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

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 Km 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 Vmax 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 Vmax 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.


Background
Kluyveromyces marxianus is a sister species to the better-known K. lactis [1]. A large number of studies on K. lactis have mainly focused on its lactose metabolism and use as a model for non-conventional yeasts [2]. In contrast, scientific literature about the fundamental aspects of K. marxianus is relatively scarce [1]. Recently, K. marxianus has gained increasing attention since some of its traits are desirable for biotechnological applications. These traits include the fastest growth rate of any eukaryotic microbe, thermotolerance, secretion of native enzymes such as inulinase, β-galactosidase and pectinase, and production of ethanol [1,3].
K. marxianus is now being investigated as an alternative to Saccharomyces cerevisiae for ethanol production, especially in simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and co-fermentation (SSCF) processes, since it can produce ethanol at higher temperatures and on a wider variety of substrates including xylose [3][4][5]. It has been reported to be able to grow at 45°С and even 52°С and to produce ethanol at temperatures above 40°C [4,6,7]. S. cerevisiae, in contrast, is unable to ferment xylose and has an optimum growth temperature ranging from 30 to 34°С [8]. The enzymatic hydrolysis during SSF or SSCF processes is usually conducted at approximately 50°C, and the products formed during the hydrolysis step in SSCF include hexoses and pentoses. The traits of K. marxianus make it suitable for use in SSCF processes involving cellulosic biomass [9,10].
Yeast alcohol dehydrogenase (ADH) catalyzes the final metabolic step in ethanol fermentation, and thus plays an important role. The ADH systems of S. cerevisiae and K. lactis were studied extensively and seven ScADH genes (ScADH1 to ScADH7) and four KlADH genes (KlADH1 to KlADH4) have reportedly been cloned [11][12][13][14]. There are only a few scientific papers on the ADH systems of K. marxianus. Recently, the complete genome sequence of K. marxianus var. marxianus KCTC 17555 was determined and four ADH-encoding genes were annotated in the genome [15]. Two genes, KmADH1 and KmADH2, were cloned from K. marxianus ATCC 12424, while other two genes, KmADH3 and KmADH4, were cloned from K. marxianus DMKU 3-1042 [12,[16][17][18]. However, heterologous expression of the four genes and the biochemical properties of the KmAdhs have not been reported yet.
The K. marxianus GX-UN120 strain obtained in our laboratory is an excellent ethanol producer at high temperature and produced 69 g/L of ethanol when fermenting 150 g/L of glucose at 40°C [19]. Determining the biochemical characteristics of the ADHs of GX-UN120 will help to explain why it can produce high levels of ethanol at high temperature. In the present study, the genes encoding the four KmAdhs of GX-UN120 were cloned and individually overexpressed in E. coli, and the biochemical characteristics of each purified KmAdh were investigated. Understanding the biochemical characteristics of the KmAdhs of K. marxianus will enhance our fundamental knowledge of the ADH systems and the metabolism of ethanol fermentation in K. marxianus.

Results
Growth and ethanol fermentation characteristics of K. marxianus GX-UN120 K. marxianus GX-UN120 is an excellent ethanol-producing mutant strain that was converted from the wild-type strain GX-15 by alternately treatment with UV irradiation and NTG for two cycles. When fermenting 150 g/l of glucose, the ethanol yield of GX-UN120 was 69 g/l which was 20% higher than that of GX-15. However, the ADH activity of GX-UN120 was not significantly higher than that of GX-15 [19]. The nucleotide sequence of KmADH1 in GX-UN120 (KF678864) was not different to that in GX-15 (JF709970). The growth and ethanol fermentation characteristics of GX-UN120 were determined compared with those of S. cerevisiae Angel, which is a commercial ethanol producer in China. The optimum temperatures for growth and ethanol fermentation of GX-UN120 were 35-40°С and 40°С, respectively, whereas that of Angel was 28-34°С. GX-UN120 grew well even at 45°С, whereas Angel was not able to grow when the temperature was over 45°С (Figure 1a and b). The time courses for ethanol formation in 150 g/L glucose by GX-UN120 at 40°С and Angel at 34°С are shown in Figure 1c. The time taken for GX-UN120 to completely consume the glucose and reach its maximum ethanol yield was the same as that for Angel. Both yeasts consumed the glucose completely within 72 h. At that time the maximum ethanol concentration and ethanol yield coefficient of GX-UN120 were 67.6 g/L and 0.45 g/g, respectively, and those of Angel were 67.7 g/L and 0.45 g/g, respectively.
Analysis of the expression of KmADHs in K. marxianus GX-UN120 The translational levels of KmADH genes in GX-UN120 were determined through the analysis of zymograms of the ADH isozymes at different fermentation phases (the lag, exponential and stationary phases) in YPD containing 150 g/L of glucose ( Figure 2). The results indicated that KmADH1 was weakly expressed at the lag phase and largely expressed at the exponential phase, and its expression level decreased at the stationary phase. KmADH4 was constitutively expressed during all phases. The expression levels of KmADH2 and KmADH3 were not detectable.
Cloning and sequence analysis of the genes encoding the four KmADHs from K. marxianus GX-UN120 The four genes encoding ADHs, KmADH1, KmADH2, KmADH3 and KmADH4, were cloned from GX-UN120 and sequenced. The open reading frames (ORFs) of the four ADH genes were, respectively, 1047, 1047, 1128 and 1140 bp and the deduced amino acid sequences were 348, 348, 375 and 379 amino acids, respectively. The deduced amino acid sequences of the four KmAdhs from GX-UN120 shared 98% to 99% identity with the corresponding four genes of ATCC 12424 and more than 80% identity with the ADHs of K. lactis, K. wickerhamii, S. cerevisiae, S. carlsbergensis, S. kluyveri, S. pastorianus and Hansenula polymorpha [11][12][13][14]16,17,[20][21][22][23]. There are five amino acid residues difference in the deduced amino acid sequence of KmADH1 in GX-UN120 and KmADH1 in ATCC 12424, they are N15H, G239V, T328S, S334V and I339V. In KmADH2, the different amino acid residues are H315N and I338V. In KmADH3, the different amino acid residues are E233D and Q240E. In KmADH4, the different amino acid residues are N268S, V360I and S378A. All these amino acid residues are not in the groups directly involved in catalysis.
The phylogenetic analysis of the amino acid sequences of the KmAdhs and yeast ADHs (Additional file 1: Table S1) in Figure 3 reveals that KmAdh1 of GX-UN120 is closely grouped with Adh1 from K. marxianus ATCC 12424, K. marxianus DMKU3-1042 and K. wickerhamii and Adh2 from K. lactis, and KmAdh2 is grouped with Adh2 from K. marxianus ATCC 12424, whereas KmAdh3 and KmAdh4 are closely grouped with Adh3 and Adh4 from K. marxianus ATCC 12424, K. marxianus DMKU3-1042, K. wickerhamii and K. lactis.
The multiple amino acid sequence alignments of KmAdhs with ADHs from other yeasts (Additional file 1: Table S1) reveal that KmAdh1 and KmAdh2 do not possess but KmAdh3 and KmAdh4 do possess the N-terminal mitochondrion targeting sequence ( Figure 4). These results indicate that KmAdh1 and KmAdh2 are cytoplasmic ADHs, whereas KmAdh3 and KmAdh4 are mitochondrial ADHs. Several conserved motifs of the microbial group I ADHs were found in the KmADHs, including Asp 202 in KmAdh1 and KmAdh2, Asp 229 in KmAdh3 and Asp 233 in KmAdh4, which determine the NAD + specificity. This suggests that KmAdhs are NAD- KmAdh4 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 Zn 2+ -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.
dependent ADHs similar to the ADHs of other yeasts [24]. The NAD + -binding motifs of the KmAdhs are GAG/ CGGLG (BoxII), similar to those in ADHs of other yeasts [12,21,25]. Zn 2+ -binding residues, which are known to be essential for enzyme catalytic activity and structure, and the Zn 2+ -binding consensuses were also found (Box I).

Expression and purification of the recombinant KmADHs
The four cloned ADH genes were expressed in E. The specific activities of KmAdhs with NAD + and NADH were 70-80 times and 50-60 times higher than those with NADP + and NADPH, respectively. These data indicate that KmAdhs prefer NAD + and NADH as cofactor. KmAdh1 showed activity in the range of pH 5.0-9.0 when acetaldehyde was the substrate and pH 6.0-9.0 when ethanol was used as the substrate. KmAdh2, KmAdh3 and KmAdh4 showed activities in the range of pH 6.0-10.0. Beyond these pH ranges, the activities of the enzymes were completely lost. The optimum pH of KmAdh1 was measured as 8.0, while those of KmAdh2, KmAdh3 and KmAdh4 were 7.0 ( Figure 6a). KmAdh1, KmAdh2, KmAdh3 and KmAdh4 were relatively stable at pH 7.0-9.0, 5.0-9.0, 6.0-9.0 and 6.0-8.0, respectively. They retained more than 60% ADH activity when incubated at the corresponding pH ranges for 24 h (Figure 6b) (Figure 6c). KmAdh1 and KmAdh3 were relatively stable in the temperature range of 30 to 45°C, while KmAdh2 and KmAdh4 were relatively stable at 30 to 40°C. The ADH activities decreased sharply above 45°C and were completely lost above 60°C (Figure 6d). The kinetic properties of the KmAdhs were determined and are summarized in Table 1. The K m values of KmAdh1 and KmAdh2 for ethanol were about 10-and 42-fold higher, respectively, than those for acetaldehyde, while the V max values for acetaldehyde were about 2and 12-fold higher, respectively, than those for ethanol. The turnover numbers (K cat ) of KmAdh1 and KmAdh2 for acetaldehyde were 2-and 12-fold and the catalytic efficiencies (K cat /K m ) were 20-and 520-fold higher than those for ethanol, respectively. These results indicate that KmAdh1 and KmAdh2 of GX-UN120 are chiefly responsible for the reduction of acetaldehyde to ethanol. KmAdh3 and KmAdh4 catalyze the oxidation reaction of ethanol to acetaldehyde but not the reduction reaction of acetaldehyde to ethanol.

Substrate specificities of the recombinant KmAdhs
The substrate specificities of the recombinant KmAdhs towards different alcohols with various chain lengths were determined and the results are shown in Figure 7a.
All four KmAdhs preferred ethanol as the best alcoholic substrate. KmAdh1 and KmAdh2 displayed high activities towards primary 1-5 carbon and 1-3 carbon alcohols, respectively, and the activities decreased with increasing chain length. Both enzymes displayed low or no activity for long chain and branched alcohols. various chain lengths and 6 aromatic aldehydes were determined and the results are shown in Figure 7b. The highest reducing activities of KmAdh1 and KmAdh2 were found with acetaldehyde. KmAdh1 displayed high activities towards most of the straight-chain aliphatic aldehydes and low or no activity towards the branchedchain aliphatic aldehydes and aromatic aldehydes. The substrate specificity of KmAdh2 towards aldehydes was similar to that of KmAdh1. But the substrate specificities of these two ADHs were different when using butyraldehyde, valeraldehyde, heptaldehyde and phenylacetaldehyde as substrates. Regarding KmAdh3 and KmAdh4, no activities were detected towards any of the tested aldehydes.

Discussion
The mechanism by which K. marxianus produces ethanol at high temperature is unknown as yet. Reports about the ethanol metabolic pathway of K. marxianus are rare. In particular, the biochemical characteristics of the ADHs from K. marxianus, which contribute to ethanol metabolism, are not understood. The growth and ethanol fermentation characteristics suggest that the fermentation capability of K. marxianus GX-UN120 at 40°С is the same as that of S. cerevisiae Angel at 34°С. In the present study, all four ADH-encoding genes of GX-UN120 were cloned and overexpressed in E. coli. The biochemical characteristics of the purified recombinant KmAdhs were investigated. To our knowledge, this is the first report of the heterologous expression of genes encoding the ADHs of K. marxianus. Amino acid sequence analysis suggests that KmAdh1 and KmAdh2 of GX-UN120 may be cytoplasmic ADHs, while KmAdh3 and KmAdh4 may be mitochondrial ADHs. All four ADHs belong to the microbial group I ADHs. Characterization of their enzymatic properties showed that KmAdhs prefer NAD + and NADH to NADP + and NADPH as cofactor, which is similar to ADHs of other yeasts [25,26,30]. With optimum temperatures of 45-55°C for ethanol and acetaldehyde, the KmAdhs are distinctly different from most reported ADHs of yeasts, which generally have optimum activities at about 30°C [28]. Perhaps this is why GX-UN120 produces its maximal yield of ethanol at 40°C, while other yeasts such as S. cerevisiae and S. carlsbergensis have maximal yields usually at 30°C [4,19].
There have been no previous reports regarding the substrate specificity of ADHs from K. marxianus. Our data indicate that the four recombinant KmAdhs of GX-UN120 have a narrow alcoholic substrate specificity, which is similar to ScAdh1 of S. cerevisiae. It was reported that the narrow substrate specificity of ScAdh1 is due to Met 271 in its substrate binding cleft, whereas there is a Leu in the corresponding position in other yeast ADHs including KmAdhs [14,31]. The alcoholic substrate specificity of the KmAdhs is similar to that of ScAdh1 but different from that of ScAdh2 [14]. The ADHs of K. lactis [26,27], Adh1 of H. polymorpha [21], ADHs of C. maltosa [30] and Adh1 of C. utilis [25] display broad alcoholic substrate specificity. KmAdh1 and KmAdh2 of GX-UN120 have a broad substrate specificity for straight-chain aliphatic aldehydes, and the specific activities towards aldehydes are more than 2-fold higher than those towards the analogous alcohols. These results suggest that KmAdh1 and KmAdh2 prefer aldehydes as their substrates and acetaldehyde was the best substrate, which is similar to ADH1s from other yeasts and KlAdh3 [21,26,27,30]. Interestingly, KmAdh1 and KmAdh2 of GX-UN120 could efficiently reduce furfural, which is formed in the pretreatment of lignocelluloses and is an inhibitor of ethanol production by S. cerevisiae. This suggests that GX-UN120 is suitable for use in the SSCF of lignocelluloses to produce ethanol.

Conclusions
Zymogram analysis showed that KmAdh1 was largely induced in K. marxianus GX-UN120 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 were cloned from strain GX-UN120 and heterologous expressed in Escherichia coli. The biochemical characteristics of the recombinant ADHs in this study indicate that KmAdh1 is the primary ADH responsible for the production of ethanol from the reduction of acetaldehyde in K. marxianus. The result that the optimum temperature of KmAdh1 was 20°C higher than that of ADH from S. cerevisiae may partially explain the ability of K. marxianus to produce ethanol at high temperature.

Strains and growth conditions
K. marxianus GX-UN120 was used in this study and grown in yeast extract, peptone, dextrose (YPD) medium at 37°С. GX-UN120 is a mutant strain that was derived from the wild-type strain GX-15 which was isolated from soil sample collected in the subtropical area of Guangxi Zhuang Autonomous Region, China [19] and stored in College of Life Science and Technology, Guangxi University, Nanning, China. E. coli DH5α (Novagen, USA) and Rosetta DE3 (Novagen, USA) strains were used as the hosts for cloning genes and overexpression of recombinant genes and were grown in LB medium with 100 mg/L of ampicillin at 37°С.
Growth and ethanol fermentation of K. marxianus and S. cerevisiae The growth and ethanol fermentation characteristics of GX-UN120 and S. cerevisiae Angel which was obtained from Angel Yeast Co., Ltd, Yichang, China were investigated in 100 mL YPD medium containing 20 g/L glucose in 250-mL Erlenmeyer flasks or 200 mL YPD medium containing 150 g/L glucose in 500-mL Erlenmeyer flasks. The flasks were incubated without shaking. Growth was measured at OD 600 and the ethanol and glucose concentrations were determined by gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively [19].  The letters italic represented the restriction sites.