| Vancomycin has been the first-line antibiotic used in clinical treatment of serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA). With the extensive use of vancomycin, the vancomycin-resistant Enterococcus faecium (VRE) and the vancomycin-resistant Staphylococcus aureus (VRSA) have emerged one after another. The glycopeptides-resistant Staphylococcus aureus (GRSA) was predicted to appear in the near future. Under this circumstance, it is necessary to develope novel glycopeptide antibiotics. With the progress of molecular biotechnology, utilization of the combinatorial biosynthesis method in the development of novel glycopeptides has become a hot research spot.The basement of the combinatorial biosynthesis is the well-understanding of the function of genes in the cluster. In this study, the function of four genes (vcm12、vcm11、vcm14 and vcm8) related to the N-methylation and the halogenation in the vancomycin gene cluster was studied by gene knockout and the activity assay of enzyme in vitro to provide correct information for combinatorial biosynthesis. The vancomycin-like compounds found from this study could be used as the lead compounds in hybrid-vancomycin development.Gene vcm12 coding for a putative N-methyltransferase was knocked out, which resulted in a mutant A. orientalis dvcm12. An expression recombinant strain E.coli BL21 (DE3)/pLYLH13 expressing gene vcm12 was constructed and induced by IPTG to produce protein Vcm12, and an activity assay of Vcm12 was then performed. The research work on the metabolites by mutant and the result of activity assay indicated that N-methyltransferase was encoded by gene vcm12. Another finding was that besides vcm12, there should be other genes involved in the N-methylation. Dimethylated products were found in the activity assay of Vcm12 with N-demethylvancomycin and other three vancomycin derivates as substrates. Mass and NMR were applied to identify the site of the dimethylation, which was located on the amino-group of the leucine.Gene vcm11 coding for a putative C3-methyltransferase was knocked out, which resulted in a mutant named A. orientalis dvcm11. The research work on the metabolites of the mutant indicated that vcm11 was a C3-methyltransferase coding gene. The existence of N-demethylvancomycin-like metabolites in the fermentation broth of the mutant suggested that gene vcm11 might have the function as N-methyltransferase. This result provides an important clue for research work on the N-methylation. Besides, an expression recombinant strain E.coli BL21 (DE3)/pLYLH16 expressing gene vcm11 was constructed and induced by IPTG to produce protein Vcm11. There are two halogenase genes in the vancomycin biosynthesis gene cluster according to gene blast, gene vcm14 (encoding a haloperoxidase) and gene vcm8 (encoding a FADH2-dependent halogenase). These two genes were disrupted separately and two mutants were obtained, named as A. orientalis dvcm14 and A. orientalis dvcm8 respectively. The research work on the metabolites of mutant ruled out the possibility of vcm14 to be involved in the halogenation of vancomycin, and indicated that there was only one halogenase, FADH2-dependent halogenase encoded by vcm8, in charg of the two chlorines . Addition ofβ-OH-Tyr in the fermentation of A. orientalis dvcm14 may lead to the emergence of vancomycin, which may suggested that vcm14 was involved in the biosynthesis ofβ-OH-Tyr. Besides, the vcm8 expression strain E.coli BL21 (DE3)/pLYLH20 was constructed and the protein Vcm8 was obtained.Twelve vancomycin-like compounds were obtained in this study and four of them are new vancomycin derivates. These compounds may be applied in the hybrid-vancomycin development as the leading compounds. |
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