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O-glycosyltransferases from Homo sapiens contributes to the biosynthesis of Glycyrrhetic Acid 3-O-mono-β-D-glucuronide and Glycyrrhizin in Saccharomyces cerevisiae.

ABSTRACT: Glycyrrhizin (GL) and Glycyrrhetic Acid 3-O-mono-β-D-glucuronide (GAMG) are the typical triterpenoid glycosides found in the root of licorice, a popular medicinal plant that exhibits diverse physiological effects and pharmacological manifestations. However, only few reports are available on the glycosylation enzymes involved in the biosynthesis of these valuable compounds with low conversion yield so far. In mammals, glycosyltransferases are involved in the phase II metabolism and may provide new solutions for us to engineer microbial strains to produce high valued compounds due to the substrate promiscuity of these glycosyltransferases. In this study, we mined the genomic databases of mammals and evaluated 22 candidate genes of O-glycosyltransferases by analyzing their catalytic potential for O-glycosylation of the native substrate, glycyrrhetinic acid (GA) for its glycodiversification. Out of 22 selected glycosyltransferases, only UGT1A1 exhibited high catalytic performance for biosynthesis of the key licorice compounds GL and GAMG. Molecular docking results proposed that the enzymatic activity of UGT1A1 was likely owing to the stable hydrogen bonding interactions and favorite conformations between the amino acid residues around substrate channels (P82~R85) and substrates. Furthermore, the complete biosynthesis pathway of GL was reconstructed in Saccharomyces cerevisiae for the first time, resulting in the production of 5.98 ± 0.47 mg/L and 2.31 ± 0.21 mg/L of GL and GAMG, respectively.

SUBMITTER: Xu K

PROVIDER: S-EPMC8283272 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the remaining nucleosomes follow a statistical positioning model.

Project description:Glycyrrhetinic acid 3-O-mono-?-d-glucuronide (GAMG), which possesses a higher sweetness and stronger pharmacological activity than those of glycyrrhizin (GL), can be obtained by removal of the distal glucuronic acid (GlcA) from GL. In this study, we isolated a ?-glucuronidase (TpGUS79A) from the filamentous fungus Talaromyces pinophilus Li-93 that can specifically and precisely convert GL to GAMG without the formation of the by-product glycyrrhetinic acid (GA) from the further hydrolysis of GAMG. First, TpGUS79A was purified and identified through matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) and deglycosylation, indicating that TpGUS79A is a highly N-glycosylated monomeric protein with a molecular mass of around 85 kDa, including around 25 kDa of glycan moiety. The gene for TpGUS79A was then cloned and verified by heterologous expression in Pichia pastoris TpGUS79A belonged to glycoside hydrolase family 79 (GH79) but shared low amino acid sequence identity (<35%) with the available GH79 GUS enzymes. TpGUS79A had strict specificity toward the glycan moiety but poor specificity toward the aglycone moiety. Interestingly, TpGUS79A recognized and hydrolyzed the distal glucuronic bond of GL but could not cleave the glucuronic bond in GAMG. TpGUS79A showed a much higher catalytic efficiency on GL (kcat/Km of 11.14 mM-1 s-1) than on the artificial substrate pNP ?-glucopyranosiduronic acid (kcat/Km of 0.01 mM-1 s-1), which is different from the case for most GUSs. Homology modeling, substrate docking, and sequence alignment were employed to identify the key residues for substrate recognition. Finally, a fed-batch fermentation in a 150-liter fermentor was established to prepare GAMG through GL hydrolysis by T. pinophilus Li-93. Therefore, TpGUS79A is potentially a powerful biocatalyst for environmentally friendly and cost-effective production of GAMG.IMPORTANCE Compared to chemical methods, the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-?-d-glucuronide (GAMG), which has a higher sweetness and stronger pharmacological activity than those of GL, via catalysis by ?-glucuronidase is an environmentally friendly approach due to the mild reaction conditions and the high yield of GAMG. However, currently available GUSs show low substrate specificity toward GL and further hydrolyze GAMG to glycyrrhetinic acid (GA) as a by-product, increasing the difficulty of subsequent separation and purification. In the present study, we succeeded in isolating a novel ?-glucuronidase (named TpGUS79A) from Talaromyces pinophilus Li-93 that specifically hydrolyzes GL to GAMG without the formation of GA. TpGUS79A also shows higher activity on GL than those of the previously characterized GUSs. Moreover, the gene for TpGUS79A was cloned and its function verified by heterologous expression in P. pastoris Therefore, TpGUS79A can serve as a powerful biocatalyst for the cost-effective production of GAMG through GL transformation.

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O-glycosyltransferases from Homo sapiens contributes to the biosynthesis of Glycyrrhetic Acid 3-O-mono-β-D-glucuronide and Glycyrrhizin in Saccharomyces cerevisiae.

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