A deviation from the universal genetic code in Candida maltosa and consequences for heterologous expression of cytochromes P450 52A4 and 52A5 in Saccharomyces cerevisiae

We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell-free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG-containing mRNAs. Moreover, we were able to transfer the non-universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.     

In vivo evidence for non-universal usage of the codon CUG in Candida maltosa

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA ^SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon. The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.     

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.     

Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa.

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.     

Cytochromes P450 of the sophorose lipid-producing yeast Candida apicola: Heterogeneity and polymerase chain reaction-mediated cloning of two genes

Candida apicola belongs to a group of yeasts producing high amounts of surface-active extracellular glycolipids consisting of sophorose and long-chain-ω- and (ω-1)-hydroxy fatty acids. The involvement of cytochrome P450 in the synthesis of sophorose lipid by the hydroxylation of long-chain fatty acids was suggested from a simultaneous increase of cellular P450 content. Hydroxylation studies indicated the existence of multiple P450 forms capable of hydroxylating not only long-chain fatty acids, but also n-alkanes. In this report, two different P450 DNA fragments amplified in a polymerase chain reaction with heterologous primers and chromosomal DNA of Candida apicola were used as homologous probes for the isolation of full-length clones from a genomic library. The open reading frames of both genes encode proteins of 519 amino acids with calculated molecular weights of 58,656 and 58,631, respectively, that contain N-terminal membrane anchor sequences and hallmark residues, in common with other eukaryotic P450s. The deduced amino acid sequences of the C. apicola P450 genes are 84·4% identical. They share 34·5 to 44·1% identity with the proteins of the yeast family CYP52 and about 25% identity with fatty acid hydroxylases of higher eukaryotes (family CYP4A) and of Bacillus megaterium (CYP102). Southern hybridization experiments revealed the existence of further P450-related genes in C. apicola. According to the P450 nomenclature system, the cloned genes were named CYP52E1 and CYP52E2, establishing a new subfamily in yeast family CYP52. The sequences were deposited in the EMBL/GenBank Library under the Accession Numbers X76225 and X87640.     

Induction of cytochrome P-450 and catalase activity in Saccharomyces cerevisiae by UV and X-ray irradiation. Possible role for cytochrome P-450 in cell protection against oxidative damage

Cytochrome P-450 was induced both in the diploid wild-type D7 strain and in two isogenic DNA-repair-deficient strains (rad3 and rad56) of Saccharomyces cerevisiae following UV- and X-irradiation. The induction occurred only in logarithmic growth phase cells and it was transient showing a peak 3 h after irradiation. The maximal amount of cytochrome P-450 was directly proportional to the radiation dose applied. Under the same experimental conditions an increase of the catalase activity was also observed, suggesting that activated oxygen species produced by irradiation might be implicated in the induction of both enzymes. The sensitivity to H2O2 of cells containing high cytochrome P-450 levels was enhanced when this enzyme was specifically inhibited by tetrahydrofuran and metyrapone. This supports the hypothesis that cytochrome P-450, as well as catalase, might be involved in cell protection against oxidative damage.     

Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.     

Analysis of copper-induced metallothionein expression using autonomously replicating plasmids in Candida glabrata

Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTIIb is more highly inducible than MTIIa and cells harboring a single MTIIb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between ? 503 and ? 292 upstream of the MT coding sequences. Expression of MTI is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.     

α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的策略

综述了宿主细胞的表达特点,外源基因的来源,表达载体和转化方法对α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的影响,并在此基础上提出了相应的策略。     

重组人凝血酶真核表达载体的构建及其在CHO细胞中的稳定表达

目的 构建含重组人凝血酶基因的重组真核表达载体,并转染哺乳动物细胞CHO,建立稳定表达重组人凝血酶的细胞株.方法 利用限制性内切酶EcoR I和XbaI将凝血酶基因插入真核表达载体pcDNA3.1（＋）中,构建含人凝血酶基因的重组质粒pcDNA3.1 （＋）/thrombin,利用双酶切和测序法鉴定.采用阳离子脂质体介导方法,将构建的表达载体转染到CHO细胞中,通过G418加压筛选出阳性细胞克隆,建立稳定表达人凝血酶的细胞株,并扩大培养.采用RT-PCR和SDS-PAGE法检测其mRNA和蛋白质的表达,并对其生物活性进行鉴定.结果 重组质粒pcDNA3.1 （＋）/thrombin经双酶切和测序鉴定证实插入序列准确无误;经RT-PCR和SDS-PAGE法鉴定,转染的CHO细胞可表达、分泌人凝血酶,得到的重组人凝血酶表观相对分子质量（Mr）为43000左右,与预测一致,且具有降解并凝固牛纤维蛋白原的活性,其比活为234.1 U/mg.结论 成功构建了真核表达载体pcDNA3.1 （＋）/thrombin,并成功地在CHO细胞中表达了具有生物活性的重组人凝血酶,该体系的建立为进一步规模化生产重组人凝血酶奠定了基础.     

白藜芦醇及其生物工程表达系统的研究进展

白藜芦醇是一种天然的植物保护素,它是许多植物受到真菌感染、紫外照射或病理状况下产生的一种植物抗毒素,可以提高植物抵抗病原体的能力,并且它也具有相当的生理活性,不但具有抗氧化、抗菌、调节血脂、抗癌的保健功能,而且研究证明白藜芦醇对于多种动物有延长寿命的功效。白藜芦醇在葡萄中的合成路径以及相关各酶的研究已经取得较好的进展。更重要的是,随着生物工程技术的发展,在植物、动物和微生物中合成白藜芦醇合酶的产物已不再困难。通过对近年来在不同表达系统中合成白藜芦醇的研究进行综述,提出利用生物工程技术来合成白藜芦醇所面对的问题及将来的发展方向。     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

Induced expression of the Candida albicans multidrug resistance gene CDR1 in response to fluconazole and other antifungals

The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5′-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus. © 1998 John Wiley & Sons, Ltd.     

黑曲霉糖化酶基因在毕赤酵母中的克隆和表达

从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。     

Candida maltosa NADPH-cytochrome P450 reductase: Cloning of a full-length cDNA,Heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion. The nucleotide sequence of the cDNA of NADPH-cytochrome P450 reductase from C. maltosa is available from the EMBL data library under Accession Number X76226.     

Effect of a high cornstarch diet on hepatic cytochrome P450 2C and 3A activity and progesterone half-life in dairy cows

In the cow, inadequate concentrations of progesterone during gestation may lead to an abrupt termination of pregnancy. The primary organ involved in progesterone catabolism is the liver, which contains an abundance of cytochrome P450 isozymes (EC 1.14.14.1; mixed-function monooxygenases). The objectives of the current experiment were to determine the effect of feeding 2 isoenergetic and isonitrogenous diets, formulated to cause divergent insulin secretion, on hepatic cytochrome P450 2C (CYP2C) and 3A (CYP3A) activity as well as the resulting biological half-life of progesterone. Twenty-two Holstein cows averaging 80 ± 7 d in milk were randomly assigned to either a high cornstarch diet or a high fiber diet in a crossover experimental design consisting of two 14-d periods. Dry matter intake, milk yield, milk lactose yield, and milk lactose percentage were similar between the 2 diets. Milk fat yield and milk fat percentage were higher in cows fed the high fiber diet, whereas milk protein yield tended to be higher and milk protein percentage was higher in cows fed the high cornstarch diet. Energy balance tended to be improved by 57% in cows consuming the high cornstarch diet. Insulin concentrations at the time of liver biopsy (3.16 ± 0.04 h post-feeding) were increased by 44% in cows consuming the high cornstarch diet compared with cows consuming the high fiber diet. Cytochrome P450 2C activity was decreased by 45%, whereas CYP3A activity tended to be lowered by 34% in cows consuming the high cornstarch diet. Cytochrome P450 2C mRNA expression tended to be decreased by 21% in cows fed the high cornstarch diet, whereas CYP3A mRNA expression was not different between the dietary treatments. The fractional rate constant of progesterone decay was not different between the 2 diets; however, the half-life of progesterone tended to be longer in cows fed the high cornstarch diet compared with cows fed the high fiber diet (85 vs. 64 min, respectively). In summary, cows consuming the high cornstarch diet had increased insulin concentrations and lower hepatic CYP2C and CYP3A activity and tended to have a longer progesterone half-life compared with cows consuming the high fiber diet. Feeding diets that stimulate insulin secretion could alter progesterone clearance during lactation, when dairy cows have increased rates of progesterone inactivation because of high energy demands and increased DMI.