included eleven strains that have been experimentally validated of microbial chain elongation (Table 1). Two metagenome-assembled genomes (MAGs; Candidatus Pseudoramibacter fermentans and Candidatus Weimeria bi da) were also included in the comparative genome analysis because their chain elongation traits were evident from metatranscriptome analyses [21].…”