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。     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

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.     

人工酵母后鲨烯路径基因对7-脱氢胆固醇合成的影响

酵母内源后鲨烯路径中的固醇类物质,是异源甾体类药物合成的重要前体。为了通过微调后鲨烯路径,与异源模块进行适配,以期达到提高异源甾体类化合物表达的目的,以维生素D₃的直接前体-7-脱氢胆固醇(7-DHC)的合成为例,首先在固醇C-24甲基转移酶(ERG6)缺失的酿酒酵母BY4742中,通过导入人源固醇C-24 还原酶DHCR24,并过表达截短的羟甲基戊二酰辅酶A还原酶tHMGR,获得可以合成7-DHC的人工酵母。在此基础上,将后鲨烯路径分割并构建成ERG1、ERG7、ERG11、ERG24-25-26-27和ERG2-3这5个模块,分别在所构建的7-DHC合成菌株中过表达。通过GC-TOF/MS分析7-DHC以及后鲨烯路径中相关代谢中间体的含量,并结合主成分分析发现,过表达不同后鲨烯模块会引起后鲨烯路径上固醇组分的变化而最终影响7-DHC的产量:与出发菌株相比,过表达ERG11模块会显著强化其他固醇物质到酵母固醇的转化;而过表达ERG2-3模块则会减少鲨烯的积累,同时显著增加羊毛固醇及其之后的固醇组分的含量,并获得迄今为止7-DHC在微生物中摇瓶水平的最高产量。因此,对ERG11和ERG2-3的表达优化对7-DHC的合成以及后鲨烯路径代谢流的强化起到了显著的作用,是后续优化人工7-DHC合成酵母的潜在靶点。为研究后鲨烯路径与其他异源甾体合成模块间的适配,提供了可供参考的案例。     

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.     

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.     

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.     

Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11).

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.     

Interplay between the cis-prenyltransferases and polyprenol reductase in the yeast Saccharomyces cerevisiae

Dolichol formation is examined in three Saccharomyces cerevisiae strains with mutations in the ERG20 gene encoding farnesyl diphosphate synthase (mevalonic acid pathway) and/or the ERG9 gene encoding squalene synthase (sterol synthesis pathway) differing in the amount and chain length of the polyisoprenoids synthesized. Our results suggest that the activities of two yeast cis-prenyltransferases Rer2p and Srt1p and polyprenol reductase are not co-regulated and that reductase may be the rate-limiting enzyme in dolichol synthesis if the amount of polyisoprenoids synthesized exceeds a certain level. We demonstrate that reductase preferentially acts on typical polyprenols with 13-18 isoprene residues but can reduce much longer polyprenols with even 32 isoprene residues.     

Isolation of the ERG2 Gene,Encoding Δ8 → Δ7 Sterol Isomerase,from the Maize Smut Pathogen Ustilago maydis

Bailey, A. M., Burden, R. S., James, C. S., Keon, J. P. R., Croxen, R., Bard, M., and Hargreaves, J. A. 1993. Isolation of the ERG2 gene, encoding Δ⁸ → Δ⁷ sterol isomerase, from the maise smut pathogen Ustilago maydis. Experimental Mycology 18, 87-92. The ERG2 gene encoding Δ⁸ → Δ⁷ sterol isomerase has been isolated from the fungal plant pathogen Ustilago maydis. This was accomplished by screening an U. maydis genomic library with a fragment of the Saccharomyces cerevisiae ERG2 gene. The identity of the U. maydis ERG2 gene was confirmed by complementation of an U. maydis Erg2 mutant and by comparing the deduced amino acid sequence encoded by the U. maydis ERG2 gene with that of the S. cerevisiae ERG2 gene product.     

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.     

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.