Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly-isolated phosphoglycerate kinase (PGK) gene.
A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1,251 base pairs encoding approximately 47-kDa polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alk1 encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis beta-galactosidase) was tested. Expression of P450alk1 gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining beta-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.
Masuda
Y
Y
Department of Agricultural Chemistry, University of Tokyo, Japan.
Park
S M
SM
Ohkuma
M
M
Ohta
A
A
Takagi
M
M
D000473:Alkanes; D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics
D000473:Alkanes; D004271:DNA, Fungal; D003577:Cytochrome P-450 Enzyme System; D010735:Phosphoglycerate Kinase; D001616:beta-Galactosidase