【作者】 牛艳；

【导师】 孔健；

【作者基本信息】 山东大学 ， 微生物， 2008， 博士

【摘要】 多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)是指含有两个或两个以上双键且碳链长为16-22个碳原子的直链脂肪酸,包括二十碳五烯酸(EPA),二十二碳六烯酸(DHA)等,其中EPA/DHA具有重要的生理作用如:营养强化,调控脂类代谢,预防和治疗动脉粥样硬化和心血管疾病,调节机体的免疫功能,调节视力和大脑发育,调节中枢神经系统功能,还可以开发为抗肿瘤药物。目前,深海鱼油是EPA及DHA等PUFA的重要商业来源,然而仅仅依靠原有的鱼油资源已经无法满足日益扩大的市场需求。另外,研究发现,鱼类并不是PUFA的真正生产者,而是通过吞食富含PUFA的海洋微藻后在体内积累。海洋微藻是EPA/DHA的初级生产者,而且从微藻中提取PUFA比生产鱼油工艺简单,产品没有鱼腥味,胆固醇含量低。因此,利用海洋微藻生产PUFA成为获取EPA和DHA的一种新的手段。但是海洋微藻多为自养藻,生长缓慢,细胞得率低,培养过程中易染杂菌,另外对微藻的规模化培养及细胞改良等方面的研究尚处于实验室阶段,所以利用微藻大规模提取生产EPA/DHA来满足社会需求还不现实。PUFA的生物合成始于C18-PUFA:LA和A LA,通过一系列的脂肪酸脱氢酶(fatty acid desaturase,FAD)的脱氢作用和脂肪酸碳链延伸酶(fatty aicdelongase,FAE)的碳链延伸作用逐步完成。FAD催化的脱氢反应是将脂肪酸链中的C-C转化为C=C,每个FAD都在酰基链的特定位置引入双键;FAE是参与C18、C22-PUFA的碳链延伸反应的一类延伸酶复合体,它催化供体(乙酰辅酶A或丙二酰辅酶A)的两个碳原子引入PUFA碳链中增加其碳链长度。随着对PUFA生物合成途径及调控机制研究的深入,从分子水平上研究微藻PUFA合成的影响因素,通过对PUFA合成途径关键酶基因的克隆,实现基因的异体高效表达,以期得到大量的PUFA,为解决EPA/DHA资源,降低生产成本,提供一条新途径。而且FAD和FAE在植物基因工程,食品工程和发酵工程等领域的广阔应用前景是毋庸置疑的,在上述领域的应用也将进一步促进对脂肪酸合成途径的认识和研究。本论文实验材料绿色巴夫藻(Pavlova viridis)为自养微藻,EPA和DHA含量丰富,分别占总脂含量的16%和9%,是研究脂肪酸合成途径中相关脱氢酶和延伸酶很好的实验材料。本文的工作和研究成果如下:1.研究了培养温度、光照强度对P.viridis生长及其EPA/DHA含量的影响,优化培养条件。结果表明,P.viridis的生长温度范围较广,在16℃～25℃均可生长,但是低温(18℃)有利于藻体内EPA和DHA的积累,经气相色谱分析此条件下的EPA和DHA含量分别为16.25%,9.226%,较25℃条件下提高了12.45%和8.226%。由此证实:尽管较高的温度有利于P.viridis的生长,但此时不饱和脂肪酸含量较低。P.viridis为自养生物,当光照强度较弱时,该藻生长停滞,此时的EPA和DHA含量较低;随着光照强度的增加,藻体生长加快,同时EPA和DHA的含量也增加到16.62%和6.79%。通过GC/MS测定18℃光照培养条件下P.viridis的脂肪酸组成,发现P.viridis不饱和脂肪酸种类齐全,含有多种不饱和脂肪酸,是克隆脱氢酶基因和延伸酶基因很好的实验材料。2.总RNA的质量直接影响到所建文库的优劣,本文在对比了Trizol试剂法,异硫氰酸胍一步法,CTAB法等方法的基础上,摸索出一种适用于P.viridis总RNA提取的有效方法—CTAB和酸酚相结合的改良CTAB法。此方法可有效去除RNA提取过程中多糖和多酚类物质的干扰,所得RNA质量较高。利用改良的CTAB法提取P.viridis总RNA,构建其cDNA文库,所建cDNA文库滴度为6×10~6pfu/mL,重组率达到98%,插入片段在250 bp—2.0 kbp之间,表明该文库达到建库要求。通过表达序列标签(expressed sequence tags,ESTs)技术对P.viridis cDNA文库进行随机序列扩增和测序,将所得200条EST序列与GenBank中已报道基因进行BLAST同源性比对,获得了许多功能基因的遗传信息,为克隆基因全长和进一步开发微藻的新功能基因奠定了基础。经过比对发现,在这200条ESTs序列中参与蛋白质合成的基因和参与能量代谢(包括光合作用和呼吸作用)的基因含量比较高,分别占41%和20%。但是通过EST技术,没有筛选到脱氢酶基因或者延伸酶基因序列,说明ESTs方法不适用于丰度较低基因的筛选。3.利用SMART RACE技术首次克隆了P.viridis C20-延伸酶基因(elkj)。利用绿色荧光蛋白(green fluorescent protein,GFP)作为elkj表达的报告基因,将克隆的C20-延伸酶基因(elkj)和GFP基因融合,实现了elkj在Escherichia coli中的表达,证实elkj编码的蛋白为膜整合蛋白,并且具有C20-延伸酶活性。elkj基因全长945 bp,编码314个氨基酸,推测蛋白质大小为34.5 kDa,经TMHMM疏水性预测分析,ELKJ蛋白七次穿膜,C末端位于胞内且具有双赖氨酸结构的内质网滞留信号。经Southern blot分析,elkj基因在P.viridis基因组中为单拷贝。C20-延伸酶催化EPA合成DPA,是从EPA到DHA合成的关键步骤。经过气相色谱分析大肠杆菌脂肪酸甲酯组成,显示重组菌E.coli DE3/pwalEL具有催化EPA产生DPA的C20-延伸酶酶活,确认了在P.viridis中从EPA到DHA的转化需要两步合成(two step conversion)。实验过程中发现,当elkj基因在大肠杆菌中表达时,重组菌E.coliDE3/pwalEL生长较对照菌株主E.coli DE3/pWaldo-gfpe弱,表明ELKJ的表达对宿主细胞产生了毒性效应,导致表达困难,无法得到目的蛋白产物。因此,将ELKJ蛋白和GFP蛋白融合表达,通过观察GFP的荧光,反映ELKJ的表达情况。结果显示,在激光共聚焦显微镜下观察到重组菌E.coli DE3/pwalEL发出绿色荧光,说明ELKJ-GFP融合蛋白正确表达,ELKJ蛋白部分插入细胞膜中,融合蛋白未形成包涵体;通过测定全细胞荧光信号,对比不同条件下ELKJ的表达量,确定ELKJ了蛋白的最优表达条件。上述实验结果说明,利用GFP的自发荧光可以作为检测膜蛋白各种表达条件的指标,为膜蛋白的研究提供了一个有效的方法,为真核生物膜蛋白的研究奠定基础。4.首次克隆了P.viridis的Δ4、Δ5脱氢酶全长基因序列,包括外显子,内含子以及基因的启动子序列。海洋微藻的分子生物学起步较晚,可供参考的遗传信息和操作工具很少,在克隆P.viridis脱氢酶基因时进行了广泛探索,对构建P.viridis cDNA文库进行大量筛选后,没有得到脱氢酶基因序列,改用SMARTRACE技术以及SEFA PCR技术成功扩增了Δ4-脱氢酶基因(pkjDes4)和Δ5-脱氢酶基因(pkjDes5)。pkjDes4其开放阅读框全长1440 bp,编码479个氨基酸,提交GenBank获得accession no.EF486526;pkjDes5其开放阅读框全长1278 bp,编码425个氨基酸,提交GenBank获得accession no.EF486527。经过Southern blot分析,pkjDes4和pkjDes5同C20-elongase基因一样,在P.viridis基因组中均为单拷贝基因,证实PUFA合成途径中的相关酶基因在基因组中的拷贝数均较低,利用大量筛选文库得到目的基因的方法比较困难。5.利用从P.viridis中克隆的C20-elongase基因elkj和Δ4-desaturase基因pkjDes4,完成了elkj在Pachia pastoris中的表达及功能验证,并构建了elkj和pkjDes4在P.pastoris中的共表达载体,为利用现代生物技术生产DHA奠定基础。利用pPIC3.5K整合型载体,构建elkj在P.pastoris中的表达载体pPEL,筛选到高拷贝整合宿主重组菌株G3,经气相色谱分析P.pastoris GS115/pPEL脂肪酸甲酯成分,显示重组菌G3具有催化EPA产生DPA的C20-elongase酶活,完成了elkj在P.pastoris中的功能验证,为利用P.pastoris构建DHA的体外合成途径奠定基础。6.以乳酸乳球菌表达载体pNZ8148为骨架在C末端融合GFP,首次构建了一个乳酸乳球菌表达载体pKj-gfp,并利用该载体,实现了ELKJ在Lactococcus lactis中的过量表达。由于NICE系统(NIsin controlled expressionsystem)的严谨性及L.lactis作为宿主表达膜蛋白的优越性,NICE系统被发展为可控制的膜蛋白质表达的理想系统。通过激光共聚焦显微镜观察nisin(乳链菌肽)诱导后L.lactis NZ9000/pKj-el绿色荧光,未发现不发荧光细胞,说明质粒在L.lactis中稳定性。利用GFP荧光检测ELKJ的表达过程,大大简化了膜蛋白表达菌株筛选和纯化的过程。更多还原

【Abstract】 Polyunsaturated fatty acids(PUFAs)are unbranched fatty acids containing more than one double bond.They are essential components of human health including EPA, DHA and so on.And EPA/DHA are associated with various physiological and pathophysiological processes,thereby affecting human health.Clinical studies show that DHA is essential for the growth and development of the brain in infants and for maintenance of normal brain function in adults.Cell growth and division,platelet aggregation,inflammatory responses,hemorrhage,vasoconstriction and vasodilation and immune functions are associated with EPA/DHA.Studies have shown their role in prevention and treatment of coronary heart disease,hypertension,type 2 diabetes, arthritis,cancer and other inflammatory and autoimmune disorders.At present,fish oil is the major source of EPA/DHA.However,the demand of EPA/DHA is constantly increasing while the fish sources producing them are depleting.Marine micralgae,the primary producers of PUFAs,are being explored as an alternative source for yielding these essential elements of health.Desaturases and elongase systems are critical enzymes involved in biosynthesis of PUFAs and occur in most living cells.Desaturases are iron-containing enzymes that introduce a double bond in a specific position counted from the carbonyl end of the fatty acids,aerobically.The elongase systems are responsible for the addition of two carbon units to the carboxyl end of a fatty acid chain.And the genes of desaturases and elongase systems have important biotechnological appeal from genetic engineering point of view.The identification and prospecting of these genes becomes a novel method for enhanced PUFA production.Pavlova viridis,a marine microalga,is rich in PUFAs,which represents an attractive production system for PUFAs such as EPA and DHA.In this dissertation,P. viridis has been used as a useful source for cloning fatty acid desaturase and elongases genes.The results and significance of this study is summarized as follows:1.Optimization of P.viridis growth conditions.P.viridis contains plenty of polyunsaturated fatty acids(PUFA).In this dissertation,we’ve studied the impacts of environmental factors on growth of P. viridis,and investigated those factors’ effects on content of EPA/DHA in total fatty acids through gas chromatograph(GC)analysis.Through mensurating the curve of growth under different environmental factors,it was indicated that the growth of P. viridis under light was visible better than which in dark,the consistency of cell reached 2.49×10~6 cells/mL after 12 days under light.However,the cell consistency was only 1.60×10~6 cells/mL after 12 days in dark.The results also indicated that high light density was helpful with accumulation of total fatty acids,especially,could increased the content of EPA/DHA.Meanwhile,the growth of P.viridis under 25℃was visible better than which under 18℃and 16℃.However,the contents of EPA/DHA declined while the growth temperature rised,which showed that lower growth temperature was helpful to the accumulation of EPA/DHA.We’ve invesgated the fatty acid composition of P.viridis through gas chromatograph mass spectra(GC/MS)analysis.The results showed there were various kind of fatty acids in P.viridis,making it an ideal candidate for studying the PUFA synthetic pathway.2.Improvation of RNA extraction and construction of P.viridis cDNA library and EST analysis.High quality RNA is critical in molecular manipulation such as the cDNA synthesis,cDNA library construction and RACE.However,P.viridis is rich in polysacchrides and polyphenols,which could interfere with the extraction of total RNA from cells.In this study,an improved CTAB method was constructed based on the traditional methods of RNA extract by Trizol agent and acid-guanidine-phenol-chloroform method.The method could effectively eliminate the interferences of polysacchrides and polyphenols.Upon the method mentioned above,total RNA was extracted from P.virids cells. Poly~+A RNA was then isolated and purified and the mRNA template was reversely transcriptionized to a single strand cDNA with modified Oligo(dT)primer using SMART(Switching Mechanism At 5’ end of RNA Transcript)technology. Consequently,the synthesized ds cDNA was ligated with vectorλTriplex2.The recombined vectors were packaged in vitro.The titer of the unamplified constructed cDNA library was 6×10~6 pfu/mL and the recombination rate was 98%.Meanwhile, the length of the inserted DNA fragment was between 500 bp and 15 kb.All of these suggested that the library could be suitable for screening low abundant mRNA for cDNA clones.The results also indicated that the improved CTAB method was appropriate for RNA extraction of microalage.The expressed sequence tags(ESTs)was a rapid method which can be used to obtain the new genes from cDNA library.The clones containing cDNA longer than 500 bp were selected for sequencing.200 ESTs were obtained by sequencing from the 5’ end of the cDNA clones.Then these ESTs were compared with sequences in the GenBank data of NCBI using Blast.Partial sequence of some functional genes was identified,including atp I and atpH genes for subunit of ATPase complex,rRNA gene, carboxypeptidase A,cytochrome b,chloroplast photosystemⅡ12 kD extrinsic protein and so on.The results provided the basis for further study of P.viridis gene expression.3.Identification,characterization and overexpression of a new C20-elongase gene elkj.A novel elongase gene was isolated for the first time from P.viridis via reverse transcriptase-polymerase chain reaction(RT-PCR)using the primers designed from conserved motifs and 5’/3’ RACE,named as elkj(GenBank accession No.486525). Sequence analysis indicated that elkj gene was with high identity with a functionally characterized C20-elongase of P.lutheri.And the elkj gene was 945 bp in length, encoding a protein of 314 amino acids with an estimated molecular weight of 34 kDa. The predicted protein ELKj contained seven transmembrane domains with its C-terminal in the cytoplasm and located in the endoplasmic reticulum.The heterologous expression in E.coli demonstrated that elkj encoded a C20-elongase that mediated the elongation of EPA into docosapentaenoic acid(DPA, 22:5n-3).Therefore,the identification and characterization of the elkj gene also confirmed the two-step conversion existed in marine microalga.When the elkj gene was overexpressed heterologously,the recombinant strains grew much slowly compared with the host strain,indicating that the ELKJ protein as a membrane protein brought toxic effects to E.coli.Thus,the ELKJ protein was fused with green fluorescent protein(GFP),to monitor the overexpression,solubilization and purification of the ELKJ-GFP protein,instead of laborious and time-consuming process.4.Identification of two novel desaturase genes:the△4-desaturase gene pkjDes4 and the△5-desaturase gene pkjDes5,respectively.The molecular biology of marine microalga hasn’t been developed until recent years and little was known about their genetic manipulation.The efforts of selecting P. viridis cDNA library by probe bloting were made to clone the desaturase genes involved in the biosynthesis of PUFA but with no progress.Thus,two partial cDNA sequences with identity to△4-and△5-desaturase genes were obtained via RT-PCR using primers designed according to the conserved histidine motifs of previously reported desaturase genes.And then the entire sequences of the two desaturase genes were isolated using SMART RACE together with SEFA PCR(self-formed adaptor PCR).Sequence analysis revealed that the△4-desaturase gene pkjDes4 was 1440 bp in length,encoding a protein with 449 amino acids(GenBank accession No.486526) and the△5-desaturase gene pkjDes5 1278 bp in length,with 425 amino acids (GenBank accession No.486527). 5.Co-expression of elkj with pkjDes4 in Pichia pastoris.In marine microalgae,the conversion of EPA to DHA involved a C20-elongase together with a△4-desaturase,which was considered as the efficient two-step conversion.In this study,the C20-elongase gene elkj was ligated with the expression vector pPIC3.5K and integrated into P.pastoris.Functional analysis through GC chromatograph indicated that the recombinant strain had the ability of elongating EPA into DPA.Then,the△4-desaturase pkjDEs4 was also ligated into the pPIC3.5K vector, which could be used to reconstructing the DHA synthesis pathway in P.pastoris.6.Construction of a novel membrane protein expression system in Lactococcus lactis.Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation.L. lactis has several properties that are ideal for enhanced expression of eukaryotic membrane proteins.In this study,a novel lactococcal vector was constructed,by inserting a green fluorescent protein sequence into the vector pNZ8148,which is a commonly used vector in the NICE system(NIsin Controlled gene Expression system).The resulting vector pKj-gfp was applied to the overexpression of an elongase-green fluorescent protein fusion in L.lactis.During expression,the fusion was monitored through in-gel fluorescence and no degradation was observed. Furthermore,the targeted protein was exclusively directed into the cytoplasmic membrane,and accounted for approximately 15%of the total membrane protein. These results indicated that the lactococcal system can be used for the overproduction of eukaryotic membrane protein.The identification of different elonagase and desaturase genes involved in the EPA/DHA production has important biotechnological applications.These genes can be used in the production of PUFA-rich transgenic plant oils for therapeutic and prophylactic use.Also,advances in understanding gene regulation in PUFA biosynthesis will also impact the single-cell oil industry,such that growth conditions of the microalgae can be manipulated to enhance the production of EPA/DHA.This in turn will impact the marine fish-farming industry which depends on microalgae for enhancing the levels of PUFAs in fish.All the above will eventually afford the public an economical source of balanced n-3/n-6 PUFA-enriched oils that will greatly impact general health and nutrition in the near future.更多还原