Breeding Of Micromonospora Olivoasterospora And Clone Of Key Gene

Fortimicins are bicyclic aminoglycoside antibiotics produced by Micromonospora olivasterospora. They have broad-spectrum antibacterial activity, low ototoxicity and nephrotoxicity. The family of aminoglycosides is insensitive to modifying enzyme because of their novel structure. Therefore, it has aroused more and more attention. But its application is limited by low yield. In order to change the status quo, the yield was increased by various means, such as mutation breeding in different ways, optimizing fermentation process with submerged culture in shake flask. Besides, the key biosynthetic genes were cloned and then expressed for directional breeding in molecular levelA stable strain FT-4was obtained by rejuvenate FT-0strain of fortimicin producer Micromonospora olivasterospora.Parent strains were treated with UV,microwave, nitrous acid, and UV+LiCl. The four ways of mutation are used in turn. The results showed that four mutation ways could enhance the fermentation ability of strains. The fortimicin yield of the high-yielding mutant strains UV60-5, W200-1,N40-0.2-3,H245-5,H2-7and H3-25were increased by43.2%,15%,23%,45.1%,18.5%and13.0%respectively. Ultimately, the yield of the strain H3-25was205.7μg/mL,2.91times of FT-4.The fermentation process for fortimicin was optimized by single factor tests and orthogonal tests. Finally, the medium composition and fermentation conditions were optimized as follows:soluble starch4%, glucose1%,yeast powder4%, MgSO40.1%, CaCO30.1%, CoCl22μg/L, glycine0.1%, pH7.0, temperature32℃, inoculum time34-36hour, inoculum volume 10%, the optimum amount in flask50/250(mL) and fermentation period8days.With these measures,The fermentation yield was increased by40.7%.Four key synthetic genes, forQ (glycyl transferase),forL (rate-limiting step enzyme),forZ(N-formimidoyl fortimicin transferase) and fmrO(self-defence gene) were amplified by PCR from Micromonospora Olivasterospora genomic DNA,then sequenced.The alignment in Genebank showed that all the four genes have above99%identity. At the same time, forQ was cloned into pET-30a plasmid. The forQ protein was expressed by IPTG induction in E.coli BL21(DE3).These studies will facilitate the further analysis and establish foundation for gene modification.