Expression Of Vitreoscilla Hemoglobin Gene In Streptomvces Diastatochromogenes1628

Toyocamycin was an important member of nucleoside antibiotics family,which displayed diverse bioactivities and distinguished application potentials forbiological control of plant diseases in the agriculture field. In this paper, in order toalleviate the dissolved oxygen (DO) limitation and improve the toyocamycinproduction of strain Streptomyces diastatochromogenes1628, we attempt to expressthe vgb gene encoding Vitreoscilla hemoglobin in S. diastatochromogenes1628.Firstly, we tried three genetic transformation methods for S.diastatochromogenes1628: PEG-mediated plasmid transformation of protoplast,electroporation and intergeneric conjugation, and optimized their conditions. In thisstudy, we optimized some key factors necessary for protoplast formation,regeneration and transformation of S. diastatochromogenes1628. When myceliumwas cultivated in CP medium with1%glycine, harvested at48h old, and then treatedwith3mg lysozyme/mL in P buffer for1h, the greatest regeneration frequency(42.5%) of protoplasts was obtained. By using1×10~9/mL protoplasts with PEG(polyethylene glycol)1000at concentration of30%(w/v), the best performance ofprotoplast transformation efficiency was4.8×10~3transformants per μg plasmidpIJ702. As for electroporation, we optimized the follow factors: the treatment ofintact cells with lysozyme was suitable for electroporation, the highest efficiency(3±0.4)×10~4/μg DNA occurred with2μg DNA and1×10~9cells under12kV/cm. Theoptimal conditions for the intergeneric conjugation were as follows: freshlygerminated spores were prepared as recipient, and mixed with donor ET12567withthe ratio of108:108, and then overlaid the plate with1mL water containing50μgapramycin and50μg nalidixic acid after16-20h, finally we got348exconjugants.Secondly, we used gfp gene as the reporter gene to test the transcription activityof erythromycin resistance gene promoter PermE*of integrative plasmid pIB139ofStreptomyces. Next, the promoter PermE*was used to heterologous expression ofgene vgb in S. diastatochromogenes1628. Green fluorescent of recombinant strain 1628-GFP was detected by using fluorescent microscopy. The result showed that thepromoter PermE*was efficient for heterologous gene expression in S.diastatochromogenes1628. The vgb gene was also successfully expressed inengineered strain1628-VHB by the CO-difference spectrum analysis. The engineeredstrain1628-VHB exhibited better effects on the improvement of toyocamycinproduction under all diverse DO conditions. Specially, the strain1628-VHB showed a48.9%and104.5%increase in toyocamycin production under middle and highlimitation DO condition, respectively, comparing with the corresponding values ofcontrol strain1628. We isolated one recombinant strain with the highest toyocamycinproduction through40recombinants, which showed a29.2%and96.7%increase inproduction under ordinary and middle limitation DO condition, respectively.Meanwhile, the engineered strain1628-VHB was genetically stable by the PCR andtoyocamycin yield test from different generation strain. To our knowledge, it is thefirst report on the improvement of toyocamycin production of strain1628viaexpression of vgb gene. Therefore, this study will pave the way for the scale-upindustrial production of toyocamycin in future.