Metabolic re-engineering of Saccharomyces cerevisiae for the conversion of xylose to ethanol

Lignocellulosic materials are such an abundant and inexpensive resource that existing suppliescould support the sustainable production of liquid transportation fuels. Xylose is the most abundant sugar in the hemicellulose (ca. 25% of dry weight) of hardwoods and crop residues, and it is thesecond only to glucose in natural abundance. Thus, the efficient utilization of the xylose component of hemicellulose offers the opportunity to significantly reduce the cost of bioethanol production.The yeastSaccharomyces cerevisiae, which is one of the most prominent ethanol producingorganisms from hexose sugars, has the drawback that it is unable to utilize pentose sugars.Our laboratory has genetically engineeredS. cerevisiae to utilize xylose by introducing the genesfor xylose reductase (XR) and xylulose dehydrogenase (XDH) fromPichia stipitis. These enzymes catalyze the sequential reduction of xylose to xylitoland oxidation of xylitol to xylulose[1].Although these strains could use xylose for growth and xylitol formation, ethanol production hasbeen rather poor. A primary reason for this poor yield has been attributed to redox cofactorimbalance. All known XDHenzymes are specific for NAD, whereas all known XR enzymes areeither specific for NADPH or have a preference for NADPH. Therefore, conversion of xylose toxylulose by this pathway results in the cellular pool of NADPH being converted to NADP and that of NAD being converted to NADH, after which further metabolism of xylose is greatly hindered.The NADH can be reoxidised under aerobic conditions, but this demands critical control of oxygenlevels to maintain fermentative metabolism and ethanol production. Recently, we have constructed different recombinant xylose utilizing S. cerevisiae strains, whichimprove xylose utilization. The first This has been achieved by the simultaneous expression of anNADH-specific glutamate dehydrogenase (GDH2) together with the primary NADPH-specific glutamate dehydrogenase (GDH1) thus creating a dual enzyme “transhydrogenase cycle”. Improvedxylose utilization and ethanol production rates we observed in batch and fed-batch cultivationswhen the GDH2 gene is overexpressed in axylose utilizing S. cerevisiae. An additionalimprovement in xylose to ethanol conversion came about by cloning and overexpressing theendogenous enzyme xylulokinase (XK), that phosphorylates xylulose to xylulose 5-phosphate.