A pathway in Escherichia coli BL21 (DE3) was constructed to produce D-allulose from D-glucose via a phosphorylation-dephosphorylation strategy. The genes related to competitive pathways were deleted using the CRISPR/Cas9 system, and allulose-6-phosphate phosphatases from different sources were selected to evaluate their effects on the synthesis of D-allulose. The expression levels of D-allulose synthetic pathway-related genes were regulated and the fermentation conditions for the engineered strain were optimized. The results showed that the titer of D-allulose increased to 0.95 g/L after the deletion of the genes encoding phosphofructokinase A (pfkA), glucose-6-phosphate dehydrogenase (zwf), allose-6-phosphate isomerase (rpiB), and mannose-6-phosphate isomerase (manA). The allulose-6-phosphate phosphatase BbA6PP from Bacteroides bouchesdurhonensis showed the best performance in D-allulose synthesis, and its use increased the titer of D-allulose to 1.21 g/L in shake flasks. The recombinant strain BE-14, obtained by regulating the expression levels of D-allulose synthetic pathway-related genes through the optimization of plasmid copy number, achieved a maximum D-allulose yield of 2.06 g/L. After optimizing the fermentation conditions, the titer of D-allulose was improved to 2.72 g/L in shake flasks. BE-14 produced the highest titer of D-allulose of 18.4 g/L in a 5 L fermenter after 46 h fermentation. Only trace amounts of glucose (0.7 g/L) and fructose (0.1 g/L) were present in the fermentation broth, which was beneficial for subsequent separation and purification. This study provides a research basis for the efficient production of D-allulose by E. coli, which is of great significance for promoting the industrial production of D-allulose
