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Comparative Studies between Streptomyces sp. FR-008 and Streptomyces Griseus IMRU3570 and Their Metabolites

Author: HuangZuo
Tutor: DengZiXin
School: Huazhong Agricultural University
Course: Molecular Biology
Keywords: Heptaene macrolide FR-008 Structure analysis HPLC Glycone Gene cloning
CLC: Q935
Type: PhD thesis
Year: 1999
Downloads: 213
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Abstract

Streptomyces sp. FR-008 and Streptomyces griseus IMRU3570 are two independently isolated producers for a similar heptaene macrolide antibiotic. Comparative studies between the two producers at chemical and biological level are the main task of the present work,No obvious difference was observed on the HPLC profiles. The main component of the products of Streptomyces sp.FR-008 was verified as candicidin D-which is also produced by S. griseus IMRU 3570 on the basis of NMR studies. Genomic analysis by normal agarose gel eleetrophoresis and pulsed-field gel electrophoresis (PFGE) showed that their chromosomal DNAs are most probably identical while Streptomyces sp. FR-008 carried two linear plasmids. e. 70kb and 130kb in size respectively. Though PKS gene cluster for antibiotics FR-008 was cloned, the genes for amino-mycosamine residue remained unknown. Thus, attempt was also made to clone the desired gene(s),Streptomyces sp. FR-008 is a strain producing a complex of heptaene macrolide antifungal antibiotics, one of which, previously named FR-008 was regarded to bis similar to candicidin, but different in sugar moiety. It was known to harbour two linear plasmids, c. 70kb and 130kb in size respectively (Xiao, et at 1997, ISBA’97). S. Griseus IMRU 3570 is another strain producing candicidin, a heptaene macrolide antibiotic. Recently, the genes for the biosynthesis of antibiotic FR-008 have been cloned in Escherichia coli using radioactively labeled pabAB genes from S. grissus IMRU3570 as probe and subsequent chromosomal walking. The pabAB genes are involved in biosynthesis of the starter unit for candicidin. The DNA sequence of the 3.8kb DNA fragment adjacent to the pabAB genes from Streptomyces sp. FR-008 was also identified to be homologous to the left end part of 4.5kb pabAB genes from Streptomyces griseus IMRU3570 (Hu Zhihao et al, personal communication). These results implied a close relationship between the two strains.Extraction and Separation of Antibiotic FR-008the culture broth of Streptomyces sp. FR-008 were extracted with 10 liters of n-butanol. After removal of n-butanol in vacua, the residue was dissolved in 60% MeOH in water and applied to a column of macroretieular neutral resin XAD-16 (Sigma). The active fraction was eluted with 80% acetone in water after washing the column with 2 bed volumes of 50% MeOHin water and concentrated in vacuo to obtain a kind of brown powder. The powder was then applied to a silica gel column chromatography charged with 80% acetone in water and eluted with the same solvent. After concentrating and drying, relatively pure FR-008 was obtained.HPLC assayA comparison between antibiotic FR-008 and candicidin has been made by studying the HPLC separation profiles of antibiotic FR-008, candicidin and a mixture of both. The retention tune of each of the three main peaks of the antibiotic FR-008 is entirety identical to candicidin, except for one peak (FR-008d), which can also be seen as a very weak signal on the HPLC profile of candicidin with the same RT. Therefore, Streptomyces FR-008 produces the same antibiotics as caadicidin. In addition, the UV spectra of FR-008a, b, c were exactly the same as candicidin. So Streptomyces sp.FR-008 is a new strain synthesizing candicidin.NMR StudiesAnalysis of the 1H-1H COSY spectrum allowed us to distinguish some important chemical shifts of the protons in FR-008b which could be identified as eandicidin D. Especially for amino-mycosamine residue, methyl protons and all the protons on the ring could be defined by correlating relation.Partial data from 13C NMR studies of the FR-008b has been described. Four C-CH3, six aromatic carbons, six carbons of mycosamine were the characteristics of candicidin D, One ketal corresponding to mycosamine and four ketons indicated that no hemiketal formed between C-15 and C-19 in FR-G08b or candicidin D. However, such hemiketal was usually thought having been formed. It might be because of the fact that the structural analysis of the most polyene macrolide antibiotics have be

Comparative Studies between Streptomyces sp. FR-008 and Streptomyces Griseus IMRU3570 and Their Metabolites

Abstract

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