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Definitions

• the present invention relates to a process for the production of various products, especially translocated polypeptides by using bacteria of the genus Bacillus which have been mutated to be incapable of sporulation, methods for producing such bacteria, and DNA constructs to be used in the processes.
• Bacteria of the genus Bacill us are being used in the production of various polypeptides and proteins for use in medicine and various industries.
• Bacillus licheniformis is used exten ⁇ sively for the production of industrial enzymes such as amylase and protease and is a popular host for the industrial prepara ⁇ tion of cloned gene products (1, 2, 3) .
• industrial enzymes such as amylase and protease
• Hundreds of tons of extracellular enzymes are manufactured annually from this orga ⁇ nism resulting in the need to dispose of a considerable tonnage of spent organisms, usually as composts.
• the first morphological change observed during the sporulation process is the synthesis of an asymmetric septum at stage II.
• Gene expression in the mother cell and maturing spore is partly governed by the ordered synthesis and activation of a cascade of sigma factors, which direct RNA polymerase to transcribe sporulation specific promoters in a temporally and spatially oriented fashion (4, 5) .
• the ⁇ F protein the product of the spoIIAC gene (8, 9) is one such sigma factor which is present in the predivisional cell but its activity is restricted to the prespore and only becomes evident after septation (14) .
• This sigma factor is crucial for establishment of compartment-specific gene expression (10) and, without it, expression of numerous genes in the developing spore is prevented.
• a major function of ⁇ F is activation of expression of the prespore specific sigma factor ⁇ G , which is responsible for gene expression in the developing spore (11) .
• the processing of pro- ⁇ E into ⁇ E which is responsible for gene expression in the mother cell, is also blocked by nonsense mutations in spoIIAC (11) .
• the deletion of the spoIIAC gene could possibly lead to total disruption of development in the spore and blockage of mother cell development, which is dependent on ⁇ E .
• the present invention provides a process for the production of a translocated polypeptide comprising i) cultivating a bacterium of the genus Bacillus which is incapable of sporulation due to a mutation, said bac ⁇ terium comprising DNA constructs encoding polypeptides involved in the production of said metabolite under conditions conducive to the expression of said DNA constructs and production of said metabolite, and ii) recovering said metabolite, with the proviso that said bacterium does not belong to the species B. subtilis.
• the invention furthermore provides host bacteria of the genus Bacillus which is incapable of sporulation due to a mutation, and a method for producing such hosts.
• the method is based on the use of information provided from B. subtilis to enable the deletion of one or more genes involved in the sporulation process.
• a deletion of the spoIIAC gene was prepared in vi tro using the splicing by overlap extension technique. This gene was introdu ⁇ ced into Bacillus licheniformis in a temperature sensitive plasmid and, following integration and excision from the chromosome, a precisely located deletion of the chromosomal gene was prepared.
• the mutated bacterium was totally asporogenous and formed abor ⁇ tively disporic cells characterized by asymmetric septa at the poles of the cell.
• alkaline protease was the same in batch cul ⁇ tures of the mutant and parent during prolonged incubation for 72 h, but ⁇ -amylase yields were reduced by about 30% by the mutation.
• the invention has been exemplified in B. licheniformis, but it is envisaged that the invention can be used in other Bacilli as well, such as B. lentus, B. amyloliquefaciens, B. thuringiensis, B. alcalophilus , B. mesentericus, etc.
• Table I shows the excision frequency of pE194ts carrying the deleted SpoIIAC allele of Bacillus licheniformis
• Figure 1 shows the splicing by overlap extension reaction used to create the deletion in spoIIA.
• Figure 2 shows the integration and excision reactions resulting in the production of a deletion in spoIIAC.
• Figure 4 shows the growth, sporulation and extracellular enzyme synthesis in B. licheniformis DN286 and DN286 spoIIACD3.
• a and B growth ( ⁇ ) and spores (D) of strain DN286 and growth of DN286 spoIIACD3 (•) in minimal medium and brain heart infusion respectively.
• C and D serine protease synthesis by strain DN286 ( ⁇ ) and strain DN286 spoIIACD3 (•) in minimal medium and brain heart infusion respectively.
• E amylase synthesis in strain DN286 (D) and in DN286 spoIIACD3 (0) , in minimal medium (D, 0) and in brain heart infusion ( ⁇ , •).
• the invention provides a process for the production of a translocated polypeptide comprising i) cultivating a bacterium of the genus Bacillus which is incapable of sporulation due to a mutation, said bac ⁇ terium comprising DNA constructs encoding polypeptides involved in the production of said metabolite under conditions conducive to the expression of said DNA constructs and production of said metabolite, and ii) recovering said metabolite, with the proviso that said bacterium does not belong to the species B. subtilis .
• the bacterium belongs to the group of species comprising B. licheniformis, B. lentus, B. amyloliquefaciens, B. thuringiensis, etc.
• the process of the invention has been exemplified by use of a Bacill us that has been made incapable of sporulation through a deletion of the SpoIIAC gene, but any other mutation effectively disrupting the sporulation process in an irrever ⁇ sible manner, such as the Spo2-mutation, and the Spo3-mutation is also comprised within the invention.
• the process of the invention should preferably make use of a bacterium wherein said mutation is irreversible.
• the process of the invention is contemplated to be used primarily for the production of any polypeptide, especially translocated polypeptides.
• Said polypeptide may be endogenous to the bacterium or exoge ⁇ nous, meaning that the polypeptide originally was produced by itself or some other organism, respectively.
• the sporulation deficient strains of the invention can be used for the production of secondary metabolites.
• the products from the process of the invention are preferably enzymes, especially industrial enzymes, such as proteases, lipases, cellulases, oxidoreductases, amylases, etc.
• translocated polypeptide is intended to indicate that the polypeptide expressed carries a signal sequence which enables it to be translocated across the cell membrane.
• the translocated polypeptide may be a secreted polypeptide or a polypeptide involved in the secretory machi ⁇ nery of the Bacillus cell in question.
• the present invention also provides a method of producing a bacterium belonging to the genus Bacillus which is incapable of sporulation due to a mutation whereby one or more genes involved in the sporulation process is deleted in part or completely.
• Gene integration and excision is a common procedure for pro ⁇ ducing specific mutations in B. subtilis and other gram positive bacteria (10, 24, 25) .
• it is also an efficient procedure for introducing defined mutations into the chromosome of other Bacillus, such as B. licheniformis.
• B. subtilis Given the close relationship between B. subtilis and B. licheniformis (26) , it was of interest to examine the possibility of using heterologous alleles for the introduction of genes into the chromosome In this way it is according to the invention possible to use the large number of sequenced genes from B. subtilis for genome manipulation in other Bacillus, such as B. licheniformis . Homology was sufficient for integration of the spoIIAC allele of B. licheniformis into the B. subtilis chromosome, albeit at lower frequency than the homologous reaction, and integration presumably occurred at the correct site since these integrants were asporogenous.
• B. licheniformis It seems likely that the reverse situation would pertain in B. licheniformis; and B. subtilis genes forced into the B. licheniformis chromosome would excise perfectly.
• amyE is subject to far fewer regulatory genes in B. subtilis than aprE. Catabolite repression and the DegSU systems are the major control systems for this gene (12), although sen (28) and pai (29) are also involved. It is not readily apparent why the synthesis of amylase was affected by the spoIIAC mutation.
• One of the major aims of the invention is to provide a bacterium suitable for industrial enzyme production purposes.
• the mutant described here is totally asporogenous under laboratory conditions and the molecular biology of sporulation predicts that it should be unable to produce endospores.
• the likelihood of reversion of a deletion spanning almost 400 bp is minimal and there is no evidence that suppression may be a problem.
• the bacterium is equivalent to its parent in e. g. alkaline protease synthesis.
• DN286 is a Bacillus licheniformis wild type strain
• NCIMB 6346 B. licheniformis NCIMB 6346 is available from NCIMB.
• Bacillus subtilis 168 was obtained from D. A. Smith (University of Birmingham, UK) .
• Escherichia coli M83 was used for all plasmid constructions.
• Luria broth and Luria broth agar (16) were used routinely with appropriate antibiotic selections; ampicillin (100 mg/ml) and erythromycin (1 mg/ml).
• Spizizen's minimal salts medium (16), Schaefer's sporulation medium (16) and brain heart infusion (BHI) broth (Oxoid) were also used. Incubation was at 37°C unless otherwise stated.
• Plasmids Plasmid pUC19 was used for cloning in E. coli JM83.
• pE194ts a mutant of pE194 which is temperature sensitive for replication was a gift from P. Youngman (University of Georgia, Athens, Ga, USA) .
• Amylase was assayed in culture supernatants using Phadebas amylase substrate (Pharmacia) at 37°C and converted to release of reducing sugars (maltose) from soluble starch using a stan- dard curve constructed with dilutions of amylase assayed by the Phadebas and Nelson-Somogyi reducing sugar assay. Units are mM maltose equivalents released/min/ml supernatant. All batch cultures were conducted at least twice; figures presented are representative of reproducible patterns of results.
• the splicing by overlap extension (SOE) reaction was used (19) .
• the promoter proximal region of the spoIIAC gene was amplified in a reaction with primers A (SEQ ID NO. 2) ;
• primers A and D represent EcoRI and BamHI sites respectively.
• the reaction mixtures comprised 200 ng of template DNA (cloned spoIIA operon of B. licheniformis) , 100 pmol of each primer, a final concentration of 125 mM for each dNTP, 6 ml of MgCl 2 (25 mM) , 10 ml of (xlO) Taq Buffer, and sterile Millipore water to 100 ml.
• the amplification programme consisted of an initial cycle of denaturation at 95°C for 10 min after which 1 unit of Taq poly ⁇ merase (Promega) was added and the reaction mixture was covered with 100 ml of light mineral oil. This was followed by 35 cycles of denaturation at 95°C for 2 min, annealing at 55°C for 2 min, and extension at 72°C for 3 min. An additional cycle of 95°C/2 min, 55°C/2 min and an extension step of 72°C/6 min completed the program.
• the 464 and 382 bp PCR products were concentrated by ethanol precipitation, purified using the Gene-Clean kit and used as a template (each at 300 ng) with primers A and D in the SOE reac ⁇ tion (same conditions as above) to generate the 806 base-pair spoIIAC deletion comprising fragment AD (Fig. 1) .
• Fragment AD was cloned into pUC19 through the BamHI and EcoRI restriction sites contained in primers A and D, respectively, transformed into E. coli JM83 and colonies containing plasmid with inserts screened on LB ampicillin plates containing 40 mg/ml X-gal.
• Recombinant plasmid from a clone was then ligated to pE194ts through the unique Pstl sites in each replicon after treating the former with calf intestinal alkaline phosphatase.
• Ligation mixes were transformed into E. coli M83 and the recombinants detected by agarose gel electrophoresis of mini-preparations.
• Plasmid prepared from E. coli was then used to transform pro ⁇ toplasts of B. licheniformis DN286 (10) and competent cells of B. subtilis 168 (20, 7) .
• B. licheniformis and B. subtilis containing the pE194-based deleted spoIIAC allele were grown overnight in 50 ml LB broth containing erythromycin at 28°C. Samples were diluted and mixed with 3 ml of soft agar containing erythromycin and overlaid onto LB agar Em plates and incubated at 28°C (permissive for replication) and 40°C (non-permissive) . The frequency of integration was the ratio of the number of colonies growing at the permissive to those growing at the non-permissive temperature. Integrants isolated at 40°C were subsequently grown routinely at 37°C.
• Excision of integrated plasmid was achieved by growing cells at 30°C in LB broth without antibiotic selection. Diluted samples were plated on LB agar and incubated overnight at 37°C and colonies replica plated onto LB agar with and without erythromycin. The frequency of excision was determined as the ratio of total number of colonies to the number of Em r colonies.
• a deletion in the spoIIAC gene of B. licheniformis was prepared using the splicing by overlap extension (SOE) technique. Two independent PCR amplifications were made from the cloned spoIIA operon using the primers described in Materials and Methods.
• One product (AB) comprised the distal end of the downstream gene (spoIIAB) and the proximal region of the spoIIAC gene.
• the second product (CD) covered the distal region of spoIIAC and part of the upstream non-coding region.
• the extensive regions of homology (overlap) in primers B and C were used to initiate a third PCR amplification which comprised products AB and CD and incorporated primers A and D. This resulted in a fragment containing the ends of the spoIIAC gene encompassing a deletion of 372 bp (fragment AD, see Fig. 1) .
• Fragment AD was cloned into pUC19 and ligated to pE194ts through the unique Pstl sites in each replicon to produce the shuttle plasmid pHWM2 (Fig. 2) which was transformed into E. coli .
• Plasmid prepared from E. coli was then used to transform protoplasts of B. licheniformis DN286 and competent cells of B. subtilis 168. Clones from each transformation were verified to contain pHWM2 by restriction enzyme analysis and used for inte ⁇ gration and excision studies.
• B. licheniformis and B. subtilis cells containing pHWM2 were grown overnight at 28°C and plated onto selective (Em-contai ⁇ ning) and non-selective media and incubated at 45°C.
• the inte ⁇ gration frequency which was estimated as the proportion of cells which could grow on erythromycin plates at 45°C, for B. licheniformis was about 10 "4 and for B. subtilis (containing the B. licheniformis allele) 100-fold lower (Table 1) .
• the frequency was between IO "2 and IO "3 for B. licheniformis .
• a deletion strain from B. licheniformis integrant 3 (Table 1) was chosen for further study. Hybridization of a labelled spoIIA operon from B. licheniformis to Southern blotted chromosomal DNA from the wild type and deleted strains which had been cut with EcoRI, revealed a smaller hybridizing fragment of 3.8 kb in the mutant compared with 4.2 kb in the parent (data not shown) . PCR amplification from chromosomal DNA of the mutant and parent strains using primers A and D showed that the mutant possessed a deletion of about 370 bp. When the 5 PCR product from the mutant was sequenced, a deletion located precisely at the junction point between primers B and C was revealed (Fig. IB) .
• the naturally low sporulation rate of DN286 was confirmed by comparing with B. licheniformis NCIMB 6346 which showed 90% to 20100% sporulation under the same three conditions.
• mutant strain showed some minor differences from the parent in the amounts of extracellular enzyme synthesized.
• hydrolysis zones from ⁇ -1, 3-glucanase and carboxymethyl cellulase were reduced in the mutant compared with the parent strain but synthesis of a-amylase, DN'ase, polygalacturonate lyase, protease, RN'ase and xylanase was largely unaffected.
• the mutant and its parent were grown in rich (BHI) and minimal media for 72 h. Sporulation and the synthesis of serine protease were monitored (Fig. 4) . Protease production was similar in both parent and mutant. In minimal medium, enzyme yield peaked after incubation for about 40 h in both strains and then remained steady. In BHI, enzyme yield was slightly repressed and peaked after incubation for about 48 h in both strains. The subsequent decline in enzyme yield was less pronounced in BHI than in the salts medium. In both media, the yield of serine protease was unaffected by the spoIIAC deletion.
• ⁇ -Amylase synthesis consistently initiated later during batch culture of the mutant compared to the parent and consequently these fermentations were continued for a longer time period than those for protease (84 h) .
• the yield of enzyme from the mutant was about 70% of that obtained from the parent strain.
• the mutant performed almost as well as the parent, particularly later in the growth cycle when biomass declined in the minimal medium.
• Bacillus subtilis and other gram-positive bacteria Biochemistry, physiology and molecular genetics. American Society for Microbiology, Washington

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