Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla haemoglobin gene (vhb).
Several Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an industrial erythromycin-producing strain of Saccharopolyspora erythraea. Cloning of vhb under the control of either the thiostrepton-inducible PtipA promoter or the constitutive PermE* promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 transformed with these constructs. However, theplasmids could not be transformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyces shuttle vector comprised of vhb under the control of the PermE* promoter, the Streptomyces plasmid pIJ350 origin of replication, the thiostrepton-resistance gene (tsr) for selection, and the oriT region which is necessary for conjugal transfer. Increased plasmid stability in Sac. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage phi C31 attachment site for chromosomal integration and vhb expressed under the PmerR promoter and was stably maintained in the chromosome of Sac. erythraea. Shake-flask cultivations of the transformed Sac. erythraea strain with the chromosomally integrated vhb gene show that vhb is expressed 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.
Brünker
Peter
P
Institute of BiotechnologyETH Zürich, 8093 ZürichSwitzerland.
Minas
Wolfgang
W
Institute of BiotechnologyETH Zürich, 8093 ZürichSwitzerland.
Kallio
Pauli T
PT
Institute of BiotechnologyETH Zürich, 8093 ZürichSwitzerland.
Baile
James E
JE
Institute of BiotechnologyETH Zürich, 8093 ZürichSwitzerland.
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D017931:DNA Primers / Q000235:genetics; D004917:Erythromycin / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics*; D006454:Hemoglobins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D016930:Saccharopolyspora / Q000235:genetics* / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000821:virology; D054793:Truncated Hemoglobins
D001426:Bacterial Proteins; D017931:DNA Primers; D006454:Hemoglobins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D004917:Erythromycin