Key regulatory elements of a strong constitutive promoter, P GCW14, from Pichia pastoris

The promoter of the Pichia pastoris gene GCW14 is strong and constitutive when glycerol is the available carbon source. To identify the cis-acting elements of this promoter (P GCW14), we constructed expression plasmids where the enhanced green fluorescent protein gene was fused to a series of mutants of P GCW14. We identified one negative (-114 to -94) and three positive regulatory regions (-426 to -152, -134 to -114, -94 to -77). The TATA box of P GCW14 was located at -48. One negative and four positive regulatory sites were identified combining error-prone PCR and directed mutation. The mutated promoter, M+20, with an increased promoter activity, was then used to express the gene for lipase B from Candida antarctica.