The development of a highly efficient fermentation process for biofuel production from lignocellulosic biomass

Abstract

In this study, bioengineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Saccharomyces cerevisiae transformed with the native genes encoding xylose reductase (XR), xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous gene xylulokinage (XK) ferments xylose to ethanol but has not yet been applied to the industrial bio-process due to the unfavorable excretion of xylitol. Intercellular redox imbalance caused by the different coenzyme specificity of XR (using NADPH/NADH with preference for NADPH) and XDH (exclusively using NAD+) has been thought to be one of the main factors of xylitol excretion. The introduction of NADP+-dependent XDH generated in this study prevented the xylitol excretion probably because of maintaining the intercellular redox balance. Recombinant yeasts were constructed with the genes encoding a wild type XR and the protein engineered XDH (with NADP) of P. stipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes performed the similar enzyme properties in S. cerevisiae cells, compared those in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared to the reference strain expressing the wild–type XDH. Measurement of intracellular coenzyme concentrations suggested that maintenance of the NADPH/NADP and NADH/NAD ratios are important for efficient ethanol fermentation from biomass sugar xylose by a recombinant S. cerevisiae.