Designing Of Fusion Protein OleT-BM3R And Its Application In Synthesis Of Linear α-olefins Utilizing Fatty Acids

Linear a-olefins(LAOs)are widely regarded as the next-generation biofuel due to its superior physical and chemical properties.They are also key feedstock chemicals for producing surfactants,detergents and polymers,etc.Currently,producing LAOs strongly depends on ethylene oligomerization that only produces even-numbered linear a-olefins.Considering decreasing fossil fuel reserves and global demand for renewable energy and green chemistry,it is of great importance to pursue renewable route to produce olefins.Currently,C(n-i)LAOs can be produced form natural-abundant and renewable fatty acids through metal-catalyzed decabonylation.While impressive,these chemical approaches usually suffer from high reaction temperature(T≥110℃).In situ distillation of products is required to maintain acceptable a-selectivity.They also require stoichiometric anhydrides for activation of the FAs.Developing eco-friendly methods for LAO production is of urgency.In 2011,it was reported that a novel P450 enzyme called OleTJE from Jeotgalicoccus sp.ATCC 8456 can catalyzed the oxidative decarboxylation of fatty acids to produce the corresponding LAOs using H2O2 as an oxidant.Millimolar concentrations of H2O2 will decativate the OleTJE,and H2O2-dependent catalytic systems typically have lower catalytic efficiency and limited substrate scope.Recent studies have shown that OleTJE can be coupled with other redox systems.These coupling systems catalyze the decarboxylation of fatty acids using O2 as an oxidant,and NAD(P)H as an electron donor.However,these system suffer from relatively lower catalytic efficiency,limited substrate scope,and relatively complicated experimental operation,which limit their application of olefin synthesis.Based on structural alignment,OleTJE was fused with the reductase domain of CYP102A 1(P450BM3),resulting in a novel fusion protein,OleT-BM3R.The activity assay showed that in this fusion protein,OleTJE is compatible with the reductase domain of CYP102A1.It can catalyze the oxidation decarboxylation of C4:0-C20:0 fatty acids with high conversion at room temperature in aqueous solution using NADPH as an electron donor and O2 as an oxidant.This oxidative decarboxylation system can also be coupled with the phosphite dehydrogenase-based NADPH regeneration system using inexpensive sodium phosphite as the final electron donor,NADPH.Compared with other OleTJE catalytic systems,OleT-BM3R exhibits better catalytic performance:a)OleT-BM3R can catalyze the decarboxylation of fatty acids into a-olefins without using H2O2,which makes it more biocompatible;b)The reactivity,substrate scopes and protein stability of fusion protein OleT-BM3R are improved:OleT-BM3R can effectively catalyze the saturated C4:0-C20:0 fatty acids,and its activity has no significant loss after 24 hours incubation at 37℃.Furthermore,lyophilized OleT-BM3R can be stored at-20℃ for several months without significant acitivity loss;c)When coupled with the NADPH regeneration system,OleT-BM3R can carry out the reaction using reduced concentration of cofactor,NADPH.Cell free lysate reaction can be carried out easily without requiring additional NADPH and protein purification,greatly reducing the cost of the reaction;d)1 gram of C18:0 fatty acid can be transformed to corresponding a-olefin with 60%conversion,demonstrating a good scability of the catalytic system.These results show that this fusion protein can be conventionally stored and transported as pre-catalyst product for in vitro synthesis of LAOs as well as a promising catalyst for biosynthesis of a-olefins in vivo.