Combinatorial Modular Pathway Engineering for Guanosine 5′-Diphosphate-<scp>l</scp>-fucose Production in Recombinant <i>Escherichia coli</i>
Li Wan, Yingying Zhu, Wen Li, Wenli Zhang, Wanmeng Mu
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.