Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coliand biochemical characterization
© Liang et al.; licensee BioMed Central Ltd. 2014
Received: 6 November 2013
Accepted: 12 May 2014
Published: 21 May 2014
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.
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.
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.
KeywordsAlcohol dehydrogenase Characterization Expression Gene cloning Kluyveromyces marxianus
Kluyveromyces marxianus is a sister species to the better-known K. lactis. A large number of studies on K. lactis have mainly focused on its lactose metabolism and use as a model for non-conventional yeasts . In contrast, scientific literature about the fundamental aspects of K. marxianus is relatively scarce . 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 [3–5]. 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 . 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 [11–14]. 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 . 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, 16–18]. 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 . 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.
Growth and ethanol fermentation characteristics of K. marxianusGX-UN120
Analysis of the expression of KmADHs in K. marxianusGX-UN120
Cloning and sequence analysis of the genes encoding the four KmADHs from K. marxianusGX-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[11–14, 16, 17, 20–23]. 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.
Expression and purification of the recombinant KmADHs
Biochemical characterization of the recombinant KmAdhs
Summary of enzymatic properties of KmAdhs and ADHs from other yeasts
Optimum temperature (°C)
8.5 × 103
2.1 × 102
1.7 × 104
4.3 × 103
2.7 × 102
3.2 × 103
2.7 × 103
2.0 × 103
0.8 × 102
9.5 × 103
5.6 × 102
2.5 × 105
9.3 × 103
3.6 × 105
3.0 × 105
2.8 × 104
1.2 × 103
3.3 × 104
2.0 × 104
8.2 × 104
3.2 × 104
8.6 × 105
8.6 × 106
1.3 × 104
8.3 × 103
2.9 × 104
9.0 × 103
2.0 × 104
1.2 × 103
1.0 × 105
9.3 × 104
7.8 × 103
9.6 × 103
6.2 × 104
6.9 × 105
2.1 × 105
8.4 × 105
2.0 × 105
1.0 × 105
2.9 × 104
1.6 × 103
2.1 × 105
1.9 × 105
Substrate specificities of the recombinant KmAdhs
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 . 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 Met271 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 . The ADHs of K. lactis[26, 27], Adh1 of H. polymorpha, ADHs of C. maltosa and Adh1 of C. utilis 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.
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  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 OD600 and the ethanol and glucose concentrations were determined by gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively .
Cloning of genes encoding KmADH from K. marxianusGX-UN120
Primers used in this study
Expression of KmADH genes in E. coliand purification of the recombinant proteins
pGXKmADH1, pGXKmADH3, pGXKmADH4 and the expression vector pET-32a(+) were separately digested with BamHI and HindIII and the target fragments were purified. pGXKmADH2 and pET-30a(+) were separately digested with Nde I and Xho I and the gene and the vector DNA were recovered. The DNA fragment containing the KmAdh genes and the corresponding expression vector were ligated with T4 DNA ligase to form pET-32a(+)-KmADH1, pET-30a(+)-KmADH2, pET-32a(+)-KmADH3 and pET-32a(+)-KmADH4. The recombinant plasmids were transformed into E. coli Rosetta DE3 to express the target proteins. KmAdh1, KmAdh3 and KmAdh4 were expressed as TrxA fusion proteins  and KmAdh2 as a His-tagged protein. The recombinant fusion proteins were purified by co-affinity chromatography using a TALON Cobalt Resin column. The purified, recombinant, fusion KmAdhs were digested with enterokinase light chain to remove the TrxA or His-tag. The digestion mixture was loaded on the TALON Cobalt Resin column. The eluted solution containing KmAdh was collected and used for further study, and the TrxA or His-tag bound to the cobalt resin remained in the column. The native molecular masses of the purified KmAdh proteins were measured by high performance gel permeation chromatography (HPGPC) using a Macrosphere GPC 1 507 μ column (250 mm × 4.6 mm, Alltech Associates, Inc.) and eluted with 0.15 M NaCl at a flow rate of 0.3 mL/min. The molecular masses were then calculated using the protein molecular weight standards ferritin horse (450 kDa), catalase bovine (240 kDa), aldolase rabbit (160 kDa) and albumin bovine (67 kDa) (SERVA Electrophoresis GmbH, Heidelberg, Germany).
The zymogram analysis of ADH isozymes and the ADH activities were assayed according to the method previously described by Cho and Jeffries  with minor modification. The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 2 mM NAD+ or 0.2 mM NADH, 0.8 M alcoholic substrates or 50 mM aldehydic substrates and the purified KmAdhs. One enzyme unit (U) was defined as the micromoles of NADH produced or consumed per minute.
Kinetic analysis of the KmAdhs was performed as previously described . The Km and Vmax values were measured using the double-reciprocal plot method of Lineweaver-Burk . Catalytic efficiency (kcat/Km) was derived from Vmax/Km [E].
DNA sequence analysis
Sequence assembly and ORF analysis were carried out using the Vector NTI program. The protein sequence similarity searches were performed with the BLAST tools (http://www.ncbi.nlm.nih.gov). Only proteins that showed significant similarity and had already been characterized as ADH were used for phylogenetic analysis and multiple sequence alignment. The nucleotide sequences of KmADH1, KmADH2, KmADH3 and KmADH4 were deposited in the GenBank database under accession numbers KF678864, KF678866, KF678865, KF678867, respectively.
This work was financially supported by a grant from the Guangxi Natural Science Foundation (2012GXNSFGA060005), and the Bagui Scholar Program of Guangxi (2011A001).
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