Cell-free enzymatic synthesis of GDP-l-fucose from mannose

Weiyang Wang,Min Wei,Yifa Zhou,Yanyun Wen,Fan Zhang,Ye Yuan,Yanbo Hu

doi:10.1186/s13568-019-0798-1

Abstract

GDP-l-fucose, the key substrate for fucosyloligosaccharide biosynthesis, has been synthesized via a de novo pathway in bacteria. In the present study, genes for GDP-l-fucose biosynthesis were cloned into the expression vector pET-28a (+) to construct five E. coli strains, with recombinant enzymes being purified by using Ni–NTA chromatography. Following optimization of the 3-step reaction, Glk, ManB and ManC were added to the reaction mixture, after which Gmd and WcaG were added to overcome feedback inhibition from the end-product to produce GDP-l-fucose at 178.6 mg/l, with a yield of 14.1%. Our studies provide the basis for using cell-free enzyme production of GDP-l-fucose.

Highlights

In human milk, fucosyloligosaccharide activates numerous biological processes, like prevention of infections, probiotic growth, and an improved immune response, in particular for infantile enteric and other pathogenic effects (Kunz and Rudloff 2006; Boehm and Stahl 2007; Chaturvedi et al 2001)
Overexpression and purification of recombinant enzymes Genes encoding mannose-1-phosphate guanyltransferase (ManC), Gmd and GDP-l-fucose synthetase (WcaG) were PCRamplified from E. coli BL21 DNA
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 GDP4-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)

Summary

Introduction

Fucosyloligosaccharide activates numerous biological processes, like prevention of infections, probiotic growth, and an improved immune response, in particular for infantile enteric and other pathogenic effects (Kunz and Rudloff 2006; Boehm and Stahl 2007; Chaturvedi et al 2001). GDP-l-fucose is an essential precursor for the biosynthesis of fucosyloligosaccharide, providing fucosyl groups to oligosaccharides (Sun et al 1995; Satoshi 2003). GDP-l-fucose is one of the most expensive and rare nucleotide sugars. It is made by using a relatively expensive chemical synthetic protocol, which is insufficient to meet demand for large scale industrial production (Khaled et al 2004). GDP-l-fucose is generated by two pathways: the major de novo metabolic pathway and the minor salvage metabolic pathway (Becker and Lowe 2003; Niittymäki et al 2004). The de novo pathway in bacteria, mammals and plants, starts from d-mannose conversion into GDP-l-fucose via a 5-step reaction (Fig. 1) (Chin et al 2013). The initial step uses glucokinase (Glk, EC 2.7.1.63) to catalyze conversion of d-mannose to mannose-6-phosphate, that is transformed to

Methods

Results

Conclusion