Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+)

A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.     

Cloning and sequencing of a cDNA encoding a copper-zinc superoxide dismutase enzyme from the marine yeast Debaryomyces hansenii

Cu-Zn superoxide dismutase (SOD-1) is a ubiquitously occurring eukaryotic enzyme with a variety of important effects on respiring organisms. A gene (dhsod-1) encoding a Cu-Zn superoxide dismutase of the marine yeast Debaryomyces hansenii was cloned using mRNA by the RT-PCR technique. The deduced amino-acid sequence shows ∼70% homology with that of cytosolic superoxide dismutase from Saccharomyces cerevisiae and Neurospora crassa, as well as lower homologies (between 55 and 65%) with the corresponding enzyme of other eukaryotic organisms, including human. The gene sequence encodes a protein of 153 amino acids with a calculated molecular mass of 15·92 kDa, in agreement with the observed characteristics of the purified protein from D. hansenii. The dhsod-1 sequence has been deposited in the public data library of the NCBI under Accession Number AFO 16383. © 1998 John Wiley & Sons, Ltd.     

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.     

A minisatellite sequence in the upstream region of the DURA3 gene from the halotolerant yeast Debaryomyces hansenii

The URA3 gene of Debaryomyces hansenii, encoding orotidine 5'-phosphate decarboxylase enzyme, was isolated by complementation in the yeast Saccharomyces cerevisiae. The deduced amino acid sequence is highly similar to Ura3 proteins from other yeast and fungal species. Analysis of the region upstream of the coding sequence revealed the presence of AG-rich minisatellite DNA sequences. In addition, upstream of the DURA3 sequence, we have found the 3'-terminal of a gene encoding a GEA2-like protein.     

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility.     

Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities.     

VII. Yeast sequencing reports. The sequence of a 27 kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals MOL1, NAT2, RPL30B,RSR1, CYS4, PEM1/CHO2, NSR1 genes and ten new open reading frames

The DNA sequence of a 26 677 bp fragment from the right arm of chromosome VII from Saccharomyces cerevisiae reveals 18 open reading frames (ORFs) longer than 300 bp. Eight ORFs correspond to previously characterized genes. G6620 is the 3′ end of the MOL1 gene coding for a polypeptide similar to stress-inducible proteins from Fusarium; G6630 is the NAT2 gene which encodes a methionine N-acetyltransferase; G6635 is the RPL30B gene coding for the ribosomal protein L30; G6658 is RSR1 encoding a ras-related protein; G6667 is CYS4, the gene for cystathionine β-synthase; G6670 is identical to ORF2 located close to CYS4; G6673 is PEM1/CHO2 encoding a phosphatidylethanolamine methyltransferase; G7001 is the NSR1 gene coding for a nuclear signal recognition protein. G6664 shares significant homology with the ORF YKR076w from chromosome XI. The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases. The sequence has been deposited in the EMBL data library under Accession Number X85807.     

The Sequence of a 36·7 kb Segment on the Left Arm of Chromosome IV from Saccharomyces cerevisiae Reveals 20 Non-overlapping Open Reading Frames (ORFs) Including SIT4, FAD1, NAM1, RNA11, SIR2, NAT1, PRP9, ACT2 and MPS1 and 11 New ORFs

A 36 688 bp fragment from the left arm of chromosome IV of Saccharomyces cerevisiae was sequenced. Sequence analysis identified 20 complete non-overlapping open reading frames (ORFs) of at least 100 amino acids. Nine of these correspond to previously identified and sequenced genes: SIT4/PPH1, FAD1, NAM1/MTF2, RNA11, SIR2/MAR1, NAT1/AAA1, PRP9, ACT2 and MPS1/RPK1. Three ORFs show homology to previously sequenced genes. One ORF exhibits a hypothetical yabO/yceC/yfiI family signature and one has the ATP-dependent helicase signature of the DEAD and DEAH box families. Six ORFs show no appreciable homology to any proteins in the database. One of these is identical to yeast expressed sequence tags and therefore corresponds to an expressed gene. In addition, two partial ORFs and 11 ORFs that are totally internal and are not likely to be functional were detected. The sequence has been submitted to the EMBL data library under Accession Number Z71781. © 1997 by John Wiley & Sons, Ltd.