Overexpression and purification of recombinant enzymes
Genes encoding ManC, Gmd and WcaG were PCR-amplified from E. coli BL21 DNA. The manB gene was amplified by using PCR with E. coli K12 strain ER3413 DNA as template. PCR products, as well as the synthesized gene product (Glk) were sub-cloned into T7-driven expression vector pET-28a (+), and recombinant plasmids were transfected into E. coli BL21 (DE3). The engineered E. coli strain was grown in Terrific Broth (TB) medium, and IPTG was added to induce protein expression. His-tagged fusion proteins were purified by using the ammonium sulfate precipitated fraction, followed by Ni–NTA chromatography. Purified proteins were found to be homogeneous by using SDS-PAGE, with molecular weights estimated to be 27.9 (Glk), 50.5 (ManB), 53.0 (ManC), 42.0 (Gmd) and 36.1 (WcaG) kDa, which corresponded to theoretical values based on amino acid sequences. Protein concentrations of Glk, ManB, ManC, Gmd and WcaG were 1.32, 1.97, 2.79, 3.63 and 3.71 mg/ml (Fig. 2).
Production of mannose-6-phosphate
According to the pathway in Fig. 1, mannose was transformed into GDP-l-fucose via mannose-6-phosphate, mannose-1-phosphate, GDP-d-mannose, and GDP-4-keto-6-deoxymannose catalyzed by glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanyltransferase (ManC), GDP-d-mannose-4,6-dehydratase (Gmd), and GDP-l-fucose synthetase (WcaG). Mannose-6-phosphate, the first intermediate in the production of GDP-l-fucose, was synthesized from mannose, catalyzed by Glk to transfer a phosphate to mannose. GTP was the phosphate donor, because it provided not only the phosphate group, but also is the cofactor for ManC production. As shown in Fig. 3a, mannose was converted into mannose-6-posphate. To improve mannose-6-phosphate production, the effect of GTP concentration, enzyme concentration, and reaction time were determined (Fig. 3b–d). Following 10 h incubation, the mannose-6-phosphate concentration was 1.2 g/l, with a conversion rate of 82.6% under optimal conditions (i.e. 10 mM GTP and 0.75 mg/ml Glk).
Production of GDP-d-mannose
GDP-d-mannose is a crucial intermediate for production of GDP-l-fucose. Here, we prepared it by conversion of mannose-6-phosphate using ManB and ManC. ManB catalyzes the reversible conversion of mannose-6-phosphate to mannose-1-phosphate. To promote the forward reaction, ManB and ManC were simultaneously added to the mannose-6-phosphate solution. Mass spectrometry (MS) analysis demonstrated a m/z of 626.05, consistent with MS data of GDP-d-mannose, illustrating that GDP-d-mannose was successfully synthesized (Fig. 4a). The yield of mannose were analyzed by HPLC, as shown in Fig. 4b, the content of GDP-d-mannose was 282.9 mg/l, with a yield of 10.4%. To optimize efficiency, Glk, ManB and ManC were simultaneously added to the mixture to initiate the reaction. Interestingly, GDP-d-mannose could still be detected by MS, and the final concentration of GDP-d-mannose reached 379.1 mg/l, with a yield of 14.6% (Fig. 4c, d). Therefore, the optimal condition for production of GDP-d-mannose was the simultaneous addition of Glk, ManB and ManC to the reaction.
Production of GDP-l-fucose
Transformation of GDP-d-mannose to GDP-l-fucose involves GDP-d-mannose-4,6-dehydratase (Gmd) and GDP-l-fucose synthetase (WcaG). Gmd catalyzes conversion of GDP-d-Mannose to GDP-4-keto-6-deoxymannose, and WcaG converts GDP-4-keto-6-deoxymannose to the end-product GDP-l-fucose with the aid of NADPH as the reducing agent. Since Gmd is inhibited by the end-product GDP-l-fucose (Sturla et al. 1997; Bisso et al. 1999), WcaG (involved in the conversion of GDP-4-keto-6-deoxymannose to GDP-l-fucose) was added separately from Gmd to the reaction mixture to overcome feedback inhibition. Initially, Gmd was added to the reaction mixture to convert GDP-d-mannose to GDP-4-keto-6-deoxymannose. After terminating the reaction by heating, WcaG was added to produce GDP-l-fucose. The negative ion mode mass spectrum showed an intense signal at m/z 610.05, corresponding to GDP-l-fucose (calculated mass 610.05). This indicates that GDP-l-fucose can be produced by this three-step method (Fig. 5a). HPLC analysis demonstrated that the final concentration of GDP-l-fucose reached 178.6 mg/l, with a yield of 14.1% (Fig. 5b).
We next tried to simplify the conversion process by using a 2-step approach in which the reaction was composed of Glk, ManB, ManC and Gmd. Following a 12 h incubation, WcaG was added to the reaction that ran for another 2 h to produce GDP-l-fucose. Although GDP-l-fucose was produced, its yield was lower than that using the 3-step approach (Fig. 5f).