Appl Microbiol Biotechnol (1990) 33:542-546

Applied Microbiology Biotechnology © Springer-Verlag 1990

Molecular cloning, nucleotide sequence and expression in Escherichia coli of the fl-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8 Lars Nitschke*, Karin Heeger, Hans Bender, and Georg E. Schulz Institut fiir Organische Chemie und Biochemie, Albertstrasse 2l, D-7800 Freiburg im Breisgau, Federal Republic of Germany Received 12 January 1990/Accepted 6 April 1990

Summary. The fl-cyclodextrin glycosyltransferase (/3CGTase) gene was isolated from a )~-library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid p T Z and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant fl-CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the threedimensional structure of the enzyme.

Introduction The cyclodextrin glycosyltransferases (CGTases, 1,4a-D-glucan: 1,4-a-D-glucopyranosyltransferase (cyclizing), EC 2.4.1.19) are of special interest because the cyclodextrins are technically important as clathrate-forming compounds (Bender 1986; Szejtli 1988). At least ten CGTase genes have been cloned (Binder et al. 1986; Sugimoto et al. 1986; Takano et al. 1986; Kimura et al. 1987a, b; Kaneko et al. 1988; Horikoshi 1988; Schmid et al. 1988) and the respective amino acid sequences have been elucidated at the D N A level. The a-, flCGTases (named after the cyclodextrin formed initially) of the bacilli have molecular weights of about 74 000 corresponding to 680 + 10 amino acid residues; they are homologous with 50%-70% identical amino acids. The * Present address: Max Planck Institut fiir Immunbiologie, Stiibeweg 51, D-7800 Freiburg im Breisgau, Federal Republic of Germany Offprint requests to: H. Bender

a-CGTase of Klebsiella p n e u m o n i a e strain M5al (mol. wt. = 69 000 corresponding to 625 residues, Binder et al. 1986) shows merely around 30% homology with the enzymes of the bacilli. Sequence similarities between aamylases and CGTases were found in four highly conserved regions that include the catalytically active amino acid residues of a-amylases (Binder et al. 1986; Kimura et al. 1987a). The three-dimensional structure o f crystalline CGTase from Bacillus circulans strain no. 8 (B. circulans) has been elucidated at a resolution of 3.4 A (Hofmann et al. 1989). The chain fold of the polypeptide has been subdivided into five domains. The fold of the three N-terminal domains resembles closely the two known folds of a-amylases (Matsuura et al. 1984; Buisson et al. 1987). This geometric homology corresponds to significant amino acid sequence homology in the first and third domains (Hofmann et al. 1989). As judged from X-ray data, the CGTase chain fold can be considered as an a-amylase chain fold with two additional domains. For a more detailed three-dimensional structure o f this CGTase it is necessary to know the amino acid sequence of the polypeptide chain. We report here the cloning of the CGTase gene of B. circulans, the expression in E. coli, the nucleotide sequence, and the primary structure of the CGTase.

Materials and methods Strains and enzymes used. Wheat bran was screened for CGTase-

synthesizing microorganisms. A positive strain producing CGTase in good yields was identified as a member of the B. circulans group and named B. circulans strain no. 8 (Hofmann et al. 1989). The BamHI arms of bacteriophage 2-EMBL 3 (Frischauf et al. 1983) and the host strain E. coli MB 406 (sup E, rec B21, rec C22, sbc B15, hfl A, hfl B, hsd R-) were purchased from Promega (Madison, USA). The plasmids pTZ18R and pTZ19R, the helper bacteriophage M13 K07 for the production of single-stranded DNA (Mead et al. 1986), the host strains E. coli NM 522 (hsdA5, A(lacpro), IF', pro +, laclqZAM15]) and E. coli JM 103 (thi, strA, endA,

543 sbcB15, hsdR4, A(lac-proAB), [F', traD36, proAB, lac IqZAM15]) were from Pharmacia (Uppsala, Sweden). The enzymes were obtained from the commercial sources stated in the text and used under conditions recommended by the supplier. All chemicals were of the highest purity available. Construction of a genomic library from B. circulans. B. circulans

DNA was prepared using a modified published procedure (Miura 1967). Washed late-log-phase cells of B. circulans (1 1 of culture in Luria Bertani [LB] medium) were lysed by lysozyme, followed by overnight treatment with proteinase 1(/sodium dodecyl sulphate (SDS) at 50°C and phenol/chloroform extraction. The genomic DNA was partially digested with Sau3AI. Fragments with lengths of 9-22 kb were isolated by sucrose gradient centrifugation (Boulnois 1987). These Sau3AI fragments were ligated into the BamHI arms of ~.EMBL 3 (treated with alkaline phosphatase from calf intestine). The recombinant phage DNA was packaged in vitro using a commercial packaging extract (Promega) and plated with E. coli MB 406 essentially as described (Rosenberg et al. 1985; Manniatis et al. 1982). Library screening and isolation of CGTase-producin9 clones.

Screening of the genomic library was performed by plating on LB plates containing 0.5% (w/v) amylopectin azure (Sigma, St. Louis, USA), which is degraded around plaques where the enzyme is expressed. Positive clones were plaque-purified, and )~-DNA was prepared from culture lysates of the recombinants (Manniatis et al. 1982). The ,~-DNA was purified by CsC1 step-gradient centrifugation. The inserts were excised from the polylinker region of AEMBL 3 with Sail and subcloned into pTZ18R and pTZ19R for restriction mapping and sequencing. The expression of the functional enzyme could still be assessed by plating transformed E. coli JM 103 onto LB agar containing amylopectin azure. DNA sequence analysis. For sequencing, the recombinant plasmids were transformed into the host strain E. coli NM 522. Single-

stranded DNA of the recombinant plasmids was prepared with M13 I(07 helper bacteriophage according to the instructions of the supplier. DNA sequencing was performed by the dideoxy method (Sanger et al. 1977; Barnes 1987), using commercial kits (Pharmacia, Uppsala, Sweden, and U.S. Biochemical Corporation, Cleveland, USA). The DNA strands were labelled with [a3sS] dATPctS (Amersham, International plc, Amersham, England). The sequencing was performed by use of synthetic oligonucleotide primers and by subcloning of restriction fragments into the pTZ vectors as well. In cases of non-compatible fragment ends, these were processed into blunt ends for proper ligation (Manniatis et al. 1982). For subcloning of the AccI/AccI fragment a commercial SalI/TaqI linker (Pharmacia) was used and the relative orientations of the inserts were analysed by hybridization of the single-stranded DNA. Two subfragments were sequenced after subcloning according to Lee and Lee (1988).

expressed CGTase exclusively, whereas the other two expressed CGTase and an amylase or a 'cyclodextrinase', as indicated by the formation of large amounts of non-cyclic saccharides (Bender 1986; DePinto and Campbell 1968). Only the former clones were used for further work. The inserts of the two CGTase-expressing clones were 11.9 kb and 11.2 kb in size. SalI digestion of both recombinants yielded two different sets of three fragments, which were all subsequently subcloned into pTZ18R. As shown by clear halos around the colonies on amylopectin azure (10%-15% of intracellular enzyme was released by lysis of the new host, Kaneko et al. 1988) two of the clones retained CGTase expression and were named pBC2 and pBC3 (insert sizes 6.0 kb and 3.4 kb, respectively). The CGTase-positive clones were cultivated in LB medium, and the enzyme was isolated and purified ( H o f m a n n 1989) from homogenized cells. Clone pBC2 yielded 1 mg C G T a s e / g wet cells and clone pBC3 yielded only 0.05 mg C G T a s e / g wet cells. The specific activities of the enzymes, their SDS-polyacrylamide gel electrophoresis (PAGE), and isoelectric focussing pattern were identical and were comparable to those of the extracellular enzyme of B. circulans ( H o f m a n n et al. 1989). Accordingly, clone pBC2 fully expressed the CGTase gene, whereas clone pBC3 was probably defective in the endogenous regulatory sequences caused by fragmentation of the genomic DNA. The CGTase gene was m a p p e d on a 3.4-kb Hind l I I / X b a I fragment of pBC2 (Fig. 1). Subcloning of this fragment into pTZ18R and p T Z 1 9 R resulted in clones pBC22 and pBC23 that expressed CGTase in the same amounts as pBC2. Sequencing and a comparison with the highly conserved C-termini of other CGTases showed that the region of the 3.4-kb insert encoding the C-terminus of the CGTase is one nucleotide upstream of the H i n d l I I site of the hybrid vector (Fig. 1). S D

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The genomic library was screened for CGTase-expressing recombinants as previously reported (Binder et al. 1986; Sugimoto et al. 1986; Takano et al. 1986; Kimura et al. 1987a, b; Kaneko et al. 1988; Horikoshi 1988; Schmid et al. 1988), i.e. for recombinants containing the endogenous regulatory sequences necessary for expression. Four positive clones were found among 8000 plaques. Assaying the CGTase activity of crude extracts of infected hosts by H P L C revealed that two of the clones

I " I b I -'Fig. 1. Strategy for cloning and determination of the nucleotide sequence of the cyclodextrin glycosyltransferase (CGTase) gene from Bacillus circulans. Numbering begins with the first codon of the open reading frame: --, inserted genomic DNA; n, signal peptide; II, gene segment corresponding to CGTase; the direction of sequencing is indicated by an arrow; O--, priming was performed with reversed primer; ~---, priming was performed with a synthetic oligonucleotide; x --, a 2',3'-dideoxy-inosine-5'-triphosphate run was done in addition. A, AccI site; 1t, HindlII site; S, Sail site; X, XbaI site

544 TTTGTTGACGTCTTTCCGGTGCATTTGTCCTSATTCTGGTATCG -167 TC,CTGTTATCAATGGCAAASAAGTATGGAGGrGC~3GGCGCTAATCAATCGASAGCCrCT -108 GCTSACTGTACACTCTCGCATCACTAGCAGAC~TTAAGATGAGgCAGTCGCCTTCCS S/D -59 AATCCC-~,CACCACAAATGCCC-CACSAACATTSTACTACATTCACGAAGGGTGGATTACC

1 ATG TTT CAA ATS GCC AAA C~C GCA TTC CTC AGC ACC ACA CTS ACC Met Phe Gln Met Ale Lys Arg Ala Phe Leu Set Thr Thr Leu Thr 46 CTC GGC TTG CTT GCC GGC AGe GCC CTG CCG TTC r ~ s CCA s e t r e 6 Leu Gly Leu Leu Ala Gly Set Ala Leu Pro Phe Leu Pro Ala Set 91

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