Analysis Of Key Enzymes In Neomycin Biosynthesis And Its In Situ Overexpression

Neomycin is an aminoglycoside antibiotic isolated from the product of Streptomyces fradiae in 1949,and is also the first aminoglycoside antibiotic containing 2-deoxystreptamine(2-DOS)to be discovered.Because of its broad-spectrum bacteriostasis,it has a good inhibitory effect on both Gram-positive and negative bacteria,and is widely used in clinical medicine,veterinary medicine,feed additives and other fields.In recent years,the neomycin biosynthesis pathway and 11 important enzymes in this pathway have been reported.In this study,we selected five kinds of enzyme genes(neoC,neoD,neoF,neoM,neoN)as the research object,screened and obtained the neomycin high-yielding starting strain SF,and constructed the overexpression plasmid and gene blocking plasmid containing the enzyme gene.Establish and optimize the E.coli-Streptomyces fradiae conjugate transfer system,and finally get 10 recombinant strains of S.fradiae-gene overexpression recombinant strains SF-neoC,SF-neoD,SF-neoF,SF-neoM,SF-neoN and Gene blocking recombinant strains SF-?neoC,SF-? neoD,SF-? neoF,SF-? neoM,SF-? neoN.The neoN gene was identified as the gene that had a great influence on neomycin production during neomycin biosynthesis,and A neomycin high-yield recombinant strain with a neomycin titer of 14150 U/mL in neomycin potency was obtained.The main research contents and results are as follows:(1)Screening and analysis of neomycin high-yielding starting strains.Establish a method for detecting neomycin by HPLC.When the titer of the neomycin standard is 0?400 U/mL,the peak area has a good linear relationship with the neomycin titer.The linear standard curve is Y=15485.838X-60137.88207(R2=0.999),and screened a strain of Streptomyces fradiae SF with a neomycin titer of 11264 U/mL.The most suitable solid medium for growth of this strain is RG medium;the most suitable liquid medium is YEME medium;When growing in YEME medium,0?24 h belongs to the growth lag period,24?60 h logarithmic growth period,60?72 h is the stable period,72h is the decay period;it is not sensitive to neomycin,ampicillin It is sensitive to chloramphenicol,amphotericin,streptomycin and kanamycin,among which it is the most sensitive.(2)Construction of recombinant plasmids.Based on the Escherichia coli-Streptomyces shuttle plasmids pSET152 and PKC1139,this study successfully constructed the recombinant plasmid Pset152-PermE*with the strong promoter ermE*gene of Streptomyces and the recombinant plasmid Pset152-PermE*with the original RBS added on this basis-RBS,and finally successfully constructed overexpression plasmids containing enzyme genes PPR-neoC,PPR-neoD,PPR-neoF,PPR-neoM,PPR-neoN and gene blocking plasmids PKC-neoC,PKC-neoD,PKC-neoF,PKC-neoM,PKC-neoN,and introducing the recombinant plasmid into E.coli ET12567(pUZ8002)for conjugation and transfer.(3)Establishment and optimization of conjugate transfer system of Streptomyces fradiae.In this study,the E.coli-Streptomyces fradiae conjugate transfer system was established and optimized.The optimal medium for conjugate transfer was A75 medium.The optimal pretreatment conditions for the recipient spores were heat shock at 50?for 10 min and 37? Pre-culture for 4 h,the optimal donor-acceptor ratio is 10:1,the optimal addition time of apramycin is 15 h after zygomatic transfer,and the frequency of zygomatic transfer is significantly increased after using the optimal conditions.And the neoC gene overexpression strain SF-neoC in neomycin biosynthesis was obtained by conjugation transfer.(4)Construction of Streptomyces fradiae gene overexpression and blocking strains.In this study,10 recombinant strains were successfully constructed and named as gene overexpression recombinant strains SF-neoC,SF-neoD,SF-neoF,SF-neoM,SF-neoN and gene-blocking recombinant strains SF-?neoC,SF-?neoD,SF-?neoF,SF-?neoM,SF-?neoN.At the same time,the apramycin resistance gene contained in the plasmid of the recombinant strain was expressed,so that the strain showed a certain degree of apramycin resistance.Gene over-expression recombinant strains SF-neoC,SF-neoD,SF-neoF,SF-neoM,SF-neoN have increased neomycin production,respectively increased by 19.27%?21.06%?20.72%?15.96%?23.07%,And the glucose concentration and viscosity in the fermentation broth are higher than the starting strain SF;the gene blocking recombinant strains SF-? neoC,SF-? neoD,SF-? neoF,SF-?neoM neomycin potency are all reduced,Only about 6000 U/mL.However SF-? neoN,because of the characteristics of neoN gene,its neomycin titer increased by 13.91%.but the glucose concentration and viscosity in all gene-blocking recombinant strains and the starting strain SF were not significantly different.And the neomycin high-yield recombinant strains SF-neoN and SF-? neoN were passaged 4 times,and their neomycin titer was stable.