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Published on: February 25, 2019
Engineering Escherichia coli BL21(DE3) for efficient production of D-allulose from D-glucose via
Junchi Zhu1, Mengting Tao1, Sanying Wang2
1State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, PR China; School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
Abstract:
D-Allulose, a low-calorie functional sweetener, is mainly produced industrially via reversible D-fructose epimerization. Recently, the thermodynamically favorable phosphorylation/epimerization/dephosphorylation pathway has attracted attention for microbial biosynthesis. In this study, we constructed the synthetic pathway by introducing Escherichia coliD-allulose-6-phosphate 3-epimerase (AlsE) and Clostridium thermocellumD-allulose-6-phosphate phosphatase (CtA6PP) into an engineered E. coli BL21(DE3) strain. To direct carbon flux toward D-allulose, competitive metabolic genes for sugar-phosphate intermediates were deleted. Furthermore, expression of AlsE and the phosphatase was precisely regulated through combined promoter and RBS engineering at both transcriptional and translational levels. The optimized strain efficiently converted D-glucose to D-allulose, achieving titers of 5.26 g/L in test-tube cultivation and 33.05 g/L in fed-batch fermentation, with a yield of 0.69 g/g D-glucose consumed. This work demonstrates the effectiveness of pathway engineering and expression tuning for enhancing D-allulose production via the phosphorylation/epimerization/dephosphorylation route.
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