Intelligent self-control of carbon metabolic flux in SecY-engineered Escherichia coli for xylitol biosynthesis from xylose-glucose mixtures.

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.

Engineering Escherichia coli for d-Allulose Production from d-Fructose by Fermentation.

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 Ni2+ 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 Ni2+. In fed-batch fermentation, the titer of d-allulose reached ≈23.3 g/L.