d-Allulose is an attractive rare sugar that can be used
as a low-calorie sweetener with significant health benefits. To meet
the increasing market demands, it is necessary to develop an efficient
and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering
strategy in Bacillus subtilis WB600
by introducing d-allulose 3-epimerase (DAEase), combined
with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for
the efficient production of d-allulose. This resulting strain
initially produced 3.24 g/L of d-allulose with a yield of
0.93 g of d-allulose/g d-fructose. We further screened
and obtained a suitable dual promoter combination and performed fine-tuning
of its spacer region. After 64 h of fed-batch fermentation, the optimized
engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and
a conversion rate of 27.6%. This d-allulose production strain
is a promising platform for the industrial production of rare sugar.