Genome Mining in Sorangium cellulosum So ce56: IDENTIFICATION AND CHARACTERIZATION OF THE HOMOLOGOUS ELECTRON TRANSFER PROTEINS OF A MYXOBACTERIAL CYTOCHROME P450*

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.The cytochrome P450 (CYP)² enzymes constitute a superfamily of external monooxygenases. The catalytic versatility of the family members explains their involvement in such diverse biological processes as biosynthesis of steroid hormones, carbon source assimilation, and metabolism of xenobiotics. In addition, cytochrome P450 enzymes have been reported to be involved in the biosynthesis of many pharmaceutically interesting secondary metabolites from a variety of microorganisms (1–4). Cytochromes P450 are usually dependent on an external electron donor. With respect to their electron transport system they can be divided into several classes, with class I (the mitochondrial/bacterial cytochrome P450 systems) being the predominant form in prokaryotes (5). In this system the electrons required for the enzymatic reaction originate from NAD(P)H and are delivered to the cytochrome P450 via a ferredoxin reductase and a ferredoxin. In a number of examples, the heterologous reconstitution of the electron transfer chain has been shown to be ineffective, if possible at all (5). Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6, 7). Interestingly, members of both the 2Fe-2S] and the non-2Fe-2S] ferredoxins have been reported to sustain cytochrome P450 catalyzed reactions. The latter group is further subdivided into mono- and dicluster ferredoxins (i.e. the 3Fe-4S] or 4Fe-4S] and the 3Fe-4S] + 4Fe-4S] or 4Fe-4S] + 4Fe-4S] ferredoxins). Remarkably, cytochrome P450 systems depending on non-2Fe-2S] ferredoxins have been found exclusively in bacteria to date (8, 9).To fulfill the role as electron mediator, the ferredoxin component of any given cytochrome P450 system has to be reduced. This reduction is achieved by a ferredoxin reductase, which in turn takes up electrons from NAD(P)H. The ferredoxin reductase is often the least characterized constituent of the cytochrome P450 system because these flavoproteins may be unstable (i.e. easily lose their cofactor) and usually show a relatively low level of expression (10).Sorangium cellulosum So ce56 is a genome-sequenced myxobacterial model strain. Because of their biotechnological potential as producers of secondary metabolites, the myxobacteria attract attention from both the academic community and the pharmaceutical industry. To date, more than 100 new basic structures and some 500 derivatives have been reported (11), with almost half of the newly discovered natural products being isolated from the genus Sorangium (11, 12). The potent anti-cancer agent epothilone, for example, was discovered from S. cellulosum So ce90 (13, 14). Epothilone is one of so far seven known myxobacterial compounds, the biosynthesis of which involves cytochromes P450 (15). Besides the epothilones, these are the antifungal leupyrrins (16) and the cytotoxic spirangienes (17) (also from S. cellulosum), the antibiotic myxovirescin from Myxococcus (18), the electron transport inhibitor stigmatellin (19) and the antibiotic aurafuron (20) from Stigmatella aurantiaca, and the antifungal ajudazols from Chondromyces crocatus (21).The recently genome-sequenced myxobacterium S. cellulosum So ce56 (12) shows great potential for biotechnological applications, as judged on the basis of its capacity for the production of secondary metabolites. Three biologically active compounds have been described so far, namely the fungicidal chivosazoles, the macrolide antibiotic etnangien, and the iron chelator myxochelin (12). Moreover, the bioinformatic analysis of the So ce56 genome has revealed numerous biosynthetic gene clusters of yet unknown function (11, 12). With a size of more than 13 Mbp, the genome of S. cellulosum So ce56 is to date the largest sequenced prokaryotic genome (12). It has been shown to harbor 21 cytochrome P450 genes. In light of the significance of S. cellulosum as a viable source of bioactive secondary metabolites (14) and the role of cytochromes P450 in the synthesis of natural products (2), it is of great interest to elucidate the function of these enzymes.Therefore, the investigation of the S. cellulosum So ce56 cytochrome P450 systems opens a fascinating field not only with regard to basic research but also to exploit the biotechnological potential of this model strain. To achieve this goal it is important to provide a functional electron transport chain. Thus, the main objective of this work was to identify a myxobacterial ferredoxin/ferredoxin reductase couple able to support reactions catalyzed by S. cellulosum So ce56 cytochromes P450.