De novo synthesis of 2'-fucosyllactose in engineered Bacillus subtilis ATCC 6051a

Microbial synthesis of 2'-fucosyllactose (2'-FL) has received much attention in recent years. In this study, Bacillus subtilis ATCC 6051a, a nonpathogenic GRAS (generally recognized as safe) organism, was engineered to produce 2'-FL. After a synthetic pathway comprising six genes was incorporated into the host cell genome, only a low level of 2'-FL (120 mg/L) was initially detected in recombinant cell culture. Comparison of heterologous lactose transporters confirmed the superior role of LacY from Escherichia coli for efficient production of 2'-FL in B. subtilis . There are two β-galactosidases, GanA and YesZ, in B. subtilis . Deletion of ganA and yesZ caused by insertion of the lacY cassette led to 2'-FL accumulation in shaking-flask culture at concentrations of 3.13 g/L and 5.56 g/L, respectively, which increased to 6.13 g/L in the double-deletion strain 164FL-GY. In addition, enhanced xylose metabolism in 164FL-GY further increased the concentration of 2'-FL to 7.14 g/L. In fed-batch fermentation, the highest productivity of 0.56 g/L·h was achieved in glycerol and xylose containing medium using engineered B. subtilis , and the accumulated 2'-FL reached 31.2 g/L. This is the first report of recombinant B. subtilis for high-level production of 2'-FL via a de novo synthesis pathway. • The first report employing Bacillus for production of 2'-fucosyllactose via a de novo synthetic pathway. • T7 RNA polymerase-based expression of 2'-fucosyllactose synthases in B. subtilis. • The production level of 2'- fucosyllactose in B. subtilis affected by xylose metabolism. • Improvement of 2'- fucosyllactose titer achieved by knockout of GanA and YesZ in B. subtilis .