Breeding Of Fidaxomicin Production Strain And Optimization Of Fermentation Conditions

Fidaxomicin,a novel narrow spectrum macrocyclic antibiotic,was recently approved by the United States Food and Drug Administration for the treatment of Clostridium difficile infection(CDI)in 2011.Compared with the same kind of antibiotics for CDI,fidaxomicin has similar clinical cure rates,lower recurrence rates and higher safety.Therefore,fidaxomicin has been confirmed to have broad application prospects and the increasing need for industrial production of fidaxomicin became a hot topic in domestic researches.Fidaxomicin was originally isolated from the fermentation broth and mycelium of Dactylosporangium aurantiacum subsp.Hamdenensis subsp.nov.(NRRL 18085)or Actinoplanes deccanesis(ATCC 21983).In this study,the strain SIIA-A2098 was conducted for optimally maximizing the fidaxomicin production using nitrogen ion implantation mutagenesis,ribosome engineering technology and Tn5 transposon insertion technique.In addition,the fermentation condition and medium composition were optimized.Furthermore,the high yield mechanism of strain was analyzed.The taxology of the strain SIIA-A2098 were investigated by morphological classification and phylogenetic analysis based on 16 S rDNA sequence.The morphological characteristic of SIIA-A2098 was similar with that of the A.deccanensis.The alignment of 16 S rDNA sequences showed that the homology between SIIA-A2098 and A.deccanesis IFO13994 is100%.Phylogenetic tree showed that SIIA-A2098 has the highest relationship with A.deccanesis IFO13994.Therefore,the strain SIIA-A2098 should be classified as A.deccanesis.Nitrogen ion implantation mutagenesis was first conducted to improve the production of fidaxomicin in SIIA-A2098,the strain SIIA-M3510 which had 8 fold production of parental strain was selected.Subsequently,we introduced ribosome engineering to improve production of fidaxomicin in strain SIIA-M3510.HPLC analysis showed that the fidaxomicin production increased by 5.4 fold in streptomycin resistance mutant SIIA-10L-2(502 μg/mL),and the ferment ability can keep stable through hereditary investigate.In addition,Tn5 transposon insertion technique was also used to select the mutant strains of SIIA-M3510 with increased production of fidaxomicin,and the results showed that although the production of fidaxomicin in different Tn5 transposon insertion mutants fluctuated significantly,the yield of fidaxomicin was not significantly improved.After the optimization of fermentation conditions and medium,the fidaxomicin production of streptomycin resistant mutant SIIA-10L-2 was increased by 66%(760 μg/mL).After cloning and sequencing of the rpsL and rsmG genes of the streptomycin resistance mutant SIIA-10L-2 and parental strains,we found that rps L gene of mutant SIIA-10L-2 had one point mutation(T114C or L38P)in ribosome streptomycin sensitive domain.In summary,a streptomycin resistance mutant of high fidaxomicin production was obtained by nitrogen ion implantation mutagenesis and ribosome engineering.Tn5 transposon plasmids were successfully inserted into genomic DNA of A.Deccanensis and expressed.Therefore,ribosome engineering and Tn5 transposon insertion technology can activate the potential gene resources of A.deccanensis,which can provide more theoretical basis for the breeding engineering strains.