Coupling xylitol dehydrogenase with NADH oxidase improves L-xylulose production in Escherichia coli culture

摘要

Escherichia coil expressing NAD-dependent xylitol-4-dehydrogenase (XDH) from Pantoea ananatis and growing on glucose or glycerol converts xylitol to the rare sugar L-xylulose. Although blocking potential L-xylulose consumption (L-xylulosekinase, lycK) or co-expression of the glycerol facilitator (gig) did not significantly affect L-xylulose formation, co-expressing XDH with water-forming NADH oxidase (NOX) from Streptococcus pneumoniae increased L-xylulose formation in shake flasks when glycerol was the carbon source. Controlled batch processes at the 1 L scale demonstrated that the final equilibrium L-xylulose/xylitol ratio was correlated to the intracellular NAD(+)/NADH ratio, with 69% conversion of xylitol to L-xylulose and a yield of 0.88 g L-xylulose/g xylitol consumed attained for MG1655/pZE12-xdh/pCS27-nox growing on glycerol. NADH oxidase was less effective at improving L-xylulose formation in the bioreactor than in shake flasks, likely as a result of an intrinsic maximum NAD+/NADH and L-xylulose/xylitol equilibrium ratio being attained. Intermittently feeding carbon source was ineffective at increasing the final L-xylulose concentration because introduction of carbon source was accompanied by a reduction in NAD+/NADH ratio. A batch process using 12 g/L glycerol and 22 g/L xylitol generated over 14 g/L L-xylulose after 80 h, corresponding to 65% conversion and a yield of 0.89 g L-xylulose/g xylitol consumed.