The SPL1 tRNA splicing gene of Candida maltosa and Candida albicans

The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.     

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.     

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.     

近平滑假丝酵母ATCC 7330羰基还原酶CpCR的表达及酶学性质研究

通过构建重组菌Escherichia coli BL21(DE3)/pACYC Duet-1-cpcr,表达带有His标签的近平滑假丝酵母Candida parapsilosis ATCC 7330羰基还原酶CpCR基因,并采用Ni-Agarose亲和层析对重组酶CpCR进行分离纯化和酶学性质研究。重组酶CpCR的基因序列全长1 107 bp,含有368个氨基酸,分子质量在41 kDa左右,比酶活力为20 U/mg;该酶在4～33℃的温度范围稳定性较好,相对酶活力在80%以上,T₅₀值为37℃;该酶的pH适宜范围在6.2～7.5,在中性条件下稳定性最好; Cu²⁺对该酶有强烈的抑制作用,在1 mmol/L条件下,相对酶活力下降30%,在5 mmol/L条件下,相对酶活下降50%;该酶对底物苯甲醛、正丁醛亲和能力强于4-氯-3-酮基-丁酸乙酯(4-chlor-3-keto-butyrate-ethyl ester,COBE),对苯甲醛或正丁醛的催化效率是对COBE的20倍左右;该酶是一种NADPH依赖型的羰基还原酶。本研究为羰基还原酶CpCR分...     

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.     

蓝莓花青素生物合成相关基因表达与分子进化分析

运用实时聚合酶链式反应方法,测定8个品种的蓝莓(包括6个高丛蓝莓:绿宝石、蓝美1号、海岸、莱格西、比洛克西、奥尼尔;2个兔眼蓝莓:括灿烂、园蓝)花青素合成途径中的VcCHS、VcCHI、VcF3H、VcDFR、VcANS结构基因及转录因子MYB基因相对表达量,分析其对花青素合成的调控作用.结果 表明:VcCHS、VcC...     

The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1

Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica, with or without overproduction of Y. lipolytica CPR expressed under the ICL promoter (pICL-CPR) or the POX2 promoter (pPOX2-CPR). Activity of cytochrome CYP1A1 was analysed by conversion of hydroxyresorufin to resorufin. Strain JMY330 and JMY330-CPR present no activity, the monocopy cytochrome CYP1A1 integrant JMY331 and JMY331-CPR derivatives present an average activity of 32.0 pM/min/dw and 48.3 and 64.6 pM/min/dw for pICL-CPR and pPOX2-CPR, respectively. Increase of CPR expression resulted in about two-fold higher activity. The multicopy 1A1 integrant JMY339 and JMY339-CPR derivatives present an activity of 129 pM/min/dw and 815-1845 pM/min/dw, respectively. Increase of CPR expression resulted in 6.3-12.8-fold higher activity, depending on the CPR transformant. We observed a 50-fold increase of activity between the monocopy integrant JMY331 as compared to the multicopies integrant JMY339-CPR in which CPR was overexpressed.     

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.     

Assessment of safety and efficacy of pine bark extract in normal and ovariectomized mice

We evaluated the influence of pine bark extract (PBE) on organs, the cytochrome-P450 (CYP) activities in liver and estrogenic effects in normal and ovariectomized (OVX) female mice. The PBE did not affect organ weights and liver-function indexes (activities of alkaline phosphatase, aspartate amino transferase, and alanine amino transferase) at doses; 0.04%, 0.4%, and 2.0% PBE in the diet, in normal and OVX female mice. In the OVX mice, CYP1A1 activity was significantly higher in the 0.4% and 2.0% PBE groups than in the OVX control group, and in the 0.4% and 2.0% PBE groups were significantly higher than in the 0.04% PBE group. CYP1A2 and 3A4 activities were significantly higher in the 2.0% PBE group than in all other groups. The PBE did not affect uterine weight and femoral bone mineral density at all PBE doses. These results showed that the dose of PBE at the recommended human intake, had no toxic and estrogenic effects in normal female and OVX mice, however, it may need attention to use the excess intake of PBE with some drugs in postmenopausal women.     

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.     

南极假丝酵母脂肪酶B在黑曲霉中的分泌表达及其硅藻土固定化应用

为提高南极假丝酵母脂肪酶B（Candida antarctica lipase B,CALB）表达量,根据黑曲霉（Aspergillus niger）密码子偏好性设计合成CALB基因,以PnaII/tpi为启动子构建CALB表达载体并转化到黑曲霉HL-1中表达,摇瓶发酵120 h后上清液中重组CALB活力为171 U/mL。研究重组CALB的酶学性质,最适反应温度和pH值分别为50 ℃和8.0,在pH 6.0～9.0和45 ℃以下具有较高的稳定性,10 mmol/L Cu2＋、Zn2＋和0.1 g/100 mL十二烷基硫酸钠强烈抑制酶活力,而1 mmol/L Ca2＋、0.1 g/100 mL山梨醇对酶活力具有非常明显的激活作用。此外,以硅藻土为载体固定CALB,固定化酶活力为187 U/g。将制备的硅藻土-CALB固定化酶用于生物合成己酸乙酯,经反应条件优化后在无溶剂体系中己酸乙酯产率达91%。     

Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis

NADPH cytochrome P450 oxidoreductase (CPR) catalyses the transfer of electrons during P450-mediated oxidation, which plays an important role in the omega-oxidation pathway of Candida tropicalis. Two putative allelic genes, CPR-a and CPR-b, were cloned from the long chain dicarboxylic acid-producing Candida tropicalis 1230, using cassette PCR methods. Both the identified open reading frames predict the gene products of 679 amino acid residues. The deduced amino acid sequences of CPR-a and CPR-b are highly homologous to CPR genes from C. tropicalis ATCC 750 and Candida maltosa. Both genes were individually expressed in a cpr mutant of Saccharomyces cerevisiae with high CPR activities, in which only a small distinction was observed between recombinant CPR-a and CPR-b. Both CPR-a and CPR-b contain one CTG codon, which codes for serine (amino acid 50) in C. tropicalis rather than universal leucine. A mutated cDNA of CPR-a with a TCG codon instead of CTG codon was constructed and expressed, resulting in little increase in CPR activity. This indicates that the alteration of Ser-50 has little effect on functional expression of CPR. Furthermore, high ketoconazole sensitivity for the cpr mutant was complemented by heterologous expression of the cloned CPR-a or CPR-b.     

杂合抗菌肽Me-HNP1真核表达载体的构建与初步表达

选择蜂毒肽(Melittin)和人体防御素(HNP-1)部分基因序列进行人工融合,通过生物信息学技术预测其理化性质和功能结构,依据毕赤酵母(Pichia pastoris)密码子偏爱性原则编码杂合肽Me-HNP1基因,聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增目的基因并定向克隆至穿梭型表达载体pPICZa A上,将构建成功的分泌表达载体pPICZαA-Me-HNP1通过电转化的方式转化到毕赤酵母感受态细胞GS115内,以甲醇为诱导剂进行诱导表达,将得到的粗蛋白进行分离纯化。Tricine-SDS-PAGE蛋白电泳在5 kDa处得到一条单一清晰条带,表明杂合抗菌肽Me-HNP1在毕赤酵母中成功表达,生物信息学分析得出杂合抗菌肽Me-HNP1分子内带有8个正电荷,等电点为9.55,脂溶性80.44,在酵母体内半衰期大于20 h。表明其具有成为优秀抗菌肽潜力。为抗菌肽的研究奠定了实验基础。     

蛋白酶基因的克隆与表达研究进展

蛋白酶在各领域的应用愈加广泛,但其大规模工业生产受到诸多因素制约。利用基因工程技术对蛋白酶基因进行改良和克隆表达,开发高产量、高纯度、高活力的蛋白酶资源成为研究的热点。本文对蛋白酶的分类、结构与功能、菌种选育等进行了概述,同时分析了蛋白酶基因在原核和真核生物中的克隆与表达研究进展,并对未来蛋白酶基因工程的发展趋势进行了展望。     

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.