Development of a genetic system in chitinase-producing streptomyces and the application of an allosamidin-insensitive chitinase gene to homologous overexpression.
A transformation system for Streptomyces sp. AJ9463 strain (allosamidin producer) was successfully developed using protoplasts and a PEG-mediated method. To prepare protoplasts, the concentration of glycine and sucrose in YEME medium were optimized to 0.5% (w/v) and 34.0% (w/v), respectively. When the protoplasts of Streptomyces sp. AJ9463 were transformed with pUWL-KS, transformants could be obtained at a high efficiency of 7.0 x 10(4) transformants per microg DNA. To ensure that the transformation system worked properly, we then constructed a constitutive expression vector pYK1, in which the ermE* promoter drives transcription of the allosamidin-insensitive chitinase gene, chiIS. Although no transformant could be obtained by the genetic system using pYK1 isolated from Escherichia coli DH5alpha, pYK1 isolated from the methylase-deficient mutant E. coli SCS110, could be introduced into Streptomyces sp. AJ9463. This indicates that Streptomyces sp. AJ9463 has a methylation-specific restriction system, and that the chiIS and/or ermE* promoter region of pYK1 includes a restriction site of its endonuclease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that pYK1 in Streptomyces sp. AJ9463 started to obviously express ChiIS from 14-h. Moreover, the pYK1-introduced strain gave a five-fold higher chitinase activity than the wild-type, suggesting that this system can be widely applied for the overexpression and gene functional analysis.
Kawachi
Ryu
R
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan. kawachi@brs.nihon-u.ac.jp
Koike
Yuko
Y
Watanabe
Yumi
Y
Nishio
Toshiyuki
T
Sakuda
Shohei
S
Nagasawa
Hiromichi
H
Oku
Tadatake
T
D000117:Acetylglucosamine / Q000031:analogs & derivatives*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D019175:DNA Methylation; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D008780:Methyltransferases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017385:Sequence Homology; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014312:Trisaccharides
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014312:Trisaccharides; C054851:allosamidin; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D002688:Chitinases; D000117:Acetylglucosamine