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 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.

Keywords:

• κ-carrageenase
• Pichia pastoris expression system
• GAP promoter
• chaperones
• transcription factors

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Public Science and Technology Research Funds Projects of Ocean [grant number 201505022-5]; Shandong Science and Technology Development Project [grant number 2017YYSP003]; and Shandong Natural Science Foundation [grant number ZR2017MD006].