Escherichia coli strain PC09 (ΔxylB, crp*) expressing an NADPH-dependent xylose reductase (CbXR) produces xylitol from xylose while growing on glucose. Resting cells give molar yields of ~3-4 xylitol produced per glucose consumed, depending on the conditions used. Studies with elevated expression levels of CbXR and xylose transporters suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. Previously, we examined the effects of deletions in key metabolic pathways (e.g. Embden-Meyerhof-Parnas and pentose phosphate) and reactions (e.g. transhydrogenase and NADH dehydrogenase) on resting cell xylitol yield. The results demonstrated the importance of direct NADPH supply by NADP⁺-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.

We will present two fundamentally different approaches to improving the coupling between glucose oxidation and NADPH-dependent xylose reduction. We first focus on improving the xylitol yield, where the NADPH requirement directly competes with growth. A metabolic model suggests that the maximum theoretical xylitol yield is 9.6 mole/mole glucose for non-growing cells. The oxidative pentose phosphate pathway (Zwf + Gnd) is predicted to supply 73% of the NADPH necessary for xylitol production by cycling glucose metabolism through the reverse reaction catalyzed by Pgi. Eliminating flux through transhydrogenase lowers the theoretical yield to 8.8. In contrast, when flux through the reverse Pgi reaction is restricted to a minimum level required for maintenance, the theoretical maximum yields drop to 9.2 and 3.6 for the wild-type and pntA deletion strains respectively. Experimentally, our approach to increase xylitol yield involves deletions in the phosphofructokinase genes and increasing expression of Zwf. Results from a variety of genetic modification scenarios will be presented.

An alternate strategy is to couple cell growth to xylitol production. Here, we begin with a growth-inhibited, NADPH-overproducing strain having deletions in pgi and sthA. Expression of NADPH-dependent xylose reductase then serves as a source of NADP⁺ regeneration, and xylitol production correlates with growth. This growth coupling was not achieved in the crp* strain. Co-expression of xylose transport genes therefore improves growth and xylitol production. This growth-coupled system allows for the evolution of strains with improved NADPH-dependent reaction productivity, and results will be presented in that context.