In this study, 11 source PS enzyme genes were cloned and expressed in Escherichia coli BL21 (DE3), and the successful strain was constructed for enzymatic property study with simultaneous fermentation and transformation experiment on the fermentation tank. The results showed that the activity of PS derived from Brevibacillus brevis was the highest (1 562.3 U/mL), and the optimal temperature was 30 ℃, the optimal pH was 7.0, and the Kcat was 115.4 s^-1. The recombinant strain was fermented and transformed in a 5 L fermenter with D-pantothenic acid and β-alanine as substrates. After the reaction for 46 h, the yield of D-pantothenic acid reached 92.2 g/L, the conversion rate reached 93.7%, and the spatiotemporal yield was 48.5 g/(L·d).