Efficient Extracellular Production of Phospholipase D in Escherichia coli via Genetic and Process Engineering Modification

Abstract

Phospholipase D (PLD) is the key enzyme in the catalytic production of rare phospholipids including phosphatidylserine. It was considered a promising method via genetic manipulation for the heterologous production of PLD in the model chassis. Few works focused on the extracellular production of PLD in engineered microbes. Herein, genetic and process engineering modification strategies were developed to achieve secretory production of PLD in Escherichia coli. The N-terminal fusion secretion signal peptide OmpA and the plasmid pBAD-gⅢC with pBAD promoter were proven to be the most effective in promoting the secretory production of PLD. Given the limitation of the cell membrane, the regulation of the key protein expression in the cell membrane as well as the addition of surfactants, were explored to accelerate the secretory production of PLD further. It was indicated that adding 0.5% (w/v) Triton X-100 was more conducive to producing PLD. Finally, fed-batch fermentation was conducted, and the maximum extracellular PLD activity achieved was 33.25 U/mL, which was the highest level reported so far. Our work demonstrated the effectiveness of genetic and process engineering strategies for the secretory production of PLD in E. coli, which provided an alternative platform for the industrial production of PLD.

Keywords

Phospholipase D / Secretory production / Signal peptide / Surfactant

Supplementary Materials

The following supporting information can be found at: https://www.sciepublish.com/article/pii/494, Table S1: Strains used in this study; Table S2: Plasmids used in this study; Table S3: Primers and synthetic oligos used in this study; Figure S1. PLD production, cell growth and glycerol consumption by the engineered ECPLD4 after knockout of mrcB and dacB; Figure S2. Comparison of the effects of different surfactants with 0.1% and 0.5% addition level on PLD production.

Author Contributions

H.L.: Conceptualization, Data curation; H.L. and Y.Y. Writing-original draft; Y.Y., T.W. and Y.N.: Investigation, Methodology; L.D. and F.W.: Writing-review & editing, Supervision, Funding acquisition, Project administration.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This work was funded by the National Key Research and Development Program of China (2022YFC2106100), the National Natural Science Foundation of China (22208011, 20308020).

Declaration of Competing Interest

The authors declared that they have no conflicts of interest to this work.

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