| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1253-2 | ||||
Resumo:Streptomyces sp. have been widely studied by the pharmaceutical industry in the development of compounds with biological activity, such as antibiotics and antitumor drugs. Among the metabolites biosynthesized by Streptomyces sp., there are polyketides that are produced by polyketide synthases (PKSs) and modified by tailoring enzymes. Recently, these enzymes have attracted attention because of their peculiarities and also of their biotechnological potential in biocatalysis. Among these enzymes, we have the spirocyclases, which are involved in the formation of spiroketal systems from natural compounds. The two hypothetic enzymes involved in the formation of these systems are Orf9 from Streptomyces violaceusniger DSM4137 and SchU2 from Streptomyces chartreusis. These enzymes are supposed to be involved in the biosynthesis of nigericin and calcymycin, respectively. Herein, we intend to obtain the recombinant hypothetic enzymes for structural determination and unveil their mechanism of catalysis. In order to do this, the genomic DNA extraction of S. violaceusniger DSM4137 and S. chartreusis was performed. We have amplified the coding region of gene orf9 and schU2 by PCR and performed the cloning into pET28a vector by the ligation-independent cloning (LIC) method. We have confirmed the cloning by colony PCR. For the expression of Orf9, the best conditions and that gave the highest fraction of soluble protein was using ArcticExpress (DE3) cells with an induction of 400 µM of IPTG for 16h at 12C. For purification, we used a buffer containing 50 mM tris-HCl, 200 mM de NaCl, and 5% glycerol in pH: 7,5. For SchU2, cell expression studies will still be performed with BL21 (DE3) to obtain the soluble fraction of the protein. With the success in protein production, we will use them to perform attempts to obtain crystals and consequently determine the structure of these two hypothetical spirocyclases and contribute to elucidating the mechanism of biosynthesis of nigericin and calcimycin. Palavras-chave: Spirocyclases, Biosynthesis, Natural Products Agência de fomento:FAPESP | |||||