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<span style="font-variant: small-caps;">d</span>-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two <span style="font-variant: small-caps;">d</span>-alanine-deficient heterologous gene expression systems were constructed, <i>B. subtilis</i> 168 D1 and <i>B. subtilis</i> 168 D2, using overlapping extension PCR and the CRE/<i>loxP</i> system. The <i>lacZ</i> gene for β-galactosidase was integrated into a specific locus of the chassis <i>B. subtilis</i> 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene <i>alrA</i> attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene <i>araA</i> of <i>E. coli</i> transform-competent <i>B. subtilis</i> 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout <i>B. subtilis</i> 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L <span style="font-variant: small-caps;">d</span>-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. <i>B. subtilis</i> 168 D1/pMA5-<i>alrA</i>-<i>araA-lacZ</i> is capable of single-cell one-step production of <span style="font-variant: small-caps;">d</span>-tagatose. This study provides a new approach to the production of functional sugars.