Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG

Journal of Biotechnology 135 (2008) 127–133

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG Young Jae Jeon, Jiunn C.N. Fong, Eny I. Riyanti, Brett A. Neilan, Peter L. Rogers, Charles J. Svenson ∗ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia

a r t i c l e

i n f o

Article history: Received 9 July 2007 Received in revised form 17 December 2007 Accepted 21 February 2008 Keywords: Thermophile Geobacillus thermoglucosidasius Thermophilic alcohol dehydrogenase Ethanol production Heterologous expression

a b s t r a c t A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterised. Inverse PCR showed that the gene (adhI) was localised with 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3 hexuloisomerase (PHI) on its genome. The deduced peptide sequence of the 1020-bp M10EXG adhI, which corresponds to 340 amino acids, shows 96% and 89% similarity to ADH-hT and ADH-T from Geobacillus stearothermophilus strains LLD-R and NCA 1503, respectively. Over-expression of M10EXG ADH-I in Escherichia coli DH5␣ (pNF303) was confirmed using an ADH activity assay and SDS-PAGE analysis. The specific ADH activity in the extract from this recombinant strain was 9.7(±0.3) U mg−1 protein, compared to 0.1(±0.01) U mg−1 protein in the control strain. The recombinant E. coli showed enzymatic activity towards ethanol, 1butanol, 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol and 2-propanol, but not methanol. In silico analysis, including phylogenetic reconstruction and protein modeling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Economic and geopolitical factors including high oil prices, environmental friendly concerns, and supply instability, have necessitated the development of sustainable processes through the conversion of renewable agricultural-based raw lignocellulosic material into bioethanol (Rogers et al., 2007; Stephanopoulos, 2007). The use of thermophilic microorganisms in a lignocellulosicbased ethanol production has been proposed to be significantly favored with a simultaneous saccharification and fermentation process (Lamed and Zeikus, 1980, 1981; Klapatch et al., 1994). The potential advantages being reduction of fermentation cooling costs following higher temperature enzymatic pre-treatment, lower contamination risks (particularly by mesophilic contaminants), and reduced energy input for distillation by comparison with the use of mesophilic yeasts and bacteria (Edwards, 1990; Lowe et al., 1993; Klapatch et al., 1994; Banat and Marchant, 1995; Banat et al., 1998). As such, the ideal thermophile ethanologen should be a facultative anaerobic microorganism that has high ethanol productivity, high ethanol tolerance and a broad substrate range. However, wild-type thermophilic facultative anaerobic bacteria have generally shown relatively low (1–5%) ethanol tolerance and a narrow substrate

∗ Corresponding author. Tel.: +61 2 9385 1213; fax: +61 2 9385 1591. E-mail address: [email protected] (C.J. Svenson). 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.02.018

range, while the thermophilic bacterium Clostridium thermocellusm, an obligate anaerobe, can be tolerated up to 8% (v/v) ethanol (Fong et al., 2006). Unlike these bacteria, a recently isolated facultative anaerobic thermophile Geobacillus thermoglucosidasius strain M10EXG has shown robust characteristics as a potential ethanologen, with highest ethanol tolerance (10%, v/v) amongst reported thermophilic facultative anaerobic bacteria and broad range substrate usage including pentose and hexose sugars (Fong et al., 2006). All microorganisms that exhibit high ethanol productivity contain genes that encode for pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). PDC is common to only plants, yeasts, and fungi, however it is absent in animals and rare in prokaryotes. This type of non-oxidative decarboxylating enzyme has only been reported in 4 prokaryotes (Ingram et al., 1999; Talarico et al., 2001; Raj et al., 2001, 2002). In contrast, ADH is widely distributed in many organisms, including animal tissues, plants, yeasts, bacteria and archaea (Ammendola et al., 1992; Cannio et al., 1994; Burdette et al., 1997). ADH is an important biocatalyst for the synthesis of chiral primary and secondary alcohols (Bradshaw et al., 1992a,b; Helmann, 1995) as well as other chemicals (Coolbear et al., 1992; Suye et al., 2002). ADHs are classified based on their cofactor specificity for such metabolites as NAD(P), the pyrroloquinoline quinine, and cofactor F420 . The NAD(P)-dependent ADHs can be further subdivided into those which are zinc-dependent, short-chain or iron-activated. A significant number of thermostable alcohol dehydrogenases have been isolated and characterised (see

128

Y.J. Jeon et al. / Journal of Biotechnology 135 (2008) 127–133

review Haki and Rakshit, 2003). This investigation is part of an ongoing research direction towards enhanced ethanol production in recombinant thermophilic bacteria through the expression of the ethanol (pet) operon, containing thermostable adhI and pdc. Therefore, we report here the isolation and characterisation of a thermostable alcohol dehydrogenase gene (adhI) from M10EXG and its over-expression in Escherichia coli. 2. Materials and methods 2.1. Bacterial strains, plasmids, and growth conditions G. thermoglucosidasius strain M10EXG (Fong et al., 2006) (ATCC BAA1067) and E. coli DH5␣ were grown in LB (Luria Bertaini) medium (Sambrook et al., 1989) at 60 ◦ C and 37 ◦ C, respectively. Media were solidified with 1.5% agar for E. coli and 3% agar for M10EXG. The plasmid pGEM® -T Easy was supplied by Promega (Wisconsin, USA). E. coli DH5␣ harbouring recombinant plasmids were selected on LB medium containing ampicillin (100 ␮g/ml), IPTG (isopropyl-␤-d-thiogalactopyranoside; 0.5 mM) and X-Gal (5bromo-4-chloro-3-indolyl-␤-d-galactoside; 80 ␮g/ml). 2.2. Fermentation studies of M10EXG To characterise carbon metabolism in strain M10EXG, fermentations were performed on thermophile minimal medium (TMM) supplemented with a mixture of glucose and xylose (Fong et al., 2006). Fermentation studies were performed in 500 ml shake flasks containing 100 ml of culture medium at 60 ◦ C with orbital shaking (200 rpm) under aerobic conditions. End-product analysis was performed as previously described (Jeon et al., 2005). Samples (1 ml) were taken from cultures grown in shake flasks. Analyses were carried out using high-performance liquid chromatography (HPLC) through an HPX-87H ion exchange column (Bio-Rad, California, USA). 2.3. DNA extractions and PCR Plasmid preparations from E. coli DH5␣, ligations and E. coli DH5␣ transformations were performed according to standard protocols (Sambrook et al., 1989). Plasmids from E. coli were isolated according to the Bio-Rad Quantum Plasmid Miniprep Kit protocol (Bio-Rad). Chromosomal DNA from M10EXG was isolated according to the method described previously (Fong et al., 2006). All PCR reactions were performed in an Eppendorf MastercyclerTM using Taq (Roche, Basel, Switzerland) and/or pfu (Promega, Wisconsin, USA) DNA polymerases. 2.4. Isolation of M10EXG adhI For the isolation of adh, degenerate primers adh98 F (5 -AAK MKT GYG GTA TGY CAT AC-3 ) and adh909 R (5 -TTW CCT TCK GCT GCA AAY TG-3 ) targeting the consensus region of ADH were designed based on the peptide sequences from Clostridium beijerinckii, Thermoanaerobacter brockii, Geobacillus stearothermophilus and Zymomonas mobilis. Thermal cycling was performed at 95 ◦ C for 5 min, followed by 25 cycles at 95 ◦ C for 30 s, 50 ◦ C for 20 s, and 74 ◦ C for 40 s, and a final extension step at 74 ◦ C for 7 min.

HindIII-digested M10EXG chromosomal DNA. Inverse PCR was carried out using primers iM10adh F (5 -GGA TTA CCG CCG GAA GAA ATG CCT ATT CC-3 ) and iM10adh R (5 -GCG TGC AAG TCC GTA TGG CAT ACA CC-3 ) on the self re-ligation mixture. 2.6. DNA sequencing and analysis Automated DNA sequencing was carried out with BigDye® Terminator V3.1 Cycle Sequencing Kit and an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, California, USA). The nucleotide sequences were analyzed with FacturaTM and AutoAssemblerTM (Applied Biosystems, California, USA) and Australian National Genome Information Service (ANGIS) software programs. Sequences were translated into peptide sequences and the molecular weight and pI values calculated using Vector NTI v6 Software Program (Invitrogen, California, USA). 2.7. Phylogenetic sequence analysis Multiple alignment of the ADH-I amino acid sequences was carried out using the Clustal X package (Version 1.83) (Thompson et al., 1994). Phylogenetic reconstruction of the newly identified ADHI was carried out using genetic distance methods based on all fully characterised prokaryotic and archaeal ADHs available. Bootstrap re-sampling with 1000 reiterations was also performed (Neilan et al., 2002). Reference strains were obtained from GenBank. 2.8. Construction of pNF303 and pNF304 for heterologus expression of ADH-I in E. coli For the heterologous expression of the ADH-I from M10EXG, the full-length adhI was amplified from chromosomal DNA with primers M10adhTpro2 F (5 -GTT TTT GTT TGC GCT GC-3 ) and M10adhT R (5 -CTT CAT TAA TAA TCG CTG GAG TGC CG-3 ). The PCR products containing the entire ORF with a putative promotor were cloned into pGEM® -T Easy. Two different plasmids were isolated from the transformation with the gene inserted in the same orientation as the lac promoter called pNF303, and also in the other direction named pNF304, respectively. Inserted PCR products were checked by sequencing for any mutations. 2.9. Expression of ADH-I in E. coli and its enzymatic assay The recombinant E. coli strains were grown to an optical density of 1.0 (600 nm) in LB medium at 37 ◦ C. The culture was then induced with 0.5 mM IPTG overnight. The overnight cultures were harvested, washed (3 times) with 1 ml of 10 mM sodium phosphate buffer (pH 7.5) and resuspended in 1 ml of the same buffer. Cells were lysed by vortexing in 1 ml of acid-washed 150–121 ␮m glass beads (Sigma, Missouri, USA) in 5 ml glass tubes. Vortexing was repeated 10 times for 1 min with 2 min incubation on ice between each vortex. Cell extracts were collected after the glass beads and cell debris was removed by centrifugation. ADH activity in the cell extracts was assayed at 60 ◦ C by monitoring ethanoldependent NAD+ reduction at 340 nm (Cannio et al., 1994). The 1.5-ml reaction mixture contained 22 mM sodium pyrophosphate buffer (pH 8.8), 3.2% (v/v) ethanol, 7.5 mM ␤-nicotinamide adenine dinucleotide (␤-NAD), and cell extracts. Total cell protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Sigma, Missouri, USA) with bovine serum albumin as the standard.

2.5. Inverse PCR 2.10. Polyacrylamide gel electrophoresis Sequencing of unknown flanking regions at the degenerate PCR product was achieved using an inverse PCR as described by Ochman et al. (1988). Template DNA was prepared by self re-ligation of

Native-PAGE and SDS-PAGE analyses were carried out using Novex® 8% Tris–glycine and NuPAGETM 4–12% bis–Tris gels, respec-

Y.J. Jeon et al. / Journal of Biotechnology 135 (2008) 127–133

tively (Invitrogen, California, USA). ADH activity staining for native-PAGE using phenazine methosulfate and nitrotetrazolium blue (Sigma) has been described elsewhere (Bryant et al., 1988). Total protein staining for SDS-PAGE was carried out using GelCode® Blue stain reagent (Pierce, Illinois, USA).

129

centration of 1.4 g l−1 and the maximum specific growth rate (max ) was approximately 0.22 h−1 . A biomass yield of about 0.27 g g−1 was achieved (based on the total sugars utilised). Acetate was produced in association with growth to a biomass concentration of 2.5 g l−1 , and l-lactate was produced at the end of growth at 1 g l−1 . Low levels of ethanol were also detected (<0.5 g l−1 ) (Fig. 1).

2.11. Protein modeling Homology modeling was performed to determine the possible differences between the putative three-dimensional structures of the ADH-I protein of M10EXG and ADH-hT of G. stearothermophilus LLDR. SWISS-MODEL (Schwede et al., 2003, http://swissmodel.expasy.org) was utilised to search for suitable templates and to produce the theoretical tertiary structure. Vector NTI v6 Software Program (Invitrogen) and Swiss-PDB Viewer (Guex and Peitsch, 1997) were used for visualisation and reproduction of the models generated from SWISS-MODEL based on the ADH-hT of G. stearothermophilus LLDR. 2.12. Nucleotide sequence accession number The nucleotide sequences of adhI, hps (encoding 3-hexulose6-phosphate synthase), and partial phi (encoding 6-phospho-3hexuloisomerase) and uorA (encoding unknown oxidoreductase) of G. thermoglucosidasius M10EXG have been deposited in the GenBank database under the accession numbers AY494991, AY494992, AY494993 and AY494990, respectively. 3. Results 3.1. Growth and metabolism of G. thermoglucosidasius M10EXG on glucose and xylose medium The growth kinetics of G. thermoglucosidasius M10EXG was investigated using defined TMM supplemented with a mixture of glucose (0.5%, w/v) and xylose (0.5%, w/v) (TGXTV). Fermentations were carried out in duplicate shake flasks at 60 ◦ C with 10% (v/v) inocula, at 200 rpm. This strain utilised both sugars simultaneously, however, more efficiently with glucose than xylose (Fig. 1). Two growth phases were observed with a higher maximum specific growth rate during the early phase. Biomass was produced to a con-

3.2. Isolation of adhI from M10EXG Based on multiple alignments of amino acids from 4 different ADHs from C. beijerinckii, T. brockii, G. stearothermophilus and Z. mobilis, degenerate primers were designed to target the conserved regions of this enzyme. Subsequently, an 809-bp adhI fragment was amplified from M10EXG chromosomal DNA. The partial adhI fragment was cloned into pGEM® -T Easy and the nucleotide sequence determined. To isolate the full-length adhI, touchdown inverse PCR was carried out, resulting in the amplification of a 2510-bp genomic fragment. The amplified fragment was cloned and the nucleotide sequence determined. The contiguous nucleotide sequences of the 2510-bp fragments and the previously cloned partial 809-bp adhI fragment indicated the presence of 2 complete and 2 partial ORFs, which have deduced peptide sequences similar to alcohol dehydrogenases from G. stearothermophilus (Sakoda and Imanaka, 1992; Cannio et al., 1994), the 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) from B. subtilis (Yasueda et al., 1999), and an uncharacterised oxidoreductase from B. cereus (Ivanova et al., 2003) and B. anthracis (Read et al., 2003). The full-length adhI (1588 bp) with a putative promotor was amplified and cloned, generating constructs with the gene in both orientations. 3.3. Comparison of the deduced peptide sequences of ADH-I from M10EXG and other ADHs Many of the conserved amino acids present in other Zndependent ADHs were also found in ADH-I from M10EXG (Fig. 2). These included C38, H61, and C148 that form the catalytic zinc ligands; C92, C95, C98 and C106 required for the structural zinc ligands; acidic residues D41 and E62 involved in binding interactions; T40 and H43 involved in the proton release system; T152, G172, G174, G177, and D195 involved in the proper positioning and binding of coenzyme NAD+ , as well as the other reported conserved residues: V70, K133, K205, G255, V258, V260, and G261 (Keshav et al., 1990; Burdette et al., 1996, 1997; Sakoda and Imanaka, 1992). Residues E7, K14 and E16, suggested to increase thermostability (Ceccarelli et al., 2004), are conserved in the ADH-hT from G. stearothermophilus strain LLD-R and ADH-I from M10EXG. 3.4. Phylogenetic analysis

Fig. 1. Growth curve of M10EXG on TGXTV (0.5% (w/v) glucose and 0.5% (w/v) xylose) medium. Legend: () glucose, (䊉) xylose, () biomass, () acetate, () ethanol, and () l-lactate. Check the culture conditions whether aerobic or anaerobic.

To further classify M10EXG ADH-I within the thermostable ADH family, a phylogenetic analysis was performed with a selection of 23 sequences covering all representatives of the NAD(P)-dependent class of this family (Fe-dependent, short-chain, and Zn-dependent) from thermophilic, mesophilic, and psychrophilic organisms. The resulting analysis revealed that three ADH subgroups are classified according to their cofactor specificity, irrespective of archaeal or bacterial origin (Fig. 3). The M10EXG ADH-I was clustered together with other enzymes from the Geobacillus genus and within the Zndependent type family. A protein similarity search, also showed that the M10EXG ADH-I is phylogenically very closely related to the ADH-hT of G. stearothermophilus strain LLD-R.

130

Y.J. Jeon et al. / Journal of Biotechnology 135 (2008) 127–133

Fig. 2. Comparison of peptide sequences of ADHs from Geobacillus thermoglucosidasius M10EXG (ADH-I), Geobacillus stearothermophilus strains LLD-R (ADH-hT) and NCA1503 (ADH-T) and ADH-I from Zymomonas mobilis strain ZM4. Amino acid positions are shown on the left while the sources are indicated on the right. Conserved amino acids are shaded (black: catalytic zinc, black asterisk: structural zinc, gray asterisk: NAD binding and gray: proton release and acidic residues) while residues that have been suggested to increase thermostability are in bold (Ceccarelli et al., 2004).

3.5. Expression of adhI in E. coli Specific ADH activity from 0.5 mM IPTG overnight-induced culture of E. coli (pNF303) was detected in the cell extracts using ethanol as the substrate (Table 1). In the absence of the lac repressor (glucose), a 20-fold higher specific ADH activity (9.7 U/mg) was observed in E. coli (pNF303) with adhI in the same orientation as the lac promoter where the start codon is located on 243 bp from the promotor. Compared to E. coli (pNF304) with adhI in the

Table 1 Expression of G. thermoglucosidasius M10EXGT adhI in E. coli Plasmida

pGEM® -T Easy pNF303 pNF304 a

ADH specific activity (U/mg protein)b −Glucosec

+Glucosec

Heat-treatedd

0.1 (±0.01)e 9.7 (±0.3) 0.5 (±0.06)

0.1 (±0.001) 1.4 (±0.06) 0.1 (±0.01)

0 9.9 (±0.4) 0.5 (±0.01)

Plasmids present in the recombinant E. coli DH5␣. Specific activity is expressed as the units (U) of enzyme per milligram of total cell protein that is required to catalyzed the oxidation of 1 ␮mol of ethanol to acetaldehyde per min. c Recombinant E. coli cultures were grown in the absence (−) or presence (+) of glucose (1%, w/v). d Recombinant E. coli cultures were grown in the absence of glucose. Samples were incubated at 60 ◦ C for 10 min followed by centrifugation (20,000 × g) at 4 ◦ C for 10 min. e Assay repeated twice. b

opposite orientation, the activity was negligible (0.5 U/mg). In the presence of glucose, a 7-fold decrease of specific ADH activity was observed in E. coli (pNF303). The decrease in specific ADH activity in E. coli (pNF304) was less significant (0.4 U/mg). Background levels of specific ADH activity in cells containing only the vector pGEM® -T Easy were also negligible (0.1 U/mg) with or without the addition of glucose to the growth media. The recombinant ADHI was stable after incubation (twice) at 60 ◦ C for 10 min without loss of activity towards ethanol. No detectable background level of specific ADH activity in E. coli (pGEM® -T Easy) was observed after similar heat-treatment of the cell extract. The over-expressed recombinant ADH-I in E. coli (pNF303) was also reactive with a range of primary alcohols (1-butanol, 1-pentanol, 1-heptanol, 1hexanol and 1-octanol) as well as the secondary alcohol 2-propanol (data not shown). Among the alcohols tested, the highest specificity ADH activity was observed with 2-propanol as the substrate, which was 1.5-fold higher than for ethanol. Specific ADH activities for the primary alcohols tested were similar to ethanol. An insignificant level of specific ADH activity towards methanol and cofactor NADP+ was also detected (<0.1 U/mg) (data not shown). On SDS-PAGE (Fig. 4A ), the over-expressed recombinant ADH-I appeared as a dense protein band, having an apparent molecular weight around 38 kDa. A single band was observed on the nondenaturating native-PAGE gel containing the cell extract from E. coli (pNF303) when stained for ADH activity (Fig. 4B). Mobility of recombinant ADH-I on native-PAGE was faster compared to the ADH from Saccharomyces cerevisiae.

Y.J. Jeon et al. / Journal of Biotechnology 135 (2008) 127–133

131

Fig. 3. Phylogenetic tree derived from NAD(P)-dependent ADH family and related amino acid sequences from a protein database (NCBI).

Fig. 4. (A) SDS-PAGE and (B) native-PAGE analyses of cell extracts from E. coli DH5␣ (pNF303) expressing G. thermoglucosidasius M10EXG adhI. Lanes: 1, protein marker; 2, DH5␣ (pGEM® -T Easy) heat-treated; 3, DH5␣ (pGEM® -T Easy); 4, DH5␣ (pNF303) heat-treated; 5, DH5␣ (pNF303); 6, ADH standard (Sigma: Saccharomyces cerevisiae) (2 ␮g); 7, DH5␣ (pNF303); 8, ADH standard (0.2 ␮g); 9, ADH standard (2 ␮g); 10, DH5␣ (pGEM® -T Easy). Positions of ADH standard and ADH-I are marked with arrows.

132

Y.J. Jeon et al. / Journal of Biotechnology 135 (2008) 127–133

Fig. 5. Theoretical model of the ADH-I monomer from (A) M10EXG, (B) ADH-hT of G. stearothermophilus strain LLD-R. (1) Catalytic Zn-binding region, (2) NAD-binding region, (3) structural Zn-binding region, and (4) putative salt bridge forming region for enhanced themostability proposed by Ceccarelli et al. (2004).

3.6. ADH-I modeling Due to the close phylogenetic relationship established between the ADH-I protein of M10EXG and ADH-hT of G. stearothermophilus strain LLD-R, a theoretical structural model of M10EXG ADH-I was generated based on the protein structure of ADH-hT obtained from the SWISS-MODEL repository. As expected from their multiplesequence alignment, the M10EXG ADH-I was almost identical to that of ADH-hT G. stearothermophilus strain LLD-R (Fig. 5). The model clearly shows that all cofactor binding sites for ADH-I including NAD, Zn-catalytic, and Zn-structural binding sites are present at the same loci as in ADH-hT. Interestingly, the salt bridge forming amino acids (E7, K14, and E16) proposed to enhance thermostability (Ceccarelli et al., 2004), is conserved in the ADH-I protein structure as also occurs in the ADH-hT structure. 4. Discussion The potential thermophilic ethanologen isolated from compost waste, a strain (M10EXG) of G. thermoglucosidasius was further characterised for the development of a next generation recombinant ethanologen. Its robust characteristics as a potential thermophilic ethanologen including high ethanol tolerance (10%, v/v) and a broad substrate range including hexose and pentose sugars (Fong et al., 2006) have driven this research. The economics of lignocellulosic-based ethanol production is significantly favored with a simultaneous saccharification and fermentation process at high temperature (60 ◦ C) due to energy saving cost from high-energy input for the post-ethanol fermentation. This Gramnegative facultative anaerobic bacterium utilised both glucose and xylose in a fully defined minimal media and produced ethanol, acetate and lactate at 60 ◦ C (Fig. 1). While some thermophilic anaerobic bacteria are able to naturally co-ferment pentoses and hexoses to ethanol including Thermoanaerobacter ethanolicus, such bacteria have low ethanol tolerance. Therefore, our research effort has been towards the isolation and/or development of thermophilic strains with high ethanol tolerance and production yield. To look for the molecular-based signatures for homoethanol production pathway in this bacterium, we tried to identify the gene coding for pyruvate decarboxylase. However, in this bacterium there were no indications of presence of the gene using degenerate primers designed based on the 4 known mesophilic pdc genes (data not shown). To date prokaryotic pdc genes have only been reported in 4 mesophilic bacteria including Zymomonas mobilis (Ingram et al., 1999), Sarcina ventriculi (Talarico et al., 2001), Acetobacter pasteurianus (Raj et al., 2001), Zymobacter palmae (Raj et al., 2002) and there are no reports on the isolation of pdc from thermophilic microorganisms. Unlike pdc, adh is widely distributed in many microorganisms, including M10EXG. This gene (adhI) encoding thermostable alcohol dehydrogenase (ADH-I) in M10EXG has been cloned and heterologously

expressed via its native promoter in recombinant E. coli. The heterologously expressed protein has shown specific ADH activity at 60 ◦ C. Reactivity with both 1-butanol and 2-propanol suggests that ADH-I may play a dual role as a primary and secondary alcohol dehydrogenase in M10EXG. Hence, this ADH-I may also be a good candidate for butanol production, which has been recently spotlighted as a second-generation biofuel. Conserved amino acid residues at significant sites, particularly the ligands for both catalytic and structural zinc atoms, compared with other thermostable ADHs (Sakoda and Imanaka, 1992; Burdette et al., 1996, 1997) suggest that ADH-I is likely to be a zinc-binding alcohol dehydrogenase. The 1588-bp full-length functional adhI with a putative promotor including 87 bp upstream from the start codon from M10EXG was cloned into pGEM® -T Easy in both orientations (pNF303 and pNF304) and expressed in recombinant E. coli DH5␣ (Table 1). Clones containing pNF303, in which the orientation of adhI is the same as the lac promoter from the vector (the distance between lac promotor from the start codon; 243 bp), produced nearly 20-fold higher specific ADH activity with ethanol compared to DH5␣ (pNF304). This is most likely due to higher expression of adhI in E. coli (pNF303) from both the adhI native promoter and the vector lac promoter. Evidence for this is further supported by a significant decrease (approximately 7-fold) in specific ADH activity in E. coli (pNF303) cultured in the medium containing glucose, a lac repressor. Similar observations have been reported for the heterologous expression of adhB from Z. mobilis in E. coli under the control of same lac promoter (Conway et al., 1987). The expression of adhI in E. coli (pNF304) indicated that the regulatory elements from the thermophilic M10EXG were functional in the mesophilic E. coli host. This might be the reason for the lower reported expression of the recombinant adhI (0.5 U/mg) with its native promoter in E. coli compared to adhA (3.3 U/mg) from Thermoanaerobacter ethanolicus strain JW200, which has an E. coli lac-like promoter (Helmann, 1995). Metabolic engineering for enhanced ethanol production has mostly focused on mesophilic microorganisms. The isolation of G. thermoglucosidasius M10EXG, a Gram-negative facultative anaerobic thermophile that utilises a wide range of pentose and hexose sugars and produce ethanol, as well as acetic and lactic acids, is part of a current strategy to evaluate ethanol production using thermophilic bacteria and/or their component enzymes. The isolation, heterologous over-expression and partial characterisation of a thermostable ADH-I from G. thermoglucosidasius M10EXG provide a basis for the construction of a complete thermophilic ethanol production operon. Acknowledgements This work was supported in part by the International Postgraduate Research Scholarship (IPRS) scheme funded by the Australia

Y.J. Jeon et al. / Journal of Biotechnology 135 (2008) 127–133

Department of Education, Science and Training (DEST), and the Australian Research Council. References Ammendola, S., Raia, C.A., Caruso, C., Camardella, L., D’Auria, S., De Rosa, M., Rossi, M., 1992. Thermostable NAD+ -dependent alcohol dehydrogenase from Sulfolobus solfataricus: gene and protein sequence determination and relationship to other alcohol dehydrogenase. Biochemistry 31, 12514–12523. Banat, I.M., Marchant, R., 1995. Characterization and potential industrial applications of five novel, thermotolerant, fermentative, yeast strains. World J. Microbiol. Biotechnol. 11, 304–306. Banat, I.M., Nigam, P., Singh, D., Marchant, R., McHale, A.P., 1998. Review: ethanol production at elevated temperatures and alcohol concentrations. Part I. Yeasts in general. World J. Microbiol. Biotechnol. 14, 809–821. Bradshaw, C.W., Fu, H., Shen, G.J., Wong, C.H., 1992a. A Pseudomonas sp. alcohol dehydrogenase with broad substrate specificity and unusual stereospecificity for organic synthesis. J. Org. Chem. 57, 1526–1532. Bradshaw, C.W., Humme, W., Wong, C.H., 1992b. Lactobacillus kefir alcohol dehydrogenase: a useful catalyst for synthesis. J. Org. Chem. 57, 1533–1536. Bryant, F.O., Wiegel, J., Ljungdahl, L.G., 1988. Purification and properties of primary and secondary alcohol dehydrogenases from Thermoanaerobacter ethanolicus. Appl. Environ. Microbiol. 54, 460–465. Burdette, D.S., Secundo, F., Phillips, R.S., Dong, J., Scott, R.A., Zeikus, J.G., 1997. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity. Biochem. J. 326, 717–724. Burdette, D.S., Vieille, C., Zeikus, J.G., 1996. Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme. Biochem. J. 316, 115–122. Cannio, R., Rossi, M., Bartolucci, S., 1994. A few acid substitutions are responsible for the higher thermostability of a novel NAD+ -dependent bacillar alcohol dehydrogenase Eur. J. Biochem. 222, 345–352. Ceccarelli, C., Liang, Z.X., Strickler, M., Prehna, G., Goldstein, B.M., Klinman, J.P., Bahnson, B.J., 2004. Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding. Biochemistry 43 (18), 5266–5277. Conway, T., Sewell, G.W., Osman, Y.A., Ingram, L.O., 1987. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J. Bacteriol. 169, 2591–2597. Coolbear, T., Daniel, R.M., Morgan, H.W., 1992. The enzyme from extreme thermophiles: bacterial sources, thermostabilities and industrial relevance. Adv. Biochem. Eng. Biotechnol. 45, 57–98. Edwards, C., 1990. Thermophiles. In: Edwards, C. (Ed.), Microbiology of Extreme Environments. McGraw-Hill Publishing Company, New York, pp. 1–32. Fong, J.C.N., Svenson, C.J., Nakasugi, K., Leong, C.T.C., Bowman, J.P., Chen, B., Glenn, D.R., Neilan, B.A., Rogers, P.L., 2006. Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost. Extremophiles 10, 363–372. Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723. Haki, G.D., Rakshit, S.K., 2003. Developments in industrially important thermostable enzymes: a review. Bioresour. Technol. 89, 17–34. Helmann, J.D., 1995. Compilation and analysis of Bacillus subtilis sA -dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23, 2351–2360. Ingram, L.O., Aldrich, H.C., Borges, A.C., Causey, T.B., Martinez, A., Morales, F., Saleh, A., Underwood, S.A., Yomano, L.P., York, S.W., Zaldivar, J., Zhou, S., 1999. Enteric bacterial catalysts for fuel ethanol production. Biotechnol. Prog. 15, 855–866. Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., Chu, L., Mazur, M., Goltsman,

133

E., Larsen, N., D’Souza, M., Walunas, T., Grechkin, Y., Pusch, G., Haselkorn, R., Fonstein, M., Ehrlich, S.D., Overbeek, R., Kyrpides, N., 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423, 87–91. Jeon, Y.J., Svenson, C.J., Rogers, P.L., 2005. Over-expression of xylulokinase in a xylosemetabolising recombinant strain of Zymomonas mobilis. FEMS Microbiol. Lett. 244, 85–92. Keshav, K.F., Yomano, L.P., An, H.J., Ingram, L.O., 1990. Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli. J. Bacteriol. 172, 2491–2497. Klapatch, T.R., Hogsett, D.A.L., Baskaran, S., Pal, S., Lynd, L.R., 1994. Organism development and characterization for ethanol production using thermophilic bacteria. Appl. Biochem. Biotechnol. 45–46, 209–223. Lamed, R., Zeikus, J.G., 1980. Ethanol production by thermophilic bacteria: relationship between fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and Thermoanaerobium brockii. J. Bacteriol. 144, 569–578. Lamed, R.J., Zeikus, J.G., 1981. Novel NADP-linked alcohol–aldehyde–ketone oxidoreductase in thermophilic ethanologenic bacteria. Biochem. J. 195, 183–190. Lowe, S.E., Jain, M.K., Zeikus, J.G., 1993. Biology, ecology, and biotechnological applications of anaerobic bacteria adapted to environmental stresses in temperature, pH, salinity, or substrates. Microbiol. Rev. 57, 451–509. Neilan, B.A., Burns, B.P., Relman, D.A., Lowe, D.R., 2002. Molecular identification of cyanobacteria associated with stromatolites from distinct geographical locations. Astrobiology 2 (3), 271–280. Ochman, H., Gerber, A.S., Hartl, D.L., 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120 (3), 621–623. Raj, K.C., Ingram, L.O., Maupin-Furlow, J.A., 2001. Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus. Arch. Microbiol. 176, 443–451. Raj, K.C., Talarico, L.A., Ingram, L.O., Maupin-Furlow, J.A., 2002. Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase gene (pdc) and comparison to bacterial homologues. Appl. Environ. Microbiol. 68 (6), 2869–2876. Read, T.D., Peterson, S.N., Tourasse, N., 2003. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423, 81–86. Rogers, P.L., Jeon, Y.J., Lee, K.J., Lawford, H.G., 2007. Zymomonas mobilis for fuel ethanol and higher value products. Adv. Biochem. Eng. Biotechnol. 108, 263–288. Sakoda, H., Imanaka, T., 1992. Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH. J. Bacteriol. 174, 1397–1402. Sambrook, J., Fritsch, E., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., 2003. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385. Stephanopoulos, G., 2007. Challenges in engineering microbes for biofuels production. Science 315, 801–804. Suye, S.I., Kamiya, K., Kawamoto, T., Tanaka, A., 2002. Efficient repeated use of alcohol dehydrogenase with NAD+ regeneration in an aqueous–organic two-phase system. Biocatal. Biotransform. 20, 23–28. Talarico, L.A., Ingram, L.O., Maupin-Furlow, J.A., 2001. Production of the gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli. Microbiology 147, 2425–2435. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Yasueda, H., Kawahara, Y., Sugimoto, S., 1999. Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression. J. Bacteriol. 181, 7154– 7160.