Modular synthase-encoding gene involved in α-olefin biosynthesis in Synechococcus sp. strain PCC 7002

Abstract

A gene involved in the production of medium-chain α-olefins was identified in the cyanobacterium Synechococcus sp. strain PCC 7002. The gene encodes a large multidomain protein with homology to type I polyketide synthases, suggesting a route for hydrocarbon biosynthesis from fatty acids via an elongation decarboxylation mechanism.

Fig. 1.

Comparison of hydrocarbon extracts from the wild-type and mutant strains of PCC 7002. GC-MS signal was normalized to the height of an internal standard peak (hexadecane, 8.3 min). Two hydrocarbons, 1,14-nonadecadiene and 1-nonadecene, were identified in extracts of the wild type and the promoter replacement Φ(P_psbA-ols) mutant but not in extracts of the Δols mutant.

Fig. 2.

(a) Domain organization and proposed mechanism of a putative olefin synthase encoded by ols. (b) Partial sequence alignments of Ols with consensus polyketide synthase domain motifs. Numbers correspond to the amino acid positions in Ols. References are in parentheses. Ppant, phosphopantetheine.

Fig. 3.

Confirmation of PCC 7002 mutants by PCR. (a) Primers amplifying the 5′ and 3′ junctions of ols (sets A and C) and the expected integrated resistance cassette (sets B and D) were used to generate PCR products specific to each strain. Gels of each PCR confirm the replacement of ols with a streptomycin resistance cassette (aadA). (b) Primers flanking the promoter of ols were used to confirm the size of the genomic sequence on both the wild type and the Φ(P_psbA-ols) mutant. The larger size of mutant PCR product is due to the presence of an aadA expression cassette positioned upstream of P_psbA. nb, no band; exp, expected.

Fig. 4.

Comparison of hydrocarbon extracts from wild type (WT), ols deletion mutant (Δols), and Φ(P_psbA-ols) strains of PCC 7002 supplemented with pentadecanoic (C_15:0) acid. The GC-MS signal was normalized to the height of an internal standard peak (hexadecane, 8.3 min). Supplementation of cultures with heptadecanoic (C_17:0) resulted in similar traces (not shown). Supplementation of odd chain fatty acids resulted in increased production of 1-octadecene (C_18:1) and a compound consistent with a doubly unsaturated 18-carbon hydrocarbon (C_18:2). No peaks corresponding to a 1-hexadecene analytical standard were observed in any of the extracts.