DD263080A5 - Nitrilasezusammensetzung - Google Patents

Abstract

The invention relates to nitrilase compositions, nucleic acid sequences encoding said nitrilase, constructions containing genes which encode said nitrilases under the control of transcriptional and translational gene regulatory genes and are recognized by a desired host foreign to the nitrilase genes. The invention also relates to host cells as well as organisms and organism parts or products containing these constructions. The nitrilase compositions of the invention have about 34 kDal with a specific activity of at least about 0.1 mmol NH 3 / min / mg and bromoxynil as the substrate. The nitrilase composition consists of bacteria, especially Klebsiella. The bacterial host of the invention comprises a foreign gene which expresses the 3,5-dihalo-4-hydroxybenzonitrile-specific nitrilase and protects bromoxynil-sensitive cells from the cytotoxic effects of the bromoxynil. The invention further relates to bromoxynilresistente plants.

Description

Field of application of the invention

The invention relates to nitrilase compositions, nucleic acid sequences encoding these nitrilases, constructions containing genes which encode these nitrilases under the control of the transcriptional and translational regulatory genes and are recognized by a desired host to which the nitrilase genes are foreign. The determination also concerns host cells containing these constructions as well as organisms and organism parts or products containing these constructions.

Characteristic of the known state of the art

The ability to confer new genetic capabilities on microorganisms and cells of higher organisms has opened up a variety of new uses. On the one hand, various agents can be prepared which are used for their cytotoxic effect; For example, in agriculture, many compounds are used to control pests and weeds. In many cases, these compounds have a relatively long residence time.

In many cases it is desirable to distinguish between the type to be maintained and the type to be controlled. So it is often desirable to destroy weeds selectively, the yield plants should be damaged as little as possible. Most broadband herbicides have a marked adverse effect on the crop, so their use is limited to preventive treatment or careful post-treatment.

There is therefore a great interest in modifying living cells to be resistant to stressors such as cytotoxic agents.

US-A-4535060 describes the use of a bacterial aroA gene conferring resistance to this compound on glyphosate (N-phosphonomethyl- (glycine) -sensitive cells. Hsu and Camper, in "Can. J. Microbiol." 22, pages 537-543 (1976), the isolation of ioxynil degrading products from soil enrichment cultures Hsu and Clemson describe in "Dissert Abstr Intrn", B 36,8, p 3708 (1976), the microwave degradation of a family Ingram and Pullin, in "Pestic.," 5, p.287-291 (1974), describe the fate of bromoxynil in three types of soil. Abstrp. Meeting Weed Soc., Am. "(1971), pages 16-17, Bromoxynil is degraded in heavy clay (Regina). Smith and Fletcher in" Hort Res. ", 4, pp. 60-62 (1964), report 3,5-di-halo-4-hydroxybenzonitriles and soil microorganisms.

Object of the invention

The aim of the invention is to provide novel agents which protect crops from the phytotoxic effect of herbicides.

Explanation of the essence of the invention

The invention has for its object to achieve protection against herbicides by genetic modification in the plant cell.

In accordance with the present invention, nitrilases, nucleic acid sequences encoding these nitrilases, will now be understood to include constructs containing genes encoding these nitrilases under regulatory and regulatory control of regulatory genes in a desired host and from a desired host to which the nitrilase genes are indicative describes host cells containing these constructions and organisms and organism parts and products containing these constructions. The bromoxynil and / or loxynil-specific nitrilases can be used to detoxify bromoxynil and related herbicide-containing sites and protect host cells from the cytotoxic effects of these herbicides. The constructions serve to distinguish between host cells containing these constructions and host cells lacking these constructions.

According to the invention, new DNA sequences, constructions, transformed cells, plants and peptides are indicated which hydrolyze halogenated hydroxybenzonitriles and in particular 3,5-dibromo- or 3,5-diiodo-4-hydroxybenzonitrile.

The invention also relates to the recovery of an enzyme which can hydrolyze the nitriles, thereby destroying the herbicidal action of the nitriles and protecting a cell or host susceptible to the herbicide or detoxifying an area contaminated with the herbicide can.

The structural gene of interest may be obtained from a unicellular microorganism and in particular a bacterium which can use the benzonitrile as the source of nitrogen or as the only source of nitrogen. The term "benzonitrile" or "nitrilase" means hereinafter that the benzonitrile is a halogenated p-hydroxybenzonitrile and especially 3,5-diiodo. or 3,5-dibromo-4-hydroxybenzonitrile (loxynil or bromoxynil) and a nitrilase is understood, which can use these halogenated benzonitriles as nitrogen source, or exclusively nitrogen source.

The enzyme can be obtained in various ways, conveniently from bacteria naturally occurring in a bromoxynil or loxynil-containing environment. Enterobacteria and in particular Klebsiella species are of particular interest. Klebsieila pneumoniae and in particular Klebsieila ozaenae can be used. Rather than isolation from the soil, these organisms can be grown in soil or other medium with increasing concentrations of benzonitrile and reduced levels of other nitrogen sources until the surviving organisms use the benzonitrile as the sole source of nitrogen.

Regardless of the bacterium containing the nitrilase, it must be selected to ensure that the nitrilase can detoxify the benzonitrile. In addition, the nitrile should be specific for the benzonitrile, rather than for analogs that do not have halogen and / or other substituents. The nitrilase according to the invention is specific for the benzonitriles indicated above and less or inactive than analogs. Conveniently, when benzonitrile is used at comparable concentrations as a source of nitrogen, the proliferation rate should not be significant, i. less than about 10%, as compared to the proliferation of a bacterium in the presence of a normal nitrogen source, such as ammonia. This result is not observed with nonspecific benzonitriles. If at least one host strain is identified, different methods can be used to identify the nitrilase coding sequence. The gene may be present on a chromosome or plasmid. The genome can be fragmented, especially with a restriction endonuclease, i. H. with one or more endonucleases that form fragments of about 5 to 50kb. These fragments can be cloned on appropriate vectors in a suitable bacterium, for example E. coli. The resulting transformants are examined for nitrilase activity, the host organism bidlet a negative background.

If at least one clone with nitrilase activity is identified, the extrachromosomal elements, plasmids or viruses containing the desired DNA fragment can be isolated in a conventional manner, for example by lysing the host, precipitating the DNA and separating the vector, plasmid or Virus DNA from the chromosomal DNA. The extrachromosomal elements can be cleaved by restriction endonuclease, the desired fragments can be isolated by various methods for separation and identification of the various large fragments, for example, by electrophoresis or density gradient centrifugation.

The fragment can generally be further manipulated, depending on size, to bring it closer to the size of the gene and its flanking control regions. Various methods can be used to manipulate the fragment containing the coding sequence for the enzyme and its regulatory flanking sequences. Partial cleavage with various restriction enzymes in different reaction mixtures, followed by cloning of the fragments to determine the fragments which still have the expression ability for the nitrilase can be used. On the other hand, the enzyme can be isolated and partially sequenced. Based on the amino acid sequence, samples can be prepared which can then be used to identify the fragments containing the gene. By combining this method with the restriction enzyme cleavage, fragments can be cloned and assayed for the presence of the desired gene. In addition, exonucleases, such as Bal31, can be used to remove the nucleotides from one or both ends of the fragment and further reduce the number of redundant nucleotides. Alternatively, the gene may be cloned into a corresponding host and the messenger RNA isolated by selection with a sample, identified in a suitable in vitro or in vivo translation system, for example Xenopus oocytes or reticulolysate. The isolated messenger RNA can then be used in a conventional manner to produce cDNA, i. H. using a reverse transcriptase and forming a complementary chain with a DNA polymerase. In this case, the resulting structural gene lacks the control regions associated with the transcription.

The DNA sequence containing the structural gene expressing the nitrilase can be linked to a variety of other DNA sequences for introduction into a corresponding host cell. The linked sequence depends on the type of host, the. Mode of introduction of the DNA sequence into the host and whether episomal maintenance or integration is desired. For prokaryotic hosts, there are a variety of vectors that can be used to introduce, by transformation, conjugation, transduction or transfection of the DNA sequence into a prokaryotic host. DNA sequences include a variety of plasmids such as pBR322, pACYCI84, pMB9 or pRK290, cosmids such as pVKIOO, or viruses such as P22.

In eukaryotic hosts, a variety of methods for introducing the DNA into the host can be used, such as transformation with Ca ⁺⁺ precipitated DNA, which is understood to mean a non-replicative DNA sequence, a plasmid or a minichromosome, transformation with a T-DNA containing Sequence in Agrobacterium, microinjection with a micropipette or electroporation. Depending on whether a competent replication system is present in the DNA construction, it depends on whether the DNA alkaline episomal element is replicated or integrated into the host genome and the structural gene is expressed in the host. Episomal elements may be used, such as tumor-inducing plasmids or fragments thereof, for example Ti or Ri, or viruses or their fragments, for example CaMV or TMV, which are non-lethal to the host and in which the structural gene is present in such episomal elements that the Expression of the structural gene is possible. Of particular interest are fragments with replication functions that lack other functions, such as ontogenesis or virulence. The fragments obtained from the nitrilase source can be cloned using a corresponding cloning vector. The cloning can be carried out in a corresponding unicellular microorganism, for example a bacterium, such as E. coli. Conveniently, a cosmid is used in which fragments of the desired size are obtained by partial or full degradation. Thus, for example, the cosmid pVKIOO with a corresponding

Restriction enzyme are partially degraded and bound to fragments that have arisen either from the partial or full degradation of a plasmid, chromosome or fragment thereof. Packaging allows only fragments of the desired size to be packaged and transduced into the host organism.

The host organism can be selected for its benzonitrile resistance. The recipient strains can be modified to have the appropriate genetic traits that allow for selection of the transductants. The transductants may be used in microorganisms for the conjugation of other microorganisms, optionally using a plasmid as a carrier. Various methods can be used to further reduce the size of the fragments containing the structural gene for the nitrilase, for example, the cosmid vector can be isolated, cleaved with a variety of restriction endonucleases, such as EcoRI, BglI or SmaI, the resulting fragments can be in a corresponding vector, conveniently with the previously used Cosmidvektor be cloned. Instead of a cosmid vector, a large number of cloning vectors of small size, such as pACYC177 or pACYC184, can be used. Thus, fragments of preferably below about 5kb, generally below about 4kb, and more preferably below about 2kb, can be cloned and confer benzonitrile resistance.

Conveniently, the fragment has about 1 kb and less than about 5kb, preferably less than about 4kb, and most preferably at least about 1047bp, and more particularly including flanking regions at least about 1100 bp and preferably less than about 1.5kb. Of particular interest is a Bglll-SmaI fragment from Klebsiella ozaenae, and more particularly a PstI HincII fragment of about 1 210bp.

The nitrilase can be expressed by any suitable source, whether prokaryotic or eukaryotic, such as bacteria, yeast, filamentous fungi or plant cells. Where no secretion occurs, the enzyme can be recovered by lysing the cells and isolating the nitrilase in a manner known per se. Suitable methods are, for example, chromatography, electrophoresis or affinity chromatography. Conveniently, bromoxynil can be attached to an insoluble carrier via an appropriate function, such as the carboxyl group, and thus immobilized to obtain the nitrilase.

The specific activity of the nitrilase according to the invention is at least about 0.1 μιηοΙ of ammonia / min / mg protein, generally at least about 0.5 or more lower than that of Harper, Biochem. J., 167, pp. 685-692 (1977).

The purified enzyme can be used in many different ways. It can be used directly in studies on bromoxynil, loxynil or other related benzonitriles. On the other hand, the enzyme can be used as a detection reagent in diagnostic assays, for example by being bound to a product of interest, for example, a hapten, antigen or antibody (see US 3654090, 3817837 and 3850752). The conjugation methods and the determination of the concentration of the product to be detected are described in detail in these patents.

The DNA sequence encoding the nitrilase can be used in a variety of ways, for example, to isolate the wild type or mutant nitrilases. On the other hand, the DNA sequence can be used for integration by recombination into a host, whereby the host obtains benzonitrile resistance.

In plant cells, the structural gene can be introduced as part of a construction into a plant cell nucleus by micropipet injection for integration through recombination into the host genome. On the other hand, one can use electroporation into which the structural gene is introduced for introduction into a plant host. If the structural gene has been obtained from a source that has regulatory signals that are not recognized by the plant host, it may be necessary to introduce appropriate regulatory signals for expression. When a virus or plasmid, such as a tumor-inducing plasmid, is used and has been mapped, a restriction site downstream of the promoter may be selected, into which the structural gene may be introduced at a suitable distance from the promoter. If the DNA sequences do not have an appropriate restriction site, they can be degraded several times with an exonuclease, such as Bal31, and a synthetic restriction endonuclease site (left) can be incorporated.

Of particular interest is the use of a tumor-inducing plasmid, such as Ti or Ri, with which the nitrilase gene can be introduced into plant cell chromosomes. The use of Ti and Ri plasmids is described in PCT-A-W084 / 02913,02919 and 02920 and EP-A-0116718 and by Matzke and Chilton in "J. MoI. App. Genetics ", 1, p. 39-49 (1981).

Using the right-hand end or both ends of the T-DNA adjacent to an expression cassette containing the nitrilase structural gene under the control of transcriptional and translational initial and terminal regulatory signals recognized by the plant host , the expression cassette can be incorporated into the plant genome and elicit the expression of nitrilase in the plant cell at various stages of development.

Various constructions can be made for expression in plant cells which provide an expression cassette which is responsible in plants for the expression of the nitrilase in the plant host.

For transcription, a variety of transcription initiation regions (promoter regions) that are either constitutive or inducible can be used.

The transcriptional initiation region is linked to the structural gene encoding the nitrilase for initiation of transcription upstream of the initiation codon, usually within about 200 bases of the initiation codon, where the 5'-untranslated region lacks an ATG.

The 3 'end of the structural gene contains one or more stop codons linked to a plant host functionally transcriptional termination region, which termination region may be linked to the same or different structural genes as the initial region.

The expression cassette has in the direction of transcription the initiation region, the structural gene under the transcriptional control of the initiation region, and the termination region responsible for the transcriptional end and the production of the messenger RNA.

As transcriptional and translational control regions, pin promoter and terminator regions may suitably be used which allow constitutive expression of the nitrilase gene. On the other hand, other promoters and / or terminators can also be used, in particular promoters which have an inducible expression or a controlled

Allow expression in a plant host. Suitable promoter regions from the Ti plasmid include opine promoters such as octopine synthetase promoter, napoline synthetase promoter, agropine synthetase proteomic or manopin synthetase promoter. Other promoters are viral promoters such as the CaMV region VI promoter or the full length (35S) promoter linked to the ribulose-1,5-bis-phosphate carboxylate genes, e.g., the small subunit, genes associated with phaseoiin-associated promoters, protein deposits, β-conglycinin or cellulose formation. The various sequences can be linked together in a manner known per se. The promoter region can be identified by the 5 'region of the structural gene, such as opine, and selected and isolated by restriction mapping and sequencing. Accordingly, the terminator region can be isolated as a 3 'region of the structural gene. The sequences can be cloned and joined in the correct orientation to allow for constitutive expression of the nitrilase gene in a plant host.

By modifying yield source cells by introducing a functional gene expressing the nitrilase, bromoxynil, loxynil or analogous herbicides can be used in a variety of crops at concentrations that ensure complete or almost complete removal of weeds and leave the crops undisturbed. In this way, considerable savings can be achieved as fertilizer and water are used more effectively and the adverse effects resulting from the presence of weeds are avoided. The expression cassette expressing the nitrilase can be introduced into a variety of plants, both monocotyledonous and dodecotyledonous plants (monocotyledons and dicotyledons) such as corn, cereals, soybeans, tobacco, cotton, tomatoes, potatoes, Brassica species, rice, peanuts, Petunias, sunflowers, sugar beets or turf. The genes may be present in cells or plant parts, such as callus, tissue, roots, tubers, offspring, plantlets, seeds, leaves, cuttings or pollen.

Because the plants are benzonitrile-resistant, many formulations can be used to protect yield plants from weeds to increase the growth of crops and reduce competition for nutrients. Thus, bromoxynil alone can be used for post-control of weeds in treated crops such as sunflowers, soybeans, cereals or cotton, or in combination preparations with other products. In the preparations, conventional amounts of pesticides are applied to the fields, i. about 0.045 to 1.8 and preferably 0.09 to 0.9g / 0.4047ha (0.1 to 4 and preferably 0.2 to 2 lb / acre) of bromoxynil, where other herbicides are present in amounts of about 0.045 to 1.8g / 0.4047ha (0.1 to 4 lb / acre) of active ingredient. The preparations may contain other additives such as detergents, auxiliaries, spray aids, adhesives or stabilizers. The preparations may be wet or dry preparations, such as flowable powders, emulsifiable and liquid concentrates, as known per se. Herbicidal solutions can be applied in a known manner, for example by spraying, sprinkling or spraying.

embodiment

The invention is explained in more detail below with reference to some examples

 Materials and methods

Restriction enzymes and T4 ligase for the linkages were used according to the manufacturers instructions. Cloning and molecular analysis were performed in standard methods according to Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982. Clone analysis was performed according to Ish-Horowitz et al, Nucl. Acids Res. , 9, p.2989-2998 (1981).

E. coli strain MM 294 was used for all cloning experiments (Hanahan in "Mol. Biol.", 1966, p.557-580 [1983]). Antibiotics were used in the following amounts:

Cm (chloramphenicol): 25 μg / ml; Tc (tetracycline): ΙΟμς / ΐηΙ; Ap (penicillin): 300μιτ ^ / ηηΙ.

The transformations of the plasmid DNA into E. coli were performed according to Mandel and Higain "J. Mol. Biol., 53, pp. 159-162 (1970).

Bacteria from a bromoxynil contaminated soil sample were isolated and selected. One such organism was Klebsiella pneumoniae, subspecies ozaenae. By partial purification and characterization of the bromoxynil-specific nitrilase from this organism, an enzyme with a dummy molecular weight of 34kDal was obtained.

After repeated culture of K. ozaenae on solid L agar, a variant was isolated which was no longer able to utilize bromoxynil as the sole source of nitrogen when grown in a liquid medium containing 1.5 g KH ₂ PO ₄ per liter. 3.5 g K ₂ HPO ₄ , 0.1 g MgSO ₄ .7H ₂ O, 50 mg yeast extract, 0.1% citrate, glycerol and succinate and trace elements (Barnett and Ingraham in J. Appl. Bacteriol, 18, p 131-143 [1975]) .This medium is known as YETE multimedium containing 0.05% bromoxynil Although this organism did not utilize the bromoxynil as the sole source of nitrogen, it was able to reach full density in L broth with 0, A K. ozaenae variant was selected and grown in 10 ml of L broth Three independent K. ozaenae colonies were selected from a bromoxynil-containing LB plate and cultured under the same conditions: these four K ozaenae colonies were co-administered in 10 ml of 0.05% Bromoxynil e bred L-broth. The cultures were grown to full density at 30 ^° C., from each culture according to Ish-Horowitz et al., Nucl. Acids Res., 9, p 2989 (1981) Plasmid DNA was prepared by electrophoresing undigested plasmid DNA on a 0.5% agarose gel and visualizing the plasmid strips by staining with ethidium bromide.

The K. ozaenae variant showed a single plasmid species (size: 68 kb), which was formed with and without bromoxynil. The three K. ozaenae colonies showed a larger plasmid species (90 kb) when grown in the presence of 0.05% bromoxynil. In the absence of bromoxynil, both forms of plasmid were present in two of the three K. ozaenae colonies. This indicates a conversion of the larger plasmid species into smaller forms with simultaneous loss of approximately 22 kb of plasmid DNA when the bromoxynil selection is abolished.

All four colonies were grown in 200 ml of L broth containing 0.05% bromoxynil. The cells were disrupted in a French press, the supernatants after high-speed centrifugation were dialyzed against a buffer containing 0.05 mol / L potassium phosphate, pH 7.5, and 2.5 mmol / l dithiothreitol (DTT). The individual crude extracts were examined for bromoxynil-specific nitrilase activity. A crude extract from the K. ozaenae variant contained no detectable nitrilase activity whereas the other K. ozaenae crude extracts had specific nitrilase activities of 0.124, 0.10 and 0.143 μηηΙ NH ₃ / min / mg protein, respectively. 200ml cells were grown at 30 ^° C until mid-log phase in M9 medium containing 0.1% glucose and 0.04% bromoxynil (Miller in "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, 1972). The crude extracts were recovered by disrupting the cells, ultracentrifuging and dialyzing the supernatant in buffer containing 0.05M potassium phosphate, pH 7.5, and 2.5 mM DTT The substrate concentration was 3 mmol / L bromoxynil in all experiments The release of ammonia was monitored according to Harper in "Biochem J.," 167, pp. 685-692 (1977). The ability of the K. ozaenae variant to grow in bromoxynil-containing L broth may result in an impermeability of the organism to that component. However, the organism can not grow in certain media utilizing bromoxynil as the sole nitrogen source.

In summary, K. ozaenae nitrilase appears to be encoded in the plasmid. The enzyme (s) encoding gene (s) appears to be located on a 22 kb plasmid DNA segment that is spontaneously lost from the K. ozaenae plasmid in the absence of the selection of bromoxynil.

The bromexynil-specific nitrilase from K. ozaenae is expressed in E. coli.

Plasmid DNA from K. ozaenae grown at 0.05% bromoxynil selection was recovered, the DNA was transformed into E. coli strain MM 294 (thi, gyrA96, endl ", hdsR17). Transform ante η was isolated on a nitrogen-depleted ( N ") solid agarose minimal medium containing per liter 1.5 g KH ₂ PO ₄ , 3.5 g K ₂ HPO ₄ , 0.1 g MgSO ₄ .7H ₂ O and 0.1% glucose, with addition of 0, 05% bromoxynil selected as sole nitrogen source. After a day's incubation, 10 colonies appeared on the selection plates. These colonies were restreaked on L agar plates containing 0.05% bromoxynil and assayed for the presence of the auxotrophic thiamine marker in MM 294. None of the colonies grew in the minimal medium in the absence of thiamine, indicating that strain E. col! MM 294 is. All colonies grew in M9 medium to which thiamine and 0.05% bromoxynil were added as the sole nitrogen source. No growth was observed in this medium in the absence of bromoxynil. Two of the colonies were selected for further analysis. Crude extract preparations from E. coli MM 294 containing the 90kb plasmid were screened for bromoxynil-specific nitrilase activity; thereby a specific activity of 216μητιοΙ released NH ₃ / min / mg was obtained. E. coli MM 294, which contained the smaller plasmid, showed no detectable nitrilase activity. The larger 90kb plasmid in E. coli was designated pBrxi, the smaller (68kb) plasmid pBrxiA.

To confirm that E. coli strain MM 294, which contains the plasmid pBrxi, generates the right as a result of Metabiliten bromoxynilspezifischen Nitrilasereaktion was added a 2 ml MM 294 culture (pBrxi) for 24 hours at 30 ⁰ C in a M9 medium to which 0.05% bromoxynil has been added. A sample of the culture filtrate was chromatographed on a C ₁ S high pressure liquid chromatography column. The total amount of bromoxynil added to the culture filtrate was converted to a new metabolic peak. The metabolite peak was identified by spectral analysis as 3 ', 5'-dibromo-4-hydroxybenzoic acid (DBHB). That is, the product of bromoxynil-specific, plasmid-encoded nitrilase expression in E. coli is the same as that of K. ozaenae

 The bromoxynil-specific nitrilase gene is cloned in E. coli.

To determine whether the DNA segment coding for the bromoxynil-specific enzyme can be cloned in E. coli, the plasmid pBrxi was digested with BamHI, resulting in two strips of 53 kb and 37 kb, respectively. The BamHI fragments were incorporated into the BamHI site of the E. coli plasmid vector pACYC 184 (Chang and Cohen in "J. Bacteriol.", 134, p. 1141 [1978]) and into E. coli strain MM Cloning into the BamHi site of pACYC 184 results in insertion-inactivation of the tetracycline resistance gene Ten chloramphenicol-resistant, tetracycline-sensitive MM294 colonies were selected, DNA was prepared by minipreparation clone analysis and digested with BamHI Four clones contained the 37 kb BamHI fragment whereas only one clone contained the larger 53 kb BamHI DNA fragment from pBrxi Five clones contained a cloned BamHI fragment which was also found in plasmid pBrx1 Δ and corresponds to the DNA segment following spontaneous All ten clones were grown in 200 ml of L broth in the presence of 20 μg / ml chloramphenicol (for selection of the plasmid), the crude extract preparations obtained were obtained Bromoxynilspezifische nitrilase activity examined. Four clones containing the 37 kb BamHI fragment exhibited nitrilase specific in the range of 0,140μιτιοΙ released NH ₃ / min / mg protein, whereas no nitrilase activity could be detected in the other six clones. These results indicate that one bromoxynil-specific nitrilase activity could be detected. These results indicate that the gene encoding a bromoxynil-specific nitrilase activity is located on a 37 kb BamHI fragment, that is cloned from plasmid pBrxi, and that the 22 kb DNA segment is lost spontaneously in the absence of selection of bromoxynil , is located within the 37 kb BamHI fragment. To confirm the orientation of the BamHI fragments relative to the pACYC 184 vector, DNA from the four clones indicated above was digested with EcoRI and electrophoresed on a 0.07% agarose gel. A combined EcoRI degradation product of plasmids pBrxi and pBrxiA was also analyzed.

Both orientations of the 37 kb BamHI fragment with respect to the vector pACYC 184 were determined and designated plasmid pBrx2 and pBrx3, respectively. It was found that the three EcoRI fragments are located within the 22kb DNA segment, which is spontaneously deleted from the plasmids pBrx2 and pBrx3. The size of these EcoRI fragments is 18.3 and 1.9 kb, respectively. The gene encoding the bromoxynil-specific nitrilase is believed to be on one of these three EcoRI fragments, provided that the nitrilase structural gene is not divided into two by an EcoRI restriction site. The localization of the bromoxynil-specific Ntrilase from E. coli (pBrx3) was investigated, the results are given in the following Table 1.

Table 1

The bromoxynil specific nitrilase is a periplasmic enzyme in E. coli

Culture conditions ^a

specific nitrilase activity

toluene-treated cells (L broth)

lysozyme-treated cells (L broth)

Total Cells (L-Broth) Total Cells (L-Broth + Brx1) Total Cells (M 9) Total Cells (M9 + BrxD Total Cells / pACYC184 (M9)

0.829

0.796 0.770 1.25

0.950 1.45

E. coli cells (WIM 294) containing pBrx3 plasmid were grown to stationary phase in 5 ml cultures at 30 ° C in the indicated medium. The cultures contained 2 (g / ml chloramphenicol and 0.04% bromoxynil (Brx 1) where indicated 1 ml from each culture was collected once with nitrilase buffer (0.1 mol / l potassium phosphate, pH 7.5). The cells were resuspended in 0.1 ml of this buffer 50 μl samples were prepared according to Ha'rper in "Biochem J.," 167, pp. 685-692 (1977), with and without 3 mmol / l Bromoxynil examined as a substrate.

b μΜοΙ NH ₃ / min / mg. The protein was determined on the basis of the optical density at 600 nm (OD ₆₀₀ ): 1.4 = 10 ⁹ cells / ml = 150 μg.

Table 1 shows that the nitrilase is located in the periplasmic space of the cell. In addition, it was found that the enzyme is expressed in the absence of bromoxynil in the medium, suggesting that no bromoxynil induction is necessary for enzyme expression.

Further purification of the bromoxynil-specific nitrilase.

The further purification of the K. ozaenae nitrilase was carried out with the following results:

Table 2

Purification of the bromoxynil-specific nitrilase from E. coli (starting material: 6 g of cells)

fraction volume protein μιτιοΙ NH ₃ / min spec. Activity ¹¹ Raw ^a 100 ml 210 mg 18.15 0.086 35-50% NH ₄ SO ₄ 6 ml 83 mg 26.77 0,250 DEAE Sephadex 56 ml 19mg 15.52 0,820

^a Cells were grown at 30 ° C until mid log phase in M 9 medium containing 0.04% bromoxynil and glucose. The crude extracts were obtained by disrupting the cells, ultracentrifuging and dialysis in buffer containing 0.05 mol / L potassium phosphate, pH 7.5, and 2.5 mmol / L DTT. The substrate concentration was 3mmol / L in all nitriase experiments.

^b μΜοΙ NH ₃ / min / mg.

A 2.5cm ² χ 10cm column was equilibrated in buffer containing 0.05M potassium phosphate, pH 7.5, 2.5mmol / L DTT and 1mmol / L EDTA. The sample was applied, the column was developed with 300 ml of a linear gradient of 0.02 to 0.40 mol / l NaCl in the buffer indicated above. At the end of the gradient, buffer containing 1 mol / l NaCl was applied. 5 ml fractions were collected, 0.075 ml samples of changing fractions were assayed for nitrilase activity. A single peak with enzyme activity was eluted at 0.22 mol / L salt. About 75% of the discontinued nitrilase activity was recovered in the active fractions.

The fractions containing the nitrilase activity from the DEAE column were added to 0.02 mol / l potassium phosphate, pH 7.5, αϊΒίνβϊβΓί, δΟμΙ (6 μg protein) of each fraction to 11.25% denatured Lämmli gel. The protein-enriched strip corresponding to DEAE column activity is a 34,000 molecular weight polypeptide. No additional polypeptides could be accumulated from the active fractions from the column. These results indicate that the bromoxyni-specific nitrilase is a polypeptide of molecular weight about 34,000 and presumably the product of a single gene.

The clone pBrx2 was completely digested with EcoRI, a fragment of about 19kb was isolated. The fragment was incorporated into the EcoRI digested vector pACYC 184 (3.9 kb) to the plasmid pBrx5, which was transformed into E. coli as described above. The plasmid was isolated in a manner known per se and digested with BglII to form an approximately 6.7 kb fragment which remained incorporated into the vector pACYC 184. The isolated plasmid pBrx7 was then digested with SmaI and BglII to a fragment of about 3.9 kb which was incorporated into Smal-BamHI digested pACYC 177 (3.7 kb) (Chang and Cohen in J. Bacteriol. 134, pp. 1141-1156 [1978]). The resulting plasmid, which had penicillin resistance, was transformed into E. coli as described above, the transformants were selected on a penicillin selection medium to the plasmid pBrx8, which contains the nitrilase gene on a 3, Carries 9 kb fragment.

pBrx8 was partially digested with PstI, the fragments were introduced into a PstI digested pUC 18 (see Yanisch-Perron et al in Gene, 33, pp. 103-119 [1985]). One clone had a 5.3kb plasmid, pBrx9, which was isolated and further digested with PstI and HinclI to a 1210 bp fragment extending in the direction from PstI to Hindi, CIaI, Sail, Seal and Phl restriction sites at relatively uniform intervals The PstI HincII fragment was sequenced according to Sanger et al in "Proc Nat Nat. Acad., USA", 74, pp. 5463-5468 (1977). The resulting sequence (with the corresponding encoded amino acids) is given in the following tables.

95 125

ATG GAC ACC ACT TTC AAA GCA GCC GCT GTT CAG GCC GAA CCG GTA TGG ATG GAT GCC GCT

Met Asp Thr Thr Phe Lys Ala Ala Ala VaI Gin Ala Giu Pro VaI Trp Met Asp Ala Ala

155. 185

GCA ACA GCC GAT AAG ACC GTG ACG CTA GTA GCT AAA GCC GCA GCG GCT GGC GCG CAG CTC

Ala. Thr Ala Asp Lys Thr VaI Thr Leu VaI Ala Lys Ala Ala Ala Ala Giy Ala Gin Leu

215 245

GTC GCA TTT CCC GAA TTG TGG ATT CCG GGC TAC CCA GGA TTC ATG CTC ACG CAC AAC CAA

VaI Ala Phe Pro Giu Leu Trp He Pro GIy Tyr Pro Giy Phe Met Leu Thr His Asn Gin

275 305

ACC GAA ACC CTA CCA TTC ATC ATT AAA TAC CGC AAG CAG GCA ATCC GCC GCC GGA CCA

Thr GIu Thr Leu Pro Phe lie Lys Tyr Arg Lys Gin Ala lie Ala Ala Asp GIy Pro

335 365

GAA ATC GAA AAA ATT CGC TGC GCG GCT CAG GAG CAT AAC ATT GCG CTC TCC TTT GGG TAC

GIu He Giu Lys He Arg Cys Ala 'Ala Gin Giu His Asn He Ala Leu Ser Phe Giy Tyc

395 425

AGC GAA CGG GCT GGC CGT ACG CTC TAC ATG TCA CAA "ATG CTT ATC GAT GCC GAT GGC ATC

Ser GIu Arg Ala GIy Arg Thr Leu Tyr Met Ser GIn Met Leu al Asp Ala Asp GIy He

, 455'485

ACC AAA ATT CGT CGT CGA AAG CTC AAA CCA ACC CGC TTT GAA CGA GAA CTC TTT GGC GAA

Thr Lys He Arg Arg Arg Lys Leu Lys Pro Thr Arg Phe GIu Arg GIu Leu Phe GIy GIu

515 545

GGT GAC GGA TCG GAC TTA CAG GTC GCC CAA ACT AGC GTT GGT CGG GTG GGT GCC CTC AAC

GIy Asp GIy Ser Asp Leu Gin VaI Ala GIn Thr Ser VaI GIy Arg VaI GIy AIa Leu Asn

575,605

TGC GCG GAG AAT TTG CAG TCG CTA AAC AAG TTT GCG CTT GCT GCC GAG GGT GAA CAG ATA

Cys Ala GIu Asn Leu GIn Ser Leu Asn Lys Phe AIa Leu Ala Ala GIu GIy GIu Gin He

635,665

CAT ATC TCC GCC TGG CCA TTC ACG CTT GGA AGC CCT GTG CTC GTC GGA GAC TCC ATC GGC

His He Ser AIa Trp Pro Phe Thr Leu GIy Ser Pro Vai Leu Vai GIy Asp Ser He GIy

695'725

GCC ATC AAC CAG GTC TAC GCG GCC GAG ACG GGG ACC TTC GTT CTC ATG TCG ACG CAG GTG

Ala He Asn Gin VaI Tyr Ala Ala Giu. Thr GIy Thr Phe VaI Leu Met Ser Thr Gin VaI

755,785

GTT GGA CCG ACC GGC ATC GCC GCC TTC GAG ATC GAA GAC AGG TAC AAC CCG AAT CAG ACT

VaI GIy Pro Thry Gly Ala Ala Phe GIu He GIu Asp Arg Tyr Asn Pro Asn GIn Tyr

815 845

CTT GGT GGT GGG TAC GCG CGG ATC TAC GGG CCT GAC ATG CAG TTG AAG AGC AAG TCG TTG

Leu GIy GIy GIy Tyr AIa Arg He Tyr GIy Pro Asp Met GIn Leu Lys Ser Lys Ser Leu

875 905

TCA CCG ACC GAA GAG GGC ATC GTC TAC GCC GAG ATC GAC CTG TCG ATG CTT GAG GCA GCA

Ser Pro Thr GIu GIu GIy lie VaI Tyr Ala Giu l Asp Leu Ser Met Leu GIu Ala Ala

935 965

AAG TAC TCG CTC GAT CCC ACG GGC CAC ACT TCG CGC CCT GAT GTG TTC AGC GTG TCG ATT

Lys Tyr Ser Leu Asp Pro Thr GIy His Tyr Ser Arg Pro Asp VaI Phe Ser VaI Ser He

995 1025

AAC CGG CAA CGG CAG CCT GCG GTG TCA GAA GTT ATC GAC TCA AAC GGT GAC GAG GAC CCG

Asn Arg GIn Arg GIn Pro Ala VaI Ser GIu VaI He Asp Ser Asn GIy Asp GIu Asp Pro

'. 1055 1085

AGA GCA GCA TGC GAG CCC GAC GAG GGG GAT CGT GAG GTC GTA ATC TCT ACG GCA ATA GGG

Arg Ala Ala Cys GIu Pro Asp GIu GIy Asp Arg GIu VaI VaI He Ser Thr Ala lie GIy

GTT CTA CCC CGT TAT TGC GGA CAT TCC.

VaI Leu Pro Arg Tyr Cys GIy His Ser

The PstI HincII fragment, which is virtually free of 5 'and 3' non-coding flanking regions, can be linked to EcoRI linkers and digested with EcoRI, then ready to be inserted into a plant expression cassette by insertion into the EcoRI. To be introduced by pCGN451.

pCGN451 comprises an octopine cassette containing about 1 566 bp of the 5 'noncoding region fused to the 3' end of the gene via an Eco RI linker and about 1349 bp of 3 'noncoding DNA. The pTI coordinates are 11207 to 12823 for the 3 'region and 13643 to 15208 for the 5' region (as defined by Öarker et al in "Plant Molecular Biology", 2, p.335 (1983). Fragment was obtained as follows: A small, under-cloned fragment containing the 5 'end of the coding region, such as a BamHI-EcoRI fragment, was cloned into pBr 322 as plasmid pCGN407 The BamHI-EcoRI fragment had an Xmnl fragment. PCGN 407 was digested with XmnI and cut with Bal31 nuclease, EcoRI linkers were added to the fragments, and after digestion with EcoRI and Bam HI, the fragments were size fractionated, the fractions were cloned and sequenced In one case, the entire coding region and 10bp of the 5 'untranslated sequences were removed using the 5' non-transcribed region, the RNA overlap site and 16bp of the 5 'untranslated region (to a BamHI site) int act remained behind. This small fragment was obtained by size fractionation on a 7% acrylamide gel, about 130 bp long fragments were eluted. This size fractionated DNA was ligated into M13mp9, various clones were sequenced; the sequence was compared to the known sequence of the octopine synthetase gene. The

M 13 construction was designated ρ 14. The plasmid was digested with BamHI and EcoRI to a small fragment attached to an Xhol-BamHI fragment, the upstream 5 'sequences of pTiA6 (Garfinkel and Nester in "J. Bacteriol.", 144, p.732 [1980]). ) and bound to an EcoRI-Xhol fragment containing the 3 'sequences.

The resulting XhoI fragment was cloned into the XhoI site of a pUC8 derivative designated pCGN426. This plasmid differs from pUC8 by the unique EcoRI site filled with DNA polymerase I, and by the loss of PstI and HindIII sites by nuclease contamination of the HincII restriction endonuclease when an XhoI linker is included in the only restriction. Place of pUC8 is installed. The resulting plasmid pCGN451 has a single EcoRI site for the insertion of the protein coding sequences between the 5 'non-coding region (the 1 550 bp of the O' non-transcribed sequence including the right border of the T-DNA, the mRNA overlap site and 16bp of the 5 'non-transcribed sequence) and the 3' region containing 267bp of the coding region, the stop codon, 196bp of the 3 'untranslated DNA, the polyA site and 1153bp of the 3' non-transcribed sequence.

The XhoI fragment containing the octopine synthetase (ocs) cassette was incorporated into the plasmid pCGN 517, which had tetracycline resistance and kanamycin resistance genes. pCGN517 was isolated from pHC79 (Hohn in "Gene", 11, p.291 [1980]) by insertion into the unique PstI site of the Kanr gene from pUC4K (Vieira in "Gene", 19, p.259 [1982]) manufactured. pCGN517 was digested with Sail, the Xhol fragment was incorporated into the unique Sall site.

The Xhol fragment was also incorporated into a second plasmid pCGN 529. pCGN 529 was prepared from pACYC 184 by insertion of the Kanr gene from Tn 5 (Rothstein et al, in "Movable Genetic Elements", p.99, Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, [1981]) BglII fragment of 2.4kb from pRiA4-T DNA (White and Nester in "J. Bacteriol.", 144, p. 710 [1980]) was introduced into the BamHI site following substitution of HindIII-BamHI Fragments of pACYC 184 with the Kan ^r gene of Tn 5 remained.

The XhoI fragment containing the ocs cassette, into which the EcoRI nitrilase gene was introduced into the unique EcoRI site of the ocs cassette, was incorporated into pCGN 517 and pCGN ~ 529, with two plasmids pN1 and pN 2, which are used for introduction into A. tumefaciens or A. rhizogenes or for integration into the T-DNA of Ti or Ri plasmids. The integration into the respective plasmids can be carried out in three steps according to Comai et al in "Plasmid", 10, pp. 30-30 (1983): A night-old cultures of E. coli hosts containing the plasmids pRK 2073, PN1 or PN 2 and A. tumefaciens A722 (Garfinkel in "J. Bacteriol.", 144, p. 732 [1980]) or A. rhizogenes A4T (White in "J. Bacteriol.", 144, p. 710 [1980] were grown overnight, the respective cultures were mixed and streaked on AB plates containing 150 μg / ml kanamycin, single colonies were streaked twice, and the correct integration was verified by Southern analysis of all Agrobacterium DNA Endonuclease digested DNA was probed with a gap-translated pBrx8.

Transformation and regeneration of tobacco leaf pieces cultivated together with A. rhizogenes. Tobacco plants were at 25 ⁰ C and 16 hours to white light in MS-medium (1 mg / l IAAundO, 15mg / l kinetin) grown. Three-week-old plants surviving grafting of the main shoot were used as tissue donors. Young leaves (up to the 4th leaf from top) were selected, from which discs were punched 2 mm in diameter and placed in Petri dishes (3 cm diameter) in 1 ml of MS medium with 1 mg / l IAA. After leaving the slices overnight in full darkness, Agrobacterium cells (A722 χ pN1 or pN 2) were added in an amount of 10 ⁸ to 10 ⁹ / ml in plant culture medium to these cultures. Cocultivation was carried out in the dark for 18 to 24 hours. The leaf pieces were freed of Agrobacterium by washing three times with MS medium lacking hormones and containing 350 mg / l cefotaxine (Boehringer, Mannheim). The leaf pieces were transferred to Petri dishes with a diameter of 9cm in 10ml of hormone-free MS medium. Petri dishes containing 0.6% Phytagar (Gibco) and 350 mg / l cefotaxin were sealed with paraffin and kept under the same conditions as the tissue donor plants. After 2 to 4 weeks, roots appeared which were taken and introduced into the same medium containing 2 mg / L IAA and 2 mg / L kinetin. The regeneration shoots became visible in the following 2 to 5 weeks.

The potted plants were sprayed with a bromoxynil solution when they had six leaves. Each plant in a 10cm diameter (4 inch) pot received 2.5ml of spray. The plants were grown in a greenhouse at 25 ⁰ C, 70% relative humidity and 60 hours exposure. The growth was checked 9 days after spraying by counting new leaves longer than 0.5 cm.

By the method described above, plants can be obtained which are bromoxynilresistant and can grow in the field in the presence of bromoxynil without adversely affecting their growth. According to the invention, it is now possible to obtain plants which are herbicide-resistant and, in particular, resistant to specific b'zonitrile herbicides. That is, the gene encoding nitrilase can be introduced into a plant host whereby the gene is expressed and confers benzonitrile resistance to the plant. In addition, the enzyme can be recovered by cloning the gene in a suitable bacterial host in which the enzyme is expressed. Active enzymes (which can be tested) find a variety of applications, for example in studies of various compounds or the benzonitrile substrate. In addition, the enzymes and enzymes expressing the enzymes can be used to remove the benzonitrile herbicides from contaminated areas.

Strain E. coli MM 294 (pBrx5) was deposited with ATCC on January 22, 1986 and released by the Release # 53435.

Claims (3)

A nitrilase composition, characterized in that it has about 34kDal with a specific activity of at least about 0.1 μηηοΙ NH ₃ / min / mg protein and bromoxynil as substrate. 2. Composition according to claim 1, characterized in that it has a specific activity of at least 0.5 μητιοΙ NH ₃ / min / mg protein. 3. Use of the nitrilase composition according to one of claims 1 to 2, characterized in that it is used for the detoxification of benzonitrile herbicides contaminated areas, for the protection of plants against the cytotoxic effects of bromoxynil and for the detection of benzonitriles and / or other analogous products becomes.