from an Amplified Genomic Sequence of Streptomyces tendaet

Transcription

1 JOURNL OF BCTERIOLOGY, Sept. 1989, p /89/ $02.00/0 Copyright D 1989, merican Society for Microbiology Vol. 171, No. 9 Heterologous Expression of the ol-mylase Inhibitor Gene Cloned from an mplified Genomic Sequence of Streptomyces tendaet KLUS-PETER KOLLER* ND GUNTHER RIESS Hoechst G, 6230 Frankfurt am Main 80, Federal Republic of Germany Received 13 March 1989/ccepted 26 May 1989 The coding region for a secreted proteinaceous inhibitor of the human a-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. fter subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. fter being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the a-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement. Inhibitors of the human a-amylase are of high potential pharmaceutical value as regulative agents in reducing digestive-starch degradation in patients suffering from diabetes and obesity (29). Proteins inhibiting human salivary gland amylases were isolated from plants (7, 22) and various microorganisms (1, 9, 23). The ac-amylase inhibitor of Streptomyces tendae is a biochemically well-characterized acidic protein of 74 amino acids (30) secreted in large amounts into the culture medium (15). Recently, the complete threedimensional structure has been elucidated by X-ray crystallography (26) and independently by nuclear magnetic resonance spectroscopy (14). Tendamistat specifically inhibits mammalian oa-amylases (30) and also some Streptomyces amylases (17). n outstanding feature of tendamistat is the almost irreversible binding to the human ax-amylase (30). However, how the inhibitor binds to the enzyme and why the binding is irreversible are unknown. To apply protein-engineering techniques to understand the mode of action of the inhibitor and to study the mechanism of protein secretion in Streptomyces spp., a group of industrially familiar, filamentous, gram-positive microorganisms, we had to clone the inhibitor gene. In the course of a strain improvement program, an overexpressing mutant of S. tendae which is characterized by amplified sequences in its genome was isolated (15). Hybridization experiments using a synthetic oligonucleotide probe suggested the presence of the a-amylase inhibitor gene on the amplified sequence (16). In the present paper we describe the gene structure coding for tendamistat and the expression of the gene following cloning into Streptomyces lividans. dditionally, we show that the nucleotide sequence and the processing of tendamistat are significantly different to those of Haim II of Streptomyces griseosporus, whose primary and secondary protein structures are similar to those of tendamistat (1, 24). MTERILS ND METHODS Organisms and media. The overproducing mutant of S. tendae (1-9362) was obtained after acriflavin-mediated muta- * Corresponding author. t Dedicated to W. Hilger on the occasion of his 60th birthday genesis as described by Koller et al. (16). S. lividans TK24, a host for expression studies, and the Streptomyces cloning vectors pij61, pij350, pij486, and pij702 were kindly provided by D.. Hopwood, Norwich, England (11, 12). Escherichia coli strains for recombinant DN techniques were HB101 (5) and JM101 (21). These strains were grown on plates and in liquid cultures by published procedures (18, 21). For monitoring inhibitor expression and secretion in liquid shaken cultures, recombinant strains of S. lividans were grown for 4 to 5 days in a complex medium (15). Recombinant DN techniques and sequencing. Chromosomal DN from S. tendae and plasmid DN from S. lividans and E. coli were prepared essentially as described elsewhere (12). Digestions of isolated DN with restriction enzymes and cloning procedures were done by standard procedures (18). Chromosomal fragments were subcloned into pbr322 (5), puc12/13, or puc18/19 plasmids (21, 33). For the identification of the inhibitor gene, a single synthetic oligonucleotide, 3' TG CTT CTG CTG TG CTT CC 5', corresponding to amino acids 37 to 43 of the inhibitor, was synthesized (19). Southern hybridizations using PstIdigested chromosomal DN of S. tendae and the 32P-labeled oligonucleotide as a probe were carried out at 35 C in 6x SSC (1x SSC is 0.15 M NaCl plus M sodium citrate)- 6x Denhardt solution (18). The nucleotide sequence of the inhibitor gene was determined by the dideoxy-chain termination method (28) after gene fragments were subcloned into M13mpl2 and M13mpl3 bacteriophages. Both orientations of the gene were read. Nucleotide and protein sequences were compared by using the lignment Score Program of Protein Identification Resource (25) and the BestFit program of the University of Wisconsin (8). ssay of inhibitor production. Selection of recombinant strains secreting the inhibitor was carried out on agar plates as described elsewhere (16). Synthesis of tendamistat in liquid cultures was measured by using an automated version of the ferricyanide reducing sugar analysis (2). For polyacrylamide gel electrophoresis, samples of culture filtrates were used without further concentration. Electrophoretic mobilities of the secreted proteins were analyzed on native 15% polyacrylamide gels by using the insulin la system (20). Gels were stained with Coomassie blue R250.

2 4954 KOLLER ND RIESS J. BCTERIOL..J Id F,8 _ *: - *+ t _- _ + - /3 1. pi 4 # _- _ a w.... i oq.j. ^ -PI' ti 1 2-S _- i I FIG. 1. Identification by Southern hybridization of the tendamistat gene on a 2.3-kb Pstl fragment of the amplified genomic sequence of S. tendae. Lane, Stained 0.8% agarose gel with PstI-digested chromosomal DN of S. tendtae; lane B, autoradiogram after hybridization with the 32P-labeled synthetic oligonucleotide probe. B RESULTS Isolation of the ot-amylase inhibitor gene. Tendamistat was originally purified from a melanin-producing Streptomyces strain, TCC 31210, classified as S. tendae. The initial yield for the secreted protein was about 10 mg/liter. To improve yields, the strain was mutagenized with N-methyl-N'-nitro- N-nitrosoguanidine and acriflavin. melanin-negative mutant, , was selected; it produced more than 1 g of protein per liter of culture filtrate. This strain is characterized by a massive amplification of a 37-kilobase (kb) genomic element (Fig. 1). Knowing the amino acid sequence of tendamistat, we designed by reverse translation a synthetic oligonucleotide probe and hybridized it to a genomic blot of PstI-digested DN of strong hybridization signal to a 2.3-kb amplified DN segment and one weaker signal to a 3.1-kb fragment were obtained (Fig. ib). The weaker signal disappeared under more stringent washing conditions at 45 C. Sequencing of the gene revealed two mismatches in the pairing of the coding strand and the oligonucleotide probe. We therefore assume that unspecific hybridization is the reason for the 3.1-kb hybridization signal. By electroelution, the 2.3-kb fragment was isolated and cloned into the PstI site of pbr322. By further hybridization studies with this synthetic oligonucleotide, a 570-base-pair (bp) HincII- SstI fragment could be identified as carrying the structural gene. Its localization on the fragment is given in Fig. 4. This fragment was subcloned into puc18, cut out with SphI-SstI, ligated into pij702 (Fig. 4C), and introduced into S. lividans TK24 by transformation. Recombinant thiostrepton-resistant clones were tested on plates for extracellular production of the inhibitor. lmost all clones were positive for ox-amylase inhibitor production (Fig. 2). Nucleotide sequence. To determine the nucleotide sequence of the tendamistat gene, the 570-bp HincII-SstI fragment was subcloned into M13mpl2 and M13mpl3 phages, and the single-stranded DN was sequenced by the dideoxy method (28). There was an open reading frame of FIG. 2. ssay for tendamistat production by recombinant S. lividans clones. () Plate test after 4 days of growth. Dark halos indicate synthesis and secretion of the inhibitor. (B) Coomassie blue-stained gel. Recombinant S. lividans was grown for 4 days in shaken culture, and samples (10,ul) of the supernatant were subjected to gel electrophoresis on native 15% polyacrylamide gels. Lane a, Mixture (10,ug each) of tendamistat, marked 2, and its B form, marked 1, which is shortened at the amino-terminal end of the molecule by the action of aminopeptidases (30); lanes b, c, d, and e, supernatants of strains carrying the tendamistat gene in pij61 (2.3-kb fragment), pij350, pij486, and pij702 (plus the melanin promoter), respectively. 312 bp (Fig. 3). It started with an TG, was terminated by a TG stop codon, and contained the coding sequence for the mature protein preceded by a 90-bp sequence which coded for a 30-amino-acid amino-terminal extension. Computer analysis revealed structural features typical for signal peptides (31). t the amino terminus there was a hydrophilic region of seven amino acids. The remaining sequence of 23 amino acids was strongly hydrophobic, containing a potential at-helical stretch of 8 residues flanked by glycine 16 and proline 9, respectively. Considering the identical amino termini of the proteins purified from S. lividans and S. tendae (see below), the cleavage site for the signal peptidase is between la Ser la and sp Thr Thr. The codon usage within the gene shows the preference for G or C in the third position, which is typical for Streptomyces genes (3). t position -16 upstream of the reading frame, the sequence 5' GGGGTG 3' had a high degree of complementarity to a sequence close to the 3' end of the 16S rrn sequence of S. lividans, suggesting a putative ribosome-binding site (4). Expression of tendamistat in S. lividans. To study the expression of the tendamistat gene, recombinant clones of S. lividans positive for inhibitor synthesis in the plate tests were transferred to liquid shaken cultures. nalysis of the proteins in the supernatants by polyacrylamide gel electrophoresis revealed secretion of the inhibitor, which was identical in size and mobility to tendamistat from S. tendae (Fig. 2B). mino acid sequencing of the purified protein revealed identity of the inhibitors at their amino termini, indicating the correct processing of tendamistat in S. lividans. To show that the 570-bp SstI-HincII fragment carried at least one promoter, we excised the fragment from the recombinant puc18 vector by digestion with EcoRI and HindlIl (Fig. 4B) and ligated it to the Streptomyces promoter probe vector pij486 (32). This vector carried a termination structure 5' to a multiple cloning site. Thus the expression of the foreign gene cloned into this site is possible only if the cloned fragment contains a promoter region. There is clearly synthesis and secretion of tendamistat in yields similar to those from pij702, from which vector pij486 was derived (Fig. 2B) (32). The expression of the tendamistat gene was also tested with different Streptomyces vectors and different

3 VOL. 171, MET RG VL RG L LEU RG I CTC T TGICC GCC TG CGC GT CGG GC CTT CG H2 C CC GT GCG TG G CGT TC GTG TGT TC MET LYS RG TYR VL CYS SER -37 LEU L L LEU VL GLY L GLY L L LW L LEU SER PRO CTT GCG GCG CTG GTG GGT GCG GGC GCC GC CTC GCC CTG TCT CCC CT TTC GTG GCC TGT GTG GT GTC CTG TGC GTG TT CCG GCG TCC THR PHE VL L CYS VL MET VL LEU CYS VL ILE PRO L SER LEU L L GLY PRO L SER L... R...SP THR THR VL SER CTC GCG GCC GGG CCG GCC TCC GCC... I... GC CG CC GTC TCC GGC GCT GCG GCC CC GG GCG GTG GCG GG GC GCG GGG C CGG GLY L L L HIS GLU L VL L GLU SP L GLY SN RGJ GLU PRO L PRO SER CYS VL THR LEU TYR GLN SER TRP RG TYR GG CCC GC CCC TCC TGC GTG CG CTC TC CG GC TGG CGG TC TC GCC GCC CCT GCC TGT GTG CC TTC CC GCG GC TGG CGC TC ILE L L PRO L CYS VL HIS PHE THR L SP TRP RG TYR 1 SER GLN L SP SM GLY CYS L GLU THR VL THR VL LYS VL TC CG GCC GC C GGC TGT GCC CG CG GTG CC GTG G GTC CC TTC GTC CC C GC TGC TCC TC GC TC TCC GTG CG GTC THR PHE VL THR SN SP CYS SER ILE SP TYR SER VL THR VL VL TYR GLU SP SP THR GLU GLY LEU CYS TYR L VL L PRO GTC TC GG GC GC CC G GGC CTG TGC TC GCC GTC GC CCG GCG TC GGC GC GGG CG GC GTC CCC TGC CGG TCG GCG C CCC L TYR GLY SP GLY THR SP VL PRO CYS RG SER L SN PRO GLY GLN ILTH THR VL GLY SP GLY TYR ILE GLY SER HIS GLY GGC CG TCI CC ICCI GTC GGC GC GGC TC TC GGC TCG CC GGC GGC GC TCI CTC CCI TTC CCG GGG TC GGC CC CGG GGC C GG GLY SP ILE LEU T PHE PRO GLY TYR GLY THR RG GLY SN GLU 74 HIS L RG TYR LEU L RG CYS LEU CC GCC CGC TC CTC GCT CGC TGC CTT TG CCC C GCC GCC TTC GTC CTG GGC GCC GTC CTG... TGC GCC CC GC GGC GC GCG CTC VL LEU GLY L VL LEU... CYS L THR SP GLY SER L LEU CG CGG CGC TGC TCC TCG GCG TCC GCG GCG CG CCC GCG TGG TGG CCG GTG GC CGG GG CGC GCT GTC CGG TG GCG GCG CCG GG CG PRO VL SP RG GLU RG L VL RG * 83 FIG. 3. Nucleotide sequence of the tendamistat gene (l) aligned with the sequence of the Haim II gene (H2) (24). Boxed in the nucleotide sequences are putative ribosomal-binding sites. sterisks mark stop codons. Large boxes show homologies in the amino acid sequences of the unprocessed proteins. rrows indicate the cleavage sites of the signal peptidase that yield tendamistat and Haim II. Cysteines involved in cystine bridge formation in the mature proteins are underlined. ***", Regions of homology between the coding strand and the oligonucleotide probe. insertion sites. mounts of secreted inhibitor were dependent on the copy number of the cloning vector, although there is no strict correlation between both (Table 1). If an additional promoter was located close and in tandem to the promoter region of the gene, a significant increase of expression occurred. This was demonstrated by cloning the 570-bp HincII-SstI fragment with the inhibitor gene into the melanin gene of pij702. Removal of the 350-bp PstI-SstI fragment containing the melanin promoter (Fig. 4D) resulted in a three to fivefold decrease in production (Table 1). DISCUSSION We have cloned the gene for the ot-amylase inhibitor from S. tendae. Expression of the gene in S. lividans results in the correct processing and folding of the gene product. Various secreted proteins such as agarase (6), different a-amylases (13, 17), endo-,b-n-acetylglucosaminidase H (27), and the amylase inhibitor Haim II of S. griseosporus (24) were expressed in S. lividans. These genes code for signal peptides which show similar structural features but different MYLSE INHIBITOR GENE OF S. TENDE 4955 PstI SstI HincII SaIlI PstI "\ bp SstI I HincII ai-gene promoter region Sstl HinclI PstJ Sphl B r> puc18 EcoRI Ia-gene HindIII promoter region C SstI tyrosinase ai-gene ^= gene D Sst I ozzz,/z4 1I PstI Sphl BclI PstI pij702 4I.. remaining tyrosinase promoter region Pst I tyrosinase ai-gene tyrosinase promoter gene region deleted pi J702 FIG. 4. Localization and orientation of the tendamistat gene (ai) on the cloned 2.3-kb PstI fragment of the amplified genomic sequence () and subcloned into puc18 (B) and the Streptomyces expression vector pij702 (C and D). cleavage sites for signal peptidases. Despite this, all of these proteins are processed by signal peptidases of S. lividans, leading to secretion of the functional proteins. Therefore, S. lividans seems to be a suitable host for the study of protein secretion. Compared with levels in the wild-type strain of S. tendae, expression levels of tendamistat were generally higher with the cloned genes. low-copy-number vector yielded a 10-fold increase in production, and a high-copynumber vector yielded a >60-fold increase in production. dditionally, the spatial arrangement of heterologous promoters in tandem stimulated the expression rate. Combining the promoters of the mel gene of pij702 and the tendamistat gene resulted in a 10-fold increase in yields. Cloning into the TBLE 1. Synthesis of tendamistat in the heterologous host, S. lividans TK24, by using different vectors, insertion sites, and tandem arrangement of promoters' Vector Cloned fragment Copy numberb (gyiteld) pij kb PstI-Pstl Low (4-5) pij kb PstI-SalI Low (4-5) pij kb PstI-SaII High (40-300) pij kb SstI-HincII High (40-300) (without melanin promoter) pij kb SstI-HincII High (40-300) (with melanin promoter) "For expression studies using pij61 and pij350 as Streptomyces vectors, the 1.56-kb Pstl-SalI fragment (Fig. 4) was converted into a PstI fragment by ligation of a PstI linker to the Sail site after fill-in with Klenow polymerase (18). The resulting PstI fragment was cloned into the unique Pstl sites of pij61 and pij350. b For the plasmid copy number, see reference 12.

4 4956 KOLLER ND RIESS high-copy-number vector pij350 gave production levels similar to those obtained with overproducing strains of S. tendae. However, we cannot yet explain why the expression rates with pij350 were so much higher compared with those of pij702. Both vectors were derived from the same replicon, pijlol (11), and should have comparable copy numbers. However, the experiments show that we need additional information to predict maximal expression levels. It may well be that unidentified promoters on the vector are responsible for the higher expression obtained with pij350. Mature tendamistat and Haim II from S. griseosporuiis seem to be similar proteins with respect to their primary and secondary protein structures (10), but there are also significant differences in the structures of the unprocessed proteins and in the coding nucleotide sequences. (i) When the Protein Identification Resource computer program LIGN (25) was used for a comparison of tendamistat and Haim II, there were 42 common residues of 149. ligning the nucleotide sequences of the unprocessed proteins by using the BestFit program (8) gave only 57% similarity. (ii) Tendamistat and Haim II had signal sequences of 30 and 37 amino acids, respectively. (iii) The signal sequences varied in their amino acid compositions. dditionally, Haim II had an unusual positively charged arginine at the cleavage site of the signal peptidase (24). (iv) Tendamistat had a short, unflexible, carboxy-terminal end following the cystine bridge of the large second loop. t the carboxy-terminal side of the second cystine bridge, there was a leucine terminating the polypeptide chain, whereas Haim II exhibited an extension of 16 amino acids. (v) During secretion through the membrane or in the culture broth, Haim II is secondarily processed and proteolytically cleaved at the carboxy terminus to give the mature protein (24). This type of processing does not occur with tendamistat. The spatial arrangement of the Trp-18, rg-19, and Tyr-20 region of tendamistat suggests an involvement in the tight binding to and irreversible inactivation of the human pancreatic o-amylase (14, 26). Using the secretion system for tendamistat in S. lividans in combination with proteinengineering techniques, we were able to show that rg-19 and Tyr-20 are indeed involved in the unique interaction between an enzyme and its inhibitor. separate publication on this subject is in preparation. CKNOWLEDGMENTS We acknowledge the help of R. Bender for determining the amounts of tendamistat in liquid cultures, D. Tripier for analyzing the amino-terminal sequence, E. Uhlmann and J. Engels (University of Frankfurt) for synthesizing the oligonucleotide probe, L. Vertesy for stimulating discussions, and H. Dornauer for his continuous support. We cordially thank C. Metz, W. Klingenberg. D. Rabold. and. Schwarz for their expert technical assistance. LITERTURE CITED 1. schauer, H., L. Vertesy, and G. Braunitzer Die Sequenz des cx-mylaseinhibitors HOE 467 (ox-mylase-inactivator HOE 467 ) aus Streptominces tenidwie Hoppe-Seyler's Z. Physiol. Chem. 362: Bender, R., and D. Sukatsch Continuous flow determination of an c-amylase inactivator using a ferricyanide reagent. nal. Biochem. 137: Bibb, M. J Gene expression in Streptorn(ces-nucleotide sequences involved in initiation of transcription and translation, p In G. Szabo, S. Biro, and M. Goodfellow (ed.). Biological and biomedical aspects of actinomycetes. kademiai Kiado, Budapest, Hungary. 4. Bibb, M. J., and S. N. Cohen Gene expression in J. BCTERIOL.. 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