Fusing the decarboxylase OleT JE and the reductase domain of P450BM3 creates a self-sufficient protein, OleT-BM3R, which is able to efficiently catalyze oxidative decarboxylation of carboxylic acids into linear α-olefins (LAOs) under mild aqueous conditions using O 2 as the oxidant and NADPH as the electron donor. The compatible electron transfer system installed in the fusion protein not only eliminates the need for auxiliary redox partners, but also results in boosted decarboxylation reactivity and broad substrate scope. Coupled with the phosphite dehydrogenasebased NADPH regeneration system, this enzymatic reaction proceeds with improved product titers of up to 2.51 g L −1 and volumetric productivities of up to 209.2 mg L −1 h −1 at low catalyst loadings (∼0.02 mol %). With its stability and scalability, this self-sufficient biocatalyst offers a nature-friendly approach to deliver LAOs.
Inspired by the catalytic mechanism of cytochrome P450 enzymes, we have evolved a fatty acid hydroxylase, P450 BSβ , into a decarboxylase after four rounds of saturation mutagenesis that now predominantly converts fatty acids to 1-alkenes. The engineered enzyme directly utilizes inexpensive H 2 O 2 to oxidatively decarboxylate C6−C18 saturated fatty acids as well as plant oil-derived unsaturated fatty acids, displaying high reactivity (e.g., 3,700 h −1 of turnover frequency for myristic acid). With in situ H 2 O 2 supplied by glucose oxidase, this enzyme delivers 1-alkenes in a more productive and scalable fashion (up to 99% yield). Our directed evolution efforts present a strategy for developing new fatty acid decarboxylase for downstream biocatalytic applications.
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