Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides.

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.

Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine.

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.

One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides.

Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20 mM MgCl2 . The maximum reaction rate of GDP-mannose achieved was 2.7 µM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP-mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2 -Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2 -Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.