Construction Of Gene Recombination System Of Amycolatopsis Mediterranei

Amycolatopsis mediterranei is a rare actinomycete that can produce clinically important antibiotics-rifamycin,whose derivatives are particularly effective for the treatment of mycobacteria.Rifamycin can also be used for the treatment of tuberculosis and leprosy caused by Mycobacterium tuberculosis and Mycobacterium leprae,respectively.Amycolatopsis mediterranei is an aerobic gram-positive bacterium belonging to the genus Actinobacteria,Nocardia,and Amycolatopsis,and it's colonies on the solid medium are orange-red.It is difficult to select markerless knock-out strain when the antibiotic resistance genes are used as markers.To establish a simple and convenient method for screening markerless genetic recombinant strain.By knocking out the enzyme-encoded gene dxs(1-deoxy-D-xylulose-5-phosphate synthase)in orange-red colony Amycolatopsis mediterranei of the first reaction in the lycopene biosynthesis pathway,a white colony mutant strain of Amycolatopsis mediterranei was obtained.Through the electroporation transformation mtf(S-malonyltransferase)gene homologous recombination vector carrying the dxs expression cassette was transformed into the white strain,the mtf gene inactivated strain in orange-red colony was obtained.The unmarked mtf homologous recombination vector was transformed into the orange-red colony strain,and then the markerless knock out of mtf strain was obtained.In industrial fermentation,due to the long fermentation period of the strains,the mycelium grows densely and the secondary metabolites increase,leading to the viscous fermentation fluid,which reduces the dissolved oxygen and further affects the production of rifamycin.Vitreoscilla lucidum hemoglobin(VHb)increase the amount of dissolved oxygen in the fermentation broth,so the VHb gene is integrated into the chromosomal DNA of Amycolatopsis mediterranei,which improves the oxygen utilization rate of the strain.Compared with the original strain,the yield of rifamycin production B increased by 14.71%,and the yield of rifamycin SV increased by 17.38%.