The ALEU2 gene--a new component for an Arxula adeninivorans-based expression platform.
The ALEU2 gene, encoding beta-isopropylmalate dehydrogenase, was isolated from the non-conventional yeast Arxula adeninivorans. The isolated gene harbours an open reading frame of 1086 bp, encoding a putative protein of 362 amino acids. The derived protein sequence shares a high degree of homology with other fungal beta-isopropylmalate dehydrogenases thus confirming the identity of the gene. The isolated ALEU2 gene was tested for its suitability to complement the auxotrophy of an A. adeninivorans aleu2 host. For this purpose the plasmid pAL-ALEU2m which contains the ALEU2 gene as a selection marker and the 25S rDNA for targeting was employed in transformation experiments. Transformants harboured a single copy of the heterologous DNA and were found to be mitotically stable. For assessment of heterologous gene expression, two model genes were incorporated into the vector: the GFP gene, encoding intracellular green fluorescent protein, and the HSA gene, encoding the secreted human serum albumin. For expression control, both gene sequences were fused to the constitutive A. adeninivorans-derived TEF1 promoter and the Saccharomyces cerevisiae-derived PHO5 terminator. In the respective recombinant strains the GFP was localised in the cytoplasm, whereas more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials using a 200-ml shake flask, maximal HSA product levels were observed after 96 h of cultivation.
Wartmann
Thomas
T
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, 06466 Gatersleben, Germany.
Stoltenburg
Regina
R
Böer
Erik
E
Sieber
Heike
H
Bartelsen
Oliver
O
Gellissen
Gerd
G
Kunze
Gotthard
G
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000235:genetics; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005656:Fungal Proteins; D005819:Genetic Markers; D008164:Luminescent Proteins; D012709:Serum Albumin; D049452:Green Fluorescent Proteins; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase