过表达三角褐指藻苹果酸酶基因提高E.coli脂肪酸合成能力研究

Overexpression of Malic Enzyme Gene from Phaeodactylum tricornutum Promotes Fatty Acids Production in Escherichia coli

为验证三角褐指藻(Phaeodactylum tricornutum)苹果酸酶基因的功能,本研究将PtME1插入pET-30a中得到重组质粒pET30a-PtME1。IPTG诱导后,携带pET30a-PtME1的大肠杆菌BL21(DE3)高效表达一分子量约为72kDa的可溶性重组蛋白。重组蛋白经Ni SephroseTM6Fast Flow系统纯化,酶活力达75.18U/mg。GC-MS分析显示表达PtME1提高了大肠杆菌脂肪酸合成能力,其C14∶0、C16∶0、C18∶1及总脂肪酸含量较对照分别提高了34.8%、69.9%、54.2%和50.2%,C16∶1产量是对照的5.6倍。研究结果表明,NADP依赖型苹果酸酶能为大肠杆菌脂肪酸合成及脂肪酸去饱和提供充足的NADPH,为进一步研究该酶在藻体内的功能奠定了基础。

The marine diatomPhaeodactylum tricornutum has been shown to be a potential producer of biodiesel due to its fast growth,lipid accumulation capability and established genetic tools.Thus,it is possible to genetically manipulate the key genes involved in fatty acids synthesis in microalgae to improve traits to achieve both high lipid and high biomass for industrial production.Malic enzyme（ME）catalyzes the oxidative decarboxylation of L-malate to yield pyruvate,CO2 and NADPH in the presence of a divalent metal ion.It is a widely distributed enzyme involved in different metabolic pathways in prokaryotic and eukaryotic microorganisms.To date,there have been a few studies that have focused on the role of MEs in lipid accumulation,mainly in plants and mammals;however,little is known about the role of these enzymes in microalgae.The full-length cDNA of malic enzyme gene was isolated from P.tricornutum and named as PtME1.It is 1 917 bp in length,encoding 437 amino acids with a molecular mass of 72 kD.In order to verify its function,the recombinant plasmid pET30a-PtME1 was built by inserting PtME1 into pET-30 a.Upon IPTG induction,soluble recombinant protein was obtained with high efficiency in E.coli BL21（DE3）harboring pET30a-PtME1.Recombinant protein was purified by Ni SephroseTM6 Fast purification Flow system and showed a single band about 72 kDa on SDS-PAGE gel.The specific activity of purified enzyme protein was measured,which reached 75.18 U per milligram protein.GC-MS analysis revealed that increased expression of the ME gene leads to increased biosynthesis of fatty acids in the recombinant strain,the contents of C14∶0,C16∶0,C18∶1and total fatty acids were increased by 34.8%,69.9%,54.2%and 50.2%,respectively.The content of C16∶1was increased by 5.6fold compared with that of the control.Research results indicated that over-expression of PtME1 in E.coli has improved the capacity of fatty acid synthesis in Escherichia coli.The NADP-ME in lipid biosynthesis is to supply enough NADPH for both biosynthesis and desaturation of fatty acids in E.coli.These results also laid foundation for further research of the malic enzyme in P.tricornutum.