Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris.
We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (P(PGK1)) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element. In the 3' downstream region we identified a tripartite element 5'-TAG-TAGT-TTT-3', which is supposed to be important for transcription termination. As in other yeasts, the PGK1 gene from P. pastoris is present as a single-copy gene. Northern blot analysis revealed that the gene is transcribed as a 1.5 kb mRNA; when cells are grown on glucose the levels of this mRNA are increased two-fold in comparison to cells grown on glycerol. The transcriptional regulation of this gene by the carbon source was further confirmed when the alpha-amylase gene from Bacillus subtilis was placed under the control of P(PGK1): higher levels of expression were obtained when cells were grown on glucose as compared to glycerol and methanol. Preliminary results related to the strength of P(PGK1) show that it represents a potential alternative to constitutive heterologous expression in P. pastoris.
de Almeida
João Ricardo Moreira
JR
Laboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF 70910-900, Brazil.
de Moraes
Lídia Maria Pepe
LM
Torres
Fernando Araripe Gonçalves
FA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D000516:alpha-Amylases / Q000235:genetics
D003062:Codon; D004271:DNA, Fungal; D005656:Fungal Proteins; D010735:Phosphoglycerate Kinase; D000516:alpha-Amylases