Establishing an enzyme cascade for one-pot production of α-olefins from low-cost triglycerides and oils without exogenous H(2)O(2) addition

BACKGROUND: Biological α-olefins can be used as both biofuels and high value-added chemical precursors to lubricants, polymers, and detergents. The prototypic CYP152 peroxygenase family member OleT(JE) from Jeotgalicoccus sp. ATCC 8456 catalyzes a single-step decarboxylation of free fatty acids (FFAs) to form α-olefins using H(2)O(2) as a cofactor, thus attracting much attention since its discovery. To improve the productivity of α-olefins, significant efforts on protein engineering, electron donor engineering, and metabolic engineering of OleT(JE) have been made. However, little success has been achieved in obtaining α-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 OleT(JE). RESULTS: In this study, a novel enzyme cascade was developed for one-pot production of α-olefins from low-cost triacylglycerols (TAGs) and natural oils without exogenous H(2)O(2) 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 H(2)O(2) generation upon glycerol oxidation, and the P450 fatty acid decarboxylase OleT(JE) for FFA decarboxylation using H(2)O(2) generated in situ. The multi-enzyme system was systematically optimized leading to the production of α-olefins with the conversion rates ranging from 37.2 to 68.5%. Furthermore, a reaction using lyophilized CRL/OleT(JE)/AldO enzymes at an optimized ratio (5 U/6 μM/30 μM) gave a promising α-olefin yield of 0.53 g/L from 1500 μM (~1 g/L) coconut oil. CONCLUSIONS: The one-pot enzyme cascade was successfully established and applied to prepare high value-added α-olefins from low-cost and renewable TAGs/natural oils. This system is independent of exogenous addition of H(2)O(2), thus not only circumventing the detrimental effect of H(2)O(2) 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.