(176q) Biosynthesis of D?Allulose Using Engineered Escherichia coli by Fermentation

D-Allulose is an ultra-low calorie sweetener with broad market prospects in the fields of food, beverage, medicine, and health care. The fermentative synthesis of D-allulose is currently under development and is considered as an ideal route, owing to its distinct advantages over the enzymatic methods that have been industrialized for D-allulose production. However, naturally occurring microorganisms capable of efficiently synthesizing D-allulose have not been found yet. Metabolic engineering is now being widely used for construction of cell factories to produce value-added chemicals. Escherichia coli, due to its well-defined genetic background and genetic manipulability, is frequently chosen as a chassis host for microbial fermentation.

Generally, D-allulose is synthesized from D-fructose through Izumoring epimerization. Here, we proposed an approach to efficiently produce D-allulose through fermentation using metabolically engineered E. coli JM109 (DE3), in which a PtsG-F transporter 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. 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. Subsequently, FryABC permease was identified to be responsible for the transport of D-allulose in E. 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 g/L of D-allulose through a unique thermo-swing fermentation process, with a yield of 0.94 g/g on D-fructose.

In contrast to Izumoring epimerization, phosphorylation−dephosphorylation pathway shows superiority in substrate conversion and even makes it possible to use D-xylose for fermentative production of D-allulose. We designed a novel route that coupled the pathways of methanol reduction, pentose phosphate (PP), ribulose monophosphate (RuMP), and allulose monophosphate (AuMP) for E. 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 reached ≈70.7 mM, with a yield of ≈0.471 mM/mM on D-xylose or ≈0.512 mM/mM on methanol. However, methanol residues in the later stage of fermentation raised serious concerns. It is widely acknowledged that methanol is toxic to humans, and its concentration in food and beverages is strictly limited. A D-xylose-sensitive translation control system was then constructed to regulate the expression of the formaldehyde detoxification operon (FrmRAB) for self-inductive detoxification by cells, achieving no methanol residue after fermentation. 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. This work provides a novel strategy for fermentative production of D-allulose from D-xylose and methanol by E. coli, and our achievements will promote the utilization of five-carbon (C5) and one-carbon (C1) substrates in biochemical industries.