Improved performance of toxic Streptomyces phospholipase D expression by combinatorial optimization in the trehalase-deficient Escherichia coli

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal Pub Date : 2025-01-14 DOI:10.1016/j.bej.2025.109640

Shaofeng Chen , Weide Xiong , Xiaoyu Lin , Xuejun Wu , Chuanyi Yao , Yinghua Lu , Xueping Ling

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引用次数: 0

Abstract

Phospholipase D (PLD) has a unique phosphatidyl catalytic site and is capable of synthesizing a variety of active phospholipids, such as phosphatidylserine, for use in the food industry. However, the microbial production of PLD is limited by its cytotoxicity. In this study, the constructed trehalase-deficient strains by CRISPR-Cas9 showed increased intracellular trehalose content and high performance in PLD production. Molecular dynamics (MD) simulations suggested that trehalose could stabilize the natural conformation of PLD, increase its solubility and expression. High PLD production (47.63 U/mL) was achieved in the recombinant strain E. coli BW25113 ΔtreA ΔtreC ΔtreF using an optimized culture strategy, with an efficiency of 5.95 U/mL/h—the highest level reported in shake flask cultures to date. Our results showed that the accumulated endogenous trehalose improved the salt tolerance of cells to alleviate PLD cytotoxicity and promote continuous PLD expression. Thus, the trehalase-deficient Escherichia coli expression system shows great potential for application in industrial PLD production.

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利用组合优化提高了海藻酶缺陷大肠杆菌中毒性链霉菌磷脂酶D的表达性能

磷脂酶D （PLD）具有独特的磷脂酰催化位点，能够合成多种活性磷脂，如磷脂酰丝氨酸，用于食品工业。然而，PLD的微生物生产受到其细胞毒性的限制。在本研究中，通过CRISPR-Cas9构建的海藻糖酶缺陷菌株显示出细胞内海藻糖含量增加和PLD生产的高性能。分子动力学（MD）模拟表明海藻糖可以稳定PLD的天然构象，增加其溶解度和表达。采用优化的培养策略，重组菌株BW25113 ΔtreA ΔtreC ΔtreF的PLD产量最高（47.63 U/mL），效率为5.95 U/mL/h，是迄今为止摇瓶培养的最高水平。我们的研究结果表明，积累的内源海藻糖提高了细胞的耐盐性，减轻了PLD的细胞毒性，促进了PLD的持续表达。因此，缺乏海藻酸酶的大肠杆菌表达系统在工业PLD生产中具有很大的应用潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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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.