For almost 100 years, phenoxy radical coupling has been known to proceed in nature. Because of the linkage of their molecular halves (regiochemistry) and the configuration of the biaryl axis (stereochemistry), biaryls are notoriously difficult to synthesize. Whereas the intramolecular enzymatic coupling has been elucidated in detail for several examples, the bimolecular intermolecular coupling could not be assigned to one single enzyme in the biosynthesis of axially chiral biaryls. As these transformations often take place regio- and stereoselectively, enzyme-catalyzed control is reasonable. We now report the identification and expression of fungal cytochrome P450 enzymes that catalyze regio- and stereoselective intermolecular phenol couplings. The cytochrome P450 enzyme KtnC from the kotanin biosynthetic pathway of Aspergillus niger was expressed in Saccharomyces cerevisiae. The recombinant cells catalyzed the coupling of the monomeric coumarin 7-demethylsiderin both regio- and stereoselectively to the 8,8'-dimer P-orlandin, a precursor of kotanin. The sequence information obtained from the kotanin biosynthetic gene cluster was used to identify in silico a similar gene cluster in the genome of Emericella desertorum, a producer of desertorin A, the 6,8'-regioisomer of orlandin. The cytochrome P450 enzyme DesC was also expressed in S. cerevisiae and was found to regio- and stereoselectively catalyze the coupling of 7-demethylsiderin to M-desertorin A. Our results show that fungi use highly specific cytochrome P450 enzymes for regio- and stereoselective phenol coupling. The enzymatic activities of KtnC and DesC are relevant for an understanding of the mechanism of this important biosynthetic step. These results suggest that bimolecular phenoxy radical couplings in nature can be catalyzed by phenol-coupling P450 heme enzymes, which might also apply to the plant kingdom.

Due to their diverse regio-and stereoselectivities, proline hydroxylases provide a straightforward access to hydroxprolines and other hydroxylated cylic amino acids, valuable chiral building blocks for chemical synthesis, which are often not available at reasonable expense by classical chemical synthesis. As yet, the application of proline hydroxylases is limited to a sophisticated industrial process for the production of two hydroxyproline isomers. This is mainly due to difficulties in their heterologues expression, their limited in vitro stability and complex product purification procedures. Here we describe a facile method for the production of cis-3-, cis-4-and trans-4-proline hydroxylase, and their application for the regio-and stereoselective hydroxylation of l-proline and its six-membered ring homologue l-pipecolic acid. Since in vitro catalysis with these enzymes is not very efficient and conversions are restricted to the milligram scale, an in vivo procedure was established, which allowed a quantitative conversion of 6 mM l-proline in shake flask cultures. After facile product purification via ion exchange chromatography, hydroxyprolines were isolated in yields of 35-61% (175-305 mg per flask). l-Pipecolic acid was converted with the isolated enzymes to prove the selectivities of the reactions. In transformations with optimized iron(II) concentration, conversions of 17-68% to hydroxylated products were achieved. The regio-and stereochemistry of the products was determined by NMR techniques. To demonstrate the applicability of the preparative in vivo approach for non-physiological substrates, l-pipecolic acid was converted with an E. coli strain producing trans-4-proline hydroxylase to trans-5-hydroxy-l-pipecolic acid in 61% yield. Thus, a synthetically valuable group of biocatalysts was made readily accessible for application in the laboratory without a need for special equipment or considerable development effort.

Phenol coupling enzymes, especially laccases and CYP-enzymes create an enormous diversity of biarylic secondary metabolites in fungi, plants, and bacteria. The enzymes and the elucidation of the corresponding metabolic pathways are presented.

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far.

The intermolecular, regio- and stereoselective phenol coupling for the biosynthesis of the bicoumarin kotanin in Aspergillus niger has been investigated. Feeding experiments with singly and doubly (13)C-labeled monomeric precursors clearly proved that it is not the coumarin siderin but its hydroxy derivative, demethylsiderin, that undergoes phenol coupling. However, siderin is demethylated regioselectively to demethylsiderin and it is the latter that is coupled to the corresponding dehydrodimer, orlandin. The product is subsequently O-methylated in a stepwise fashion to demethylkotanin and kotanin. Crude extracts were analysed by HPLC with chemically synthesized bicoumarins as reference compounds. This and a stereochemical analysis of the isolated bicoumarins revealed that A. niger produces exclusively the (P)-atropisomers of the three 8,8'-bicoumarins, kotanin, demethylkotanin, and orlandin. The absence of other monomeric or dimeric coumarins strongly suggests an intermolecular, regio- and stereoselective mode for the phenol-coupling step.