Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine
| Autor: | Anna Schildbach, Thomas Rexer, Markus Pietzsch, Jan Klapproth, Erdmann Rapp, Reza Mahour, Udo Reichl, Steffen Klamt |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Bioengineering Nucleotide sugar 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound Polyphosphate kinase Escherichia coli chemistry.chemical_classification Phosphotransferases (Phosphate Group Acceptor) Uridine Diphosphate N-Acetylglucosamine Cell-Free System 010405 organic chemistry Temperature General Medicine Phosphoric Monoester Hydrolases Uridine Biosynthetic Pathways Enzymes 0104 chemical sciences carbohydrates (lipids) De novo synthesis Kinetics 030104 developmental biology Uridine diphosphate N-acetylglucosamine Enzyme chemistry Biochemistry Inorganic diphosphatase Nucleoside-Phosphate Kinase Flux (metabolism) Biotechnology |
| Zdroj: | Journal of Biotechnology |
| Popis: | In spite of huge endeavors in cell line engineering to produce glycoproteins with desired and uniform glycoforms, it is still not possible in vivo. Alternatively, in vitro glycoengineering can be used for the modification of glycans. However, in vitro glycoengineering relies on expensive nucleotide sugars, such as uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) which serves as GlcNAc donor for the synthesis of various glycans. In this work, we present a systematic study for the cell-free de novo synthesis and regeneration of UDP-GlcNAc from polyphosphate, UMP and GlcNAc by a cascade of five enzymes (N–acetylhexosamine kinase (NahK), Glc–1P uridyltransferase (GalU), uridine monophosphate kinase (URA6), polyphosphate kinase (PPK3), and inorganic diphosphatase (PmPpA). All enzymes were expressed in E. coli BL21 Gold (DE3) and purified using immobilized metal affinity chromatography (IMAC). Results from one-pot experiments demonstrate the successful production of UDP-GlcNAc with a yield approaching 100%. The highest volumetric productivity of the cascade was about 0.81 g L−1 h−1 of UDP-GlcNAc. A simple model based on mass action kinetics was sufficient to capture the dynamic behavior of the multienzyme pathway. Moreover, a design equation based on metabolic control analysis was established to investigate the effect of enzyme concentration on the UDP-GlcNAc flux and to demonstrate that the flux of UDP-GlcNAc can be controlled by means of the enzyme concentrations. The effect of temperature on the UDP-GlcNAc flux followed an Arrhenius equation and the optimal co-factor concentration (Mg2+) for high UDP-GlcNAc synthesis rates depended on the working temperature. In conclusion, the study covers the entire engineering process of a multienzyme cascade, i.e. pathway design, enzyme expression, enzyme purification, reaction kinetics and investigation of the influence of basic parameters (temperature, co-factor concentration, enzyme concentration) on the synthesis rate. Thus, the study lays the foundation for future cascade optimization, preparative scale UDP-GlcNAc synthesis and for in situ coupling of the network with UDP-GlcNAc transferases to efficiently regenerate UDP-GlcNAc. Hence, this study provides a further step towards cost-effective in vitro glycoengineering of antibodies and other glycosylated proteins. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|