Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase

Abstract

Aliphatic medium-chain 1-alkenes (MCAEs, ∼10 carbons) are "drop-in" compatible next-generation fuels and precursors to commodity chemicals. Mass production of MCAEs from renewable resources holds promise for mitigating dependence on fossil hydrocarbons. An MCAE, such as 1-undecene, is naturally produced by Pseudomonas as a semivolatile metabolite through an unknown biosynthetic pathway. We describe here the discovery of a single gene conserved in Pseudomonas responsible for 1-undecene biosynthesis. The encoded enzyme is able to convert medium-chain fatty acids (C10-C14) into their corresponding terminal olefins using an oxygen-activating, nonheme iron-dependent mechanism. Both biochemical and X-ray crystal structural analyses suggest an unusual mechanism of β-hydrogen abstraction during fatty acid substrate activation. Our discovery unveils previously unidentified chemistry in the nonheme Fe(II) enzyme family, provides an opportunity to explore the biology of 1-undecene in Pseudomonas, and paves the way for tailored bioconversion of renewable raw materials to MCAE-based biofuels and chemical commodities.

Keywords: biofuel; biosynthesis; hydrocarbon; iron-dependent desaturase/decarboxylase; terminal olefin.

Fig. 1.

Identification of the enzyme responsible for 1-undecene biosynthesis in Pseudomonas. (A) Demonstration of 1-undecene production from headspace SPME–GCMS analysis. (i) 1-undecene production was observed during the library screening in the E. coli EPI300 expressing the gene undA. (ii) P. aeruginosa PA14 naturally produces 1-undecene. (iii) Disruption of the gene PA14_53120 (an undA homolog) completely abolished 1-undecene production in the P. aeruginosa ∆PA14_53120 mutant. (iv) E. coli EPI300 does not naturally produce 1-undecene. (B) UndA-containing two-gene operons conserved in Pseudomonas. (C) Production titer of extracellular 1-undecene by overexpressing undA (P. fluorescens Pf-5), PSPTO_1738 (P. syringae pv. tomato DC3000), PA14_53120 (P. aeruginosa PA14), and Pput_3952 (P. putida F1) in E. coli BL21 Star. Error bars represent SDs from at least three independently performed experiments. (D) The biosynthesis of 1-undecene from LA catalyzed by UndA, an oxygen-activating, nonheme, iron-dependent desaturase/decarboxylase.

Fig. 2.

UndA activity toward selected substrates: A, [1-¹³C]LA; B, [12-¹³C]LA; C, AHDA; D, [α,α-D2]LA; E, [D23]LA; F, BHDA; G, DEA. Both deuterium atoms of [α,α-D2]LA are proposed to retain at the α-carbon position forming [1,1-D2]undecene in D.

Fig. 3.

Structures of UndA and proposed 1-undecene synthesis mechanism. (A) Overall structure of UndA with helices in blue and loops in salmon. Iron is shown in black, and DEA is shown in yellow for carbons and red for oxygens. (B) Substrate binding pocket of UndA. DEA, presented as a ball-and-stick model, is surrounded by a simulated annealing omit map in blue, contoured at 3.0 σ. Pocket-forming residues are displayed as sticks and the hydrophobic residues are colored in orange. (C–F) Weighted electron density maps surrounding the active site of the holo-, DEA-, and BHDA-bound structures with 2mF_o-DF_c (in gray, at 1.8 σ) and mF_o-DF_c (green and red, at ±3.0 σ). The distal oxygen atoms of the dioxygen species in E and F are surrounded by the simulated annealing omit maps in green, contoured at 3.0 σ. (G) Proposed mechanism for 1-undecene biosynthesis by UndA. The molecular oxygen is proposed to be reduced to H₂O likely by some reductant as shown in SI Appendix, Fig. S14A.