Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)

Abstract

The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wildtype strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.Biosynthetic pathways for many antibiotics in bacteria and plants are often associated with the presence of cytochromes P450 (P450, CYP).3 The CYPs perform late-stage stereo-and regiospecific metabolic reactions, such as hydroxylation, epoxidation, dehydrogenation, and C-C bond coupling, to modify the parent structural skeletons to give biosynthetic intermediates and/or bioactive products (1-4). Members of the genus Streptomyces produce a wide variety of bioactive secondary metabolites used in human and veterinary therapeutics, including a majority of the medically useful antibiotics (5, 6). Although terpenoids are one of the largest classes of naturally occurring, biologically active substances, with more than 50,000 known compounds, including anti-cancer drugs, antiparasitic, and antibacterial agents, only a relative handful of such terpene metabolites have been isolated from Streptomyces spp (7,8).P450 monooxygenases are a superfamily of heme proteins utilizing atmospheric dioxygen and electrons supplied by NAD(P)H almost always through redox partner proteins to generate, most commonly, oxidized organic products and a molecule of water (9). P450s play an important role in drug metabolism and in the biosynthesis of steroids, lipids, vitamins, antibiotics, and other natural secondary metabolites.Streptomyces coelicolor A3 (2) is genetically one of the most studied actinomycetes and has been a model for genetic analysis of antibiotic production. The complete genome sequence of S. coelicolor has revealed the presence of 18 CYP, 6 ferredoxin, and 4 ferredoxin reductase genes (10, 11). The endogenous biological function of CYP170A1, belonging to the 18-member P450 CYPome of S. coelicolor, has not previously been identified (12). 4 The sco5223 gene encoding CYP170A1 in S. coelicolor is part of a two-gene cluster with a sesquiterpene cyclase gene (sco5222) with which it shares a four-nucleotide ATGA transcriptional overlap at its 5Ј-end. The sco5222 gene product has been shown to catalyze the synthesis of a novel sesquiterpene, epi-isozizaene (1, Fig. 1), by cyclization of the universal sesquiterpene synthase substrate farne...

2-Alkyl-4-hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers discovered by Streptomyces coelicolor genome mining

“…Here we report the discovery of the MMFs, the fifth family of previously unknown natural products of the model actinomycete S. coelicolor A3 (2) to be discovered by a genome mining approach (21)(22)(23)(24). One advantage of the genome mining approach for natural product discovery is that the genes and enzymes involved in the biosyn- thesis of the newly discovered natural product are also identified.…”

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

“…In the case of CYP158A2, a three-gene operon was identified encoding a Type III polyketide synthase. In other examples, CYPs of S. coelicolor A3(2) appeared in operons with a non-ribosomal peptide synthase (CYP105N1, 33) and another (CYP170A1) adjacent to a terpene synthase, subsequently determined to be involved in albaflavanone biosynthesis [134]. The operons can often provide clues to enzyme activities and by combining experimental approaches, e.g.…”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…These silent BGCs showed indications of possible biosynthesis of secondary metabolites, but interestingly, no corresponding compounds were detected under standard laboratory growth conditions. In the following years, many of these silent BGCs were successfully activated and their corresponding secondary metabolites were characterized, including iron-chelators desferrioxamine [5] and coelichelin, [6,7] sesquiterpene antibiotic albaflavenone, [8] and polyketide (PK) alkaloid coelimycin. [9] The initial successes in identifying novel secondary metabolites from silent BGCs have led to the subsequent increases in the genome sequencing of streptomycetes in the last decade ( Figure 1).…”