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.

We screened for microorganisms able to use flavonoids as a carbon source; and one isolate, nominated Stilbella fimetaria SES201, was found to possess a disaccharide-specific hydrolase. It was a cell-bound ectoenzyme that was released to the medium during conidiogenesis. The enzyme was shown to cleave the flavonoid hesperidin (hesperetin 7-O-alpha-rhamnopyranosyl-beta-glucopyranoside) into rutinose (alpha-rhamnopyranosyl-beta-glucopyranose) and hesperetin. Since only intracellular traces of monoglycosidase activities (beta-glucosidase, alpha-rhamnosidase) were produced, the disaccharidase alpha-rhamnosyl-beta-glucosidase was the main system utilized by the microorganism for hesperidin hydrolysis. The enzyme was a glycoprotein with a molecular weight of 42224 Da and isoelectric point of 5.7. Even when maximum activity was found at 70 degrees C, it was active at temperatures as low as 5 degrees C, consistent with the psychrotolerant character of S. fimetaria. Substrate preference studies indicated that the enzyme exhibits high specificity toward 7-O-linked flavonoid beta-rutinosides. It did not act on flavonoid 3-O-beta-rutinoside and 7-O-beta-neohesperidosides, neither monoglycosylated substrates. In an aqueous medium, the alpha-rhamnosyl-beta-glucosidase was also able to transfer rutinose to other acceptors besides water, indicating its potential as biocatalyst for organic synthesis. The monoenzyme strategy of Acremonium sp. SES201 = DSM 24697, [corrected] as well as the enzyme substrate preference for 7-O-beta-flavonoid rutinosides, is unique characteristics among the microbial flavonoid deglycosylation systems reported.

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The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-β-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-L-rhamnopyranosylβ-D-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ࣘ6.0. The structure of 4-hydroxyphenyl-β-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-β-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 • C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pK a value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds. C 2018 International

Most aroma compounds exist in vegetal tissues as disaccharide conjugates, rutinose being an abundant sugar moiety in grapes. The availability of aroma precursors would facilitate analytical analysis of plant-based foods. The diglycosidase α-rhamnosyl-β-glucosidase from Acremonium sp. DSM 24697 efficiently transglycosylated the rutinose moiety from hesperidin to 2-phenylethanol, geraniol, and nerol in an aqueousÀorganic biphasic system. 2-Phenethyl rutinoside was synthesized up to millimolar level with an 80% conversion regarding the donor hesperidin. The hydrolysis of the synthesized aroma precursors was not detected in an aqueous medium. However, in the presence of ethanol as a sugar acceptor, the enzyme was able to transfer the disaccharide residue forming the alkyl-rutinoside. The aroma precursors were significantly hydrolyzed (up to 3À4% in 2 h at 30 °C), which indicated the potential use of the enzyme for biotechnological applications, for example, in aroma modulation of fermented foods.

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Chorismate and isochorismate constitute branch‐point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli . We propose a model for the binding of isochorismate to the active site of MenD ((1 R ,2 S , 5 S ,6 S )‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐3‐ene‐1‐carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α‐ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double‐variant Asn117Arg–Leu478Thr preferentially converts (5 S ,6 S )‐5,6‐dihydroxycyclohexa‐1,3‐diene‐1‐carboxylate (2,3‐ trans ‐CHD), the hydrolysis product of isochorismate, with a >70‐fold higher ratio than that for the wild type. The single‐variant Arg107Ile uses (5 S ,6 S )‐6‐amino‐5‐hydroxycyclohexa‐1,3‐diene‐1‐carboxylate (2,3‐ trans ‐CHA) as substrate with >6‐fold conversion compared to wild‐type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L −1 ). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild‐type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.