Enhancing the expression of recombinant κ-carrageenase in Pichia pastoris using dual promoters, co-expressing chaperones and transcription factors

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 PAOX1 in combination with the constitutive PGAP 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.