Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.     

Heterologous expression of Aspergillus oryzae xylose reductase and xylitol dehydrogenase genes facilitated xylose utilization in the yeast Saccharomyces cerevisiae

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

一种来源于链格孢菌的MAPK激酶AtPBS2基因的克隆及功能分析

通过遗传学手段,构建了细极链格孢菌（Alternaria tenuissima）cDNA酵母表达文库并筛选获得MAPK激酶AtPBS2基因,该基因全长2 492bp,编码683个氨基酸。AtPbs2p与来源于烟曲霉（Aspergillus fumigatus）的AtPbs2p（XP_752961）、粗糙脉孢菌（Neurospora crassa）的NcPbs2p（XP_965727）、稻瘟菌（Magnaporthe grisea）MGCH7（XP_001522946）及酵母（Saccharomyces cerevisiae）ScPbs2p（EDN63254）序列分别具有52%、52%、49%和47%的相似性。在高渗环境下,AtPBS2基因与酵母的ScPBS2基因功能相同,具有耐高渗透压环境的能力。细极链格孢菌（A.tenuissima）中存在HOG通路信号途径,AtPBS2基因可能与链格孢菌（A.tenuissima）的逆境适应性调节密切相关。     

Cloning and functional characterization of the Rhizopus oryzae high affinity iron permease (rFTR1) gene

Rhizopus oryzae is the most common etiologic agent of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. Therefore, the high affinity iron permease (rFTR1) which encodes a protein required to scavenge iron from the environment, is highly likely to be a critical determinant of virulence for R. oryzae. We have cloned rFTR1 by using a PCR approach relying on degenerate primers designed from the conserved regions of Saccharomyces cerevisiae high affinity iron permease. Sequence analysis of a 2.0 kb EcoRI genomic clone revealed a single open reading frame of 1107 bp that lacked introns. The putative rFtr1p had significant homology to known fungal high affinity iron permeases from Candida albicans (46% identity) and S. cerevisiae (44% identity). In R. oryzae, rFTR1 was expressed in iron-depleted and not in iron-rich media. Finally, rFTR1 restored the ability of an ftr1 null mutant of S. cerevisiae to grow on iron-limited medium and to take up radiolabeled iron, whereas S. cerevisiae transformed with the empty vector did not. These data demonstrate that we have cloned the gene encoding a R. oryzae high affinity iron permease and the putative rFtr1p is involved in assimilation of iron from iron-depleted environments.     

Identification of Delta12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae.

Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S-4, which is used industrially to produce arachidonic acid. Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal omega6-desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal omega3-desaturase (from soybean). The evolutionary implications of these enzymes will be discussed. The cloned cDNA was confirmed to encode a Delta12-desaturase, which was involved in the desaturation of oleic acid (Delta9-18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S. cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%). Genomic Southern blot analysis of the transformants with the Mortierella Delta12-desaturase gene as a probe confirmed integration of this gene into the genome of A. oryzae. The M. alpina 1S-4 Delta12-desaturase is the first example of a cloned nonplant Delta12-desaturase.     

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Bioenergetic properties of the thermoalkaliphilic Bacillus sp. strain TA2.A1

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (Deltap) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum DeltapH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Deltapsi) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth. In contrast, the Deltap declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0. An inwardly directed sodium motive force (DeltapNa(+)) of -100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Deltap. The phosphorylation potential of strain TA2.A1 was maintained between -300 mV and -418 mV, and the calculated H(+)/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the DeltapNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Deltap. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.     

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.     

Identification of the Xenopus laevis homolog of Saccharomyces cerevisiae DNA2 and its role in DNA replication

The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication. S. cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2). Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs. XDna2p is even more closely related (60% identical) to a partial human cDNA. The Xenopus Dna2 (XDna2) gene was able to complement an S. cerevisiae dna2-1 mutant strain for growth at the nonpermissive temperature, suggesting that XDna2p is a functional as well as a structural homolog of the yeast protein. Recombinant XDna2p was expressed in insect cells and purified. Like the ScDna2p purified from yeast, it is a single-stranded DNA endonuclease and a DNA-dependent ATPase, suggesting that both of these activities are part of the essential function of Dna2p. However, unlike ScDna2p from yeast, recombinant XDna2p showed no DNA helicase activity. When XDna2 was immunodepleted from interphase egg extracts, chromosomal DNA replication was almost completely inhibited. From the size of the residually synthesized DNA from the XDna2-depleted egg extracts, it seems that initiation of DNA replication may be impaired. This interpretation is also supported by the normal DNA replication of M13 single-stranded DNA in the XDna2-depleted egg extracts.     

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

The Trichoderma atroviride seb1 (stress response element binding) gene encodes an AGGGG-binding protein which is involved in the response to high osmolarity stress

The chitinase genes of Trichoderma spp. (ech42, chit33, nag1) contain one or more copies of a pentanucleotide element (5'-AGGGG-3') in their 5'-noncoding regions. In Saccharomyces cerevisiae, this motif is recognized and bound by the stress response regulator proteins Msn2p/Msn4p. To test whether this motif in the chitinase promoters is bound by a Trichoderma Msn2/4p homolog, we have cloned a gene (seb1) from T. atroviride which encodes a C2H2 zinc-finger protein that is 62 (64)% identical to S. cerevisiae Msn2p (Msn4p) in the zinc-finger region, and almost identical to the G-box binding protein from Haematonectria haematococca and to polypeptides encoded by uncharacterized ORFs from Neurospora crassa and Aspergillus nidulans. Its zinc-finger domain specifically recognizes the AGGGG sequence of the ech42 and nag1 promoter in band-shift assays. However, a cDNA clone of seb1, when overexpressed in S. cerevisiae, was unable to complement a Delta msn2/4 mutant of S. cerevisiae. Levels of seb1 mRNA increased under conditions of osmotic stress (sorbitol, NaCl) but not under other stress conditions (cadmium sulfate, pH, membrane perturbance). A T. atroviride Delta seb1 strain, produced by transformation with a seb1 copy disrupted by insertion of the A. nidulans amdS gene, showed strongly reduced growth on solid medium, but grew normally in liquid medium. In liquid medium, growth of the disruption strain was significantly more inhibited by the presence of 1 M sorbitol and 1 M NaCl than was that of the wild-type strain. Despite the presence of AGGGG elements in the promoter of the chitinase gene nag1, no differences in its expression were found between the parent and the disruption strain. EMSA analyses with cell-free extracts obtained from the seb1 disruption strain showed the presence of proteins that could bind to the AGGGG-element in nag1 and ech42. We therefore conclude that seb1 encodes a protein that is involved in the osmotic stress response, but not in chitinase gene expression, in T. atroviride.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

Complementation of Saccharomyces cerevisiae ccc2 mutant by a putative P1B-ATPase from Brassica napus supports a copper-transporting function

Copper transport across membranes plays an important role in plant growth and survival. P(1B)-type ATPases participate in transmembrane transport of copper in various organisms. A Brassica napus cDNA (BnRAN1) encoding a putative Cu(2+)-ATPase was cloned in this study. A complementation assay demonstrated that the protein encoded by this cDNA could functionally replace Ccc2p, a Saccharomyces cerevisiae Cu(2+)-ATPase, rescuing growth of ccc2 mutant under iron-limited conditions. Our results suggest that this rescue likely resulted from restoration of copper delivery, mediated by BnRAN1, to Fet3p. This study is amongst the first to demonstrate that a putative plant P(1B)-ATPase is functional and to examine its substrate specificity.