Two Novel Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus japonicus

Abstract

In the present study, two novel phenolic UDP glycosyltransferases (PUGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level.IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.

Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis

“…The easily scalable total biosynthesis of such molecules is achieved by: (i) expressing appropriate PKS subunit pairs to afford natural or unnatural BDL congeners in a yeast chassis; and (ii) coexpressing a novel fungal type II/III detoxification module to decorate the polyketide scaffolds with a 4-O-methylglucose biosynthon. Although enzymes for the biosynthetic glycosylation of various bioactive aglycones have been characterized from bacterial or plant sources (1, 7), the first fungal enzymes with significant aglycone promiscuity were only identified in 2017 (6,9). Considering the biosynthetic and biodegraditive capacities of filamentous fungi, and their proven proficiency in glycosylating various scaffolds (11-16, 20, 41), extending the pool of biocatalysts with novel fungal enzymes, such as the B. bassiana GT-MT methylglucosylation module for the glycodiversification (3,57) of various aglycones, is highly promising.…”

“…narrow-spectrum sterol 3β-glucosyltransferases were identified from various yeast strains until recently. While this work was in progress, the discovery of two disparate, nonorthologous phenolic GT groups from three basidiomycete fungi were reported (MhGT1 from Mucor hiemalis, and UGT58A1/UGT59A1 from Rhizopus japonicus and Absidia coerulea, respectively) (6,9). These discoveries indicated that despite their very low similarity to known enzymes from other organisms (6,9,10), promiscuous yet regio-and stereospecific GTs from the kingdom Fungi can indeed be identified and harnessed for synthetic biology (6,9).…”

“…While this work was in progress, the discovery of two disparate, nonorthologous phenolic GT groups from three basidiomycete fungi were reported (MhGT1 from Mucor hiemalis, and UGT58A1/UGT59A1 from Rhizopus japonicus and Absidia coerulea, respectively) (6,9). These discoveries indicated that despite their very low similarity to known enzymes from other organisms (6,9,10), promiscuous yet regio-and stereospecific GTs from the kingdom Fungi can indeed be identified and harnessed for synthetic biology (6,9). Nevertheless, fungal genome sequence data clearly indicate that a much larger variety of diverse GTs remain to be discovered, especially from the filamentous ascomycetes that dedicate 1-2% of their genomes to as yet uncharacterized GTs (6,9).…”

Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System

“…The purified UGT88E18 was applied to the in vitro reaction with calycosin (Compound 1) or formononetin (Compound 2, another important isoflavonoid present in R. astragali) as the Chemical glucosylation is notoriously complicated because of various disadvantages such as low efficiency, poor stereospecificity, a series of protection and deprotection steps, and environmental pollution [14]. In this respect, enzymatic glucosylation using regio-and stereoselective UGTs can alleviate these disadvantages [15,16]. Extensive studies indicate that sucrose synthase (SuSy) is a versatile biocatalyst that can synthesize costly uridine diphosphate glucose (UDPG) from abundant and cheap sucrose and catalytic amounts of uridine diphosphate (UDP) [17,18].…”

Biocatalytic Synthesis of a Novel Bioactive Ginsenoside Using UDP-Glycosyltransferase from Bacillus subtilis 168

“…The poor aqueous solubility of natural products resulted in a low absorption and a short retention time in the intestine [21]. Glycosylation mediated by UGTs transfers the hydrophilic sugar moieties to natural products, which can significantly enhance their solubility and thus improve their bioavailability and pharmacodynamics [29,30]. The aqueous solubility of ginsenoside Rg3 was limited to 90 µM.…”