Metatranscriptomic and Thermodynamic Insights into Medium-Chain Fatty Acid Production Using an Anaerobic Microbiome

Abstract

Biomanufacturing from renewable feedstocks can offset fossil fuel-based chemical production. One potential biomanufacturing strategy is production of medium-chain fatty acids (MCFA) from organic feedstocks using either pure cultures or microbiomes. While the set of microbes in a microbiome can often metabolize organic materials of greater diversity than a single species can and while the role of specific species may be known, knowledge of the carbon and energy flow within and between organisms in MCFA-producing microbiomes is only now starting to emerge. Here, we integrated metagenomic, metatranscriptomic, and thermodynamic analyses to predict and characterize the metabolic network of an anaerobic microbiome producing MCFA from organic matter derived from lignocellulosic ethanol fermentation conversion residue. A total of 37 high-quality (>80% complete, <10% contamination) metagenome-assembled genomes (MAGs) were recovered from the microbiome, and metabolic reconstruction of the 10 most abundant MAGs was performed. Metabolic reconstruction combined with metatranscriptomic analysis predicted that organisms affiliated with Lactobacillus and Coriobacteriaceae would degrade carbohydrates and ferment sugars to lactate and acetate. Lachnospiraceae- and Eubacteriaceae-affiliated organisms were predicted to transform these fermentation products to MCFA. Thermodynamic analyses identified conditions under which H₂ is expected to be either produced or consumed, suggesting a potential role of H₂ partial pressure in MCFA production. From an integrated systems analysis perspective, we propose that MCFA production could be improved if microbiomes were engineered to use homofermentative instead of heterofermentative Lactobacillus and if MCFA-producing organisms were engineered to preferentially use a thioesterase instead of a coenzyme A (CoA) transferase as the terminal enzyme in reverse β-oxidation. IMPORTANCE Mixed communities of microbes play important roles in health, the environment, agriculture, and biotechnology. While tapping the combined activities of organisms within microbiomes may allow the utilization of a wider range of substrates in preference to the use of pure cultures for biomanufacturing, harnessing the metabolism of these mixed cultures remains a major challenge. Here, we predicted metabolic functions of bacteria in a microbiome that produces medium-chain fatty acids from a renewable feedstock. Our findings lay the foundation for efforts to begin addressing how to engineer and control microbiomes for improved biomanufacturing, how to build synthetic mixtures of microbes that produce valuable chemicals from renewable resources, and how to better understand the microbial communities that contribute to health, agriculture, and the environment.

Keywords: anaerobic digestion; biorefining; carboxylate platform; hexanoic acid; medium-chain fatty acids; metagenomics; metatranscriptomics; octanoic acid.

FIG 1

Transformation of materials in lignocellulosic ethanol conversion residues by an anaerobic microbiome and abundance of MAGs. During 120 days of reactor operation, compounds in conversion residues (CR) were converted to medium-chain fatty acids. In panels A and B, the bars in the first set of bars in the figure indicate the concentrations in the feed (CR), whereas the rest of the bars describe concentrations in the reactor. A more detailed description of the operation of this reactor is presented elsewhere (4). Samples were taken for metagenomic (MG) analysis from five time points (day 12, day 48, day 84, day 96, and day 120) and for metatranscriptomic analysis (MT) from one time point (day 96). Overall, the bioreactor transformed xylose, uncharacterized carbohydrates, and uncharacterized COD to acetic (C2), butyric (C4), hexanoic (C6), and octanoic (C8) acids. The microbial community was enriched in 10 MAGs.

FIG 2

Relative abundance and expression of the 10 most abundant MAGs in the bioreactor at day 96. Relative abundance was determined by mapping DNA sequencing reads to the MAG and normalizing to the length of the MAG genome. Relative transcript abundance (expression) was determined by mapping cDNA sequencing reads to the MAG and normalizing to the length of the MAG genome.

FIG 3

Phylogenetic analysis of 10 MAGs obtained from reactor biomass. Draft genomes from this study are shown in bold text. Red text indicates an organism that has been shown to produce MCFA. National Center for Biotechnology Information assembly accession numbers are shown in parentheses. Node labels represent bootstrap support values, with solid circles representing a bootstrap support value of 100. The phyla and class of genomes are shown in shaded boxes, and families are indicated by brackets.

FIG 4

Relative expression of genes involved in the conversion of xylose and lactate to MCFA. Expression levels of key enzymes involved in (A) utilization of xylose and (B) acyl chain elongation are indicated. Dashed lines represent the existence of multiple enzyme reactions between the indicated molecules. The relative RPKM (relRPKM) values are normalized to the median RPKM for the MAG. Gene expression data are presented as log₂(relRPKM) values. The color intensities of the heatmaps represent the relative gene expression levels, with red color intensity indicating expression below median levels and blue intensity indicating expression above median levels. Gray indicates that the gene is absent from the genome. For genes that are not predicted to be expressed by any MAGs, the associated enzyme product is grayed out in the pathway figure. Key pathway intermediates include xylulose, xylulose-5-phosphate (X-5-P), ribose-5-phosphate (R-5-P), sedoheptulose-7-phosphate (S-7-P), erythyrose-4-phosphate (E-4-P), glyceraldehyde-3-phosphate (G-3-P), fructose-6-phosphate (F-6-P), acetate (Ac), acetyl-phosphate (Ac-P), acetyl-CoA (Ac-CoA), lactate, and ethanol. Enzyme abbreviations are as follows. (A) XylT = xylose transporter, XI = xylose isomerase (EC 5.3.1.5); XK = xylulose kinase (EC 2.7.1.17), R5PE = ribulose-5-phosphate epimerase (EC 5.1.3.1), R5PI = ribose-5-phosphate isomerase (EC 5.3.1.6), TA = transaldolase (EC 2.2.1.2), TK = transketolase (EC 2.2.1.1), PK = d-xyulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (EC 4.1.2.9), GluT = glucose transporter, FruT = fructose PTS transporter, HK = hexokinase (EC 2.7.1.1, EC 2.7.1.2), G6PI = glucose-6-phoshphate isomerase (EC 5.3.1.9), PFK = phosphofructokinase (EC 2.7.1.11), PDH = pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4), PFOR = pyruvate flavodoxin oxidoreductase (EC 1.2.7.-), ADA = acetaldehyde dehydrogenase (EC 1.2.1.10), AD = alcohol dehydrogenase (EC 1.1.1.1), PTA = phosphate acetyltransferase (EC 2.3.1.8), ACK = acetate kinase (EC 2.7.2.1), and LDH = lactate dehydrogenase (EC 1.1.1.27). (B) LacT = lactate permease, ACS = acetyl-CoA synthetase (EC 6.2.1.1), ACAT = acetyl-CoA C-acyltransferase (EC 2.3.1.16, EC 2.3.1.9), HAD = 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.157, 1.1.1.35), ECH = enoyl-CoA hydratase (EC 4.2.1.55, EC 4.2.1.17), ACD = acyl-CoA Dehydrogenase (EC 1.3.99.2, EC 1.3.99.-), EtfA = electron transfer flavoprotein A, EtfB = electron transfer flavoprotein B, TE = thioesterase (EC 3.1.2.20), CoAT = 4-hydroxybutyrate CoA transferase (EC 2.8.3.-), Rnf = ferredoxin–NAD⁺ oxidoreductase–Na+ translocating (EC 1.18.1.8), H2ase = ferredoxin hydrogenase (EC 1.12.7.2), HydABC = bifurcating [Fe-Fe] hydorgenase (EC 1.12.1.4), Ech = energy-conserving hydrogenase (EchABCDEF).

FIG 5

Predicted transformations of major substrates in conversion residues to MCFA by this anaerobic microbiome. The microbes in the LAC and COR bins are predicted to produce sugars from complex carbohydrates. Simple carbohydrates, including xylose remaining in conversion residues, are converted to lactate and acetate (C2) by Lactobacillus (LAC) and Coriobacteriaceae (COR) MAGs. The Lachnospiraceae (LCO1) MAG converts pentoses directly to butyric acid (C4). The Eubacteriaceae (EUB1) MAG produces hexanoic acid (C6) and octanoic acid (C8) from lactate. Further, LCO1 may utilize hydrogen to elongate C2 and C4 to MCFA, as represented by dashed lines. Additionally, EUB1 may elongate C2, C4, and C6 to C8.