CA2096284A1 - Process for stable chromosomal gene amplification - Google Patents
Process for stable chromosomal gene amplificationInfo
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- CA2096284A1 CA2096284A1 CA002096284A CA2096284A CA2096284A1 CA 2096284 A1 CA2096284 A1 CA 2096284A1 CA 002096284 A CA002096284 A CA 002096284A CA 2096284 A CA2096284 A CA 2096284A CA 2096284 A1 CA2096284 A1 CA 2096284A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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Abstract
Abstract of the Disclosure A simple process for stable chromosomal gene amplification of DNA sequences which encode a polypeptide in procaryotic microorganisms, particularly bacillus strains.
Description
.
209628~
PROCESS FOR ST~BLE CHROMOSOMAL GENE AMPLI FICATION
Backaround of the Invention The invention relates to a simple process for stable chromosomal gene amplification of DNA sequences which encode a polypeptide in procaryotic microorganisms, in particular 5 in bacillus strains.
Microorganisms, in particular bacillus strains, find widespread use in industry for the microbiological preparation of polypeptides, in particular for the pr~duction of important b~c~llus enzymes, such as, e.g., proteases or amylases etc. An important aim in the opti~ization of the preparation ~of such enzymes is to increase the expression of these enzymes by the badlli. To achieve this, for example, the regulation of the expression can ~e optimized, or the genetic information for the enzyme and its expression in the bac~lus can be a~pl~fied. To achieve amplifioation of the genetic information, the state of~the~art, on the one hand, proposes an increase in the acil:l us of the number of plasmids with the neces~a~y genetic information, or, on t~e other hand, attempts to increase the expression of the polypeptide o~ interest by multiple incorporatlon;of the genetic information encoding it into the chromQsomal DNA o~ the~bacillus. For reasons of stability, howeve~r,~increasing the plasmid number is not suitable~for producing ~the~ enzymes of interest on an indus~rial saale. ~It i8 therefore pre~erred in the art to seek to achiéve increase of expression: by multiple --:
..
.......
~, " : '::
.. ..
:
2096~84 incorporation of the genetic information into the chromosomal DNA.
Summary of the Invention It is therefore the object of the invention to provide a simple process for amplifying a DNA sequence which codes for a polypeptide of interest.
Another object of the invention is to provide a process for achieving stable chromosomal i~tegration of additional copies of a DNA sequence coding for a polypeptide of interest into the genome of an organism.
A further object of the invention is to provide a process for amplifying a DNA sequence which is generally applicable to DNA sequences coding for various polypeptides.
It is also an object of the invention to provide a process for amplifying a DNA sequence coding for an enzyme.
An additional object of the invention is to provide a process particularly suitable for stably incorporating a DNA
seguence coding for a peptide of interest into the genome of a bacillus.
These and other ob;ects of the invention are achieved by providing a process for preparing a transformed procaryotic host cell containing a chromosome which comprises at least two copies of a DNA sequence encoding a polypeptide of interest, the process comprising the steps of:
- transforming a procaryotic host cell which already contains chromosomal DNA co~prising at least one copy of a DNA 6equence encoding the polypeptide of interest, with a circular DNA suitable for transformation of such a host cell, the circular DNA consisting essentially of a) a DNA fragment containing the DNA sequence encoding the polypeptide of interest and necessary DNA sequences for expression thereof, and 3S b) at least one DNA fragment containing a DNA sequence encoding a æelectable marker, 209628~
the circular DNA being free of DNA sequences which enable it to replicate autonomously in the host cell, - subsequently selecting with the aid of the selectable marker, cells which have chromosomally integrated at least one copy of the circular DNA, and - isolating the selected transformed cells.
~etailed Descri~tion of Preferred Embodiments The invention thus relates to a process for preparing a transformed procaryotic ho~t cell which conta;ns in the chromosome at least two copies of the DNA sequence encoding a polypeptide of interest, which process is characterized in that a procaryotic host cell, which alre~dy contains in its chromosomal DNA at least one copy of a DNA sequence encoding a polypeptide of interest, is transformed with a circular DNA, which is suitable for the transformation of the host cell, essentially comprising a) a DNA fragment, which contains the DNA sequence encoding a polypeptide of interest, including the necessary sequences for the expression and, where appropriate, for the secretion of the polypeptide of interest, and comprising b) at least one DNA fragment with a DNA sequence which encodes a selectable marker, without this circular DNA containing any DNA sequence which ena~les it to replicate autonomously in the host cell, and such cells, which have chromosomally integrated at least one copy of the clrcular DNA employed, subsequently are selected with the aid of the selectable marker, and the cells thu~ transformed are isolated.
That the circular DNA, which is suitable for the transformation, consists essentially of fragment a) and fragment b) signifies, according to the invention, that the circular DNA may additionally contain short DNA sequences, for exa~ple linker sequences, ~uch as those which were required for the con8truction of the circular DNA.
.~, The sequences contained in fragment a) which are necessary for the expression and, where appropriate, for the secretion of the polypeptide of interest are the promoter, c,perator, enhancer, terminator and signa sequences which are required for this purpose, and additionally, where appropriate, are also pre-sequences and pro-sequences such as those which are necessary, for example, for the secretion of a protease.
All customary DNA sequences which permit selection of the transformed cells may be employed as DNA sequences encoding selectable markers in fragment b). Customary selection procedures of this type are, for example, selections based on auxotrophic mutations or, preferably, antibiotic resistances of the cells employed.
Bac~lli, preferably Bacillus alcalophilus, Bacillus licheniformis or Bacillus subtilis, are expediently employed as host organisms in ~he process according to the invention.
Examples of suitable polypeptides include proteases, preferably alkaline proteases, and in particular highly 2n alkaline proteases, as well as ~-amylases, lipases or cellulases.
In the process according to the invention, integration of the DNA sequence encoding the polypeptide of interest into t~e geno~e of the host cell is achieved by simple ~5 homologous recombination. For this purpose, a host cell, which at least already contains one DNA sequence for the polypeptide of interest and the DNA sequences required for the expression of this polypeptide, is tran~formed according to the invention with a circular DNA which possesses DNA
sequence~ wh~ch are homologous to the chromosomal DNA of the host cells. In this connection, these homoloqous DNA
sequences correspond at the same time to the DNA fragment a), which contains the DNA sequence encoding the polypeptide of interest. According to the invention therefore no further ~pecial target DNA sequence, which i~ homologous to the chromoso~al DNA of the host organism, is necessary for 209~28~
the homologous recombination, since the function of such a sequence is concomitantly fulfilled by the above DNA
fragment a) with the DNA sequence encoding a polypeptide of interest. Furthermore, the circular DNA employed here S contains at least one marker DNA sequence! for example one which encodes an antibiotic resistance. After the host organism has been transformed with this circular DNA, the marker sequence serves for the selection of those transformants which contain more than one DNA sequence encoding the polypeptide of interest. In order to ensure that only those transformants are obtained and selected in which the circular DNA has been integrated into the chromosomal DNA of the host organism, the circular DNA
employed possesses no DNA sequences which permit autonomous lS replication of this DNA in the host organism. Transmission of the circular DNA in the form of plasmids is thus excluded. By the process according to the invention, the transformed host cells contain the DNA sequence encoding the polypeptide of interest stably integrated into their genome.
Known methods may ~e used in the process according to the invention for preparing the circular DNA which is necessary for the transformation. For this purpose, the ~NA
sequence encoding the polypeptide of interest, as well as the sequences which are required for polypeptide expression and, where appropriate, polypeptide secretion in a host organism, i.e. regulatory sequences, signal sequences, promoter sequences, terminator sequences and the DNA
sequences encoding the pre- and pro-units of the polypeptide, are ~ir~t isolated from a microorganism which itsel~ produces the polypeptide of interest. For example, the chromosomal DNA from a bacterium ("donor bacterium~), in particular from a Bac~llus species which produces the polypeptide of interest, e.g. a protease or amylase, is isolated by known methods and partially hydrolyzed using ~uitable restriction endonucleases. The resulting restriction fragments of the donor DNA can be separated ,'' ' ~, ' ~ .. ' ~ .
209628~
according to size, e.g. by gel electrophoresis, and the fragments of the desired size can then be recombined with a suitable vector DNA, e.g. a plasmid DNA. Bacteria, preferably a ~acillus species, can be transformed with the in-vitro recombined plasmid (vector DNA ~ restriction fragments of the donor DNA), and the transformants selected according to the known marker property of the vector DNA
(antibiotic resistance). Among these transformants, those clones are sought which secrete the polypeptide of interest, e.g. a protease or amylase. Finally, the plasmid DNA
introduced into these transformants is isolated from a clone with the desired polypeptide activity. Renewed transformation of a bacterium is used to examine whether the polypeptide activity is plasmid-bound, i.e. whether the polypeptide activity is coupled to the marker property. In addition to the vector DNA with known restriction sites, the resulting isolated plasmid contains the desired structural gene for the polypeptide of interest as well as the regulatory seguences, signal sequences, promoter sequences and terminator sequences, which are required for polypeptide expression, and, where appropriate, the DNA sequences encoding the pre- and pro-units of the polypeptide. If the vector contains further DNA sequences from the donor bacterium, which are not required here, it is advisable, where appropriate, to eliminate these unreguired sequences prior to further use of the vector, and to reduce the donor DNA sequence essentially to the structural gene for the polypeptide and to the DNA sequences which are reguired for its expression and, where appropriate, for its secretion.
To do this, for example, the plasmid encompassing the additional, unrequired DNA sequences is restricted or cut with a number of different restriction endonucleases; the resulting DNA fragments are separated according to size by gel electrophoresis, and a restriction map is constructed on the ba~is of the band pattern which has been found. In this way, the restriction sites which are located in the region 2~9628~
of the donor DNA sequence are determined. At this point, knowledge of the restriction map of the plasmid makes it possible to excise from this plasmid, by cutting with selected restriction endonucleases, a DNA fragment of the S donor DNA sequence in which essentially only the required sequences from the donor bacterium remain. By reincorporating this donor DNA sequence, which has ~een reduced in size, into a suitable vector, a new plasmid can be obtained which is capable of polypeptide expression and which can se~ve as the basis for the production of the circular DNA used in the process according to the invention.
The restriction map in Figure 1 represents an example of such a reduced plasmid which has the designation pCLEAN4.
In this plasmid, the polypeptide of interest is a highly al~aline protease from a Bacillus alc~lophilus.
To produce circular DNA which can be e~ployed in the process according to the invention, those DNA sequences are removed from the plasmid of the above type pCLEAN4 which would permit replication of the plasmid in the host organism. If the starting vectors contain several DNA
sequences which encode, for example, different antibiotic resistances, the antibiotic resistance DNA sequences which are not reguired can also be removed from the vector. In the case of the above example of the plasmid pCLEAN4, this plasmid contains, for example, t~e functions repU and oriB
as well as resistances against the antibiotics neomycin and kanamycin. Based on a knowledge of the restriction map, these unwanted DNA sequences can be removed ~rom the plasmid by cutting with 8u1tably selected restriction endonucleases.
In the case of the plasmid pCLEAN4, a re~triation endonuclease Nsi I is used, for example, to delete fro~ the vector a fragment which contains the functions for the replication of the plasmid in Bacillus species as well as the resistances against neomycin and kanamycin, and the remaining DNA is then converted into a circular DNA uæi ng DNA ligase. The DNA sequence encoding a polypeptide of .;nterest, as well as the sequence for the antibiotic resistance phleomycin, are still retained from this deleted vector as circular DNA (pCLEAN4-del). With this circular DNA, and using t~e polypeptide gene of interest (in this case a highly alkaline protease), integration into the chromosome of e.g. Bacillus alcalophilus can be achieved by homologous recombination.
The circular DNA construction according to ~he invention can also be prepared by in vitro recombination, using DNA ligase, of a) the DNA sequence, which encodes the polypeptide of interest and which can be obtained e.g. by excising the corresponding DNA sequence with restriction endonucleases from a plasmid containing this sequence, with b) a resistance marker, which was obtained by excising with restriction endonucleases a corresponding DNA
sequence from a plasmid which contains this resistance marker, so that a circular DNA results.
Preferably, any DNA sequence encoding an antibiotic resistance is suitable as a resistance marker which a) is expressed in the microorganism to be transformed and b) encodes resistance against an antibiotic against which the microorganism to be transformed is not naturally resistant.
For;example, the resi~tance gene can encode resistance against an antibiotic selected from the group consistinq of chloramphenicol, ampicillin, tetracycline, kanamy¢in, neomycin, mitomycin C, bleomycin or phleomycin. A DNA
~equence from the plasmid pUBllO which encodes resistance against the antibiotic phleomycin is particularly suitable.
8y transformation of a microorganism which already contains in the chromoso~al DNA at least one DNA æequence encoding a pol~peptide of interest, a further DNA sequence encoding this polypeptide together with the pertinent ~ - 8 -, . .
209628~
sequences which are necessary for expression and, where appropriate, secretion, can be integrated by homologous recombination into the chromosome of the host organism at the site of the gen~ for the polypeptide of interest. When this takes place, the additional DNA sequence encoding a polypeptide of interest and the pertinent sequences necessary for expression and, where appropriate, secretion are incorporated into the chromosome ~y simple cross-over in such a way that the DNA sequence encoding the antibiotic resistance comes to lie between the original DNA encoding the polypeptide which is already present and the newly introduced DNA which likewise encodes this polypeptide.
Subsequently, the transformed microorganisms are selected using this antibiotic resistance by cultivating them on a lS nutrient medium to which the relevant antibiotic has additionally been added at a particular concentration.
Under these conditions, only those transformants are capable of replication which have the circular DNA according to the invention integrated in their chromosomal DNA. The concentration of the antibiotic added to the culture medium is chosen in such a way that at least such transformants are selected as contain, ~n the chromosomal DNA, at least two DNA sequences encoding the polypeptide of interest and one DNA sequence encoding the resistance marker.
By cultivating transformants of this nature at higher antibiotic concentrations, those descendants can be isolated which possess in their genomes several copies of the DNA
~equence encoding the antibiot~c resistance, and thus al80 several copies of the DNA sequence encoding the polypeptide of interest. A feature of such clones may be a further elevation in the synthesis of the polypeptide of interest.
As is known, constructions of this nature, with adjacent repetitive DNA sequences in the genome, prove to be unstable in common laboratory strains. In industrial high-performance strains, which have generally been isolatedafter multiple cycles of mutation, and which therefore _ g _ 2~9628~
generally possess a diminished recombination capability, which is recognized e.g. by increased sensitivity towards the antibiotic mitomycin C, repetitive sequences of this nature in the chromosome are sufficiently stable for carrying out large-scale industrial production in th~
absence of antibiotic. If instabilities are observed, it is possi~le to isolate recombination-deficient descendants by searching, after mutation with a common mutagen, for those mutants which show increased sensitivity towards the antibiotic mitomycin C.
Brief Description of the Drawinas m e invention will be described in further detail with reference to the accompanying drawings in which:
Fig. 1 shows a restriction map of the plasmid pCLEAN4;
Fig. 2 shows a restriction map of the plasmid pUB 131;
Fig. 3 shows a restriction map of the plasmid pLI l;
and Fig. 4 is a graph of the proteolytic activities in the culture supernatants of cultures of B. alcalophilus HA
139/28 and HA 139/28-INTl.
ExamPles The following description of typical experimental emboaiments is intended to further illustrate the invention without limiting its scope.
Unless otherwise indicated, the work was generally carried out according to methods such as thosa d~scribed in T. Maniatis, E.F. Frit~ch, J. Sambrook, Molecul~r Clon~ng, A Labor~tory Nanual, Cold Spxing Harbor Laboratory (1982~.
The starting vectors which were used ~re commercially available on an unrestricted basis, or they may be prepared from available vectors ~y known methods. In this way, the vector M13gl31 was obtained from Amersham, Buckinghamshire, England.
The different restriction endonucleases which were used are known in the art and are commercially available. ~he reaction conditions which are required on each occasion when using these Xnown restriction endonucleases are likewise S known.
After incubation with a restriction endonuclease, the protein was removed by extraction in a known manner (e.g.
with phenol and chloroform~, and the cleaved DNA isolated in a known ~anner (e.g. by precipitation with ethanol from the aqueous fraction) and used further.
If desired, the cutting of vectors with restriction endonucleases may be followed by hydrolysis of the terminal 5'-phosphate radical with an alkaline phosphatase (de-phosphorylation). When dephosphorylation of the 5'-end was undertaken in the examples, it was effected in a known manner. Further details regarding the dephosphorylation procedure and the reagents which are required for it, may be obtained from Mania~is et al. (pp. 133 - 134).
Partial hydrolysis denotes incomplete digestion of DNA
by a restriction endonuclease. In this case, the reaction conditions are chosen so that, in a DNA substrate, cutting takes place at some, but not all, of the recognition sites for the restriction endonuclease employed.
To obtain and isolate particular DNA fragments, e.g.
after treatment of DNA with reætriction endonucleases, the resulting DNA fragments were ~eparated in a known manner by gel electrophoresis (e.g. on agarose gel) and subsequently identified based on their molecular weight (determination by comparison with reference DNA fragments of known molecular weight). The desired DNA fragments were then isolated from the corresponding gel ~ones.
Ligations can be carried out under known conditions, e.g. in a buffer with about lQ units of T4-DNA ligase per 0.5 ~g of an approximately equimolar amount of the DNA
fragmenta to be ligated (see e.g. Maniatis et al., p. 146).
2~(3628~
Transformation is understood to mean the introduction of DNA into a microorganism so that the DNA replicates autonomously in the microorganism as plasmid DNA, or is incorporated chromosomally, and can, where appropriate, be expressed. For the transformation of Bacillus species, the method described by Anagnostopoulos et al. [J. Bact. 81 :741-46 (1961)], is suitable, for example.
A polycloning site (polylinker) is a short to average-sized double-stranded DNA fragment which contains multiple reco~nition ~ites for restriction endonucleases in close proximity to each other. For example, one polycloning site used in the examples, which originates from the vector M13tgl31, has a size of about 0.07 kB (kilobases) and contains recognition sites for 14 different restriction endonucleases.
The Bacillus alcalophilus ~train employed in Example 1 and designated as Bacillus alcalophilus HA 1 was deposited July 28, 1989 in the Deutsche Sammlung von Mikroorganismen (DSM) with the DSM number 5466. Other microorganisms which were used are available commercially, e.g. Bacillus subtilis BD 244 (Bacillus Genetic Stock Center 1 A 46) or Bacillus su~tilis BD 366 (B~cillus Genetic Stock Center 1 E 6).
Further microorganisms used within the scope of the invention can be produced from Bac~llus DSM 5466 or from commercially available microorganisms according to the procedures given below:
1. ~acillus alcalophilus HA 139/28 and HA 28/12.
Both strains are descendants o~ Bacillus alcalophilus HA 1. They secrete a highly alkaline protease at an increased le~el, as described e.g. in Published German Patent Application No. DE 40 23 458, and can be employed as industrial production strains. Strains of this nature may be obtained by treating Bacillus alcalophilus HA 1 with mutagenic ~ubstances, such as, e.g., 1-methyl-3-nitro-1-nitrosoguanidine, according to conventional and well-known 209628~
methods, and subsequently searching on protein-agar plates for those mutants which are able to form larger areas of hydrolysis than the starting strain. By repeated cycles of mutation, strains can be isolated exhibiting additionally increased protease formation.
2. Bacillus licheniformis KC 14/1-422.
This strain is a Bacillus licheniformis which secretes an alkaline pxotease at an elevated level and can be used as an industrial production strain. A strain of this nature can be obtained by, e.g. treating a Bacillus lichenifo~mis Type Strain (obtainable from the Deut~che Sammlung von Mikroorganismen under the number DSM 13) with mutagenic substances, such as, e.g., l-methyl-3-nitro-1-nitrosoguanidine, according to conventional and well-known methods, and subsequently searching on protein-agar plates for those ~utants which are able to form larger areas of hydrolysis than the starting strain. By repeated cycles of mutation, strains can be isolated exhibiting additionally increased protease formation.
209628~
PROCESS FOR ST~BLE CHROMOSOMAL GENE AMPLI FICATION
Backaround of the Invention The invention relates to a simple process for stable chromosomal gene amplification of DNA sequences which encode a polypeptide in procaryotic microorganisms, in particular 5 in bacillus strains.
Microorganisms, in particular bacillus strains, find widespread use in industry for the microbiological preparation of polypeptides, in particular for the pr~duction of important b~c~llus enzymes, such as, e.g., proteases or amylases etc. An important aim in the opti~ization of the preparation ~of such enzymes is to increase the expression of these enzymes by the badlli. To achieve this, for example, the regulation of the expression can ~e optimized, or the genetic information for the enzyme and its expression in the bac~lus can be a~pl~fied. To achieve amplifioation of the genetic information, the state of~the~art, on the one hand, proposes an increase in the acil:l us of the number of plasmids with the neces~a~y genetic information, or, on t~e other hand, attempts to increase the expression of the polypeptide o~ interest by multiple incorporatlon;of the genetic information encoding it into the chromQsomal DNA o~ the~bacillus. For reasons of stability, howeve~r,~increasing the plasmid number is not suitable~for producing ~the~ enzymes of interest on an indus~rial saale. ~It i8 therefore pre~erred in the art to seek to achiéve increase of expression: by multiple --:
..
.......
~, " : '::
.. ..
:
2096~84 incorporation of the genetic information into the chromosomal DNA.
Summary of the Invention It is therefore the object of the invention to provide a simple process for amplifying a DNA sequence which codes for a polypeptide of interest.
Another object of the invention is to provide a process for achieving stable chromosomal i~tegration of additional copies of a DNA sequence coding for a polypeptide of interest into the genome of an organism.
A further object of the invention is to provide a process for amplifying a DNA sequence which is generally applicable to DNA sequences coding for various polypeptides.
It is also an object of the invention to provide a process for amplifying a DNA sequence coding for an enzyme.
An additional object of the invention is to provide a process particularly suitable for stably incorporating a DNA
seguence coding for a peptide of interest into the genome of a bacillus.
These and other ob;ects of the invention are achieved by providing a process for preparing a transformed procaryotic host cell containing a chromosome which comprises at least two copies of a DNA sequence encoding a polypeptide of interest, the process comprising the steps of:
- transforming a procaryotic host cell which already contains chromosomal DNA co~prising at least one copy of a DNA 6equence encoding the polypeptide of interest, with a circular DNA suitable for transformation of such a host cell, the circular DNA consisting essentially of a) a DNA fragment containing the DNA sequence encoding the polypeptide of interest and necessary DNA sequences for expression thereof, and 3S b) at least one DNA fragment containing a DNA sequence encoding a æelectable marker, 209628~
the circular DNA being free of DNA sequences which enable it to replicate autonomously in the host cell, - subsequently selecting with the aid of the selectable marker, cells which have chromosomally integrated at least one copy of the circular DNA, and - isolating the selected transformed cells.
~etailed Descri~tion of Preferred Embodiments The invention thus relates to a process for preparing a transformed procaryotic ho~t cell which conta;ns in the chromosome at least two copies of the DNA sequence encoding a polypeptide of interest, which process is characterized in that a procaryotic host cell, which alre~dy contains in its chromosomal DNA at least one copy of a DNA sequence encoding a polypeptide of interest, is transformed with a circular DNA, which is suitable for the transformation of the host cell, essentially comprising a) a DNA fragment, which contains the DNA sequence encoding a polypeptide of interest, including the necessary sequences for the expression and, where appropriate, for the secretion of the polypeptide of interest, and comprising b) at least one DNA fragment with a DNA sequence which encodes a selectable marker, without this circular DNA containing any DNA sequence which ena~les it to replicate autonomously in the host cell, and such cells, which have chromosomally integrated at least one copy of the clrcular DNA employed, subsequently are selected with the aid of the selectable marker, and the cells thu~ transformed are isolated.
That the circular DNA, which is suitable for the transformation, consists essentially of fragment a) and fragment b) signifies, according to the invention, that the circular DNA may additionally contain short DNA sequences, for exa~ple linker sequences, ~uch as those which were required for the con8truction of the circular DNA.
.~, The sequences contained in fragment a) which are necessary for the expression and, where appropriate, for the secretion of the polypeptide of interest are the promoter, c,perator, enhancer, terminator and signa sequences which are required for this purpose, and additionally, where appropriate, are also pre-sequences and pro-sequences such as those which are necessary, for example, for the secretion of a protease.
All customary DNA sequences which permit selection of the transformed cells may be employed as DNA sequences encoding selectable markers in fragment b). Customary selection procedures of this type are, for example, selections based on auxotrophic mutations or, preferably, antibiotic resistances of the cells employed.
Bac~lli, preferably Bacillus alcalophilus, Bacillus licheniformis or Bacillus subtilis, are expediently employed as host organisms in ~he process according to the invention.
Examples of suitable polypeptides include proteases, preferably alkaline proteases, and in particular highly 2n alkaline proteases, as well as ~-amylases, lipases or cellulases.
In the process according to the invention, integration of the DNA sequence encoding the polypeptide of interest into t~e geno~e of the host cell is achieved by simple ~5 homologous recombination. For this purpose, a host cell, which at least already contains one DNA sequence for the polypeptide of interest and the DNA sequences required for the expression of this polypeptide, is tran~formed according to the invention with a circular DNA which possesses DNA
sequence~ wh~ch are homologous to the chromosomal DNA of the host cells. In this connection, these homoloqous DNA
sequences correspond at the same time to the DNA fragment a), which contains the DNA sequence encoding the polypeptide of interest. According to the invention therefore no further ~pecial target DNA sequence, which i~ homologous to the chromoso~al DNA of the host organism, is necessary for 209~28~
the homologous recombination, since the function of such a sequence is concomitantly fulfilled by the above DNA
fragment a) with the DNA sequence encoding a polypeptide of interest. Furthermore, the circular DNA employed here S contains at least one marker DNA sequence! for example one which encodes an antibiotic resistance. After the host organism has been transformed with this circular DNA, the marker sequence serves for the selection of those transformants which contain more than one DNA sequence encoding the polypeptide of interest. In order to ensure that only those transformants are obtained and selected in which the circular DNA has been integrated into the chromosomal DNA of the host organism, the circular DNA
employed possesses no DNA sequences which permit autonomous lS replication of this DNA in the host organism. Transmission of the circular DNA in the form of plasmids is thus excluded. By the process according to the invention, the transformed host cells contain the DNA sequence encoding the polypeptide of interest stably integrated into their genome.
Known methods may ~e used in the process according to the invention for preparing the circular DNA which is necessary for the transformation. For this purpose, the ~NA
sequence encoding the polypeptide of interest, as well as the sequences which are required for polypeptide expression and, where appropriate, polypeptide secretion in a host organism, i.e. regulatory sequences, signal sequences, promoter sequences, terminator sequences and the DNA
sequences encoding the pre- and pro-units of the polypeptide, are ~ir~t isolated from a microorganism which itsel~ produces the polypeptide of interest. For example, the chromosomal DNA from a bacterium ("donor bacterium~), in particular from a Bac~llus species which produces the polypeptide of interest, e.g. a protease or amylase, is isolated by known methods and partially hydrolyzed using ~uitable restriction endonucleases. The resulting restriction fragments of the donor DNA can be separated ,'' ' ~, ' ~ .. ' ~ .
209628~
according to size, e.g. by gel electrophoresis, and the fragments of the desired size can then be recombined with a suitable vector DNA, e.g. a plasmid DNA. Bacteria, preferably a ~acillus species, can be transformed with the in-vitro recombined plasmid (vector DNA ~ restriction fragments of the donor DNA), and the transformants selected according to the known marker property of the vector DNA
(antibiotic resistance). Among these transformants, those clones are sought which secrete the polypeptide of interest, e.g. a protease or amylase. Finally, the plasmid DNA
introduced into these transformants is isolated from a clone with the desired polypeptide activity. Renewed transformation of a bacterium is used to examine whether the polypeptide activity is plasmid-bound, i.e. whether the polypeptide activity is coupled to the marker property. In addition to the vector DNA with known restriction sites, the resulting isolated plasmid contains the desired structural gene for the polypeptide of interest as well as the regulatory seguences, signal sequences, promoter sequences and terminator sequences, which are required for polypeptide expression, and, where appropriate, the DNA sequences encoding the pre- and pro-units of the polypeptide. If the vector contains further DNA sequences from the donor bacterium, which are not required here, it is advisable, where appropriate, to eliminate these unreguired sequences prior to further use of the vector, and to reduce the donor DNA sequence essentially to the structural gene for the polypeptide and to the DNA sequences which are reguired for its expression and, where appropriate, for its secretion.
To do this, for example, the plasmid encompassing the additional, unrequired DNA sequences is restricted or cut with a number of different restriction endonucleases; the resulting DNA fragments are separated according to size by gel electrophoresis, and a restriction map is constructed on the ba~is of the band pattern which has been found. In this way, the restriction sites which are located in the region 2~9628~
of the donor DNA sequence are determined. At this point, knowledge of the restriction map of the plasmid makes it possible to excise from this plasmid, by cutting with selected restriction endonucleases, a DNA fragment of the S donor DNA sequence in which essentially only the required sequences from the donor bacterium remain. By reincorporating this donor DNA sequence, which has ~een reduced in size, into a suitable vector, a new plasmid can be obtained which is capable of polypeptide expression and which can se~ve as the basis for the production of the circular DNA used in the process according to the invention.
The restriction map in Figure 1 represents an example of such a reduced plasmid which has the designation pCLEAN4.
In this plasmid, the polypeptide of interest is a highly al~aline protease from a Bacillus alc~lophilus.
To produce circular DNA which can be e~ployed in the process according to the invention, those DNA sequences are removed from the plasmid of the above type pCLEAN4 which would permit replication of the plasmid in the host organism. If the starting vectors contain several DNA
sequences which encode, for example, different antibiotic resistances, the antibiotic resistance DNA sequences which are not reguired can also be removed from the vector. In the case of the above example of the plasmid pCLEAN4, this plasmid contains, for example, t~e functions repU and oriB
as well as resistances against the antibiotics neomycin and kanamycin. Based on a knowledge of the restriction map, these unwanted DNA sequences can be removed ~rom the plasmid by cutting with 8u1tably selected restriction endonucleases.
In the case of the plasmid pCLEAN4, a re~triation endonuclease Nsi I is used, for example, to delete fro~ the vector a fragment which contains the functions for the replication of the plasmid in Bacillus species as well as the resistances against neomycin and kanamycin, and the remaining DNA is then converted into a circular DNA uæi ng DNA ligase. The DNA sequence encoding a polypeptide of .;nterest, as well as the sequence for the antibiotic resistance phleomycin, are still retained from this deleted vector as circular DNA (pCLEAN4-del). With this circular DNA, and using t~e polypeptide gene of interest (in this case a highly alkaline protease), integration into the chromosome of e.g. Bacillus alcalophilus can be achieved by homologous recombination.
The circular DNA construction according to ~he invention can also be prepared by in vitro recombination, using DNA ligase, of a) the DNA sequence, which encodes the polypeptide of interest and which can be obtained e.g. by excising the corresponding DNA sequence with restriction endonucleases from a plasmid containing this sequence, with b) a resistance marker, which was obtained by excising with restriction endonucleases a corresponding DNA
sequence from a plasmid which contains this resistance marker, so that a circular DNA results.
Preferably, any DNA sequence encoding an antibiotic resistance is suitable as a resistance marker which a) is expressed in the microorganism to be transformed and b) encodes resistance against an antibiotic against which the microorganism to be transformed is not naturally resistant.
For;example, the resi~tance gene can encode resistance against an antibiotic selected from the group consistinq of chloramphenicol, ampicillin, tetracycline, kanamy¢in, neomycin, mitomycin C, bleomycin or phleomycin. A DNA
~equence from the plasmid pUBllO which encodes resistance against the antibiotic phleomycin is particularly suitable.
8y transformation of a microorganism which already contains in the chromoso~al DNA at least one DNA æequence encoding a pol~peptide of interest, a further DNA sequence encoding this polypeptide together with the pertinent ~ - 8 -, . .
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sequences which are necessary for expression and, where appropriate, secretion, can be integrated by homologous recombination into the chromosome of the host organism at the site of the gen~ for the polypeptide of interest. When this takes place, the additional DNA sequence encoding a polypeptide of interest and the pertinent sequences necessary for expression and, where appropriate, secretion are incorporated into the chromosome ~y simple cross-over in such a way that the DNA sequence encoding the antibiotic resistance comes to lie between the original DNA encoding the polypeptide which is already present and the newly introduced DNA which likewise encodes this polypeptide.
Subsequently, the transformed microorganisms are selected using this antibiotic resistance by cultivating them on a lS nutrient medium to which the relevant antibiotic has additionally been added at a particular concentration.
Under these conditions, only those transformants are capable of replication which have the circular DNA according to the invention integrated in their chromosomal DNA. The concentration of the antibiotic added to the culture medium is chosen in such a way that at least such transformants are selected as contain, ~n the chromosomal DNA, at least two DNA sequences encoding the polypeptide of interest and one DNA sequence encoding the resistance marker.
By cultivating transformants of this nature at higher antibiotic concentrations, those descendants can be isolated which possess in their genomes several copies of the DNA
~equence encoding the antibiot~c resistance, and thus al80 several copies of the DNA sequence encoding the polypeptide of interest. A feature of such clones may be a further elevation in the synthesis of the polypeptide of interest.
As is known, constructions of this nature, with adjacent repetitive DNA sequences in the genome, prove to be unstable in common laboratory strains. In industrial high-performance strains, which have generally been isolatedafter multiple cycles of mutation, and which therefore _ g _ 2~9628~
generally possess a diminished recombination capability, which is recognized e.g. by increased sensitivity towards the antibiotic mitomycin C, repetitive sequences of this nature in the chromosome are sufficiently stable for carrying out large-scale industrial production in th~
absence of antibiotic. If instabilities are observed, it is possi~le to isolate recombination-deficient descendants by searching, after mutation with a common mutagen, for those mutants which show increased sensitivity towards the antibiotic mitomycin C.
Brief Description of the Drawinas m e invention will be described in further detail with reference to the accompanying drawings in which:
Fig. 1 shows a restriction map of the plasmid pCLEAN4;
Fig. 2 shows a restriction map of the plasmid pUB 131;
Fig. 3 shows a restriction map of the plasmid pLI l;
and Fig. 4 is a graph of the proteolytic activities in the culture supernatants of cultures of B. alcalophilus HA
139/28 and HA 139/28-INTl.
ExamPles The following description of typical experimental emboaiments is intended to further illustrate the invention without limiting its scope.
Unless otherwise indicated, the work was generally carried out according to methods such as thosa d~scribed in T. Maniatis, E.F. Frit~ch, J. Sambrook, Molecul~r Clon~ng, A Labor~tory Nanual, Cold Spxing Harbor Laboratory (1982~.
The starting vectors which were used ~re commercially available on an unrestricted basis, or they may be prepared from available vectors ~y known methods. In this way, the vector M13gl31 was obtained from Amersham, Buckinghamshire, England.
The different restriction endonucleases which were used are known in the art and are commercially available. ~he reaction conditions which are required on each occasion when using these Xnown restriction endonucleases are likewise S known.
After incubation with a restriction endonuclease, the protein was removed by extraction in a known manner (e.g.
with phenol and chloroform~, and the cleaved DNA isolated in a known ~anner (e.g. by precipitation with ethanol from the aqueous fraction) and used further.
If desired, the cutting of vectors with restriction endonucleases may be followed by hydrolysis of the terminal 5'-phosphate radical with an alkaline phosphatase (de-phosphorylation). When dephosphorylation of the 5'-end was undertaken in the examples, it was effected in a known manner. Further details regarding the dephosphorylation procedure and the reagents which are required for it, may be obtained from Mania~is et al. (pp. 133 - 134).
Partial hydrolysis denotes incomplete digestion of DNA
by a restriction endonuclease. In this case, the reaction conditions are chosen so that, in a DNA substrate, cutting takes place at some, but not all, of the recognition sites for the restriction endonuclease employed.
To obtain and isolate particular DNA fragments, e.g.
after treatment of DNA with reætriction endonucleases, the resulting DNA fragments were ~eparated in a known manner by gel electrophoresis (e.g. on agarose gel) and subsequently identified based on their molecular weight (determination by comparison with reference DNA fragments of known molecular weight). The desired DNA fragments were then isolated from the corresponding gel ~ones.
Ligations can be carried out under known conditions, e.g. in a buffer with about lQ units of T4-DNA ligase per 0.5 ~g of an approximately equimolar amount of the DNA
fragmenta to be ligated (see e.g. Maniatis et al., p. 146).
2~(3628~
Transformation is understood to mean the introduction of DNA into a microorganism so that the DNA replicates autonomously in the microorganism as plasmid DNA, or is incorporated chromosomally, and can, where appropriate, be expressed. For the transformation of Bacillus species, the method described by Anagnostopoulos et al. [J. Bact. 81 :741-46 (1961)], is suitable, for example.
A polycloning site (polylinker) is a short to average-sized double-stranded DNA fragment which contains multiple reco~nition ~ites for restriction endonucleases in close proximity to each other. For example, one polycloning site used in the examples, which originates from the vector M13tgl31, has a size of about 0.07 kB (kilobases) and contains recognition sites for 14 different restriction endonucleases.
The Bacillus alcalophilus ~train employed in Example 1 and designated as Bacillus alcalophilus HA 1 was deposited July 28, 1989 in the Deutsche Sammlung von Mikroorganismen (DSM) with the DSM number 5466. Other microorganisms which were used are available commercially, e.g. Bacillus subtilis BD 244 (Bacillus Genetic Stock Center 1 A 46) or Bacillus su~tilis BD 366 (B~cillus Genetic Stock Center 1 E 6).
Further microorganisms used within the scope of the invention can be produced from Bac~llus DSM 5466 or from commercially available microorganisms according to the procedures given below:
1. ~acillus alcalophilus HA 139/28 and HA 28/12.
Both strains are descendants o~ Bacillus alcalophilus HA 1. They secrete a highly alkaline protease at an increased le~el, as described e.g. in Published German Patent Application No. DE 40 23 458, and can be employed as industrial production strains. Strains of this nature may be obtained by treating Bacillus alcalophilus HA 1 with mutagenic ~ubstances, such as, e.g., 1-methyl-3-nitro-1-nitrosoguanidine, according to conventional and well-known 209628~
methods, and subsequently searching on protein-agar plates for those mutants which are able to form larger areas of hydrolysis than the starting strain. By repeated cycles of mutation, strains can be isolated exhibiting additionally increased protease formation.
2. Bacillus licheniformis KC 14/1-422.
This strain is a Bacillus licheniformis which secretes an alkaline pxotease at an elevated level and can be used as an industrial production strain. A strain of this nature can be obtained by, e.g. treating a Bacillus lichenifo~mis Type Strain (obtainable from the Deut~che Sammlung von Mikroorganismen under the number DSM 13) with mutagenic substances, such as, e.g., l-methyl-3-nitro-1-nitrosoguanidine, according to conventional and well-known methods, and subsequently searching on protein-agar plates for those ~utants which are able to form larger areas of hydrolysis than the starting strain. By repeated cycles of mutation, strains can be isolated exhibiting additionally increased protease formation.
3. Bacillus licheniformis SEETT 18.
This strain is a Bacillus licheniformis which secretes ~-amylase at an elevated level and can be used as an industrial production strain. A strain of this nature can be obtained by treating, e.g. a Bacillus licheniform~s ~train (obtainable from the American Type Culture Collection under the number ATCC 27811) with mutagenic substances, such a~, e.g., 1-methyl-3-nitro-1-nitrosoguanidine, according to conventional and well-Xnown methode, and subsequently searching on starch-agar plates for those mutants which are capable of forming larger areas of hydrolysis (detected after layering with Lugol's solution3 than the starting strain. By repeated cycles of mutation, strains can be isolated exhibiting additionally increased amylase for~ation.
20962~
Example 1: Preparation of a genomic DNA library from B.
alcalophilus HA 1 and isolation of the gene for a highly alkaline protease.
Chromosomal DNA was isolated according to the method of Saito et al. [Biochim. Biophys. Acta. 72:619-629 (1963) ]
from the natural isolate Bacillus alcalophilus ffA
(deposited in the Deutsche Sammlung von Mikroorganismen under the number DSM 5466) and subjected to partial hydrolysis with the restriction endonuclease Sau3A.
The restriction fragments were separated by electrophoresis on an agarose gel, and the fragments having sizes of 3 to 8 kB were isolated.
The isolated and size-selected DNA fragments from Bacillus alcalophilus HA 1 were recombined in vitro with vector DNA of the plasmid pUB 110 (prepared as described in Example 2). For this purpose, plasmid pUBllO was first restricted with the restriction endonuclease BamH I and su~sequently dephosphorylated with calf intestinal alkaline phosphatase. Subsequently, 2 ~g of the restricted and dephosphorylated vector DNA were incubated with 8 ~g of the Bacillus ~lcalophilus DNA fragments, and with T4 DNA ligase, in a total volume of 100 ~1 at 16C for 24 hours~
Protoplasts of the strain Bacillus subtilis BD 224 were transformed according to the method described by S. Chang 25 and N. Cohen ~Mol . Gen. Genet. 168~ 15 (1979)3 with the DNA obtained by the in vitro recombination. The transformants were selected on plates containing neomycin and subsequently transferred to skimmed-milk agar. Among 13,800 trans~ormants which were examined, one was found which formed a recognizably larger area as a result of proteolysis of the skimmed-milk agar. The plasmid DNA was isolated from this clone according to Maniatis et al. The cloned fragment of B. alcalophilus DNA, which was contained in this plasmid, had a size of 4.1 kB and contained the complete infor~ation for the highly alkaline protease from Bacillus alcalophilus HA 1.
.
. ' ~ ' .
To simplify the subsequent procedure, the 4.1 kB DNA
fragment was first reduced in size. For this purpose, the recognition sites for restriction endonucleases which were located on the DNA fragment were determined by cutting the plasmid with different restriction endonucl~ases and separating the fraqments of restricted DNA by - electrophoresis on an agarose gel. A 2.3 kB DNA fragment, which could be obtained by cutting with the restriction endonucleases Ava I and Hind III, was identified which possessed the complete information for the highly alkaline protease and which was used for the subsequent procedure.
For this, the above plasmid with the 4.1 kB fraqment was restricted with the restriction endonucleases Ava I and Hind III. The 2.3 kB DNA fragment was isolated and ligated with the vector pUB 131 (prepared as described in Example 2) which had likewise previously been cut with Ava I and Hind III.
The resulting plasmid, which was designated pCLEAN4, was introduced into the strain B. subtilis BD 224. ~he transformants were able to secrete the highly alkaline protease, which showed that the Ava I/Hind III fragment contains the complete structural gene for the highly alkaline protease from B. alcalophilus HA 1. The restriction map of the plasmid pCLEAN4 is shown in Fig. 1.
ExamPle 2: Isolation and purification of the plasmid pUBllO
and construction of the vector pUB 131.
Plasmid pUB 110 was isolated from the strain ~ac~llus subtll~s BD 366 (pVB 110) according to the method o~ T. J~
Gryczan et al. [J. Bacter~ol. ~34:318-29 (1978)~ and subsequently purified by cesium chloride density gradient centrifugation accordinq to Maniatis et al. (p. 93). Vector pUB 110 contains unique restriction sites for the restriction endonucleases BamH I and EcoR I and marker DNA
sequences which encode antibiotic resistances against neomycin and phleomycin, as well as DNA sequences (norigin ~96284 of replication~) which are required for replication in Baci~lus species.
Plasmid pUB 110, obtained as described above, was restricted with EcoR I and BamH I. The smaller fragment of 790 bp (base pairs) was replaced by a polylinker consisting of 67 base pairs, which had previously been isolated as an EcoR I/Bgl II fragment from the vector M13tgl31. The resulting vector, designated pUB 131, is thus a derivative of pUB 110 in which the approximately 0.8 kB EcoR I/BamH I
fragment had been deleted and replaced by a polycloning site. ~he restriction map of the vector with the designation pUB 131 is reproduced in Fig. 2.
Example 3: Transformation of B. alcalophilus species.
B. alca70philus HA 139/28 and HA 28/12 were transformed by the protoplast method descri~ed by Chang and Cohen tMol.
~en. Genet. 168~ 15 (1979)] which was modified as follows:
The growth medium contained 1% of 1 molar carbonate buffer (53 g Na2CO3, 42 g NaH2CO3 in 1000 ml). The pH of the ~DN3~ regeneration plates was adjusted to pH 7.8 with NaOH
before pouring. For selection of the transformants, the regeneration plates contained 30 ~g/ml of phleomycin.
Example 4: Chromosomal gene amplification of the highly alkaline protease from B. alcalophilus HA 1.
As the restriction map of the vector pU8 131 in Figure 2 shows, a cleavaga site for the restriction endonuclease Nsi I is located at both 1976 bp and 2862 bp. By remov~ng the intervening 886 bp fragment, the funations repU, oriB
and neo'/kanar can consequently be deleted, so that the res~lting DNA is no longer capable of autonomous replication in a host cell. Plasmid pCLEAN4 (see Fig. 1) contains no recognition sites for Nsi I in the 2.3 kB DNA fragment for the highly alkaline protease from B. alcalophilus HA 1. By deleting the 886 Bp Nsi I fragment from plasmid pCLEAN4, a .' ' ' ' 209628~
DNA was obtained which could not ~e replicated autonomously in a host cell, but which could be used to transform B. alcalophilus and other bacilli since - this DNA still contained the phleomycin resistance marker and was thus selectable and - using the protease gene, it was possible to integrate this DNA in the chromosome of e.g. B. alcalophilus by homologous recombination.
10 ~g of plasmid pCLEAN4 were cut with Nsi I. The hydrolyzed DNA was separated on an agarose gel and the larger fragment ~5173 bp) was isolated. This fragment was converted into circular DNA using T4 DNA ligase.
This circular DNA, with the designation pCLEAN4-del, no longer encoded resistance against the antibiotic neomycin/kanamycin and, since the functions repU and oriB
were missing, could no longer be replicated autonomously and thus could no longer exist as a plas~id in bacterial cells.
The circular DNA pCLEAN4-del was subsequently introduced into protoplasts of the strains B. alcalop~ilus HA 28/12 and B. alcalophilus HA 139/28. Selection was effected based on phleomycin resistance (see Example 3).
Among the transformants obtained, the transformant HA
28/12-INT 542, in the case of strain HA 28/12, and the transformant HA 139128-INT 1, in the case of strain HA
139/28, were selected for further investigation. Even with repeated attempts at plasmid isolation, no plasmid DNA could be detected in either of the two transformants. Using DNA
isolated from these transformants, it was not possible to transform B. subt~lis to phleomycin or kanamycin/neomycin resistance. It was consequently established that the transformants were free of episomal elements~
By means of Southern blotting, it was possible to show that the pCLEAN4-del DNA had been integrated by homologous recombination into the chromosome of the respective host orqanism HA 28tl2 or HA 139/28 at the site of the gene for the highly alkaline protease.
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The strains B. alcalophilus H~ 139/28-INT 1 and B.
alcalophilus HA 28/12-INT 542 were isolated on "DM3"
regeneration plates which contained 30 ~g~ml phleomycin.
The extent of the resistance towards phleomycin was dependent on the growth medium being used. The aforementioned transformants were only resistant to a maximum phleomycin concentration of 3 ~g/ml on customary laboratory media such as, e.g., tryptose blood agar base plates or Luria broth agar plates. Adaptation to higher phleomycin concentrations was possible by repeatedly culturing the strains in liquid media containing increafiing concentrations of phleomycin. Strains adapted in this way contained several copies of the phleomycin resistance gene per genome, as well as several copies o~ the gene for the highly alkaline protease from B. alcalophilus HA 1.
Exam~le 5: Determination of the protease yields in shaker flas~s.
The strains B. alcalophilus HA 139/28-INT 1 and B.
alcalophilus HA 28/12-INT 542 from Example 4, the corresponding starting strains, and strains which had been adapted to higher phleomycin concentrations (Example 4), were cultured under the conditions described below. After culturing for 31 hours, the proteolytic activities in the 25 culture supernatants were measured. The results are listed in Table 1. In each case the yield of t~e starting strain was set at 100%.
Culture conditions:
50 ml of pre-culture medium (15 g of tryptone, 7.S g of yeast extract, 5 g of NaCl, 50 g of starch and 10 ml of corn steep liquor in 1000 ml) were inoculated with a single colony of the strain to be tested and incubated at 37C for 18 hours, and at 320 rpm, in conical flas~s with baffles.
50 ml of primary culture ned~um (40 g of soya bean meal, 100 g of starch and 20 ml of corn steep liquor in 1000 ml) were ,, . , , , ~ .
, 209628~
inoculated with 2.5 ml of this culture and incubated at 37C, and at 320 rpm, in conical flasks with baffles. Both media additionally contained 150 ml of carbonate buffer (s3 g of Na2Co3 and 42 g of NaHCO3 in 1000 ml) per 1000 ml of c~lture medium. The results are listed in Table 1.
Table 1 B. ~lcalophilusPhleomycin Activity in the strain Resistance culture supernatant HA 139/28 o ~g/ml 100 %
HA 139/28-INT l3 ~g/ml 170 %
HA 139/28-INT l5 ~g/ml 175 HA 139/28-INT 110 ~g/ml 180 %
HA 139/28-INT 120 ~/ml 160 %
HA 139/28-INT 125 ~g/ml 120 %
HA 28/12 0 ~g/ml 100 %
HA 28/12-INT 5423 ~g/ml 135 %
HA 28/12-INT 54210 ~g/ml 135 %
Examnle 6: Stability of the DNA integrations.
a) B~ alcalophilus HA 28jl2-INT 542 from Example 4 was cultured for 50 generations in Luria broth (10 g of tryptone, 5 g of yeast extract and 5 g of NaCl in 1000 ml of H20 - 10 ml of carbonate buffer / 1 liter of medium). Subsequently, dilutions of the bacteria were spread on agar plates without antibiotics and, after incubation of the Petri dishes, 100 colonies were transferred to agar plates which contained 3 ~g/ml phleomycin. No colonies were found which were unable to grow in the presence of phleomycin. The results show that the phleomycin resistance marker in the chromosome exhibits a high degree of stability.
2~39628~
b) The strains B. alcalophilus HA 139/28 and ~. alcalo-philus HA 139/28-INT 1 from Example 4 were cultured as in Example 5 (media recipe and culture conditions).
20 ml aliquots of pre-culture medium were each inoculated with a singlé colony of the strain from an agar plate. The ~edium for B. alcalophilus HA 139/28-INT 1 contained 10 ~gjml phleomycin (the strain had previously been adapted to the corresponding antibiotic concentration). After incubating for 18 hours, S0 ~l of this culture were transferred into 50 ml of primary culture medium:without phleomycin. After incubating for 24 hours, the cells were transferred into fresh primary culture medium at a dilution of 1:1000. A
total of four subcultures of this nature were carried lS out in the absence of phleomycin. The proteolytic activities in the culture supernatants in each of the individual cultures were determined after 31 hours.
The result of this experiment is shown in Figure 4.
The proteolytic activity in the first culture of B.
alcalophilus HA 139/28 was set at 100%. The experiment shows that the phenotype "amplified protease secretion"
of B. alcalophilus HA 13g/28-INT 1 is extremely stable.
Example 7: Chromosomal gene amplification of alkaline protease fro~ Bacillus licheni~ormis.
Chromosomal a~plification of the gene for the alkaline protease of ~. Iicheniformis was carried out in a protease-overseoreting B. lichen~ormis. The experiments were carried out in a corresponding manner to those for the amplification of the gene for the highly alkaline protease of B. ~lcaloph~lus ( see Example~ 3 to 5)~
Figure 3 shows the restriction map of the plasmid pLI
1. m e plasmid is constructed in a manner similar to plasmid pCLEAN4, except that the insert corresponding to the 2.3 kB DNA fragment in pCLEAN4 encodes the alkaline protease 20'~6284 from B. licheniformis instead of the highly alkaline protease from B. alcalophilus, as in pCLEAN4.
In the case of plasmid pLI 1, it was not possible to u~e the restriction endonuclease NsiI to delete the DNA
sequences which are required for autonomous replication, since there is a further recognition site for Nsi I in the DNA SeQ~ence which encodes the alkaline protease.
Plasmid pLI 1 was therefore restricted with the restriction endonucleases BspHl and Nco 1, and agarose gel electrophoresis was used to isolate the 4479 ~ fragment, which was subsequently converted into circular DNA using T4 DNA ligase. The ligation was possible since both nucleases produce the same protruding single strands. The circular DNA was introduced into the strain B. licheniformis KC 1411-422 by means of protoplast transformation. The transformation was carried out as described in Chang and Cohen l~ol. Gen. Genet. 168~ 15 (1979)~. The selection was effected on l'DM3~ regeneration plates, which contained 3S ~g/ml phleomycin. Among the transformants obtained, the strain B. licheniformis KC 14/1-422-INT 14 was selected.
B. licheniformis KC 14/1-422 INT 14 and B.
licheniformis KC 14/1-422 were cultured as described in Example 5. However, the media did not contain any carbonate buffer. The proteolytic activity in the culture supernatant o~ B. licheniformis XC 14/1-422-INT 14 was higher by 20%
than that in the case of B. licheniformis KC 14/1-422.
~3~1QL~: Chromosomal gene amplification of ~-amylase from Baclllus l~cheniformis.
A chromosomal amplification of the gene for the ~-amylase of B. licheniformis was described in an amylase-oversecreting B. licheniformis~
The experiments were carried out in a corresponding manner to those for the amplification of the gene for the highly alkaline protease of B. alcalophilus (see Examples 3 to 5).
209~2~
The plasmid pSEH 1 was constructed in a similar manner to plasmid pCLEAN4, except that the insert corresponding to the protease in pCLEAN4 in this case encoded the ~-amylase from B. licheniformis ATCC 27811.
The DNA sequences of plasmid pSEH 1 which are required for autonomous replication were deleted using the restriction endonuclease Nsi I. The circular DNA was introduced by means of protoplast transformation into the strain B. licheniformis SEETT 18. Among the transformants obtained, the strain B. licheniformis SEETT 18-INT 13 was selected. B. licheniformis SEETT 18-INT 13 and B.
licheniformis SEETT 18 were cultured in a medium containing lactose, cottonseed meal, soya bean meal and corn steep liquor. The amylolytic activity in the culture supernatant of B. licheniformis SEETT 18-INT 13 was 25% higher than that in the case of B. licheniformis SEETT 18.
The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.
This strain is a Bacillus licheniformis which secretes ~-amylase at an elevated level and can be used as an industrial production strain. A strain of this nature can be obtained by treating, e.g. a Bacillus licheniform~s ~train (obtainable from the American Type Culture Collection under the number ATCC 27811) with mutagenic substances, such a~, e.g., 1-methyl-3-nitro-1-nitrosoguanidine, according to conventional and well-Xnown methode, and subsequently searching on starch-agar plates for those mutants which are capable of forming larger areas of hydrolysis (detected after layering with Lugol's solution3 than the starting strain. By repeated cycles of mutation, strains can be isolated exhibiting additionally increased amylase for~ation.
20962~
Example 1: Preparation of a genomic DNA library from B.
alcalophilus HA 1 and isolation of the gene for a highly alkaline protease.
Chromosomal DNA was isolated according to the method of Saito et al. [Biochim. Biophys. Acta. 72:619-629 (1963) ]
from the natural isolate Bacillus alcalophilus ffA
(deposited in the Deutsche Sammlung von Mikroorganismen under the number DSM 5466) and subjected to partial hydrolysis with the restriction endonuclease Sau3A.
The restriction fragments were separated by electrophoresis on an agarose gel, and the fragments having sizes of 3 to 8 kB were isolated.
The isolated and size-selected DNA fragments from Bacillus alcalophilus HA 1 were recombined in vitro with vector DNA of the plasmid pUB 110 (prepared as described in Example 2). For this purpose, plasmid pUBllO was first restricted with the restriction endonuclease BamH I and su~sequently dephosphorylated with calf intestinal alkaline phosphatase. Subsequently, 2 ~g of the restricted and dephosphorylated vector DNA were incubated with 8 ~g of the Bacillus ~lcalophilus DNA fragments, and with T4 DNA ligase, in a total volume of 100 ~1 at 16C for 24 hours~
Protoplasts of the strain Bacillus subtilis BD 224 were transformed according to the method described by S. Chang 25 and N. Cohen ~Mol . Gen. Genet. 168~ 15 (1979)3 with the DNA obtained by the in vitro recombination. The transformants were selected on plates containing neomycin and subsequently transferred to skimmed-milk agar. Among 13,800 trans~ormants which were examined, one was found which formed a recognizably larger area as a result of proteolysis of the skimmed-milk agar. The plasmid DNA was isolated from this clone according to Maniatis et al. The cloned fragment of B. alcalophilus DNA, which was contained in this plasmid, had a size of 4.1 kB and contained the complete infor~ation for the highly alkaline protease from Bacillus alcalophilus HA 1.
.
. ' ~ ' .
To simplify the subsequent procedure, the 4.1 kB DNA
fragment was first reduced in size. For this purpose, the recognition sites for restriction endonucleases which were located on the DNA fragment were determined by cutting the plasmid with different restriction endonucl~ases and separating the fraqments of restricted DNA by - electrophoresis on an agarose gel. A 2.3 kB DNA fragment, which could be obtained by cutting with the restriction endonucleases Ava I and Hind III, was identified which possessed the complete information for the highly alkaline protease and which was used for the subsequent procedure.
For this, the above plasmid with the 4.1 kB fraqment was restricted with the restriction endonucleases Ava I and Hind III. The 2.3 kB DNA fragment was isolated and ligated with the vector pUB 131 (prepared as described in Example 2) which had likewise previously been cut with Ava I and Hind III.
The resulting plasmid, which was designated pCLEAN4, was introduced into the strain B. subtilis BD 224. ~he transformants were able to secrete the highly alkaline protease, which showed that the Ava I/Hind III fragment contains the complete structural gene for the highly alkaline protease from B. alcalophilus HA 1. The restriction map of the plasmid pCLEAN4 is shown in Fig. 1.
ExamPle 2: Isolation and purification of the plasmid pUBllO
and construction of the vector pUB 131.
Plasmid pUB 110 was isolated from the strain ~ac~llus subtll~s BD 366 (pVB 110) according to the method o~ T. J~
Gryczan et al. [J. Bacter~ol. ~34:318-29 (1978)~ and subsequently purified by cesium chloride density gradient centrifugation accordinq to Maniatis et al. (p. 93). Vector pUB 110 contains unique restriction sites for the restriction endonucleases BamH I and EcoR I and marker DNA
sequences which encode antibiotic resistances against neomycin and phleomycin, as well as DNA sequences (norigin ~96284 of replication~) which are required for replication in Baci~lus species.
Plasmid pUB 110, obtained as described above, was restricted with EcoR I and BamH I. The smaller fragment of 790 bp (base pairs) was replaced by a polylinker consisting of 67 base pairs, which had previously been isolated as an EcoR I/Bgl II fragment from the vector M13tgl31. The resulting vector, designated pUB 131, is thus a derivative of pUB 110 in which the approximately 0.8 kB EcoR I/BamH I
fragment had been deleted and replaced by a polycloning site. ~he restriction map of the vector with the designation pUB 131 is reproduced in Fig. 2.
Example 3: Transformation of B. alcalophilus species.
B. alca70philus HA 139/28 and HA 28/12 were transformed by the protoplast method descri~ed by Chang and Cohen tMol.
~en. Genet. 168~ 15 (1979)] which was modified as follows:
The growth medium contained 1% of 1 molar carbonate buffer (53 g Na2CO3, 42 g NaH2CO3 in 1000 ml). The pH of the ~DN3~ regeneration plates was adjusted to pH 7.8 with NaOH
before pouring. For selection of the transformants, the regeneration plates contained 30 ~g/ml of phleomycin.
Example 4: Chromosomal gene amplification of the highly alkaline protease from B. alcalophilus HA 1.
As the restriction map of the vector pU8 131 in Figure 2 shows, a cleavaga site for the restriction endonuclease Nsi I is located at both 1976 bp and 2862 bp. By remov~ng the intervening 886 bp fragment, the funations repU, oriB
and neo'/kanar can consequently be deleted, so that the res~lting DNA is no longer capable of autonomous replication in a host cell. Plasmid pCLEAN4 (see Fig. 1) contains no recognition sites for Nsi I in the 2.3 kB DNA fragment for the highly alkaline protease from B. alcalophilus HA 1. By deleting the 886 Bp Nsi I fragment from plasmid pCLEAN4, a .' ' ' ' 209628~
DNA was obtained which could not ~e replicated autonomously in a host cell, but which could be used to transform B. alcalophilus and other bacilli since - this DNA still contained the phleomycin resistance marker and was thus selectable and - using the protease gene, it was possible to integrate this DNA in the chromosome of e.g. B. alcalophilus by homologous recombination.
10 ~g of plasmid pCLEAN4 were cut with Nsi I. The hydrolyzed DNA was separated on an agarose gel and the larger fragment ~5173 bp) was isolated. This fragment was converted into circular DNA using T4 DNA ligase.
This circular DNA, with the designation pCLEAN4-del, no longer encoded resistance against the antibiotic neomycin/kanamycin and, since the functions repU and oriB
were missing, could no longer be replicated autonomously and thus could no longer exist as a plas~id in bacterial cells.
The circular DNA pCLEAN4-del was subsequently introduced into protoplasts of the strains B. alcalop~ilus HA 28/12 and B. alcalophilus HA 139/28. Selection was effected based on phleomycin resistance (see Example 3).
Among the transformants obtained, the transformant HA
28/12-INT 542, in the case of strain HA 28/12, and the transformant HA 139128-INT 1, in the case of strain HA
139/28, were selected for further investigation. Even with repeated attempts at plasmid isolation, no plasmid DNA could be detected in either of the two transformants. Using DNA
isolated from these transformants, it was not possible to transform B. subt~lis to phleomycin or kanamycin/neomycin resistance. It was consequently established that the transformants were free of episomal elements~
By means of Southern blotting, it was possible to show that the pCLEAN4-del DNA had been integrated by homologous recombination into the chromosome of the respective host orqanism HA 28tl2 or HA 139/28 at the site of the gene for the highly alkaline protease.
209628~
The strains B. alcalophilus H~ 139/28-INT 1 and B.
alcalophilus HA 28/12-INT 542 were isolated on "DM3"
regeneration plates which contained 30 ~g~ml phleomycin.
The extent of the resistance towards phleomycin was dependent on the growth medium being used. The aforementioned transformants were only resistant to a maximum phleomycin concentration of 3 ~g/ml on customary laboratory media such as, e.g., tryptose blood agar base plates or Luria broth agar plates. Adaptation to higher phleomycin concentrations was possible by repeatedly culturing the strains in liquid media containing increafiing concentrations of phleomycin. Strains adapted in this way contained several copies of the phleomycin resistance gene per genome, as well as several copies o~ the gene for the highly alkaline protease from B. alcalophilus HA 1.
Exam~le 5: Determination of the protease yields in shaker flas~s.
The strains B. alcalophilus HA 139/28-INT 1 and B.
alcalophilus HA 28/12-INT 542 from Example 4, the corresponding starting strains, and strains which had been adapted to higher phleomycin concentrations (Example 4), were cultured under the conditions described below. After culturing for 31 hours, the proteolytic activities in the 25 culture supernatants were measured. The results are listed in Table 1. In each case the yield of t~e starting strain was set at 100%.
Culture conditions:
50 ml of pre-culture medium (15 g of tryptone, 7.S g of yeast extract, 5 g of NaCl, 50 g of starch and 10 ml of corn steep liquor in 1000 ml) were inoculated with a single colony of the strain to be tested and incubated at 37C for 18 hours, and at 320 rpm, in conical flas~s with baffles.
50 ml of primary culture ned~um (40 g of soya bean meal, 100 g of starch and 20 ml of corn steep liquor in 1000 ml) were ,, . , , , ~ .
, 209628~
inoculated with 2.5 ml of this culture and incubated at 37C, and at 320 rpm, in conical flasks with baffles. Both media additionally contained 150 ml of carbonate buffer (s3 g of Na2Co3 and 42 g of NaHCO3 in 1000 ml) per 1000 ml of c~lture medium. The results are listed in Table 1.
Table 1 B. ~lcalophilusPhleomycin Activity in the strain Resistance culture supernatant HA 139/28 o ~g/ml 100 %
HA 139/28-INT l3 ~g/ml 170 %
HA 139/28-INT l5 ~g/ml 175 HA 139/28-INT 110 ~g/ml 180 %
HA 139/28-INT 120 ~/ml 160 %
HA 139/28-INT 125 ~g/ml 120 %
HA 28/12 0 ~g/ml 100 %
HA 28/12-INT 5423 ~g/ml 135 %
HA 28/12-INT 54210 ~g/ml 135 %
Examnle 6: Stability of the DNA integrations.
a) B~ alcalophilus HA 28jl2-INT 542 from Example 4 was cultured for 50 generations in Luria broth (10 g of tryptone, 5 g of yeast extract and 5 g of NaCl in 1000 ml of H20 - 10 ml of carbonate buffer / 1 liter of medium). Subsequently, dilutions of the bacteria were spread on agar plates without antibiotics and, after incubation of the Petri dishes, 100 colonies were transferred to agar plates which contained 3 ~g/ml phleomycin. No colonies were found which were unable to grow in the presence of phleomycin. The results show that the phleomycin resistance marker in the chromosome exhibits a high degree of stability.
2~39628~
b) The strains B. alcalophilus HA 139/28 and ~. alcalo-philus HA 139/28-INT 1 from Example 4 were cultured as in Example 5 (media recipe and culture conditions).
20 ml aliquots of pre-culture medium were each inoculated with a singlé colony of the strain from an agar plate. The ~edium for B. alcalophilus HA 139/28-INT 1 contained 10 ~gjml phleomycin (the strain had previously been adapted to the corresponding antibiotic concentration). After incubating for 18 hours, S0 ~l of this culture were transferred into 50 ml of primary culture medium:without phleomycin. After incubating for 24 hours, the cells were transferred into fresh primary culture medium at a dilution of 1:1000. A
total of four subcultures of this nature were carried lS out in the absence of phleomycin. The proteolytic activities in the culture supernatants in each of the individual cultures were determined after 31 hours.
The result of this experiment is shown in Figure 4.
The proteolytic activity in the first culture of B.
alcalophilus HA 139/28 was set at 100%. The experiment shows that the phenotype "amplified protease secretion"
of B. alcalophilus HA 13g/28-INT 1 is extremely stable.
Example 7: Chromosomal gene amplification of alkaline protease fro~ Bacillus licheni~ormis.
Chromosomal a~plification of the gene for the alkaline protease of ~. Iicheniformis was carried out in a protease-overseoreting B. lichen~ormis. The experiments were carried out in a corresponding manner to those for the amplification of the gene for the highly alkaline protease of B. ~lcaloph~lus ( see Example~ 3 to 5)~
Figure 3 shows the restriction map of the plasmid pLI
1. m e plasmid is constructed in a manner similar to plasmid pCLEAN4, except that the insert corresponding to the 2.3 kB DNA fragment in pCLEAN4 encodes the alkaline protease 20'~6284 from B. licheniformis instead of the highly alkaline protease from B. alcalophilus, as in pCLEAN4.
In the case of plasmid pLI 1, it was not possible to u~e the restriction endonuclease NsiI to delete the DNA
sequences which are required for autonomous replication, since there is a further recognition site for Nsi I in the DNA SeQ~ence which encodes the alkaline protease.
Plasmid pLI 1 was therefore restricted with the restriction endonucleases BspHl and Nco 1, and agarose gel electrophoresis was used to isolate the 4479 ~ fragment, which was subsequently converted into circular DNA using T4 DNA ligase. The ligation was possible since both nucleases produce the same protruding single strands. The circular DNA was introduced into the strain B. licheniformis KC 1411-422 by means of protoplast transformation. The transformation was carried out as described in Chang and Cohen l~ol. Gen. Genet. 168~ 15 (1979)~. The selection was effected on l'DM3~ regeneration plates, which contained 3S ~g/ml phleomycin. Among the transformants obtained, the strain B. licheniformis KC 14/1-422-INT 14 was selected.
B. licheniformis KC 14/1-422 INT 14 and B.
licheniformis KC 14/1-422 were cultured as described in Example 5. However, the media did not contain any carbonate buffer. The proteolytic activity in the culture supernatant o~ B. licheniformis XC 14/1-422-INT 14 was higher by 20%
than that in the case of B. licheniformis KC 14/1-422.
~3~1QL~: Chromosomal gene amplification of ~-amylase from Baclllus l~cheniformis.
A chromosomal amplification of the gene for the ~-amylase of B. licheniformis was described in an amylase-oversecreting B. licheniformis~
The experiments were carried out in a corresponding manner to those for the amplification of the gene for the highly alkaline protease of B. alcalophilus (see Examples 3 to 5).
209~2~
The plasmid pSEH 1 was constructed in a similar manner to plasmid pCLEAN4, except that the insert corresponding to the protease in pCLEAN4 in this case encoded the ~-amylase from B. licheniformis ATCC 27811.
The DNA sequences of plasmid pSEH 1 which are required for autonomous replication were deleted using the restriction endonuclease Nsi I. The circular DNA was introduced by means of protoplast transformation into the strain B. licheniformis SEETT 18. Among the transformants obtained, the strain B. licheniformis SEETT 18-INT 13 was selected. B. licheniformis SEETT 18-INT 13 and B.
licheniformis SEETT 18 were cultured in a medium containing lactose, cottonseed meal, soya bean meal and corn steep liquor. The amylolytic activity in the culture supernatant of B. licheniformis SEETT 18-INT 13 was 25% higher than that in the case of B. licheniformis SEETT 18.
The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.
Claims (10)
1. A process for preparing a transformed procaryotic host cell containing a chromosome which comprises at least two copies of a DNA sequence encoding a polypeptide of interest, said process comprising the steps of:
- transforming a procaryotic host cell which already contains chromosomal DNA comprising at least one copy of a DNA sequence encoding said polypeptide of interest, with a circular DNA suitable for transformation of said host cell, said circular DNA consisting essentially of a) a DNA fragment containing the DNA sequence encoding said polypeptide of interest and necessary DNA
sequences for expression thereof, and b) at least one DNA fragment containing a DNA sequence encoding a selectable marker, said circular DNA being free of DNA sequences which enable it to replicate autonomously in said host cell, - subsequently selecting with the aid of said selectable marker, cells which have chromosomally integrated at least one copy of said circular DNA, and - isolating the selected transformed cells.
- transforming a procaryotic host cell which already contains chromosomal DNA comprising at least one copy of a DNA sequence encoding said polypeptide of interest, with a circular DNA suitable for transformation of said host cell, said circular DNA consisting essentially of a) a DNA fragment containing the DNA sequence encoding said polypeptide of interest and necessary DNA
sequences for expression thereof, and b) at least one DNA fragment containing a DNA sequence encoding a selectable marker, said circular DNA being free of DNA sequences which enable it to replicate autonomously in said host cell, - subsequently selecting with the aid of said selectable marker, cells which have chromosomally integrated at least one copy of said circular DNA, and - isolating the selected transformed cells.
2. A process according to claim 1, wherein said host cell is a bacilli.
3. A process according to claim 2, wherein said host cell is selected from the group consisting of Bacillus alcalophilus and Bacillus licheniformis.
4. A process according to claim 1, wherein the DNA
sequence in fragment a) encoding a polypeptide of interest encodes a protease.
sequence in fragment a) encoding a polypeptide of interest encodes a protease.
5. A process according to claim 4, wherein said DNA
sequence encodes an alkaline protease.
sequence encodes an alkaline protease.
6. A process according to claim 1, wherein the DNA
sequence in fragment a) encoding a polypeptide of interest encodes an amylase.
sequence in fragment a) encoding a polypeptide of interest encodes an amylase.
7. A process according to claim 6, wherein said DNA
sequence encodes an .alpha.-amylase.
sequence encodes an .alpha.-amylase.
8. A process according to claim 1, wherein said selectable marker in fragment b) is a DNA sequence which encodes resistance to an antibiotic.
9. A process according to claim 8, wherein said selectable marker in fragment b) is a DNA sequence which encodes resistance to phleomycin.
10. A process according to claim 1, wherein said DNA
fragment a) further comprises DNA necessary for secreting said polypeptide of interest.
fragment a) further comprises DNA necessary for secreting said polypeptide of interest.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4216246A DE4216246A1 (en) | 1992-05-16 | 1992-05-16 | Simple procedure for stable chromosomal gene amplification |
| DEP4216246.7 | 1992-05-16 |
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| CA2096284A1 true CA2096284A1 (en) | 1993-11-17 |
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ID=6459063
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002096284A Abandoned CA2096284A1 (en) | 1992-05-16 | 1993-05-14 | Process for stable chromosomal gene amplification |
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| EP (1) | EP0570787A3 (en) |
| JP (1) | JPH06303986A (en) |
| KR (1) | KR930023461A (en) |
| BR (1) | BR9301900A (en) |
| CA (1) | CA2096284A1 (en) |
| DE (1) | DE4216246A1 (en) |
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| EP1002109A1 (en) * | 1997-07-03 | 2000-05-24 | Novo Nordisk A/S | Protein producing cell containing multiple copies of a desired gene and a screenable marker but no selection marker |
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| GR82200B (en) * | 1983-07-06 | 1984-12-13 | Gist Brocades Nv | |
| FR2563533B1 (en) * | 1984-04-27 | 1986-08-22 | Centre Nat Rech Scient | METHOD FOR AMPLIFYING THE EXPRESSION OF A DETERMINED GENE IN SUBACTIVE BACILLUS AND STRAINS OBTAINED |
| FR2569723B1 (en) * | 1984-08-31 | 1986-09-19 | Centre Nat Rech Scient | APPLICATION OF ANTIBIOTICS FROM THE PHLEOMYCIN FAMILY AS A SELECTION AGENT IN THE FIELD OF GENETIC ENGINEERING |
| AU620326B2 (en) * | 1987-02-27 | 1992-02-20 | Dsm Ip Assets B.V. | Stable gene amplification in chromosomal dna of prokaryotic microorganisms |
-
1992
- 1992-05-16 DE DE4216246A patent/DE4216246A1/en not_active Withdrawn
-
1993
- 1993-05-06 KR KR1019930007771A patent/KR930023461A/en not_active Application Discontinuation
- 1993-05-08 EP EP9393107517A patent/EP0570787A3/en not_active Withdrawn
- 1993-05-14 JP JP5112830A patent/JPH06303986A/en active Pending
- 1993-05-14 CA CA002096284A patent/CA2096284A1/en not_active Abandoned
- 1993-05-14 FI FI932215A patent/FI932215A/en unknown
- 1993-05-14 BR BR9301900A patent/BR9301900A/en not_active Application Discontinuation
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| JPH06303986A (en) | 1994-11-01 |
| FI932215A (en) | 1993-11-17 |
| DE4216246A1 (en) | 1993-11-18 |
| FI932215A0 (en) | 1993-05-14 |
| EP0570787A2 (en) | 1993-11-24 |
| KR930023461A (en) | 1993-12-18 |
| EP0570787A3 (en) | 1994-08-17 |
| MX9302822A (en) | 1993-11-01 |
| BR9301900A (en) | 1993-11-23 |
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