| Nemadectin,produced by Streptomyces cyaneogriseus subspecies noncyanogenus,is a 16-membered macrocyclic lactones antibiotic.The industrial production of moxidectin after synthesizing by four steps chemical reactions,can be widely used as pesticide for its broad-spectrum,high efficient and safe anthelmintic activity.In this paper,seven genetic engineered strains were constructed to solve the present production problems including low output,high by-products and complex chemosynthesis,which can also supplies an idea host strains for further studies.First,though homologous recombination,the methyltransferase was knocked out from nemadectin biosynthetic gene cluster.So accordingly,the LL-F28249Y and LL-F28249λ components would be wiped off from fermetation broth.plasmid pSET-nemD was constructed and introduced into the initial strain MOX-101 to delete the gene nemD based on homologous double cross-over method.A gene knockout strain MOX-AnemD149 was obtained,which has been proved that it no longer produced LL-F28249Y and LL-F28249λcomponents.In the meanwhile,the precursor,nemadectin,showed a slightly increase(12%)compared to the initial strain.Second,another knockout plasmid pKC1139-oli was constructed and introduced into the initial strain MOX-101 to delete the 90kb gene oli,which is also the polyketide synthase function genetype.Differently,the I-Scel enzyme was used to promote the second homologous double cross-over.Then,a gene knockout strain MOX-△oli-72 was obtained.Not only the oligomycin analogs component disappeared,but also an 39%increase(165mg/l)in production of nemadectin was found by compared to the initial strain MOX-101(119mg/l).Then,five strains which harbored mutate KR3 domain of C23 keto-recductase in nemadectin biosynthesis pathway were constructed,which would provide a more efficient process to synthesize moxidectin with fewer chemical reaction steps in the future. |
