An Enzyme Module System for in situ Regeneration of Deoxythymidine 5′‐Diphosphate (dTDP)‐Activated Deoxy Sugars

Abstract

A highly flexible enzyme module system (EMS) was developed which allows for the first time the in situ regeneration of deoxythymidine 5'-diphosphate (dTDP)-activated deoxy sugars and furthermore enables us to produce novel sorangiosides in a combinatorial biocatalytic approach using three enzyme modules. The SuSy module with the recombinant plant enzyme sucrose synthase (SuSy) and the deoxy sugar module consisting of the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-b-l-rhamnose were combined with the glycosyltransferase module containing the promiscuous recombinant glycosyltransferase SorF from Sorangium cellulosum So ce12. Kinetic data and the catalytic efficiency were determined for the donor substrates of SorF: dTDP-a-d-glucose, dTDP-b-l-rhamnose, uridine diphosphate (UDP)-ad-glucose (Glc), and dTDP-6-deoxy-4-keto-a-d-glucose. The synthesis of glucosyl-sorangioside with in situ regeneration of dTDP-Glc was accomplished by combination of SuSy and SorF. The potential of the EMS is demonstrated by combining SuSy, RmlB, RmlC, RmlD with SorF in one-pot for the in situ regeneration of dTDP-activated (deoxy) sugars. The HPLC/MS analysis revealed the formation of rhamnosyl-sorangioside and glucosyl-sorangioside, demonstrating the in situ regeneration of dTDP-b-l-rhamnose and dTDP-a-d-glucose and a cycle number for dTDP higher than 9. Furthermore, NADH (reduced form of nicotinamdie adenine dinucleotide) regeneration with formate dehydrogenase in the reduction step catalyzed by the 4-ketoreductase RmlD could be integrated in the one-pot synthesis yielding similar conversion rates and cycle numbers. In summary, we have established the first in situ regeneration cycle for dTDP-activated (deoxy) sugars by a highly flexible EMS which allows simple exchange of enzymes in the deoxy sugar module and exchange of glycosyltransferases as well as aglycones in the glycosyltransferase module to synthesize new hybrid glycosylated natural products in one-pot.

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

“…In our previous work we demonstrated feasible separation of nucleotides and nucleotide sugars with borate buffer (50 mM Na 2 B 4 O 7 · 10H 2 O/64 mM boric acid, pH 8.9) in single capillary CE‐UV . Recently, we accomplished separation of nucleotide oligosaccharides using ammonium acetate buffer (50 mM, pH 9.2) .…”

“…Therefore, enzyme modules for nucleotide sugar synthesis (N(M)DP‐sugar module) and glycan synthesis (GT‐module) afford fast and efficient analytical methods to define optimum reaction conditions. In this way, N(M)DP‐sugar modules and GT‐modules are combined to generate production modules in assembly lines for the synthesis of glycoconjugates . In our approach to build up a ”Golgi Glycan Factory (GGF)“ sequential and one‐pot enzyme cascades were established for the synthesis of novel hybrid or natural glycosylated products .…”

“…In this way, N(M)DP‐sugar modules and GT‐modules are combined to generate production modules in assembly lines for the synthesis of glycoconjugates . In our approach to build up a ”Golgi Glycan Factory (GGF)“ sequential and one‐pot enzyme cascades were established for the synthesis of novel hybrid or natural glycosylated products . Enzyme cascade reactions require intensive investigations on parameters influencing enzyme performance such as temperature optima, pH‐optima, kinetics, cofactor dependency, inhibition by products or intermediate products, substrate spectrum, and enzyme ratios.…”

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

“…(Poly)phosphate can drive the glycosylation reaction towards completion by the exergonic cleavage of the phosphoanhydride bond (ΔG° = −30 − −32 kJ·mol −1 [158]) upon phosphorylation of nucleosides with polyphosphate kinase (PPK). Mono- or diphosphorylation with PPK have been reported for ATP [184,188,189,190,191,192,193], UTP [189,194,195], CTP [196,197], tTMP [198], often showing broad promiscuity towards different nucleotides [187].…”

Glycosyltransferase cascades for natural product glycosylation: Use of plant instead of bacterial sucrose synthases improves the UDP‐glucose recycling from sucrose and UDP

“…However, because the majority of the known glucosyltransferases requires UDP-glucose, this was also the clear focus of past efforts of using SuSy for donor substrate recycling [15][16][17][18][19][20]. To expand the scope of SuSy-GT cascade reactions beyond glucosyl transfer, additional NDP-glucose-modifying enzymes were added to enable regeneration of UDP-galactose [21][22][23][24][25], UDP-glucuronic acid [26] and dTDP-rhamnose [27].…”