Summary auto-generated
Researchers successfully engineered an industrial erythromycin-producing strain of Saccharopolyspora erythraea to stably express the Vitreoscilla haemoglobin gene (vhb). Initial attempts to express vhb using plasmid-based systems in S. lividans worked well, producing active hemoglobin, but direct transformation of S. erythraea with these plasmids failed. A conjugative shuttle vector was constructed but proved unstable in S. erythraea, likely due to homologous recombination with chromosomal erythromycin biosynthesis genes. The researchers overcame this by creating an integration vector using the Streptomyces phage φC31 attachment site for stable chromosomal insertion, with vhb expressed under the PmerR promoter. This chromosomally integrated construct proved genetically stable and successfully produced active hemoglobin in S. erythraea. Critically, shake-flask cultivations demonstrated that vhb-expressing strains produced approximately 60% more erythromycin than the original strain, with increased production rates particularly evident during days 1-3 and days 7-9 of fermentation, despite similar biomass production between strains.
Key findings
- Vitreoscilla haemoglobin gene was successfully integrated into the S. erythraea chromosome using φC31 attachment site-mediated integration, achieving stable maintenance over fermentation periods
- VHb was expressed in biologically active form in the recombinant S. erythraea strain, confirmed by CO-difference spectrum assays
- Erythromycin production increased by approximately 60% in the vhb-expressing strain compared to the wild-type, with no reduction in cell biomass
- Plasmid-based vhb expression systems proved unstable in S. erythraea due to recombination with chromosomal erythromycin biosynthesis genes, necessitating chromosomal integration approach
- The recombinant strain maintained genetic stability with >97% of cells retaining thiostrepton resistance marker throughout 9-day cultivation without selection pressure
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Abstract
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.