Cloning And Expression Of Vitreoscilla Hemoglobin Gene (vgb) In Penicillium Chrysogenum

The Vitreoscilla hemoglobin gene(vgb) with inartificial promoter was cloned and expressed in E.coli and penicillium chrysogenum.On the works of the predecessor,we know that the temperature induced promotor, the sucrose induced promoter to vgb expressed not very well in the E.coli. Therefore we looks up the naturally induced promoter to vgb - low oxygen promoter from Gene Bank, then synthesized the low oxygen promoter sequence and added into the vgb structural gene then cloned to pUC18, has constructed recombined plasmid pUC18-vgb. In order to clone vgb to penicillium chrysogenum,The shuttle plasmid pMK₄-vgb in E.coli-Bacillus subtilis was constructed on the recombined plasmid pUC18-vgb, and both plasmids were transformed into E. coli. The pUC 18-vgb, and pMK₄-vgb expressed Vitreoscilla hemoglobin(VHb) in E.coli ,with the inspections of the protein electrophoresis and CO difference spectrum.The constructed shuttle plasmid pMK₄-vgb with chloramphenicol-resistance was transformed into penicillium chrysogenum protoplast by the use of electroshock. Some chloramphenicol-resistance fungus were filtrated on the chloramphenicol culture medium(chloramphenicol thickness 50mg/ml).The vgb gene was successfully recombined into the penicillium chrysogenum genome through the PCR inspection, and the transformed successfully expressed the Vitreoscilla hemoglobin protein in low-oxygen ferment. The transformed penicillium chrysogenum was more ascendant than the original fungus in hight-oxygen ferment on output of the aimed production. Forther more mycelium growth quantity and the fermentation value enhanced 9.76% and 9.68%.The vgb was expanded in application area through the study, and the research can also afford the foundation for vgb research in other filaceous fungus. The results are also beneficial to reconstruct other antibiotic-production fungus.