Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter

We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P-GAP) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P-GAP in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P-AOX1) in methanol-grown cells. As an example of the use of P-GAP, we showed that beta-lactamase synthesized under transcriptional control of P-GAP is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P-GAP has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P-AOX1 in P. pastoris.