三角褐指藻D5-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

D5-脂肪酸脱氢酶是合成花生四烯酸的关键酶。根据已报道的D 5-脂肪酸脱氢酶基因设计引物, 分别从三角褐指藻基因组DNA和总cDNA中扩增得到1520 bp和1410 bp的特异片段, 序列分析结果显示, 结构基因中含有一个大小为110 bp的内含子, 这是国内外首次报道。将D5-脂肪酸脱氢酶基因亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0中, 在大肠杆菌中筛选到含有目的片段的重组质粒pYPTD5, 用电击转化的方法将重组质粒pYPTD5转化到营养缺陷型酿酒酵母菌株INVSc1中, 在缺省培养基中筛选得到酿酒酵母转化菌株YPTD5。在合适的培养条件下, 添加外源底物双高g-亚麻酸和诱导物半乳糖, 培养并收集菌体。通过脂肪酸甲酯气相色谱分析, 表明三角褐指藻D5-脂肪酸脱氢酶基因在酿酒酵母中获得了高效的表达, 将双高g-亚麻酸转化为花生四烯酸, 其底物转化率达到了45.9%。     

雅致枝霉△6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

根据真菌△^6 -脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉（Thamnidium elegans）As3．2806△^6 -脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术（RACE）向两端延伸得到1504bp的△^6 -脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的△^6 -脂肪酸脱氧酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSel的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSel中异源表达。通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸（GLA）的含量占酵母总脂肪酸的7．5％。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的△^6 -脂肪酸脱氢酶基因（GenBank．AY941161）。     

花生油酸脱氢酶基因在酿酒酵母中的高效表达

将克隆的油酸脱氢酶基因(AF900663)亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES6/CT,从大肠杆菌中筛选到含有目的基因的重组质粒pYES/HO-A,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,经半乳糖诱导后,收集菌体,用气相色谱质谱(GC-MS)仪分析转化酵母的脂肪酸色谱的结果表明,HO-A所编码的酶具有油酸脱氢酶活性,能将酵母内源性油酸转化为亚油酸,油酸脱氢酶的表达量为15.6%,高于已有的报道。     

小眼虫藻△8-脂肪酸脱氢酶基因的克隆及其在酿酒酵母中的表达

△8途径是合成多不饱和脂肪酸的替代途径,△8-脂肪酸脱氢酶是该途径的关键酶之一.根据已报道的△8-脂肪酸脱氢酶基因设计引物,分别从小眼虫藻基因组DNA和cDNA中扩增得到该基因片段,序列分析表明:结构基因长1 266 bp,编码421个氨基酸;该基因没有内含子,比已经报道的△8-脂肪酸脱氢酶基因长6bp,并且N末端序列也有所不同.利用酿酒酵母的载体pYES2.0构建△8-脂肪酸脱氢酶表达载体pYEFD,并转化到营养缺陷型酿酒酵母菌株INVSc1中,在选择培养基中筛选得到酿酒酵母转化菌株YD8.YD8在合适的培养条件下,添加外源底物二十碳二烯酸和二十碳三烯酸并诱导基因表达.脂肪酸甲酯气相色谱分析表明小眼虫藻△8-脂肪酸脱氢酶基因在酿酒酵母中获得了高效表达,将二十碳二烯酸和二十碳三烯酸分别转化成二高-γ-亚麻酸和二十碳四烯酸,其底物转化率分别达到了31.2%和46.3%.     

卷枝毛霉△^6—脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ—亚麻酸的酿酒酵母工程菌株,应用RT—PCR技术,从卷枝毛霉中扩增出△^6—脂肪酸脱氢酶基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2．0,在大肠杆菌中筛选到含有目的基因的重组质粒pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScl中,在SC—ura合成培养基中筛选到转化酵母,在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸色谱分析,结果表明：γ—亚麻酸占总脂肪的50．07％。迄今为止,这是国内外△^6—脂肪酸脱氢酶基因在酿酒酵母表达量最高的报道。     

玉米△12脂肪酸脱氢酶基因（FAD2）在酿酒酵母中的表达

玉米△12脂肪酸脱氢酶是催化油酸形成亚油酸的关键酶。将其编码基因FAD2（GenBank登陆号：DQ496227）克隆到酿酒酵母表达载体pYES2．0中,构建成重组质粒pYE／FAD2,转化到酿酒酵母进行诱导表达,同时以pYES2．0转化子为对照。气相色谱（Gc）分析表明,重组转化子亚油酸的含量占酵母总脂肪酸的1．54％,而对照未检测到亚油酸。表明FAD2基因具有编码△12脂肪酸脱氢酶的功能。为探索转译起始密码子周边序列的改变对FAD2基因表达产生的影响,将该基因的起始密码子上游序列进行修改,构建重组表达载体pYE／FAD2—1,转化酿酒酵母进行表达。GC分析表明,pYE／FAD2—1转化子的亚油酸含量占总脂肪酸含量的8．81％,是对照pYE／FAD2转化子的近5倍。     

雅致枝霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

根据真菌Δ6-脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉(Thamnidium elegans)As3.2806Δ6-脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术(RACE)向两端延伸得到1504bp的Δ6-脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的Δ6-脂肪酸脱氢酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSc1的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSc1中异源表达。通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸(GLA)的含量占酵母总脂肪酸的7.5%。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的Δ6-脂肪酸脱氢酶基因(GenBank,AY941161)。     

卷枝毛霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ 亚麻酸的酿酒酵母工程菌株,应用RT PCR技术,从卷枝毛霉中扩增出△6 脂肪酸脱氢酶 基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒 pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,在SC ura合成培养基中筛选到转化酵母,在合适 的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸 色谱分析,结果表明:γ 亚麻酸占总脂肪的50.07%。迄今为止,这是国内外△6 脂肪酸脱氢酶基因在酿酒酵母表 达量最高的报道。     

深黄被孢霉M6-22△6-脂肪酸脱氢酶基因在酿酒酵母中的表达

应用PCR技术,从含有深黄被孢霉△6-脂肪酸脱氢酶基因的重组质粒pTMICL6中,扩增出1.38kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMID6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INVSc1中,在SC-Ura合成培养基中,选择到酵母工程株YMID6.在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体.通过GC-MS对酵母工程株所含的全部脂肪酸进行色谱分析,结果表明,γ-亚麻酸的含量占酵母总脂肪酸的8.69%.     

小眼虫藻Δ8-脂肪酸脱氢酶基因的克隆及其在酿酒酵母中的表达

Δ8途径是合成多不饱和脂肪酸的替代途径,Δ8-脂肪酸脱氢酶是该途径的关键酶之一。根据已报道的Δ8-脂肪酸脱氢酶基因设计引物,分别从小眼虫藻基因组DNA和cDNA中扩增得到该基因片段,序列分析表明：结构基因长1 266 bp,编码421个氨基酸;该基因没有内含子,比已经报道的Δ8-脂肪酸脱氢酶基因长6 bp,并且N末端序列也有所不同。利用酿酒酵母的载体pYES2.0构建Δ8-脂肪酸脱氢酶表达载体pYEFD,并转化到营养缺陷型酿酒酵母菌株INVSc1中,在选择培养基中筛选得到酿酒酵母转化菌株YD8。YD8在合适的培养条件下,添加外源底物二十碳二烯酸和二十碳三烯酸并诱导基因表达。脂肪酸甲酯气相色谱分析表明小眼虫藻Δ8-脂肪酸脱氢酶基因在酿酒酵母中获得了高效表达,将二十碳二烯酸和二十碳三烯酸分别转化成二高-γ-亚麻酸和二十碳四烯酸,其底物转化率分别达到了31.2％和46.3％。     

Overexpression of endogenous delta-6 fatty acid desaturase gene enhances eicosapentaenoic acid accumulation in Phaeodactylum tricornutum

The effect of overexpression of endogenous delta-6 fatty acid desaturase gene (ER<DELTA>6FAD) on eicosapentaenoic acid (EPA) production and total lipid content was investigated in Phaeodactylum tricornutum. All three randomly selected transformants exhibited significant increase (47.66%) in their EPA content, which reached up to 38.101 mg/g DW in algal strain D63. The total lipid content of all the three transformants was significantly higher (16.40–18.64%) than that of the wild-type strain. These findings suggested that the reaction catalyzed by ER<DELTA>6FAD is a limiting step for EPA biosynthesis, overexpression of endogenous ER<DELTA>6FAD gene is an effective method for improving EPA production in eukaryotic microalgae, and fatty acid metabolic pathway in microalgae can be genetically modified.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Identification and characterization of an animal delta(12) fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae

We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae. The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C. elegans omega-3 fatty acid desaturase. In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions. This is the first report of a cloned animal Delta(12) desaturase gene. Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids. The increase of membrane fluidity brought about by this change in unsaturation was measured. The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress.     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.

Phaeodactylum tricornutum is an unicellular silica-less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N-terminus and the three histidine clusters common to all front-end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Delta5- and Delta6-fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Delta6-specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the omega3- and omega6-pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the omega3- and omega6-fatty acids with similar efficiencies, indicating that none of them was specific for either the omega3- or the omega6-pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.