Establishing an enzyme cascade for one-pot production of alpha-olefins from low-cost triglycerides and oils without exogenous H2O2 addition.

Background: Biological alpha-olefins can be used as both biofuels and high value-added chemical precursors to lubricants, polymers, and detergents. The prototypic CYP152 peroxygenase family member OleTJE from Jeotgalicoccus sp. ATCC 8456 catalyzes a single-step decarboxylation of free fatty acids (FFAs) to form alpha-olefins using H2O2 as a cofactor, thus attracting much attention since its discovery. To improve the productivity of alpha-olefins, significant efforts on protein engineering, electron donor engineering, and metabolic engineering of OleTJE have been made. However, little success has been achieved in obtaining alpha-olefin high-producer microorganisms due to multiple reasons such as the tight regulation of FFA biosynthesis, the difficulty of manipulating multi-enzyme metabolic network, and the poor catalytic performance of OleTJE. Results: In this study, a novel enzyme cascade was developed for one-pot production of alpha-olefins from low-cost triacylglycerols (TAGs) and natural oils without exogenous H2O2 addition. This artificial biocatalytic route consists of a lipase (CRL, AOL or Lip2) for TAG hydrolysis to produce glycerol and free fatty acids (FFAs), an alditol oxidase (AldO) for H2O2 generation upon glycerol oxidation, and the P450 fatty acid decarboxylase OleTJE for FFA decarboxylation using H2O2 generated in situ. The multi-enzyme system was systematically optimized leading to the production of alpha-olefins with the conversion rates ranging from 37.2 to 68.5%. Furthermore, a reaction using lyophilized CRL/OleTJE/AldO enzymes at an optimized ratio (5 U/6 muM/30 muM) gave a promising alpha-olefin yield of 0.53 g/L from 1500 muM (~1 g/L) coconut oil. Conclusions: The one-pot enzyme cascade was successfully established and applied to prepare high value-added alpha-olefins from low-cost and renewable TAGs/natural oils. This system is independent of exogenous addition of H2O2, thus not only circumventing the detrimental effect of H2O2 on the stability and activity of involved enzymes, but also lower the overall costs on the TAG-to-olefin transformation. It is anticipated that this biotransformation system will become industrially relevant in the future upon more engineering efforts based on this proof-of-concept work.