| Ramoplanin is a depsipeptide antibiotic that is active against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE).Orf20 is the only halogenase gene in the biosynthesis gene cluster of ramoplanin, it is likely to get a dechlorin ramoplanin by inactiving orf20 gene, and offer condtions for heterogenous expression of halogenases to produce new glycopeptide antibiotics. In this study, we constructed a mutant SIPI-GE.2006 dorf20 (â–³orf20) in which a fragment encoding 64 amino acids has been deleted, and in resulting exconjugant we had detected a compound which has consistent molecular weight with dechlorin ramoplanin. On these basis, we constructed mutant strains SIPI-GE.2006 dorf20(â–³orf20)Rorf20(orf20-1) and SIPI-GE.2006 dorf20(â–³orf20)Rorf20(orf20-2). In both resulting exconjugants we had detected ramoplanin by HPLC and LC-MS. This study proved the function of orf20, and laid the foundation of ramoplanin reconstitution by combinatorial biowynthesis.Refer to the similarity of glycon between ramoplanin and, glycopeptide antibiotic mannopeptimycin, we presume that the mechanism of ramoplanin glycosylation is similar to mannopeptimycin. The core structure transported to the out of membrane after its synthesis in the cytoplasm, and then glycosylated to become the end product. Therefore, transporter gene duplication might promote ramoplanin synthesis. In this study, pSM-11 and pSM-12 were transformed into SIPI-A.2006 chromosome, constructed mutant strains SIPI-GE.2006 Rorf8(orf8) and SIPI-GE.2006 Rorf23(orf23). RT-PCR showed that the transcriptional level of two genes had increased, but in the resulting exconjugants ramoplanin yields were not improved, and the overexpression of orf8 and orf23 had not improved the self-resistence of ramoplanin. Consequently, we presume that enzymes encoded by orf8 and orf23 have other unknown functions.
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