Terminal olefin (1-alkene) biosynthesis by a novel p450 fatty acid decarboxylase from Jeotgalicoccus species

Abstract

Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleT(JE)), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleT(JE) is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

FIG. 1.

Terminal olefins identified in Jeotgalicoccus sp. ATCC 8456. (A) GC/MS trace of hexane-extracted Jeotgalicoccus cells. (B) Mass spectrum of the peak at 9.45 min, which was identified as 1-nonadecene. (C) Mass spectrum of the authentic 1-nonadecene standard. For designations, see Table 1.

FIG. 2.

Fatty acid feeding of Jeotgalicoccus sp. ATCC 8456 cultures. The changes in olefin distribution upon feeding of three different fatty acids (0.5%, wt/vol) are shown. Straight-chain olefins are in dark gray and branched-chain olefins in light gray. For designations, see Table 1. The values are averages of five and two independent experiments for the unfed and fed cultures, respectively.

FIG. 3.

Demonstration of in vitro activity and purification of a fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456. (A) GC/MS trace of in vitro reactions of ATCC 8456 cell-free lysate without (top) and with (bottom) eicosanoic acid as a substrate. The peak at 8.85 min was identified as 1-nonadecene. (B) Coomassie-stained SDS-PAGE of the active enzyme fraction after a three-step purification protocol.

FIG. 4.

Analysis of recombinant OleT_JE. (A) In vivo activity upon expression in E. coli with and without stearic acid feeding. (B) In vitro activity of purified OleT_JE with stearic acid as substrate. 1-C₁₅-ene, 1-pentadecene; 1-C₁₇-ene, 1-heptadecene; 1,10-C₁₇-diene, 1,10-heptadecadiene. TIC, total ion count.

FIG. 5.

Homology model of OleT_JE. (A) Overview of homology model of OleT_JE obtained by threading the OleT_JE sequence with the crystal structure of P450_BSβ from B. subtilis (Protein Data Bank entry 1IZO). (B) A view of the active sites of OleT_JE (purple) overlaid with the one of P450_BSβ (green). In red is the free fatty acid cocrystallized with P450_BSβ. Most amino acid residues in the active site are conserved between the two enzymes, except for His85 in OleT_JE, which corresponds to Gln in P450_BSβ.

FIG. 6.

In vitro hydroxylation and decarboxylation of palmitic acid by purified OleT_JE, P450_BSβ, and P450_BSβ Q85H proteins. (A) Rate of product formation. (B) Ratio of fatty acid decarboxylation over hydroxylation. 1-PE, 1-pentadecene; α-OH PA, α-hydroxy palmitic acid; β-OH PA, β-hydroxy palmitic acid.

FIG. 7.

Proposed mechanism for fatty acid decarboxylation and α- or β-hydroxylation carried out by OleT_JE and related cyp152 P450 enzymes.