(176r) Reprogramming of Sugar Transport Pathways in Escherichia coli using Secy (?P) Channel and Its Applications

As the initial step in sugar metabolism, the sugar-specific transporters in cells play a decisive role in the passage of sugars through the plasma membrane into the cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work demonstrates that the permeabilized SecY channel can be used as a nonspecific sugar transporter for Escherichia coli. The SecY with deletion of the plug domain allowed passage of glucose, fructose, mannose, xylose, and arabinose, and the channel with further pore ring mutations achieved lactose transport, indicating that the sugar passage via the SecY (ΔP) channel was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides, and was benefited in bypassing transport saturation, improving sugar tolerance, reducing competitive inhibition, and avoiding carbon catabolite repression, which are usually encountered with the native sugar uptake systems.

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 the SecY (ΔP) channel in xylitol-producing E. 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.

D-Allulose is considered as an ideal alternative to sucrose and has shown tremendous application potential in many fields. Here, we proposed an approach to efficiently produce D-allulose through fermentation using a metabolically engineered E. coli JM109 (DE3), in which a SecY (ΔP) channel and a DPEase were co-expressed, ensuring that D-fructose could be transported in its non-phosphorylated form and then converted into D-allulose by cells. Further deletion of fruA, manXYZ, mak, galE, and fruK, and use of Ni²⁺ in 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 by use of E. coli (DPEase, SecY [ΔP], ΔFruA, ΔManXYZ, ΔMak, ΔGalE, ΔFruK) and 8 μM Ni²⁺. In fed-batch fermentation, the titer of D-allulose reached ≈ 23.3 g/L.

The SecY (ΔP) channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system to take up sugars. The SecY (ΔP) channel can thus provide a unique sugar passageway in future development of robust cell factories for biotechnology applications.