Production of erythromycin C Saccharopolyspora erythraea mutant A226G-Construction.docVIP

 PAGE \* MERGEFORMAT 23 Production of erythromycin C Saccharopolyspora erythraea mutant A226G-Construction Author: Zhang Yuan HUANG Xun-end department Kong Chang Liu Tao Qin Zhao Hao Xiang Xiao-Wei Zhang [Abstract] eryG Saccharopolyspora erythraea gene encoding the product is erythromycin 3 amp;quot; O  methyltransferase (EryG), inactivation eryG genes can block the biosynthesis of erythromycin A, accumulation of the intermediate product of erythromycin Su C. to Saccharopolyspora erythraea A226 genomic DNA as a template, using overlap PCR gene was amplified by eryG on both sides of about 1600bp DNA fragment (Δ G), which removes the EryG on S  adenosyl-L-methionine-binding domain corresponds to The 290bp DNA fragment. the fragment was cloned in the plasmid pWHM3, construct a homologous recombination plasmid pWHMΔ G. PEG-mediated protoplast transformation method will be pWHMΔ G into Saccharopolyspora erythraea A226, through chromosomal homologous recombination, gene mutation on chromosome eryG . using thin-layer chromatography, PCR and mass spectrometry method of filtering out a eryG gene mutations in Saccharopolyspora erythraea A226G-mutant, this mutant was mainly accumulated erythromycin C intermediate products rather than synthetic erythromycin A. through the eryG gene function compensation Saccharopolyspora erythraea genetically engineered bacteria A226G-G can be restored erythromycin A, synthesis. [Keywords:] Saccharopolyspora erythraea; erythromycin; 3 amp;quot; O  methyltransferase; homologous recombination; functional compensation ABSTRACT Saccharopolyspora erythraea erythromycin 3 amp;quot; O  methyltransferase (EryG), encoded by the eryG gene, could play an important role in erythromycin biosynthesis, and the inactivation of eryG gene would lead to erythromycin C accumulation in the eryG mutant fermentation culture. Taking Sac . erythraea genomic DNA as a template, about 1,600 bp DNA fragment with deletion of a 290bp fragment which con