pTONA5: a hyperexpression vector in Streptomycetes.
We constructed the Streptomyces hyperexpression vector pTONA5 based on pIJ702 vector; it includes a metalloendopeptidase (SSMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from Streptomyces aureofaciens TH-3. The vector contains recognition sites for restriction enzymes NdeI and EcoRI/XbaI/HindIII between the promoter and terminator to facilitate heterologous gene cloning. The plasmids were transferred from Escherichia coli to streptomycetes via conjugation from oriT; the transformants were able to be selected using kanamycin and/or thiostrepton. The SSMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. We constructed expression plasmids including three Streptomyces aminopeptidases-leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP)-using the pTONA5 vector and Streptomyces lividans. Although they lack signal peptides for secretion, PAP and APP were secreted at high levels in the culture broth.
Hatanaka
Tadashi
T
Research Institute for Biological Sciences, Okayama, Kaga-gun, Okayama, Japan. hatanaka@bio-ribs.com
Onaka
Hiroyasu
H
Arima
Jiro
J
Uraji
Misugi
M
Uesugi
Yoshiko
Y
Usuki
Hirokazu
H
Nishimoto
Yukifumi
Y
Iwabuchi
Masaki
M
D000626:Aminopeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D013728:Terminator Regions, Genetic
D005982:Glutathione Transferase; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; C022450:prolyl aminopeptidase; C022750:X-Pro aminopeptidase