Construction of Recombinant Saccharomyces cerevisiae with Ethanol and Aldehydes Tolerance via Overexpression of Aldehyde Reductase

Abstract

Furfural and hydroxy-methyl-furfural (HMF) are produced by lignocellulosic biomass during heat or acid pretreatment and are toxic to yeast. Aldehyde reductase is the main enzyme to reduce furfural and HMF. To improve the conversion efficiency of lignocellulosic biomass into ethanol, we constructed Saccharomyces cerevisiae with overexpression of aldehyde reductase (encoded by ari1). The gene of aldehyde reductase (encoded by ari1) was cloned via polymerase chain reaction (PCR) and ligated with the expression vector pGAPZαC. Western blot coupled with anti-His tag confirmed overexpression of the ari1 gene. The growth curves of the wild and ari1-overexpressed strain in the YPD medium were found to be almost identical. Compare to the ari1-overexpressed strain, the wild strain showed a longer doubling time and lag phase in the presence of 20 mM furfural and 60 mM HMF, respectively. The real-time PCR results showed that furfural was much more potent than HMF in stimulating ari1 expression, but the cell growth patterns showed that 60 mM HMF was more toxic to yeast than 20 mM furfural. S. cerevisiae with ari1 overexpression appeared to confer higher tolerance to aldehyde inhibitors, thereby increasing the growth rate and ethanol production capacity of S. cerevisiae in an aldehyde-containing environment.

Keywords: Saccharomyces cerevisiae; aldehyde reductase; ethanol production; furfural; hydroxy-methyl-furfural.

Figure 1

Comparison of the ari1 DNA sequence for ari1 obtained in this study with that published by Liu et al., 2009. Query: The ari1 DNA sequence published by Liu et al., 2009. Subject: The ari1 DNA sequence from this study. It is shown that the DNA sequence of this study is different from that of Liu et al.

Figure 2

Comparison of the amino acid sequence for the ARI protein obtained in this study with that published by Liu et al., 2009. Query: The ARI amino acid sequence published by Liu et al., 2009. Subject: The ARI amino acid sequence in this study. ¹ Cofactor binding site, ² Active site, ^s DNA sequences were different from those of Liu et al. but their amino acids were the same. ^d Both DNA sequences and amino acids were different from those of Liu et al., 2009.

Figure 3

(A) PCR confirmation of the recombinant expression vector pGAPZC-ari1 in SC and SCA. (B) Overexpression of ARI in SC and SCA. SC (S. cerevisiae) and SCA (S. cerevisiae with ari1 gene overexpression).

Figure 4

Furfural tolerance analysis of Saccharomyces cerevisiae. Solid lines represent engineered strains; dashed lines represent wild strains.

Figure 5

HMF tolerance analysis of Saccharomyces cerevisiae. Solid lines represent engineered strains; dashed lines represent wild strains.

Figure 6

Ari gene expression (bar chart) and cell growth (graph) of SCA (filled symbol) and SC (open symbol) on the YPD medium (A), YPD + 20 mM furfural (B) and YPD + 60 mM HMF (C). SC (S. cerevisiae) and SCA (S. cerevisiae with ari1 gene overexpression).

Figure 6

Ari gene expression (bar chart) and cell growth (graph) of SCA (filled symbol) and SC (open symbol) on the YPD medium (A), YPD + 20 mM furfural (B) and YPD + 60 mM HMF (C). SC (S. cerevisiae) and SCA (S. cerevisiae with ari1 gene overexpression).

Figure 7

Ari gene expression (bar chart) and aldehyde degradation (line chart) of SCA (filled symbol) and SC (open symbol) in the presence of 60 mM HMF (Blue color) or 20 mM Furfural (Red color) on the YPD medium. SC (S. cerevisiae) and SCA (S. cerevisiae with ari1 gene overexpression).

Figure 8

Ethanol conversion rate and production using SCA (filled symbol) and SC (open symbol) in the presence of 10% glucose on the YPD medium. SC (S. cerevisiae) and SCA (S. cerevisiae with ari1 gene overexpression).