Integrative transformation of the dimorphic yeast Arxula adeninivorans LS3 based on hygromycin B resistance

Rösel, Harald; Kunze, Gotthard

doi:10.1007/s002940050322

Cited by 66 publications

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“…Transformation procedures, isolation and characterization of nucleic acids E. coli strains were transformed according to the procedure of Hanahan (1983) and A. adeninivorans transformants were generated according to Rösel and Kunze (1998). Plasmid DNA isolation, restriction fragment isolation, labelling of fragments and Southern transfer were carried out as previously described (Wartmann et al, 2002a).…”

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confidence: 99%

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

Böer¹,

Bode²,

Mock³

et al. 2009

Yeast

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The tannase-encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587-amino acid protein, preceded by an N-terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild-type and recombinant strains were essentially indistinguishable. A molecular mass of ∼320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35-40• C and a pH optimum at ∼6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wildtype strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks.

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confidence: 99%

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

Böer¹,

Bode²,

Mock³

et al. 2009

Yeast

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“…Target sites for integration should be carefully chosen to prevent or minimize negative effects on yeast physiology. If desired, the copy number of integrated DNA can be increased by choosing repeating target sequences within the genome, for example ribosomal DNA [63,64] or the delta sequences of the yeast retrotransposon Ty1 [65,66].…”

Section: Methods Of Recombinant Dna Technology In Yeastmentioning

confidence: 99%

Fermentation of Food by Means of Genetically Modified Yeast and Filamentous Fungi

Leisegang

Nevoigt

Spielvogel

et al. 2006

Genetically Engineered Food

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“…Transformation procedure, isolation and characterization of nucleic acids E. coli strains and A. adeninivorans cells were transformed according to Hanahan (1983), and Rösel and Kunze (1998), respectively. Isolation of plasmid DNA and restriction fragments, labelling of fragments and Southern transfer were carried out as previously described (Wartmann et al, 2002).…”

Section: E Böer Et Almentioning

confidence: 99%

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

Böer

Schröter

Bode

et al. 2009

Yeast

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In Arxula adeninivorans nitrate assimilation is mediated by the combined actions of a nitrate transporter, a nitrate reductase and a nitrite reductase. Single-copy genes for these activities (AYNT1, AYNR1, AYNI1, respectively) form a 9103 bp gene cluster localized on chromosome 2. The 3210 bp AYNI1 ORF codes for a protein of 1070 amino acids, which exhibits a high degree of identity to nitrite reductases from the yeasts Pichia anomala (58%), Hansenula polymorpha (58%) and Dekkera bruxellensis (54%). The second ORF (AYNR1, 2535 bp) encodes a nitrate reductase of 845 residues that shows significant (51%) identity to nitrate reductases of P. anomala and H. polymorpha. The third ORF in the cluster (AYNT1, 1518 bp) specifies a nitrate transporter with 506 amino acids, which is 46% identical to that of H. polymorpha. The three genes are independently expressed upon induction with NaNO 3 . We quantitatively analysed the promoter activities by qRT-PCR and after fusing individual promoter fragments to the phytase (phyK ) gene from Klebsiella sp. ASR1. The AYNI1 promoter was found to exhibit the highest activity, followed by the AYNT1 and AYNR1 elements. Direct measurements of nitrate and nitrite reductase activities performed after induction with NaNO 3 are compatible with these results. Both enzymes show optimal activity at around 42 • C and near-neutral pH, and require FAD as a co-factor and NADPH as electron donor.

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Integrative transformation of the dimorphic yeast Arxula adeninivorans LS3 based on hygromycin B resistance

Cited by 66 publications

References 0 publications

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

Fermentation of Food by Means of Genetically Modified Yeast and Filamentous Fungi

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

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