P450 in C-C coupling of cyclodipeptides with nucleobases.

Bacterial cytochrome P450 enzymes catalyze various and often intriguing tailoring reactions during the biosynthesis of natural products. In contrast to the majority of membrane-bound P450 enzymes from eukaryotes, bacterial P450 enzymes are soluble proteins and therefore represent excellent candidates for in vitro biochemical investigations. In particular, cyclodipeptide synthase-associated cytochrome P450 enzymes have recently gained attention due to the broad spectrum of reactions they catalyze, i.e. hydroxylation, aromatization, intramolecular C-C bond formation, dimerization, and nucleobase addition. The latter reaction has been described during the biosynthesis of guanitrypmycins, guatrypmethines and guatyromycines in various Streptomyces strains, where the nucleobases guanine and hypoxanthine are coupled to cyclodipeptides via C-C, C-N, and C-O bonds. In this chapter, we provide an overview of cytochrome P450 enzymes involved in the C-C coupling of cyclodipeptides with nucleobases and describe the protocols used for the successful characterization of these enzymes in our laboratory. The procedure includes cloning of the respective genes into expression vectors and subsequent overproduction of the corresponding proteins in E. coli as well as heterologous expression in Streptomyces. We describe the purification and in vitro biochemical characterization of the enzymes and protocols to isolate the produced compounds for structure elucidation.

Production of Diketopiperazine Derivatives by Pathway Engineering with Different Cyclodipeptide Synthases from Various Streptomyces Strains.

Cyclodipeptides from fungi and bacteria are often modified by different tailoring enzymes. They display various biological and pharmacological activities, and some derivatives are used as drugs. In a previous study, we elucidated the function of the silent guatrypmethine gene cluster from Streptomyces cinnamoneus containing a cyclodipeptide synthase (CDPS) core gene gtmA and four genes gtmB-gtmE for tailoring enzymes. The latter are used in this study for the design of modified cyclodipeptides by genetic engineering. Addition of six different cyclodipeptides to the Streptomyces albus transformant harboring gtmB-gtmE led to the detection of different pathway products. Coexpression of five CDPS genes from four Streptomyces strains with gtmB-gtmE resulted in the formation of diketopiperazine derivatives, differing in their modification stages. Our results demonstrate the potential of rational gene combination to increase structural diversity.

Biosynthesis of Xylariolide D in Penicillium crustosum Implies a Chain Branching Reaction Catalyzed by a Highly Reducing Polyketide Synthase.

Fungi are important sources for the discovery of natural products. During the last decades, technological progress and the increasing number of sequenced genomes facilitated the exploration of new secondary metabolites. Among those, polyketides represent a structurally diverse group with manifold biological activities. In this study, we successfully used genome mining and genetic manipulation for functional proof of a polyketide biosynthetic gene cluster from the filamentous fungus Penicillium crustosum. Gene activation in the native host and heterologous expression in Aspergillus nidulans led to the identification of the xil cluster, being responsible for the formation of the 6-methyl-2-pyrone derivative xylariolide D. Feeding with 13C-labeled precursors supported the hypothesis of chain branching during the backbone formation catalyzed by a highly reducing fungal polyketide synthase. A cytochrome P450-catalyzed hydroxylation converts the PKS product to the final metabolite. This proved that just two enzymes are required for the biosynthesis of xylariolide D.