Enzymatic tools for engineering natural product glycosylation

Glycosylated natural products have served as reliable platforms for the development of many existing front-line drugs. In an effort to explore the contribution of the sugar constituents of these compounds, research groups have focused upon the development of chemical and enzymatic tools to diversify natural product glycosylation. Among the complementary routes available, in vivo pathway engineering, also referred to as 'combinatorial biosynthesis', is an emerging method that relies upon the co-expression of sugar biosynthetic gene cassettes and glycosyltransferases in a host organism to generate novel glycosylated natural products. An overview of recent progress in combinatorial biosynthesis is highlighted in this review, emphasizing the elucidation of nucleotide-sugar biosynthetic pathways and recent developments on glycosyltransferases.     

A strategy for cloning glycosyltransferase genes involved in natural product biosynthesis

The soil-borne and marine gram-positive Actinomycetes are a particularly rich source of carbohydrate-containing metabolites. With the advent of molecular tools and recombinant methods applicable to Actinomycetes, it has become feasible to investigate the biosynthesis of glycosylated compounds at genetic and biochemical levels, which has finally set the basis for engineering novel natural product derivatives. Glycosyltransferases (GT) are key enzymes for the biosynthesis of many valuable natural products that contain sugar moieties and they are most important for drug engineering. So far, the direct cloning of unknown glycosyltransferase genes by polymerase chain reaction (PCR) has not been described because glycosyltransferases do not share strongly conserved amino acid regions. In this study, we report a method for cloning of novel so far unidentified glycosyltransferase genes from different Actinomycetes strain. This was achieved by designing primers after a strategy named consensus-degenerate hybrid oligonucleotide primer (CODEHOP). Using this approach, 22 novel glycosyltransferase encoding genes putatively involved in the decoration of polyketides were cloned from the genomes of 10 Actinomycetes. In addition, a phylogenetic analysis of glycosyltransferases from Actinomycetes is shown in this paper.     

A bacterial glycosyltransferase gene toolbox: Generation and applications

The bioactivity of many natural products produced by microorganisms can be attributed to their sugar substituents. These substituents are transferred as nucleotide-activated sugars to an aglycon by glycosyltransferases. Engineering these enzymes can broaden their substrate specificity and can therefore have an impact on the bioactivity of the secondary metabolites.In this review we present the generation of a glycosyltransferase gene toolbox which contains more than 70 bacterial glycosyltransferases to date. Investigations of the function, specificity and structure of these glycosyltransferases help to understand the great potential of these enzymes for natural product biosynthesis.     

Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution

Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels, and/or even the molecular mechanism of action. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.     

The impact of enzyme engineering upon natural product glycodiversification

Glycodiversification of natural products is an effective strategy for small molecule drug development. Recently, improved methods for chemo-enzymatic synthesis of glycosyl donors has spurred the characterization of natural product glycosyltransferases (GTs), revealing that the substrate specificity of many naturally occurring GTs as too stringent for use in glycodiversification. Protein engineering of natural product GTs has emerged as an attractive approach to overcome this limitation. This review highlights recent progress in the engineering/evolution of enzymes relevant to natural product glycodiversification with a particular focus upon GTs.     

Glycosyltransferases as biocatalysts

Glycosyltransferases are useful synthetic tools for the preparation of natural oligosaccharides, glycoconjugates and their analogues. High expression levels of recombinant enzymes have allowed their use in multi-step reactions, on mg to multi-gram scales. Since glycosyltransferases are tolerant with respect to utilizing modified donors and acceptor substrates they can be used to prepare oligosaccharide analogues and for diversification of natural products. New sources of enzymes are continually discovered as genomes are sequenced and they are annotated in the Carbohydrate Active Enzyme (CAZy) glycosyltransferase database. Glycosyltransferase mutagenesis, domain swapping and metabolic pathway engineering to change reaction specificity and product diversification are increasingly successful due to advances in structure-function studies and high throughput screening methods.     

Altering the glycosylation pattern of bioactive compounds

Many bioactive natural products are glycosylated compounds in which the sugars are important or essential for biological activity. The isolation of several sugar biosynthesis gene clusters and glycosyltransferases from different antibiotic-producing organisms, and the increasing knowledge about these biosynthetic pathways opens up the possibility of generating novel bioactive compounds through combinatorial biosynthesis in the near future. Recent advances in this area indicate that antibiotic glycosyltransferases show some substrate flexibility that might allow us to alter the types of sugar transferred to the different aglycons or, less frequently, to change the position of its attachment.     

天然产物的糖基化及糖基侧链改造

抗生素和抗癌药物等多种天然产物的活性都依赖于其糖基侧链,糖基侧链结构的变化对母体化合物的生物活性、底物适应性及药理学性质具有重要影响。糖基侧链结构变化多端,修饰、改变天然产物的糖基侧链已成为获得临床候选药物的重要方法。利用化学法和酶法,研究者创造了多种改造天然产物糖基化的方法。详细介绍了天然产物的糖基化过程,并从组合生物学、糖基转移酶改造、糖类随机化及新型糖类随机化和糖基转移酶可逆性四方面阐述了糖基侧链的改造方法。     

微生物合成黄酮糖苷类天然产物研究进展

黄酮糖苷类天然产物是植物中黄酮类化合物的主要存在形式,通过糖基化修饰,可以改变其水溶性、稳定性等,赋予其新的生物活性和功能。黄酮类化合物的糖基化修饰通常由植物源或微生物源的糖基转移酶催化,根据糖基的位置、类型和数量的不同,可形成多种类型的黄酮糖苷类产物。随着合成生物学和代谢工程的快速发展,在微生物中合成植物源黄酮糖苷类天然产物取得了重要进展。综述了糖基转移酶的聚类分析及糖基供体的途径改造,并对代谢工程优化黄酮糖苷类天然产物的微生物合成进行了分析讨论,并对其发展前景进行了展望。     

Glycosyltransferases involved in the biosynthesis of biologically active natural products that contain oligosaccharides

A unique characteristic of carbohydrates is their structural diversity which is greater than that of many other classes of biological compounds. Carbohydrate-containing natural products show many different biological activities and some of them have been developed as drugs for medical use. The biosynthesis of carbohydrate-containing natural products is catalysed by glycosyltransferases. In this review we will present information on the function of glycosyltransferases involved in the biosynthesis of oligosaccharide antibiotics focusing especially on urdamycins and landomycins, two angucycline antibiotics with interesting antitumor activities. We will also discuss the use of glycosyltransferases in combinatorial biosynthesis to generate new "hybrid" antibiotics.     

Structure, mechanism and engineering of plant natural product glycosyltransferases

Glycosylation is a key mechanism in determining chemical complexity and diversity of plant natural products, and influencing their chemical properties and bioactivities. Uridine diphosphate glycosyltransferases (UGTs) are the central players in these glycosylation processes for decorating natural products with sugars. Crystal structures of plant UGTs have revealed their exquisite architectures and provided the structural basis for understanding their catalytic mechanism and substrate specificity. Structure-based UGT engineering can alter substrate specificity; compromise or enhance catalytic efficiency; and confer reversibility to the glycosylation reaction. This review highlights the structural insights on plant UGTs and successes in glycosylation engineering.     

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure sugar nucleotide donor substrates is required. Herein, the rapid synthesis and purification of eight sugar nucleotides is described by a facile 30 min activation of nucleoside 5'-monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxysugar nucleotides, and hexose or pentose-derived sugar nucleotides, respectively.     

放线菌天然产物糖基化改造研究进展

放线菌可以产生结构多样的天然产物, 其中包括很多重要的抗菌和抗肿瘤药物。糖基化修饰在天然产物中广泛存在, 糖基侧链的变化往往会影响天然产物的生物活性。本文综述了放线菌来源天然产物糖基化改造的研究进展。糖基侧链改造的方法主要分为体内基因工程和体外酶学法。运用这两种方法已经成功对多种天然产物进行了糖基侧链改造, 获得了大量带有新糖基修饰的天然产物, 其中有些生物活性得以提高。天然产物糖基侧链改造为新药开发提供了一个重要的途径。     

Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes: isolation, characterization and generation of novel glycosylated derivatives

Many bioactive natural products synthesized by actinomycetes are glycosylated compounds in which the appended sugars contribute to specific interactions with their biological target. Most of these sugars are 6-deoxyhexoses, of which more than 70 different forms have been identified, and an increasing number of gene clusters involved in 6-deoxyhexoses biosynthesis are being characterized from antibiotic-producing actinomycetes. Novel glycosylated compounds have been generated by modifying natural deoxysugar biosynthesis pathways in the producer organisms, and/or the simultaneous expression in these strains of selected deoxysugar biosynthesis genes from other strains. Non-producing strains endowed with the capacity to synthesize novel deoxysugars through the expression of engineered deoxysugar biosynthesis clusters can also be used as alternative hosts. Transfer of these deoxysugars to a multiplicity of aglycones relies upon the existence of glycosyltransferases with an inherent degree of 'relaxed substrate specificity'. In this review, we analyze how the knowledge coming out from isolation and characterization of deoxysugar biosynthesis pathways from actinomycetes is being used to produce novel glycosylated derivatives of natural products.     

Engineering the glycosylation of natural products in actinomycetes

Bioactive natural products are frequently glycosylated with saccharide chains of different length, in which the sugars contribute to specific interactions with the biological target. Combinatorial biosynthesis approaches are being used in antibiotic-producing actinomycetes to generate derivatives with novel sugars in their architecture. Recent advances in this area indicate that glycosyltransferases involved in the biosynthesis of natural products have substrate flexibility regarding the sugar donor but also, less frequently, with respect to the aglycon acceptor. Therefore, the possibility exists of altering the glycosylation pattern of natural products, thus enabling an increase in the structural diversity of natural products.     

Phenylpropanoid glycosyltransferases from osage orange (Maclura pomifera) fruit

Flavonoids and isoflavonoids are well known for their beneficial effects on human health and their anti-insect and anti-microbial activities in plants. Osage orange fruit is rich in prenylated isoflavones and dihydrokaempferol and its glucoside. Four glycosyltransferases were identified from a collection of osage orange fruit expressed sequence tags. Biochemical characterization suggested that the glycosyltransferase UGT75L4 might be responsible for glucosylation of dihydrokaempferol in vivo, although this enzyme exhibited broad substrate recognition toward isoflavonoids and flavonoids in vitro. UGT88A4 was active on coumarin substrates. Identification of highly active phenylpropanoid glycosyltransferases will facilitate the metabolic engineering of glycosylated natural products in plants.     

Characterization of TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase from the D-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies

Deoxysugars are critical structural elements for the bioactivity of many natural products. Ongoing work on elucidating a variety of deoxysugar biosynthetic pathways has paved the way for manipulation of these pathways for the generation of structurally diverse glycosylated natural products. In the course of this work, the biosynthesis of d-mycaminose in the tylosin pathway of Streptomyces fradiae was investigated. Attempts to reconstitute the entire mycaminose biosynthetic machinery in a heterologous host led to the discovery of a previously overlooked gene, tyl1a, encoding an enzyme thought to convert TDP-4-keto-6-deoxy-d-glucose to TDP-3-keto-6-deoxy-d-glucose, a 3,4-ketoisomerization reaction in the pathway. Tyl1a has now been overexpressed, purified, and assayed, and its activity has been verified by product analysis. Incubation of Tyl1a and the C-3 aminotransferase TylB, the next enzyme in the pathway, produced TDP-3-amino-3,6-dideoxy-d-glucose, confirming that these two enzymes act sequentially. Steady state kinetic parameters of the Tyl1a-catalyzed reaction were determined, and the ability of Tyl1a and TylB to process a C-2 deoxygenated substrate and a CDP-linked substrate was also demonstrated. Enzymes catalyzing 3,4-ketoisomerization of hexoses represent a new class of enzymes involved in unusual sugar biosynthesis. The fact that Tyl1a exhibits a relaxed substrate specificity holds potential for future deoxysugar biosynthetic engineering endeavors.     

Recent progress in synthesis of carbohydrates with sugar nucleotide-dependent glycosyltransferases

Sugar nucleotide-dependent glycosyltransferases (GTs) are key enzymes that catalyze the formation of glycosidic bonds in nature. They have been increasingly applied in the synthesis of complex carbohydrates and glycoconjugates with or without in situ generation of sugar nucleotides. Human GTs are becoming more accessible and new bacterial GTs have been identified and characterized. An increasing number of crystal structures elucidated for GTs from mammalian and bacterial sources facilitate structure-based design of mutants as improved catalysts for synthesis. Automated platforms have also been developed for chemoenzymatic synthesis of carbohydrates. Recent progress in applying sugar nucleotide-dependent GTs in enzymatic and chemoenzymatic synthesis of mammalian glycans and glycoconjugates, bacterial surface glycans, and glycosylated natural products from bacteria and plants are reviewed.     

Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.     

Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.