文献类型：期刊文献

英文题名：Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris

作者：Huang, Mengmeng[1];Gao, Yanyun[1];Zhou, Xiangshan[1];Zhang, Yuanxing[1,2];Cai, Menghao[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2017

卷号：40

期号：3

起止页码：341

外文期刊名：BIOPROCESS AND BIOSYSTEMS ENGINEERING

收录：;EI(收录号：20164502974397);WOS:【SCI-EXPANDED(收录号:WOS:000394372600002)】；

基金：This work was financially supported by the Chinese National High Technology Research and Development Program (No. 2014AA093501; 2012AA092103), Fundamental Research Funds for the Central Universities (22A201514040), Special Project for "Significant New Drugs Development" from the Ministry of National Science and Technology (2012ZX09102101) and Grants of Young and Middle-aged Leading Science and Technology Innovation Talents from Ministry of Science and Technology of China.

语种：英文

外文关键词：Unfolded protein response; HAC1p; Heterologous expression; P. pastoris; Raw-starch hydrolyzing alpha-amylase

摘要：Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing alpha-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.