Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

Abstract

Background: The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions.

Results: Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of Bud31 and Hpr1 was found to lead to the increase of both ethanol yield and fermentation rate, while Pho85, Vrp1 and Ygl024w expression is required for maximal ethanol production in VHG fermentations. Five genes, Erg2, Prs3, Rav1, Rpb4 and Vma8, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate.

Conclusions: The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers.

Figure 1

Comparison of the yeast genes described as determinants of resistance to inhibitory concentrations of (A) ethanol, glucose and acetic acid or of (B) ethanol, acetic acid and furfural or vanillin. The genes in the intersection of these datasets are highlighted. This comparative analysis was based on published genome-wide phenotypic screenings carried out in the presence of the referred stressors [15-19].

Figure 2

(A) Comparison between the concentration of CO₂ (Δ[CO₂]_corr) at mid fermentation point (49 h) and of the final amount of ethanol (Δ[Ethanol]_corr) produced by cells of the parental strain S. cerevisae BY4741 and by mutants deleted for the Anp1, Bud31, Hpr1, Pho85, Ppa1, Rpl1B, Vrp1 and Ygl024w genes during fermentation of a growth medium optimized for VHG technology. The Δ[CO₂]_corr and Δ[Ethanol]_corr parameters were calculated using equations 3 and 4, which are detailed in Methods. (B) The profile of CO₂ production by wild-type cells or by the selected deletion mutants (all mentioned above except for Δrpl1b mutant). Those deletion mutants found to start the fermentation at the same time as wild-type cells (shown on left) were separated from those which started the fermentation later (shown on right). Error bars represent the range between independent biological duplicates.

Figure 3

Comparison between the concentration of CO₂ (Δ[CO₂]_corr) at mid fermentation point (14 h) and of the final concentration of ethanol (Δ[Ethanol]_corr) produced by cells of the parental strain S. cerevisae BY4741 and by the mutants deleted for genes conferring resistance against ethanol, acetic acid and vanillin or furfural in the fermentation of WSH. The Δ[CO₂]_corr and Δ[Ethanol]_corr parameters were calculated using equations 3 and 4, as described in Methods. Error bars represent the error propagation associated with arithmetic operations used to determine the global relative variation of each mutant strain.

Figure 4

Profile of CO₂ production by wild-type cells or by the mutants deleted for genes providing resistance against ethanol, acetic acid and vanillin or furfural. Those deletion mutants found to produce much lower levels of CO₂ than those achieved by cells of the parental strain (panel A) were separated from those producing lower, but more similar concentrations (panel B). Error bars represent the range between independent biological duplicates.