Abstract
Objectives
To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.
Results
P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.
Conclusions
A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.
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Acknowledgments
This work was financially supported by FAPDF (Grant# 193.000.582/2009) and CNPq. The authors are thankful to Alexsandro Sobreira Galdino for his assistance in the zymogram methodology.
Supporting Information
Supplementary Fig. 1–Sequence of the 270-bp synthetic DNA fragment used to remove the PvuI and SmaI sites present in the kan coding sequence
Supplementary Fig. 2–Relevant sequences required for heterologous gene expression present on pPICK2
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Arruda, A., Reis, V.C.B., Batista, V.D.F. et al. A constitutive expression system for Pichia pastoris based on the PGK1 promoter. Biotechnol Lett 38, 509–517 (2016). https://doi.org/10.1007/s10529-015-2002-2
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DOI: https://doi.org/10.1007/s10529-015-2002-2