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Characterization of the potential of selected monooxygenases of Sorangium cellulosum So ce56 for the biosynthesis of complex compounds and analysis of the reaction mechanisms

Subject Area Metabolism, Biochemistry and Genetics of Microorganisms
Term from 2011 to 2019
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 209236536
 
Final Report Year 2019

Final Report Abstract

Based on our results on the identification and cloning of the whole CYPome (meaning all cytochromes P450 of an organism), from Sorangium cellulosum So ce56 (21 cytochromes P450 (CYPs), among them nine new CYP families), the aim of the project was (i) to identify new reaction types of CYPs, especially from novel CYP families, (ii) to study the function of novel CYPs, (iii) to investigate CYPs which might have potentially interesting physiological functions and (iv) to get insight into the structural basis of the reaction mechanism of myxobacterial CYPs. Taken together, the following main progress was achieved during the two periods of project support by the DFG: 1) The first steroid 1α-hydroxylase known to date was characterized. The resolution of its 3D structure was performed. 2) CYPs from S. cellulosum So ce56 were shown to be efficient and (in part) highly selective enzymes for terpene hydroxylation displaying bacterial CYPs as interesting new tools for terpene decoration. 3) A highly efficient redox system for epothilone epoxidation has been established and novel reactions on epothilones were found using CYPs from S. cellulosum So ce56. 4) CYPs from So ce56 were shown to efficiently hydroxylate drugs leading to products as found in human and displaying this way their possible involvement in xenobiotic degradation in the soil. 5) The 3D structures of 3 different cytochromes P450s, CYP260A1, CYP260B1, CYP267B1 (with and without substrates) have been resolved and gave valuable hints and explanations concerning substrate binding, selectivity of hydroxylation and product formation. 6) Studying and expanding the substrate scope of CYP267B1 demonstrated this P450 as an unusual enzyme with very broad substrate specificity. This might be of importance for screening potential new substrates for novel products. 7) Mutants of CYP260A1 have been produced with hydroxylation selectivity at C1 or C17 position, respectively. Two mutants have been crystallized with the substrate, progesterone, und demonstrated changed binding of the substrate (flipped around by about 180°) to allow for the different hydroxylation positions. This data gives an excellent insight and explanation of the structural basis for the steroid hydroxylation selectivity. 8) First promising results for the chemical derivatization of hydroxylated steroids have been obtained but need further optimization concerning the yield of the product. 9) Besides, 3 terpene synthases type I found in the genome of S. cellulosum Soce56, have been cloned and characterized and several of their sesquiterpene products have also been identified in the supernatant of the bacterial cells demonstrating their production under physiological conditions. 10) Cascade reactions of various terpene synthases with different P450s have been shown and a combinatorial platform was used to demonstrate the usefulness of this approach to produce known and novel products.

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