Advances in Engineering Nucleotide Sugar Metabolism for Natural Product Glycosylation in Saccharomyces cerevisiae

Abstract

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

Keywords: Saccharomyces cerevisiae; Uridine diphosphate sugar metabolism; glycosides; glycosylation; natural products; nucleotide sugar.

Figure 1

Structure of uridine diphosphate α-d-glucose (UDP-d-Glc) consisting of uracil, ribose, and diphosphate (which compose UDP) and d-glucose. This is a common activated form of d-glucose which is essential for recognition by UDP-dependent glycosyltransferases and its use in glycosylation.

Figure 2

Major pathways to produce different UDP-sugars from simple sugars in vivo. Pathways that start from individual sugars with known enzymes are included. Gray dashed arrows indicate transporters from the cytosol into the lumen of the Golgi apparatus. Many UDP-sugars are transported into the Golgi apparatus by specific UDP-sugar transporters and can be interconverted in the Golgi lumen, but only a few are shown as examples. Enzymes in the cytosol that interconvert or salvage UDP-sugars can be found either purely cytosolic or anchored to the cell membrane, Golgi apparatus, etc.S. cerevisiae’s native metabolism is depicted with black arrows, and non-native pathways to access other UDP-sugars are depicted with red arrows. A full list of enzymes needed for each part of this metabolic network can be found in Table S1.

Figure 3

Salvage pathway. The salvage pathway consists of the phosphorylation of a sugar by an associated kinase and then the transfer of a UMP moiety from UTP by a UDP-sugar pyrophosphorylase.

Figure 4

De novo synthesis of UDP-sugars from UDP-d-Glc/UDP-d-Gal starting with d-Gal and d-Glc feed-in. S. cerevisiae’s native metabolism is shaded in orange. Free glucose can be phosphorylated by glucokinase (GlcK) or hexokinase to d-Glc-6-P, which is then mutated by phosphoglucomutase (PGM) to d-Glc-1-P, which in turn is converted to UDP-d-Glc by UDP-d-Glc pyrophosphorylase (UGP). Free galactose can be phosphorylated by GALK and converted by GALT with UDP-d-Glc to form UDP-d-Gal and d-Glc-1-P. UDP-d-Gal is also interconverted with UDP-d-Glc by UDP-d-Glc 4-epimerase (UGE). UDP-l-Rha is made from UDP-d-Glc using UDP-rhamnose synthase (RHM), a three-domain enzyme composed of a 4,6-dehydratase, denoted as RHM (N), and a 3,5-epimerase and 4-keto-reductase, denoted as RHM (C). UDP-d-Fuc is made by UDP-glucose-4,6-dehydratase (UG46DH) via a common keto-sugar intermediate, UDP-4-keto-6-deoxy-d-Glc, which is then reduced by neomenthol dehydrogenase (NMD). UDP-l-Rha is a known inhibitor of several UG46DH domains of RHM. UDP-d-GlcA is synthesized from UDP-d-Glc by UDP-d-Glc 6-dehydrogenase (UGD). UDP-d-GlcA 4-epimerase (UglcAE) interconverts UDP-d-GlcA and UDP-d-GalA by C4 epimerization. The C6 carboxylic acid of UDP-d-GlcA is decarboxylated by UDP-d-Xyl synthase (UXS) to yield UDP-d-Xyl. UDP-d-Api/Xyl synthase (AXS) can convert UDP-d-GlcA to a mixture of UDP-d-Xyl and UDP-d-Api. UDP-l-Arap and UDP-l-Araf are the pyranose/furanose isomers of UDP-l-Ara. UDP-d-Xyl 4-epimerase (UXE) synthesizes UDP-l-Arap from UDP-d-Xyl by C4 epimerization, and UDP-l-Ara mutase (UAM) performs a ring mutation of UDP-l-Arap to form UDP-l-Araf. Similarly, UDP-d-Gal mutase (UGM) interconverts UDP-d-Gal and UDP-d-Galfvia ring rearrangement.

Figure 5

Examples of glycosylated natural products from flavonoids to polyketides and terpenoids. Sugars are labeled and highlighted in red.

Figure 6

Examples of glycosylated terpene products made in S. cerevisiae and their biosynthetic pathways. Native yeast metabolism is shaded in orange, steviol glycoside biosynthesis in blue, and saponin biosynthesis in green.

Figure 7

Examples of flavonoid glycosides glycosylated in S. cerevisiae.