Enhance protein secretion pathway and energy metabolism to improved protein activity in recombinant Pichia pastoris

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal Pub Date : 2025-09-23 DOI:10.1016/j.bej.2025.109935

Hao Shi , Hao Chen , Tianwei Tan

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Abstract

Methanol, as an inexpensive and clean energy source in the context of carbon neutrality and the methylotrophic yeast Pichia pastoris offers a unique pathway for methanol utilization. This study engineered the secretory pathway in Pichia pastoris GS115 to optimize the methanol-driven production of Penicillium amazonicum-derived glucose oxidase (GOX). Initially, high-copy α-SP was identified as the dominant expression strain from the six signal peptides. Subsequently, systematic overexpression of secretory pathway-related factors revealed that signal α-SP-mediated ER membrane targeting (SEC12) and its synergistic interplay with ER-resident folding (ERO1) machinery critically determined secretion capacity and methanol bioconversion efficiency. PPG-ERO1-SEC12 achieved a yield of 198.73 U/mL in shake-flask cultures. An imbalance between central carbon metabolism and energy level was found, which revealed low energy utilization efficiency and prolonged oxidative stress. Key energy metabolism genes (noxE, FDH1, PYK1, and IDH1) were overexpressed and enhanced the activities of key enzymes (AOX, FLD, FDH, CAT, and IDH) in the energy metabolism pathway, but also increased protein activity by 3.2-fold. Finally, this approach achieved a glucose oxidase yield of 2604.20 U/mL (5.09 g/L protein titer) in 5-L batch fermentation, with a specific activity of 511.63 U/mg and > 95 % purity. This groundbreaking strategy not only offers valuable technical insights for industrial applications but also has the potential to reduce costs and enhance efficiency.

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改善重组毕赤酵母的蛋白质分泌途径和能量代谢，提高蛋白质活性

甲醇作为一种廉价的清洁能源，在碳中和的背景下，毕赤酵母为甲醇的利用提供了一条独特的途径。本研究设计了毕赤酵母GS115的分泌途径，以优化甲醇驱动的亚马逊青霉衍生葡萄糖氧化酶（GOX）的生产。最初，高拷贝α-SP被鉴定为6种信号肽的优势表达菌株。随后，分泌途径相关因子的系统性过表达表明，信号α- sp介导的ER膜靶向（SEC12）及其与ER-resident folding （ERO1）机制的协同相互作用对分泌能力和甲醇生物转化效率至关重要。PPG-ERO1-SEC12在摇瓶培养中产率为198.73 U/mL。中心碳代谢与能量水平不平衡，能量利用效率低，氧化应激持续时间长。关键能量代谢基因（noxE、FDH1、PYK1、IDH1）过表达，能量代谢途径关键酶（AOX、FLD、FDH、CAT、IDH）活性增强，蛋白质活性提高3.2倍。最后，该方法在5-L批量发酵中获得了2604.20 U/mL（5.09 g/L蛋白滴度）的葡萄糖氧化酶产率，比活性为511.63 U/mg，纯度为>； 95 %。这一突破性的战略不仅为工业应用提供了有价值的技术见解，而且具有降低成本和提高效率的潜力。

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来源期刊

Biochemical Engineering Journal 工程技术-工程：化工

CiteScore

7.10

自引率

5.10%

发文量

380

审稿时长

34 days

期刊介绍： The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology. The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields: Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics Biosensors and Biodevices including biofabrication and novel fuel cell development Bioseparations including scale-up and protein refolding/renaturation Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells Bioreactor Systems including characterization, optimization and scale-up Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis Protein Engineering including enzyme engineering and directed evolution.