Background: The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast. Results: Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4ʹ-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity. Conclusions: This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.

Chalcones, including 4′-hydroxychalcones, have garnered significant attention in the area of drug discovery due to their diverse pharmacological properties, such as anti-inflammatory, antioxidative, and anticancer effects. However, their low water solubility and bioavailability limit their efficacy in vivo. Glycosylation presents a promising approach to enhance the water solubility, stability, and metabolic properties of chalcones. This study investigates the enzymatic glycosylation of eight chemically synthesized 4′-hydroxychalcones using a diverse set of sugar glucosyltransferases from bacterial, plant, and fungal sources, alongside Glycine max sucrose synthase (GmSuSy) in a cascade reaction. Among the tested enzymes, five exhibited a remarkable versatility for glycoside production, and for large-scale biotransformation, flavonoid 7-O-glucosyltransferase Sbaic7OGT from Scutellaria baicalensis was selected as the most effective. As a result of the experiments conducted, eight trans-chalcone glycosides were obtained. During the purification of the reaction products, we also observed the isomerization of the products by simple sunlight exposure, which resulted in eight additional cis-chalcone glycosides. This study highlights the novel use of a cascade reaction involving Glycine max sucrose synthase (GmSuSy) for the efficient glycosylation of trans-4′-hydroxychalcones, alongside the unexpected discovery of cis-chalcone glycosides during the purification process.

Plant cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the regioselective hydroxylation of aromatic compounds. However, their expression in bacterial hosts is hampered by poor solubility, membrane anchoring and the requirement for redox partners. In this work, we report the design and characterization of modular expression systems that enable the functional production of SbCYP82D1.1 from Scutellaria baicalensis (SbF6H) in Escherichia coli. Both independent expression and synthetic fusion systems were evaluated by combining a CYP with a compatible reductase (ATR2_tr from Arabidopsis thaliana) to catalyze the conversion of chrysin into baicalein. A combinatorial library of N-terminal variants, host strains, media, and induction strategies was constructed and screened. Among the tested host, E. coli DH 10-beta provided the highest product titers, particularly when cultures were supplemented with 5-aminolevulinic acid. Truncation of the native transmembrane anchor significantly improved catalytic performance, whereas the addition of the heterologous MALLLAVF tag decreased activity. Fusion systems outperformed separate expression formats, showing approximately two-fold higher activity, with the flexible glycine–serine linker (L_GS) supporting the highest hydroxylation product formation. The corresponding fusion construct showed an apparent conversion of 0.1 mM chrysin to baicalein of up to 90% under the applied whole-cell reaction and analytical conditions, although this value should be interpreted with caution due to the concurrent instability of baicalein observed in all reactions and culture conditions. This result nevertheless indicates a marked improvement in whole-cell baicalein formation compared with previously reported bacterial systems. Together, these results demonstrate that rational N-terminal engineering combined with fusion protein design can enable efficient bacterial expression of plant CYPs, representing a promising step toward scalable production of hydroxylated flavonoids.

Steroid glycosides constitute an important class of bioactive molecules, yet their selective synthesis remains challenging. Here, we established a screening platform for nucleotide sugar-dependent glycosyltransferases (GTs) coupled with sucrose synthase (SuSy) for in situ UDP-glucose regeneration, enabling cost-efficient steroid glucosylation. A library of GTs comprising literature-derived enzymes and newly mined archaeal and fungal candidates was constructed using sequence filtering, AlphaFold3 modeling, and docking-guided prioritization. The resulting panel was screened against 31 structurally diverse steroids (androgens, estrogens, pregnanes, and corticosteroids) using crude Escherichia coli lysates as catalysts and UPLC-DAD, LC-MS and NMR analytics. YjiC and OleD glycosyltransferases emerged as the most promiscuous biocatalysts, while Sbaic7OGT and SgUGT74AC1_M7 displayed greater selectivity toward estrogens and selected testosterone derivatives. Product assignment for representative reactions was validated using authenticated reference standards or NMR (1D/2D) analysis, confirming regioisomeric estradiol monoglucosides (3-O- and 17-O-), estrone 3-O-glucoside, and an unexpected product diversification for 17α-testosterone by endogenous E. coli enzyme, where the major product was identified as a 6′-O-acetylated glucoside. Finally, SuSy-coupled cascades were applied in semi-preparative scale and evaluated under optimized conditions and co-immobilization formats.