甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。     

甾醇C-22去饱和酶高表达对酵母细胞麦角甾醇合成的影响

通过PCR扩增克隆到酵母菌甾醇C-22去饱和酶基因(ERG5)的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pYPE5。以铜离子螯合蛋白基因CUP1替换ERG5基因内部序列获得ERG5破坏菌株YSE5,其中麦角甾醇的合成被阻断,而积累了甾醇中间体Ergosta-5,7-dien-3β-ol。表达质粒pYPE5转化破坏菌株后使细胞恢复了合成麦角甾醇的能力。说明表达质粒上的ERG5基因得到了功能性的表达。将表达质粒pYPE5转化酿酒酵母单倍体菌株YS58,通过营养缺陷互补筛选到重组菌株YS58(pYPE5)。对重组菌株、破坏菌株和互补菌株细胞甾醇组分和含量进行测定,发现重组菌株和互补菌株的麦角甾醇和总甾醇含量明显低于对照菌YS58(YEp352)。测定不同培养时间细胞的麦角甾醇含量,发现重组菌株的麦角甾醇含量始终低于对照菌YS58(YEp352)。可见,ERG5在酵母中的高表达导致细胞麦角甾醇含量降低。     

甾醇酰基转移酶基因高表达对酵母菌麦角甾醇合成的影响

通过PCR扩增克隆到含酵母菌甾醇酰基转移酶基因ARE2编码序列和上游调控序列的DNA片段ARE21及仅含编码序列的DNA片段ARE22。分别以ARE2启动子,乙醇脱氢酶基因ADH1启动子和铜抗性基因CUP1启动子及ADH1终止子为调控元件构建了酵母菌表达质粒pHX2,pHXA2和pHXC2。表达质粒分别转化酿酒酵母单倍体菌株YS58和以前通过细胞杂交构建的麦角甾醇高产菌株YEH56。通过营养缺陷互补和铜抗性筛选到转化子,质粒上的ARE2基因在YS58和YEH56中都实现了活性表达,使细胞内甾醇酯化水平升高,并导致细胞麦角甾醇含量的提高。对转化菌株的培养条件进行了初步研究,在优化条件下,重组转化菌株YEH56(pHX2)、YEH56(pHXA2)和YEH56(pHXC2)的麦角甾醇含量分别是受体菌YEH56 的13、13和14倍。     

采用基因敲除和反义技术抑制酿酒酵母ERG7基因表达

酿酒酵母(Saccharomyces cerevisiae)固有的甲羟戊酸(MVA)/麦角甾醇代谢途径生成的中间体2,3-氧化鲨烯是三萜类化合物的合成前体,以酿酒酵母为底盘细胞通过合成生物学技术组建这些化合物的代谢途径时,需要下调2,3-氧化鲨烯流向麦角甾醇的代谢流。在酿酒酵母中由羊毛甾醇合酶(ERG7)催化的2,3-氧化鲨烯环化是麦角甾醇和三萜类化合物生物合成分支形成的关键位点。采用基因敲除和反义RNA 2种技术对ERG7基因的表达进行下调。设计含有与ERG7基因ORF两侧序列同源的长引物,以质粒PUG66为模板进行PCR扩增,构建带有loxP-Marker-loxP的ERG7基因敲除组件,采用LiAc/SS Carrier DNA/PEG方法转化双倍体酿酒酵母INVSc1,通过同源重组的方式获得酿酒酵母ERG7基因单倍体缺失突变株,并对其进行了分子生物学确证。大量培养野生型和突变型菌株,菌体冷干后在碱醇溶液中90℃回流1h,正己烷萃取后旋蒸干溶剂,甲醇溶解残留物麦角甾醇。通过TLC和HPLC方法比较麦角甾醇含量,结果表明:与野生型菌株相比,突变型菌株的麦角甾醇含量明显降低。     

酿酒酵母adh2和ald6双基因缺失突变株的构建

酿酒酵母乙醇合成代谢过程中, 阻断或削弱乙醛至乙酸代谢流不但能增强乙醇合成流, 同时还能降低发酵乙酸含量。本研究以乙醇脱氢酶Ⅱ(adh2)基因缺陷型酿酒酵母YS2-Dadh2为出发菌株, 应用长侧翼同源两步PCR(LFH-PCR)策略构建乙醛脱氢酶Ⅵ(ald6)基因敲除组件, 转化酿酒酵母YS2-Dadh2敲除ald6基因, 之后转入表达质粒pSH65到阳性克隆中, 半乳糖诱导表达Cre重组酶切除Kanr基因筛选标记, 最后, 传代丢失质粒pSH65获得单倍体ald6基因缺失突变株。利用同样的敲除组件和技术再次敲除其等位基因, 最终获得双基因缺失突变株YS2-△adh2-Dald6。发酵实验表明与出发菌株YS2相比, 突变株乙酸合成量降低18%, 乙醇最高产量提高12.5%。     

ADH7启动子精细调控表达MSN2酿酒酵母菌株对 糠醛耐受的研究

【目的】构建自我精细调控表达应激转录调控基因MSN2的酿酒酵母(Saccharomyces cerevisiae)基因工程菌株,提高其对糠醛的耐受能力。【方法】以酿酒酵母BY4742基因组DNA为模板,采用PCR技术扩增获得ADH7启动子、CYC1终止子以及MSN2编码框序列,以pUG6质粒为载体构建含ADH7p-MSN2-CYC1t表达盒的重组表达质粒pUG6-AM。通过醋酸锂法,将线性化后的质粒pUG6-AM转入酿酒酵母BY4742,筛选阳性转化子,初步分析其对糠醛的耐受能力,采用荧光定量PCR技术检测MSN2基因及其调控代表基因的转录变化。【结果】构建了在ADH7启动子控制下表达MSN2的酿酒酵母基因工程菌株AM01,该菌株对糠醛耐受能力明显增强,MSN2基因的转录得到了自我精细调控,并提高了其调控基因的转录水平。【结论】以糠醛诱导表达基因的启动子精细调控应激转录调控基因MSN2的转录表达,既可提高酿酒酵母工程菌株对糠醛的耐受能力,又能避免其持续高效表达带来的副作用。     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

SSU1多拷贝表达对酿酒酵母二氧化硫生成量的影响

【目的】在不影响酵母正常代谢前提下,构建亚硫酸盐分泌量提高的基因工程菌株,增加二氧化硫生成量,有效地解决啤酒老化问题。【方法】以适量高产二氧化硫工业啤酒酵母突变株M8总DNA为模板,PCR方法得到带有不同长度5′端非编码区的基因片段SSU1-1、SSU1-2,以大肠杆菌-酿酒酵母穿梭质粒YEp352构建表达载体pSU1和pSU2,转化实验室酵母YS58,验证SSU1多克隆表达对其二氧化硫生成量的影响。进而将pSU2转化工业啤酒酵母M8,利用亚硫酸盐抗性筛选转化子,并对其二氧化硫和硫化氢生成量及其啤酒抗老化性能进行测定和分析。【结果】实验室酵母转化子pSU1-4和pSU2-3二氧化硫生成量较原株明显提高而硫化氢生成量基本不变,工业啤酒酵母转化子Y2二氧化硫生成量比原株M8提高74.4%,TBA值下降14.9%,DPPH自由基清除率提高38.2%,硫化氢生成量基本不变。【结论】SSU1基因的多拷贝表达有效提高了亚硫酸盐转运蛋白Ssu1p表达量,增加了亚硫酸盐分泌量,啤酒抗氧化能力得到明显增强,而对酵母硫代谢途径中亚硫酸盐还原为硫化物代谢过程没有影响。     

酒类酒球菌mleP基因的克隆及其在酿酒酵母中的表达

苹果酸通透酶具有协助苹果酸 乳酸发酵 (MLF)的重要功能。以酒类酒球菌 (Oenococcusoeni)优良菌系Oenococcus Lee SD 2a的总DNA为模板 ,用PCR方法克隆到苹果酸通透酶基因mleP ,构建了重组质粒pBMmleP。序列分析表明克隆到的基因序列与已报道的序列同源性为 99%。为使目的基因在酿酒酵母中表达 ,以大肠杆菌 酿酒酵母穿梭质粒YEp35 2为载体 ,以PGK1强启动子和ADH1终止子为调控元件 ,构建了重组表达质粒YEpmleP ,并转化酿酒酵母 (Saccharomycescerevisiae)YS5 8。酵母转化子用含有亮氨酸、组氨酸和色氨酸的YNB平板筛选鉴定。获得的转化子在添加了L 苹果酸 (5g L)的培养基中培养 4d ;取培养液上清用HPLC检测 ,结果显示重组转化子YSP的培养液中L 苹果酸剩余含量均低于空载体转化子YS35 2 ,因此所得酵母重组转化子对苹果酸的转运能力有所提高     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

Overexpression of a sterol C-24(28) reductase increases ergosterol production in Saccharomyces cerevisiae

Three plasmids, pHX4, pHXA4 and pHXC4, containing sterol C-24(28) reductase gene (ERG4) under the control of ERG4, ADH1 or CUP1 promoters, respectively, and the copper resistance gene as the selection marker were constructed, and they were then introduced into Saccharomyces cerevisiae. Ergosterol production in recombinant strains was enhanced. Under the optimal culture condition, ergosterol content in recombinant strains YEH56(pHX4), YEH56(pHXA4) and YEH56(pHXC4) was 1.2, 1.4 and 1.5-fold (47 mg g^–1) of that in the original strain.     

Nucleotide sequence of the gene encoding yeast C-8 sterol isomerase.

The ERG2 gene encoding the Saccharomyces cerevisiae C-8 sterol isomerase, an enzyme involved in plant, animal, and fungal sterol biosynthesis was sequenced. A large open reading frame comprising 222 amino acids was observed.     

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.     

Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase

The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open reading frame comprising 365 amino acids. Gene disruption resulting from a deletion/substitution demonstrates that ERG3 is not essential for cell viability or the sparking function.     

A simple method for the isolation of zymosterol from a sterol mutant of Saccharomyces cerevisiae.

A simple method is described for the direct isolation of zymosterol (5 alpha-cholesta-8,24-dien-3 beta-ol) of high purity from a sterol mutant of Saccharomyces cerevisiae. This yeast strain, which is a double mutant of the ERG6 (sterol transmethylase) and ERG2 (C-8 sterol isomerase) genes, accumulates zymosterol as its major sterol component.     

粗糙脉孢菌中甾醇还原酶基因erg24对麦角甾醇合成的影响

粗糙脉孢菌为子囊菌中的高效纤维素降解菌,可以直接以纤维素为营养源进行生长。本研究以粗糙脉孢菌为实验对象,利用基因工程技术构建甾醇还原酶基因erg24的高表达菌株,分别以蔗糖、麦麸、玉米秸秆、小麦秸秆、杨树木屑、水稻秸秆6种物质的粉末为碳源培养野生型粗糙脉孢菌和erg24高表达菌株,利用半定量RT-PCR测定在不同培养条件下erg2、erg24和erg6 3个麦角甾醇合成相关基因的表达水平,采用HPLC方法测定不同培养条件下麦角甾醇的积累量。研究结果表明,分别以玉米秸秆、杨树木屑、水稻秸秆这3种粉末为碳源时,培养物中的erg2、erg24和erg6 3个基因表达量较高。在不同培养条件下erg24高表达菌株合成麦角甾醇量显著高于野生型粗糙脉孢菌的合成量,且以杨树木屑粉末为碳源培养时,所获得的麦角甾醇产量最高,为30.53μg/mg。结果表明erg24基因是粗糙脉孢菌合成麦角甾醇的关键基因之一,利用玉米秸秆、小麦秸秆、杨树木屑或水稻秸秆粉末为碳源培养粗糙脉孢菌时,可获得较高产量的麦角甾醇。研究结果为以农业废弃物为营养源,利用真菌生产麦角甾醇奠定了基础。     

The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) is essential for growth

The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) was cloned by complementing a Saccharomyces cerevisiae erg26 mutant with a C. albicans genomic library. Sequence analysis showed a 70% identity between the C. albicans and S. cerevisiae ERG26 genes at the amino acid level. Sequential disruption of both copies of the ERG26 gene in the presence of an integrated rescue cassette containing a third copy of the ERG26 gene under the control of the inducible pMAL2 promoter, resulted in cells capable of growing only in the presence of the inducer. The results establish that the ERG26 gene is essential for growth and that inhibitors of the Erg26p may represent a new and highly effective class of antifungal agents.     

The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.

In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem. 258:4472-4476, 1983), a cell cycle event that may be required to enter G1 (C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. USA 84:4012-4016, 1987). The sterol biosynthetic pathway in S. cerevisiae was genetically altered to assess the functional role of the C-28 methyl group of ergosterol. ERG6, the putative structural gene for S-adenosylmethionine: delta 24-methyltransferase, which catalyzes C-24 methylation, was cloned, and haploid strains containing erg6 null alleles (erg6 delta 1 and erg6 delta ::LEU2) were generated. Although erg6 delta cells are unable to methylate ergosterol precursors at C-24, they exhibit normal vegatative growth, suggesting that C-28 sterols are not essential in S. cerevisiae. However, erg6 delta cells exhibit pleiotropic phenotypes that include defective conjugation, hypersensitivity to cycloheximide, resistance to nystatin, a severely diminished capacity for genetic transformation, and defective tryptophan uptake. These phenotypes reflect the role of ergosterol as a regulator of membrane permeability and fluidity. Genetic mapping experiments revealed that ERG6 is located on chromosome XIII, tightly linked to sec59.