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D-allulose, a naturally occurring monosaccharide, is present in small quantities in nature. It is considered a valuable low-calorie sweetener due to its low absorption in the digestive tract and zero energy for growth. Most of the recent efforts to produce D-allulose have focused on in vitro enzyme catalysis. However, microbial fermentation is emerging as a promising alternative that offers the advantage of combining enzyme manufacturing and product synthesis within a single bioreactor. Here, a novel approach was proposed for the efficient biosynthesis of D-allulose from glycerol using metabolically engineered Escherichia coli. FbaA, Fbp, AlsE, and A6PP were used to construct the D-allulose synthesis pathway. Subsequently, PfkA, PfkB, and Pgi were disrupted to block the entry of the intermediate fructose-6-phosphate (F6P) into the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Additionally, GalE and FryA were inactivated to reduce D-allulose consumption by the cells. Finally, a fed-batch fermentation process was implemented to optimize the performance of the cell factory. As a result, the titer of D-allulose reached 7.02 g/L with a maximum yield of 0.287 g/g., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 an ultra-low-calorie sweetener with broad market prospects in the fields of food, beverage, health care, and medicine. The fermentative synthesis of D-allulose is still under development and considered as an ideal route to replace enzymatic approaches for large-scale production of D-allulose in the future. Generally, D-allulose is synthesized from D-fructose through Izumoring epimerization. This biological reaction is reversible, and a high temperature is beneficial to the conversion of D-fructose. Mild cell growth conditions seriously limit the efficiency of producing D-allulose through fermentation. FryABC permease was identified to be responsible for the transport of D-allulose in Escherichia coli by comparative transcriptomic analysis. A cell factory was then developed by expression of ptsG-F, dpe, and deletion of fryA, fruA, manXYZ, mak, and galE. The results show that the newly engineered E. coli was able to produce 32.33 ± 1.33 g L -1 of D-allulose through a unique thermo-swing fermentation process, with a yield of 0.94 ± 0.01 g g -1 on D-fructose., (© 2023 Wiley-VCH GmbH.)

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.

Xylitol is a salutary sugar substitute that has been widely used in the food, pharmaceutical, and chemical industries. Co-fermentation of xylose and glucose by metabolically engineered cell factories is a promising alternative to chemical hydrogenation of xylose for commercial production of xylitol. Here, we engineered a mutant of SecY protein-translocation channel (SecY [ΔP]) in xylitol-producing Escherichia coli JM109 (DE3) as a passageway for xylose uptake. It was found that SecY (ΔP) channel could rapidly transport xylose without being interfered by XylB-catalyzed synthesis of xylitol-phosphate, which is impossible for native XylFGH and XylE transporters. More importantly, with the coaction of SecY (ΔP) channel and carbon catabolite repression (CCR), the flux of xylose to the pentose phosphate (PP) pathway and the xylitol synthesis pathway in E. coli could be automatically controlled in response to glucose, thereby ensuring that the mutant cells were able to fully utilize sugars with high xylitol yields. The E. coli cell factory developed in this study has been proven to be applicable to a broad range of xylose-glucose mixtures, which is conducive to simplifying the mixed-sugar fermentation process for efficient and economical production of xylitol., (© 2021 Wiley Periodicals LLC.)

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.