Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization

Abstract

Background: Kluyveromyces marxianus has recently become a species of interest for ethanol production since it can produce ethanol at high temperature and on a wide variety of substrates. However, the reason why this yeast can produce ethanol at high temperature is largely unknown.

Results: The ethanol fermentation capability of K. marxianus GX-UN120 at 40°С was found to be the same as that of Saccharomyces cerevisiae at 34°С. Zymogram analysis showed that alcohol dehydrogenase 1 (KmAdh1) was largely induced during ethanol production, KmAdh4 was constitutively expressed at a lower level and KmAdh2 and KmAdh3 were almost undetectable. The genes encoding the four alcohol dehydrogenases (ADHs) were cloned from strain GX-UN120. Each KmADH was expressed in Escherichia coli and each recombinant protein was digested with enterokinase to remove the fusion protein. The optimum pH of the purified recombinant KmAdh1 was 8.0 and that of KmAdh2, KmAdh3 and KmAdh4 was 7.0. The optimum temperatures of KmAdh1, KmAdh2, KmAdh3 and KmAdh4 were 50, 45, 55 and 45°C, respectively. The K(m) values of the recombinant KmAdh1 and KmAdh2 were 4.0 and 1.2 mM for acetaldehyde and 39.7 and 49.5 mM for ethanol, respectively. The V(max) values of the recombinant KmAdh1 and KmAdh2 were 114.9 and 21.6 μmol min⁻¹ mg⁻¹ for acetaldehyde and 57.5 and 1.8 μmol min⁻¹ mg⁻¹ for ethanol, respectively. KmAdh3 and KmAdh4 catalyze the oxidation reaction of ethanol to acetaldehyde but not the reduction reaction of acetaldehyde to ethanol, and the K(m) values of the recombinant KmAdh3 and KmAdh4 were 26.0 and 17.0 mM for ethanol, respectively. The V(max) values of the recombinant KmAdh3 and KmAdh4 were 12.8 and 56.2 μmol min⁻¹ mg⁻¹ for ethanol, respectively.

Conclusion: These data in this study collectively indicate that KmAdh1 is the primary ADH responsible for the production of ethanol from the reduction of acetaldehyde in K. marxianus. The relatively high optimum temperature of KmAdh1 may partially explain the ability of K. marxianus to produce ethanol at high temperature. Understanding the biochemical characteristics of KmAdhs will enhance our fundamental knowledge of the metabolism of ethanol fermentation in K. marxianus.

Figure 1

The growth and ethanol fermentation characteristics of K. marxianus GX-UN120 and S. cerevisiae Angel. Exponential phase cultures of GX-UN120 and Angel were used to inoculate YPD medium containing 20 g/L glucose to a final OD₆₀₀ of 0.2 or fermentation medium containing 150 g/L glucose with 10% of the inoculum. (a) Growth in YPD medium containing 20 g/L glucose for 24 h without shaking. (b) Ethanol fermentation was carried out in 150 g/L glucose at different temperatures for 72 h without shaking. (c) The time course of ethanol fermentation was recorded in 150 g/L glucose at 40°C (GX-UN120) or 34°C (Angel) without shaking. Experiments were performed in triplicate and the results are given as mean values with error bars indicating standard deviations.

Figure 2

Zymogram analysis of ADH isozymes from GX-UN120 during ethanol fermentation. Fermentation was performed in YPD containing 150 g/L glucose at 40°C. Cells were harvested from the broth at the lag (4 h), exponential (24 h) and stationary phases (72 h) and disrupted by grinding on ice. The cell extracts were separated on 7.5% polyacrylamide gel and the gels were stained for ADH activity with ethanol as the substrate.

Figure 3

Phylogenetic analysis of KmAdhs of GX-UN120. The sequences were aligned to generate an unrooted phylogenetic tree with MEGA 4.0 using the neighbor-joining method. Branch support values from 1000 bootstrap replications are presented beside each node and a Poisson correction was carried out. GenBank accession numbers are shown in brackets after each enzyme name. All proteins included in the analysis were enzymatically characterized as alcohol dehydrogenases. References are listed in Additional file 1: Table S1 in the supplementary materials.

Figure 4

Alignment of the conserved amino acid residues and structurally conserved regions of the KmAdhs. Alignment was done using the Vector NTI program. The protein codes correspond to those listed in Additional file 1: Table S1 in the supplementary material. Asp residues in deep grey determine the specificity for NAD⁺. Residues in box I and box II indicate Zn²⁺-binding and NAD⁺-binding moieties, respectively. KmAdh, alcohol dehydrogenase from K. marxianus; KlAdh, alcohol dehydrogenase from K. lactis; KwAdh, alcohol dehydrogenase from K. wickerhamii; ScAdh, alcohol dehydrogenase from S. cerevisiae.

Figure 5

Electrophoresis of KmAdhs from K. marxianus GX-UN120. SDS-PAGE analysis on 10% polyacrylamide gel stained with Coomassie light blue. a, KmAdh1; b, KmAdh2; c, KmAdh3; d, KmAdh4. Lane M, protein molecular weight markers (116.0, 66.2, 45.0, 35.0 25.0 and 18.4 kDa). Lane 1, proteins of E. coli Rosetta DE3 harboring the empty plasmid pET-32a(+) in a, c, d and empty pET-30a(+) in b; lane 2, proteins of E. coli Rosetta DE3 harboring the plasmids pET-32a(+)-KmADH1, pET-32a(+)-KmADH3 and pET-32a(+)-KmADH4 in a, c and d and pET-30a(+)-KmADH2 in b; lane 3, the purified recombinant KmAdhs fusion proteins; lane 4, the purified KmAdhs. The recombinant fusion proteins and proteins after digestion with enterokinase light chain were purified with Co–NTA chromatography.

Figure 6

Effects of pH and temperature on enzyme activities of KmAdhs. a Determination of the optimal pH of the KmAdhs. Enzyme assays were performed at the indicated pH at 40°C using ethanol (closed symbols) and acetaldehyde (open symbols) as substrates. squares, 50 mM citrate-phosphate buffer (pH 4.0-7.0); circles, 50 mM sodium-phosphate buffer (pH 6.0-8.0); triangles, 50 mM Tris–HCl buffer (pH 8.0-9.0); diamonds, 50 mM glycine-NaOH buffer (pH 9.0-10.0). b pH stability of the KmAdhs. Enzyme activity was measured under optimal conditions (50 mM sodium-phosphate buffer of pH 7.0, 40°C) after the enzyme was incubated in the indicated buffers at 4°C for 24 h. c Determination of the optimal temperatures of the KmAdhs. Activity was measured at pH 7.0 (50 mM sodium-phosphate buffer) at the indicated temperatures. d Thermal stability of KmAdhs. Enzyme activity was measured under optimal conditions (50 mM sodium-phosphate buffer of pH 7.0, 40°C) after the enzyme had been incubated at the indicated temperature for 30 min. The error bars represent the standard deviations of triplicate measurements.

Figure 7

The activities of the KmAdhs on various alcohols and aldehydes. a Relative activities on various alcohols; b Relative activities on various aldehydes. Experiments were conducted in reaction mixtures containing 50 mM sodium phosphate buffer (pH 7.0), 2 mM NAD⁺ for alcohols or 0.2 mM NADH for aldehydes, 0.8 M alcohols or 50 mM aldehydes and the purified KmAdhs (1–20 μg of protein) at pH 7.0 and 40°C.