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Over-expression system for secretory phospholipase D by Streptomyces lividans
C Ogino1, M Kanemasu, Y Hayashi
1Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, 657-8501 Kobe, Japan.
Applied Microbiology and Biotechnology
|January 24, 2004
Summary
Researchers developed a highly efficient system for producing phospholipase D (PLD) using genetic engineering. This novel method significantly enhances PLD secretion in Streptomyces lividans compared to the original producer, Streptoverticillium cinnamoneum.
Area of Science:
- Biotechnology
- Molecular Biology
- Microbial Fermentation
Background:
- Phospholipase D (PLD) is an enzyme with various industrial applications.
- Streptoverticillium cinnamoneum naturally produces PLD, but in low yields.
- Efficient production of secretory PLD is desirable for biotechnological applications.
Purpose of the Study:
- To develop a heterologous expression system for enhanced production of phospholipase D (PLD).
- To optimize culture conditions for maximizing PLD secretion in a recombinant host.
Main Methods:
- Cloning the structural gene and promoter region of PLD from Streptoverticillium cinnamoneum.
- Introducing the gene construct into Streptomyces lividans using a shuttle vector.
- Optimizing fermentation parameters including initial glucose concentration and nutrient feeding.
Main Results:
- The transformant Streptomyces lividans secreted significantly higher amounts of PLD (up to 5.5x10(4) U/l, 118 mg/l) compared to the wild-type producer (1.1x10(3) U/l, 2 mg/l).
- Optimal conditions involved an initial glucose concentration of 17.5 g/l and controlled feeding of carbon and nitrogen sources.
- Maximum PLD yield was achieved after approximately 60 hours of cultivation.
Conclusions:
- Heterologous expression of PLD in Streptomyces lividans provides a markedly more efficient system for secretory PLD production.
- Optimized fermentation strategies are crucial for maximizing enzyme yield in recombinant microbial systems.
- This engineered system offers a promising platform for industrial-scale PLD manufacturing.