Improving the Response of Saccharomyces cerevisiae to Lignocellulosic Hydrolysate Inhibitors in Ethanolic Fermentation

Abstract: The production of ethanol based on lignocellulosic biomass requires the fermentation of a hydrolysate containing hexose and pentose sugars in an inhibitory environment. In fact, the lignocellulosic hydrolysate obtained from pretreatment and hydrolysis of the raw material contains a variety of inhibitory compounds, including (i) the furaldehydes 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde (furfural), (ii) weak acids (e.g. acetic acid) and (iii) phenolic compounds. These compounds reduce ethanol yield and the specific ethanol productivity, extend the lag phase, and reduce the growth rate and viability of microorganisms. In this work, the enzymes ADH6, mut-ADH1 and XR were identified as being responsible for NADPH-, NADH-, and NAD(P)H-dependent HMF and furfural reduction, respectively, in yeast. The tolerance and fermentation rates of Saccharomyces cerevisiae laboratory strains on defined medium and/or on lignocellulosic hydrolysate were improved by overexpression of the genes encoding for these enzymes. It was also shown that overexpression of furaldehyde reductases benefits product distribution in recombinant xylose-utilizing S. cerevisiae strains carrying the xylose reductase/xylitol dehydrogenase pathway during xylose fermentation. Finally, strains with higher furaldehyde conversion rates were shown to grow faster and ferment lignocellulosic hydrolysates faster. Evaluation of industrial strains of S. cerevisiae showed that the selection of a robust strain and its evaluation under representative conditions are essential for lignocellulosic hydrolysate fermentation, since strains perform differentially depending on the hydrolysate and the conditions employed. The use of fed-batch mode is advantageous not only because the inhibitors are kept at low concentrations and the capacity of the yeast to convert them is not surpassed, but also because it allows cells to adapt to the inhibitors in the lignocellulosic hydrolysate. Indeed, using microarray analysis, and in vitro and in vivo activity measurements it was demonstrated that short-term adaptation of the yeast to lignocellulosic hydrolysate increased detoxifying activities and induced the expression of genes related to the repair of cellular damage. The results presented in this work show that integration of process design and strain improvement strategies could be used to improve the yeast performance in ethanolic fermentation of lignocellulosic hydrolysates.