The Disruption Of EryF Gene In Saccharopolyspora Erythraea And The Influence Of Biosynthetic Of Erythromycin

eryF encodes erythromycin C-6 hydroxylase,in order to produce C-6 dehydroxylation erythromycin,we disrupt this gene by means of inframe-deletion strategy.An 144 bp internal fragment of eryF was deleted through the PCR method.The disruption plasmid pSPU261,which was successfully constructed basing on the plasmid vector pHZ132,can not be transferred into erythromycin producing strain-Saccharopolyspora erythraea.So we construced the disruption plasmid pSPU263,it was transferred into S.erythraea by conjugation.The single-crossover strains C6,F3 and J8 was verified by PCR.After then,six apramycin-sensitive strains were selected.F3-1014,F3-1018, F3-1110,J8-760 were double-crossover disruption mutants,confirmed by PCR and sequencing.F3-125,J8-251 were back mutanted to wildtype.Thin-layer chromatography(TLC)analysis indicates that the production of double-crossover disruption strain F3-1110 have significant change compared with the original strain.Separation and purification of the production,molecular weight of the four bioactive components was analysed by mass spectrometry(MS),all of them are 16 smaller than the compared erythromycin A,B,C,D.That is mean,erythromycin A,B,C,D were transformed into hydroxy erythromycin A,B,C,D.The eryF gene was successfully disrupted, but the titer of F3-1110 strain was far belower than the original strain because of the decrease of hydroxy erythromycin A.