Uncovering the substrate of olefin synthase loading domains in cyanobacteria Picosynechococcus sp. strain PCC 7002

Cyanobacteria are widespread, photosynthetic, gram-negative bacteria that generate numerous bioactive secondary metabolites via complex biosynthetic enzymatic machinery. The model cyanobacterium Picosynechococcus sp. strain PCC 7002, hereafter referred to as PCC 7002, contains a type I polyketide synthase (PKS), termed olefin synthase (OlsWT), that synthesizes 1-nonadecene and 1,14-nonadecadiene: α-olefins that are important for growth at low temperatures. The putative biochemistry encoded by the PKS domains suggests that OlsWT will create an olefin with one additional carbon relative to the original substrate (+1 mechanism). The first domain in the multi-module OlsWT protein has homology to fatty acyl-AMP ligases (FAALs) that typically activate free fatty acids prior to creating novel thioester linkages. Paradoxically, unmodified wildtype PCC 7002 is not known to maintain a substantial pool of free fatty acids, and prior work demonstrated conversion of exogenous pentadecanoic acid to 1-octadecene instead of the expected 1-hexadecene. In this study, we developed PCC 7002 as a heterologous host to facilitate the expression and study of Ols proteins in effort to discover their true substrates. Here, we report the successful expression of two Ols homologs from Geminocystis sp. NIES-3709 and Xenococcus sp. PCC 7305 in PCC 7002 that generated 1-heptadecene and 1-pentadecene, respectively. Through the additional deletion of a gene encoding an acyl-acyl carrier protein (ACP) synthetase (Aas) responsible for activation of exogenous free fatty acids, we demonstrated the expected conversion of exogenously provided odd-chain fatty acids to α-olefins containing one additional carbon. These data suggest that short-lived fatty acids liberated from lipid membranes are the Ols substrate. We subsequently confirmed OlsWT activity on octadecanoic acid via in vitro chrome azurol S assay using a purified FAAL module. Collectively, this work clarifies the in vivo substrate of Ols FAAL domains and identifies the FAAL module as a target for future bioengineering to allow access to desired α-olefins.