Transporter mining and metabolic engineering of Escherichia coli for high?level D?allulose production from D?fructose by thermo?swing fermentation
Graphical Abstract and Lay SummaryA novel cell factory was introduced which was capable of efficiently producing D?allulose from D?fructose through thermo?swing fermentation. The regular change of fermentation temperature improved the efficiency of Izumoring epimerization and reduced the heat damage to cells. More importantly, deletion of D?allulose transporter ensured that the target product accumulated continuously.AbstractD?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.