Use of xylose reductase as a cofactor enhancing system for in vivo biocatalysis

Cofactor imbalance is a common challenge in whole-cell bioconversion and thus limits the efficiency of biocatalysts. Various approaches have been employed to enhance cofactor availability, including specific engineering of pathways to increase intracellular levels of NAD(P)H, FMN, FAD, ATP and CoA. Recently, we have demonstrated that addition of xylose reductase (XR) in and supplying lactose to metabolically engineered cells can enhance levels of their sugar phosphates, leading to greater synthesis of NAD(P)H, FMN, FAD, ATP, and CoA in these cells, and thus a higher yield of bioconversion products. We propose that the XR/lactose system can be used as a generic tool to enhance precursor pools for cofactor synthesis for various in vivo biocatalysts. Here, we provide a protocol for the use of the XR/lactose system in fatty alcohol biosynthesis by Escherichia coli BL21(DE3). Step-by-step protocols and remarks should allow readers to adapt the use of XR/lactose for their engineered cells which should alleviate the problem of cofactor supply in whole-cell biocatalysis.