Journal
BIOTECHNOLOGY LETTERS
Volume 35, Issue 12, Pages 2113-2119Publisher
SPRINGER
DOI: 10.1007/s10529-013-1312-5
Keywords
Constitutive promoter; GCW14 promoter; Lipase B; Pichia pastoris; TATA box; Transcriptional regulation
Categories
Funding
- National Natural Science Foundation of China [20976062, 31000027]
- Ministry of Science and Technology of the People's Republic of China [2011AA100905]
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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.
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