Overexpression of arginase gene CAR1 renders yeast Saccharomyces cerevisiae acetic acid tolerance

Abstract

Acetic acid is a common inhibitor present in lignocellulose hydrolysate, which inhibits the ethanol production by yeast strains. Therefore, the cellulosic ethanol industry requires yeast strains that can tolerate acetic acid stress. Here we demonstrate that overexpressing a yeast native arginase-encoding gene, CAR1, renders Saccharomyces cerevisiae acetic acid tolerance. Specifically, ethanol yield increased by 27.3% in the CAR1-overexpressing strain compared to the control strain under 5.0 g/L acetic acid stress. The global intracellular amino acid level and compositions were further analyzed, and we found that CAR1 overexpression reduced the total amino acid content in response to acetic acid stress. Moreover, the CAR1 overexpressing strain showed increased ATP level and improved cell membrane integrity. Notably, we demonstrated that the effect of CAR1 overexpression was independent of the spermidine and proline metabolism, which indicates novel mechanisms for enhancing yeast stress tolerance. Our studies also suggest that CAR1 is a novel genetic element to be used in synthetic biology of yeast for efficient production of fuel ethanol.

Keywords: Acetic acid tolerance; Amino acid metabolism; Arginase; CAR1; Ethanol production; Saccharomyces cerevisiae.

Fig. 1

Impact of CAR1 overexpression on the growth of S. cerevisiae 4126 strains on YPD agar plates supplemented with 5.0 g/L acetic acid to test their tolerance to the inhibitor and without acetic acid supplementation as the control (A), and ethanol fermentation by S. cerevisiae 4126 engineered with CAR1 overexpression using YPD medium composed of 4 g/L yeast extract, 3 g/L peptone, 100 g/L glucose and 5 g/L acetic acid (B–E). The data for ethanol fermentation were expressed as the means for triplicate with standard deviations. 4126-WT, S. cerevisiae 4126; 4126-HO, S. cerevisiae 4126 engineered with the empty plasmis; 4126-CAR1, S. cerevisiae 4126 engineered with CAR1 overexpression, respectively.

Fig. 2

An overview on the profile of amino acid metabolism (A, B, C and D) and the heat map (E) highlighting the fold changes of intracellular amino acids. Ctr-CAR1 and Ctr-HO represent S. cerevisiae 4126 engineered with CAR1 overexpression and the empty plasmid cultured in the YPD medium without acetic acid supplementation, and HAc-CAR1 and HAc-HO represent those two recombinat strains cultivated Detailed data for amino acids are available in Table S2. Abbreviations: Ala, alanine; Arg, arginine; Asn, asparaginate; Asp, asparate; Cys, cysteine; Di-Cys, dicyseine; Gln, glutamine; Glu, glutamate; Gly, glycine; His, histidine; Hylys, hydroxylysine; Hypro, hydroxyproline; Ile, isoleusine; Lys, lysine; Leu, leucine; Met, methionine; Orn, ornithine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, thronine; Tyr, tyrosine; Val, valine; α-ABA, α-aminobutyric acid. Yeast cells were harvested at their exponential growth phase for the analysis with triplicate under the statistic significance *p < 0.05 and **p < 0.01.

Fig. 3

Transcription of genes related to the arginine metabolism (A) and intracellular spermidine contents of the recombinant strains (B). Relative gene expression was determined by the 2^−ΔΔCt method using the expression of ALG9 as the internal control, which is highlighted by the fold change (Log₂FC). Pathways for arginine metabolism was constructed based on information from the KEGG Pathway (http://www.genome.jp/kegg/). Intracellular spermidine contents of the recombinat strains were determined dansyl chloride derivatization based HPLC method as described in the Material and Method section. Yeast cells grew to exponential-phase in fermentation medium were harvest and used for the determination of transcription and intracellular spermidine contents.

Fig. 4

Intracellular ATP (A) and plasma membrane permeability (B) for S. cerevisiae 4126-CAR1 and S. cerevisiae 4126-HO. The yeast cells were harvested at their exponential growth phase at 36 h and 48 h, respectively, with their OD₆₀₀ of 0.46 and 0.63 for the analysis with triplicate under the statistical significance ***p < 0.001.