The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001

Abstract

Saccharomyces cerevisiae is able to ferment xylose,when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However,xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1),ribose 5-phosphate ketol isomerase (EC 5.3.1.6),transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1),as well as all four enzymes simultaneously,were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001,expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026,overproducing all four non-oxidative PPP enzymes,ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l31 xylose or chemostat cultures of 20 g l31 glucose and 20 g l31 xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However,the specific growth rate on xylulose increased from 0.02^0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026,suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l31 xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP,whereas control of the xylose fermentation rate is situated upstream of xylulokinase,in xylose transport,in xylose reductase,and/or in the xylitol dehydrogenase.