Construction Of Multi-enzymatic System For Efficient Synthesis Of UDP-N-GlcNAc

Uridine 5'-diphospho N-acetylglucosamine (UDP-GlcNAc), an activated nucleotide sugar, plays an important role in the biochemistry of all living organisms. And it is an essential substrate in the synthesis of oligosaccharides, which possesses high potential to be developed into pharmaceuticals and functional materials. However, due to the lack of an efficient method to synthesize this product, UDP-GlcNAc is in great demand and its price is notoriously high. Therefore, the aim of this work was to develop an efficient method to produce UDP-GlcNAc at low cost.Firstly, three recombinant His6-tagged plasmids were constructed, pET28-yqgR (Glucokinase), pET28-agml (N-acetylglucosamine-phosphate mutase) and pET28-glmU (N-acetylglucosamine-1-phosphate uridyltransferase), and transformed into E. coli Rosetta(DE3). After induction, the above three enzymes could be efficiently overexpressed as soluble form, which was combined with dried yeast to construct one multi-step enzymatic systems, being able to produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, including substrate concentration, reaction temperature and reaction duration,31.5 mM UDP-GlcNAc was produced from 50 mM GlcNAc and 50 mM UMP.Compared with the traditional in vivo expression system, cell-free protein synthesis (CFPS) provides a much more rapid and convenient strategy for recombinant protein production. Therefore, CFPS was employed as an alternative to investigate the reconstruction of the UDP-GlcNAc biosynthetic pathway containing the above three enzymes. Six recombinant His6-tagged in vitro vectors were constructed, including pIVEX2.4c-yqgR, pIVEX2.4c-agml and PIVEX2.4c-glmU as well as pSJ2-yqgR. pSJ2-agml and pSJ2-glmU. If the three enzymes were produced in cell-free system separately and then added to the reaction system together,4.1 mM UDP-GlcNAc could be accumulated with 40 mM GlcNAc as the substrate at 22℃after 24 h reaction. If the three enzymes were expressed in one cell-free reaction simultaneously, 1.9 mM UDP-GlcNAc could be synthesized under the same conditions. These results indicated that YqgR, Agml and GlmU were functionally expressed in one cell-free reaction simultaneously, and these enzymes were coupled to function as a complete UDP-GlcNAc biosynthetic pathway. Therefore, CFPS promises an ideal platform for the reconstruction of biosynthetic pathways, and even metabolic networks.