Abstract
Guanosine 5'-diphosphate (GDP)-l-fucose is an important nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. Here, a combinatorial modular pathway engineering strategy was implemented to efficiently increase the intracellular titers of GDP-l-fucose in engineered Escherichia coli. The de novo GDP-l-fucose synthesis pathway was partitioned into two modules and fine-tuned at both transcriptional and translational levels, which remarkably improved the GDP-l-fucose production. In addition, the gene encoding the UDP-glucose lipid carrier transferase (WcaJ) was inactivated to eliminate the competing metabolite pathway from GDP-l-fucose to colanic acid. Furthermore, cofactors were regenerated to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve a titer of 18.33 mg/L in shake-flask cultivation, showed 106.21 mg/L intracellular GDP-l-fucose accumulation and a DCW-specific GDP-l-fucose content of 4.28 mg/g through fed-batch cultivation. In general, this study demonstrated that the utilization of combinatorial modular pathway engineering significantly improved the de novo synthesis of GDP-l-fucose in engineered E. coli.
Highlights
Guanosine 5′-diphosphate (GDP)-L-fucose is a vital nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes
To date, this is the first utilization of a modular pathway optimization approach to increase the production potential of the de novo synthesized GDP-L-fucose
Given that GDP-D-mannose is a pivotal precursor in the target pathway, ManB and ManC were placed as the upstream module responsible for converting mannose-1-P into GDP-D-mannose
Summary
Guanosine 5′-diphosphate (GDP)-L-fucose is a vital nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. GDP-L-fucose is primarily harnessed as an important glycosyl donor for protein glycosylation and as a precursor for the synthesis of other sugar nucleotides in eukaryotes. It is mainly involved in the biotransformation of fucosylated oligosaccharides in prokaryotes [2]. Large-scale production of fucosylated HMOs can be an arduous task due to the limitation in the availability of GDP- L-fucose. As an indispensable precursor in the biosynthesis of fucosylated HMOs, efficient production of GDP- L-fucose is very important
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