[Expression of Vitreoscilla hemoglobin gene in Streptomyces avermitilis]

Two expression vectors, pWY101 and pWY102, were constructed by cloning Vitreoscilla hemoglobin gene(vhb) with its native oxygen-regulated promoter into E. coli-Streptomyces shuttle vector pIJ653. They were introduced into Streptomyces avermitilis, but Western blotting experiment failed to detect vhb gene expression. pHZ1252 is another shuttle vector for expressing VHb, in which vhb structural gene is controlled by a strong, thiostrepton-inducible Streptomyces promoter PtipA. pHZ1252 was transformed into S. avermitilis and expressed VHb which had biological activity after thiostrepton induction. pHZ1252 was structurally unstable in S. avermitilis, occurring deletion recombination. However, the rest part of pHZ1252 was stable in S. avermitilis and still contained vhb gene and PtipA. Plasmid pHZ1252 isolated from S. avermitilis was unable to transform E. coli, showing the loss of part of E. coli plasmid from pHZ1252.