Directed Evolution Of P450 Hydroxylase For Production Of 1-alkenes From Fatty Acids

As an ideal next-generation fuels and the important chemical material,the synthetic method of 1-olefin has received extensive attention.Currently,these olefins are mainly accessed via ethylene oligomerization in the industry,which only yields even-numbered terminal olefins.Recently,there has been broad interest in the oxidative decarboxylation of renewable fatty acid substrates for the synthesis of 1-olefins.Moreover,the fatty acids in nature are dominated by even-numbered carbon chain lengths,therefore 1-olefins with odd number of carbon lengths can be directly obtained through oxidative decarboxylation,thereby filling the gap in the production of odd-numbered carbon long olefins in the polyethylene industry.Given the harsh reaction conditions and the pollution risks of the organic synthetic methods,it is urgent to find a environment friendly way to produce 1-olefins.Several groups including us have developed a variety of enzymatic systems for 1-olefin production,however these systems all suffer low enzymatic activities.Based on the mechanistic potential of wild-type P450BS? in the sysnthesis of oxidative decarboxylation and its high activity of hydroxylation towards fatty acids,we aim to change its bais between the rebound process of HO·to the fatty acid radical and the rearrangement of the fatty acid radical for decarboxylation by directed evolution.We selected some potential sites related to the catalytic process near the substrate,and used iterative saturation mutation methods to conduct multiple rounds of screening,successfully transforming this hydroxylase into decarboxylase.When steatic acid was used as the substrate,the TOF of the reaction was as high as 3,744 h-1,which is better than the reaction rate of other reaction systems.And the range of substrates was extended to C6-C18 saturated fatty acids and oleic and linoleic unsaturated fatty acids.In addition,in order to reduce the amount of ketone byproduct produced by the over-oxidation of fatty acids in the H2O2 system,a glucose oxidase(GO)was introduced to the reaction system to ensure a slow,continuous,and mild supply of H2O2.The GO/engineered P450BS? coupled system successfully suppressed the formation of ketones and further improved 1-olefin production.This study opens up a new path for the biocatalytic synthesis of 1-olefins,breaks the catalytic boundary of CYP152 members,and sets a new direction for the field of P450 peroxidase enzyme engineering.