Over-expression system for secretory phospholipase D by Streptomyces lividans.
The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces lividans using a shuttle vector-pUC702-for Escherichia coli and S. lividans. The transformant was found to secrete a large amount of PLD (about 2.0x10(4) U/l, 42 mg/l) when cultured in a jar fermentor. Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5x10(4) U/l, 118 mg/l) after about 60 h. In contrast to the original producer, Stv. cinnamoneum, which secretes only a small amount of PLD (about 1.1x10(3) U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.
Ogino
C
C
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, 657-8501 Kobe, Japan.
Kanemasu
M
M
Hayashi
Y
Y
Kondo
A
A
Shimizu
N
N
Tokuyama
S
S
Tahara
Y
Y
Kuroda
S
S
Tanizawa
K
K
Fukuda
H
H
D039903:Actinobacteria / Q000201:enzymology*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose; D010739:Phospholipase D / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D010739:Phospholipase D; D005947:Glucose