Preliminary Study On Genetic Engineered Strain Of Vancomycin

Vancomycin, a glycopeptide antibiotic, was used as the first-line drug in treatment of serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, with vancomycin-resistant Enterococcus faecium (VRE) came into being in the 1980s, the number of VRE has been increasing. People are afraid that MRSA would finally become resistant to vancomycin. Under this circumstance, it is necessary to carry out research and development on novel glycopeptide antibiotics for treating serious infections. With the increasing knowledge about the biosynthesis of some glycopeptide antibiotics including vancomycin, glycosyltransferases (Gtfs) are used to generate new hybrid compounds. Another promising way is to use gene recombination technology, based on the DNA transformation system for vancomycin producing strains, to develop novel compounds.This paper concerns with several aspects on developing new vancomycin analogues including: the expression of glycosyltransferase gene gtfD and gtfE; the analysis of hydrolysis products; the DNA transformation system of vancomycin-producing strain and the optimization of fermentation.Through PCR, gene gtfD and gtfE encoding glycosyltransferase were subcloned from the plasmid pLY24 and pLY21 into an expression plasmid pET-29a to constuct new plasmid pLY24ET and pLY21ET. The de-vancomsamine vancomycin could be gained through hydrolysis, which might be used as the substrate for enzyme transformation. This work made preparations for the further in-vitro study on the activity of glycosyltransferase and the usage of different glucosyl.Several important factors for protoplast preparation from Amycolatopsis-orientalis were studied and optimized such as the type of substrate, the concentrations of the addition of glycin, the concentrations of lysozyme and so on. The results showed that about 3.18 x l0~7/ml of protoplasts were formed when the mycelia were incubated for 1 hour at 28 °C in TSB medium containing 2% glycin, and treated with 2 mg/ml lysozyme. The frequency of protoplast regeneration was 3.1%. PCR identification confirmed that the Amycolatopsis mediterranei-E. coli shuttle plasmid pRLAM containing apramycin resistance gene was introduced into A. orientalis by PEG-assisted transformation of protoplasts. This result paved the way for the construction of genetic engineered strain.After the optimization of fermentation conditions, the productivity of vancomycin could be maintained 1000 μg /ml. The soluble medium might be chosen as the potential substrate to screen the novel compounds.