文献类型：期刊文献

英文题名：Increased Expression of Recombinant Chitosanase by Co-expression of Hac1p in the Yeast Pichia pastoris

作者：Han, Minghai Wang, Weixian Gong, Xun Zhou, Jianli Xu, Cunbin Li, Yinfeng

第一作者：韩铭海

通信作者：Han, MH[1]|[144402eaf094eaf27b6d2]韩铭海;

机构：[1]Guizhou Inst Technol, Coll Food & Pharmaceut Engn, Guiyang, Peoples R China;[2]South China Normal Univ, Guangdong Prov Key Lab Fermentat & Enzyme Engn, Guangzhou, Peoples R China

第一机构：贵州理工学院食品药品制造工程学院

通信机构：corresponding author), Guizhou Inst Technol, Coll Food & Pharmaceut Engn, Guiyang, Peoples R China.|贵州理工学院食品药品制造工程学院;贵州理工学院;

年份：2021

卷号：28

期号：12

起止页码：1434-1441

外文期刊名：PROTEIN AND PEPTIDE LETTERS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000735388700012)】；

基金：This study has been funded by the National Natural Science Foundation of China (Grant No. 31760022) and by Guizhou Provincial Science and Technology Foundation (Grant No. [2019]1146).

语种：英文

外文关键词：Unfolded protein response; Hac1p; Pichia pastoris; chitosanase; secretory expression; co-expression

摘要：Background: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR). Objective: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX(1) (PAOX(1)) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris. Method: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZ alpha A, and the resulting pPICZ alpha A/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1(i) cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1(i) cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDS-PAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. Results: Hac1p co-expression with PAOX(1) enhanced rCBS secretion by 41% at 28 degrees C. Although the level of UPR resulting from Hac1p co-expression with PAOX(1) was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX(1) further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression with PAOX(1) or PGAP promoted rCBS secretion from 22 to 30 degrees C and raised the optimum induction temperature. Conclusion: The study indicated that Hac1p co-expression with PAOX(1 )or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.