The Study Of Liplase Til And The P450 Decarboxylase Olet_JEFusion Protein To Biosynthesis Aliphatic Alkenes

The irreversibly diminishing reserves,the fluctuating price of crude oil,togther with the serious environmental problems,all these vexing and pressing problems suggested the unpleasant of fossil fuels.Within this context,it is imperative for us to find biofuels with properties highly mimic fossil fuels from renewable resources through biosynthetic or metabolic pathways.Aliphatic alka(e)nes best suited the criteria and proved to be the ideal substitute for fossil fuels,α-alkenes are special microbial natural products.These biohydrocarbons have attracted much attention due to their potential application as biofuels and biomaterials.Herein,the gene of lipase TIL and the P450 decarboxylase OleTjE were fused as fusion protein,overexpressed,purified,and characterized.By coupling the activity of lipase and P450 decarboxylase,lipids can be transformed into a-alkenes via free fatty acid intermediates.In this study,by adjusting the order of fusion components and the location of his-tag,four kinds fusion plasmids were constructed:pET28b-his-TlL-OleTJE、pET28b-TlL-OleTjE-his、pET28b-his-OleTJE-TlL and pET28b-OleTjE-TlL-his;all were successfully overexpressed in E.coli BL21(DE3)under IPTG induction.Their in vitro activities were compared,fusion protein his-OleTjE-TIL and his-TIL-OleTjE worked well,but the other two fusion protein did not show any obvious activity.The fusion protein his-OleTjE-TILand his-TIL-OleTjE catalysis trimyristin(C14)to produce trimyritic acid with yield 21.3%and 19.5%,catalysised trimyristic acid to produce 1-tridecene with yield 62.3%and 15.1%.Fusion protein his-OleTjE-TILwith higher catalysis activity.The optimal fusion enzyme his-OleTjE-TIL was able to convert trimyristin(C14),the 31.7%yield of 1-tridecene from trimyristin demonstrates good substrate channeling effect.The mixed system of two free enzyme converted trimyristin to trimyristic acid with yield 3.3±0.3%,FusC could convert trimyristic acid with yeild14.8± 1.9%;in the mixed system,the convert yield of 1-tridecene was 29.9±3.3%,FusC was 61.2±3.9%.Due to the substrate channeling effect,the two mixed enzyme system could produce 1-tridecenewith yeild 14.7±1.1/%,FusC was 31.7±2.9%.As report before,the two steps would take 9 hours to produce 1-tridecene 32.7± 1.4%,however,FusC only need to take 3 hours.The optimal fusion enzyme his-OleTjE-TIL was also able to convert other triacylglycerols including trilaurin(C12)and tripalmitin(C16)into corresponding a-alkenes via the coupled hydrolysis and decarboxylation.The mixed system of two enzyme could convert trilaurin(C12)and tripalmitin(C16)into corresponding a-alkenes 18.3±2.1%14.7±1.1%and 8.46±0.3%,but FusC was 28.1±4.1%31.7±2.9%and 26.6±0.4%.Due to the higher catalytic efficiency compared to the mixed TIL and OleTjE,as well as the simplified enzyme purification and hence the lower cost,this fusion strategy may hold great potential of application.To save the cost of purification,in this study,fusion proteins gene TIL-OleTjE with codon optimization were constructed and cloned into genome of Pichia Pastoris.Whereas the mutant did not expressed the fusion protein,even with the successfully secretory expression of lipase TIL,its transcription and expression are being investigated.