Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a ''PnitA-NitR'' system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, -caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as Ϸ40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the P nitA-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes.
Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19 -2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.
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