Flexible Glycosyltransferase Research Articles

Enzymatic synthesis of novel unnatural phenoxodiol glycosides with a glycosyl donor flexible glycosyltransferase MeUGT1

Isoflavonoids are of great interest due to their human health-promoting properties, which have resulted in studies on exploiting these phytochemicals as hotspots in diverse bio -industries. Biocatalytic glycosylation of isoflavonoid aglycones to glycosides has attracted marked interests because it enable the biosynthesis of isoflavonoid glycosides with high selectivity under mild conditions, and also provide an environmentally friendly option for the chemical synthesis. Thus, these inspired us to exploit new flexible and effective glycosyltransferases from microbes for making glycosides attractive compounds that are in high demand in several industries. Most recently, we have reported the functional characterization of a bacterial-origin recombinant glycosyltransferase (MeUGT1). Herein, more detailed kinetic characteristics of this biocatalyst, using a number of glycosyl donor substrates, were examined for further investigation of its biocatalytic applicability, enabling it feasible to biosynthesize new glycosides; phenoxodiol-4′-O-α-glucuronide, phenoxodiol-4′-O-α-(2''-N-acetyl)glucosaminide, phenoxodiol-4′-O-α-galactoside, phenoxodiol-4′-O-α-(2''-N-acetyl)galactosaminide and phenoxodiol-4′-O-α-(2''-deoxy)glucoside. The thorough kinetic analyses revealed that while the recombinant enzyme can utilize, albeit with different substrate preference and catalytic efficiency, a total five different nucleotide sugars as glycosyl donors, exhibiting its promiscuity towards glycosyl donors. This is the first report that a recombinant glycosyltransferase MeUGT1 that can regio-specifically glycosylate C4′-hydroxyl function of semi-synthetic phenoxodiol isoflavene to biosynthesize a series of unnatural phenoxodiol-4′-O-α-glycosides.

Heterologous Expression Leads to Discovery of Diversified Lobophorin Analogues and a Flexible Glycosyltransferase.

The heterologous expression of the lobophorin biosynthetic gene cluster from marine-derived Streptomyces pactum SCSIO 02999 has led to the discovery of new analogues that carry d-kijanose (or its variants with a 3-amino or hydroxyl group) at C-9. The identification of intermediates resulting from the inactivation of lobG1 (encoding C-17 kijanosyltransferase) and lobG3 (encoding C-9 digitoxosyltransferase) demonstrates the substrate flexibility of LobG3 to also accept d-kijanose and its variants. Notably, two lobophorins with d-kijanose at C-9 show improved bioactivities.

Mutational analyses for product specificity of YjiC towards α-mangostin mono-glucoside

Glycosyltransferases (GTs) are key enzymes for the post-modification of secondary metabolites in drug development processes. In our prior research, an one-pot enzymatic system produced α-mangostin 3,6-di-O-β-D-glucopyranoside (Mg1) at a higher proportion using wild-type glycosyltransferase (YjiC) but α-mangostin 3-O-β-D-glucopyranoside (Mg2) exhibited markedly higher anti-bacterial activities. This study focuses on a Bacillus licheniformis-originated flexible glycosyltransferase by mutagenesis to examine the active site residues involved in glycosylation for a product specificity towards Mg2. The generated H298A, H298S, and H298C mutants of YjiC exhibited a regiospecificity towards glycosylated product (Mg2) and were targeted in this study. The production pattern of Mg1 decreased to 63 (H298A), 85 (H298S) and 95% (H298C) yields compared to the wild-type YjiC. The increase of uridine 5′-diphosphate (UDP) leading to the inhibition of enzyme activity and production of uridine 5′-diphosphate glucose (UDP-glucose) in overall system was critical for the specific glycosylated product formation rate. H298A, H298S, and H298C mutants and YjiC exhibited 244, 251, and 186% increases in Mg2 production yields, respectively. And also H298A, H298S, and H298C showed 281, 279, and 251% increases in yield of Mg3 compared with wild type YjiC, respectively. There was improved conversion of both mono-glucosides product (Mg2a and Mg3) than di-glucosides products. The H298 mutants were found to overcome the limitation of the wild-type YjiC for regioselective synthesis of Mg2 by an enzymatic system.

Donor specificity of YjiC glycosyltransferase determines the conjugation of cytosolic NDP-sugar in in vivo glycosylation reactions

Escherichia coli BL21 (DE3) was engineered by blocking glucose-1-phosphate utilizing glucose phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf) and uridylyltransferase (galU) genes to produce pool of four different rare dTDP-sugars. The cytosolic pool of dTDP-l-rhamnose, dTDP-d-viosamine, dTDP-4-amino 4,6-dideoxy-d-galactose, and dTDP-3-amino 3,6-dideoxy-d-galactose was generated by overexpressing respective dTDP-sugars biosynthesis genes from various microbial sources. A flexible glycosyltransferase YjiC, from Bacillus licheniformis DSM 13 was also overexpressed to transfer sugar moieties to 3-hydroxyl group of 3-hydroxyflavone, a core unit of flavonoids. Among four rare dTDP-sugars generated in cytosol of engineered strains, YjiC solely transferred l-rhamnose from dTDP-l-rhamnose and tuned to rhamnosyltransferase.

Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

Saccharothrix espanaensis is a member of the order Actinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase from Saccharothrix espanaensis which is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.

Ketoolivosyl-tetracenomycin C: A new ketosugar bearing tetracenomycin reveals new insight into the substrate flexibility of glycosyltransferase ElmGT

A new tetracenomycin analog, 8-demethyl-8-(4′-keto)-α-l-olivosyl-tetracenomycin C, was generated through combinatorial biosynthesis. Streptomyces lividans TK 24 (cos16F4) was used as a host for expression of a ‘sugar plasmid’ (pKOL) directing the biosynthesis of NDP-4-keto-l-olivose. This strain harbors all of the genes necessary for production of 8-demethyl-tetracenomycin C and the sugar flexible glycosyltransferase ElmGT. To the best of our knowledge, this report represents the first characterization of a tetracenomycin derivative decorated with a ketosugar moiety. Also, as far as we know, 4-keto-l-olivose has only been described as an intermediate of oleandomycin biosynthesis, but has not been described before as an appendage for a polyketide compound. Furthermore, this report gives further insight into the substrate flexibility of ElmGT to include an NDP-ketosugar, which is unusual and is rarely observed among glycosyltransferases from antibiotic biosynthetic pathways.

Development of a Streptomyces venezuelae-Based Combinatorial Biosynthetic System for the Production of Glycosylated Derivatives of Doxorubicin and Its Biosynthetic Intermediates

Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.

미생물 유래 특이당을 이용한 플라보노이드 당화반응

Glycosylation is a key mechanism in determining diversity of natural products, and influencing their bioactivities. This approach requires a core set of glycosyltransferase that synthesizes the diverse sugar structures observed in nature. Recently, the researchers have begun to alter the sugar moiety and glycosylation patterns of natural products both in vivo E. coli system and in vitro for their glycodiversification. This review highlights new glycosylation tools using microbialproduced deoxysugar and a flexible glycosyltransferase on natural plant-flavonoids to generate novel glycoforms with useful biological activity.

Cloning and Heterologous Expression of the Aranciamycin Biosynthetic Gene Cluster Revealed a New Flexible Glycosyltransferase

Cloning and Heterologous Expression of the Aranciamycin Biosynthetic Gene Cluster Revealed a New Flexible Glycosyltransferase

Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin

Background: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC).Results: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC.Conclusions: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.