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-24甲基转移酶和甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成中的调控作用

摘要：【目的】研究ERG6基因编码的甾醇C-24甲基转移酶和ERG2基因编码的甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成代谢中的调控作用。【方法】通过PCR扩增克隆到酿酒酵母甾醇C-8异构酶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pPERG2;同时,在本实验室已构建的ERG6表达质粒pPERG6的基础上,构建了ERG2和ERG6共表达的重组质粒pPERG6-2。将表达质粒转化酿酒酵母单倍体菌株YS58,依据营养缺陷互补筛选到重组菌株YS58(pPERG2)和YS58(pPERG6-2)。通过紫外分光光度法和气相色谱法分析重组菌株甾醇组分和含量。【结果】在ERG6高表达的重组酵母菌中,甾醇中间体和终产物麦角甾醇的含量均比对照菌高;而在ERG2高表达的酵母菌株中,无论甾醇中间体,还是麦角甾醇的含量均明显降低。ERG6和ERG2共表达重组菌株YS58(pPERG6-2)的麦角甾醇含量是对照菌株YS58(YEp352)的1.41倍,是ERG2单独高表达菌株YS58(pPERG2)的1.92倍,是ERG6单独高表达菌株YS58(pPERG6)的1.12倍。【结论】本研究首次证明甾醇C-24甲基转移酶催化的反应是酿酒酵母麦角甾醇合成代谢途径中的一个重要的限速步骤,该酶活性提高不但补偿了ERG2高表达对甾醇合成的负效应,而且使麦角甾醇含量进一步提高,为构建麦角甾醇高产酵母工程菌株提供了实验依据。     

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。     

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。     

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     

Oenococcusoeni苹果酸-乳酸酶基因克隆及其在酿酒酵母中的表达

苹果酸-乳酸酶是苹果酸-乳酸发酵过程中负责苹果酸转化为乳酸的功能酶。在进行酒酒球菌SD2a的苹果酸-乳酸酶基因(mleA)克隆测序基础上,以PGK1强启动子和ADH1终止子为调控元件,以大肠杆菌-酵母菌穿梭质粒YEp352为载体,构建了重组表达质粒并转化酿酒酵母YS58。酵母转化子用SD/Ura平板筛选鉴定。斑点杂交检测表明目的基因mleA转化到受体菌中,SDSPAGE检测表明获得的转化子表达了约60kDa的目标蛋白。获得的转化子在添加了L苹果酸的培养基中培养4d;取培养液上清用HPLC检测L苹果酸及L乳酸含量,采用t检验进行差异显著性分析,结果表明mleA基因进行了功能性的表达,将L苹果酸转化成L乳酸,L苹果酸和L乳酸含量分别与对照差异极显著和显著,苹果酸的相对降低率平均为20.95%。在有选择压力条件下,重组质粒相对稳定,而在无选择压力条件下,传代培养10d后大约有65%的重组质粒丢失。     

角鲨烯合成酶特异性mU6-siRNA真核表达载体的构建及鉴定

利用RNA干扰技术靶向构建角鲨烯合成酶基因ERG9特异性真核表达载体。将针对粟酒裂殖酵母(Schizosaccharomyces pombe Linder)ERG9不同部位所设计的3对siRNA序列通过重组技术克隆到质粒mU6 pro中构建真核表达重组体mU6 ERG9 siRNA1、2、3,转化DH5α菌株扩增,提取质粒通过限制性酶切和测序分析对重组表达载体进行鉴定,分析结果表明3个表达载体的设计基因插入正确,成功构建了ERG9特异性真核表达载体mU6 ERG9 siRNA,重组体的成功构建为在裂殖酵母细胞中靶向RNA干扰角鲨烯的合成从而提高辅酶Q10合成途径的代谢通量打下基础。     

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%。     

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.     

Aerobic isolation of an ERG24 null mutant of Saccharomyces cerevisiae.

The ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae. We report here, however, that strains with null mutations in the ERG24 gene can grow on defined synthetic media in aerobic conditions. These sterol mutants produce ignosterol (ergosta-8,14-dienol) as the principal sterol, with no traces of ergosterol. In addition, we mapped the ERG24 gene to chromosome XIV between the MET2 and SEC2 genes. Our results indicate that ignosterol can be a suitable sterol for aerobic growth of S. cerevisiae on synthetic media and that inactivation of ERG24 is only conditionally lethal.     

Cloning and characterization of a gene complementing the mutation of an ethanol-sensitive mutant of sake yeast

Ethanol-sensitive mutants (esl to es10) were isolated from sake yeast, Saccharomyces cerevisiae SY-32. These mutants were unable to grow at 7% ethanol at which the wild type strain SY-32 does grow. The mutants had a variety of fermentation rates and viabilities in the presence of ethanol. The gene ERG6, complementing the ethanol-sensitive mutation of es5, was cloned from an SY-32 gene library. ERG6 encodes S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) in the ergosterol synthetic pathway. Mutant es5 had a reduced ability to synthesize ergosterol. An erg6 disruptant was also ethanol-sensitive. These results suggested that ERG6 plays an important role in the ethanol tolerance of S. cerevisiae.     

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.     

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.