Enhancing the expression of recombinant κ-carrageenase in Pichia pastoris using dual promoters, co-expressing chaperones and transcription factors. Issue 2 (3rd March 2020)

• Record Type: Journal Article
• Authors: Yu, Yuan; Liu, Zhemin; Chen, Meng; Yang, Min; Li, Li; Mou, Haijin
• Is Part Of: Biocatalysis and biotransformation. Volume 38:Issue 2(2020)
• Page Start: 104
• Page End: 113
• Publication Date: 2020-03-03
• Subjects: κ-carrageenase -- Pichia pastoris expression system -- GAP promoter -- chaperones -- transcription factors; Enzymes -- Biotechnology -- Periodicals; Enzymes -- Industrial applications -- Periodicals; Biotransformation (Metabolism) -- Periodicals; 660.63
• DOI: 10.1080/10242422.2019.1655001
• Languages: English
• ISSNs: 1024-2422

Abstract

Abstract: In this study, with the aid of a constitutive promoter, and the co-expression of chaperone and transcription factor (TF) genes, the expression and enzymatic activity of recombinant κ-carrageenase in Pichia pastoris containing truncated κ-carrageenase gene cgkZΔPst (GS115/pPIC9K- cgkZΔPst ) was enhanced. The recombinant P. pastoris strain containing constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter enabled the expression of recombinant κ-carrageenase without methanol induction, the enzymatic activity was 2.73 U/mL after 96 h of shake flask fermentation at 22 °C. The enzymatic activity increased to 7.96 U/mL under methanol induction during P. pastoris growth, showing a 1.4-fold increase compared to that of the control group. With the co-expression of a series of chaperone genes and TFs that could promote protein folding, prevent protein aggregation, and counteract oxidative stress, the expression level of cgkZΔPst showed a 1.29- to 1.93-fold increase from that in the control group. The enzymatic activity of the recombinant κ-carrageenase increased to 7.07–7.70 U/mL. The use of the inducible P AOX1 in combination with the constitutive P GAP can further improve the productivity of recombinant κ-carrageenase. The rational selection of molecular chaperones and TFs can also promote recombinant κ-carrageenase secretion in P. pastoris . This work can be useful for the heterologous expression of other marine-origin glycoside hydrolases in P. pastoris .