Abstract
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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Acknowledgments
This work was supported by National Natural Science Foundation of China (No. 20976062 and 31000027) and the Ministry of Science and Technology of the People’s Republic of China (National “863” Project No. 2011AA100905).
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Fig. S1
Structure of plasmid pGEL. Supplementary material 1 (TIFF 3795 kb)
Fig. S2
Relative activity of 22 promoters with random mutations. The promoter activities were normalized relative to the activity obtained from the wild-type (WT) promoter (dark column). Supplementary material 2 (TIFF 7857 kb)
Table S1
Supplementary material 3 (DOCX 19 kb)
Table S2
Supplementary material 4 (DOCX 16 kb)
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Zhang, X., Zhang, X., Liang, S. et al. Key regulatory elements of a strong constitutive promoter, P GCW14 , from Pichia pastoris . Biotechnol Lett 35, 2113–2119 (2013). https://doi.org/10.1007/s10529-013-1312-5
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DOI: https://doi.org/10.1007/s10529-013-1312-5