| Erythromycin, a clinically useful macrolide antibiotic, is produced by Saccharopolyspora erythraea. An increasing proportion of pathogenic bacteria have developed drug resistance to erythromycins for the long-term use. Thus, there is a growing need to develop new derivatives of erythromycin to combat the worsening problem of antibiotic resistance among potential life-threatening pathogens. The third generation of erythromycin which modified by semi-synthesis has been on market, but the procedure of chemosynthesis is tedious and that would pollute environment. Therefore, exploration of the biosynthetic restructuring pathway engineering of erythromycin is a good choice for novel antibiotic productions.Glycosyl groups are essential for the biological activities of antibiotics and play important roles in bacteriostasis and protection of production bacteria. They would have great influence on products if glycons are changed in bacteria. Thus, producing new antibiotics according to changing glycosyl are hot field recently. The main ways for modifying aglycons are changing glycosyltransferases and feeding with different sugar substrates. Changing glycosyltransferases includes inactivation of glycosyltransferases, heterologous expression of glycosyltransferases, over expression of glycosyltransferases, and so on.There are two glycosylation procedures in erythromycin biosynthetic pathway. One is the glycosylation reaction happened in C-3 of macrolide, another is happened in C-5. EryBV and EryCⅢglycosyltransferases are responsible for the two glycosylation reactions respectively. In order to change the sugar residue, construction of glycon synthesis or glycosyltransferase mutant is a very important process. Thus, glycosyltransferases EryBⅤand EryCⅢwere inactived respectively according to gene knock-out and the two mutants A226-⊿eryBⅤand A226-⊿eryCⅢwere successfully constructed. They paved the way for modifying sugar structures and synthesizing erythromycin analogs through combinatorial biosynthesis.In order to inactivate eryBⅤand eryCⅢgene,130bp was deleted in both of them,⊿eryBⅤand⊿eryCⅢwere obtained respectively. 1000bp upstream and 1000bp downstream homologous arms from the deletion points were amplification by PCR, and then cloned into plasmid pUC18 to construct pUC18-⊿eryBⅤand pUC18-⊿eryCⅢrespectively. After identification,⊿eryBⅤand⊿eryCⅢwere subcloned into pWHM3 to construct homologous recombinant vectors pWHM3-⊿eryBⅤand pWHM3-⊿eryCⅢ. Then the new vectors were transferred into Saccharopolyspora erythraea A226 respectively. One integrate A226/pWHM3-⊿eryBⅤand one integrate A226/pWHM3-⊿eryCⅢwere screened out by Thiostrepton. The bioassay test and thin-layer chromatography analysis of the two integrates showed that the biosynthetic pathway of erythromycin was disrupted in both of them and erythromycin couldn't be produced. According to the instability of plasmid pWHM3, the two mutants which EryBⅤinactivation or EryCⅢinactivation in Saccharopolyspora erythraea were screened out respectively, and they were designated as A226-⊿eryBⅤand A226-⊿eryCⅢ. Bioassay test of them indicated that their fermentation products didn't have Bacillus subtilis-inhibiting abilities. The results of TLC showed that there were no erythromycins. At last, MS identifications supplied that there was EB or MEB accumulated in the two mutants respectively. Then, eryBⅤand eryCⅢgene were cloned into expression plasmid pZMW separately and transferred into the corresponding mutant, and the mutant strains restored abilities to produce erythromycins.The Saccharopolyspora erythraea mutants A226-⊿eryBⅤand A226-⊿eryCⅢwith glycosyltransferase EryBⅤor EryCⅢinactivated were successfully constructed, and they paved the way for modifying sugar structures and synthesizing erythromycin analogs. The genes eryBⅤand eryCⅢwere cloned correctly, and this prepared for the studying of their enzymology activities.
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