An Enzyme Module System for in situ Regeneration of Deoxythymidine 5′-Diphosphate (dTDP)-Activated Deoxy Sugars
✍ Scribed by Carsten Rupprath; Maren Kopp; Dennis Hirtz; Rolf Müller; Lothar Elling
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 150 KB
- Volume
- 349
- Category
- Article
- ISSN
- 1615-4150
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✦ Synopsis
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‐β‐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‐α‐D‐glucose, dTDP‐β‐L‐rhamnose, uridine diphosphate (UDP)‐α‐D‐glucose (Glc), and dTDP‐6‐deoxy‐4‐keto‐α‐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‐β‐L‐rhamnose and dTDP‐α‐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.