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.

Keywords: Ech complex; Firmicutes; MCFA; Pseudoramibacter; RNF complex; hydrogenase; “Ca. Weimeria,” chain elongation.

FIG 1

Phylogenetic analysis of two metagenome-assembled genomes (shown in bold text) predicted to perform chain elongation in an anaerobic microbiome converting lignocellulosic residues to medium-chain fatty acids. (A) A phylogenetic tree was created based on concatenated amino acid sequences for 120 single-copy marker genes from the GTDB using RAxML. Bootstrap support values are indicated at the nodes. Closed circles indicate bootstrap support values of 100. Organisms that have representative isolates are indicated by an “I” superscript, and organisms representing NCBI type strains are indicated with a “T” superscript. Genome identifiers are provided in parentheses. (B) BLAST average nucleotide identity (ANIb) comparison for LCO1.1 (“Ca. Weimeria bifida”) to related genomes. (C) ANIb comparison for EUB1.1 (“Ca. Pseudoramibacter fermentans”) to related genomes.

FIG 2

Metabolic pathways involved in chain elongation. Reverse β-oxidation is a four-step process using acyl-CoA acetyltransferase (ACAT), 3-hydroxy-acyl-CoA dehydrogenase (HAD), enoyl-CoA dehydratase (ECoAH), and acyl-CoA dehydrogenase (ACD). The reduction of enoyl-CoA with NADH can be combined with the reduction of ferredoxin through an electron-bifurcating acyl-CoA dehydrogenase complex containing EtfA and EtfB. The terminal enzyme of reverse β-oxidation can be either a CoA transferase (CoAT) or thioesterase (TE). Reverse β-oxidation is coupled with proton-translocating enzymes to generate ATP with reduced ferredoxin. This figure is partly adapted from previous work (8).

FIG 3

Organization of selected genes in organisms related to “Ca. Weimeria bifida,” “Ca. Pseudoramibacter fermentans,” and known chain-elongating organisms. Reverse β-oxidation genes are shown in red, and genes involved in lactate utilization, if present, are shown in blue. Reverse β-oxidation genes include acyl-CoA acetyltransferase (ACAT), 3-hydroxy-acyl-CoA dehydrogenase (HAD), enoyl-CoA dehydratase (ECoAH), acyl-CoA dehydrogenase (ACD), electron transfer flavoprotein A (etfA), and electron transfer flavoprotein B (etfB). Lactate utilization genes include lactate permease (lctP) and lactate dehydrogenase (LDH). Other genes included in the figure are prephenate dehydrogenase (PRDH) (J, K, and L), 2-hydroxyglutaryl-CoA dehydratase (hgd) involved in propionate production in Megasphaera elsdenii (G), and acetyl-CoA carboxylase (acc) and fatty acid biosynthesis (fab) genes when they are adjacent to reverse β-oxidation genes (J, K, and L). nt, nucleotides.

FIG 4

Transcript abundance for predicted chain elongation transcripts by “Ca. Weimeria bifida.” “Prefed” represents the time point prior to feeding the reactor. Error bars show 95% confidence levels determined with CuffLinks. Transcript abundance data are shown for transcripts encoding enzymes used for reverse β-oxidation (A), the RNF complex (B), hydrogenases (C), and terminal enzymes of reverse β-oxidation (D). The roles of the designated enzymes in chain elongation are provided in Fig. 2. FPKM, fragments per kilobase per million.

FIG 5

Predicted routes for xylose utilization by “Ca. Weimeria bifida.” (A) After xylose activation to xylulose-5-phosphate, “Ca. Weimeria bifida” can consume pentoses via the pentose phosphate pathway or the phosphoketolase pathway. Key enzymes involved in xylose consumption include ribulose 5-phosphate epimerase (R5PE), ribose-5-phosphate isomerase (R5PI), transketolase (TK), transaldolase (TA), transketolase (TK), phosphoketolase (PK), and acetate kinase (AK). (B) Transcript abundance for 36 h after providing the bioreactor with conversion residue. “Prefed” represents the time point prior to feeding the reactor. Error bars show 95% confidence levels determined with CuffLinks.

FIG 6

Transcript abundance for chain elongation transcripts by “Ca. Pseudoramibacter fermentans.” “Prefed” represents the time point prior to feeding the reactor. Error bars show 95% confidence levels determined with CuffLinks. Transcript abundance data are shown for genes encoding enzymes used for reverse β-oxidation (A), the RNF complex (B), hydrogenases (C), and terminal enzymes of reverse β-oxidation (D). The roles of enzymes in chain elongation and enzyme definitions are shown in Fig. 2.

FIG 7

Pairwise linear correlation analyses for expression of “Ca. Pseudoramibacter fermentans” genes encoding lactate dehydrogenase (LDH), acetyl-CoA dehydrogenase (ACD), prephenate dehydrogenase (PRDH), electron transfer flavoprotein A (EtfA), and electron transfer flavoprotein B (EtfB). Values on the on x and y axes represent the number of fragments per kilobase per million. Numbers within the plots are coefficients of determination.

FIG 8

Glycerol utilization by “Ca. Pseudoramibacter fermentans.” (A) “Ca. Pseudoramibacter fermentans” contains genes for three glycerol utilization routes. Key enzymes in these pathways include glycerol kinase (GK), glycerol-3-phosphate dehydrogenase (G3PD), glycerol dehydrogenase (GDH), dihydroxyacetone kinase (DHAK), glycerol dehydratase (GDT), and propionaldehyde dehydrogenase (PAD). (B) Transcript abundance for glycerol transport and utilization. Subunits of a single enzyme complex are indicated in parentheses. “Prefed” represents the time point prior to feeding the reactor. Error bars show 95% confidence levels determined with CuffLinks.

FIG 9

Glycerol utilization gene clusters in “Ca. Pseudoramibacter fermentans” (A), related Pseudoramibacter species (B and C), and Acetonema longum (D). Blue shading indicates a gene predicted to encode an enzyme involved in route 3 (Fig. 8A), and gray shading indicates a gene predicted to encode a microcompartment protein.

FIG 10

Transcript abundance for a lactate utilization gene cluster in “Ca. Pseudoramibacter fermentans.” Genes include a transcriptional regulator, lactate permease (lctP), a potential flavodoxin, lactate dehydrogenase (LDH), and acetyl-CoA dehydrogenase (ACD). “Prefed” represents the time point prior to feeding the reactor. Error bars show 95% confidence levels determined with CuffLinks.

FIG 11

A maximum-likelihood phylogenetic tree of lactate dehydrogenase amino acid sequences among different members of the Firmicutes phylum. Anaerobic lactate consumers contain lactate dehydrogenase and form a distinct cluster from lactate producers, such as those found among the lactobacilli.