解脂耶氏酵母(Yarrowia lipolytica)ATCC30162脂肪酶基因Yllip1和Yllip2的结构及表达特征

以目前报道油脂产量最高的解脂耶氏酵母菌株(Yarrowia lipolytica) ATCC 30162为对象,采用逆转录PCR扩增到脂肪酶编码基因Yllip1和Yllip2,编码产物分别为816和549个氨基酸。保守结构域预测表明,Yllip1包含Patatin类磷脂酶和功能未知的DUF3336结构域,而Yllip2包含lipase＿3类脂肪酶结构域,且这两个蛋白都具有1～4个跨膜区域。与不同物种来源的脂肪酶同源蛋白的多序列比对表明Yllip1和Yllip2分别包含8和6个保守区域,这些生物信息学分析表明这两个来源于解脂耶氏酵母的脂肪酶作用底物可能分别为细胞内膜磷脂和酰基甘油酯。荧光定量PCR分析表明:培养基中添加油酸在短期内(6 h)诱导了这两个脂肪酶基因Yllip1和Yllip2的显著上调表达,表明它们可能参与了酵母分解利用油酸的生化过程。     

解脂耶氏酵母脂肪酶LIP4和LIP5在毕赤酵母中的异源表达及酶学性质

【目的】克隆解脂耶氏酵母(Yarrowia lipolytica)脂肪酶LIP4和LIP5的cDNA序列,研究其基因结构,并实现其在毕赤酵母中的功能表达,以探讨其酶学性质。【方法】利用反转录PCR首次扩增LIP4和LIP5的编码基因,用SignalP 3.0分析其基因序列,然后分别构建胞内表达载体pPIC3.5K-Lip4、pPIC3.5K-Lip5和胞外表达载体pPIC9K-Lip4、pPIC9K-Lip5,将其转入毕赤酵母GS115中表达,以NTA树脂纯化酶蛋白,研究其酶学性质。【结果】cDNA序列测序结果显示两者均不含内含子,酶蛋白的氨基酸序列中含有典型脂肪酶的活性三联体结构和五肽保守区;酶学性质研究表明,两者的最适底物均为癸酸(C8)对硝基苯酚酯,最适pH为7.0,最适温度为40℃,但LIP4对pH和温度更敏感;两者均能被Ca2+激活,且LIP5还能为Mg2+激活,但均被Hg2+、乙二胺四乙酸(EDTA)和苯甲基磺酰氟(PMSF)强烈抑制。【结论】首次克隆了解脂耶氏酵母脂肪酶LIP4和LIP5编码基因,实现了其在毕赤酵母中的活性表达,并初步研究了其酶学性质,为上述脂肪酶的应用及进一步深入研究解脂耶氏酵母脂肪酶家族奠定了基础。     

解脂耶氏酵母tsr1突变体的构建

将解脂耶氏酵母与蛋白质分泌有关的TSR1基因编码区部分缺失的DNA片段转化一株解脂耶氏酵母,通过体内同源重组,部分缺失的外源tsr1片段取代了酵母染色体上的正常的TSR1基因,从而获得tsr1的转化子。Southern杂交结果表明,用该法成功地构建了tsr1突变体,这为进一步研究解脂耶氏酵母TSR1基因的功能奠定了基础。     

黑曲霉中内切菊粉酶基因及其全合成基因在解脂耶氏酵母中表达的比较

利用酵母密码子偏爱性将黑曲霉(Aspergillus niger)中的内切菊粉酶(Endoinu linase)基因通过基因全合成的方式合成为酵母密码子偏爱性的内切菊粉酶基因。然后将原始和全合成的内切菊粉酶基因克隆到解脂耶氏酵母表达载体PINA1296上,得重组解脂耶氏酵母表达载体pHBM2020、pHBM2021,将两种质粒分别转化解脂耶氏酵母(Yarrowia lipolytica)CLIB725,筛选得到重组解脂耶氏酵母CLIB725(pHBM2020)、CLIB725(pHBM2021),将两种重组酵母摇瓶培养,经SDS-PAGE、测酶活检测表明两种基因在解脂耶氏酵母中都有表达,全合成菊粉酶比原始菊粉酶酶活要高。     

毕氏海蓬子SbDREB基因的克隆与表达分析研究

以毕氏海蓬子的基因组为模板,通过PCR技术扩增到一个编码DREB蛋白AP2保守结构域的基因片段;根据该片段序列设计引物,以毕氏海蓬子经NaCl处理的植株肉质茎cDNA为模板,应用RACE技术获得该基因的cDNA全长,命名为SbDREB(GenBank登录号:JF894301)。SbDREB基因cDNA全长1206bp,包含一个编码284个氨基酸的完整开放阅读框。对氨基酸序列比对分析表明,该蛋白在靠近N端具有典型的AP2/EREBP保守结构域,且该结构域与一些高等植物DREB类转录因子的AP2区域具有高度同源性。进化树分析表明SbDREB属于DREB亚家族中的A-6亚族。实时荧光定量PCR结果显示:干旱、高盐和ABA能够诱导其表达,而低温则使其表达下调,表明该基因在毕氏海蓬子植株对干旱、盐和低温等非生物胁迫的应答中起作用。     

解脂耶氏酵母TSR1基因的定点诱变

对解脂耶氏酵母与蛋白质分泌有关的TSR1基因进行寡核苷酸介导的定点诱变,限制性内切酶切割的拼接,得到了该基因的一系列缺失突变体。这为进一步研究TSR1基因不同结构域的功能奠定了基础。     

解脂耶氏酵母脂肪酶基因lip1的克隆、密码子优化及功能表达

摘要：【目的】克隆解脂耶氏酵母（Yarrawia lipolytica）脂肪酶基因lip1,并通过密码子优化,首次实现其在毕赤酵母(Pichia pastoris)中的诱导型和组成型表达。【方法】通过PCR扩增Y. lipolytica脂肪酶基因lip1,根据P. pastoris密码子偏爱性,运用重叠延伸PCR合成改造后基因MLip1,将其分别克隆至诱导型分泌载体pPIC9K和新构建的组成型分泌载体pGAP9K上,电转至P. pastoris GS115中,G418抗性筛选得到高拷贝转化重组子,摇瓶发酵     

解脂耶氏酵母新型表达载体构建及癌基因rho在其中的表达

为了简化解脂耶氏酵母表达载体构建过程、消除抗生素污染,将mel基因(编码酪氨酸酶)作为新型报告基因用于构建新型酵母表达载体,利用组装PCR人工合成基因mel,并用重叠PCR将其与同源组成型强启动子p TEF、分泌性信号肽XPR2pre及强终止区LIP2t融合,构建新型胞外及胞内表达载体,并利用其在解脂耶氏酵母野生菌株中表达人源癌基因rho.成功获得mel全基因并将其与启动子、信号肽和终止区融合,得到融合片段TXML,用其替换原有表达载体的筛选标记基因ura3d4,构建得到新型胞外及胞内表达载体pINA1297-M和pINA1297-a-M,转化后的酵母阳性转化子性状明显,随后利用此新型表达系统获得可溶性异源蛋白Rho.首次实现了将mel作为一种便捷、价廉、无污染的新型筛选标记基因运用于非常规酵母表达系统中,更为mel在其它真核表达系统中的运用奠定了技术基础;获得的可溶性Rho蛋白可为研究其性质、结构、功能及与Rho癌基因家族其它成员的相互作用提供条件.     

牦牛MT-I/-Ⅱ cDNA分子克隆及其蛋白质结构分析

利用基因特异引物YMTSP1和YMTSP2,通过RT-PCR从牦牛肝脏组织RNA中克隆出了牦牛MT-Ⅰ(Genbank Accession NoAY513744)和MT-Ⅱ(Genbank Accession NoAY513745)基因编码区全长.将牦牛MT-Ⅰ和MT-Ⅱ cDNA序列在CBI上进行同源性搜索发现,牦牛MT-Ⅰ/-Ⅱ编码区序列在不同哺乳动物中相当保守.牦牛MT-Ⅰ和MT-Ⅱ编码的MT-Ⅰ和MT-Ⅱ蛋白分别由61个氨基酸组成,其具有保守的短肽结构如C-X-C,C-C-X-C-C,C-X-X-C等,其决定MT蛋白分子的整个三维结构,在分子进化上十分保守.同时对牦牛MT的疏水性和跨膜区分析表明,牦牛MT蛋白可能不存在跨膜区,也不存在信号肽,是1种非分泌蛋白.并通过同源比较模建,预测和构建了牦牛MT-Ⅰ和MT-Ⅱ蛋白的分子空间结构,表明牦牛MT-Ⅰ和MT-Ⅱ由α-和β-两个结构域组成,在α-结构域含有5个Cys短肽结构,β-结构域有4个Cys短肽结构,且2个结构域由保守的三肽序列KKS相连.     

解脂耶氏酵母TSR1基因的定点诱变*

对解脂耶氏酵母与蛋白质分泌有关的TSR1基因进行寡核苷酸介导的定点诱变 ,限制性内切酶切割和拼接 ,得到了该基因的一系列缺失突变体。这为进一步研究TSR1基因不同结构域的功能奠定了基础。     

YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid.     

SOA genes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeast Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene ( LIP2 ) was monitored using a LIP2 -β-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol.     

Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica

AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. METHODS AND RESULTS: The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. SIGNIFICANCE AND IMPACT OF THE STUDY: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. CONCLUSIONS: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.     

Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode an extracellular lipase. The growth of a Deltalip2 strain on triglycerides as sole carbon source suggest an alternative pathway for triglycerides utilisation in this yeast. Here, we describe the isolation and the characterisation of the LIP7 and LIP8 genes which were found to encode a 366 and a 371-amino acid precursor protein, respectively. These proteins which belong to the triacylglycerol hydrolase family (EC 3.1.1.3) presented a high homology with the extracellular lipase CdLIP2 and CdLIP3 from Candida deformans. The physiological function of the lipase isoenzymes was investigated by creating single and multi-disrupted strains. Lip7p and Lip8p were found to correspond to active secreted lipases. The lack of lipase production in a Deltalip2 Deltalip7 Deltalip8 strain suggest that no additional extracellular lipase remains to be discovered in Y. lipolytica. The substrate specificity towards synthetic ester molecules indicates that Lip7p presented a maximum activity centred on caproate (C6) while that of Lip8p is in caprate (C10).     

Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54-S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins.     

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).     

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.     

解脂耶氏酵母脂肪酶Lip2的表面展示及其酶学性质

表面展示酶作为全细胞催化剂具备诸如能提高酶的稳定性、省去纯化过程、节约成本等优点。脂肪酶是应用最为广泛的工业酶之一。本研究利用酿酒酵母细胞壁蛋白Cwp2作为锚定蛋白,将解脂耶氏酵母脂肪酶Lip2展示在酿酒酵母细胞表面,以制备脂肪酶全细胞催化剂。Lip2被融合到Cwp2的N端,Cwp2通过其C端的GPI锚定信号共价结合到细胞壁上。表面展示的Lip2可以水解三丁酸甘油酯及对硝基苯酚辛酸酯(pNPC),其pNPC水解酶活达到4.6U/g干细胞。作为全细胞催化剂,表面展示的Lip2具备良好的催化特征,其最适温度为40°C,最适pH为8.0,同时还具备良好的有机溶剂稳定性。     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

Yarrowia lipolytica cells mutant for the PEX24 gene encoding a peroxisomal membrane peroxin mislocalize peroxisomal proteins and accumulate membrane structures containing both peroxisomal matrix and membrane proteins

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. Functional complementation of the oleic acid-nonutilizing strain mut1-1 of the yeast Yarrowia lipolytica has identified the novel gene, PEX24. PEX24 encodes Pex24p, a protein of 550 amino acids (61,100 Da). Pex24p is an integral membrane protein of peroxisomes that exhibits high sequence homology to two hypothetical proteins encoded by the open reading frames YHR150W and YDR479C of the Saccharomyces cerevisiae genome. Pex24p is detectable in wild-type cells grown in glucose-containing medium, and its levels are significantly increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex24 mutants are compromised in the targeting of both matrix and membrane proteins to peroxisomes. Although pex24 mutants fail to assemble functional peroxisomes, they do harbor membrane structures that contain subsets of peroxisomal proteins.