Cloning and Expression of the Tyrosinase Gene from Streptomyces antibioticus in Streptomyces lividans

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Abstract
In 2 separate studies, a BclI-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from S. antibioticus DNA into 2 SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using S. lividans 66 as the host. The fragment (1.55 kilobases) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has 2 non-essential BclI sites) to generate 4 new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All 6 plasmids conferred melanin production (Mel+ phenotype) on their host. As in the S. antibioticus parent, S. lividans strains carrying the tyrosinase synthesis gene possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of S. antibioticus; the majority remained intracellular in the S. lividans clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in S. antibioticus. Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for BglII, SphI and SstI that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any of these sites abolished the Mel+ phenotype. Thus, pIJ702 is a useful cloning vector with insertional inactivation of the Mel+ character as the basis of clone recognition.