Mechanism Of Salinomycin Over-production And Metabolic Engineering Of High-yield Strains

Salinomycin,a member of polyether antibiotics,has been widely used in animal husbandry and veterinary medicine as food additive and growth promoter for 30 years.In addition,salinomycin has recently been identified as a potential agent to inhibit leukemia stem cell and epithelial cancer stem cells.The commercial fermentation of salinomycin is highly productive,reaching yield of up to 100 g/L.In order to elucidate the overproduction mechanism,the genome of the high-yielding strain BK 3-25 was fully sequenced and compared with the wild-type DSM 41398.Strain BK 3-25 has a 75-kb large deletion and 60 additional Indels and SNVs.Subsequently,individual or combined deletions of the 75-kb region and PKS-6in the wild-type resulted in improved salinomycin yields from 2.60 to 5.20,6.90 and9.50 g/L?53 % of BK 3-25?,respectively.Moreover,13 putative regulatory genes among the 55 CDSs were individually inactivated,and 6 were proved to be negatively involved in the transcription of sln genes.Combined deletions of two major negative regulatory genes SLNWT₃357 and SLNWT₇015 caused further improved transcription of sln genes as well as the yield,from 2.60 to 7.30 g/L?40 % of BK 3-25?.Additionally,8 active competitive gene clusters were individually deleted in BK3-25 and the deletion of PKS-NRPS-2 and PKS-4 significantly enhanced salinomycin production.Ethylmalonyl-Co A was identified to be the rate-limiting precursor of salinomycin biosynthesis and tandem over-expression of the key genes ccr,orf11 and orf12 in the ethylmalonyl-Co A biosynthetic pathway successfully enhanced the salinomycin yield by 39.3 % compared to BK 3-25,from 18.0 g/L to 25.1 g/L.It is interesting that the salinomycin industrial fermentation medium contains 15 %soybean oil which is the highest soybean oil proportion in fermentation medium among the common antibiotics.The transcriptome analysis of BK 3-25 elucidated that the transcription level of salinomycin PKS genes,?-oxidation genes and precursorbiosynthesis genes were enhanced under the increased concentration of soybean oil in the fermentation medium,which mean the increased adding amount of soybean oil induced the genes to strengthen the transcription.However,the genes related to TCA cycle showed opposite trend,and higher adding amount of soybean oil reduced cellular growth.The deletion of genes SLNHY₅015?glt A?and SLNHY₆740?fum?which are putatively involved in primary metabolism reduced cellular growth and improved the titer of salinomycin.In conclusion,we clarified the mechanism for salinomycin overproduction via genomics,transcriptomics and comparative genomics,and a few derivatives of the high-yielding strain BK 3-25 with higher yield were constructed via genetic engineering.The strategies described here are helpful for improving the production of many other antibiotics.