Genetic engineering of S. cerevisiae for pentose utilization

The two most widespread pentose sugars in our biosphere are D-xylose and L-arabinose. The pentose catabolic pathways are relevant for microorganisms living on decaying plant material and also in biotechnology when cheap raw materials such as plant hydrolysates are fermented to ethanol. In fungi, i.e. in yeast and mold, L-arabinose is sequentially converted to L-arabinitol, L-xylulose, xylitol and D-xylulose and enters the pentose phosphate pathway as D-xylulose 5-phosphate. In molds the reductions are NADPH-linked and the oxidations are NAD+-linked. We recently identified the two missing genes in this pathway. The functional overexpression of all the genes of the pathway in S. cerevisiae led to growth on L-arabinose and ethanol production under anaerobic conditions however at very low rates. In this communication we show that in a yeast species the L-arabinose pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by L-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. To our knowledge this is the first report of an NADH-linked L-xylulose reductase. D-xylose fermentation to ethanol with recombinant S. cerevisiae is often slow and has a low yield. One reason is that the catabolism of these pentoses through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH which must be regenerated in a separate process. To facilitate the NADPH regeneration, the recently discovered gene GDP1 coding for a fungal NADP GAPDH was expressed in a S. cerevisiae strain with the D-xylose pathway. Glucose 6-phosphate dehydrogenase is the main path for NADPH regeneration, however it causes futile CO2 production and creates a redox imbalance on the pathway for anaerobic fermentation to ethanol. The deletion of the corresponding gene, zwfl, in combination with overexpression of GDP1 could stimulate D-xylose fermentation with respect to rate and yield; i.e. less CO2 and xylitol were produced. Through redox engineering a yeast strain, which was mainly producing xylitol and CO2 from D-xylose, was converted to a strain producing mainly ethanol.