d-Allulose is a desirable sucrose substitute
with potential
applications in food and health care. d-Allulose can be synthesized
using d-glucose as a substrate through coupling glucose isomerase
with d-allulose 3-epimerase (DAEase); however, the product
yield is typically less than 20% at reaction equilibrium and thus
limits its use in industrial applications. Here, a 3R-ketose phosphorylation
pathway coupled with an adenosine triphosphate (ATP) regeneration
system was developed for the efficient synthesis of d-allulose
in Escherichia coli using d-glucose as a substrate. The l-rhamnulose kinase (RhaB)
was used to break the inherent reaction equilibrium due to its substrate
specificity, resulting in increases in d-allulose titer by
69.9% to 4.96 ± 0.49 g/L. By optimizing the whole cell transformation
conditions and designing an ATP regeneration module, d-allulose
production reached 17.62 ± 0.77 g/L from 30 g/L d-glucose
with a final yield of 0.73 g/g without the addition of exogenous ATP.
To evaluate the potential industrial application of this multienzyme
cascade system, d-allulose was produced from cane molasses
(124.16 ± 2.69 g/L glucose equivalent) with a final d-allulose titer of 62.60 ± 3.76 g/L. The present study provides
a practical enzymatic approach for the economical synthesis of d-allulose.
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