Study On Gene Knockout Of Glucose-6-phosphate Dehydrogenase In Amycolatopsis Mediterranei

At present,rifamycin as an important antibiotic in the treatment of tuberculosis and leprosy,mainly rely on the fermentation of Amycolatopsis mediterranei.With the outbreaks of TB in recent years,more rifamycin derivatives were required to be produced.While the traditional mutation breeding can not meet requirement.To build high yield industrial bacteria,This paper used genetic engineering to explore the knockout method of A.mediterranei.In order to facilitate the structure of gene inactivation methods of A.mediterranei,this paper constructed a method of triparental mating for the gene from E.coli to A.mediterranei.An induced conjugational plasmid pULVK1-Am-OriT was constructed for the study.Some related factors such as collection period of mycelia,the ratio of three strains and the incubation time of mixed strains.The results indicated that when the receptor bacteria OD₆₀₀ up to 0.7,donor bacteria:assist bacteria:receptor bacteria to be 1:1:1.5,the incubation time was 20 h,the conjugation rate can be best--7.3×10^-7.In this paper,the homologous arms of glucose 6-phosphate dehydrogenase gene were connected to pMD102,a plasmid without the ability of independent replication in A.mediterranei,to construct the recombinant vector.The recombinant vector was transferred to A.mediterranei by electroporation,and XGYY?G6PDH was obtained by antibiotic marker and verified by PCR.As the result,a mutant bacteria of A.mediterranei with glucose 6-phosphate dehydrogenase gene knockout was constructed.