脂肪酶-P450脂肪酸脱羧酶融合蛋白催化的α烯烃合成

Production of α-alkenes catalyzed by the fused lipase and P450 fatty acid de-carboxylase

α烯烃是一种独特的微生物天然产物.由P450脂肪酸脱羧酶催化的中长链(C_(12)-C_(20))脂肪酸脱羧反应生成的α烯烃因其在生物燃料和生物材料领域的潜在应用前景备受关注.通过将脂肪酶TlL的甘油三酯水解活性与P450脱羧酶OleT_(JE)的脂肪酸脱羧活性偶联,可将油脂原料两步转化为α烯烃.为了提高油脂到α烯烃的转化效率,并降低两种酶的分步制备成本,本研究通过20个氨基酸的linker序列将双酶共价连接形成重组融合蛋白NHis6-OleT_(JE)-linker-TlL(FusC).在大肠杆菌中表达的FusC经镍柱层析纯化后成功实现双功能活性.经气相色谱分析,融合蛋白催化的甘油三酯顺序水解-脱羧偶联反应的产烃率达到31.7%,催化效率相对于双酶游离混合体系明显提升,展现出良好的底物通道效应,具有潜在的实际应用前景.

The irreversibly diminishing reserves and the fluctuating price of crude oil, together with the serious concerns on the global environment and energy security, all suggests an unpleasant future of fossil fuel. Within this context, it is imperative to find alternative eco-friendly fuel with properties that highly mimic fossil fuel. Aliphatic alka（e）nes generated from renewable resources via biosynthetic pathways perfectly suits the criteria and represents the ideal ＂drop-in＂ biofuels. α-Alkenes are special microbial natural products. These biohydrocarbons have attracted much attention due to their potential application as biofuels and biomaterials. By coupling the activity of lipase and P450 decarboxylase, lipids（i.e. triglycerides） can be transformed into α-alkenes via free fatty acid intermediates. To improve the conversion efficiency of lipids to α-alkenes, in this study, four different versions of fusion proteins of the lipase TlL from Thermomyces lanuginosus and the P450 decarboxylase OleTJE from Jeotgalicoccus sp. ATCC 8456, including FusA（His6-TlL-linker-OleTJE）, Fus B（TlL-Linker-OleTJE-His6）, FusC（His6-OleTJE-linker-TlL） and FusD（OleTJE-linker-TlL-His6）, were constructed and comparatively evaluated. The optimal fusion enzyme FusC was able to convert a number of triacylglycerols including trilaurin（C12）, trimyristin（C14） and tripalmitin（C16） into corresponding α-alkenes via the coupled hydrolysis and decarboxylation. The 31.7% yield of 1-tridecene from trimyristin demonstrates good substrate channeling effect. Due to the higher catalytic efficiency compared to the mixed TlL and OleTJE, as well as the simplified enzyme purification and hence the lower cost, this fusion strategy may hold significant potential of application.