Discovery and implementation of a novel pathway for n-butanol production via 2-oxoglutarate

Ferreira, Sofia; Pereira, Rui; Liu, Filipe; Vilaça, Paulo; Rocha, Isabel

doi:10.1186/s13068-019-1565-x

Biotechnology for Biofuels and Bioproducts

Table 1 Estimated change in Gibbs energy and experimental validation for the reactions constituting the 2-oxoglutarate pathway

From: Discovery and implementation of a novel pathway for n-butanol production via 2-oxoglutarate

Step	Reaction	Δ_rG′^m (KJ mol⁻¹)	K′_eq	E.C. number	Enzyme	Gene NCBI accession no	Enzymatic activity^a	Exp. validation in E. coli
1	2-Oxoglutarate + NADH + H⁺⟺ 2-Hydroxyglutarate^b + NAD⁺	− 22.6 ± 3.6	9.2 × 10³	EC 1.1.1.399	2-Oxoglutarate reductase	hgdH [AF] WP_012938338	[19]	[20, 21]
2	Acetyl-CoA + 2-Hydroxyglutarate ⟺ Acetate + 2-Hydroxyglutaryl-CoA	− 8.6 ± 15.3	32.1	EC 2.8.3.12	Glutaconate CoA-transferase (subunits α and β)	gctA [AF] WP_012939156.1	[22]
2		− 8.6 ± 15.3	32.1	EC 2.8.3.12	Glutaconate CoA-transferase (subunits α and β)	gctB [AF] WP_012939155.1	[22]
3	2-Hydroxyglutaryl-CoA ⟺ Glutaconyl-CoA + H₂O	0.7 ± 2.9	0.751	EC 4.2.1.167	(R)-2-Hydroxyglutaryl-CoA dehydratase Subunits α, β and activator	hgdA [CS] WP_003501726.1	[23]
						hgdB [CS] WP_003501727.1	[23]
						hgdC [AF] WP_012939153.1	[24]
4	Glutaconyl-CoA ⟺ Crotonyl-CoA + CO₂	− 35.8 ± 17.4	1.9 × 10³	EC 1.3.8.6	Glutaryl-CoA dehydrogenase	gcdH [PA] WP_042912665.1	[25]	–
4	Glutaconyl-CoA ⟺ Crotonyl-CoA + CO₂	− 35.8 ± 17.4	1.9 × 10³	EC 4.1.1.70	Glutaconyl-CoA decarboxylase α subunit	gcdA [CS] WP_003501720.1	[26]	–
5	Crotonyl-CoA + NADH + H⁺ ⟺ Butanoyl-CoA + NAD⁺	− 40.0 ± 17.0	1.0 × 10⁷	EC 1.3.1.44	Trans-2-enoyl-CoA reductase	ter [TD] WP_002681770.1	[27]	[5, 28,29,30]
6	Butanoyl-CoA + NADH + H⁺⟺ Butanal + CoA + NAD⁺	− 57.0 ± 16	9.6 × 10⁹	EC 1.2.1.57	Aldehyde/alcohol dehydrogenase	adhE1 [CA] WP_077851567.1	[31]	[32, 33]
7	Butanal + NADH + H⁺ ⟺ Butanol + NAD⁺	− 24.2 ± 4.8	1.8 × 10⁴	EC 1.1.1.1	Aldehyde/alcohol dehydrogenase	adhE1 [CA] WP_077851567.1	[31]	[32, 33]

The estimated change in Gibbs free energy (Δ_rG′^m) values were computed with eQuilibrator [17] for each reaction constituting the novel pathway to produce butanol from 2-oxoglutarate (assumptions: reactant/product concentrations of 1 mM), as well as equilibrium constant (K′_eq) values at a pH of 7 and an ionic strength of 0.1 M. The encoding gene and respective microorganism, as well as references of enzymatic activity validation with the substrate and experimental validation in Escherichia coli are also shown, plus the EC number and NCBI accession number
Exp experimental, EC enzyme commission, AF Acidaminococcus fermentans, CS Clostridium symbiosum, PA Pseudomonas aeruginosa, TD Treponema denticola, CA Clostridium acetobutylicum
^aThis refers to the substrate in italics in the reaction column
^bOnly the enzymes converting the (R)-isomer were considered. There are two enantiomers of 2-hydroxyglutarate; however, in KEGG (the database from which the reactions were retrieved), these variations are not represented. Depending on the enzyme, one of these forms can be preferably synthesized. The two subsequent reactions are part of the glutamate degradation pathway, existent is some microorganisms, where the (R)-isomers of the other compounds are preferably consumed

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ISSN: 2731-3654

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