We constructed the Streptomyces hyperexpression vector pTONA5a. It includes a metallo-endopeptidase (SCMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from S. aureofaciens TH-3. The pTONA5a plasmid, which also contains oriT/mob, can be transferred into streptomycetes through conjugation with Escherichia coli. The resulting transformants can be selected with kanamycin and/or thiostrepton. The SCMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. Using pTONA5a and S. lividans, we constructed a series of plasmids carrying Streptomyces genes encoding aminopeptidases, asparaginases, and proteases. It is particularly interesting that despite the lack of N-terminal Sec and Tat secretion signals, some of these enzymes were secreted into the medium in high amounts. A useful method for the industrial production of these enzymes was constructed until the unusual extracellular expression remained unknown.
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