Harnessing a P450 fatty acid decarboxylase from for microbial biosynthesis of odd chain terminal alkenes.

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a direct microbial biosynthesis of terminal alkenes from fermentable sugars by harnessing a P450 fatty acid (FA) decarboxylase from (OleT). We first characterized OleT and demonstrated its in vitro HO-independent activities towards linear C10:0-C18:0 FAs, with higher activity for C16:0-C18:0 FAs. Next, we engineered a alkene biosynthesis pathway, consisting of OleT and an engineered thioesterase (TesA) with compatible substrate specificities, and introduced this pathway into for terminal alkene biosynthesis from glucose. The recombinant EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose. To improve alkene production, we identified and overcame the electron transfer limitation in OleT, by introducing a two-component redox system, consisting of a putidaredoxin reductase (CamA) and a putidaredoxin (CamB) from into EcNN101, and demonstrated the alkene production increased ~2.8 fold. Finally, to better understand the substrate specificities of OleT observed, we employed protein modeling to illuminate the functional role of FA binding pocket.