Enhanced Expression of Alcohol Dehydrogenase I in Pichia pastoris Reduces the Content of Acetaldehyde in Wines

Abstract

Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.

Keywords: Pichia pastoris; acetaldehyde; alcohol dehydrogenase I; cofactor regeneration; multicopy; wine.

Figure 1

Diagram of the plasmids with a single expression cassette for the expression of ADH1_N-6×His or ADH1_C-6×His with promoter P_AOX1 (a) and for the expression of ADH1_N-6×His with promoters P_AOX1, P_AOXm, or P_ADH3 (b).

Figure 2

Enzymatic activity (a–c) and SDS-PAGE analysis (d) of different strains of Pichia pastoris, including the control strain GS115/pPIC9K (G/9K) and the recombinant strains GS115/pPIC9K-ADH1_N-6×His (N-His) and GS115/pPIC9K-ADH1_C-6×His (C-His) (a), as well as other strains represented by promoter name of the expression cassette (b) and the copy number of target gene (c). The strains G/9K(E) and ADH3 are cultured with ethanol as the carbon source, and G/9K(M) and other strains are cultured with methanol as the carbon source. Lane M represents the protein marker. Lanes 1 to 6 correspond to the methanol-induced strains GS115/pPIC9K, GS115/pHKA-ADH1_N-6×His, GS115/pHKA-ADH1_N-6×His-2Copies, GS115/pHKAOXm-ADH1_N-6×His, GS115/pHKAOXm-ADH1_N-6×His-2Copies, and GS115/pHKAOXm-ADH1_N-6×His-3Copies, respectively. Lanes 7 and 8 correspond to strains GS115/pPIC9K and GS115/pHKADH3-ADH1_N-6×His, respectively, cultured with ethanol as the carbon source. The bands of ADH1 are circled in red squares. Each experimental measurement of enzymatic activity is repeated three times and expressed as the mean ± standard deviation. The statistical significance is determined by Student’s t test based on p < 0.05 (**) and p < 0.01 (***), respectively.

Figure 3

Growth curves of strains of Pichia pastoris with ethanol (a) or methanol (b) as the carbon sources. G/9K, the control strain GS115/pPIC9K; the remaining strains are represented by the promoter names and copy number of target gene of the expression cassette.

Figure 4

SDS-PAGE analysis of purified ADH1. Lane M: protein marker; lanes 1–4: 5%, 10%, 20%, and 50% buffer B gradient, respectively. The bands of ADH1 are circled in a red square.

Figure 5

Variations in the content of acetaldehyde in wine by ADH1. (a) Concentration of acetaldehyde in reaction systems at 0.5 h. (b) Concentration of acetaldehyde in wine samples with the addition of ADH1 and NADH of different concentrations over time. Each measurement of the content of acetaldehyde is repeated three times and expressed as the mean ± standard deviation. The statistical significance is determined by Student’s t test based on p < 0.05 (**) and p < 0.01 (***), respectively.

Figure 6

Diagram of cofactor regeneration (a) and the reduction in the content of acetaldehyde in four groups of wine samples by both ADH1 and GDH (b). Symbols “+” and “−” represent the presence and absence of the corresponding substances, respectively. Each measurement of the content of acetaldehyde is repeated three times and expressed as the mean ± standard deviation. The statistical significance is determined by Student’s t test based on p < 0.01 (***), respectively.