Abstract

Biorenewables have the potential to move society’s dependence away from petrol-based industrial products, towards cleaner and more sustainable sources. The technology has moved from focussing on low energy density ethanol- or butanol-based fuel sources to more complex,
energy rich hydrocarbons. Recently, a heme-dependent P450 decarboxylase enzyme, OleTJE, was discovered in the bacterium Jeotgalicoccus sp 8456. This enzyme catalyzes the decarboxylation
of a broad-range of long-chain fatty acids producing terminal alkenes that are key energy-rich hydrocarbons valuable for biofuels. Here, we developed an absorbance-based high-throughput assay that facilitated the determination of the activity and Michaelis-Menten kinetics of OleTJE. We screened OleTJE decarboxylation by detecting CO2, a product of decarboxylation. In this assay, CO2 was converted to bicarbonate and then catalyzed by phosphoenolpyruvate carboxylase and malate dehydrogenase to malate, leading to a decrease in NADH, the detecting
molecule. This assay was then used to screen a mutant OleTJE and a non-P450 decarboxylase, to showcase its versatility to screen mutant variants and other decarboxylases.