constitutive expression system for Pichia pastoris based on the PGK1 promoter

• Published in: Biotechnology letters 2016-03, Vol.38 (3), p.509-517
• Main Authors: Arruda, Andrelisse, Reis, Viviane Castelo Branco, Batista, Vinícius Daniel Ferreira, Daher, Bruno Sahim, Piva, Luiza Cesca, De Marco, Janice Lisboa, de Moraes, Lidia Maria Pepe, Torres, Fernando Araripe Gonçalves
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Bacteria; Bearing; Biochemistry; Biomedical and Life Sciences; Biotechnology; cell growth; Enzymes; Gene Expression; Gene Targeting - methods; Genetic engineering; Genetic Vectors; Genetics, Microbial - methods; glycolysis; Life Sciences; loci; Mathematical analysis; metabolic engineering; Metabolism; Microbiology; Original Research Paper; Phosphoglycerate Kinase - genetics; Pichia - enzymology; Pichia - genetics; Pichia pastoris; Plasmids; promoter regions; Promoter Regions, Genetic; Protein expression; Proteins; recombinant proteins; secretion; Vectors (mathematics); Yeast; Yeasts
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-015-2002-2

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.