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Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine

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Abstract

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates l-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL l-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application.

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 21676121), the National first-class discipline program of Light Industry Technology and Engineering (No. LITE2018-18), the Six talent peaks project in Jiangsu Province (No. 2015-SWYY-006), and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (No. PPZY2015B146).

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Authors and Affiliations

  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Hai-Juan Hou, Jin-Song Gong, Yu-Xiu Dong, Heng Li, Hui Li, Zhen-Ming Lu, Xiao-Mei Zhang, Zheng-Hong Xu & Jin-Song Shi

  2. National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Zhen-Ming Lu & Zheng-Hong Xu

  3. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kongens Lyngby, Denmark

    Jiufu Qin

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  1. Hai-Juan Hou
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Hou, HJ., Gong, JS., Dong, YX. et al. Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine. Bioprocess Biosyst Eng 42, 1185–1194 (2019). https://doi.org/10.1007/s00449-019-02116-7

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