Cloning, nucleotide sequence, and expression of the Clostridium thermocellum cellodextrin phosphorylase gene and its application to synthesis of cellulase inhibitors

JOURNALOF FERMENTATION AND BIOENCINEERINC Vol. 85, No. 2, 144-149. 1998

Cloning, Nucleotide Sequence, and Expression of the Clostridium thermocehm Cellodextrin Phosphorylase Gene and Its Application to Synthesis of Cellulase Inhibitors TAKASHI

KAWAGUCHI, YASUO IKEUCHI, NORIKO TSUTSUMI, JUN-ICHI SUMITANI, AND MOT00 ARAI*

AKIHIKO

KAN,

Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 5994531, Japan Received 30 September 1997/Accepted

14 November 1997

The cellodextrin phosphorylase (CDP) gene of Clostridium thermocellum was cloned and sequenced. The nucleotide sequence of the insert of a positive clone contained an open reading frame of 2940 bp encoding a polypeptide of 980 amino acid residues with a calculated molecular mass of 111,182 daltons. Escherichia coli cells harboring the plasmid for expression produced CDP protein accounting for 30% of the total cellular proteins with an activity of 59.9 units/ml culture, which corresponded to 0.93 mg/ml culture. The expressed CDP could be used to synthesize cellulase inhibitors as cellooligosaccharide analogues using glucose-lphosphate as a glucose donor and 4-O-&o-ghtcopyranosyl-l-deoxynojirimycin or 6-O-&cellobiosyl-l-deoxynojirimycin as an acceptor. [Key words:

Clostridium thermocellum, cellodextrin phosphorylase,

cloning, expression,

cellulase inhibitor,

enzymatic synthesis]

analogues were enzymatically synthesized by the transglycosylation reaction using cellooligosaccharides as a glucose donor and 1-deoxynojirimycin (DNJ), a potent inhibitor against a-glucosidase, P-glucosidase and trehalase (8), as an acceptor (9). However, the efficiency of the synthesis was relatively low, and inhibitors as analogues of longer cellooligosaccharides could not be obtained. There have been a few molecular genetic studies on aglucan phosphorylases. However, the first I-glucan phosphorylase genes to be cloned and sequenced-the cellobiose phosphorylase (cepA) and cellodextrin phosphorylase (cepB) genes of Clostridium stercorariumhave recently been reported (10). Here, we describe the cloning and nucleotide sequence of the cdp gene from C. thermocellum and its expression in E. coli. We also demonstrate the possibility that the expressed CDP can be used for the synthesis of cellulase inhibitors.

Clostridium thermocellum, a thermophilic anaerobic bacterium, produces cellulose-degrading enzymes when grown on cellulose as a major carbon source. During growth, the concerted action of endoglucanases and exoglucanases on cellulose produces cellodextrins and cellobiose. In many cellulolytic microorganisms, the conversion of these products to glucose is usually considered to be catalyzed by ,9-glucosidase. However, it is known that C. thermocellum intracellularly produces cellobiose phosphorylase (CBP) (cellobiose : orthophosphate (Y-Dglucosyl-transferase, EC 2.4.1.20) and cellodextrin phosphorylase (CDP) (1,4-/3-D-oligoglucan : orthophosphate a-D-glucosyl-transferase, EC 2.4.1.49), which work on the conversion of cellooligosaccharides to fermentable sugars. CBP and CDP catalyze reversible phosphorolysis of cellobiose and cellodextrins, respectively. Phosphorolysis is energetically advantageous and might constitute the primary route of cellooligosaccharide utilization, particularly in anaerobic environments. We previously reported the purification and some properties of CBP (1) and CDP (2), and revealed the possibility that cellooligosaccharides were enzymatically synthesized from cellobiose and glucose-l-phosphate using the reverse reaction of CDP (2). Recently, many primary structures of cellulases have been elucidated on the basis of gene cloning (3). However, our understanding of cellulase-cellulose interaction, including the active site of cellulases and the mechanism of cellulose hydrolysis is still limited in comparison with the extensive information available on amylases. One reason why the study of cellulase is less advanced seems to be a lack of specific inhibitors of cellulase, only a few inhibitors reported. One of them, epoxycellooligosaccharide, was described as a modifying substrate analogue (4, 5), and another, thiooligosaccharide, as a nonhydrolyzable substrate analogue (6, 7). We also reported previously that cellulase inhibitors as substrate

MATERIALS

AND METHODS

Bacteria1 strains, plasmids, and media C. thermocellum ATCC 27405 was used as a DNA donor. E. coli DHSa (supE44, AlacU169($801acZAM15), hsdR17, recA 1, endA 1, gyrA96, thi-1 , relA 1) was used as a cloning host for genomic library construction. E. coli MV1184 (ara, A(lac-proAB), rpsL, thi, (A#801acZAM15), A(srl-recA)306:: TnlO(tet’), F’[traD36, proAB+, la@, lacZAMlS]) was used as a host for the propagation of Ml3 bacteriophage. The plasmid pUC18 or pUC118 was used for gene library construction and pUC118/119 for subcloning and sequencing studies. C. thermocellum was cultured anaerobically in K medium as described previously (2). E. coli strains were cultured and maintained appropriately in LB, 2~ YT, or M9 medium supplemented with ampicillin (100 pg/ml) if necessary. Chromogenic substrate X-gal (40pg/ml) and IPTG (10 pg/ml) were added to the medium to detect recombinant plasmids or Ml3 phage.

* Corresponding author. 144

VOL. 85, 1998

CELLODEXTRIN

Amino acid sequencing and preparation of synthetic oligonucleotide probe The N-terminal amino acid sequence of CDP purified according to the procedure previously reported (2) was identified by Edman degradation with a PSQ-1 protein sequencer system (Shimadzu). Purified CDP was modified by S-carboxymethylation according to the standard method and digested with TPCK-trypsin (Boehringer Mannheim). The fragments were separated by reversed phase HPLC and the seven independent peptide fragments obtained also were sequenced as described above. On the basis of the resultant amino acid sequence of a tryptic peptide, the following oligonucleotide was designed as a probe for hybridization experiments: 5’-GCIGATCCIAATATIGA TGGIGTITATTT-3’ (29 mer; I, inosine; encodes the peptide ADPNIDGVYF). The oligonucleotide was synthesized by Biologica Co. (Nagoya). DNA manipulations For general molecular biological techniques such as plasmid purification, enzyme digestion, E. coli transformation, propagation and purification of Ml3 bacteriophage, etc, the methods described in Molecular Cloning (11) or Current Protocols in Molecular Biology (12) were employed. Southern blot analysis and colony hybridization Southern blotting was principally done according to the method of Southern (13). DNA fragments after digestion of an appropriate restriction endonuclease were separated on an agarose gel. The fragments were then transferred and fixed onto a nylon membrane (Hybond N-t, Amersham) by the capillary method using 0.4 N NaOH as recommended by the supplier. Colony hybridization was principally done by the method of Grunstein and Hogness (14). A nylon membrane (Hybond Nf) on which cells were grown overnight at 37°C was treated with 0.5 N NaOH containing 1.5 M NaCl and then neutralized with 0.5 M Tris-HCl (pH7.2) containing 1.5 M NaCl and 1 mM EDTA. DNA was fixed on the membrane by treatment with 0.4N NaOH for 20min. To remove cell debris, the filter was washed several times in 3 x SSC/O.l% SDS at room temperature followed by one washing in the same buffer for 4-6 h at 65°C. The synthetic oligonucleotide probe was labeled by [T-~~P]ATP (> 185 TBq/mmol, Amersham) and T4 polynucleotide kinase. DNA fragments were labeled by [a-32P]dATP (> 110 TBq/mmol, Amersham) and a Random Primed DNA Labeling Kit (Boehringer Mannheim). Prehybridization was done at 37°C for 1 b and hybridization was done overnight at 42°C for the synthetic oligonucleotide probe or 65°C for the random-labeled probe. Genomic library construction Total DNA of C. thermocellum was purified by the method of Saito and Miura (15). The first library was constructed as follows. Chromosomal DNA, which was completely digested by HindIII, was electrophoresed on an agarose gel. Fragments around 1.5 kb long were recovered and purified by the method of Chen and Thomas, Jr. (16) or that of Wang and Rossman (17). The fragments were then ligated in the dephosphorylated Hind111 site of pUC18, and E. coli DHSa was transformed by the resultant recombinant plasmids. The second library was constructed as follows. Chromosomal DNA was completely digested by PstI and the fragments were separated on an agarose gel. Fragments of about 6.0 kb were ligated in the dephosphorylated PstI site of pUC119, and E. coli DH5a was transformed by the resultant plasmids.

PHOSPHORYLASE

GENE FROM C. THERMOCELLUM

145

DNA sequencing and analysis DNA fragments were subcloned in pUCl18 or 119 and a series of nested deletion mutants was generated according to the method of Henikoff (18). Ml3 recombinant phages were propagated after infection of M13K07 helper phage and single-stranded DNA was purified as described by Vieira and Messing (19). The sequencing reaction was done by the dideoxy chain termination method (20) using a T7Sequencing kit (Pharmacia). Nucleotide sequences were determined on both strands. Construction of expression vectors pDEH1 and The plasmid pDH7 (see Results and Fig. pTEH1 I), in which a 6.0-kb PstI fragment containing the cdp gene was inserted, was double-digested with SspI and Hind111 and the 2.0-kb fragment encoding the N-terminal half of the CDP protein was introduced between SmaI and Hind111 of pUC118 to yield pDSH1. The plasmid pDH7 was digested with Hind111 and the 1.5kb fragment was ligated with HindIII-digested pDSH1. The clone in which the fragment was inserted in the correct orientation was selected by restriction endonuclease digestion, giving the plasmid pDEH1. pDEH1 was double-digested with EcoRI and Hind111 and the 2.0-kb fragment was inserted between the EcoRI and Hind111 sites of pTrc99A (21) to yield pTE1. pDEH1 was digested with Hind111 and the 1.5-kb fragment was ligated with HindIII-digested pTE1, giving pTEH1. Accordingly, the expression vectors pDEH1 and pTEH1 should express the cdp gene under the control of lac and trc promoter, respectively. Preparation of cell-free extract E. coli cells cultured overnight in an appropriate medium were harvested by centrifugation (12,00Orpm, 1 min, 4°C) and resuspended in the 50 mM Tris-HCl (pH 7.5) containing 0.1% 2-mercaptoethanol. The cells were disrupted by sonication using an Ultrasonic Disruptor UD-201 (Tomy Seiko). The suspension was centrifuged (12,000 rpm, IOmin, 4°C) to save the supernatant as a crude enzyme solution. Enzymatic activity CDP activity was measured as described previously (2). One unit of activity was defined as the amount of the enzyme that liberated 1 ,umol of Pi per 15min. Electrophoresis of protein SDS-polyacrylamide gel electrophoresis was done by the method of Laemmli (22) using the molecular weight marker “Daiichi”.II (Daiichi Pure Chemicals) as a standard. Protein bands were detected by staining with Coomassie brilliant blue R-250. Synthesis and analysis of cellulase inhibitors The cell-free extract prepared from E. coli DHSa harboring pTEH1 was incubated at 50°C for 30min and centrifuged to remove precipitated material. The resultant supernatant was used as a crude enzyme solution for the synthesis of cellulase inhibitors. The acceptors for the enzyme reaction, 4-0-/%o-glucopyranosyl-l-deoxynojirimytin (Gl(pl-4)DNJ) and 6-O-,%cellobiosyl-l-deoxynojirimycin (G2@1-6)DNJ), were enzymatically synthesized and purified as described previously (9). The reaction was performed according to the method previously reported for cellooligosaccharide synthesis using CDP (2). The reaction mixture (0.25 ml) consisting of 20mM Gl(IJ14)DNJ (or G2(Pl-6)DNJ), 10 mM cu-D-glucose-lphosphate, 1 mM EDTA, 1OmM DTT, 50mM Tris-HCI (pH 7.5), and 10 units CDP was incubated at 50°C. The reaction was stopped by heating for 20min in boiling water and centrifuged to remove insoluble material. The

146

KAWAGUCHI

ET AL.

supernatant was put on a Dowex SOW column (H-form, 1.0 x 5.0 cm) and the column was washed with distilled water. The adsorbed fraction was eluted with 0.5 N NH40H, and the eluent was concentrated in vucuo. The concentrated solution was spotted onto a silica gel plate (Kiesel gel 60FZs4, Merck) and developed twice with the following solvent system: 1-butanol/ethanol/CHCls/25% NH,OH, 4 : 5 : 2 : 8. After spraying cont. H2S04 containing 1% vanillin followed by heating for several min at llO”C, the products were identified using GlQ314)DNJ to G6@1-4)DNJ and G2(pl-6)DNJ as standards; these compounds were prepared as previously reported (9) and used. RESULTS Screening of the first library Cloning of cdp gene by colony hybridization using the synthetic oligonucleotide probe yielded six positive clones. The clones all contained the same 1,5-kb inserted fragment. The nucleotide sequence of the inserted fragment was determined. In the amino acid sequence deduced from the nucleotide sequence, two of the amino acid sequences which had been determined by Edman degradation were found, indicating that the cloned fragment contained a part of the cdp gene. However, the fragment was obviously not long enough to contain the complete structural gene. Since brief restriction endonuclease mapping on the chromosome using the cloned fragment as a probe revealed that a 6.0-kb PstI fragment contained the entire coding region (data not shown), a second library was constructed as described in Materials and Methods. The second colony hybridization using the SphI-Hind111 fragment of the insert obtained by the first screening as a probe gave two positive clones, both of which had the same inserted fragment. The resultant plasmid was named pDH7, and the restriction sites of the inserted fragment were determined (Fig. 1). Nucleotide and deduced amino acid sequences The nucleotide sequence of the coding region and its adjacent regions was determined. The nucleotide and deduced amino acid sequences are shown in Fig. 2. The fragment contained a sole open reading frame (ORF) of 2940 bp that encoded a polypeptide of 980 amino acids with a calculated molecular mass of 111,182 Da. A putative Shine-Dalgarno sequence (SD) and possible promoter region including - 10 and -35 sequences were found upstream of the start codon of the ORF. The 3’flanking region contained a pair of inverted repeat sequences capable of forming potential stem-and-loop structure, a possible transcriptional terminator. The first 25 amino acids deduced from the nucleotide sequence of the ORF perfectly coincided with those of the purified enzyme identified by Edman degradation. Additionally, all of the seven internal amino acid sequences were found. These results provide strong proof that the ORF is the true cdp gene. Expression of cdp gene in E. coli Two kinds of expression vectors were constructed as described in Materials and Methods. In plasmid pDEH1, the cdp gene was transcribed under the control of lac promoter and the copy number of the plasmid was high, while in pTEH1 the gene was transcribed under the trc promoter and the copy number was low. E. coli DH5a was transformed by these plasmids. Each transformant was cultured overnight at 37°C with shaking in 2 x YT medium containing

J. FERMENT.BIOENG.,

I FIG. 1. Restriction map of the fragment inserted in pDH7. The white arrow indicates the position and orientation of the CC@gene. Bg, BgAI; Hd, HindIII; Ps, P&I; Sp, SphI; Ss, SspI.

ampicillin and the enzyme activity of the cell-free extract was measured. The activities of the transformants harboring pDEH1 and pTEH1 were 13.5 and 41.3 units/ml medium, respectively. When IPTG was added, the transformant (pDEH1) produced 21.6 units/ml, 1.8-fold higher than that without IPTG. In contrast, with the transformant (pTEH1) the addition of IPTG led to a loss of activity. No activity was detected in either the culture supernatant or the insoluble fraction after cell disruption in the case of both plasmids with/without IPTG. As E. coli often produces an exogenous protein intracellularly as an insoluble inclusion body or an inactive soluble protein because of overexpression stress, whole cellular protein of the transformant (pTEH1) was examined on SDS-PAGE. As shown in Fig. 3A, no protein band at the position corresponding to CDP was observed in either the intracellular insoluble fraction or the soluble fraction of the transformant with IPTG induction. A small amount of the CDP was found to be expressed in the insoluble fraction of the transformant without IPTG induction (Fig. 3B), though the protein produced should be inactive. The CDP protein band was still observed in the insoluble fraction, and higher productivity was not obtained even when the cells were grown at a lower temperature of 25 or 30°C in order to reduce the over-expression stress. To improve the CDP productivity in E. coli, pTEH1 was introduced into several kinds of host strains and the activities of the transformants in the cellular soluble fraction were measured. Among the hosts tested, strain JM103 gave the highest activity (59.9 units/ml). Synthesis of cellulase inhibitors Aliquots (50 111) were taken out of the reaction mixture at intervals of 2 h, and the products of each sample were analyzed by thin-layer chromatography (TLC). As shown in Fig. 4, using Gl(Pl-4)DNJ as an acceptor, at least five spots apart from the substrate were detected and identified as G2Q314)DNJ to G6(Pl-4)DNJ judging from their Rf values. When Gl(fil-6)DNJ was used as an acceptor, the same results were obtained (data not shown), indicating that CDP can recognize Gl(pl-6)DNJ as well as Gl@l-4)DNJ as an acceptor. DISCUSSION A DNA fragment containing the cdp gene was cloned and sequenced. The cdp gene consisted of 2940 bp encoding a 980-amino acid protein with a calculated molecular mass of 111,182 Da, which is almost identical to the value of the native CDP monomer determined on SDSPAGE (2). The coding region was accompanied by putative promoter and SD sequences in the 5’-flanking region and a stem-and-loop structure in the 3’-flanking region. The SD sequence was perfectly identical to that of the C. thermocellum celS gene (23). The predicted amino acid sequence was compared with those of the C. ster-

VOL. 85, 1998

CELLODEXTRIN

PHOSPHORYLASE

GENE FROM C. THERMOCELLUM

147

AATGACCGTTGTTTTATTGCAAAGTTGTTGTAG~TGA~TTGATTTCAC~TTAGTTTATAG~C~~~C~GTTT~~~T~TTGAC~TTGCAGGGATTG~T~TGTAT~G

120

GAACCAAAAATCGCACTCAATTATCTTATATTGACRAGTRCT

240

I****** =z= SD ATGATTACTAAAGTAACAGCGAGAAATAATAAGATAACACCTGTTGAGTTGTTG~TC~GTTT~~C~GATT~TCTG~C~TTTTGCGGATCGTGTTTTTACTGACGCGGCG MITKVTARNNKITPVELLNQKFGNKINLGNFADRVFTDAA =z-=

360 40

TTCAAAAATGTGGCAGGCATTGCARATTTTGCCTATG~GCGCCGGT~TGCAGGTTCTTATffi~CTGCATTGTTTC~TATCTG~CAGTTTGTACCTGACC~TCTGTTTGT FKNVAGIANLPMKAPVMQ VLMENCIVSKYLKQ FVPDRSVC

480 80

TTTGTTGAAGAAGGACAGAAATTTTACATAGTACTTGAAG FVEEGQKFYIVLEDGQKI

600 120

TTGAAGTGCCTGAGGATGTAAACAAGGCTCTCAGGGCTACGGT~GTGATGT~GCATTGGGCTGGT EVPEDVNKALRATVSDVKHWAG

TATTTGACGGRAGACGGGGAGCATGTAATCGCCCTTTT~CC~CTCC~TCC~ATTTTTATGTG~TTTGCTTATAGG~CAGGCTTGGTTTT~GGACATTGCAGAC~CT YLTEDGEHVIALLKPAPGPHFYVNLLIGNRLGFKRTLQTT

720 160

CCGAAAAGTGTGGTTGACAGGTTCGGAAOAGGTTCGTTCCGTTCCCATGCTGC~CCCAGGTGCTGGC~CGAGATTTGACATGCGCCAGGAGG~CGGTTTTCCTGCG~CAGACAG PKSVVDRFGRGSFRSHAATQVLATRFDMRQE ENGFPANRQ

-

TTCTATTTGTATGAAGACGGCAAACAGATTTTTTATTCCGCATT~TTGATGAC~CATTGTTGAGGCTACCTGC~CATTCATGC~TCGTACGGT~T~TAT~GACGGCATGT FYLYEDGKQ IFYSALIDDNIVEATCKHSCNRTVI KYKTAC

840 200 960 240

AATCTGGAAATTACAAGAACCATCTTCCTGGTGCCTCACATTTGTCCATT NLEITRTIFLVPHKKGFPLATELQRIEIKNASDKARNLSI

1080 280

ACATATACGGGAATGTTTGGAACGGGTGCCGTTCATGCGATATTTGAGGACGT~CATACAC~TGTTATCATGC~GTGCCGCCCTTTAC~TGAC~GGGTGAGTTTATCGG~TA TYTGMFGTGAVHAIFEDVTYTNVIMQSAALYNDKGEFIGI

1200 320

ACTCCTGATTATTATCCTGAAGAATTTAAACAGGATRCA~ATAC~GATTTGTCACGATGATTGTCCGC~CGGGGACGAG~TCATTCCCGCAGAGTTTCTGCACGGACTAC~CGACTTTGTA TPDYYPEEFKQDTRFVTMIVRNGDEKSFPQS FCTDYNDFV

1320 360

GGCACAGGAACATTGGAGCATCCGGCAGCGGATGTAATGT~TTG~C~C~GCTG~CCGC~~TCCGGGATTCTTTGCCCTG~TGCGCCGTTTACGTTG~CCGGGC~GACAGTCATA GTGTLEHPAADVIEQQAEPQRSGILCPGCAVYVEPGKTVI

1440 400

ATAGACACTTTCACCGGTTTGTCTTCGAGCARGGATAATGTTTG IDTFTGLSSSKDNENYSDAVMLRELDNLLRYFEKSESVEE

1560 440

AAARAAGCGAATCTGTGGAAGAA

ACATTGAATGAAATTATCAACTTCCATGAAAATTAT~C~TACTTCCAGTTC~TCCC~~C~GCTGTTTGATTCCGGATTT~CAGG~TTTGGCGTTGGAGGTATTGTATCAG TLNEIINFHENYGKYFQF N P G NK L F D S G F N R N LA L E V 1, Y

Q

1800 520

ACATTTATGATTTGTTCTTTCGGACAAACACAGAAAGGATATCGAG~TCGGTTCA~~TTCA~CCTGTTTGCATCCATGTACTATTTTAT~CATA~ATATCAGGATTTTGTA EIGSGNSGLFASMYYF T FM I CSFGQTQKGYR INIGYQDFV AAGGAATTGTTGTTTGAGTGGACGGCAAACGTATATAAAACAGCCTGTCTCTTGCAGGCA KELLFEWTANVYKMGYANHNFYLVGKQRDCI

PM

T

A

C

L

TATTACAGATATATTATTTATACAAAAGATACTTCGGTATT~TGAGG~GTACC~TT~CGACGG~C~TG~GAG~TGT~GAG~CGCTG~~CTATCATCCAGTAT PVADGNNEKRAVRETLKAI Y Y R Y I IYTKDTSVLNEEV

1680 480

I. Q

A

1920 560

I

Y

2040 600

Q

TCCGCTTGTATTTCTGTCGGTGATCATGGCCTTCCG~TGCT~ATCTT~AGACT~~TGACTGCCTG~GATTGACAGC~CAGTATAGACGGTGC~CC~G~GT~TACTAC SACISVGDHGLPLLDLADWNDCLKIDSNSIDGATKEKLYY

2160 640

GAACAGTTGRAGAAGACAAACGGCAAATATGGAGATCGCTTTATGAGCGATTATTC~~GCGTGATG~TGCTTTCCTCTTG~GTTGGC~TTGACCATTTGGCTG~TTGC~CT IDHLAEIAT E Q I. KKTNGKYGDRFMSDYSESVMNAFLLKLA

2280 680

TTGGATRATGACACTCARCTGGCCCAACAAATGAGTG~TTGTC~GAGGTTACAGACCGCATTCAG~CATGCCT~~G~CTTCTTTGCCCGTGTTCTTAT~CCGTTAC KHAWKENFFARVLINRY LDNDTQLAQQMS ELSKEVTDRIQ

2400 720

CCGCTGATCCGAACATTGACGGCGTGTACTTCTTAAACAGTTTTGCATGGTC~TGCTGTCCGATGTTGC~CC FAWSVLSDVAT

AAAGACGGTTCCTATACTTATTTGGGAGGGCGA KDGSYTYLGAKGDKLSADPNIDGVYFLNS

2520 760

GATGAGCAAATAGCAATAATGGTGGATGTCATCAAAAAATATTTGTTAACTCCGTACGGCTTGCGTTTGGTAACACCTGCCGATTTG~C~TTGC~TGATACTGC~CAGGGCAT IANDTATGH DE Q I AIMVDVIKKYLLTPYGLRLVTPADLNK

2640 800

TACTTCTTTGGTGACAffiGACGGTGCTGTCTTC~CATGCTTC~TGATffiCAGTTGTTGCGCTTATC~GGCTGC~G~GT~GAC~TGAGCTTGCC~G~TGGcA LAKEMA YFFGDRENGAVFKHASMMAVVALIKAAKKVKDNE

2760 840

AGAATAGCGTACTTTATGATAGACTTGGTACTGCCATACA RIAYFMIDLVLPYKTEIR

2880 880

SRLQEIQGYALNISILTQEKIL

ACCTTTGTTGAGCCGGGAACGGCAACCTGGCTTAACTTG~TCTTATTTCCCTGGCAGG~TAGAGTACACCAGGGATGG~TTTCCTTC~TCCGATACTTCGGG~GAGG~CTCAG LREEETQ EYTRDGISFNPI TFVEPGTATWLNLNLISLAGI

3000 920

TTGAATTTCACTTTGAAAGCGCCGAAATGCTCATATAAGTTTAGTATTAC~CC~TTGGTTTTGCTAG~TGG~GTTC~~TATG~CTTTTTGTTGATGGAC~GATTGAC LNFTLKAPKCSYKFSITKPVGFARMESSEYELFVDGQKID

3120 960

AACACTGTCATTCCAATGTATACGGATG~G~CATATAGTGACTCTT~GTTT~T~GTGCTAT~T-CATTGCATTG~TATTGGGTTGTT~G~CCTTGACCA NT V I P MYTDEKEHIVTLKFK*

3240 980

CTACTCTTTTTT

3286

FIG. 2. Nucleotide and deduced amino acid sequences of cdp gene. The putative - 10 and - 35 promoter regions and SD sequence are shown by double-underlines and asterisks, respectively. Arrows indicate the position of a palindrome sequence capable of forming a stem-and-loop structure. The amino acid sequences of CDP identified by Edman degradation are indicated by underlines. The SspI and Hind111 sites which were used for the construction of expression vectors are shown by white letters on a black background. The nucleotide sequence data reported in this paper have been deposited the DDBJ, EMBL, and GenBank nucleotide sequence databases under the accession number ABO06822.

148

KAWAGUCHI

J. FERMENT. BIOENG.,

ET AL.

6 1

23

45

67

FIG. 3. SDS-PAGE of proteins produced by E. coli DHSa harboring pTEH1 and CDP localization in the cellular fraction. Cells in a 1 ml overnight culture at 37°C were harvested by centrifugation, resuspended in 0.5 ml buffer containing 50 mM Tris-HCl and 5 mM EDTA (pH 7.5). and disrupted by sonication. The suspension was used as a total cellular protein fraction. The suspension was centrifuged again and the supernatant was used as a soluble cellular fraction. The precipitates was resuspended in 0.5 ml of the same buffer and the resultant suspension was used as an insoluble cellular fraction. From these fractions, 20 ~1 aliquots were taken and electrophoresed on 7.5% SDS-polyacrylamide gel. (A) Total cellular protein. Lanes: 1, molecular weight marker; 2, purified CDP (standard); 3, pTEH1 without IPTG induction; 4, pTEH1 with IPTG induction; 5, pTrc99A. (B) CDP localization in cellular fraction. Lanes: 1, purified CDP; 2 (soluble fraction) and 3 (insoluble fraction), pTrc99A; 4 (soluble) and 5 (insoluble), pTEH1 without IPTG induction; 6 (soluble) and 7 (insoluble), pTEH1 with IPTG induction.

corarium cellobiose phosphorylase gene (cepA) and the cellodextrin phosphorylase gene (cepB), which are the only ,&glucan phosphorylase genes cloned and sequenced to date and which show a high degree of similarity (41.7% identical residues) on the protein primary sequence level (10). C. thermocellum CDP was longer than C. stercorarium CepA and CepB by 169 and 200 amino acid residues, respectively. Unexpectedly, the overall primary structure of CDP had a low degree of similarity with that of either CepA (21% identity by optimized alignment) or CepB (20% identity), though these two thermophilic bacteria, classified as being of the same genus, could be closely related and their cellulase-related enzymes revealed relatively high similarity. Only a small region where amino acids conserved among the three proteins were noticeably concentrated was found (Fig. 5). An amino acid residue(s) within this region might be involved in catalytic action. We attempted to find proteins related to CDP by searching through the protein se-

Gl(fil-4)DNJ G2(fil-4)DNJ G3( /j l-4)DNJ G4@1-4)DNJ GS(\jl-4)DNJ G6(Sl-4)DNJ

I

I

I

I

I

0

2

4

6

8

Incubation time (h) FIG. 4. Thin-layer chromatogram of cellulase inhibitors synthesized from DNJ and glucose-l-phosphate using synthetic reaction of CDP. Cl, Glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose, G6, cellohexaose; DNJ, I-deoxynojirimycin.

quence databases. However, no significant similarity was found, even to any other known amino acid sequence of related enzymes such as a-glucan phosphorylases, nucleoside phosphorylases, and glycosyl transferases. In order to express the cdp gene in E. coli cells, two kinds of expression vectors were constructed. The E. coli transformant harboring pDEH1 (under the control of the lac promoter) produced CDP at 13.5 and 21.6 units/ml culture with and without IPTG induction, respectively. The intensity of the protein band corresponding to CDP on SDS-PAGE suggested that all the CDP protein was produced in an active form in the intracellular soluble fraction (data not shown). The transformant harboring pTEH1 (under the control of trc promoter) produced CDP at 41.3 units/ml culture without IPTG, but IPTG addition led to a complete loss of activity. No protein band corresponding to CDP or its degraded product was observed in the analysis of the whole intracellular protein by SDS-PAGE, indicating that the loss of activity was not due to CDP produced in an inactive form such as insoluble inclusion body. Though the reason is unclear, the growth of the transformant induced by IPTG was extremely slow, and the plasmid could hardly be restored from the cells after a lengthy period of incubation. Therefore, it was concluded that the loss of production resulting from IPTG induction was due to the defection or deletion of the plasmid because of over-expression stress. Though the cells were grown at a lower temperature of 25°C to reduce the stress, the productivity was not recovered. The highest activity was obtained by E. coli JM103 harboring pTEH1 grown at 37°C without IPTG induction. Under CDP

:

(607)-GDHGLPLLDLADWNDCLKID-(626)

CepA

:

(469)-GPHGLPLIGRADWNDCLNLN-(488)

CepB

:

(460)-GPHALP-YSRADWNDTLNLD-(478) *. -. **** - *****

FIG. 5. Amino acid sequence alignment of highly homologous region among C. thermocellum cellodextrin phosphorylase (CDP), C. stercorarium cellobiose phosphorylase (CepA), and cellodextrin phosphorylase (CepB). Asterisks and dots represent identical and similar amino acids, respectively.

Vot. 85, 1998

CELLODEXTRIN

these conditions, the CDP protein produced accounted for 30% of the total cellular protein and its activity was 59.9 units/ml culture, corresponding to 0.93 mg/ml culture. This activity is 440-fold higher than that of C. thermocellum. As reported previously, cellulase inhibitors can be enzymatically synthesized from water-soluble cellooligosaccharides and DNJ using the transglycosylation activity of Meicelase CMB-5003, a cellulase mixture derived from Trichoderma viride (9). In this case, we obtained sufficient amounts of Gl(Bl-4)DNJ, G2@1-4)DNJ, and G2@1-6)DNJ but less of the longer derivatives. To obtain longer analogues of the cellooligosaccharides, we investigated the possibility that the CDP activity promoting the synthesis of cellooligosaccharides could be used for the synthesis of cellulase inhibitors. Although the role of CDP could be the degradation of cellooligosaccharides in C. thermocellum cells, the enzyme reaction is reversible and the equilibrium of the reaction was found to favor the synthesis reaction under the conditions used here. By comparing the intensity of the spot derived from the starting material with those of products, the yield of total products from the starting material can be calculated to be more than SO%, which is much higher than that of Meicelase transglycosylation activity. Furthermore, we purified each analogue to homogeneity on HPLC and all of them had inhibitory activity against the hydrolyzing activity of FI-CMCase (24), one of the endo-cellulases from Aspergillus aculeatus, on carboxymethylcellulose. Cloning and high expression of the cdp gene in E. coli made it possible to easily prepare a large quantity of CDP. In addition we developed an easy method of enzymatically synthesizing cellulase inhibitors, which could make an important contribution to studies on the molecular mechanism of cellulase action. We have also been studying the cellobiose phosphorylase of C. thermoceilum which catalyzes the phosphorolysis of cellobiose. We anticipate that cellulase inhibitors will be able to be synthesized easily and at low cost from DNJ and glucose-l-phosphate in combination with CBP and CDP if CBP can use DNJ as an acceptor for the synthetic reaction. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES Tanaka, K., Kawaguchi, T., Imada, Y., Ooi, T., and Arai, M.: Purification and properties of cellobiose phosphorylase from Clostridium thermocellum. J. Ferment. Bioeng., 79, 212216 (1995). Arai, M., Tanaka, K., and Kawaguchi, T.: Purification and properties of cellodextrin phosphorylase from Clostridium thermocellum. J. Ferment. Bioeng., 77, 239-242 (1994). Gilkes, N. R., Hen&sat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A.: Domains in microbial ,%l,Cglycanases: sequence conservation, function, and enzyme families. Microbiol. Rev., 55, 303-315 (1991). Hoj, P. B., Rodriguez, E. B., Iser, J.R., Stick, R. V., and Stone, B.A.: Active site-directed inhibition by optically pure epoxyalkyl cellobiosides reveals differences in active site geometry of two 1,3-l ,4-p-o-glucan 4-glucanohydrolases. The impor-

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