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d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

d-Allulose is a rare hexose with great application potential, owing to its moderate sweetness, low energy, and unique physiological functions. The current strategies for d-allulose production, whether industrialized or under development, utilize six-carbon sugars such as d-glucose or d-fructose as a substrate and are usually based on the principle of reversible Izumoring epimerization. In this work, we designed a novel route that coupled the pathways of methanol reduction, pentose phosphate (PP), ribulose monophosphate (RuMP), and allulose monophosphate (AuMP) for Escherichia coli to irreversibly synthesize d-allulose from d-xylose and methanol. After improving the expression of AlsE by SUMO fusion and regulating the carbon fluxes by knockout of FrmRAB, RpiA, PfkA, and PfkB, the titer of d-allulose in fed-batch fermentation reached ≈70.7 mM, with a yield of ≈0.471 mM/mM on d-xylose or ≈0.512 mM/mM on methanol.

d-Allulose is considered an ideal alternative to sucrose and has shown tremendous application potential in many fields. Recently, most efforts on production of d-allulose have focused on in vitro enzyme-catalyzed epimerization of cheap hexoses. Here, we proposed an approach to efficiently produce d-allulose through fermentation using metabolically engineered Escherichia coli JM109 (DE3), in which a SecY (ΔP) channel and a d-allulose 3-epimerase (DPEase) were co-expressed, ensuring that d-fructose could be transported in its nonphosphorylated form and then converted into d-allulose by cells. Further deletion of fruA , manXYZ , mak , galE , and fruK and the use of Ni 2+ in a medium limited the carbon flux flowing into the byproduct-generating pathways and the Embden-Meyerhof-Parnas (EMP) pathway, achieving a ≈ 0.95 g/g yield of d-allulose on d-fructose using E. coli (DPEase, SecY [ΔP], ΔFruA, ΔManXYZ, ΔMak, ΔGalE, ΔFruK) and 8 μM Ni 2+ . In fed-batch fermentation, the titer of d-allulose reached ≈23.3 g/L.