Polyketides form a structurally diverse and pharmaceutically important class of secondary metabolites. Both diversity and biological activity are largely facilitated by post‐polyketide synthase tailoring including methylation, oxidation, reduction, glycosylation, and dimerization. Cytochrome P450 enzymes (CYPs), flavin‐dependent monooxygenases (FMOs), and laccases are known to catalyze phenol coupling in the biosynthesis of polyketide dimers. Polyketide homodimers resulting from enzyme catalysis are often formed in a highly regio‐ and stereoselective manner, in contrast to analogous nonenzymatic dimerization. Although it is known that CYPs and FMOs are capable of selectively generating one of several putative isomers, hitherto described laccases depend on auxiliary proteins to achieve similar selectivity. Herein, regioselective phenol coupling catalyzed by a fungal laccase is demonstrated. The heterologously produced Av‐VirL from Aspergillus viridinutans selectively generated the 6,6′‐homodimer of (R)‐semivioxanthin. Genome analysis is used to show that laccase‐based phenol‐coupling systems are widespread in fungi. Homologues of Av‐VirL were identified in the putative biosynthetic gene clusters of vioxanthin, xanthomegnin, and xanthoepocin, and of the perylenequinones hypocrellin A, elsinochrome A, and cercosporin. These findings show that laccases are capable of selective phenol coupling in the absence of auxiliary proteins.

Biaryl compounds are ubiquitous metabolites that are often formed by dimerization through oxidative phenol coupling. Hindered rotation around the biaryl bond can cause axial chirality. In nature, dimerizations are catalyzed by oxidative enzymes such as laccases. This class of enzymes is known for non‐specific oxidase reactions while inherent enantioselectivity is hitherto unknown. Here, we describe four related fungal laccases that catalyze γ‐naphthopyrone dimerization in a regio‐ and atropselective manner. In vitro assays revealed that three enzymes were highly P‐selective (ee >95 %), while one enzyme showed remarkable flexibility. Its selectivity for M‐ or P‐configured dimers varied depending on the reaction conditions. For example, a lower enzyme concentration yielded primarily (P)‐ustilaginoidin A, whereas the M atropisomer was favored at higher concentration. These results demonstrate inherent enantioselectivity in an enzyme class that was previously thought to comprise only non‐selective oxidases.

WolfgangS teglich, [b] and MichaelM üller* [a] DedicatedtoProf. Rolf Huisgen on his 100 th birthday.Dimeric polyketides are widespreadf ungal secondary metabolites. They occur in both ascomycetes and basidiomycetes and, therefore, across fungal phyla.H ere we report the isolation of an ew binaphthalene, named rufoschweinitzin, from the basidiomycete Cortinarius rufoolivaceus. Rufoschweinitzin consists of two symmetrically 4,4'-coupled torachrysone-8-O-methyl ether moieties. Furthermore, we have identified ab inaphthalene biosynthetic gene clusteri na nu nrelated fungus, the ascomycete Xylaria schweinitzii. Heterologous expression of the encoded cytochrome P450 enzymev erifiedi ts coupling activity:d imerization of torachrysone-8-O-methyl ether led to the formation of rufoschweinitzin alongside ah itherto unknown regioisomer,n ow named alloschweinitzin. We have thusd emonstrated enzymatic formation of the basidiomycete's metabolite rufoschweinitzin and made the regiochemistry of alloschweinitzin accessible with an ascomycete-derived enzyme.Naturally occurring binaphthalenes have attracted the interest of scientists due to their auspicious bioactivity and their similarity to BINOL-type catalysts. [1][2][3][4][5] Nevertheless,t heir biosynthesis has not been investigated as yet, especially with regardt o the regio-ands tereoselective oxidative phenol coupling of the monomeric precursors.Recent research hasd emonstrated that different sets of enzymes are responsible for the dimerizationo fp olyketides in fungi. Laccases were shown to mediate regioselective CÀC bond formation,s ometimes depending on auxiliary proteins to be active or to introduce stereoinformation at the biaryl axis. [6][7][8][9] CytochromeP 450 enzymes (CYPs)a re also associated with phenol-coupling reactions.I nt he biosynthesis of bicoumarins, they control both regio-and stereoselectivity on their own, without the need for auxiliary proteins. [10,11] Further,C YPs are known to catalyze the coupling reactioni nt he biosynthe-sis of the dimericn aphthopyrones nigerone (5)a nd aurasperone A( 7,S cheme 1). [12] These two metabolites are regioisomers, each exclusively formed by ad istinct Aspergillus strain and not occurring as ar egioisomericm ixture. This means that enzymes of one class (CYPs) can catalyzes elective dimerizations leading to different regioisomers. The mechanismsb ehind this are unknown.Here, the isolation and characterization of an ew binaphthalene, named rufoschweinitzin (2), from the basidiomycete Cortinarius rufoolivaceus are presented.F urthermore, we also demonstrate enzymatic formation of 2 through the action of aC YP that is encoded in ab iosynthetic gene cluster( BGC) identified in the genome of the ascomycete Xylaria schweinitzii BCC 1337. Besides the coupling activity of the CYP,w eh ave verifiedt he activity of two other encoded enzymes-namely, two O-methyltransferases-through heterologous expression. As hypothesized, the two O-methyltransferasesm ethylate the monomeric precursor 6-hydroxymusizin (8)t op rovide torachry...

Pre-)anthraquinones are widely distributed natural compounds and occur in plants, fungi, microorganisms, and animals, with atrochrysone (1) as the key biosynthetic precursor. Chemical analyses established mushrooms of the genus Cortinarius-the webcaps-as producers of atrochrysone-derived octaketide pigments. However, more recent genomic data did not provide any evidence for known atrochrysone carboxylic acid (4) synthases nor any other polyketide synthase (PKS) producing oligocyclic metabolites. Here, we describe an unprecedented class of non-reducing (NR-)PKS. In vitro assays with recombinant enzyme in combination with in vivo product formation in the heterologous host Aspergillus niger established CoPKS1 and CoPKS4 of C. odorifer as members of a new class of atrochrysone carboxylic acid synthases. CoPKS4 catalyzed both heptaand octaketide synthesis and yielded 6-hydroxymusizin (6), along with 4. These first mushroom PKSs for oligocyclic products illustrate how the biosynthesis of bioactive natural metabolites evolved independently in various groups of life.

The biaryl scaffold, often showing axial chirality, is a common feature of various fungal natural products. Their biosynthesis requires an oxidative phenol-coupling reaction usually catalyzed by laccases, cytochrome P450 enzymes, or peroxidases. The combination of a laccase and a fasciclin domain-containing (fas) protein is encoded in many biosynthetic gene clusters of biaryls from ascomycetes. However, such phenol-coupling systems including their regio- and stereoselectivity have not been characterized so far. Elucidating the biosynthesis of the antiparasitic binaphthalene sporandol from Chrysosporium merdarium, we demonstrate the combination of a laccase and a fas protein to be crucial for the dimerization reaction. Only the heterologous coproduction of the laccase and the fas protein led to a functional phenol-coupling system, whereas the laccase alone showed no coupling activity. Thus, the laccase/fas protein combination forms an independent group of phenol-coupling enzymes that determines the coupling activity and selectivity of the reaction concurrently and applies to the biosynthesis of many fungal natural products with a biaryl scaffold.