DK159282B - PROCEDURE FOR STABILIZING EXTRA-CHROMOSOMAL ELEMENTS IN A GROWTH BACTERIA IN CULTURE, TRANSFORMED BACTERIES AND A PROCEDURE FOR MANUFACTURING A DESIRED PRODUCT IN SUCH TRANSFORMED BACTERIES - Google Patents

Description

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This invention relates to a method for stabilizing extrachromosomal elements in a host bacterium during culture, transformed bacteria, and a method for producing a desired product in such transformed bacteria.

Microorganisms containing extra-chromosomal genetic elements, exemplified here by plasmids, are generally unstable in that the plasmid can often be irreversibly dropped. This instability is particularly pronounced if the plasmid does not originate from the host organism, because it e.g. contains genes from other organisms or is engineered by genetic engineering.

To enhance plasmid stability, antibiotics or other bioactive compounds against which the plasmid, but not the chromosome, exhibit resistance are usually added to the medium used for culture of the microorganism. In such a medium, only those cells that retain the plasmid with the antibiotic resistance gene will multiply. The major disadvantage of this method is that it requires large scale cultivation of antibiotic resistant micro-organisms, addition of expensive antibiotics to the growth medium with possible adverse effect on the environment and subsequent extensive purification to remove the antibiotic from the desired product.

Complementation of an auxotropic mutation of the host chromosome is another known method for stabilizing plasmids. However, this method places severe restrictions on the composition of the culture medium and requires cultivation in a growth medium which does not contain the nutrient required by the host organism, thereby limiting the potential for improving productivity.

The object of the present invention is to provide a method for stabilizing extra-chromosomal elements in transformed bacteria without the need to use antibiotics and without imposing severe restrictions on the composition of the growth medium.

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It is a further object of the present invention to provide transformed bacteria containing such stabilized extra-chromosomal elements, as well as a method for producing desired products in transformed bacteria without the need to use antibiotics or to impose special restrictions on the composition of the growth medium. .

The invention is based on the recognition that extra-chromosomal elements can be retained in host bacteria during culture in conventional media if the extra-chromosomal elements contain a specific function that under the given conditions is a requirement for normal growth of the host.

When a host bacterium having a mutation, deletion or other defect in a gene encoding a function necessary under the given conditions for normal growth of the host is transformed by e.g. a plasmid containing a gene encoding this function will survive only such cells that are transformed with and retain the plasmid because the host requirement in such cells is supplemented only by the plasmid. However, permanent plasmid conservation is assured only if the transfer of genetic information 20 from plasmid to chromosome cannot occur and if the degree of spontaneous mutation of the host bacterium to a mutant without such a requirement is immaterial.

According to a first aspect of the present invention, there is provided a method for stabilizing extra-chromosomal elements in a host bacterium during culture, and this method is characterized by introducing into the extra-chromosomal element a DNA sequence encoding D, L-alanine race, and that the extra-chromosomal element containing said DNA sequence is transformed into the host bacterium, using a host bacterium with a defect in the chromosomal valley gene for D, L-alanine racemase. The requirement caused by the chromosomal gene defect in the host bacterium is thereby suppressed by the extra-chromosomal element and loss of the extra-chromosomal element during cultivation is immaterial.

As used herein, the term "extra-chromosomal elements" means plasmids, bacteriophages, or any genetic material not normally found in the host bacterium either as itself 3

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constant molecules or integrated into the chromosome. Preferred extra-chromosomal elements are plasmids, but bacteriophages or other vector systems can also be used.

According to a further aspect of the present invention there is provided a transformed bacterium which is characterized in that it is derived from a host bacterium which has a defect in the chromosomal valley gene for D, L-alanine racemase and contains an extra-chromosomal element containing a DNA sequence encoding D, L-alanine racemase.

The present invention further provides a method for producing desired products (e.g., DNA, RNA, peptides or proteins) in transformed bacteria, and this method is characterized in that a bacterium that has a defect in the chromosomal valley gene and contains an extra-chromosomal element containing a DNA sequence encoding D, L-alanine racemase, and a DNA sequence encoding the desired product is grown in a suitable culture medium upon which the desired product is extracted from the culture medium.

Suitable host bacteria belong to Bacillus or Enterobacteriaceae species, e.g. Bacillus subtilis and Escherichia coli, although other suitable bacteria will be obvious to those skilled in the art.

In the specification, the following terms are used: dal gene: the gene for D, L-alanine racemase 25 dal + gene: a functional gene (wild type) for D, L-alanine racemase dal-1 gene: a gene with a mutation in the D, L-alanine racemase gene that normally requires external D-alanine dal host host: a host with a mutation in the dal gene (which normally requires external delivery of D-alanine for growth dal + host: a host with a wild-type dal gene “j **

Valley host: a host with no external D-alanine requirement

Valley host: a host with requirements for external D-alanine

R

Cam: chloramphenicol resistance 35 Can: kanamycin resistance

Amp; ampicillin resistance

o R

bla: gene for beta-lactamase causing Amp 4

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cat: chloramphenicol acetyltransferase gene causing Cam amyM: gene for a maltogenic amylase 5 For most bacteria, the ability to grow and divide depends on a well-preserved cell delineation (i.e., cell membranes, cell walls, and related structures). Explosion or degradation of the cell boundary usually leads to dissolution or cessation of growth.

10 One of the components necessary for stable cell delineation in Bacillus subtilis and in most other bacteria is D-alanine. D-alanine is an essential part of the cell wall in which it serves to crosslink polysaccharide chains and thus provides the cell with the necessary rigidity. D-alanine is not found in most common growth media, and many bacteria comprising B. subtilis and E. coli synthesize this amino acid from L-alanine by the enzyme D, L-alanine racemase and do not require an external source of D-alanine. for growth. Some mutants need an external supply of D-alanine for growth, e.g. dal-1 mutants of B.

20 subtilis in which the D, L-alanine racemase gene has been damaged due to the mutation (Freese et al., Proc.Natl.Acad.Sci.

51: 1164-72, 1964 and Dul et al., J. Bacteriol. 15: 1212-14, 1973).

According to the present invention, it has been shown that plasmids containing a functional gene for D, L-alanine racemase ensure their conservation in a dal host if the dal + gene cannot be transferred from plasmid to chromosome and if the frequency of spontaneous mutation of the host to a Dal + phenotype is of no significance, as well as if external D-alanine is not available to the cell.

Thus, by inserting a gene for a desired product into a suitable dal + plasmid and culturing the plasmid in a suitable dal host, the dal + plasmid will be conserved in the cell population during growth, thereby ensuring high yields of the desired product expressed. during the autonomous replication of the plasmid.

Since D-alanine is not found in many usual growth media, no restrictions are placed on the culture medium.

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Thus, the present invention provides a convenient method for stabilizing extra-chromosomal elements carrying genes for desired products during cultivation.

To ensure that the degree of spontaneous mutation of the host organism to a Dal + phenotype is insignificant, it may be necessary to remove part of the dal gene. By combining such a host containing a deletion of a portion of the dal gene + + necessary for expression of the Dal phenotype, with a dal plasmid containing the entire dal + gene and segments homologous to the DNA flanking both sides of the deletion on the chromosome, transfer of the dal + allele from plasmid to chromosome can occur by homologous crossover. To avoid such a homologous crossover, a host was constructed in which the dal deletion continued into segments flanking the dal gene on the chromosome. A plasmid also containing a functional dal + gene was constructed but not DNA homologous to the DNA flanking both sides of the deletion on the chromosome. The combination of this plasmid with the aforementioned valley host constitutes the preferred host vector pair.

Alternatively, dal * allelic transfer can be avoided by using a recombinant-deficient host or by using an extra-chromosomal dal + gene, which has no or little DNA homology to the host bacterium's chromosome, the dal is preferably phased from a B. subtilis strain as explained in further detail in the following. In addition to the dal + gene, the plasmid should also contain a sequence of plasmid replication, e.g. replication functions of the high-copy plasmid pUB110 for replication in Bacillae or other Gram-positive bacteria or the replication function of pBR322 for replication in Enterobacteriaceae or other gram-negative bacteria.

Production of a desired product according to the present invention will be illustrated by the production of a maltogenic amylase from a Bacillus strain (NCIB 11837) expressed by its own promoter. Other examples of desired products that can be prepared according to the present invention are other amylases, amylo-glycosidases, pullulanases, proteases, lipases, hormones and other kinds of enzymes or eukaryotic proteins and peptides.

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The invention will be further explained with reference to the drawing, in which 1 shows the construction of plasmids pDN691, pDN770, pDN820 and pDN1050; 2 shows the construction of pDN1122; FIG. Figure 3 shows the construction of plasmids pDN1090, pDN1120, pDN1222 and pDN12277 and a restriction enzyme sites map on pDN1010. 4 shows the construction of plasmids pDN1130 and 10 pDN1290; 5 shows the construction of plasmid pDN1274, and fig. 6 shows the construction of plasmid pDN1800.

The dal + gene was obtained from DN497, a derivative of Bacillus subtilis 168 (Spizizen, Proc.Natl.Acad.Sci. 44, 1072 -78, 1958). Chromosomal DNA was completely digested with appropriate restriction enzymes and spliced with a plasmid pDN691 which exhibits resistance to chloramphenicol and kanamycin and is able to replicate in B. subtilis. The spliced DNA was transferred into a D-alanine-requiring B. subtilis dal-1, selecting for the complementation of the D-alanine requirement. A Dal + transformant that

ISLAND

at the same time, Cam (chloramphenicol resistant) has contained a recombinant plasmid derived from pDN691 to which the Dal + and CamR pheriotypes are linked. Probably due to homologous recombination between chromosome and plasmid, only negligible amounts of the plasmid could be detected in the transformant.

The present invention allows the cultivation of transformed bacteria in antibiotic-free media. However, it is to be understood that this is not a condition of the feasibility of the invention but a consequence thereof. Of course, if it is to be useful or preferred to grow the transformed bacteria of the invention in the presence of antibiotics, this can be done.

To avoid recombination and subsequent integration of the plasmid into the chromosome, plasmid was prepared from this Dal + R and Cam transformant and transformed into a strain DN733 dal recE requiring D-alanine and not prone to recombination.

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bination, selecting for Dal +. The transformants became “I * R R

at the same time Dal Cam and Kan (kanamycin resistant). The recE gene is a gene present in normal B. subtilis strains. It expresses an enzyme that allows genetic recombination between 5 homologous (i.e., similar or nearly identical) DNA sequences. (Dubnau D. et al., J. Bacteriol. 117 (1974), 488-493).

The transformants contained a small amount of a plasmid of ca. 16 kb. From this plasmid, a 2.0 kb Clal-Sphl fragment exhibiting a Dal + phenotype was cloned onto a 2.6 kb 10 fragment on plasmid pDN820: Cam in strain DN608: dal, selecting for Cam Dal, obtained a 4.6 kb recombinant plasmid pDN1000. However, this plasmid could replace the dal-1 mutation on the chromosome of recombinant strains (eg DN608) with the dal + allele by homologous recombination, whereby the selection of a Dal phenotype without simultaneous Cam selection will no longer ensure the preservation of the plasmid.

To prevent transmission of the dal + allele to the chromosome by homologous recombination and subsequent loss of plasmid and to reduce the frequency of mutations causing a 20 dal + phenotype, a deletion was performed in both the dal gene in the host and in a neighboring segment of the host chromosome. : The cloned valley gene was cut with restriction enzymes EcoRl and EcoR5 and then digested with exonuclease Bal31. The digested mixture became

spliced and transformed into the DN608 valley, becoming Selek-R

25 for Cam. Transformants containing plasmids with deletions in the dal gene were identified as Dal Cam. To construct a host strain with the appropriate deletion in the dal gene, a dal + host was transformed with one of the deletion plasmids, R _ p pDN1274 Cam dal. When selected for Cam, approx. 0.1% 30 of the transformants are Dal- probably due to the replacement of the chromosomal dal + gene with the dal deletion from pDN1274 by homologous recombination. Following spontaneous loss of pDN1274 during culture in the presence of D-alanine and in the absence of chloramphenicol, the plasmid-free strain DN1280 Dal-. Southern blotting analysis of chromosomal DNA from DN1280 revealed that it actually contained the expected dal deletion. This valley deletion host, strain DN1280, was transformed with a dal + plasmid (e.g.

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pDN1090) containing both the whole dal + gene and segments homologous to the DNA flanking both sides of the deletion on the chromosome. Therefore, the transfer of the dal + gene from plasmid to the chromosome by homologous recombination could take place and the host could be transformed into dal. Thus, preservation of the plasmid was no longer a condition of a Dal + phenotype and the plasmid was frequently lost. A dal plasmid pDN1277, containing the entire functional dal + gene but not DNA homologous to the DNA flanking the deletion in the host chromosome as explained above, was then constructed. Consequently, transfer of dal + from plasmid to chromosome by homologous recombination could not occur after transformation of the dal deletion host by pDN1277. Thus, DN1280 is a suitable dal host in D-alanine-free media for dal plasmids that do not contain DNA comprising the EcoR5 site, which normally flanks the dal 15 gene on the chromosome but has been removed in DN1280.

To ensure that cloned genes of scientific or commercial interest can really be conserved on plasmids in D-alanine-free media by the above host vector system, the gene for the Bacillus C599 maltogenic amylase (NCIB 20 11837) was transferred to a dal + plasmid. Two plasmids pDNH30 and pDNl290 were constructed. Both plasmids contain the replication functions of plasmid pUB110 and functional dal and amyM genes. None of the plasmids exhibit resistance to any antibiotics. The plasmids pDNH30 and pDNl290 were transformed into the aforementioned B. subtilis strain DN1280. Strain DN1280, as mentioned above, contains a chromosomal deletion that includes both a portion of the dal gene required for expression of the Dal + phenotype and an adjacent segment not required for the Dal + phenotype, whether or not part of the dal gene senso strictu. This last 30 part of the deletion is not contained in pDN1290. Accordingly, the dal gene cannot be transferred from pDN1290 to the chromosome by double homologous recombination. Plasmid pDNH30, on the other hand, contains both the entire segment removed on the chromosome and adjacent segments. Accordingly, transfer of the dal + gene from 35 plasmid to chromosome and subsequent plasmid loss can occur.

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The transformed strains obtained DN1297 (= DN1280 pDN1130) and DN1300 (= DN1280 pDN1290) were tested for plasmid stability during culture (cf. Example 7). During the cultivation of DN1297, homologous crossover occurred, which restored the dal'1 'gene 5 on the chromosome, and both the plasmid and Amy + phenotype were frequently lost.

When culturing DN1300, no loss of plasmid Amy + phenotype was observed during culture in a medium without D-alanine. However, if D-alanine was added to the medium, cells 10 often lost the plasmid and became Amy Dal ”. Thus, stable conservation of a plasmid containing a gene not derived from the host organism of commercial interest in antibiotic-free medium was demonstrated.

To demonstrate that plasmids can also be conserved in 15 gram-negative organisms by suppressing a D-alanine requirement, the dal gene from B. subtilis was cloned onto a plasmid in a D-alanine-requiring Escherichia coli mutant and shown to complement the requirement.

The invention will be further explained by the following 20 examples.

Production of plasmids and chromosomal DNA and transformation of Bacillus subtilis and E. coli were performed according to the following general procedures. Digestion with the restriction enzymes, Bal31 nuclease treatment, oligo-DNA linker insertion and splicing with T4 ligase of DNA was performed with enzymes from New England Biolabs under the conditions recommended by the dealers.

Originates

All strains were derivatives of Bacillus subtilis 168 (Spizizen, Proc.Natl.Acad.Sci., 44: 1072-78, 1958) RUB200: aroI906, amyE07, amyR2 was obtained from Dr. Frank Young,

University of Rochester, New York. SL438: trpC2 (sporulation and protease deficient) was obtained from Dr. Kim Hardy, Biogen, 4 "

Geneva. DN497: amyE07, amyR2 is an aro transformant of RUB200 35 with chromosomal DNA from SL438, QB1133: aroI906, metB5, sacA321, amyE was from Dr. Georges Rapoport, IRBM, Paris. QB1130: valley, metB5, sacA331, amyE 'was obtained from Bacillus Genetic Stock 10

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Center, Columbus, Ohio. DN608: dal-1, metB, sacA, amyE was an aro +, dal-1 transformant of QB1133 with chromosomal DNA from QB1130.

MT120: leuB6, recE4 r m was obtained from Dr. Teruo Tanaka, - -----— mm

Mitsubishi-Kasei Institute of Life Sciences, Tokyo. DN773: dal-1,5 amyE, recE, sacA was a met + recE transformant of DN608 with chromosomal DNA from MT120. DN606 is DN608 transformed with plasmid pUB110.

Escherichia coli strain TKL10: thr-1 leuB6 codAl trp64 pyrFlOl his-108 thyA6 argG66 ilvA634 thi-1 alr-1 deoCl lacYl 10 tonA21 tsx95 supE44 (Wijsman, Genet.Res., Camb. 20: 269-77, 1972) was obtained. from Dr. Barbara Bachmann E. coli Genetic Stock Center Connecticut, U.S.A. (CGSC 5466).

Plasmids 15 pUC9 of 2.7 kb exhibit resistance to ampicillin and were obtained from pBR322 (Vieira et al., Gene 19: 259-68, 1982) pBR322 of 4.4 kb exhibits resistance to ampicillin and tetracycline (Bolivar et al. , Gene 2: 95 - 113, 1977).

Plasmids pUB110 and pBD64 (Gryczan et al., J.

Bacteriol. 134: 318-329, 1978, and Gryczan et al., J. Bacteriol. 141: 246-53, 1980) were isolated from B. subtilis strain BD366 and BD624, respectively. pUB110 and pBD64 both exhibit resistance to kanamycin and pBD64 also to chloramphenicol. B. subtilis strains BD366 and BD624 are available from Bacillus Genetic Stock Center, 25 Columbus, Ohio, USA (strain Nos. BGSC 1E6 and 1E22). 7.6 kb plasmid pDN452 exerts resistance to chloramphenicol and kanamycin and contains the structural gene for a maltogenic amylase from Bacillus subtilis NCIB 11837. The construction of pDN452 is described in published European Patent Application No. 120,693.

30 I._Transformation of B. subtilis

Competent Bacillus subtilis cells were prepared according to Yasbin et al (J. Bacteriol. 21: 296-304, 1975). The cells were then harvested by centrifugation (7000 rpm, 3 min), resuspended in 1/10 volume of the supernatant containing 20% glycerol, frozen in liquid nitrogen and stored at -70 ° C. For transformation, frozen cells were thawed at 42 ° C and mixed with 1 room-11

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capture buffer (Spizizen's minimal medium (Spizizen, Proc.Natl.Acad. Sci. USA 44: 1072-78, 1958)) with 0.4% glucose, 0.04 M MgCl 2 and 0.002 m EGTA). DNA was added and the mixture was incubated with shaking at 37 ° C for 20 minutes. The cells were then spread on suitable selection media.

II. Transformation of E. coli

A culture of E. coli K-12 strain # 802 stored overnight in LB (10 g Bactotrypton, 5 g Bacto yeast extract 10 and 10 g NaCl per liter of water, pH 7.0) was diluted 100 times in 5 00 ml of LB and grown at 37 ° C to OD ^ q = 0.4. The culture was cooled, placed 15 minutes on ice, centrifuged for 15 minutes at 3000 rpm (in a Sorvall GS3 rotor), resuspended in 200 ml cold 0.1 M CaCl2, placed on ice for 20 minutes, centrifuged for 10 15 minutes at 3000 rpm, resuspended in 5 ml of cold 0.1 M CaCl 2 ° 9 placed on ice for 20 hours. Then, cold glycerol was added to a content of 10% and samples were frozen in liquid nitrogen and stored at -70 ° C. Frozen cells were thawed on ice, DNA was added, the mixture was incubated for 45 minutes on ice, for 2 minutes at 37 ° C and then spread on a suitable selection medium.

III. Production of plasmids from E. coli E. coli was grown overnight in 250 ml of LB, 0.4% glucose and an appropriate antibiotic. Cells were harvested by centrifugation and resuspended in 4 ml of buffer 1 (0.025 M Tris-HCl, pH = 8.0, 0.01 M EDTA, 0.05 M glucose, 2 mg / ml lysozyme). The suspension was incubated at 0 ° C for 15 minutes and then mixed with 8 ml of buffer 2 (0.2 M NaOH, 1% SDS). Then 6 ml of buffer 3 (3M NaAcetate, pH = 4.8) was added and the mixture was kept at 0 ° C for 60 min followed by centrifugation for 20 min at 19000 rpm (about 45000 g in Sorvall SS34 rotor). The supernatant was precipitated with 0.6 volumes of cold isopropanol and resuspended in 1.2 ml of 5TE (0.05 M Tris-HCl, pH = 8.0, 0.005 M EDTA), plus 20 μΐ of boiled RNase A (Boehringer) (2 mg / ml). Thirty minutes later, the solution was spread on top of 4.0 ml of buffer 4 (80 g of CsCl plus 56 ml of 5TE) and 0.1 ml of EtBr (10 mg / ml of ethidium bromide) in a VTi65

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12 tubes. The mixture was then centrifuged at 45000 rpm for 20 hours. Plasmid was then removed from the tube, dialyzed and extracted as described in section VI.

5 IV._ Production of plasmids from B. subtilis

The plasmids were prepared as described for E. coli strains (see section III) but with the following modifications. Cultivation was done in LB containing 0.01 M potassium phosphate, pH = 7.0 and an appropriate antibiotic (eg 6 µg / ml chloramphenicol) 10 and if required 100 µg / ml D-alanine. After harvest, the cells were incubated at 37 ° C with lysozyme. Buffer 2 was replaced by a mixture of one volume of buffer 2 and three volumes of buffer 5 (0.2 M glycine, 0.2 M NaCl and 1% SDS). The following steps were the same as in III.

15 V. Small-scale production of plasmids from B. subtilis

Plasmid from 5 ml of B. subtilis in LB (containing 0.01 M phosphate pH = 7.0 and appropriate antibiotics and D-alanine if required) was prepared as described in section IV except that 1: the volume of the buffers was reduced four times, 2: 0.5 ml phenol and 0.5 ml chloroform were added after buffer 3, 3: after centrifugation at 19000 rpm, the supernatant was precipitated with ethanol, resuspended in 400 μΐ buffer 6 (0.05 M Tris-HCl pH = 8.0, 0.1 M NaAcetate), the plasmid was again precipitated, resuspended in 400 μΐ buffer 6, precipitated, washed and resuspended in 100 μΐ TE (0.01 M Tris-HCl, pH = 8.0, 0.001 M EDTA) with 1 μg / ml boiled RNase A (Boehringer).

WE. Preparation of B. subtilis chromosomal DNA A pellet of frozen cells from ca. 50 ml of culture was

resuspended in 1.1 ml buffer (0.05 M Tris-HCl, pH = 7.4, 0.1 M NaCl, 25% sucrose). 100 μΐ lyzosym (25 mg / ml) and 150 μΐ EDTA (0.5 M, pH = 8.0) were added. The mixture was incubated at 37 ° C for 30 minutes and 2 ml of 0.2% SDS was added followed by incubation for 35 30 minutes at 37 ° C. 1 g of CsCl and 0.05 ml of EtBr (10 mg / ml) were added per 0.95 ml of mixture and the mixture was centrifuged at 45000 rpm at 15 ° C for 20 hours in a VTi65 rotor (Beckman). DNA

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13 was located under a long wave UV lamp and removed by puncturing the tube with a needle. EtBr was extracted with isopropanol and the solution was dialyzed for 2 hours against TEE (0.01 M Tris-HCl, pH = 8.0, 0.01 M EDTA). The solution was then adjusted to 8 ml with TEE and extracted twice with phenol and once chloroform. DNA was precipitated with 0.1 M NaCl and cold ethanol and dissolved in 1 ml TE (0.01 M Tris-HCl, pH = 8.0, 0.001 M EDTA). The chromosomal DNA solution was stored at 4 ° C.

Example 1

Construction of plasmid pDN1050 (Fig. 1)

Plasmid pBD64 was cut with restriction enzymes Eco RI and SphI, and a 3.6 kb fragment was spliced to a 0.56 kb Eco RI-SphI fragment from E. coli plasmid pBR322. The resulting 4.2 kb plasmid pDN691 showed chloramphenicol and kanamycin resistance. A 0.4 kb Hind3-BamH1 fragment of pDN691 was replaced with a 0.02 kb Hind3-BamH1 fragment from E. coli plasmid pUC9. The resulting 3.8 kb plasmid pDN720 exhibited chloramphenicol and kanamycin resistance.

A 2.8 kb NcoI-NcoI fragment of pDN720 was replaced with a 1.8 kb NcoI-NcoI fragment from pDN770. The 3.6 kb pDN770 was a spontaneous deletion of pBD64 and exhibited chloramphenicol resistance. The resulting 2.8 kb plasmid pDN820 exhibits chlorine-amphenicol resistance. pDN820 was opened at the single HgiAl site 25 and digested with Bal31 for 30 seconds at 30 ° C, removing a 0.1 kb fragment. The obtained linear fragments were spliced with a Bgl2 oligonucleotide linker from New England Nuclear (no. 1001). Plasmid pDN1050 of 2.7 kb containing a Bgl2 linker and exhibiting chloramphenicol resistance was isolated from the ligation mixture.

Example 2

Construction of plasmid pDN1122 (Fig. 2)

7.6 kb plasmid pDN452 and exhibiting chloramphenicol-35 and kanamycin resistance and containing the structural gene amyM of a Bacillus C599 maltogenic amylase was digested with Aval treated with exonuclease Bal31 spliced with EcoRl oligonucleotide 14

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time linker (Biolab # 1004), digested with EcoRl, spliced with T4 ligase and transformed into B. subtilis DN497; amyE, with R + being selected for Cam. An Amy transformant contained 6.6 kb plasmid p520-20. The amylase yield from p520-20 was not less than the yield from pDN452.

A 2.7 kb NcoI-NcoI fragment from p520-20 was then replaced with a 1.7 kb NcoI-NcoI fragment from pDN770 (prepared as in Example 1). The 5.6 kb plasmid pDN808 obtained contained amyM and exhibited resistance to chloramphenicol.

A 2.6 kb EcoRl-Sphl fragment from pDN808 was replaced with a 2.4 EcoRl-Sphl fragment from pDN1050 (prepared as described in Example 1). The resulting 5.4 kb plasmid pDNH22 contained amyM and exhibited resistance to chloramphenicol.

Example 3

Cloning of the valley gene

Ca. 3 μg of chromosomal DNA from B. subtilis strain DN497 and R R

1 μΘ of plasmid pDN691 Cam Kan was completely digested with restriction enzymes BamH1 and Sphl. Chromosomal and plasmid DNA were mixed and spliced with T4 ligase and transformed into strain “R + DN606; dal pUB110: Kan. Among the approx. 200 Dal transformants had an R + simultaneously become Cam as an indication that the Dal phenotype was linked to a recombinant plasmid derived from pDN691. Plasmids T5 were prepared from this Dal Cam transformant and transformed into the strain DN773 dal recE-, selecting for Dal. Valley transformants were simultaneously Cam and Kan. The resistance to kanamycin is built into the cloning vector pDN691. This is not affected by the cloning of the dal gene, so that the recombinant plasmid transformed into DN733 is Dal Cam Kan.

The transformants contained only a very small amount of a plasmid of ca. 16 kb.

From this plasmid, a 2.0 kb Clal-Sphl fragment exhibiting a Dal + phenotype was cloned into the Clal and Sphl site of plasmid pDN820 in strain DN608; dal to form a recombinant 35 plasmid, pDN10OO of 4.6 kb.

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A restriction enzyme sites map on pDN1000 is shown in FIG. 3. The transformed strain DN1000 = DN608 pDN1000 was deposited at The National Collection of Industrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, on December 7, 1984, and was assigned reference number NCIB 12029.

NCIB, an international depositary recognized by the Budapest Treaty of 1977, ensures the permanence of the depository and public access thereto in accordance with Rules 9 and 11 of this Treaty.

10 The following observations prove that the cloned chromo somal fragment did indeed contain the dal gene (as defined by the dal-1 mutation) and not another gene capable of suppressing the D alanine requirement: 1: Linearized (non-replicative) plasmid transferred a dal recipient to dal but not to cam as an indication of homologous recombination between the dal gene on the chromosome and the cloned chromosomal fragment.

2: Approx. 0.2% of plasmids made from a host bacterium dal-1 on the chromosome were dal (though with a restriction enzyme pattern indistinguishable from the original dal + plasmid). These dal plasmids could not complement the chromosomal dal-1 mutation as an indication that the chromosomal mutation had been transferred to dal plasmids by homologous recombination.

3: By Southern blotting analysis of chromosomal B.

subtilis DNA, it was demonstrated that a chromosomal Clal-SphI fragment hybridized with a fragment of the same size from 30 pDN1000. Thus, no significant rearrangement of the dal gene occurred prior to its cloning on pDN1000.

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Example 4

Construction of plasmid pDN1130 (Figs. 3 and 4) pDN1000 (from Example 3) containing the dal + gene was opened at the ClaI site, digested with exonuclease Bal31 and spliced 5 with a BamH1 oligonucleotide linker (no. 1017 from Biolabs). The resulting plasmid pDN1090 contained the dal + gene and exhibited chloramphenicol resistance.

pDN1122 (from Example 2) was cut with Sphl and Bgl2, and a 4.4 kb fragment was spliced with a 2.0 kb Sphl-BamHl 10 fragment from pDN1090.

• + +

The resulting plasmid pDN1130 contained the amyM and dal gene but did not show resistance to chloramphenicol.

Example 5 Construction of plasmid pDNl290 (Figs. 3 and 4)

Plasmid pDN1010 dal Cam of 4.5 kb was constructed by exonuclease Bal31 digestion of pDN10OO digested with SphI and introduction of both a Bgl2 and a Sacl oligonucleotide linker (# 1001 and # 1005 from Biolabs, respectively).

20 pDN1110 was opened at the single EcoR5 site, exo nuclease digested with Bal31 and spliced with two Bgl2 oligonucleotide linkers (no. 1001 from Biolabs). The fusion of links with a plasmid forming a Bell site. The resulting plasmid pDN1222 contained the dal + gene and exhibited chloramphenicol resistance.

A 0.7 kb BcII-BcII fragment from pDN1022 was spliced with a 3.5 kb BamH1-BcII fragment from pDN1090 (from Example 4).

The resulting 4.2 kb plasmid pDNl277 contained the dal + gene and exhibited chlormaphenicol resistance. Plasmid pDN1227 was transformed into strain DN1280 (see Example 6) and the resulting strain DN1517 (= DN1280 pDN1227) was deposited at the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen,

Scotland, December 7, 1984 and received reference number NCIB 12030.

pDN1090 was further converted to plasmid pDN1120 35 dal Cam of 4.5 kb by replacing a 2.6 kb Sphl-BamH1 fragment with a 2.5 kb Sphl-BamH1 fragment from pDN1050 (prepared as described in Example 1 ).

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+ R

PDN1265 amyM Cam was constructed by Bal31 exo-nuclease digestion of pDN1120 digested with EcoR5 and subsequent digestion with SphI. A 2.9 kb fragment was then spliced with SacI oligonucleotide linker (Biolabs # 1005) and a 2.9 kb fragment from pDN1130 obtained by digestion with Eco RI and Bal31 exonuclease treatment and subsequent digestion with SphI.

The resulting 5.8 kb plasmid pDN1265 contained the amyM + gene and exhibited chloramphenicol resistance.

pDN1265 was then cut with Sphl-Bgl2 and a 4.7 kb 10 fragment was spliced with a 1.6 kb Sphl-Bgl2 fragment from PDN1277.

The resulting plasmid pDNl290 of ca. 6.3 kb contained the amyM'1 'and dal + gene, but no antibiotic resistance marker.

Example 6

Construction of a host with a deletion in the valley gene (Fig. 5)

By cutting 5 μg of pDN1090 in the cloned dal + gene with restriction enzymes EcoRl and EcoR5 and subsequent digestion of the fragments with exonuclease Bal31 for 60 seconds, splicing 20 and transformation in DN608: dal-l, deletions were observed, 4 · which destroyed Dal the phenotype of the plasmid. Transforming the

R

strain DN497 with one of these deletion plasmids pDNl274 Cam _ R

valley and selection for Cam gave approx. 0.1% transformants that were

Dal, presumably due to replacement of the chromosomal valley + 25 gene with the dal deletion from pDN1274. After spontaneously losing R - pDNl274, the transformant became Cam, but remained Dal. Southern blotting analysis of chromosomal DNA of this strain DN1280 showed that it really contained the expected valley deletion. The frequency of mutations that could cause the return of this dal 30 deletion mutant to a dal phenotype was less than 10. DN1280 containing plasmid pDNl277 = DN1517 (see Example 5) was deposited.

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Example 7

Stability of dal + amyM + plasmids in a dal 'host

To demonstrate both the stability of host - plasmid combination DN1300 (= DN1280 pDN1290) and the importance of absence 5 of overlapping chromosomal and plasmid DNA segments flanking the chromosomal deletion, the following experiment was performed:

Single colonies of strains DN1297 (= DN1280 pDN1130) and DN1300 (= DN1280 pDNl290) were resuspended in LB medium sup-10 pierced with 10 mM potassium phosphate buffer pH = 7.0 and 0.2% glucose. Half of each resuspension was seeded in the above medium, the other half in identical media supplemented with 200 μg / ml D-alanine. By diluting the cultures either 100 or 1000 times, the strains were grown a number of generations at 37 ° C under good aeration either in the presence or absence of D-alanine in the medium. For . 7 each dilution, no less than 10 cells were transferred. At a 1-day interval, the frequency of Amy + colonies (a total of about 100) was tested on LB media plates with or without D-alanine according to the growth conditions. The results are shown in Table 1. Ten randomly selected Amy colonies were found to contain no plasmid.

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Table 1

Frequency of Amy cells after growth in LB medium with and without D-alanine.

Generations 5 Strains Plasmid 51 68 85 __% Amy ~ DN1297 PDN1130 2% 20% DN1297 + D-ala1 pDN1130 60% DN1300 PDN1290 0% 0% 0% 10 DN1300 + D-ala1 pDN1290 60% 90% 1 First 20 generations in absence of D-alanine 15 The results shown in Table 1 demonstrate that a plasmid pDNl290 is very unstable under non-selective conditions {addition of D-alanine to the growth medium) but becomes stable during growth in a selective medium (without addition of D -alanine). It has been further demonstrated that homologous double crossing, 20 restoring the dal + gene on the host chromosome, and subsequent plasmid loss did not occur in DN1300 (no Amy cells after 85 generations of culture), whereas the frequency of Amy cells was 20% after 68 generations of DN1297 grown in a D-alanine-free medium, presumably due to transfer of the dal + gene from plasmid to chromosome and subsequent plasmid loss.

Example 8

Construction of plasmid pDN1800 (Fig. 6) 30 pDN1284 was constructed by combining the 1.4 kb Bgl2-Bgl2 fragment from pDN1222 (see Fig. 3) with the 1.5 kb BamH1-Bgl2 fragment from pDN1050 (see Fig. 3).

From pDN1284, the dal + gene was transferred on a 1.4 kb Bgl2-SalI fragment to the classical E. coli plasmid vector 35 pBR322 cut with BamH1 and SalI. The recombinant plasmid pDN1800: Amp (ampicillin resistant) was transformed into E. coli strain TKL10, selected for AmpR, to give 20

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strain DN1800: pDNl800. Strain DN1800 was deposited at NCIB d.

November 22, 1985 and was given the reference number NCIB 12181. The permanence of the repository and the public's access thereto are thus secured (see above). TKL10 exhibits a D-alanine-requiring phenotype at 5 42 ° C, but the requirement is suppressed in DN1800. Thus, pDN1800 containing the dal gene from B. subtilis is capable of complementing the D-alanine requirement in an E. coli strain that has a defect in the alanine racemase activity (Wild et al., Molec.Gen.Genet. 198: 315- 22, 1985).

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Claims (9)

21 DK 159282 B 5 A method of stabilizing extra-chromosomal elements in a host bacterium during culture characterized by introducing into the extra-chromosomal element a DNA sequence encoding D, L-alanine racemase and the extra-chromosomal element containing said DNA sequence is transformed into the host bacterium using a host bacterium with a defect in the chromosomal valley gene for D, L-alanine racemase. - 15 Method according to claim 1, characterized in that the host bacterium has a mutation in the chromosomal dal gene for D, L-alanine racemase, or a deletion comprising at least part of the dal gene. The method of claim 2, characterized in that the mutation is a deletion of both a portion of the dal gene required for expression of the Dal + phenotype and a portion of direct flanking DNA unnecessary for the expression of the Dal + phenotype, whether it is part of the dal gene or not. 25 Transformed bacterium, characterized in that it is derived from a host bacterium that has a defect in the chromosomal valley gene for D, L-alanine racemase and that it contains an extra-chromosomal element containing a DNA sequence containing codes for D, L-alanine racemase. 5 The Dal + phenotype, whether or not part of the dal gene, and contains an extra-chromosomal element containing a DNA sequence encoding D, L-alanine racemase. Transformed bacterium according to claim 4, characterized in that it contains a mutation in the chromosomal dal gene for D, L-alanine racemase or a deletion comprising at least 35 part of the dal gene. 22 DK 159282 B Transformed bacterium according to claim 5, characterized in that it has a deletion of both a portion of the dal gene necessary for expression of the Dal + phenotype and of a portion of direct flanking DNA which is unnecessary for expression of the dal + phenotype. Process for the preparation of a desired product in transformed bacteria, characterized in that a bacterium which has a defect in the chromosomal valley gene for D, L-alanine race and contains an extra-chromosomal element containing a DNA sequence encoding D, L-alanine racemase, and a DNA sequence encoding the desired product are grown in a suitable culture medium and the desired product is recovered from the culture medium. Method according to claim 7, characterized in that the host bacterium contains a mutation in the chromosomal dal gene 20 for D, L-alanine racemase or a deletion comprising at least part of the dal gene. The method according to claim 8, characterized in that the host bacterium has a deletion of both a portion of the dal gene required for expression of Dal + phenotype and of a portion of direct flanking DNA unnecessary for expression of Dal + phenotype. , whether or not part of the dal gene, and that the extra-chromosomal element contains a DNA sequence encoding D, L-alanine racemase.