Enhancing the expression of recombinant kappa-carrageenase in Pichia pastoris using dual promoters, co-expressing chaperones and transcription factors
文献类型： 外文期刊
作者： Yu, Yuan 1 ; Liu, Zhemin 2 ; Chen, Meng 2 ; Yang, Min 2 ; Li, Li 2 ; Mou, Haijin 2 ;
作者机构： 1.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Qingdao, Shandong, Peoples R China
2.Ocean Univ China, Coll Food Sci & Engn, Qingdao 266003, Shandong, Peoples R China
关键词： kappa-carrageenase; Pichia pastoris expression system; GAP promoter; chaperones; transcription factors
期刊名称：BIOCATALYSIS AND BIOTRANSFORMATION （ 影响因子：2.181； 五年影响因子：2.104 ）
ISSN： 1024-2422
年卷期：
页码：
收录情况： SCI
摘要： 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 kappa-carrageenase in Pichia pastoris containing truncated kappa-carrageenase gene cgkZ Delta Pst (GS115/pPIC9K-cgkZ Delta Pst) was enhanced. The recombinant P. pastoris strain containing constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter enabled the expression of recombinant kappa-carrageenase without methanol induction, the enzymatic activity was 2.73 U/mL after 96 h of shake flask fermentation at 22 degrees 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 cgkZ Delta Pst showed a 1.29- to 1.93-fold increase from that in the control group. The enzymatic activity of the recombinant kappa-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 kappa-carrageenase. The rational selection of molecular chaperones and TFs can also promote recombinant kappa-carrageenase secretion in P. pastoris. This work can be useful for the heterologous expression of other marine-origin glycoside hydrolases in P. pastoris.
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