DE4231764A1 - Chromosomal integration of many copies of product gene - esp. into bacteria, by double crossing over, using thymidylate synthase gene as indicator, giving transformants of high prodn. capacity. - Google Patents

Abstract

Integration of a product gene (A) in the chromosome of a recipient (e.g. bacterium), which is effected (and selected) using a thy (thymidylate synthase) gene and involved multiple, stepwise individual copies of (A) comprises, (1) general recombination by the mechanism of double crossing over (2CO); (2) use of a cloned they gene, alternately in functional and inactivated form, for integration and selection of (A); (3) detection of 2CO through antibiotic sensitivity and single crossing over (1CO) by antibiotic resistance and (4) confirmation of (A) integration by 2CO by detecting additional gene copies and lack of plasmid DNA. USE/ADVANTAGE - The method is used to produce bacterial strains for stable prodn. of proteins encoded by (A), specifically alpha amylase or neutral protease. Many copies of highly expressed (A) can be introduced, providing transformants of high productivity. These are genetically stable and environmentally safe.

Description

The invention relates to a method for integrating a Product gene in the chromosome of a recipient, for example as a bacterial strain serving as a recipient strain mes, according to the preamble of claim 1.

Chromosomal gene integration and amplification procedures Bacillus were used repeatedly for genetic stabilization lization and for the stable amplification of cloned Product genes tested (Mountain 1989) to increase to achieve the gene dose and thus the product formation. Advantages of these methods compared to the use of ex trachromosomal (autonomously replicating) recombinant Plasmids were mostly for highly expressed and hetero loge product genes as well as under industrial conditions high product formation and long-term cultivation expect, among which a destabilizing selection pressure is applied against recombinant production strains.

For Bacillus, advantages were particularly high waits for extrachromosomal recombinant plasmids in Ba cillus due to structural and segregative plasmid insta often not or only to a limited extent. This one  The previously applied integration Procedures correspond only to a very limited extent.

Contradictory results lie for procedures using Amplification units in which the product gene and a closely linked antibiotic resistance gene within an egg DNS repeat structure under increasing selection pressure of an antibiotic are amplified. The one with so The degree of amplification achievable according to expenditure is after withdrawal of the antibiotic mostly not stable. This instability is due to recombination processes direct DNS repeats (Vagner / Ehrlich 1988) and DNS single strand formation upon replication of amplified plasmid DNA in the presence of the plasmid replication region ori-rep (Young / Ehrlich 1989). Another disadvantage this method consists in the amplification of genes for resistance to human therapeutic antibiotics such as Chloramphenicol and kanamycin.

In contrast, a high level of genetic stability was found in Ver drive with multiple, gradual integration of individual gene copies at randomly distributed locations of the bacterial chromosome can be obtained, and after repeated integration of highly expressed product genes product formation increased significantly (Kallio 1987, Steinborn 1988/1) and this result also during a longer cultivation (100 generations) without selection Maintain antibiotic pressure (Steinborn 1988/1). However, the disadvantages of these methods are their use the simple crossing-over, the resulting integra tion of complete integration plasmids, including genes for resistance to human therapeutic antibiotics tika, and in low selectivity.

This low selectivity is due to the fact that A product gene is usually not selective, a step wise increase in the number of copies is difficult to prove,  a certain selection characteristic is not immediately for the next integration usable and the number of avail ren selection features is limited. Congression (Kallio 1987) and mutative inactivation (Steinborn 1988/1) are helpful, but only increase the selectivity partially and require a significant technical up wall.

It has also been suggested that a thy gene for thymidylate Synthetase as a selection feature for the multiple, step-by-step integration (Steinborn 1988/2). Of the particular advantage is its double selectivity, by a functional thy gene based on thymidine Prototrophy and an inactivated thy gene using Tri resistance to methoprim can be selected. But this was also Proposal is limited to the simple crossing-over, which is disadvantageous for the integration. This takes place between the recipient's chromosome and an integration Plasmid in circular form with a chromosomal DNA Fragment in lateral position to the product gene, which is attached to ho mologer position of the chromosome the general recombination initiated (Niaudet 1985). The product gene is included the entire plasmid DNA integrated, so that with multiple Integration also several complete plasmid copies inte be grated. This is disadvantageous because the plasmid Ko pien as a foreign DNA burden the recipient and a desta bilizing recombination and uncontrolled release of plasmid DNA.

These shortcomings are said to be after another proposal (Kallio 1987) by using the double crossing over are fixed, in contrast to the simple Crossing-over the integration is limited to the product gene can be. Studies on double crossing-over be have been limited to a few theoretical papers B. subtilis 168, with only one antibiotic resistance gene  has simply been integrated (Niaudet 1985). Stable ha ploid integration strains could under certain conditions (transformation of linearized integration Plasmids in competent recipient cells, flanking the DNA to be integrated in the integration plasmid by ho mologous, not immediately adjacent DNA fragments with the same relative orientation as in the chromosome of the Rezi clients and deletion of the recipient 's chromosome in the Position of double crossing-over and loss of le viability upon deletion of an essential gene function) be isolated with increased frequency. This method but is based on recipients of high transformation ability limits, so that also linearized integration plasmids to be transformed in large numbers - a prerequisite for an effective double crossing-over.

All other Bacillus species have a much smaller one Ability to transform, so that additional measures and other methods of plasmid transformation, selection or Induction must be taken. An industrially so interpretive species such as B. amyloliquefaciens have so far been simple chem crossing-over accessible, which also only by means of trans formed, in vivo replicated integration plasmids could be managed (Steinborn 1988/2, Vehmaanperä 1991).

The invention has set itself the task of specifying To eliminate disadvantages of the known prior art gene and a method described in more detail at the outset stalten that the benefits of multiple copies number the integration of a highly expressed product gene and there with the high productivity of the recipient with his ho her genetic stability and also with environmental organic logical security.

According to the invention the object is achieved in that gene Real recombination according to the double mechanism Crossing-over is applied to a cloned thy gene  in functional form and - alternating with this - in inactivated form for integration and selection of the pro duct gene is used that double crossing-over due to antibiotic sensitivity and simple crossing-over proven by antibiotic resistance, and that the integration that took place through the double crossing-over of the product gene by detecting additional gene copies of this product gene and the absence of plasmid DNA is done. It is particularly advantageous if a functional thy gene in individual gene copies as by means of different integration plasmids for example using different pIT integration Plasmids integrated into the recipient's chromosome and its integration based on thymidine prototrophy selek and if the product gene continues in individual Gene copies gradually using an integration plas mids, for example using a pIG integration plas mids, after a previous integration of a Gene copy of the functional thy gene into this thy gene integrated, thereby inactivating the thy gene and the so he produced thymidine auxotrophy using correlated trime resistance to thoprim is selected. It is particularly good stig if the pIT integration plasmids functional thy gene through chromosomal DNA fragments of the recipient is flanked at homologous positions of the chromosome the integration of the thy gene by simple or double crossing-over, and if in doing so the pIG integration plasmids the product gene of DNA Fragments of the thy gene are flanked by the integration of the product gene through single or double crossing cause over, and if further in the integration plasmi the thy gene or the product gene and theirs flanking DNA fragments through closely linked antibio tika resistance genes are flanked. It is beneficial that the antibiotic resistance genes in double crossing not be integrated and the antibiotic sensitivity  activity of the recipient to prove the double crossing over is used, and that the antibiotic resi stenz genes integrated in a simple crossing-over and the Antibiotic resistance of the recipient for the detection of a fold crossing-over is used.

Sub-claims 9 to 55 contain further features.

Because of the double crossing-over, only DNS is used with the Pro duct gene integrated while the antibiotic resistance genes and other plasmid DNA excluded from integration are. It is therefore quite remarkable and essential Ches result of the invention that the method initially used genetic engineering to make a recipient appropriate changes, but this in the result the status of conventional, not genetically engineered processed integration strains. The invention combines two essential aspects: with Mit Genetic engineering can be highly productive, for example bacterial production strains are created, however no more features of genetic engineering and therefore also strict environmental biological requirements suffices.

The thy gene is used as a selective reporter gene for multiple, step-by-step integration in such a way that in a first integration step using a first pIT integration plasmid, a copy of the functional thy gene is first obtained by double crossing-over ( DC01) is integrated and this integration is selected on the basis of genetically stable thymidine prototrophy (Thy⁺). In a second integration step, a pIG integration plasmid is used to integrate the product gene into this integrated thy gene by double crossing-over (DC02), the thy gene being inactivated and the thymidine auxotrophy (Thy Th) thus generated on the basis of correlated, stable trimethoprim resistance (Tmp ^r ) can be used for selection for integration of the product gene. Then a second copy of the functional thy gene is integrated using a second, different pIT integration plasmid at a different, randomly distributed location of the chromosome and a second copy of the product gene is integrated into it using the same pIG integration plasmid . In an alternating manner, the integration of the product gene is continued up to such a number of copies and corresponds to the gene dose corresponding to the performance and the physiological tolerance of the recipient. In addition to the particular suitability of the thy gene as a selective reporter gene for multiple step-by-step integration through double crossing-over, it is also noteworthy because of its epidemiological and toxicological safety.

Serve as flanking DNA fragments for the pIT integra plasmid sections of non-preselected chromo somal DNA fragments that indicate the general recombination random homologous sites on the chromosome init ieren. Both sections must be one for this recom bin have sufficient length so that bilateral one multiple crossing-over leads to double crossing-over.

In the pIG integration plasmids, on the other hand, are sections of the thy gene used as flanking DNA fragments, which the general recombination in a previously integrated Initiate thy gene. The flanking DNA fragments have the same re in the pIT and pIG integration plasmids relative orientation as in the recipient's chromosome, a Precondition for the generation of stable haploid integra trunks by means of double crossing-over.

Because of the immediate vicinity of the flanking DNA fragments can be used with relative integration plasmids little effort can be made, and large deletions in the recipient's chromosome are avoided, so that the  Viability of these integration tribes is promoted. For the same reason, pIT integration plasmids from excluded the integration, the DNA fragments with a Gene for an essential gene function included in Integration to inactivate the homologous chromosomal Gene can lead. The spectrum of all other integrati ons plasmids, on the other hand, stand for the integration of ver different product genes. It is advisable these integration plasmids initially when integrating a homologous product gene with easily measurable and detectable Gene function (e.g. a product gene for an extracellular res enzyme) and only afterwards for integration other product genes, especially heterologous genes, to use.

With the aid of the method according to the invention, different product genes can be integrated into different recipients by means of different thy genes as reporter genes, provided the product genes are expressed by the recipient and their thymidine auxotrophy using tri methoprim resistance is selectable. For example, a thyL gene from B. licheniformis can be used as a reporter gene and the homologous, highly expressed genes amyA and nprA for alpha-amylase and neutral protease, respectively, can be integrated into bacterial strains of B. amyloliquefaciens, which because of their high potency to form extracellular enzymes, their traditional and widespread industrial application and their toxicological safety are particularly suitable for the process according to the invention. However, it was already pointed out that B. amyloliquefaciens is only slightly transformable, like most Bacillus species except B. subtilis, so that linearized integration plasmids are not transformed frequently enough. It is therefore preferred according to the invention to use circular integration plasmids which are replicated in plasmid transformants themselves. The increased occurrence of simple crossing-over is selected after segregation of the integration plasmid by means of the flanking resistance genes; the selection is particularly secure due to the use of two resistance genes and their close coupling to the product gene. In contrast, these resistance genes are not integrated with double crossing-over, so that after segregation of the integration plasmid, effective screening for integration strains with double crossing-over via antibiotic sensitivity, for example against chloramphenicol Cm ^s and MLS antibiotic MLS ^s is made possible. Integration strains with double crossing-over for the thy gene and subsequently for a product gene can therefore be selected and detected very effectively using the phenotypes Thy + Cm ^s MLS ^s and Tmp ^r C ^ms MLS ^s using the method according to the invention .

Prerequisite for the selection of thymidine prototrophs Integration strains are stable thymidine auxotrophic bacteria rien strains by deletion due to double Crossing-over by means of pID deletion plasmids the. This creates reversion-stable deletion mutan ten, where the isolation of integration strains not by spontaneous thymidine-prototrophic revertants is impaired. In the host strains, the Chromosomally coded formation of by-products, in particular of highly expressed extracellular enzymes, redu be decorated or eliminated, so that education, purity and Stability of the product can be improved (Steinborn 1988, Vehmaanperä 1991). Such derivatives can be chemi shear mutagenesis are generated; to eliminate one Product formation, deletion mutants are generated.

The thyA gene becomes advantageous and an alternative to deletion and other unwanted chromosomal genes of the bacterial Strain by integrating the product gene into these genes deactivated, which means at the same time and in addition to the Integra  tion using pIT and pIG integration plasmids the Pro duct gene also multiple and step by step in single Copies can be integrated by double crossing-over can. According to the invention, pIP integration plasmids are used for this constructed and applied using pIG integration plas are structurally and functionally comparable. In the Un In contrast to these, those contain sections of the inactivating genes as flanking DNA fragments, and the integration takes place in the chromoso to be deactivated paint genes so that the integration of the product gene on Reason of double crossing-over using pIP integration Plasmids based on gene inactivation, antibiotic Sensitivity and the absence of plasmid DNA detected will.

Because of the general application of double crossing-over after all, there is no plasmid DNA in the integration strains men included.

For integration into other bacterial strains of B. amyloliquefaciens, for example ATCC23844, with hosts specific plasmid stability are conditional replicas integration-incapable integration plasmids in a comparable manner applied. For this purpose, temperature-sensitive pIT, pIG and pIP integration plasmids by in vitro mutagenesis testifies that after integration at non-permissive tempera allow effective plasmid segregation.

Finally, the integration strains isolated in this way Characterized by the Southern DNA hybridization method be taken, the number of integrated gene copies and the absence of plasmid DNA as criteria for dop peltes crossing-over. The genetic stabilizer lity is due to constant product formation and number of the integrated gene copies measured.

The invention is based on the drawing two exemplary embodiments explained in more detail.

Show in the drawing

Fig. 1, the production and use of pit and PIG Integrationsplasmiden,

Fig. 2 shows the restriction map of a plasmid pSB319 with a cloned gene Thyl,

Fig. 3 shows the restriction map of a plasmid pSB411 with cloned thyA gene

Fig. 4 shows the restriction map of plasmid pSB351,

Fig. 5, the restriction map of an integration Plasmi of Pit1,

Fig. 6 shows the restriction map of an integration of the pIG1 Plasmi,

Fig. 7 shows the restriction map of an integration Plasmi of pIG2,

Fig. 8 shows the restriction map of a deletion plasmid PID1 and

Fig. 9 shows the restriction map of an integration of the Plasmi PIP1.

The following abbreviations are used in the drawing and symbols used:

A, B, C: non-preselected chromosomal DNA fragments of a recipient strain;
C / 1, C / 2: sections of a chromosomal DNA fragment C,
thy: cloned thy gene for thymidylate synthetase;
thy / 1, thy / 2: sections of the thy gene;
Cm ^r : antibiotic resistance gene for resistance to chloramphenicol;
MLS ^r : Antibiotic resistance gene for resistance to macrolide linkosamide and streptogramin B antibiotics;
BI: BclI interface;
EI: EcoRI interface;
P: product gene;
DCO: double crossing-over;
a, b, c, d; successive positions in C in natural and equally relative orientation a ′, b ′, c ′, d ′ as in the homologous region in the chromosome of the recipient strain;
e, f, g, h: successive positions in thy in natural and equally relative orientation a ′, b ′, c ′, d ′ as in the integrated thy gene;
repR: repR gene for an essential replication protein RepR;
thyL: thyL gene for thymidylate synthetase from B. licheniformis,
thyA: thyA gene for thymidylate synthetase from B. amyloliquefaciens.

Continuous double lines without an arrow mark chromosomal Genes with unknown DNA sequence; with sections the These genes are supposed to have an arrow indicating their same relative orientation compared to the full DNA fragment. In the practice The representations correspond to the usual symbolism  when displaying restriction maps and general my the schematic representation in genetic engineering.

On an explicit description of common genetic engineering African or other procedural steps will be described below renounced. Knowledge of the isolation of plasmid DNA, DNA fragments or chromosomal DNA, cleavage of DNA by restrictases, "fill in" reaction, gel electro phoresis, plasmid transformation, Southern hybridization, Cultivation, chemical mutagenesis or selection provided and can appropriate publications (Sambrook 1989).

The following starting materials, auxiliary substances or methods frequently used:

6.2 kb vector plasmid pGB354 with Cm ^r (Behnke 1982) for cloning and as starting plasmid for all integration plasmids;
Bacterial strain B. subtilis GSB26 - as recipient strain for plasmid constructions, derived as a spontaneous streptomycin-resistant mutant from the amylase-defective bacterial strain QB1133 sacA321 aroI906 metB5 amyE (Steinmetz 1976) from the strain line of B. subtilis 168;
Bacterial strain (thymidine auxotroph) B. subtilis 168 TT thyA thyB trpC2 (Steinmetz 1976) as recipient strain for the cloning and genetic engineering of thy genes for Bacillus thymidylate synthesis;
Bacterial strain B. amyloliquefaciens BE71 with strain-specific high segregative plasmid instability (Steinborn 1988) as the starting strain for recipient strains for integration;
Bacterial strain B. amyloliquefaciens ATCC23844 (In gle / Boyer 1976) as a starting strain for recipient strains for conditionally replication-incapable (ts) integration plasmids and as a gene donor strain for the cloning of a thyA gene;
PEG protoplast method (Chang / Cohen 1979) for plasmid transformation in Bacillus with modifications of the media (Steinborn 1988) for plasmid transformation in B. amyloliquefaciens;
Cultivation at 37 ° C in liquid TBY complete medium with 1% bactotryptone, Difco - 0.5% yeast extract, Difco - 0.5% NaCl and with pH = 7.0 or on TBY - agar (1.8% agar Agar);
Selection for plasmid-coded antibiotic resistance is carried out by adding 10 micrograms / ml chloramphenicol Cm or 5 micrograms / ml erythro mycin Em, selection for trimethoprim resistance (Gryczan / Dubnau 1982) at 10 micrograms / ml trimethoprim Tmp in the presence of 50 micrograms / ml thymidine in MSM minimal medium (Demain 1958); in the case of auxotrophy of the bacterial strains, essential amino acids are supplemented at 50 micrograms / ml and thymidine at 100 micrograms / ml;
Southern hybridization for the detection of DNA homology, in particular of integrated gene copies of the product gene, according to the non-radioactive digoxigenin method (Boehringer) when using a vacuum blotter (Bio-Rad);
Detection and semi-quantitative determination of the formation of extracellular alpha-amylase and neutral protease by means of degradation yards on agar plates with the addition of 1% insoluble maize starch or 0.5% skim milk powder; the quantitative determination of the enzyme activities in culture supernatants is carried out according to methods described in detail (Steinborn / Hofemeister 1984, Steinborn 1988);
DNA isolations from bacterial strains of Bacillus for chromosomal DNA and plasmid DNA on a large scale according to the methods of Saito / Miura 1963 and Gryczan 1978.

1. Primary cloning and restriction mapping of thy Genes for thymidylate synthetase from B. licheniformis and B. amyloliquefaciens

The primary cloning of thy genes for thymidylate synthesis tase from B. licheniformis and B. amyloliquefaciens - successor referred to as the thyL or thyA gene - is "shot gun "using a donor strain B. licheniformis ATCC9789 (Ingle / Boyer 1976) or B. amyloliquefaciens ATCC23844, a thymidine auxotrophic recipient strain B. subtilis TT, a plasmid vector pGB354 or pSB3 (Steinborn 1983) and the restrictions BclI and PstI leads.

After transformation of the ligation mixtures, the Se lesson on DM3 agar without thymidine addition, on which the Re recipient strain grows in small, thin colonies. When Transformants with potentially recombinant plasmids for thy genes become iso rapidly growing, prototrophic colonies and on enlarged plasmids with additional BclI- or PstI DNA fragment gel-electrophoretically character siert. You will then be in the plasmid-free recipient Strain retransformed and in plasmid-encoded thymidine Prototrophy as recombinant plasmids with cloned thy- Genes identified.  

In one experiment, 16 or 4 recombinant plasmids were isolated, each with an identical additional DNA fragment, each of which a representative plasmid is designated as plasmid pSB319 or pSB411 and is characterized by restriction analysis (one to three times restriction cleavage) ( FIG. 2 and 3). According to the restriction maps, the location and size of the functional thy genes are localized within the cloned DNA fragments by means of deletion or insertion analysis.

The identity of the cloned DNA fragments as chromosomal DNA fragments of the gene donor strains used are by means of Southern hybridization based on DNA homology where the cloned BclI and PstI DNA fragments from the plasmids pSB319 and pSB411 against BclI and PstI-ge cleaved chromosomal DNA from the gene donor strains ATCC9789 or ATCC23844 and from B. subtilis GSB26 (Kon trolls) can be hybridized.

The cloning of thy genes in the plasmids pSB319 and pSB411 is first functionally proven by the fact that these recombinant plasmids followed in B. subtilis TT indicated defect in the formation of thymidylate synthetase (Gryczan / Dubnau 1982) complement and in thymidine-auxo trophic bacterial strains of B. subtilis 168 and B. amyloliquefaciens thymidine prototrophy and correlated Cause sensitivity to trimethoprim. These functions are we for the inventive use of thy genes considerable.

2. Generation of plasmids pSB351, pSB352 and pSB355 as Starting plasmids for integration and deletion plasmids

Starting from the plasmid vector pGB354 (Cm ^r ), plasmids are constructed which contain different numbers of unique restrictionase interfaces between two closely adjacent antibiotic resistance genes (Cm ^r , MSL ^r ). First, the plasmid vector pGB354 is cleaved in its unique BclI interface and a HindIII DNA fragment with the MLS ^r gene from a plasmid pDB101 (Behnke 1979) is cloned using the "fill in" kit according to the instructions (from Stratagene). This creates a plasmid pSB351 ( FIG. 4) which contains a unique BclI site between the resident Cm ^r gene and the closely adjacent MSL ^r gene. The MSL ^r gene encodes, among other things, antibiotic resistance to erythromycin (Em ^r ), which is used for selection in the method according to the invention.

Then the BclI site of the plasmid pSB351 a linker with an internal EcoRI interface and distal Interfaces for the BglII and BamHI restrictionases from ei a plasmid pIC20H (Marsh 1984) to form a plasmi of the pSB352 subcloned. Finally, the EcoRI Interface of the plasmid pSB352 a polylinker with dista len EcoRI sites from a plasmid pIC20R (Marsh 1984), so that a plasmid pSB355 with numerous unique restrictionase interfaces between the flankie antibiotic resistance genes.

3. Generation of pIT integration plasmids

The pIT integration plasmids are obtained by recloning one non-preselected chromosomal DNA fragment of B. amyloliquefaciens in the EcoRI site of Plasmid pSB352 and subsequent recloning of the thyL gene from the plasmid pSB319 into a medium-sized restrictase Chromosomal DNA fragment cut. The overall size and relative orientation remain the sections of the cloned chromosomal DNA fragment unchanged compared to the chromosome of the donor strain.  

The recloning of different chromosomal DNA fragments from the plasmids pSB370 to pSB379 creates a plasmid series with the names pIT1 to pIT10. The chromosomal DNA fragments in the plasmids pSB370 to pSB379 were previously shot-shot using a donor strain B. amyloliquefaciens ATCC23844, an amylase-defective recipient strain B. subtilis GSB26, the restrictase EcoRI and a plasmid vector pSB3 ( Steinborn 1983 ) cloned and characterized for suitable medium-sized restriction tase sites. The chromosomal DNA fragments were inserted into an internal EcoRI site of the cloned gene for an extracellular amylase in the plasmid vector pSB3, so that the cloning of the chromosomal DNA fragments is detected by means of insertion inactivation of the amylase gene.

The identity of the cloned chromosomal DNA fragments as fragments from B. amyloliquefaciens is by Southern Hybridization confirmed due to DNA homology, where the cloned chromosomal DNA fragments against EcoRI-ge cut chromosomal DNA of the donor strain ATCC23844 and from B. subtilis GSB26 (control).

In FIG. 5, the integration plasmid pIT1 contains a chromosomal 3.5 kb long DNA fragment with a central BclI site into which the thyL gene from the plasmid pSB319 is located within the BclI DNA cloned into this plasmid. Fragments was cloned. In the integrati on plasmid pIT1, the thyL gene is flanked by sections of the chromosomal DNA fragment and these sections, because of the starting plasmid pSB352, are flanked by the antibiotic resistance genes Cm ^r and MSL ^r .

4. Generation of pIG integration plasmids

It first becomes the thyL gene using the BclI restrictionase from plasmid pSB319 using the recipient Strain B. subtilis into the BclI site of the plasmid pSB351 is cloned, creating a plasmid pSB360. On concluding the product gene into an internal cut place the functional thyL gene in plasmid pSB360 right cloned so that the thyL gene is inactivated by insertion and the product gene is flanked by sections of the thyL gene which is one on both sides for general recombination have sufficient length. The in is suitable for this tern PvuII interface of the thyL gene, which because of the smooth ends for recloning using various re is strictly suitable.

Analogous to the pIT integration plasmids, the total size and the relative orientation of the sections of the thyL gene compared to the functional thyL gene are not changed when the product gene is recloned, and the product gene and the flanking sections are not changed of the thyL gene are flanked by the antibiotic resistance genes Cm ^r and MLS ^r .

The highly expressed amyA and nprA genes for the extracellular enzymes alpha-amylase or neutral protease from B. amyloliquefaciens recloned. Here the recombinant plasmids pSB6 (Stein born / Hofemeister 1984) as gene donors and B. subtilis TT used as recipient and the recloning on the basis of Thymidine auxotrophy or correlated trimethoprim resi stenz and high formation of the plasmid-coded, extracellu detected enzymes.  

The amyA and the nprA genes are recloned within a PvuII or BclI DNA fragment, in the BclI DNA fragment the "fill in" reaction using a kit (from Stratagene) is used. FIGS. 6 and 7 show the thus prepared integration plasmids pIG1 and pIG2.

5. Generation of deletion plasmids

The starting point for the production of deletion plasmids for inactivating the chromosomal thyA gene from B. amyloliquefaciens is the cloned thyA gene in the plas mid pSB411 (cf. 1.) according to FIG. 3. Within this functional thyA gene there is a small one , medium-sized DNA fragment removed in such a way that the thyA gene is inactivated and a portion of the chromosomal DNA fragment from B. amyloliquefaciens cloned into the plasmid pSB411 remains sufficiently long for the general recombination on both sides of the deletion site. Then the deleted DNA fragment remaining in the plasmid is recloned using the PstI restrictionase into a PstI interface in the polylinker of the plasmid pSB355, so that it is flanked by the antibiotic resistance genes Cm ^r and MSL ^r .

Because of two closely adjacent PvuII and SacI sections in the thyA gene, the described deletion plasmids can be easily prepared for this, so that deletion plasmids pID1 ( FIG. 8) and pID2 are obtained. When B. subtilis TT is used as a recipient, the selection can be made on the basis of the trimethoprim resistance and plasmid-coded antibiotic resistance. Deletion plasmids for inactivating other chromosomal genes are produced in the same way.

6. Generation of pIP integration plasmids

The pIP integration plasmids are created by recloning of the product gene into a cloned gene of the recipient, which inactivated in its chromosome and at the same time the pro product gene can be integrated by double crossing-over should. It is here on the inactivation of the chromosomal thyA and nprA genes in the integration of the amyA gene raised.

An integration plasmid pIP1 ( FIG. 9) is formed when the amyA gene within a PvuII DNA fragment from the plas mid pSB6 into the internal EcoRV site of the thyA gene in the plasmid pSB411 and thus the thyA gene is inactivated by insertion. The inactivated thyA gene is then cloned into the PstI site in the polylinker of the plasmid pSB355 by means of the restriction PstI, so that this integration plasmid pIP1 also contains the flanking antibiotic resistance genes Cm ^r and MLS ^r .

The integration plasmid pIP2 is obtained in the same way if the amyA gene is in an internal EcoRV interface of the nprA gene is inserted in the plasmid pSB92.

7. Generation of conditional replication-incapable inte generation plasmids

There are temperature-sensitive (ts) integration plasmids isolated, the permissive temperature (30 ° C) in the Re replicate and at elevated temperature (42 °) are not capable of replication. They are from the integrati ons plasmids or starting plasmids pSB351, pSB352 and pSB355 derived by chemical mutagenesis by mutagenized by ethyl methanesulfonate EMS and an closing in the bacterial strain B. subtilis GSB26 transfor  be lubricated. Treatment time and concentration of the muta gens are chosen so that the mutagenized Plas mide still a transformation frequency of about 10% in Achieve comparison to untreated plasmids. The scree Temperature sensitive plasmids are used according to the Replication technology: the plasmid Transformants first at 30 ° C, after replication is at Incubated at 42 ° C. The ones not growing at this temperature Transformants are un on temperature sensitive plasmids tries.

8. Multiple, step-by-step integration through double Crossing-over using pIT and pIG integration plasmids

First, an integration plasmid pIT1 from 3. ( FIG. 5) is transformed into a stably thymidine-auxotrophic bacterial strain of B. amyloliquefaciens BE71, and plasmid transformants are selected using thymidine prototrophy and then in without pressure by antibiotics thymi din-free minimal medium MSM passages over 10 to 30 generations, whereby the integration plasmid segregates and integration strains with integrated thyL gene are selectively enriched. The integration takes place before or during the journey, and the integration of the thyL gene by double crossing-over is determined phenotypically via stable thymidine prototrophy and antibiotic sensitivity to chloramphenicol and erythromycin. For this purpose, MSM is plated after the passenger on thymidine-free minimal medium and antibiotic sensitivity is demonstrated using the replication technique. For this, as well as all subsequent integrations, double crossing-over is confirmed biochemically by means of Southern hybridization, in that an additional copy of the integrated gene is detected in the chromosome of the integration strain and no plasmid DNA of the integration plasmid is detected. For this, who integrated the integrated gene and the starting plasmid of the integration plasmid (for example pSB351 for pIT1) hybridized against the chromosomal DNA of the integration strain.

After integration of a copy of the thyL gene by double crossing-over, the integration plasmid pIG1 is transformed according to 4 ( FIG. 6) into such an integration strain and plasmid transformation by means of plasmid-encoded antibiotic resistance Cm ^r Em ^r selected. Subsequently, plasmid transformants are passed through antibiotics in thymidine-containing minimal medium MSM for 10 to 30 generations without selection pressure, the amyA gene being integrated. When integrated by double crossing-over, the previously integrated thyL gene is inactivated and plasmid DNA remains excluded from the integration, so that integration strains with double crossing-over phenotypically due to trimethoprim resistance and antibiotic sensitivity to chloramphenicol and erythromyin can be selected. This selection is again carried out using replication technology, the MSM master plates containing additions of thymidine and trimethoprim and the replication plates additionally being supplemented with chloramphenicol and erythromycin.

Finally, the genetic stability of the integrier ten amyA gene and its gene dose effect on amylase Education, and such integration Tribes as recipients for the further gradual integration gration of the amyA gene selected after passengers without Selection pressure over 100 generations no loss of integrated amyA gene and a constant increased Show amylase formation.

Now a second copy of the thyL gene is made using a second, different pIT integration plasmids that, also randomly distributed part of the chromosome and into this using the same pIG integration plas  mids pIG1 integrates a second copy of the amyA gene. In the same, alternating way, the integration of the amyA gene up to such a copy number and gene dose continued, the performance and physiological Tolerance of the recipient.

The multiple, step-by-step integration of the nprA gene by double crossing-over is carried out in the same way as for the amyA gene. The same pIT integration plasmids and recipient strains are used; the main difference is the use of an integration plasmid pIG2 ( Fig. 7) with nprA gene.

Conditional replication-capable (ts) integration plasmids out of 7. are for integration into recipient strains of B. amyloliquefaciens, for example ATCC23844, used in contrast to the recipient strain BE71 the integration is not a strain-specific high segregative Plasmid instability can be used for plasmid segregation can. The segregation of these ts integration plasmids will efficient by inhibiting plasmid replication 42 ° C reached. Otherwise there are no differences Use of normal replicating integration plasmids.

9. Isolation of stable thymidine-auxotrophic receptacles ten tribes

Stable thymidine-auxotrophic bacterial strains of B. amyloliquefaciens (see FIG. 10) can be generated by means of double crossing-over using deletion plasmids (see 5.) pID1 ( FIG. 8) or pID2. After plasmid transformation and segregation, the deletion in the chromosomal thyA gene is selected or detected on the basis of double crossing-over via trimethoprim resistance and antibiotic sensitivity to chloramphenicol and erythromycin, as well as when integrating with pIG integration - plasmids are used (cf. 8.).

10. Inactivation of the thyA chromosomal gene and others chromosomal genes using pIP integration plasmids

By means of pIP integration plasmids (see FIG. 6), the product gene is integrated into unwanted or nonessential chromosomal genes of a recipient strain B. amyloliquefaciens by double crossing-over and thereby inactivates the chromosomal gene in question. Thus, at the same time as when using pID deletion plasmids, the copy number of the integrated product gene is increased, so that in addition to the integration by means of pIT and pIG integrati ons plasmids, its gene dose is increased. Using the integration plasmids pIP1 ( FIG. 9) and pIP2, for example, the thyA and nprA genes are inactivated and thus two copies of the amyA gene are integrated.

Integration through double crossing-over is also applied here Hand of inactivation of the chromosomal genes, of anti biotic sensitivity to chloramphenicol and erythromy cin and the integrated copies of the amyA gene sen.

11. Mutagen-induced plasmid-chromosome recombination

To increase the frequency of double crossing overs who the possibly carried out experiments for UV induction. in the Contrary to the usual method (Mudgett 1991) transformed cells and not plasmid DNA in vitro delt. The UV dose is chosen so that 30 to 50% of the be beamed bacterial cells survive.

Appendix: Literature 92.223PN

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Chang, S./Cohen, SN Molec. genet. 168, 111-115 (1979) (Chang / Cohen 1979)
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Gryczan, TJ / Dubnau, D., Gene 20, 459-469 (1982) (Gry czan / Dubnau 1982)
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Kallio, P. et al., Appl. Microbiol. Biotechnol. 27, 64-71 (1987) (Kallio 1987)
Marsh, JL et al., Gene 32, 481-485 (1984) (Marsh 1984)
Mountain, A., in CR Harwood (Ed.), Biotechnology Hand books, vol. 2; Plenum Press, New York, 73-114 (1989) (Moun tain 1983)
Mudgett, JS et al., Gene 97, 131-136 (1991) (Mudgett 1991)
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Claims (55)

1. A method for integrating a product gene into the chromosome of a recipient, for example a bacterial strain serving as a recipient strain, which is caused and selected by a thy gene for thymidylate synthetase and repeatedly and stepwise in individual gene copies of the product gene, characterized in that

• general recombination is used according to the double crossing-over mechanism,
• - a cloned thy gene is used in a functional form and - alternating with this - in inactivated form for the integration and selection of the product gene,
• - the double crossing-over by antibiotic sensitivity and simple crossing-over by antibiotic resistance are detected and
• - The double crossing-over of the integration of the product gene is confirmed by the detection of additional gene copies of this product gene and the absence of plasmid DNA.

2. The method according to claim 1, characterized in that a functional thy gene in individual gene copies step by step using different integration plas mide, for example using different pIT integra plasmids integrated into the recipient's chromosome and its integration using thymidine prototrophy is read. 3. The method according to claim 1 and 2, characterized in net that the product gene step in individual gene copies as an integration plasmid, for example using a pIG integration plasmid, after one each previous integration of a gene copy of the function capable thy gene integrated into this thy gene, thereby the thy gene inactivated and the thymidine auxotro thus generated selectivity based on correlated trimethoprim resistance is tiert. 4. The method according to claim 2, characterized in that in the pIT integration plasmids the functional thy- Gene flan through chromosomal DNA fragments of the recipient the is at the homologous positions of the chromosome Integration of the gene through single or double Cause crossing-over. 5. The method according to claim 3, characterized in that in the pIG integration plasmids the product gene of DNA Fragments of the thy gene are flanked by the integration of the product gene through single or double crossing over effect. 6. The method according to any one of claims 4 or 5, characterized characterized in that in the integration plasmids the thy- Gene or the product gene and its flanking DNA fragments through tightly coupled antibiotic resistance Genes are flanked.   7. The method according to claim 6, characterized in that the antibiotic resistance genes in double crossing-over not be integrated and the antibiotic sensitivity of the recipient to prove the double crossing-over is used. 8. The method according to claim 6, characterized in that the antibiotic resistance genes with a simple crossing-over be integrated and the antibiotic resistance of the recipient ducks used to demonstrate simple crossing-over becomes. 9. The method according to any one of claims 1 to 8 thereby indicates that the recipient is a general recombiner nation capable, especially for the production of extracellu bacterial strain of Bacillus amylo liquefaciens is used. 10. The method according to any one of claims 1 to 9, characterized characterized that a conditional replication incapable Integration plasmid is used. 11. The method according to claim 10, characterized in that a derivative of Bacillus amyloliquefaciens ATCC23844 is used as a recipient. 12. The method according to any one of claims 1 to 9, characterized characterized in that a recipient with a strain-specific high segregative plasmid instability is used. 13. The method according to claim 12, characterized in that used a derivative of Bacillus amyloliquefaciens BE71 becomes. 14. The method according to any one of claims 1 to 13, characterized characterized in that the recipient is a stable thymidine auxo trophes derivative of a bacterial strain is used.   15. The method according to any one of claims 1 to 14, characterized characterized in that a recipient of a bacterium as recipient rien tribe is used, in which the not by the Product gene chromosomally encoded generation of in particular highly expressed extracellular enzymes greatly reduced or is eliminated. 16. The method according to any one of claims 1 to 15, characterized characterized in that the product gene is a highly expressed homologous or heterologous product gene. 17. The method according to claim 16, characterized in that that the product gene is an amyA gene for alpha-amylase from Ba cillus amyloliquefaciens. 18. The method according to claim 16, characterized in that that the product gene is an nprA gene for neutral protease of Bacillus amyloliquefaciens is. 19. The method according to any one of claims 1 to 18, characterized characterized in that a plasmid vector is used which leads to structurally stable integration plasmids. 20. The method according to any one of claims 1 to 19, characterized characterized in that a plasmid pGB354 as a plasmid vector is used. 21. The method according to any one of claims 1 to 20, characterized characterized that a cloned thy gene from Bacillus is used. 22. The method according to claim 21, characterized in that a thyL gene from Bacillus licheniformis ATCC9789 ver is applied.   23. The method according to claim 22, characterized in that that the thyL gene by "shot gun" using the restrictase BclI cloned into the plasmid vector pGB354 and a plasmid pSB319 is obtained. 24. The method according to claim 22, characterized in that an MLS ^r gene is cloned for resistance to MLS antibiotics from a plasmid pDB101 in the plasmid vector pGB354. 25. The method according to claim 24, characterized in that the plasmid pSB351 obtained by the recloning, in which a unique BclI site is flanked by the MLS ^r gene and a resident Cm ^r gene for resistance to chloramines, is used as a starting point Plasmid is used for the generation of integration plasmids for double crossing-over. 26. The method according to claim 23 to 25, characterized records that the thyL gene from the plasmid pSB319 in the unique BclI site of the plasmid pSB351 under profit A plasmid pSB360 is cloned. 27. The method according to claim 23 to 26, characterized records that the product gene into an internal interface of the thyL gene in plasmid pSB360 to form one pIG integration plasmids inserted and the thyL gene is deactivated. 28. The method according to claim 27, characterized in that the product gene enters the PvuII site of the thyL Gene is inserted. 29. The method according to claim 23 to 28, characterized records that the amyA gene for alpha-amylase from a Plasmid pSB6 into the PvuII site to form a pIG integration plasmid pIG1 is inserted.   30. The method according to claim 23 to 28, characterized records that the neutral protease nprA gene is from a Plasmid pSB92 into the PvuII site to form egg pIG integration plasmid pIG2 is inserted. 31. The method according to claim 25, characterized in that a BamHI-BglII DNA fragment from the synthetic Po lylinker of a plasmid pIC20H in the unique BclI section site is subcloned so that a plasmid pSB352 with egg A unique EcoRI interface is created. 32. The method according to claim 31, characterized in that a non-preselected DNA fragment from B. amyloliquefaciens in the EcoRI site and thereby plasmids pSB370 to pSB379 with one un different DNA fragment from B. amyloliquefaciens be. 33. The method according to any one of claims 23 to 32, characterized characterized in that the thyL gene from the plasmid pSB319 in a medium-sized interface of the chromosomal DNA Fragments in the plasmids pSB370 to pSB379 is inserted and thereby obtained pIT integration plasmids pIT1 to pIT10 become. 34. The method according to claim 32 and 33, characterized records that the pIT integration plasmids are chromosomal DNA fragments whose recombination with the Chro mosom does not affect the recipient's viability does. 35. The method according to any one of claims 23 to 32, characterized characterized in that the chromosomal DNA fragments by "shot gun" from B. amyloliquefaciens ATCC23844 into an in tern EcoRI site of the amyA gene in the plasmid pSB6 be cloned and the cloning by insertion Inak activation is proven.   36. The method according to claim 1 to 35, characterized in net that serving as recipient, stable thymidine auxotro phe bacterial strains of B. amyloliquefaciens using Dele tion in their chromosomal thyA gene for thymidylate synthesis tase by double crossing-over with the help of suitable De lettion plasmids are generated and the thyA gene in is activated. 37. The method according to claim 36, characterized in that that the deletion plasmids by deletion of an internal DNA fragment of the cloned thyA gene in a plasmid pSB411 and recloning of the deleted thyA gene in one Plasmid pSB355 can be generated. 38. The method according to claim 37, characterized in that that the plasmid pSB411 by "shot gun" cloning of the thyA gene from B. amyloliquefaciens ATCC23844 by means of Re Strictase PstI is obtained in a plasmid pSB3. 39. The method according to claim 37, characterized in that the plasmid pSB355 by recloning the synthetic Polylinkers from a plasmid pIC20R into plasmid pSB352 is obtained by means of the EcoRI restrictionase. 40. The method according to any one of claims 37 to 39, characterized in that the deleted thyA gene is flanked in the deletion plasmids for the detection of double crossing-over of one MLS ^r and one Cm ^r gene. 41. The method according to any one of claims 37 to 40, characterized characterized in that a deletion plasmid pID1 by Dele tion of the internal PvuII DNA fragment in the cloned thyA gene is generated. 42. The method according to any one of claims 37 to 40, characterized characterized in that a deletion plasmid pID2 by Dele tion of the internal SacI-DNA fragment in the cloned thyA gene is generated.   43. The method according to claim 15, characterized in that unwanted formation of chromosomal genes by chemical mutagenesis is reduced. 44. The method according to claim 15, characterized in that unwanted product formation is eliminated by the these coding chromosomal genes at double cros sing-over by deletion using deletion plasmids inak be activated. 45. The method according to claim 1 to 35, characterized in net that the thyA gene and / or other unwanted chromo somale genes of the recipient with double crossing-over Be inactivated with the aid of pIP integration plasmids, which at the same time as the pIT and pIG integration plasmids for the multiple step-by-step integration of the product gene to be used. 46. The method according to claim 45, characterized in that the pIP integration plasmids by insertion of the Pro duct gene in the cloned thyA gene and / or in other un desired cloned genes and subsequent recloning of the produced by inactivated genes in the plasmid pSB355 become. 47. The method according to any one of claims 45 or 46, characterized in that the gene inactivated by the insertion in the pIP integration plasmids for the detection of double crossing-over is flanked by one MLS ^r - and one Cm ^r gene . 48. The method according to any one of claims 45 to 47, characterized characterized in that a pIP integration plasmid pIP1 by Insertion of the amyA gene into the internal EcoRV interface of the thyA gene is generated in the plasmid pSB411.   49. The method according to any one of claims 45 to 47, characterized characterized by a pIP integration plasmid pIP2 by Insertion of the amyA gene into the internal EcoRV interface of the nprA gene is generated in the plasmid pSB92. 50. The method according to any one of claims 1 to 49, characterized characterized in that by in vitro mutagenesis of Integra tion plasmids conditionally unable to replicate, tempera tursensitive integration plasmids are generated. 51. The method according to any one of claims 1 to 50, characterized characterized in that the integration and deletion plas mide in a suitable manner, for example according to the PEG-Pro toplast method or by means of electroporation, in recipients duck strains of B. amyloliquefaciens transformed and Select plasmid transformants via antibiotic resistance be animals. 52. The method according to any one of claims 1 to 50, characterized characterized in that plasmid transformants with conditional Replication-incapable, temperature-sensitive integration Plasmids for integration and plasmid segregation at not permissive temperature and without selection pressure by anti biotics are cultivated. 53. The method according to any one of claims 1 to 52, characterized characterized in that plasmid transformants with strain spec fish high segregative plasmid instability to integra tion and plasmid segregation without selection pressure by An tibiotics are passed through. 54. The method according to any one of claims 1 to 53, characterized characterized in that the crossing-over by mutagen-indu graced plasmid-chromosome integration is promoted.   55. The method according to any one of claims 1 to 54, characterized characterized that the integration, the number of inte free gene copies and the absence of plasmid DNA in the Chromosome of the recipient by Southern hybridization be detected.