GENETIC-ENGINEERING OF AN INDUSTRIAL STRAIN OF SACCHAROPOLYSPORA-ERYTHRAEA FOR STABLE EXPRESSION OF THE VITREOSCILLA HEMOGLOBIN GENE (VHB)

Several Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an indust rial erythromycin-producing strain of Saccharopolyspora erythraea. Clo ning of vhb under the control of either the thiostrepton-inducible Pti pA promoter or the constitutive PermE* promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 t ransformed with these constructs, however, the plasmids could not be t ransformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyce s shuttle vector comprised of vhb under the control of the PermE* prom oter, the Streptomyces plasmid pIJ350 origin of replication, the thios trepton-resistance gene (tsr) for selection, and the oriT region which is necessary far conjugal transfer. Increased plasmid stability in Sa c. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage phi C31 atta chment site for chromosomal integration and vhb expressed under the Pm erR promoter and was stably maintained in the chromosome of Sac. eryth raea, Shake-flask cultivations of the transformed Sac. erythraea strai n with the chromosomally integrated vhb gene show that vhb is expresse d in an active form. The corresponding amount of erythromycin produced in the vhb-expressing strain was approximately 60% higher relative to the original VHb-negative strain.