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Gene expression using Bacillus
Gene expression using Bacillus Georges Rapoport and Andr~ Klier D e p a r t m e n t of Biotechnology, Institut Pasteur, 75724 Paris Cedex 15, France Current Opinion in Biotechnology 1990, 1:21-27
Introduction The search for systems to synthesize proteins has led to renewed interest in the species of the Bacillus genus, largely because of their ability to produce large amounts of proteins. The hope is to use Bacilli to produce foreign proteins in secreted or in non-secreted forms. Fortunately, the Bacillus species are well characterized in terms of genetics and physiology, and their manipulation by recombinant DNA technology is well developed. In addition, most Bacilli are non-pathogenic to humans, animals and plants and do not produce endotoxins, which is a considerable advantage when the protein to be purified is for medical purposes. Finally, some species of Bacillus secrete a variety of proteins, some in large quantities, indicating the presence of developed and efficient machinery for protein secretion and also the presence of strong promoters that could be used for gene overexpression. Moreover the cell envelope of Gram-positive bacteria is more amenable to protein secretion than that of Gram-negative bacteria, because the exported proteins only have to cross the cytoplasmic membrane to be released from the cells. Several host-vector systems have been developed in Bacilli for the expression and in some cases secretion of homologous and heterologous gene products. This short review will be limited to a discussion of developments in the overexpression of some heterologous proteins in Bacillus subtilis and Bacillus brevis that have been reported in the last 2 years. The ef~ciencies of differently constructed vectors will be compared with regard to yield, the type of the foreign products and their biological activities. The limitations of each system will also be discussed with particular reference to their stability, their ability to secrete cytoplasmic proteins and the degradation of their recombinant gene products by proteases, which are produced in high concentrations by Bacillus species.
Expression in B. subtilis The alkaline protease system of B. amyloliquefacMns The subtilisin of Bacillus amyloliquefaciens ( apr BamP ) is synthesized as a preproenzyme of 275 amino acid
residues and contains a signal peptide of 30 amino acids. The cleavage site is at position 107 of the pro-enzyme [1]. Using oligonucleotide-directed mutagenesis Vasantha and Filpula [2*] have recently fused in-frame a complete synthetic gene bpr, coding for the mature bovine pancreatic ribonuclease A, to the signal peptide coding region of apr and placed it under the control of the apr promoter. The resulting gene fusion was introduced into an Escherichia colgB, subtilis shuttle vector derived from pBR322 and pC194. The eukaryotic gene product (13 kD) was shown to be expressed in B. subtilis and was identified, by westem blotting, in both the cellular and supernatant fractions. The fusion gene product was processed at a single site located between the C-terminal Ala residue of the signal peptide and the N-terminal Lys residue of the mature protein. In addition, the secreted enzyme was active, suggesting that the disulfide bridges had formed correctly. The yield of the recombinant ribonuclease A in the culture medium was estimated to be several mg per liter. This low level is probably a result of proteolysis rather than poor production and does not reflect the ability of B. subtilis to secrete eukaryotic heterologous proteins. Another fusion, which included four additional amino acids between regions encoding the signal sequence of apr and the beginning of the gene for mature bpr, was constructed by inserting a useful BamHI site two codons from the end of the signal peptide. The resulting protein was also processed at a site located downstream of the signal peptide. This 'imperfect' fusion led, nevertheless, to the production of a ribonuclease A variant that had detectable activity, as shown by a novel plate assay. This expression system has already been used successfully by the same group for the secretion of the protein A from Staphylococcus aureuswith a yield of up to 3 g/liter [3].
The (z-amylase system of B. amyloliquefaciens It has been proposed that the diphtheria toxin, when joined to a specific antibody or a hormone, might be used to attack cancer cells. The production of diphtheria toxin in B. subtiliswas recently reported by Palva and colleagues [4]. The toxin gene was cloned from the chromo somal DNA of a non-toxic mutant of Corynebacterium diphtheriae tox228 that produces the inactive toxin, CRM
Abbreviations CAT--chloramphenicol acetyltransferase; HIV~human immunodeficiency virus; hEGF~human epidermal growth factor; MWP--middle wall protein; mDHFR--mouse dihydrofolate reductase; ODu,0--optical density at 660 nm; OWP---outer wall protein; RT--reverse transcriptase; SDS-PAGE--sodium dodecyl sulfate polyacrylamide gel electrophoresis; tPA--tissue plasminogen activator. (~ Current Biology Ltd ISSN 0958-1669
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Expressionsystems 228. The structural gene of the mature protein was inserted into a pUB110-derived a-amylase secretion vector, which contains the promoter and the encoding region of the signal sequence of wamylase (31 amino acids) followed by a HindIII linker. Two truncated forms of the toxins, lacking 151 C-terminal amino acids, were constructed by site-directed mutagenesis. These truncated forms may be useful for toxin conjugates. Western blotting experiments indicated that both the full-size CRM 228 polypeptide (58 kD) and the truncated forms (42 kD) were expressed and secreted by B. subtilis. However, no details were reported of how the hybrid proteins were processed. The maximum amount of the full-size toxin present in the supernatant of flask culture was about 4 mg/liter. The truncated forms were less stable than the whole protein; it was also shown that the culture conditions affected the yield of the CRM 228 production and that the use of a B. subtilis host lacking the main extracellular proteases did not improve the yield. The same a-amylase vector system has already been used successfully for the production of the pertussis toxin subunit $1 from Bordetella pertussis [5]. Pertussis toxin, which is composed of five subunits, is a promising candidate for an acellular vaccine against whooping cough. The $1 product recovered from the supernatant of B. subtills had a similar ADP-fibosylation activity to that of the native pertussis toxin. The total amount of the protein found in the medium was about 100 mg/liter, but most o f this was truncated or proteolysed forms of the toxin. More recently [6"], all the pertussis subunits were produced in the culture supematant of a low-exoproteaseproducing strain of B. subtilis, but the amounts obtained varied considerably: Sl and S5 were produced in large quantities (100 mg/liter and 60mg/liter, respectively), whereas much lower amounts (a few mg per liter) of S2, S3 and S4 were obtained. Membrane-associated subunits were also found; these were mainly S5. The introduction of a HindlII site, useful for DNA manipulations by site-directed mutagenesis, caused some minor changes in the N-terminal amino acids of the mature pertussis subunits. In the cases of $2 and $4, a Lys residue was introduced at position + 2. This was thought to explain the poor expression observed but the replacement of these Lys residues by the Thr and Asp residues found in the wild-type did not noticeably change the yield of the corresponding proteins. The precise locations of the processing sites in the different constructions were not reported. Another application of the a-amylase system has been the production of pneumolysin, a promising diagnostic antigen from Streptococcuspneumoniae that is not normally secreted [7"]. Pneumolysin was produced at low level by B. subtilis when expressed from the p U B l l 0 vector with its own promoter. The protein was active but not secreted into the rfi'~dium. Higher level expression was achieved when the entire coding sequence or the two regions encoding the C-terminus (corresponding to amino acids 265-471 and 55-471) were fused to the promoter and signal sequence of the a-amylase gene of B. am)* loliquefaciens. The fusion proteins containing the C-ter-
minus were not active and remained cell associated. But when the coding sequence was fused, a hemolytically active form was recovered from the culture medium in the expected processed form (50 kD). After partial purification, the total yield of pneumolysin was about 10 mg/liter. This material was produced in sufficient amounts and in a suitable form for use in enzyme-linked immunosorbent assays. The first description of the secretion in B. subtilis of a plant enzyme, the a-galactosidase from Cyamopsis tetragonoloba, was recently reported by Overbeeke et al. [8--]. The vector used included the SPO2 promoter from plasmid pPL608, a synthetic ribosome-binding site and the a-amylase gene signal sequence from B. amylolique faciens. The expression was analyzed in cell extracts and in the supernatant of a npr E apr A strain of B. subtilis. Extracellular a-galactosidase concentrations reached a level of 1700U/ml (which represents about 10mg per liter) after 12 h of cultivation in a super-rich medium. One specific band with molecular mass of 40 kD, compatible with the deduced amino-acid composition of the mature plant enzyme (39 777 D), was detected by western blot analysis. This value is slightly lower than that of the enzyme obtained from the original plant or from Saccharomyces cervisiae, and reflects the absence of protein glycosylation in B. subtilis. Identification of the first 20 amino acids of the secreted a-galactosidase showed that the processing of the a-amylase signal sequence had been correctly performed, with specific cleavage between two Ala residues. Furthermore, the enzyme recovered from the B. subtilis medium behaved in the same way with the natural substrate (guar gum) as the enzyme derived from the plant.
The levansucrase system of B. subtilis Levansucrase is one of many enzymes that are secreted by B. subtilis. It is specifically induced by sucrose, and is expressed during the logarithmic growth phase. Levansucrase is encoded by the sacB gene and expressed from a constitutive promoter in the closely linked sacR locus. The sacR locus contains a palindromic structure acting as a transcription terminator. In the presence of sucrose, an anti-terminator, the sacYgene product that belongs to the sacS operon, allows transcription of the sacB gene. The expression of this gene is also controlled by other regulatory genes such as the two-component system degS/degU and also by degQ (formerly sacU and sacQ). Several upmutations are available: degUb, degff °, sacSb and sacs c. These factors make the sacB system a suitable candidate for the construction of an efficient secretion system for heterologous proteins. An expression and secretion vector has been constructed which includes the sacB promoter, the regulatory sequence sacR and the entire signal sequence of levansucrase, which was reconstructed with a synthetic oligonucleotide. The inclusion of a useful restriction site, NaeI, at the end of the signal sequence allows fusions in the three possible reading frames [9]. As a model system, the TEM-]3-1actamase gene from E. coli
Gene expression using Bacillus Rapoport and Klier was fused in phase with the signal sequence. The expression and secretion of the enzyme was tested in a sacSb strain of B. subtilis after introduction of the construction in a shuttle vector, E. coli/B, subtilis, derived from pC194. Assays of ]3-1actamase activity demonstrated substantial sucrose-inducible secretion of the enzyme during the vegetative phase. More than 80% of the gene product was found in the supematant of the cell culture. The inducible and enhancible expression and secretion system based on sacB has recently been further improved by introduction of the regulatory gene degQ [10"]. The degQ gene was expressed constitutively from a strong promoter, P43, during both the vegetative and stationary phases. The presence of the P43-degQ cassette on a multicopy plasmid increased the activity of [3-1actamase by about 17-fold. This was similar to the activity measured when the sacRB-~-lactamase fusion lacking the degQ gene was introduced into a degUb-containing strain of/3. subtilis. No additive effect was observed when the construct containing the degQ cassette was introduced into the degOr° strain. The sacB system has also been used for the inducible secretion of a cellulase (EGA; encoded by the celA gene) from Clostridium thermocellum [11.]. This protein is another example of a naturally secreted protein. Expression of EGA in different B. subtilis genetic backgrounds ( degUh, sacSb, sacS c) showed that its regulation is similar to that of levansucrase. The highest enzyme activity was recovered, as expected, from the culture supematant of a degUb strain. Concentration corresponded to approximately I mg/liter per unit of optimal density at 660 nm (OD660) , and compares favorably with EGA production in C. thermocellum or in E. coll. The molecular weight of 46 kD, calculated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the secreted polypeptide, is compatible with the calculated molecular weight of the mature enzyme. In these experiments, the celA gene was carried on a multicopy vector that was derived from pC194. However, such hybrid plasmids are often unstable in B. subtilis. To avoid instability in the structural plasmid, the foreign gene was more recently inserted into the chromosome of B. subtilis and allowed to amplify [12,.]. It has been shown already that a construct that consists of a genetic marker (a gene encoding antibiotic resistance) flanked by directly repeated sequences can be amplified in the B. subtilis chromosome by increasing the selective pressure and that the resulting amplified structure contains tandemly repeated amplification units (about 20 to 50 per chromosome). It had also been shown that gene amplification can be induced by the presence of an active origin of replication of a plasmid integrated into the B. subtills chromosome. This phenomenon, which takes place in the absence of selective pressure, has been applied [12-.] to the previously described sacRDcelA fusion. The construct includes plasmid pE194, which is thermosensitive for replication, and an amplification unit comprising direct repeated sequences of pBR322, the cat gene encoding the chloramphenical acetyltransferase (CAT) and a 2.7kb DNA fragment, which carries the sacR-celA fu-
sion. A two-step amplification procedure yielded up to 250 amplification units per chromosome and these were stably maintained in the absence of selective pressure. The cellulase, EGA, was the major secreted protein, and analysis of the N-terminal processing site revealed three different cleavage sites in the vicinity of the signal peptidase recognition sequence; 30% of the molecules were cleaved at the predicted site. Judging from the specific activity of the secreted enzyme, it seems likely that all three processed forms were active. The kinetics of the cat gene expression were followed in parallel with EGA production. CAT activity, which is found intracellularly, increased 50-fold in 25 h, whereas EGA activity increased only 10-fold, suggesting that other limiting factors modulate the expression of the cela gene and/or the secretion of its product. A level of 10 mg/liter per OD660 unit was reached after 25 h in a degUb B. su~ tills mutant cultivated in rich medium, corresponding to a 10-fold increase in EGA production compared with the multicopy plasmid strategy. Induction of DNA amplification into the chromosome seems to be a valuable alternative to multicopy expression vectors, especially as plasmid instability is often observed in 13. subtilis. The secretion of an eukaryotic protein using the sacB system has also been tried but with much less success [13]. The mouse a7 interferon gene, the signal sequence of which had been removed, was introduced into the levansucrase secretion vector. The interferon protein was found at low levels in cell extracts from both the wildtype strain and the degUb mutant. Modification of the amino acid composition of the signal peptide and mature interferon junction, which restored an a-helix conformation had no effect on the secretion of the product. These results suggest that, in t3. subtilis, polypeptide structures other than those found around the cleavage site are involved in the secretion of eukaryotic proteins.
The staphylokinase expression system Staphylokinase (Sak) is a 15.5kD enzyme secreted by some strains of Staphylococcus. This enzyme is responsible for the plasminogen activation that leads to fibrinolysis. The structural gene encoding staphylokinase (Sak42D) is located on phage genomes and has been cloned and sequenced from phages S~C and 42D. When a DNA fragment carrying the gene for Sak42D was introduced into the Gram-positive hosts B. subtilis and Strep tococcus sanguis, active Sak was detectable in culture supematants of both Gram-positive hosts [14]. The yield of Sak at the end of the log phase of B. subtilis reached 25 mg/liter of culture supematanc Sak purified from cub ture supernatant yielded two protein fractions after analysis by SDS-PAGE. The larger, minor polypeptide was probably unprocessed Sak [14]. Breitling et al. [15".] have used a DNA fragment carrying the signal sequence coding region, the ribosome-binding site and the putative promoter of the sak 42D gene as a portable expression secretion unit. Insertion of this unit in front of the genes for human interferon a l (hlFNa) and a hybrid h I F N a l / a 2 resulted in the expression and
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Expressionsystems secretion or export of the heterologous gene products in both Gram-positive and Gram-negative hosts. Expression of these constructs in E. cull led to some instability in the recombinants strains, but no such instability was observed in B. subtilis although the expression level was higher. In B. subtilis, the IFNcz secretion started in the early log phase and continued constitutively until growth ceased. The concentration of hlFNcz in the culture at the end of the log phase was about 15 mg/liter. However, prolonged incubation resulted in a significant drop in production caused by residual protease activity. One natural vector used by Breitling et al. [15 °°] for interferon expression was highly stable even in the absence of selective pressure. The hlFNczl was purified from culture supematants of B. subtilis and then analysed. A single band, with no detectable degraded products, was visualized and the determination of the N-terminal amino-acid sequence confirmed the homogeneity of the preparation and the accurate processing of the sak signal peptide at the expected site. The mature recombinant IFNczl protein carried the expected 11 amino-acid N-terminal extension that resulted from DNA manipulation, but this additional tail did not change the specificity of the IFNal protein. Curiously, the same recombinant plasmid when introduced into S. sanguis, another Gram-positive host, did not lead to significant synthesis of hlFNczl. The main limitations of this system are its relatively narrow host range, and the synthesis of mature heterologous polypeptides with a N-terminal extension.
Lac-based inducible systems High-level expression of a foreign gene might be detrimental to the host. In E. cull, this disadvantage has been overcome by using inducible expression cassettes, the most widely used systems being derived from the E. coli /ac, trp and lambda CI repressors. In order to increase the potential of B. subtilis as a host for the high-level expression of foreign genes, the use of similar regulatory elements would be an advantage. Yansura and Henner [16] used the E. cull lac operator and repressor, together with a promoter from the phage SPO1, to control gene expression of human leukocyte interferon in B. subtilis. This system has also been shown to be a very useful tool for studying the expression, in the original host, of some regulatory genes that had their own promoters deleted [17"]. Some promoters and terminators of Gram-negative ori gin are recognized by purified 0 -43 RNA polymerase from B. sublilis. One such promoter, PN25, from the bacteriophage T5 genome was shown to be able to initiate the transcription of a foreign gene leading to an accumulation of the gene product at a level up to 25% of the total cellular protein [18,1,9]. This promoter was fused to a synthetic DNA fragment carrying the E. coli lac operator generating a regulatable promoter PN25/O [20"°]. This promoter was linked to a synthetic ribosome-binding site followed by a multicloning site. This represents the first component of a lac-based inducible system for B. sub tills. The second component is a lac repressor gene that
is able to be transcribed and translated in B. subtilis. This was constructed by replacing the authentic promoter and ribosome-binding site with the equivalent Gram-positive elements. Two expression systems were created [20.°]. The first one was made up of an E. colgB, subtilis shutfie vector pREP9 harboring the two components of the lac-based inducible system. The second expression system was composed of a B. subtilis strain constitutively producing the lac repressor from a derivative of the S. a u r e u s plasmid pE194 (pBL1) and a compatible shuttie expression vector containing the regulatable PN25/O (p602/20). The plasmid pREP9 was used to overproduce bacterial CAT after addition of the inducer IPTG. After 2 to 3 h of induction, the CAT protein was the major cellular protein, accounting for up to about 15% of the total cell protein. The dual plasmid expression system was also used to produce eukaryotic polypeptides such as the mouse dihydrofolate reductase (mDHFR), the human immunodeficiencyvirus (H1V)-I, reverse transcriptase (RT) and the mature part of human tissue plasminogen activator (tPA) [20o.,21,22.]. All three polypeptides were synthesized intracellularly by B. subtilis after induction and were biologically active. The tPA was localized in inclusion bodies, and was therefore easily purified. The enzymatic ac tivity could be partially recovered after complete reduction and slow oxidation of solubilized inclusion bodies. As with melanoma tPA, the recombinant tPA exhibited very high levels of plasminogen activation in the presence of fibrin, even though the recombinant product had a 3 amino-acid N-terminal extension. However, the total amount of tPA produced by B. subtilis was relatively low (about 0.5% of the total cell weight) compared with the values reported for the synthesis of the mDHFR (10% of the total protein) [20..]. A translational fusion between the E. coli cat gene and the open reading frame of HIV p o l gene was constructed [21]. After 2 h of induction, a high-molecular-weight polypeptide was synthesized, consistent in size with the pol encoded precursor polyprotein. After prolonged incubation, the precursor polypeptide disappeared and two polypeptides of molecular weights 64 and 52 kD were produced. They corre sponded in size to H1V-1 RT polypeptides, suggesting a correct processing in B. subtilis. These polypeptides were purified to near homogeneity and displayed high levels of RT activity. However, the expression level was relatively low, and the yield of pure enzyme was not sufficient for industrial use. In order to increase the potential of the dual plasmid inducible system and to overcome the disadvantages of using two compatible plasmids, the lacI gene with its Gram-positive control elements was intro duced into the chromosome of B. subtilis, using phage (I)105 as a cloning vehicle [20"]. This system is now under evaluation. In the T5-based expression system [18, 19,20°',21,22"], the foreign genes were expressed during logarithmic growth, when the production of extracellular proteases is low. Expression during later growth phase requires modification of the expression cassettes by introducing promoters recognized by the transcriptional machinery
Gene expression using Bacillus Rapoport and Klier present during the stationary phase. Such twin promoters would ensure constant synthesis of lac repressor and the foreign polypeptides. Use of B. subtilis mutants devoid of the major protease genes, as in the example of tPA, would allow an accumulation of the heterologous gene product during the stationary phase. Alternatively, these systems could be modified to secrete the foreign polypeptides to the culture supemfttant by inserting a cartridge containing a DNA fragment encoding an exoenzyme signal peptide downstream of a regulatable promoter. This type of approach has already been used, employing the signal sequence of the cz-amylase to secrete the E. coli 13-1actamase [20-.]. Although B. subtilis is an efficient extracellular enzyme producer and has been shown to be a good proteinsecreting host, some mammalian proteins produced by this bacterium are often degraded by its extracellular proteases, even in those B. subtilis mutants that lack the major proteases. Another Bacillus species, B. brevis secretes large amounts of protein but little or no detectable proteases.
Expression in B. brevis One particular strain, B. brevis47, accumulates 12-25 g of extracellular proteins per liter of medium under appropriate growth conditions [23"]. These extracellular proteins have been shown to derive from the cell-wall proteins, based on morphological, immunological and biochemical criteria. In the case of B. brevis 47, the secreted proteins consist mainly of two polypeptides of 130 kD (OWP; outer wall protein) and 150kD (MWP; middle wall protein) [23"]. These proteins are synthesized, during the log phase and form hexagonal arrays on the cell surface of the bacteria. During the stationary phase, these proteins continue to be synthesized, but instead of being deposited on the cell surface they are secreted into the medium. The MWP and OWP genes of B. brevis have recently been cloned and sequenced [23"]. They are organized in an operon (cpw operon) and interrupted by an untranslated sequence of 130 bp. Both MWP and OWP are synthesized in precursor forms with signal peptides of 23 and 24 amino-acid residues, respectively. Five transcriptional start points were identified by primer extension and Sl analysis [23",24"]. The corresponding frye promoters were located in the 300 bp upstream from the MWP coding sequence. One of them, the P2 promoter, led to the efficient and constitutive expression of the c w p operon in B. brevis 47. Interestingly, this promoter was not used efficiently in B. subtilis and it does not show homology with the other promoter sequences reported for Gram-positive bacteria. This result suggests that the P2 promoter is a specific promoter that is used preferentially in B. brevis [24"]. The 5' region of the cwp operon contains two possible translational start sites located tandemly in the same read-
ing frame [23"]. Each of these sites contains a sequence that is highly complementary to the 3' end of B. brevis
rRNA, and an initiation codon. The amino-acid sequence that stretches from Met-23, the N-terminus of the translational product from the second initiation site, to Ala-1 shows all the characteristics of signal peptides of secretory precursors. The translational product from the first site contains an extra peptide of 31 amino-acid residues preceding a typical MWP signal sequence. Fusion experiments indicate that both initiation sites are used in B. brevis. However, translation appears to start at the second site much more frequently than at the first site. An expression vector was constructed for use in heterologous protein production in B. brevis [23"]. Its replication origin came from either a natural plasmid identified in some B. brevis strains (pWT481) or the pUB110 plasmid of S. aureus. A 600 bp DNA fragment was inserted into the vector and contained the 5' region of the B. br~ vis 47 MWP gene, including the multiple promoters and ribosome-binding sites, as well as the region encoding the signal peptide and the N-terminal portion of the mature form of MWP. A synthetic human epidermal growth factor (hEGF) gene was cloned into this expression vector, so that the fused gene encodes the MWP signal peptide directly followed by the mature hEGF sequence [25"]. The recombinant plasmid was introduced into both B. brevis 47 and HPD 31 using a novel Tris hydrochloride buffer/polyethylene glycol transformation method [23"]. B brevis strain HPD 31 carrying the recombinant plasmid produced as much as 240 mg of extracellular hEGF per liter of culture. The amount of hEGF increased markedly during the early stationary phase and, interestingly, remained almost constant after 3 days of incubation. Most of the hEGF was found in the culture supematant. This polypeptide was purified and had the same N-terminal and C-terminal amino-acid sequences, and amino-acid composition as natural hEGF. Moreover, the recombinant hEGF showed almost the same specific biological activity as authentic hEGF. These results suggest that the hEGF synthesized in B. brevis is correctly processed and efficiently secreted, and that the correct disulfide bridges are formed. In order to demonstrate that the B. brevis host vector system is useful for large-scale production of any mammalian protein, the cDNA of human cz-amylase and the cDNA of porcine pepsinogen A were independently fused to the MWP signal peptide coding sequence [23.,26.]. In both cases, the foreign polypeptides were secreted from B. brevis, although the amount was about 6 and 20 times less, respectively, than that of hEGF. Also, in both cases, proteins were found in the culture supematant and were similar in size and in function to the authentic polypeptides. Under acidic conditions, the rate of the activation process of the extracellular pepsinogen was the same as that of the natural proenzyme. In conclusion, the genes for several bacterial and mammalian secretory proteins have been expressed efficiently with the 13. brevis expression system and large quantities of biologically active proteins have been easily purified
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Expressionsystems from the culture medium. Proteins produced by 13. brevis usually remain stable, even after prolonged cultivation. This stability may be a result of the absence of extracellular proteases. The different proteins were expressed at different levels which suggests that the secretory machinery of this bacterium has some limitations. It also remains to be seen whether 13. brevis can secrete non-secretory polypeptides.
Annotated references and recommended reading • o•
Of interest Of outstanding interest
1.
VASANTHAN, THOMPSON LD, RHODES C, BANNER C, NAGLE J, FILPUIA D: G e n e s for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large ORF bet w e e n the regions coding for signal s e q u e n c e and m a t u r e protein. J Bacteriol 1984, 159:811-819.
VASANTHAN, FILPULA D: Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus sutr this. Gene 1989, 76:53~50. Expression of a small eukaryotic synthetic gene using the apr system of B. amyloiiquefaciens. The product was secreted by B. subtilis in an 2.
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active form. 3.
VASANTHAN, THOMPSON LD: Secretion of a heterologous protein from Bacillus subt~'lis with t h e aid of protease signal sequences. J Bacteriol 1986, 165:837-842.
4.
HEMILAH, GLODE LM, PALVAI: Production o f diphtheria toxin CRM228 in B. subtilis. FEMS Microbiol Lett 1989, 65:193-198.
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RUNEBERG-NYMANK, ENGSTROM O, LOFDAHL S, YLOSTALO S, SARVASM: Expression and secretion of pertussis toxin subunit SI in Bacillus subtills. Microbiol Pathogenesis 1987, 3:461468.
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SARIS P, TAIRA S, AIRAKSINEN U, PALVAA, SARVASM, PALVA I, • RUNEBERG-NYMANK: Production and secretion of pertussis toxin subunits in Bacillus subtilis. FEMS Microbiol lett 1990, 68:143-148. The five subunits of the pertussis toxin were secreted in B. subillis with the c~-amylase of B. amyloliquefaciens, but the level of their expression varied considerably. 7.
TA1RA S, JALONEN E, PATON JC, SARVAS M, RUNEBERG-NYMAN • K: Production of pneumolysin, a p n e u m o c o c c a i toxin, in Bacillus subtilis. Gene 1989, 77:211-218. Production of an active staphylococcal hemolysin with the a-amylase expression and secretion system of B. amyloliquefaciens. OVERBEEKE N, TERMORSHUIZEN GHM, GUISEPPIN MLF, o• UNDERWOODDR, VERRIPSCT: Secretion of t h e a-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis. Appl Environ Microbiol 1990, 56:1429-1434. First report of the expression and secretion of a plant enzyme with the 0t-amylase system of B. amyloliquefaciens. There was correct process ing of the hybrid precursor and the non-glycosylated enzyme secreted by B. subtilis was active.
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JOLIFF G, EDELMAN A, KLIER A, RAPOPORT G: Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Appl Environ Microbio11989, 55:2739-2744. inducible expression of a normally secreted enzyme using the levansuerase expression and secretion system in high-producing mutants of B. subtilis. 12. ••
PETITMA~ JOLIFF G, MESASJM, KLIERA~ RAPOPORTO, EHRLICH SD: Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtills by induction of chromosomal DNA amplification. Bio/Technology 1990, 8:559-563. Improvement of the chromosomal amplification procedure in 13. subtilis by the introduction of a plasmid origin of replication into the chromo some. Up to 250 copies of the amplified unit per chromosome were obtained in the absence of selective pressure. High inducible expression of a heterologous cellulase was studied as a model. 13.
DION M, RAPOPORT G, DOLY J: Expression of the MulFNa7 gene in Bacillus subtilis using t h e levansucrase system, Biochimie 1989, 71:747-755.
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BEHNKE D, GERLACH D: Cloning and expression in Escherichia coli, Bacillus subtilis and Streptococcus sanguis of a g e n e for staphylokinase - a bacterial plasminogen activator. Mol Gen Genet 1987, 210:528-534.
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BREITLINGR, GERLACHD, HARTMANNM, BEHNKE D: Secretory expression in Escherichia coli and Bacillus subtilis of hum a n interferon c~ g e n e s directed by staphylokinase signals. Mol Gen Genet 1989, 217:384-391. This paper describes the construction of a secretion vector based on the promoter and signal sequence of the sak gene. Significant expression level of hIFNcO or hlFN~zl/2 was obtained at the end of the log phase. Precise processing of the precursor form was also demonstrated. 16.
17. HENNER DJ: Inducible expression of regulatory g e n e s in • Bacillus subtilis. Methods Enzymol 1990, 185:223-228. Description of the utilization and integration into the chromosome of the inducible spac system in B. subtilis. 18.
PESCHKEU, BEUCK V, BUJARD H, GENTZ R, LE GRICE S: Efficient utilization of Escherichia coli transcriptional signals in Bacillus subt~'lis. J Mol Biol 1985, 186:547-555,
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ZUKOWSKIMM, MILLERL.' Hyperproduction of an intracellular heterologous protein in a sac Uo m u t a n t of B. subtilis. Gene 1986, 46:247 255.
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LE GPaCE SFJ: Regulated p r o m o t e r for highqevel expression of heterologous g e n e s in Bacillus subtilis. Methods Enzymol 1990, 185:201-214. A review of the construction of the /aobased inducible systems for B. subtilis and their use for the expression of heterologous proteins. 21.
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EDELMANA, JOLIFF G, KLIER A, RAPOPORT G: A system for the inducible secretion of proteins from Bacillus s u b tills during logarit vl~ ic growth. FEMS Microbiol Lett 1988,
52:117-120. 10. •
WONGSL: Development of an inducible and enhancible expression and secretion system in Bacillus sublllis. Gene 1989, 83:215-223. Improvement of the levansucrase expression and secretion system by introduction into the vector of a cassette containing the regulatory degQ gene. Use of TEM-[3-1actamase as a model protein for secretion.
YANSURADG, HENNER DJ: Use of Escherichia coli lac repressor and operator to control g e n e expression in Bacillus subtilis. Proc Natl Acad Sci USA 1984, 81:439443.
LE GRICE SFJ, BEUCK V, MOUS J: Expression of biologically active h u m a n T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis. Gene 1987, 55:95-103.
22. •
WANGLF, HUM x,X/W,KALYANNK, LEE SG, HUNG PP, DOI RH: Synthesis and refolding of h u m a n tissue-type plasminogen activator in Bacillus subtills. Gene 1989, 84:127-133. Use of a dual plasmid inducible system for the synthesis of tPA in /3. subtilis. The recombinant tPA was purified from inclusion bodies and was biologically active. 23, •
UDAKAS, TSUKAGOSHI N, YAMAGATAH: Bacillus brevis, a host bacterium for efficient extracellular production of useful proteins. Biotechnol and Gen Eng Rev 1989, 7:113-146. This review describes the construction of secretory vectors based on the MWP gene of 13. brevis. 24. •
ADACHIT, YAMAGATAH, TSUKAGOSH1N, UDAKA S: Multiple and tandemly arranged p r o m o t e r s of the cell wall protein gene o p e r o n in Bacillus brevis 47. J Bacteriol 1989, 171:1010-1016.
Gene expression Identification by primer extension and Sl analysis of five different promoters of the CWP operon. They are all differently regulated depending on the growth phase. 25. .•
YAMAGATA H, NAKAHAMAK, SUZUKIY, KAKINUMAA, TSUKAGOSHI N, UDAKAS: Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci USA 1989, 86:3589-3593. Construction of a highly emcient secretion vector based on the 5' region of a cell-wallprotein gene, leading to the high-level secretion (240 mg/1) of hEGF.
26.
using Bacillus R a p o p o r t a n d Klier
T ~ o M, MORIOKAT, YAMAGATAH, TSUKAGOSHIN, UDAKA S: Production of swine pepsinogen by protein-producing Bacillus brevis carrying swine pepsinogen cDNA. Appl Mi crobiol Biotecbnol 1989, 30:75-80. Ei~icient production of the porcine pepsinogen A in t3. brevis. The preenzyme is autocatalytic~y converted into the active form.
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Introduction The search for systems to synthesize proteins has led to renewed interest in the species of the Bacillus genus, largely because of their ability to produce large amounts of proteins. The hope is to use Bacilli to produce foreign proteins in secreted or in non-secreted forms. Fortunately, the Bacillus species are well characterized in terms of genetics and physiology, and their manipulation by recombinant DNA technology is well developed. In addition, most Bacilli are non-pathogenic to humans, animals and plants and do not produce endotoxins, which is a considerable advantage when the protein to be purified is for medical purposes. Finally, some species of Bacillus secrete a variety of proteins, some in large quantities, indicating the presence of developed and efficient machinery for protein secretion and also the presence of strong promoters that could be used for gene overexpression. Moreover the cell envelope of Gram-positive bacteria is more amenable to protein secretion than that of Gram-negative bacteria, because the exported proteins only have to cross the cytoplasmic membrane to be released from the cells. Several host-vector systems have been developed in Bacilli for the expression and in some cases secretion of homologous and heterologous gene products. This short review will be limited to a discussion of developments in the overexpression of some heterologous proteins in Bacillus subtilis and Bacillus brevis that have been reported in the last 2 years. The ef~ciencies of differently constructed vectors will be compared with regard to yield, the type of the foreign products and their biological activities. The limitations of each system will also be discussed with particular reference to their stability, their ability to secrete cytoplasmic proteins and the degradation of their recombinant gene products by proteases, which are produced in high concentrations by Bacillus species.
Expression in B. subtilis The alkaline protease system of B. amyloliquefacMns The subtilisin of Bacillus amyloliquefaciens ( apr BamP ) is synthesized as a preproenzyme of 275 amino acid
residues and contains a signal peptide of 30 amino acids. The cleavage site is at position 107 of the pro-enzyme [1]. Using oligonucleotide-directed mutagenesis Vasantha and Filpula [2*] have recently fused in-frame a complete synthetic gene bpr, coding for the mature bovine pancreatic ribonuclease A, to the signal peptide coding region of apr and placed it under the control of the apr promoter. The resulting gene fusion was introduced into an Escherichia colgB, subtilis shuttle vector derived from pBR322 and pC194. The eukaryotic gene product (13 kD) was shown to be expressed in B. subtilis and was identified, by westem blotting, in both the cellular and supernatant fractions. The fusion gene product was processed at a single site located between the C-terminal Ala residue of the signal peptide and the N-terminal Lys residue of the mature protein. In addition, the secreted enzyme was active, suggesting that the disulfide bridges had formed correctly. The yield of the recombinant ribonuclease A in the culture medium was estimated to be several mg per liter. This low level is probably a result of proteolysis rather than poor production and does not reflect the ability of B. subtilis to secrete eukaryotic heterologous proteins. Another fusion, which included four additional amino acids between regions encoding the signal sequence of apr and the beginning of the gene for mature bpr, was constructed by inserting a useful BamHI site two codons from the end of the signal peptide. The resulting protein was also processed at a site located downstream of the signal peptide. This 'imperfect' fusion led, nevertheless, to the production of a ribonuclease A variant that had detectable activity, as shown by a novel plate assay. This expression system has already been used successfully by the same group for the secretion of the protein A from Staphylococcus aureuswith a yield of up to 3 g/liter [3].
The (z-amylase system of B. amyloliquefaciens It has been proposed that the diphtheria toxin, when joined to a specific antibody or a hormone, might be used to attack cancer cells. The production of diphtheria toxin in B. subtiliswas recently reported by Palva and colleagues [4]. The toxin gene was cloned from the chromo somal DNA of a non-toxic mutant of Corynebacterium diphtheriae tox228 that produces the inactive toxin, CRM
Abbreviations CAT--chloramphenicol acetyltransferase; HIV~human immunodeficiency virus; hEGF~human epidermal growth factor; MWP--middle wall protein; mDHFR--mouse dihydrofolate reductase; ODu,0--optical density at 660 nm; OWP---outer wall protein; RT--reverse transcriptase; SDS-PAGE--sodium dodecyl sulfate polyacrylamide gel electrophoresis; tPA--tissue plasminogen activator. (~ Current Biology Ltd ISSN 0958-1669
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Expressionsystems 228. The structural gene of the mature protein was inserted into a pUB110-derived a-amylase secretion vector, which contains the promoter and the encoding region of the signal sequence of wamylase (31 amino acids) followed by a HindIII linker. Two truncated forms of the toxins, lacking 151 C-terminal amino acids, were constructed by site-directed mutagenesis. These truncated forms may be useful for toxin conjugates. Western blotting experiments indicated that both the full-size CRM 228 polypeptide (58 kD) and the truncated forms (42 kD) were expressed and secreted by B. subtilis. However, no details were reported of how the hybrid proteins were processed. The maximum amount of the full-size toxin present in the supernatant of flask culture was about 4 mg/liter. The truncated forms were less stable than the whole protein; it was also shown that the culture conditions affected the yield of the CRM 228 production and that the use of a B. subtilis host lacking the main extracellular proteases did not improve the yield. The same a-amylase vector system has already been used successfully for the production of the pertussis toxin subunit $1 from Bordetella pertussis [5]. Pertussis toxin, which is composed of five subunits, is a promising candidate for an acellular vaccine against whooping cough. The $1 product recovered from the supernatant of B. subtills had a similar ADP-fibosylation activity to that of the native pertussis toxin. The total amount of the protein found in the medium was about 100 mg/liter, but most o f this was truncated or proteolysed forms of the toxin. More recently [6"], all the pertussis subunits were produced in the culture supematant of a low-exoproteaseproducing strain of B. subtilis, but the amounts obtained varied considerably: Sl and S5 were produced in large quantities (100 mg/liter and 60mg/liter, respectively), whereas much lower amounts (a few mg per liter) of S2, S3 and S4 were obtained. Membrane-associated subunits were also found; these were mainly S5. The introduction of a HindlII site, useful for DNA manipulations by site-directed mutagenesis, caused some minor changes in the N-terminal amino acids of the mature pertussis subunits. In the cases of $2 and $4, a Lys residue was introduced at position + 2. This was thought to explain the poor expression observed but the replacement of these Lys residues by the Thr and Asp residues found in the wild-type did not noticeably change the yield of the corresponding proteins. The precise locations of the processing sites in the different constructions were not reported. Another application of the a-amylase system has been the production of pneumolysin, a promising diagnostic antigen from Streptococcuspneumoniae that is not normally secreted [7"]. Pneumolysin was produced at low level by B. subtilis when expressed from the p U B l l 0 vector with its own promoter. The protein was active but not secreted into the rfi'~dium. Higher level expression was achieved when the entire coding sequence or the two regions encoding the C-terminus (corresponding to amino acids 265-471 and 55-471) were fused to the promoter and signal sequence of the a-amylase gene of B. am)* loliquefaciens. The fusion proteins containing the C-ter-
minus were not active and remained cell associated. But when the coding sequence was fused, a hemolytically active form was recovered from the culture medium in the expected processed form (50 kD). After partial purification, the total yield of pneumolysin was about 10 mg/liter. This material was produced in sufficient amounts and in a suitable form for use in enzyme-linked immunosorbent assays. The first description of the secretion in B. subtilis of a plant enzyme, the a-galactosidase from Cyamopsis tetragonoloba, was recently reported by Overbeeke et al. [8--]. The vector used included the SPO2 promoter from plasmid pPL608, a synthetic ribosome-binding site and the a-amylase gene signal sequence from B. amylolique faciens. The expression was analyzed in cell extracts and in the supernatant of a npr E apr A strain of B. subtilis. Extracellular a-galactosidase concentrations reached a level of 1700U/ml (which represents about 10mg per liter) after 12 h of cultivation in a super-rich medium. One specific band with molecular mass of 40 kD, compatible with the deduced amino-acid composition of the mature plant enzyme (39 777 D), was detected by western blot analysis. This value is slightly lower than that of the enzyme obtained from the original plant or from Saccharomyces cervisiae, and reflects the absence of protein glycosylation in B. subtilis. Identification of the first 20 amino acids of the secreted a-galactosidase showed that the processing of the a-amylase signal sequence had been correctly performed, with specific cleavage between two Ala residues. Furthermore, the enzyme recovered from the B. subtilis medium behaved in the same way with the natural substrate (guar gum) as the enzyme derived from the plant.
The levansucrase system of B. subtilis Levansucrase is one of many enzymes that are secreted by B. subtilis. It is specifically induced by sucrose, and is expressed during the logarithmic growth phase. Levansucrase is encoded by the sacB gene and expressed from a constitutive promoter in the closely linked sacR locus. The sacR locus contains a palindromic structure acting as a transcription terminator. In the presence of sucrose, an anti-terminator, the sacYgene product that belongs to the sacS operon, allows transcription of the sacB gene. The expression of this gene is also controlled by other regulatory genes such as the two-component system degS/degU and also by degQ (formerly sacU and sacQ). Several upmutations are available: degUb, degff °, sacSb and sacs c. These factors make the sacB system a suitable candidate for the construction of an efficient secretion system for heterologous proteins. An expression and secretion vector has been constructed which includes the sacB promoter, the regulatory sequence sacR and the entire signal sequence of levansucrase, which was reconstructed with a synthetic oligonucleotide. The inclusion of a useful restriction site, NaeI, at the end of the signal sequence allows fusions in the three possible reading frames [9]. As a model system, the TEM-]3-1actamase gene from E. coli
Gene expression using Bacillus Rapoport and Klier was fused in phase with the signal sequence. The expression and secretion of the enzyme was tested in a sacSb strain of B. subtilis after introduction of the construction in a shuttle vector, E. coli/B, subtilis, derived from pC194. Assays of ]3-1actamase activity demonstrated substantial sucrose-inducible secretion of the enzyme during the vegetative phase. More than 80% of the gene product was found in the supematant of the cell culture. The inducible and enhancible expression and secretion system based on sacB has recently been further improved by introduction of the regulatory gene degQ [10"]. The degQ gene was expressed constitutively from a strong promoter, P43, during both the vegetative and stationary phases. The presence of the P43-degQ cassette on a multicopy plasmid increased the activity of [3-1actamase by about 17-fold. This was similar to the activity measured when the sacRB-~-lactamase fusion lacking the degQ gene was introduced into a degUb-containing strain of/3. subtilis. No additive effect was observed when the construct containing the degQ cassette was introduced into the degOr° strain. The sacB system has also been used for the inducible secretion of a cellulase (EGA; encoded by the celA gene) from Clostridium thermocellum [11.]. This protein is another example of a naturally secreted protein. Expression of EGA in different B. subtilis genetic backgrounds ( degUh, sacSb, sacS c) showed that its regulation is similar to that of levansucrase. The highest enzyme activity was recovered, as expected, from the culture supematant of a degUb strain. Concentration corresponded to approximately I mg/liter per unit of optimal density at 660 nm (OD660) , and compares favorably with EGA production in C. thermocellum or in E. coll. The molecular weight of 46 kD, calculated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the secreted polypeptide, is compatible with the calculated molecular weight of the mature enzyme. In these experiments, the celA gene was carried on a multicopy vector that was derived from pC194. However, such hybrid plasmids are often unstable in B. subtilis. To avoid instability in the structural plasmid, the foreign gene was more recently inserted into the chromosome of B. subtilis and allowed to amplify [12,.]. It has been shown already that a construct that consists of a genetic marker (a gene encoding antibiotic resistance) flanked by directly repeated sequences can be amplified in the B. subtilis chromosome by increasing the selective pressure and that the resulting amplified structure contains tandemly repeated amplification units (about 20 to 50 per chromosome). It had also been shown that gene amplification can be induced by the presence of an active origin of replication of a plasmid integrated into the B. subtills chromosome. This phenomenon, which takes place in the absence of selective pressure, has been applied [12-.] to the previously described sacRDcelA fusion. The construct includes plasmid pE194, which is thermosensitive for replication, and an amplification unit comprising direct repeated sequences of pBR322, the cat gene encoding the chloramphenical acetyltransferase (CAT) and a 2.7kb DNA fragment, which carries the sacR-celA fu-
sion. A two-step amplification procedure yielded up to 250 amplification units per chromosome and these were stably maintained in the absence of selective pressure. The cellulase, EGA, was the major secreted protein, and analysis of the N-terminal processing site revealed three different cleavage sites in the vicinity of the signal peptidase recognition sequence; 30% of the molecules were cleaved at the predicted site. Judging from the specific activity of the secreted enzyme, it seems likely that all three processed forms were active. The kinetics of the cat gene expression were followed in parallel with EGA production. CAT activity, which is found intracellularly, increased 50-fold in 25 h, whereas EGA activity increased only 10-fold, suggesting that other limiting factors modulate the expression of the cela gene and/or the secretion of its product. A level of 10 mg/liter per OD660 unit was reached after 25 h in a degUb B. su~ tills mutant cultivated in rich medium, corresponding to a 10-fold increase in EGA production compared with the multicopy plasmid strategy. Induction of DNA amplification into the chromosome seems to be a valuable alternative to multicopy expression vectors, especially as plasmid instability is often observed in 13. subtilis. The secretion of an eukaryotic protein using the sacB system has also been tried but with much less success [13]. The mouse a7 interferon gene, the signal sequence of which had been removed, was introduced into the levansucrase secretion vector. The interferon protein was found at low levels in cell extracts from both the wildtype strain and the degUb mutant. Modification of the amino acid composition of the signal peptide and mature interferon junction, which restored an a-helix conformation had no effect on the secretion of the product. These results suggest that, in t3. subtilis, polypeptide structures other than those found around the cleavage site are involved in the secretion of eukaryotic proteins.
The staphylokinase expression system Staphylokinase (Sak) is a 15.5kD enzyme secreted by some strains of Staphylococcus. This enzyme is responsible for the plasminogen activation that leads to fibrinolysis. The structural gene encoding staphylokinase (Sak42D) is located on phage genomes and has been cloned and sequenced from phages S~C and 42D. When a DNA fragment carrying the gene for Sak42D was introduced into the Gram-positive hosts B. subtilis and Strep tococcus sanguis, active Sak was detectable in culture supematants of both Gram-positive hosts [14]. The yield of Sak at the end of the log phase of B. subtilis reached 25 mg/liter of culture supematanc Sak purified from cub ture supernatant yielded two protein fractions after analysis by SDS-PAGE. The larger, minor polypeptide was probably unprocessed Sak [14]. Breitling et al. [15".] have used a DNA fragment carrying the signal sequence coding region, the ribosome-binding site and the putative promoter of the sak 42D gene as a portable expression secretion unit. Insertion of this unit in front of the genes for human interferon a l (hlFNa) and a hybrid h I F N a l / a 2 resulted in the expression and
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Expressionsystems secretion or export of the heterologous gene products in both Gram-positive and Gram-negative hosts. Expression of these constructs in E. cull led to some instability in the recombinants strains, but no such instability was observed in B. subtilis although the expression level was higher. In B. subtilis, the IFNcz secretion started in the early log phase and continued constitutively until growth ceased. The concentration of hlFNcz in the culture at the end of the log phase was about 15 mg/liter. However, prolonged incubation resulted in a significant drop in production caused by residual protease activity. One natural vector used by Breitling et al. [15 °°] for interferon expression was highly stable even in the absence of selective pressure. The hlFNczl was purified from culture supematants of B. subtilis and then analysed. A single band, with no detectable degraded products, was visualized and the determination of the N-terminal amino-acid sequence confirmed the homogeneity of the preparation and the accurate processing of the sak signal peptide at the expected site. The mature recombinant IFNczl protein carried the expected 11 amino-acid N-terminal extension that resulted from DNA manipulation, but this additional tail did not change the specificity of the IFNal protein. Curiously, the same recombinant plasmid when introduced into S. sanguis, another Gram-positive host, did not lead to significant synthesis of hlFNczl. The main limitations of this system are its relatively narrow host range, and the synthesis of mature heterologous polypeptides with a N-terminal extension.
Lac-based inducible systems High-level expression of a foreign gene might be detrimental to the host. In E. cull, this disadvantage has been overcome by using inducible expression cassettes, the most widely used systems being derived from the E. coli /ac, trp and lambda CI repressors. In order to increase the potential of B. subtilis as a host for the high-level expression of foreign genes, the use of similar regulatory elements would be an advantage. Yansura and Henner [16] used the E. cull lac operator and repressor, together with a promoter from the phage SPO1, to control gene expression of human leukocyte interferon in B. subtilis. This system has also been shown to be a very useful tool for studying the expression, in the original host, of some regulatory genes that had their own promoters deleted [17"]. Some promoters and terminators of Gram-negative ori gin are recognized by purified 0 -43 RNA polymerase from B. sublilis. One such promoter, PN25, from the bacteriophage T5 genome was shown to be able to initiate the transcription of a foreign gene leading to an accumulation of the gene product at a level up to 25% of the total cellular protein [18,1,9]. This promoter was fused to a synthetic DNA fragment carrying the E. coli lac operator generating a regulatable promoter PN25/O [20"°]. This promoter was linked to a synthetic ribosome-binding site followed by a multicloning site. This represents the first component of a lac-based inducible system for B. sub tills. The second component is a lac repressor gene that
is able to be transcribed and translated in B. subtilis. This was constructed by replacing the authentic promoter and ribosome-binding site with the equivalent Gram-positive elements. Two expression systems were created [20.°]. The first one was made up of an E. colgB, subtilis shutfie vector pREP9 harboring the two components of the lac-based inducible system. The second expression system was composed of a B. subtilis strain constitutively producing the lac repressor from a derivative of the S. a u r e u s plasmid pE194 (pBL1) and a compatible shuttie expression vector containing the regulatable PN25/O (p602/20). The plasmid pREP9 was used to overproduce bacterial CAT after addition of the inducer IPTG. After 2 to 3 h of induction, the CAT protein was the major cellular protein, accounting for up to about 15% of the total cell protein. The dual plasmid expression system was also used to produce eukaryotic polypeptides such as the mouse dihydrofolate reductase (mDHFR), the human immunodeficiencyvirus (H1V)-I, reverse transcriptase (RT) and the mature part of human tissue plasminogen activator (tPA) [20o.,21,22.]. All three polypeptides were synthesized intracellularly by B. subtilis after induction and were biologically active. The tPA was localized in inclusion bodies, and was therefore easily purified. The enzymatic ac tivity could be partially recovered after complete reduction and slow oxidation of solubilized inclusion bodies. As with melanoma tPA, the recombinant tPA exhibited very high levels of plasminogen activation in the presence of fibrin, even though the recombinant product had a 3 amino-acid N-terminal extension. However, the total amount of tPA produced by B. subtilis was relatively low (about 0.5% of the total cell weight) compared with the values reported for the synthesis of the mDHFR (10% of the total protein) [20..]. A translational fusion between the E. coli cat gene and the open reading frame of HIV p o l gene was constructed [21]. After 2 h of induction, a high-molecular-weight polypeptide was synthesized, consistent in size with the pol encoded precursor polyprotein. After prolonged incubation, the precursor polypeptide disappeared and two polypeptides of molecular weights 64 and 52 kD were produced. They corre sponded in size to H1V-1 RT polypeptides, suggesting a correct processing in B. subtilis. These polypeptides were purified to near homogeneity and displayed high levels of RT activity. However, the expression level was relatively low, and the yield of pure enzyme was not sufficient for industrial use. In order to increase the potential of the dual plasmid inducible system and to overcome the disadvantages of using two compatible plasmids, the lacI gene with its Gram-positive control elements was intro duced into the chromosome of B. subtilis, using phage (I)105 as a cloning vehicle [20"]. This system is now under evaluation. In the T5-based expression system [18, 19,20°',21,22"], the foreign genes were expressed during logarithmic growth, when the production of extracellular proteases is low. Expression during later growth phase requires modification of the expression cassettes by introducing promoters recognized by the transcriptional machinery
Gene expression using Bacillus Rapoport and Klier present during the stationary phase. Such twin promoters would ensure constant synthesis of lac repressor and the foreign polypeptides. Use of B. subtilis mutants devoid of the major protease genes, as in the example of tPA, would allow an accumulation of the heterologous gene product during the stationary phase. Alternatively, these systems could be modified to secrete the foreign polypeptides to the culture supemfttant by inserting a cartridge containing a DNA fragment encoding an exoenzyme signal peptide downstream of a regulatable promoter. This type of approach has already been used, employing the signal sequence of the cz-amylase to secrete the E. coli 13-1actamase [20-.]. Although B. subtilis is an efficient extracellular enzyme producer and has been shown to be a good proteinsecreting host, some mammalian proteins produced by this bacterium are often degraded by its extracellular proteases, even in those B. subtilis mutants that lack the major proteases. Another Bacillus species, B. brevis secretes large amounts of protein but little or no detectable proteases.
Expression in B. brevis One particular strain, B. brevis47, accumulates 12-25 g of extracellular proteins per liter of medium under appropriate growth conditions [23"]. These extracellular proteins have been shown to derive from the cell-wall proteins, based on morphological, immunological and biochemical criteria. In the case of B. brevis 47, the secreted proteins consist mainly of two polypeptides of 130 kD (OWP; outer wall protein) and 150kD (MWP; middle wall protein) [23"]. These proteins are synthesized, during the log phase and form hexagonal arrays on the cell surface of the bacteria. During the stationary phase, these proteins continue to be synthesized, but instead of being deposited on the cell surface they are secreted into the medium. The MWP and OWP genes of B. brevis have recently been cloned and sequenced [23"]. They are organized in an operon (cpw operon) and interrupted by an untranslated sequence of 130 bp. Both MWP and OWP are synthesized in precursor forms with signal peptides of 23 and 24 amino-acid residues, respectively. Five transcriptional start points were identified by primer extension and Sl analysis [23",24"]. The corresponding frye promoters were located in the 300 bp upstream from the MWP coding sequence. One of them, the P2 promoter, led to the efficient and constitutive expression of the c w p operon in B. brevis 47. Interestingly, this promoter was not used efficiently in B. subtilis and it does not show homology with the other promoter sequences reported for Gram-positive bacteria. This result suggests that the P2 promoter is a specific promoter that is used preferentially in B. brevis [24"]. The 5' region of the cwp operon contains two possible translational start sites located tandemly in the same read-
ing frame [23"]. Each of these sites contains a sequence that is highly complementary to the 3' end of B. brevis
rRNA, and an initiation codon. The amino-acid sequence that stretches from Met-23, the N-terminus of the translational product from the second initiation site, to Ala-1 shows all the characteristics of signal peptides of secretory precursors. The translational product from the first site contains an extra peptide of 31 amino-acid residues preceding a typical MWP signal sequence. Fusion experiments indicate that both initiation sites are used in B. brevis. However, translation appears to start at the second site much more frequently than at the first site. An expression vector was constructed for use in heterologous protein production in B. brevis [23"]. Its replication origin came from either a natural plasmid identified in some B. brevis strains (pWT481) or the pUB110 plasmid of S. aureus. A 600 bp DNA fragment was inserted into the vector and contained the 5' region of the B. br~ vis 47 MWP gene, including the multiple promoters and ribosome-binding sites, as well as the region encoding the signal peptide and the N-terminal portion of the mature form of MWP. A synthetic human epidermal growth factor (hEGF) gene was cloned into this expression vector, so that the fused gene encodes the MWP signal peptide directly followed by the mature hEGF sequence [25"]. The recombinant plasmid was introduced into both B. brevis 47 and HPD 31 using a novel Tris hydrochloride buffer/polyethylene glycol transformation method [23"]. B brevis strain HPD 31 carrying the recombinant plasmid produced as much as 240 mg of extracellular hEGF per liter of culture. The amount of hEGF increased markedly during the early stationary phase and, interestingly, remained almost constant after 3 days of incubation. Most of the hEGF was found in the culture supematant. This polypeptide was purified and had the same N-terminal and C-terminal amino-acid sequences, and amino-acid composition as natural hEGF. Moreover, the recombinant hEGF showed almost the same specific biological activity as authentic hEGF. These results suggest that the hEGF synthesized in B. brevis is correctly processed and efficiently secreted, and that the correct disulfide bridges are formed. In order to demonstrate that the B. brevis host vector system is useful for large-scale production of any mammalian protein, the cDNA of human cz-amylase and the cDNA of porcine pepsinogen A were independently fused to the MWP signal peptide coding sequence [23.,26.]. In both cases, the foreign polypeptides were secreted from B. brevis, although the amount was about 6 and 20 times less, respectively, than that of hEGF. Also, in both cases, proteins were found in the culture supematant and were similar in size and in function to the authentic polypeptides. Under acidic conditions, the rate of the activation process of the extracellular pepsinogen was the same as that of the natural proenzyme. In conclusion, the genes for several bacterial and mammalian secretory proteins have been expressed efficiently with the 13. brevis expression system and large quantities of biologically active proteins have been easily purified
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Expressionsystems from the culture medium. Proteins produced by 13. brevis usually remain stable, even after prolonged cultivation. This stability may be a result of the absence of extracellular proteases. The different proteins were expressed at different levels which suggests that the secretory machinery of this bacterium has some limitations. It also remains to be seen whether 13. brevis can secrete non-secretory polypeptides.
Annotated references and recommended reading • o•
Of interest Of outstanding interest
1.
VASANTHAN, THOMPSON LD, RHODES C, BANNER C, NAGLE J, FILPUIA D: G e n e s for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large ORF bet w e e n the regions coding for signal s e q u e n c e and m a t u r e protein. J Bacteriol 1984, 159:811-819.
VASANTHAN, FILPULA D: Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus sutr this. Gene 1989, 76:53~50. Expression of a small eukaryotic synthetic gene using the apr system of B. amyloiiquefaciens. The product was secreted by B. subtilis in an 2.
•
active form. 3.
VASANTHAN, THOMPSON LD: Secretion of a heterologous protein from Bacillus subt~'lis with t h e aid of protease signal sequences. J Bacteriol 1986, 165:837-842.
4.
HEMILAH, GLODE LM, PALVAI: Production o f diphtheria toxin CRM228 in B. subtilis. FEMS Microbiol Lett 1989, 65:193-198.
5.
RUNEBERG-NYMANK, ENGSTROM O, LOFDAHL S, YLOSTALO S, SARVASM: Expression and secretion of pertussis toxin subunit SI in Bacillus subtills. Microbiol Pathogenesis 1987, 3:461468.
6.
SARIS P, TAIRA S, AIRAKSINEN U, PALVAA, SARVASM, PALVA I, • RUNEBERG-NYMANK: Production and secretion of pertussis toxin subunits in Bacillus subtilis. FEMS Microbiol lett 1990, 68:143-148. The five subunits of the pertussis toxin were secreted in B. subillis with the c~-amylase of B. amyloliquefaciens, but the level of their expression varied considerably. 7.
TA1RA S, JALONEN E, PATON JC, SARVAS M, RUNEBERG-NYMAN • K: Production of pneumolysin, a p n e u m o c o c c a i toxin, in Bacillus subtilis. Gene 1989, 77:211-218. Production of an active staphylococcal hemolysin with the a-amylase expression and secretion system of B. amyloliquefaciens. OVERBEEKE N, TERMORSHUIZEN GHM, GUISEPPIN MLF, o• UNDERWOODDR, VERRIPSCT: Secretion of t h e a-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis. Appl Environ Microbiol 1990, 56:1429-1434. First report of the expression and secretion of a plant enzyme with the 0t-amylase system of B. amyloliquefaciens. There was correct process ing of the hybrid precursor and the non-glycosylated enzyme secreted by B. subtilis was active.
11. •
JOLIFF G, EDELMAN A, KLIER A, RAPOPORT G: Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Appl Environ Microbio11989, 55:2739-2744. inducible expression of a normally secreted enzyme using the levansuerase expression and secretion system in high-producing mutants of B. subtilis. 12. ••
PETITMA~ JOLIFF G, MESASJM, KLIERA~ RAPOPORTO, EHRLICH SD: Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtills by induction of chromosomal DNA amplification. Bio/Technology 1990, 8:559-563. Improvement of the chromosomal amplification procedure in 13. subtilis by the introduction of a plasmid origin of replication into the chromo some. Up to 250 copies of the amplified unit per chromosome were obtained in the absence of selective pressure. High inducible expression of a heterologous cellulase was studied as a model. 13.
DION M, RAPOPORT G, DOLY J: Expression of the MulFNa7 gene in Bacillus subtilis using t h e levansucrase system, Biochimie 1989, 71:747-755.
14.
BEHNKE D, GERLACH D: Cloning and expression in Escherichia coli, Bacillus subtilis and Streptococcus sanguis of a g e n e for staphylokinase - a bacterial plasminogen activator. Mol Gen Genet 1987, 210:528-534.
15. •°
BREITLINGR, GERLACHD, HARTMANNM, BEHNKE D: Secretory expression in Escherichia coli and Bacillus subtilis of hum a n interferon c~ g e n e s directed by staphylokinase signals. Mol Gen Genet 1989, 217:384-391. This paper describes the construction of a secretion vector based on the promoter and signal sequence of the sak gene. Significant expression level of hIFNcO or hlFN~zl/2 was obtained at the end of the log phase. Precise processing of the precursor form was also demonstrated. 16.
17. HENNER DJ: Inducible expression of regulatory g e n e s in • Bacillus subtilis. Methods Enzymol 1990, 185:223-228. Description of the utilization and integration into the chromosome of the inducible spac system in B. subtilis. 18.
PESCHKEU, BEUCK V, BUJARD H, GENTZ R, LE GRICE S: Efficient utilization of Escherichia coli transcriptional signals in Bacillus subt~'lis. J Mol Biol 1985, 186:547-555,
19.
ZUKOWSKIMM, MILLERL.' Hyperproduction of an intracellular heterologous protein in a sac Uo m u t a n t of B. subtilis. Gene 1986, 46:247 255.
20. ••
LE GPaCE SFJ: Regulated p r o m o t e r for highqevel expression of heterologous g e n e s in Bacillus subtilis. Methods Enzymol 1990, 185:201-214. A review of the construction of the /aobased inducible systems for B. subtilis and their use for the expression of heterologous proteins. 21.
8.
9.
EDELMANA, JOLIFF G, KLIER A, RAPOPORT G: A system for the inducible secretion of proteins from Bacillus s u b tills during logarit vl~ ic growth. FEMS Microbiol Lett 1988,
52:117-120. 10. •
WONGSL: Development of an inducible and enhancible expression and secretion system in Bacillus sublllis. Gene 1989, 83:215-223. Improvement of the levansucrase expression and secretion system by introduction into the vector of a cassette containing the regulatory degQ gene. Use of TEM-[3-1actamase as a model protein for secretion.
YANSURADG, HENNER DJ: Use of Escherichia coli lac repressor and operator to control g e n e expression in Bacillus subtilis. Proc Natl Acad Sci USA 1984, 81:439443.
LE GRICE SFJ, BEUCK V, MOUS J: Expression of biologically active h u m a n T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis. Gene 1987, 55:95-103.
22. •
WANGLF, HUM x,X/W,KALYANNK, LEE SG, HUNG PP, DOI RH: Synthesis and refolding of h u m a n tissue-type plasminogen activator in Bacillus subtills. Gene 1989, 84:127-133. Use of a dual plasmid inducible system for the synthesis of tPA in /3. subtilis. The recombinant tPA was purified from inclusion bodies and was biologically active. 23, •
UDAKAS, TSUKAGOSHI N, YAMAGATAH: Bacillus brevis, a host bacterium for efficient extracellular production of useful proteins. Biotechnol and Gen Eng Rev 1989, 7:113-146. This review describes the construction of secretory vectors based on the MWP gene of 13. brevis. 24. •
ADACHIT, YAMAGATAH, TSUKAGOSH1N, UDAKA S: Multiple and tandemly arranged p r o m o t e r s of the cell wall protein gene o p e r o n in Bacillus brevis 47. J Bacteriol 1989, 171:1010-1016.
Gene expression Identification by primer extension and Sl analysis of five different promoters of the CWP operon. They are all differently regulated depending on the growth phase. 25. .•
YAMAGATA H, NAKAHAMAK, SUZUKIY, KAKINUMAA, TSUKAGOSHI N, UDAKAS: Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci USA 1989, 86:3589-3593. Construction of a highly emcient secretion vector based on the 5' region of a cell-wallprotein gene, leading to the high-level secretion (240 mg/1) of hEGF.
26.
using Bacillus R a p o p o r t a n d Klier
T ~ o M, MORIOKAT, YAMAGATAH, TSUKAGOSHIN, UDAKA S: Production of swine pepsinogen by protein-producing Bacillus brevis carrying swine pepsinogen cDNA. Appl Mi crobiol Biotecbnol 1989, 30:75-80. Ei~icient production of the porcine pepsinogen A in t3. brevis. The preenzyme is autocatalytic~y converted into the active form.
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