期刊文献+

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 被引量:2

Overexpression of Malic Enzyme Gene from Phaeodactylum tricornutum Promotes Fatty Acids Production in Escherichia coli
下载PDF
导出
摘要 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.
出处 《中国海洋大学学报(自然科学版)》 CAS CSCD 北大核心 2016年第5期65-69,共5页 Periodical of Ocean University of China
基金 国家重点基础研究发展计划项目(2011CB200901) 国家科技支撑计划项目(2011BAD14B01)资助~~
关键词 三角褐指藻 苹果酸酶 脂肪酸 原核表达 P.tricornutum malic enzyme gene fatty acid prokaryotic expression
  • 相关文献

参考文献25

  • 1Chang G G, Tong L. Structure and function of malic enzymes, a new class of oxidative decarboxylases [J].Biochemistry, 2003, 42 (44) : 12721~12733.
  • 2Edwards G E, Andreo C S. NADP-malic enzyme from plants [J]. Phytochemistry, 1992, 31(6): 1845-1857.
  • 3Drincovich M F, Casati P, Andreo C S. NADP malic enzyme from plants: A ubiquitous enzyme involved in different metabolic path- ways [J]. FEBS Letters, 2001, 490(1): 1 6.
  • 4Casati P, Drincovich M F, Edwards G E, et al. Malate metabolism by NADP-malic enzyme in plant defense [J]. Photosynthesis Re- search, 1999, 61(2): 99-105.
  • 5Smith R G, Gauthier D A, Dennis D T, et al. Malate~and pyru- rate-dependent fatty acid synthesis in leucoplasts from developing castor endosperm [J].Plant Physiology, 1992, 98 (4): 1233- 1238.
  • 6Shearer H L, Turpin D H, Dennis D T. Characterization of NADP-dependent malic enzyme from developing castor oil seed en- dosperm[J].Archives of Biochemistry and Biophysics, 2004, 429 (2) : 134-144.
  • 7Wheeler M C G, Tronconi M A, Drincovich M F, et al. A compre- hensive analysis of the NADP-malie enzyme gene family of Arabi- dopsis [J].Plant Physiology, 2005, 139(1): 39 51.
  • 8Song Y, Wynn J P, Li Y, et al. A pre-genetic study of the iso- forms of malic enzyme associated with lipid accumulation in Mucor circinelloides [J]. Microbiology, 2001, 147(6): 1507-1515.
  • 9Zhang Y, Adams I P, Ratledge C. Malic enzyme: The controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2. 5-fold increase in lipid accumula- tion[J].Microbiology, 2007, 153(7): 2013-2025.
  • 10Wynn J P, bin Abdu[ Hamid A, Ratledge C. The rote of matic enzyme in the regulation of lipid accumulation in filamentous fungi [J]. Microbiology, 1999, 145(8):1911-1917.

二级参考文献13

  • 1刘波,孙艳,刘永红,赵宗保.产油微生物油脂生物合成与代谢调控研究进展[J].微生物学报,2005,45(1):153-156. 被引量:44
  • 2王金霞,谭海东,赵宗保.大肠杆菌K12苹果酸酶的克隆、表达与纯化[J].生物加工过程,2006,4(1):35-38. 被引量:3
  • 3姜岷,谢鑫,许琳,严明.过量表达苹果酸酶对E.coli FMJ39厌氧混合酸发酵的影响[J].中国生物工程杂志,2007,27(1):69-74. 被引量:7
  • 4Dolezal P, Vanacova S. Malic enzymes of Trichomonasvaginalis:two enzyme families,two distinct origins[J]. Gene,2004,329:81-92.
  • 5Wynn JP, Ratledge C. Malic enzyme is a major source ofNADPH for lipids accumulation by Aspergillus nidulans [J].Microbiology,1997,143 : 253-257.
  • 6Li Z,Sun H,Mo X. Overexpression of malic enzyme (ME) ofMucor circinelloides improved lipid accumulation in engineeredRhodotorula glutinis[J]. Appl Microbiol Biotechnol,2012 ,doi :10.1007/s00253-012-4571-5.
  • 7Zhang Y, Ratledge C. Multiple isoforms of malic enzyme inthe oleaginous fungus yMortierella alpine[J\. Mycol Research, 2008,112:725-730.
  • 8Zhang Y, Adams LP,Ratledge C. Malic enzyme:the controllingactivity for lipid production. Overexpression of malic enzymein Mucor circinelloides leads to a 2.5 -fold increase in lipidaccumulation[J], Microbiology,2007,153 : 2013-2025.
  • 9Ratledge C. Fatty acid biosynthesis in microorganisms beingused for single cell oil production [J]. Biochimie, 2004,86 : 807-815.
  • 10Mat-Jan F, Alam KY, Clark DP. Mutants of E. coli deficientin the fermentative lactate dehydmgenase [J]. J Bacteriology ,1989,171(1):342-348.

共引文献9

同被引文献29

引证文献2

二级引证文献5

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部