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Published on: June 27, 2017
pTONA5: a hyperexpression vector in Streptomycetes.
Tadashi Hatanaka1, Hiroyasu Onaka, Jiro Arima
1Research Institute for Biological Sciences, Okayama, Kaga-gun, Okayama, Japan. hatanaka@bio-ribs.com
Protein Expression and Purification
|September 26, 2008
Summary
A new Streptomyces hyperexpression vector, pTONA5, was developed for cloning and expressing genes. This vector enables high-level secretion of proline aminopeptidase (PAP) and aminopeptidase P (APP) in Streptomyces lividans.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Streptomyces species are crucial for producing secondary metabolites and industrial enzymes.
- Efficient expression systems are needed to harness the biosynthetic potential of Streptomyces.
- Existing vectors may have limitations in heterologous gene expression and protein secretion.
Purpose of the Study:
- To construct a novel Streptomyces hyperexpression vector, pTONA5.
- To evaluate the functionality of the pTONA5 vector for heterologous gene expression.
- To assess the secretion efficiency of specific Streptomyces aminopeptidases using the pTONA5 vector.
Main Methods:
- Construction of the pTONA5 vector using pIJ702, incorporating a metalloendopeptidase (SSMP) promoter and terminator.
- Ligation of restriction enzyme sites (NdeI, EcoRI/XbaI/HindIII) for gene cloning.
- Conjugation of plasmids from Escherichia coli to Streptomyces species, with selection markers (kanamycin, thiostrepton).
- Construction of expression plasmids for leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP).
- Expression and analysis of PAP and APP secretion in Streptomyces lividans.
Main Results:
- The pTONA5 vector was successfully constructed, featuring a constitutive SSMP promoter active under specific conditions (phosphate and glucose).
- The vector facilitates heterologous gene cloning via multiple restriction enzyme sites.
- Proline aminopeptidase (PAP) and aminopeptidase P (APP) were secreted at high levels in the culture broth of Streptomyces lividans, despite lacking signal peptides.
- The SSMP promoter demonstrated constitutive function in the presence of inorganic phosphate and glucose.
Conclusions:
- The pTONA5 vector is a valuable tool for Streptomyces gene expression and protein production.
- High-level secretion of specific aminopeptidases (PAP, APP) can be achieved using the pTONA5 system in Streptomyces lividans.
- The developed vector system holds potential for the biotechnological production of enzymes and other valuable compounds from Streptomyces.
