Journal
ELECTRONIC JOURNAL OF BIOTECHNOLOGY
Volume 50, Issue -, Pages 1-9Publisher
UNIV CATOLICA DE VALPARAISO
DOI: 10.1016/j.ejbt.2020.12.002
Keywords
Streptomyces chromofuscus; Phospholipase D (PLD); Soluble expression; Lactose induction; Combinatorial optimization
Categories
Funding
- China Postdoctoral Science Foundation [2016M600417, 2017T100373]
- 333 project of Jiangsu Province [BRA2017458]
- Six Talent Peaks Project in Jiangsu Province [2015-JY-016]
- Priority Academic Program Development of Jiangsu Higher Education Institutions, PAPD
- Doctorate Fellowship Foundation of Nanjing Forestry University
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By optimizing the gene and cultivation conditions, high-level soluble expression of PLD was achieved in E. coli, enabling the production of high-activity PLD. The use of inexpensive cultivation media and materials from waste sources can optimize expression efficiency.
Background: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. Results: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 +/- 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 +/- 1.49 U/mL by feeding lactose and inducing at 20 degrees C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 +/- 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. Conclusions: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD. (C) 2020 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V.
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