Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200

Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200

FEMS Microbiology Letters 190 (2000) 57^62 www.fems-microbiology.org Cloning, sequencing and expression in Escherichia coli of the primary alcohol d...

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FEMS Microbiology Letters 190 (2000) 57^62

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Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200 Peter J. Holt *, Richard E. Williams, Keith N. Jordan, Christopher R. Lowe, Neil C. Bruce Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK Received 28 April 2000; received in revised form 27 June 2000; accepted 28 June 2000

Abstract The structural gene, adhA, for a thermostable primary alcohol dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. Constitutive expression from its own promoter was observed in Escherichia coli. The nucleotide sequence of adhA corresponded to an open reading frame of 1197 bp, encoding a polypeptide of 399 amino acids with a calculated Mr of 43 192. Amino acid sequence analysis showed 67^69% identity with alcohol dehydrogenases from two archaeal species and 29^37% identity with bacterial type III alcohol dehydrogenases. This represents the first reported cloning of an alcohol dehydrogenase from a bacterial species that is both thermostable and active against primary long-chain alcohols. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Type III alcohol dehydrogenase ; Thermostable alcohol dehydrogenase; Thermoanaerobacter ethanolicus

1. Introduction Two strains of the species Thermoanaerobacter ethanolicus are known, the type strain T. ethanolicus JW200 and T. ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E), both isolated originally from thermal springs in Yellowstone National Park, USA [1,2]. Work on these extremely thermophilic, non-spore-forming, chemoorganotrophic anaerobic strains has been largely concerned with the potential to ferment a variety of biopolymers, and to produce ethanol, under harsh conditions. Consequently, two alcohol dehydrogenases have been identi¢ed in T. ethanolicus, one active against primary long-chain alcohols (1³ Adh) and one active against secondary alcohols (2³ Adh) [3,4]. Each of these enzymes has been puri¢ed and characterised to some extent, although prior to this work, only the 2³ Adh had been cloned and studied further [5^7]. In contrast, the T. ethanolicus 1³ Adh has received no reported attention since its initial puri¢cation and characterisation, which showed the protein to be a zinc-contain-

* Corresponding author. Tel. : +44 (1223) 334160; Fax: +44 (1223) 334162 E-mail : [email protected]

ing, NADP(H)-dependent homotetramer, with an approximate subunit Mr of 44 500 [3,4]. Other than possible uses in industrial biotransformations, an NADP(H)-dependent alcohol dehydrogenase active against long-chain alcohols has the potential to couple NADPH-dependent enzymes to bacterial luciferase in the detection of analytes such as arsenate [8] and mercuric ions [9]. 2. Materials and methods 2.1. Organisms and growth media Type strain T. ethanolicus was obtained from the German Culture Collection (DSM 2246; ATCC31550). T. ethanolicus DSM 2246 was cultured using a 0.8% (w/v) glucose medium, as recommended by the DSM and ATCC. Starter cultures were grown in 500 ml sealed anaerobic bottles at 55³C for 72 h. 50 l of medium were inoculated with 1.5 l of starter culture and grown anaerobically at 60³C for 24 h to an estimated optimum 1³ Adh level [3^5]. Cells were harvested by centrifugation and stored at 380³C until required. For 16S rDNA analysis, puri¢ed genomic DNA was prepared from a culture of T.

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 1 9 - 0

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ethanolicus DSM 2246 by a standard method [10] and used as a template for polymerase chain reaction (PCR) with the primers fD1 and rD1 [11]. The PCR product was T/A cloned into pCR0 2.1-TOPO0 (Invitrogen) and sequenced on both strands. Escherichia coli strains BL21(DE3), DH5K and JM109 from laboratory stocks were made competent by a standard method and transformed by electroporation [10]. All recombinant E. coli strains were grown on LB medium with 100 Wg ml31 carbenicillin in a shaking incubator at 37³C and 220 rpm. Cells were grown to late exponential phase when no induction was used, or until 2 h after the addition of isopropylthiogalactoside at mid-exponential phase growth. Cells were harvested by centrifugation and stored at 380³C until required. 2.2. Protein puri¢cation Frozen cells were thawed at 4³C and resuspended to 0.25 g ml31 in 20 mM 3-(N-morpholino)propanesulfonic acid pH 6.5 with 0.1 mM ZnSO4 and 0.2 mM L-mercaptoethanol (bu¡er A). Resuspended cells were disrupted by ultrasound and clari¢ed by centrifugation. This cell extract was heat-treated (70³C for 50 min) and clari¢ed by centrifugation. The enzyme was further puri¢ed using octyl agarose column chromatography (loaded in 1.0 M ammonium sulfate in bu¡er A, eluted with a gradient from 1.0 to 0 M ammonium sulfate in bu¡er A), and Resource1-Q (Amersham Pharmacia Biotech Ltd.) anion exchange column chromatography (loaded in bu¡er A, eluted with a gradient from 0^0.1 M potassium chloride in bu¡er A). Activity from the ¢rst column stage was dialysed overnight against bu¡er A and concentrated to approximately 20 ml using a Dia£o0 Ultra¢lter device with a YM30 30kDa cut-o¡ membrane, prior to the second column stage. Activity eluted from the second column stage was pooled and dialysed overnight against bu¡er A, dialysate concentrated to 10 ml and stored in 1 ml aliquots at 380³C. All column chromatographic and ultra¢ltration procedures were carried out at 4³C. 1³ Adh puri¢ed from T. ethanolicus was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS^PAGE) and electroblotted onto a PVDF membrane. Partial digestion of the enzyme by trypsin was used to obtain a fragment with an approximate Mr of 26 000 on SDS^PAGE, which was also electroblotted onto a PVDF membrane. The N-terminal amino acid sequence of the denatured polypeptide and of the tryptic fragment were determined to be Met-Try-Glu-Thr-Ile-Asn-Pro-Asn-LysVal-Phe-Glu-Leu-Arg and Asn-Tyr-Lys-Pro-Ala-Ile-AsnTyr-Asp, respectively. 2.3. Enzyme assays 1³ Adh activity was determined spectrophotometrically against the optimum substrate pentanol (2 M) [3,4], by

monitoring the optical density change at 340 nm due to the reduction of NADP‡ in Tris^HCl (100 mM; pH 8.9) with ZnSO4 (0.1 mM). Protein concentrations were measured using Pierce Coomassie1 Protein Reagent according to the manufacturer's instructions. 2.4. Oligonucleotides, sequencing and sequence analysis Custom oligonucleotides were obtained from SigmaGenosys. All DNA and protein sequencing was done by the Protein and Nucleic Acid Sequencing facility, Department of Biochemistry, University of Cambridge, UK. Nucleotide and protein database searching was done using BLAST [12]. 2.5. Cloning and overexpression Genomic DNA from T. ethanolicus was digested with a range of restriction enzymes, subjected to agarose gel (1%) electrophoresis and Southern blotted. The Southern blots were then probed with a labelled 510-bp oligonucleotide corresponding to the N-terminal section of adhA. This 510-bp oligonucleotide sequence was obtained by PCR, with oligonucleotide primers designed from the AdhA partial amino acid sequences determined in this work (Section 2.2); the forward primer 5P-ATG TGG GAR ACN AAR ATH and the reverse primer 5P-AAT TCR TCR TAN GCD ATN GCN GG. T. ethanolicus genomic DNA was used as template. The PCR product was labelled with an enhanced chemiluminescence random prime labelling kit and then used to analyse the Southern blots. The adhA gene was cloned into pGEM7Zf‡ on both a 1.8-kb NsiI^EcoRI fragment and a 6.5-kb EcoRI fragment, to give pNEadh and pEadh, respectively. PCR was used to introduce an NdeI restriction site at the translation start codon and a SalI restriction site downstream of adhA, the PCR product was T/A cloned into pCR0 2.1TOPO0 , subcloned into pT7-7 using the NdeI and SalI restriction sites, and the construct (pT7-7adh) transformed into E. coli strain BL21(DE3). 2.6. Promoter analysis Fortuitously, adhA was cloned on DNA fragments with extensive upstream sequence, enabling some analysis of the promoter sequences to be performed. PCR was used to obtain adhA with 374 bp of DNA immediately upstream of the translation start codon, to introduce an NdeI restriction site at 3374 bp from the start codon and to create a SalI restriction site downstream of the gene. The PCR product was T/A cloned into pCR0 2.1TOPO0 and then subcloned into pT7-7 using the NdeI and SalI restriction sites, creating pT7-7adhp. The pT77adhp and pT7-7adh constructs were transformed into E. coli strains DH5K and JM109. Heat-treated crude cell extracts were prepared from cultures of each strain and

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Table 1 Comparison of Thermoanaerobacter ethanolicus adhA with related enzymes Enzyme

Identity with T. ethanolicus AdhA (%)

Residues per monomer

Monomers per native enzyme

Optimum substrate

Cofactor

Metal (per subunit)

Acc. No./ Reference

(1) (2) (3) (4) (5) (6)

100 67 32 30 30 30

399 406 383 390 465 383

4 4 4 2 x x

NADP‡ NADP‡ NAD‡ NAD‡ 6 NADP‡ NAD‡ NAD‡

4 Zna x 1 Zn Zn Zn Fe

AF178965 AAB63011 P06758 Q04945 P10127 P11549

(7) Clostridium acetobutylicum Adh1 (8) Clostridium acetobutylicum AdhA (9) Bacillus methanolicus Mdh (10) Thermococcus hydrothermalis Adh (11) Pyrococcus furiosus Adh (i) (12) Escherichia coli Adh2 (13) Schizosaccharomyces pombe Adh (14) Citrobacter freundii Pdh (15) Klebsiella pneumoniae Pdh

29 29 29 69 37 31 31 30 30

388 389 381 406 390 382 422 387 387

x 2 10 x x x x 8 (8)

NADP‡ NAD‡ 6 NADP‡ NAD‡ x (NADP‡ ) x x NAD‡ (NAD‡ )

x Zn 1 Zn+2 Mg x (Fe) (Fe) x Fe (Fe)

P13604 Q04944 P31005 CAA74334 AAC25557 P37686 Q09669 P45513 Q59477

(16) (17) (18) (19)

N N N N

N N N N

4 4 6 4

NADP‡ NADP‡ gNAD‡ NADP‡ gNAD‡ NAD‡

None 1 Fe 1 Zn+1 Fe 1 Zn

AAB30263 AAB35052 [36] [37]

22 21

352 352

4 4

pentan-1-ol butan-1-olR ethanolR butan-1-olR x (1,3propanediol) butan-1-olR butan-1-olR methanol x (methanol) x x 1,3-propanediol (1,3propanediol) hexan-1-ol hexan-1-ol pentan-1-ol C3^C10 1³ alcohols propan-2-ol butan-2-ol

NADP‡ NADP‡

1 Zna 1 Zn

S71131 P14941

Thermoanaerobacter ethanolicus AdhA Thermococcus AN1/zilligii Adh Zymomonas mobilis Adh2 Clostridium acetobutylicum AdhB Saccharomyces cerevisiae Adh4 Escherichia coli fuco

Thermococcus litoralis Adh Thermococcus strain ES-1 Adh Pyrococcus furiosus Adh (ii) Pseudomonas putida K23-1 Adh

(20) Thermoanaerobacter ethanolicus 2³Adh (21) Thermoanaerobacter brockii 2³Adh

Thermoanaerobacter ethanolicus adhA is described [1] in comparison with: Thermococcus AN1/zilligii Adh [2] and the seven other members of the type III family described by Li and Stephenson [3^9]; six further enzymes for which complete DNA sequences have been reported and which contain the type III family strictly conserved residues and motifs [10^15]; four enzymes described with N-terminal amino acid sequence and protein data only, but which exhibit characteristics of the type III enzymes [16^19] and the two 2³ Adhs described from Thermoanaerobacter spp. [20,21]. Adh, alcohol dehydrogenase; Mdh, methanol dehydrogenase ; Pdh, propanediol dehydrogenase; N, only N-terminal amino acid sequence reported; x, not reported ; xR , restricted range of substrates tested, optimum substrate may be di¡erent ; (xxx) conceptual data only; Zna , both T. ethanolicus 1³ Adh/adhA and 2³ Adh were initially reported as having possibly four Zn per subunit, although cloning and further analysis of the 2³ Adh revealed just one Zn per subunit, the metal-ion composition of the 1³ Adh has not been con¢rmed; Acc. No., the accession numbers given are those employed by Entrez at the NCBI for protein search and identi¢cation purposes. Where an accession number is not available, known references are given.

AdhA speci¢c activity determined. The 374-bp sequence of DNA immediately upstream of adhA with the ¢rst 49 bp of the gene was obtained using PCR. This PCR product was T/A cloned into pCR0 2.1-TOPO0 and subcloned into the pQF50-derived [13] promoter probe vector pQF52, which contains lacZ as a reporter gene, using BamHI and XbaI restriction sites, to give pQF52prom. Three cultures of E. coli JM109 with pQF52prom were grown to an OD600 of V0.7. Promoter strength was analysed by measuring L-galactosidase activity against o-nitrophenyl-L-Dgalactopyranoside (ONPG) by the method of Miller [14]. Additionally, three cultures were grown to an OD600 of V0.7, from which cell extracts were prepared and L-galactosidase activity against ONPG measured spectrophotometrically at 420 nm. 3. Results 3.1. T. ethanolicus culture and enzyme puri¢cation T. ethanolicus has been reported to exist in co-culture

with Clostridium species [15,16] known to contain alcohol dehydrogenases related to the 1³ Adh of T. ethanolicus [16,17]. 16S rDNA analysis con¢rmed that the culture was T. ethanolicus. Zn2‡ was found to be an essential requirement of the 1³ Adh in all solutions, as enzyme activity was lost rapidly in the absence of Zn2‡ , and only partially recoverable by dialysis into Zn2‡ -containing bu¡er. Enzyme preparations were puri¢ed to apparent homogeneity as judged by SDS^ PAGE analysis, with a yield of 0.17 mg g31 wet weight of T. ethanolicus cells at a speci¢c activity of 51 U mg31 . 3.2. Cloning and overexpression Two genomic DNA fragments containing the complete adhA gene were cloned, a 6.5-kb EcoRI fragment and a 1.8-kb NsiI^EcoRI fragment, identical to the 3P end of the EcoRI fragment. An 1197-bp open reading frame was identi¢ed within the NsiI^EcoRI fragment, located 198 bp downstream of the 3P NsiI restriction site, extending from a GTG initiation codon (+1 bp) to a TAA stop codon (1198 bp). Downstream of the stop codon were

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inverted repeats typical of intrinsic transcription termination sequences, at bp 1201^1217 and bp 1287^1308. The open reading frame encoded a 399-amino acid polypeptide with a calculated Mr of 43 192. The molar GC content of adhA was 42%, higher than the 37^39% GC content typical of a Thermoanaerobacter genome [18]. Deduced amino acids 1^13 concur with the N-terminal amino acid sequence of the mature protein determined previously, suggesting that the initial GTG-encoded methionine is not processed. The N-terminal amino acid sequence of the tryptic fragment, Asn-Tyr-Lys-Pro-Ala-Ile-Asn-Tyr-Asp, corresponds to deduced amino acids 171^179 in a fulllength polypeptide sub-unit. Puri¢ed AdhA was noted as having an apparent subunit Mr of 44 000 as judged by SDS^PAGE, directly equivalent to the wild-type enzyme. Speci¢c activity of the recombinant enzyme was highest with pentanol as substrate and equivalent to 100% of the speci¢c activity of the wild-type enzyme. With the speci¢c activity against pentanol taken as 100%, speci¢c activities against ethanol (73%), propanol (75%) and hexanol (70%) were also directly comparable with the wild-type enzyme. Comparable thermostabilities were also observed, with heating at 70³C for 180 min giving no observable loss of speci¢c activity in either the wild-type or the recombinant enzyme. 3.3. Sequence analysis AdhA had 67% amino acid sequence identity with a thermostable 1³ Adh from the archaeal species Thermococcus zilligii (formerly Thermococcus strain AN1) [19,20], which is similar to an enzyme reported from Thermococcus litoralis [21], 69% identity to another thermococcal enzyme [22], 37% identity to a pyrococcal enzyme [23] and 29^32% identical to a number of non-thermostable Adhs from mesophilic bacterial species [19]. All the enzymes most similar to AdhA at sequence level fall into category 1, group III of Reid and Fewson [24], also called the type III Adh family [19]. A summary of enzymes most similar to AdhA is given in Table 1. Nucleotide sequence data have been submitted to GenBank, accession No. AF178965. 3.4. Promoter analysis Analysis of the 374 bp of sequence immediately upstream of adhA revealed a Shine^Dalgarno sequence compatible with E. coli, strong homology to an E. coli lac-like promoter and several putative BoxA^BoxB type archaeal promoters (Fig. 1) [18,25^27]. Constructs in pT7-7 were made both with and without this promoter region (pT77adhp and pT7-7adh, respectively) in order to test for possible promoter function in E. coli. The ¢rst 49 bp of the gene were included in the construct pT7-7adhp, as signi¢cant homology to a potential transcription-enhancing downstream box described for E. coli [28] was

Fig. 1. T. ethanolicus adhA promoter elements. The DNA sequence upstream of adhA, from the 3P end of a putative transposase-encoding open reading frame (TAA, bold), to the ¢rst 50 bp of adhA are shown. (a) E. coli-like sequences: a putative lac-like promoter (lac prom.) with 335, 310 sequences and putative translation start site, TSS (bold, underscored) ; putative lac repressor binding site (lac rep.; bold); Shine^ Dalgarno sequence (S-D; bold, underscored) ; 5P end of NsiI^EcoRI clone, marking the truncated promoter region (NsiI; bold); GTG transcription start (bold) and putative downstream box (DB; bold, underscored). (b) Archaeal BoxA^BoxB type promoter-like sequences: 1A and B (bold); 2A and B (bold, underscored); 3A and B (bold, italicised); Shine^Dalgarno sequence (S-D; bold, underscored); 5P end of NsiI^EcoRI clone, marking the truncated promoter region (NsiI; bold); and GTG transcription start (bold).

apparent in this sequence (Fig. 1). Expression from the promoterless construct (pT7-7adh) was minimal, AdhA activity in DH5K being 6 0.07 U (mg protein)31 and not detectable in JM109. However, with the promotercontaining construct (pT7-7adhp), AdhA levels in DH5K and JM109 were 3.3 and 3.8 U (mg protein)31 , respectively. With the promoter probe construct pQF52, the adhA promoter gave rise to L-galactosidase expression at a level of 210 ( þ 11) Miller units. L-Galactosidase activity measured in crude cell extracts from pQF52 cultures gave a level of 0.14 ( þ 0.02) U (mg protein)31 , where 1 U = 1 Wmol ONP produced per min from ONPG. These values indicate that the nucleotide sequence immediately upstream of adhA comprises a constitutive promoter in E. coli, as well as being presumably an active promoter sequence in T. ethanolicus.

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BNFL plc and the EPSRC. The authors gratefully acknowledge Alison Inskip for technical assistance and Dr. Susan Rosser, Dr. Deborah Rathbone and Ms. Sarah de Jager for comments and advice.

References Fig. 2. 12.5% SDS^PAGE gel showing puri¢cation of wild-type and recombinant T. ethanolicus ADH. Lane 1, crude cell-free extract of E. coli JM109 expressing recombinant T. ethanolicus ADH ; lane 2, puri¢ed recombinant ADH ; lane 3, crude cell-free extract of T. ethanolicus ; lane 4, molecular weight markers ; lane 5, puri¢ed T. ethanolicus (wild-type) ADH.

4. Discussion The 2³ Adh cloned from T. ethanolicus has been reported as di¡ering in only three residues from T. brockii 2³ Adh and as being 75% identical to a 2³ Adh from the Clostridium beijerinckii [6,7]. However, T. ethanolicus AdhA is more similar to alcohol dehydrogenases from other species than to T. ethanolicus 2³ Adh. Sequence analysis indicates that T. ethanolicus 1³ Adh is a member of the type III family [19], and is clearly much closer in identity to alcohol dehydrogenases from archaeal Thermococcus spp. (67^69%) than to the rest of the family (29^ 37%). A small number of Thermoanaerobacter promoter sequences have been identi¢ed [16,29^32], most by homology to known promoters in E. coli. Although this process may itself have in£uenced the observation, most of the known and putative sequences seem similar to the E. coli consensus promoter sequences [26,33]. This work shows that the adhA promoter from T. ethanolicus is capable of directing expression in E. coli, in both vlacI and lacIq strains. It is worth noting that with the truncated promoter present in pNEadh, expression was seen in only the vlacI strain DH5K and not at all in the lacIq strain JM109. Homology to an E. coli lac repressor binding site centred at 374, is present in both full-length and truncated promoters, whereas only the complete promoter sequence has homology to a strong E. coli lac promoter also, immediately upstream of the truncated sequence, between 3242 and 3206 (Fig. 2). Upstream of the 374-bp promoter region was the end of a putative open reading frame with 32% identity (50% similarity), at amino acid level, to the C-terminus of an IS3/IS904 family transposase. Interestingly, an IS element has been reported as in£uencing expression of an E. coli enzyme closely related to AdhA [34,35]. Acknowledgements This work was funded by a DTI LINK grant with

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