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Anaerobic production of medium-chain fatty alcohols via a ?-reduction pathway.

ABSTRACT: In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse ?-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and ?-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse ?-oxidation pathway produced n-alcohols at titers of 1.8?g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

SUBMITTER: Mehrer CR

PROVIDER: S-EPMC6072553 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Anaerobic production of medium-chain fatty alcohols via a β-reduction pathway.

Mehrer Christopher R CR Incha Matthew R MR Politz Mark C MC Pfleger Brian F BF

Metabolic engineering 20180525

In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse β-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify sev ...[more]

PMID: 29807110

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Project description: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.

Project description:Fatty acid-derived biofuels and biochemicals can be produced in microbes using β-oxidation pathway engineering. In this study, the β-oxidation pathway of Saccharomyces cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs) when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain Δpox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which encodes an acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed in the Δpox1 strain. The resulting Δpox1 [pox2+] strain exhibited a growth defect because the β-oxidation pathway was blocked in peroxisomes. To unblock the β-oxidation pathway, the gene CROT, which encodes carnitine O-octanoyltransferase, was expressed in the Δpox1 [pox2+] strain to transport the accumulated medium chain acyl-coAs out of the peroxisomes. The obtained Δpox1 [pox2+, crot+] strain grew at a normal rate. The effect of these genetic modifications on fatty acid accumulation and profile was investigated when the strains were grown on oleic acids-containing medium. It was determined that the engineered strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] had increased fatty acid accumulation and an increased ratio of MCFAs. Compared to the wild-type (WT) strain, the total fatty acid production of the strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in Δpox1 [pox2+] and Δpox1 [pox2+, crot+] increased 2.26- and 1.87-fold compared to the WT strain, respectively. In addition, MCFAs in the culture medium increased 3.29-fold and 3.34-fold compared to the WT strain. These results suggested that fatty acids with an increased MCFAs ratio accumulate in the engineered strains with a modified β-oxidation pathway. Our approach exhibits great potential for transforming low value fatty acid-rich feedstock into high value fatty acid-derived products.

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Anaerobic production of medium-chain fatty alcohols via a ?-reduction pathway.

Publications

Anaerobic production of medium-chain fatty alcohols via a β-reduction pathway.

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