Nucleotide Sugar is an activated form in the process of sugar conversion and synthesis, widely present in eukaryotes and prokaryotes. It is essential for the operation of physiological functions and even survival of organisms as a glycosyl donor in polysaccharide synthesis. Structurally, nucleotide sugars can be divided into nucleoside monophosphate (NMP)-sugar and nucleotide diphosphate (NDP)-sugar, among which NDP-sugar is the more common nucleotide sugar, and uridine diphosphate (UDP)-sugar is the most common nucleotide sugar donor, which is transferred to the glycosyl acceptor by Glycosyltransferase or synthetase in the polysaccharide biosynthesis pathway to build glycosidic bonds.
Focusing on the Biosynthesis of nucleotide sugars, Professor Jay Keasling's laboratory at the University of California, Berkeley, engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases, and obtained a variety of UDP-sugars from simple starting substrates (such as glucose, Galactose, etc.), providing a new biosynthetic platform for the future production of various glycosylated new natural products and proteins.
Currently, this research result has been published in ACS Synthetic Biology under the title " Engineered Saccharomyces cerevisiae as a Biosynthetic Platform of Nucleotide Sugars".
Glycosylation is a common modification in proteins, lipids, and Natural Products. The sugars on these molecules can greatly change their biological activity and stability. Today, the industry's chemical research on glycosylation has begun to explore the synthetic space of glycoconjugates and the physiological role of sugars in biology.
In nature, the addition of sugars in the glycosylation process is achieved by highly specific glycosyltransferases, among which NDP-sugars are the monomer substrates for more than 90% of glycosylation reactions and are important components of natural polysaccharides and glycoconjugates. Developing new biosynthetic pathways to more simply produce nucleotide sugars will greatly accelerate the characterization of new glycosylation reactions, the elucidation of their potential regulatory mechanisms, and the production of glycosylated molecules.
Fig. 1 Biosynthesis of UDP-D-Glc and UDP-D-Gal. (Crowe, et al., 2024)
Nucleotide sugars can be synthesized de novo, a process involving nucleotide sugar synthetases, which provide a biosynthetic network that converts existing nucleotide sugars into NDP-sugar monomers of various structures through reactions such as oxidation, decarboxylation, and isomerization.
As a model microorganism, yeast has genetic operability and scalability, can express the entire biosynthetic pathway of complex natural products, and produce new natural products, etc., and is often used to study the function of new enzymes in vivo. In this study, Jay Keasling and his team chose yeast as a biosynthetic platform chassis to build this enzyme network.
Yeast can produce UDP-D-glucose (UDP-D-Glc) and UDP-D-galactose (UDP-D-Gal) by itself, and these UDP-sugar substrates can be converted into other UDP-sugars by expressing the corresponding heterologous nucleotide sugar synthases.
In this study, they demonstrated that nucleotide sugar synthases from plants and microorganisms can be expressed individually and in combination in engineered S. cerevisiae, thereby obtaining various types of UDP-sugars.
Specifically, UDP-D-Glc can be converted into UDP-D-glucuronic acid (UDP-D-GlcA), UDP-D-xylose (UDP-D-Xyl), UDP-d-apiose (UDP-d-Api), UDP-D-fucose (UDP-D-Fuc), UDP-L-rhamnose (UDP-L-Rha), UDP-L-arabinopyranose (UDP-L-Arap), and UDP-l-arabinofuranose (UDP-L-Araf) by nucleotide sugar synthase.
Notably, the interconversion of various types of UDP-sugars is allosterically regulated by certain nucleotide sugars, for example, the downstream product UDP-D-Xyl strongly inhibits UDP-D-Glc 6-dehydrogenase (UGD).
In order to overcome the inhibitory effect and better regulate the intracellular levels of UDP-D-GlcA and UDP-D-Xyl, they explored multiple strategies to delay the production of UDP-D-Xyl by generating UGD mutants, screening functional homologs, and orthogonal induction systems (Tet-On) to maintain the production and accumulation of upstream UDP-sugars, thereby circumventing this inhibitory effect and allowing both UDP-D-GlcA and UDP-D-Xyl to accumulate in yeast.
In addition to de novo synthesis, another synthesis strategy for nucleotide sugars is through the salvage pathway, in which free sugars are activated by kinases to produce sugar-1-phosphate, which is then converted to NDP-sugar by NDP pyrophosphorylase. In this study, Jay Keasling and his team expressed genes encoding the salvage pathway to directly activate free sugars, thereby achieving the biosynthesis of UDP-l-Arap and UDP-l-Alaf.
In addition, they conducted a time course study based on this engineered yeast strain containing a biosynthetic pathway for producing multiple non-natural UDP-sugars to analyze the time-dependent interconversion of these sugars and the role of UDP-D-Xyl in regulating UDP-sugar metabolism. Over the course of 48 hours after induction, they observed the role of UDP-D-Xyl in regulating UDP-sugar synthesis to maintain high intracellular concentrations of UDP-D-Glc.
The experimental results show that the concentration of UDP-sugar in yeast ranges from 62 μM to 3.4 mM, and the high concentration of nucleotide sugars can be used for intracellular glycosylation. The research team said that the concentration of nucleotide sugars can be further increased by testing different nucleotidase homologs or by increasing the natural pool of UDP-D-Glc.
Fig. 2 The biosynthesis pathway of UDP-sugar in engineered yeast. (Crowe, et al., 2024)
In general, this study transformed S. cerevisiae into a UDP-sugar biosynthetic platform by utilizing its natural UDP-D-Glc and UDP-D-Gal metabolism, as well as plant and microbial nucleotide sugar synthases. In addition to the UDP-D-Glc and UDP-D-Gal that yeast itself possesses, they synthesized seven heterologous UDP-sugars (including UDP-D-GlcA, UDP-D-Xyl, UDP-D-Api, UDP-L-Rha, UDP-D-Fuc, UDP-L-Arap and UDP-L-Araf) through de novo pathways and salvage pathways, greatly increasing the accessibility and availability of UDP-sugars.
In addition, based on this biosynthetic platform, sugar synthases can be functionally characterized in vivo, and the regulation of intracellular UDP-sugar interconversion and the production of glycosylated secondary metabolites and proteins can be detected, which will help to gain a deeper understanding of the biosynthesis, regulation, and physiological role of glycosylated biomolecules in biology.
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