Efficient conversion of Lignocellulosic biomass to bioethanol by recombinant Saccharomyces Keisuke Cerevisiae

Abstract

Today, bioethanol is a promising alternative liquid fuel for gasoline in the transportation sector. However, economically bioethanol produced from lignocellulosics biomass is not competitive with gasoline. Thus, development and optimization for processing techniques are required to realize an extensive fuel ethanol utilization. Xylose is one of the major fermentable sugars present in lignocellulosics biomass, the second most abundant carbohydrate polymer in nature after glucose. The efficient fermentation of xylose is required to develop economically viable processes for producing ethanol from biomass. A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD+-dependent XDH. In this study, we focused on the effect of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52-fold and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency compared with the wild-type (WT). Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20% increase of ethanol production and 52% decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP+ and NADH/NAD+ ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.