Study On The Molecular Mechanism Of The Glycosylation Step During Macrolactins Biosynthesis In Bacillus

Bacillus marinus B-9987 was isolated from the rhizosphere of halophyte Suaeda s alsa collected in the intertidal zone of Bohai Bay of Eastern China. B. marinus B-9987 is able to produce various macrolactins, which are 24-membered macrolides with anti bacterial, antiviral and antitumor activitie. Most macrolactins were isolated from mari ne-derived bacteria, such as Actinomadura sp. and Bacillus sp. While the backbone of macrolactins is asembled by polyketide synthetase (PKS), their tailoring steps, such a s glycosylation, is utterly unknown. Attachment of sugar moieties to aglycone scaffolds is usually performed by glycosyltransferases (GTs), which catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules,forming glycosidic bonds. For the purpose of elucidating the glycosylation mechanism of macrolactins, our studies were carried out as follows:Firstly, macrolactins were isolated from the fermentation broth of B. marinus B-9 987 by repeated column chromatography on silica gel and semi-preparative HPLC, an d were identified by modern spectroscopy methods of NMR, MS and UV. Three com pounds(1-3) were identified:macrolactinA,7-O-malonyl macrolactin A and macrolact in B. These three compounds were used as sugar acceptor of the purified glycosyltran sferase in the following study.Secondly, we have identified a GT gene bmmGT1 from the genome of B. marinu s B-9987 by comparative. The recombinant plasmid for bmmGT1 expression was constructed, and was successfully expressed in E. coli BL21. The catalytic activity of BmmGTl was characterized, and the result revealed BmmGTl is able to catalyze the glycosylation of all the tested MLNs.Thirdly, the in vitro assays of BmmGT1 were conducted. We examined its activities at different pH. temperatures, and cations. Under the optimized conditions, the sugar donor specilicity and acceptor specificity for BmmGT1 were tested. Furthermore, we performed steady-state kinetic characterizations of BmmGT1-catalyzed reactions. These results provide basic datas of the structure and function of enzymes.Finally, we have demonstrated that BmmGT1 exhibits broad substrate specificity with regards to the aglycons and sugar donors, and totally three novel glycosylated macrolactins were generated. With the benefit of the sugar moiety bestowed on the compounds, we would expect improvement of their solubility and proably bioactivity. Moreover, our results demonstrated the promising application of BmmGT1 in structure diversification of natural products.