title: Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.
abstract: 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 α-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.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Mengmeng
authors: M
authors: Huang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Yanyun
authors: Y
authors: Gao
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Xiangshan
authors: X
authors: Zhou
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.|Shanghai Collaborative Innovation Center for Biomanufacturing, 130 Meilong Road, Shanghai, 200237, China.
authors: Yuanxing
authors: Y
authors: Zhang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. cmh022199@ecust.edu.cn.
authors: Menghao
authors: M
authors: Cai
mesh_terms: D017398:Alternative Splicing; D000681:Amylases / Q000737:chemistry; D003062:Codon; D005656:Fungal Proteins / Q000737:chemistry*; D018628:Gene Dosage; D005786:Gene Expression Regulation; D006868:Hydrolysis; D007218:Industrial Microbiology / Q000379:methods; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D060888:Real-Time Polymerase Chain Reaction; D013213:Starch / Q000737:chemistry; D013696:Temperature; D056811:Unfolded Protein Response; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D003062:Codon; D005656:Fungal Proteins; D013213:Starch; D000681:Amylases; D000516:alpha-Amylases
keywords: HAC1p; Heterologous expression; P. pastoris; Raw-starch hydrolyzing α-amylase; Unfolded protein response