Construction method of recombinant Bacillus subtilis strain for expressing D-allulose 3-epimerase based on D-alanine defective selection marker

The invention relates to a construction method of a recombinant Bacillus subtilis strain for expressing D-allulose 3-epimerase based on D-alanine defective selection marker, belonging to the technical field of enzyme gene engineering. The method comprises the following steps: by using Bacillus subtilis 1A751 as the initial strain, knocking out D-alanine racemase gene (dal) on the chromosome to obtain D-alanine defective 1A751(dal-); by using DPE enzyme gene of clostridium ATCC 35704 as the parent, fusing a P43 promoter on the upstream to construct P42-DPE, constructing into a plasmid pUB110 (NCBI-Gene ID:9507338) to obtain pUB-P43-DPE, substituting antibiotic-resistant genes Kan and Blm on the pUB-P43-DPE with the dal to construct pUB-P43-DPE-dal; transforming into the 1A751(dal-) to obtain the recombinant Bacillus subtilis 1A751-pUB-P43-DPE-dal which is named Bacillus subtilis SK38.001, and the collection number is CCTCC NO: M2015257. The total enzyme activity of the fermentation liquid is up to 16U/mL. The method has important industrial application value.