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Journal of General Microbiology (1987), 133, 1775-1782. Printed in Great Britain 1775 Secretion of Bacillus subtilis a-Amylase in the Periplasmic Space of Escherichia coli By K O H - I C H I T A C H I B A N A , ' K O J I Y O D A , ' S A T O R I W A T A N A B E , ' HIROSHI KADOKURA,' YOSHIHIRO KATAYAMA,' K U N I O YAMANE,* MAKARI YAMASAKI'* AND G A K U Z O TAMURA' Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Institute of Biological Sciences, University of Tsukuba, Sakura, Ibaraki-ken 305, Japan (Received 27 October 1986 ;revised 23 February 1987) The Bacillus subtilis a-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA)signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis a-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide. INTRODUCTION When useful proteins are produced in Escherichia coli, it is preferable that they are secreted into the periplasmic space or the culture medium because (1) their detection, recovery and purification will be easier, (2) the amount of the product may increase, and (3) degradation of the product by cytoplasmic proteases will be avoided. Secretory or membrane proteins are generally synthesized as large precursors with a signal peptide at the amino-terminus. The early steps of secretion are initiated by the function of the signal peptide. During translocation across the plasma membrane, the signal peptide is proteolytically cleaved to yield the mature protein (Blobel & Dobberstein, 1975; Davis & Tai, 1980; Michaelis & Beckwith, 1982). Hence, in the secretory production of proteins, it is necessary to endow them with a signal peptide. A series of secretion vectors for the expression and secretion of foreign gene products in E. coli has been constructed and are named 'Psi vectors', because they contain the promoter and the signal sequence of the E. coli alkaline phosphatase gene (phoA).They also have unique cloning sites just at the end of the signal sequence. The human a-interferon gene (Miyake et al., 1985) and the human epidermal growth factor gene (Oka et al., 1985) have been inserted just behind thephoA signal sequence. In both cases the protein is found in the periplasmic space. B. subtilis a-amylase is a well-characterized enzyme that has industrial uses. The structural gene (amyE), including the promoter and the signal sequence, has been cloned in PUB1 10 (Takeichi et al., 1983)and the sequence determined (Ohmura et al., 1983). It is of interest to elucidate whether the mature polypeptide of the B. subtilis a-amylase can be translocated to the periplasm with the aid of the E. coli signal peptide, and whether the E. coli signal peptidase can correctly process the fusion protein or not. In this paper, the coding sequence of the a-amylase has been subcloned into the 'Psi vector', and expression and secretion of the protein into the periplasm has been examined. METHODS Bacterialstrains,media andplasmids. E. coli K12 MC1061 [F-araD139 A(ara-le~)~~~~lacX74(AlacIPOZY) galU galK hsdM+hsdR- rpsL] was used as the general host for transformation, expression and experiments. E. coli K12 LE392 [F-supE supF hsdM+ hsdR- lac+]was also used as the host for expression and secretion experiments. 0001-3780 0 1987 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41 1776 K.-I. TACHIBANA A N D OTHERS E. coli K12 YK660 [F196+thi-1 leuB6 metE70 hisF860 lacZ36 xyl-5 rpsLlO5 rpsElSphoA8 recAl] was used for production of the single-stranded plasmid DNA derivatives. Transformation and plasmid construction were done as described by Maniatis et al. (1982). Nutrient broth containing 1.0% (w/v) peptone, 0.3% (w/v) meat extract and 0.5% (w/v) NaCl was purchased from Eiken and used as rich medium generally. The inducing medium forphoAdirected production of the gene product was the MOPS medium of Neidhardt et al. (1974) supplemented with about 0.4 mM-sodium phosphate as an endogenous component of 0.2% (w/v) Difco vitamin-free Casamino acids. For induction of b-galactosidase the medium was also supplemented with 1 m-isopropyl b-thiogalactoside (IPTG) and 5 mM cyclic AMP. The cells consume sodium phosphate in the medium as they grow. When the ODSso reaches about 0.6, the concentration of sodium phosphate becomes low enough to derepress the phoA promoter. The repressing medium was the same as above except that 1 mwsodium phosphate was added. The a-amylase structural gene (amyE) of B. subtilis Marburg NA64 has been cloned into PUB1 10 (Takeichi et al., 1983). This plasmid was designated pTUB4 and the DNA sequence of the inserted 2.3 kb fragment is known (Ohmura et al., 1983). pYK33 I(a 'Psi vector') is a derivative of pBR322 containing the same multiple cloning sites as M13mplO (Norrander et al., 1983) at the end of the promoter-signal sequence and the intergenic region of the filamentous fl phage (Beck & Zink, 1981). The construction of pYK331 will be reported elsewhere. The singlestranded circular DNAs were prepared from filamentous-phage-like particles which were produced after the infection of male E. coli harbouring pYK331 or its derivatives with fd phage. Enzymes and chemicals. All enzymes were purchased from Takara Shuzo (Kyoto, Japan). Reaction conditions used were those described by the manufacturer. IPTG, cyclic AMP, MOPS and Tricine were purchased from Sigma. Vitamin-free Casamino acids were from Difco. [3SS]Methionine[400 Ci mmol-l (14.8 TBq mmol-l)] was purchased from Amersham. The B. subtilis a-amylase antibodies were a kind gift from Dr Y.Nagata (Institute of Applied Microbiology, University of Tokyo). Preparation of theperiplasmic fraction. This was done by the osmotic shock method of Neu & Heppel (1965). SDS-PAGE. The buffer system was that of Laemmli (1970). Proteins were detected by staining with Coomassie Brilliant Blue or by autoradiography. Immunological detection of a-amylase. The labelling of cells with [3sS]methionineand the detection of a-amylase by immunoprecipitation was done as described by Ito et al. (1981). Synthesis of oligonucleotide and site-specijc deletion. A 2 1-mer oligonucleotide (S'TTCGCCGTTTCAGCTTTTGTC3') was prepared by a manual solid-phase method (Miyoshi et al., 1980). The site-specificdeletion was done as described by Wallace et al. (1980) except that single-stranded DNA was prepared from single-stranded phage particles. Assay of enzymes. a-Amylase was assayed by the method of Fuwa (1954). Alkaline phosphatase was assayed by the method of Brickman & Beckwith (1975). b-Galactosidase was assayed by the method of Miller (1972). Purification and amino acid sequence analysis of the secreted a-amylase. The a-amylase secreted into the periplasmic space was purified as described by Matsuzaki et al. (1974). Amino acid sequence analysis was done by Edman degradation using a solid-phase automated sequencer (Beckman system 890). The phenylthiohydantoinamino acid derivatives were analysed by HPLC (Beckman Model 1 IOA). RESULTS Cloning of the amyE gene in the E. coli expression vector In the course of studies on secretion of foreign products in E. coli, expression of the amyEgene with the aid of the promoter and signal sequence of the phoA gene was attempted. First pTUB4 was cleaved with BglII and XbaI, and the 4.2 kb fragment containing the amyE gene was inserted between the BumHI and XbaI sites in the multiple cloning sites of pYK331. E. coli K 12 MC 1061 was transformed by the ligation mixture and ampicillin-resistant transformants were selected. They were then transferred to a nutrient broth agar plate containing 1% (w/v) soluble starch and, after growth at 37 "C overnight, the plate was exposed to iodine vapour. Colonies carrying the new plasmid, designated pYK338, were detected by the formation of large haloes. E. coli K12 MC1061 carrying pYK331 or pYK338 was grown in a liquid culture under inducing or repressing conditions and the cells and supernatant were separated by centrifugation. Cells were subjected to osmotic shock to obtain the periplasmic fraction. a-Amylase activity of MC1061/pYK338 was 2.4 units ml-I (ODssounit)-' and increased to 9.2 units ml-I (ODsso unit)-' during induction. Of the total activity 54% was detected in the periplasm and little in the medium. From the sequence analysis of DNA (Ohmura et al., 1983; Yang et al., 1983) and protein (Mantsala & Zalkin, 1979), the product of umyE in B. subtilis was predicted to have an amino-terminal extension of 41 amino acid residues not found in the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41 1777 Secretion oja-amylase in E. coli Xbal Xhl (b) + BamHI Bglll + Xbal phoA signal (21a.a) ------Intervening .-b sequence- +.let Lys C l n Ser . . . . . . . . . . Thr Lys Ala T r p Ala Ala G l y Arg k u Lhnb C T C AAA CAA ACC . . . . . . . . . . ACA T-A TCC C C T CCA C C T CCA C T C TAG Hind111 -About 1 kb - om) E I ... ... signal (33a a) ___ . . . . . . . . . . . . . . . . . . . . . . . . . . MCt Phe Ala Lys . . . . . . . . . . . Pro Ala Ala Ala ............... 33 A 34 A C T C T C A A C A A T C T T T CCA AAA ........... C F C C C C C T CCC Hpal I unt.,’€ 41 42 Ser A 1 a ) G l u Thr Ala Asn Lys Ser Asn G 1 u ’ L . e ~ Thr Ala A C T C C T CAA ACC CCC AAC AAA TCG A A T GAG C T T ACA C C A AIul .................. .................. Fig. 1 . Construction and structure of pYK338. (o)phoA’ represents the promoter and the coding regions for the signal peptide of the E. colialkaline phosphatase gene; amyErepresents the structural gene of the B. subtilis a-amylase; sfp represents the intergenic region of the filamentous fl phage (see Methods); MCS represents the multiple cloning sites. (b)Translational reading frame of the phoA’-umyE chimera gene in pYK338. The amino-terminus of the extracellular mature a-amylase is indicated by the arrow labelled B; the predicted cleavage site of the signal peptidase is indicated by the arrow labelled A. secreted enzyme. This sequence contains a signal sequence for secretion and is first cleaved between Ala and Glu (33rd and 34th amino acid from the amino-terminus, position A in Fig. 1 b) during the secretion process and further cleaved between Glu and Leu (position B in Fig. 1b) to form the mature extracellular a-amylase. If position A of the amyE gene is joined to just after the end of the phoA signal codons to form an ‘in frame’ gene fusion, a-amylase should be expressed and secreted under the control of the promoter and signal sequence of thephoA gene. A deletion between the end of the phoA signal sequence and position A of the amyE gene in pYK338 was therefore sought by a site-specific deletion using a synthetic oligonucleotide (Fig. 2). Restriction endonuclease trimming oj’ the intervening sequence Several attempts to delete about 1000 bases from pYK338 directly with the aid of a mutagenic 21-mer oligonucleotide failed. This might be explained by a marked lowering of the probability of annealing between the primer and the template, so, to facilitate the deletion, a major part of the intervening DNA was deleted with the aid of restriction enzymes (Fig. 2). pYK338 was cut Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41 1778 K.-I. T A C H I B A N A A N D O T H E R S H p u n SUII l ? EcoRl (partially) T,-ligase I 17h ( Hbtd I I I iHnal I 1 Helper phage fd stranded ApY K 3.78 Fig. 2. Construction of pAMYA. phoA’ represents the promoter and the coding regions for the signal peptide of the E. coli alkaline phosphatase gene; amyE represents the structural gene of the B. subtilk aamylase; sfp represents the intergenic region of the filamentous f t phage; MCS represents the multiple cloning sites. pYK338 was converted to ApYK338 by restriction enzyme trimming and it was further converted to pAMYA by oligonucleotide-directed deletion. The latter deletion was facilitated by making ApYK338 into a single-stranded circular form by fd phage function. with Sun and a 1900 bp fragment containing the amino-terminus of the amyEgene was purified. This fragment was cut with HpaII and the staggered ends were filled in using DNA polymerase Klenow fragment. After cutting with EcoRI, the 450 bp fragment was purified; it has one blunt and one EcoRI-compatible end. Separately, pYK338 was cut with HindIII, the staggered ends filled in using DNA polymerase Klenow fragment, and, after cutting partially with EcoRI, ligated to the 450 bp fragment. This new plasmid (ApYK338) is about 1000 bp shorter than pYK338. The strain carrying ApYK338 did not produce a-amylase because of the reading frame shift for the amyE gene. Oligonucleotide-directed site-specific deletion The single-stranded derivative of ApYK338 was prepared as described in Methods. The synthetic 21-mer oligonucleotide was 5’-phosphorylated and used as a primer for DNA synthesis on the single-stranded circular DNA. E. coZi MC1061 was transformed with the reaction mixture Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41 1779 Secretion of a-amylase in E. coli Table 1. Distribution of a-amylase and marker enzymes in E. coli K12 LE392 (lac+) All three enzymes were assayed in the same culture. The results are mean values f SD of five independent experiments. Enzyme activity* Enzyme Plasmid a- Amylase None pYK331 pYK338 pAMYA Alkaline phosphatase None pYK331 pYK338 pAMYA B-Galactosidase None pYK331 pYK338 pAMYA Total Periplasmic 0.1 0 0.1 0.1 1.1 f 0.1 8.6 f 1.0 0.7 f 0.1 56.5 f 3.0 0.1 10.1 f 0.7 0.2 9.7 f 0.5 0.1 11.4 f 1.1 0 0.1 0 0 1.0 f 0.1 7.8 f 0.5 0.7 f 0.1 55.4 f 2.8 0 9.8 f 0.5 0.2 9.3 f 0.5 0.1 10.9 f 1.0 0 10.4 f 0.9 0.1 0-2 0.1 10.9 f 1.0 33.3 f 1.5 47.3 f 3.6 42-9 f 2.6 58.8 f 3-8 43.5 f 2.2 49.3 f 3.1 45.4 f 2.6 61.4 f 2-9 0.1 0 0.1 0.7 f 0-1 0.1 0.2 * a-Amylase activity, units ml-l (ODSSounit)-'; x pgalactosidase activity, units ml-l (ODSS0)-l; alkaline phosphatase activity, AAd2,-, min-' ml-I (ODSso unit)-'. and transformants were selected for ampicillin resistance. Some transformants should carry mutant plasmids. Transformants were mixed, plasmid DNAs prepared from them, and digested to completion with Hind111 which should not cut the deletion plasmid. This mixture was used to transform E. coli YK660; ampicillin-resistant transformants were selected and tested for aamylase on inducing agar plates containing 1% (w/v) starch. Of fifty transformants tested three gave large haloes on exposure to iodine vapour. Characterization of the mutant plasmid The three transformants were purified and plasmid DNA prepared from them. Singlestranded derivatives of the plasmids were prepared by superinfection with fd phage. The DNA sequence covering the junction of the phoA and the amyE genes was determined by the dideoxy chain-termination method (Sanger et al., 1977) and was found to be identical for all three plasmids. The direct and 'in frame' linkage of the two genes was also verified. One of these plasmids (pAMYA) was examined further. Enzyme localization a-Amylase and other marker enzyme activities were assayed in each culture after subcellular fractionation : alkaline phosphatase, a typical periplasmic enzyme, was found entirely in the periplasmic fraction (Table 1) and /?-galactosidase,a cytoplasmic enzyme, was absent both from periplasm and medium. As E. coli K12 MC1061 is a lacX74 deletion mutant, E. coli K12 LE392 (lac+)transformed with pYK331, pYK338 or pAMYA was used in this experiment. In the inducing medium, the strain carrying pAMYA produced 7-fold higher activity than those carrying pYK338. Almost all the activity was found in the periplasmic fraction and not in the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41 1780 K.-I. TACHIBANA A N D OTHERS Fig. 3. Autoradiogram of 12.5% (w/v) SDS-PAGE of [3SS]methionine-labelledwhole cells or the periplasm fraction from strains LE392/pYK33 1 or LE392/pAMYA. Cells were labelled with [3sS]methioninefor 2 h, harvested, and used for enzyme assay and product analysis by SDS-PAGE. Lanes 1, 2, 5, 6, 9 and 10, pYK331; 3, 4, 7, 8, 11 and 12, pAMYA; 1-4, whole cells; 5-8, periplasm fraction obtained by osmotic shock ; 9-1 2, anti-a-amylase antiserum immunoprecipitate of periplasm fractions; 2,4,6,8,10 and 12, inducing conditions; 1,3,5,7,9 and 11, repressing conditions. The bands of a-amylase are indicated by the arrow. medium after subcellular fractionation. The a-amylase activity was entirely in the periplasm. These results show that the promoter and the signal sequence ofphoA in pAMYA can efficiently direct the production and secretion of the B. subtilis a-amylase into the periplasm of E. coli. To examine the products from pAMYA, cells carrying pAMYA or pYK331 were labelled with [3SS]methionineand subjected to SDS-PAGE after subcellular fractionation. Each periplasmic fraction was immunoprecipitated with rabbit anti-a-amylase antiserum and was analysed by SDS-PAGE (Fig. 3). In lanes of the whole cell lysate and the periplasmic fraction of pAMYA under inducing conditions, aspecific band with an M , value corresponding to the B. subtilis aamylase (59 x lo3) was found on the autoradiogram (Fig. 3, lanes 4 and 8). In the autoradiogram of immunoprecipitates, a specific band was detected only in the cells carrying pAMYA under inducing conditions (lane 12). The a-amylase in the periplasmic fraction was purified and the five amino-terminal residues shown to be Glu-Thr-Ala-Asn-Lys-. This sequence is that predicted if the precursor polypeptide is correctly processed by the E. coli signal peptidase. DISCUSSION As shown in Table 1, a small amount of B. subtilis a-amylase was synthesized and secreted into the periplasm of E. coli carrying pYK338 even under the repressed condition of the phoA promoter. It is quite possible that the promoter of the B. subtilisamyEgene can function in E . coli Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41 Secretion of a-amylase in E. coli 1781 because the sequence of the promoter does not differ from the consensus sequence of the E. coli promoter (Hawley & McClure, 1982). The B. amyloliquefaciensa-amylase gene is also expressed in E. coli (Palva et al., 1981). The increased a-amylase activity in strain LE392/pYK338 in derepressing conditions may be due to considerable readthrough from the phoA promoter. The trace of a-amylase activity recorded for strains which should not possess the amyE gene is probably due to some reducing substances which influence colour development in the assay. This is a disadvantage of this assay method. It is a curious phenomenon that b-galactosidase activities increased slightly in low-phosphate media (Table 1). The promoter of the lac operon may be further activated or some upstream promoter may be derepressed in this strain. A single large protein (M,54 x lo3) is present in the periplasmic extract in repressing conditions (Fig. 3). It should be different from elongation factor Tu ( M , 44 x lo3), which is released by osmotic shock and represents a major protein in some cases (Jacobson & Rosenbusch, 1976), but its nature is as yet unknown. The extra bands at an M , of about 24 x lo3 in lane 6 should be products of some open reading frame downstream of thephoA promoter in pYK33 1, because their expression is enhanced under derepressed conditions and eliminated when the amyE gene is inserted behind the phoA promoter. Our results show that B. subtilis a-amylase was efficiently expressed and secreted into the periplasm of E. coli with the aid of the phoA promoter and signal sequence. Production of aamylase on inducing agar plates containing starch could be easily detected by halo formation on exposure to iodine vapour. This halo formation was probably due to some cell lysis, because aamylase was not detected in the liquid culture medium (Table 1). The DNA sequence of the B. subtilis a-amylase is known so the amino acid sequence of its precursor protein can be deduced (Ohmura et al., 1983). It has an amino-terminal extension of 41 amino acid residues. If this sequence is the signal peptide, it is much longer than signal peptides found in other exported proteins of prokaryotes and eukaryotes (Kreil, 1981). Possibly only a part of this sequence forms a signal peptide. For this reason it has been suggested that the precursor protein of the B. subtilis a-amylase may be cleaved twice to form the mature extracellular enzyme (Ohmura et al., 1983; Yamazaki et al., 1983), as reported for the formation of exopenicillinase of Bacillus licheniformis (Nielsen & Lampen, 1982) and Staphylococcus aureus (McLaughlin et al., 1981). In the present study we constructed a plasmid, pAMYA, in which the end of the phoA signal sequence is directly linked to the predicted first cleavage site of the B. subtilis a-amylase sequence, using the technique of oligonucleotide-directed site-specific deletion, and examined whether the precursor protein synthesized by thephoA’-amy’E gene is cleaved twice or not in E. coli like the extracellular a-amylase in B. subtilis. 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Nucleic Acids Journal of Bacteriology 156, 327-331. Research 8, 5473-5489. YANG, M., GALIZZI, A. & HENNER,D. (1983). NEIDHART, F. C., BLOCH,P. L. &SMITH, D. F. (1974). Nucleotide sequence of the amylase gene. Nucleic Culture medium for enterobacteria. Journal of Acids Research 11, 237-249. Bacteriology 199, 736-747. NEU, H. C. & HEPPEL,L. A. (1965). The release of enzymes from Escherichia coli by osmotic shock and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 17:55:41