FUNGAL RESISTANCE MARKERS

Abstract: A drug resistant strain of P.chrysogenum has said drug resistance conferred by heterologuns DNA, in particular a heterologous gene(s) under the control of a Penicillium control sequence(s). Drug resistance is especially to sulpho­ nomides, or to trimethoprim or methotrexate by a gene derived from plasmid R388. A vector capable of conferring resistance to methotrexate or sulphonamide on P.chry­ sogenum, comprises at least one said resistance gene under the control of trpC controlling sequences.

Description: The present invention relates to drug-resistance genes useful as selection markers for P.chrysogenum. More precisely the present invention relates to a drug resistant strain of Penicilium chrysogenum wherein the said drug resistance is conferred by a segment of heterologous DNA capable of conferring resistance to sulfonamide, trimethoprim or methotrexate. Although transformation in P.chrysogenum with auxotrophic markers has previously been reported (Sanchez, F., et al "Transformation in P.Chrysogenum. " Gene 51: 97-107 (1987) ), dominant resistance markers (universal for all strains) not requiring any particular mutation of the host strain, have not so far been described. Sulfonamides (e.g. sulfamethoxazole) are structural analogues of p-aminobenzoic acid. As far as is known, they act as drugs by reducing the amount of synthesis of functional dihydrofolate. Sulfonamides are active against bacteria, yeasts, protozoa and plants but not mammals, as folic acid is a vitamin requirement for mammals. Methotrexate (MTX) and trimethoprim (Tp), structural analogues of dihydrofolic acid, inhibit the action of the enzyme dihydrofolic acid reductase (DHFR). This enzyme catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid (THF). THF is a coenzyme in 1-carbon transfer reactions (biosynthesis of purines, vitamins and amino acids) but, from the pharmacological viewpoint, its most important function is in the reaction whereby deoxyuridylate (dUMP) is converted to deoxythymidylate (dTMP). In this latter reaction THF acts not only as a coenzyme but also as a reductant, being oxidized to DHF, and THF must be regenerated via the DHFR-catalyzed reaction. Thus inhibition of DHFR leads then to a deficiency of dTMP and, accordingly, to inhibition of DNA synthesis. Although MTX and Tp both inhibit DHFR, Tp has a several fold lower affinity for eukaryotic as opposed to bacterial DHFR, so MTX is the drug of choice for the inhibition of eukaryotic DHFR. Plasmid mediated resistance to sulfonamides is widespread and in some instances this resistance is due to a new dihydropteroate synthetase specified by the plasmid (e.g. plasmid R388 - Svedberg. G. et al "Plasmid-borne sulfonamide resistance determinants studied by restriction enzyme analysis." J. Bacteriol. 153 : 1228-1237 (1983)). At least two varieties of plasmid-specified DHFR have been described (Fling, M.E., et al " Monitoring of plasmid-encoded, trimethoprim-resistant dihydrofolate reductase genes: detection of a new resistant enzyme." Antimicrob. Agents Chemother. 22 : 882-888 (1982) ), accounting for Tp/MTX resistances. Type II DHFRs are essentially totally insensitive to Tp or MTX. Plasmid R388 specifies type II DHFR. Resistance to MTX has been used as a marker for vectors in eukaryotic organisms (yeast, Drosophila cells, mammalian cells). In several cases the DHFR gene used is the one present in pR388 where the original bacterial promoter has been removed and substituted by a promoter homologous to the eukaryotic organism used (Miyajima, A., et al . " Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae." Mol. Cell. Biol. 4 : 407-414 (1984). Bourois, M. et al . "Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance." EMBO J. 2 : 1099-1104 (1983). O'Hare, K., et al . "Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expressing a prokaryotic dihydrofolate reductase." Proc. Natl. Acad. Sci. USA 78 : 1527-1531 (1981) ). P.chrysogenum is an industrially important filamentous fungus of the ascomycetes family, and is used in particular in the commercial production of penicillins. Much research has been directed towards obtaining high-yielding strains by classical mutagenesis and brute-force selection. It has now been found that it is possible to apply the techniques of molecular cloning to P.chrysogenum by the introduction of drug-resistance markers into the P.chrysogenum chromosome, or as satellite DNA. Thus, in a first aspect of the present invention, there is provided a drug resistant strain of P.chrysogenum wherein the said drug resistance is conferred by heterologous DNA, and a method of transformation using said resistance. In the co-pending application (DK 515/87; EP 87300916.1; JP 21653/1987; US 009713) examples of auxotrophic markers are given for P.chrysogenum , and the text thereof is incorporated herein by reference. Particularly, strains of E. coli containing the trpC gene have been deposited under the Budapest Treaty with the National Collection of Industrial Bateria, Scotland, on 14 March, 1986, and have the deposit numbers NCIB 12222 and NCIB 12223. These deposits correspond to pPctrpC1 and pPctrpC6, as described herein, and it should be appreciated that references to these plasmids include references to corresponding plasmids and derivatives as described in the co-pending application. Using promoters and terminators associated with P.chrysogenum , such as are present in pPctrpC1 and pPctrpC6, it is possible to use exogenous DNA conferring drug resistance on other organisms, in P.chrysogenum . Thus, resistance to any desired drug can be conferred upon P.chrysogenum by use of homologous control sequences with an appropriate exogenous resistance gene. For example, it has been found that the resistance genes of plasmid R388 can be incorporated in vectors for P.chrysogenum to confer resistance to Tp and Su. The sequence of the plasmid R388-dihydrofolate reductase gene has been published (Swift. G., et al "DNA sequence of a plasmid-encoded dihydrofolate reductase." Mol. Gen. 181 : 441-5447 (1981). Zolg. J. W. et al "Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene" Nucl. Acids Res. 9 : 697-710 (1981) ). Details concerning the determination of the plasmid R388-dihydropteroate synthetase gene, substitution of the original bacterial promoters in both genes by a promoter from P.chrysogenum , and frequencies of transformation of the different constructions, are described in the Examples. The present invention will be further illustrated with reference to the following Figures and Examples in which: Figure 1. Shows the construction of an EcoRI cassette fragment containing the structural gene of DHFR from R388. o SaU3AI sites. Δ TaqI sites. Dotted box is open reading frame (ORF) of DHFR. Figure 2. Shows the nucleotide sequence of the EcoRI cassette fragment containing the DHFR gene from R388. ORF of the DHFR gene is boxed. AluI recognition sequences are underlined. Figure 3. Shows a physical map of pPctrpC1. Symbols are as follows: E EcoRI; H HindIII; Ha HaeIII; N NcoI; Ns NsiI; P PstI; S SalI; Sm SmaI; X XbaI. Box is the Sau3AI fragment of P.chrysogenum DNA containing the trpC gene. Dotted box is the trpC ORF. p: trpC promoter. t: trpC terminator. Single line represents pUC13. Figure 4. Shows the construction of pFS1 and pFS2. Light dotted box is the trpC ORF gene, dark dotted box is the Mtx r gene. Enzyme nomenclature and symbols are as in Figure 3. ( ) represents the filled end of the corresponding restriction cut. A AluI. Figure 5. Shows a physical map of the 1.7 Kb BamHI fragment from R388 containing the dihydropteroate synthetase gene. Figure 6. Shows the construction of pML1 and pML2. Light dotted box is the trpC ORF, dark dotted box is Su r gene. Enzyme nomenclature and symbols are as in Figure 3. ( ) represents the filled end of the corresponding restriction cut. EXAMPLES Example 1 Vectors With The R388-Dihydrofolate Reductase Gene Under The Control Of The P.chrysogenum trpC Promoter And Terminator Construction of an EcoRI cassette fragment containing the R388-dihydrofolate reductase gene. The dihydrofolate reductase structural gene from plasmid R388 is 236 bp long, and is contained in a PvuII-EcoRI fragment of 1160 bp (Swift, G., McCarthy, et al "DNA sequence of a plasmid-encoded dihydrofolate reductase." Mol. Gen. Genet. 181: 441-5447 (1981). Zolg, J.W. et al "characterization of a R plasmid-associated. trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene." Nucl. Acids Res. 9 : 697-710 (1981)). In order to obtain a fragment with the complete nucleotide sequence of the structural gene and where most of the 5' upstream sequences had been deleted, there was purified from the PvuII-EcoRI fragment a Sau3AI-EcoRI fragment of 278 bp containing 156 bp of the structural gene (3' end plus additional 122 bp) and the adjacent TaqI-Sau3AI fragment of 84 bp containing the 5' 80 bp of the structural gene plus an additional 4 bp upstream from the initial ATG. The ligation of the two fragments enabled the reconstitution of the structural gene with TaqI-EcoRI ends. To obtain the cassette EcoRI fragment, two oligonucleotides were synthesized that, when reannealed, had EcoRI-TaqI ends. pUC13, digested with EcoRI and treated with phosphatase was mixed with the two fragments and the reannealed oligonucleotides, and ligase was added. E. coli DH1 competent cells were transformed with the mixture. Clones containing the appropriate construct were selected. Figure 1 shows the construction of this EcoRI cassette, and the sequence is shown in Figure 2. An AluI fragment of the EcoRI cassette. The 378 bp EcoRI fragment was digested with AluI (see Fig. 2). A 284 bp fragment was separated by electrophoresis on polyacrylamide (PAA) gel and purified by extraction with 0.5 M ammonium acetate 1mM EDTA. P.chyrsogenum trpC promoter and terminator. The coding sequence of the P.chrysogenum trpC gene has been published (Sänchez. F. et al "The complete nucleotide sequence of the trpC gene from P.chrysogenum " Nucl. Acids Res. 15 1874 (1987). Plasmid pPctrpC1 (Sänchez, F., et al "Molecular cloning and characterization of the trpC gene from P.chrysogenum ", Mol. Gen. Genet. 205: 248-252 (1986)) contains 350 bp upstream from the initial ATG of the trpC gene and 289 bp downstream from the stop codon. Figure 3 shows a map of pPctrpC1 where the relevant characteristics of this construct are shown. Plasmid pPctrpC1 has been deposited (NCIB 12222). There is a single HaeIII restriction site in the 5' 350 bp (other HaeIII sites are placed in pUC13, trpC gene and trpC terminator sequences), situated 2 bp upstream from the initial ATG. To obtain a fragment containing the promoter sequence of this gene an EcoRI-HaeIII fragment of 346 bp was purified from pPctrpCl. A unique NsiI recognition sequence is present in pPctrpC1, 16 bp downstream from the stop codon. Plasmid pPctrpC1 was digested with NsiI, the sticky ends converted to blunt ends by T4 DNA polymerase digestion and the linearized plasmid digested with EcoRI. Two fragments were thus obtained, one containing the promoter and trpC gene (2.6 Kb) and the other (3Kb) containing the original vector (pUC13) plus the terminator sequence of the trpC gene. This latter fragment was separated from the 2.6 Kb by PAA gel electrophoresis and purified by extraction with 0.5M ammonium acetate 1mM EDTA. Ligation of promoter, terminator and gene: vector pFS1. Fragments of the Mtx r gene (284 bp), trpC promoter (350 bp) and pUC13-trpC terminator (3 Kb) were mixed in a 1:1:1 molar ratio and treated with T4 DNA ligase. E. coli DH1 competent cells were transformed with the mixture and clones containing the appropriate sequence in the appropriate orientation were selected. The plasmid from one of these clones was named pFS1. Figure 4 shows the construction and map of pFS1. A vector containing the trpC and Mtx r markers: pFS2. A SmaI-XbaI fragment containing the sequence trpC promoter-Mtx r gene-trpC terminator was obtained from pFS1 by digestion with the relevant enzymes. The XbaI sticky end was blunted, using Klenow polymerase, and the said fragment was ligated to SmaI-digested pPctrpC1. E. coli DH1 competent cells were transformed with the Oixture and clones containing inserts were selected. All the clones analyzed contained the Mtx r gene with the same direction of transcription as the trpC gene. The plasmid from one of these clones was named pFS2. The construction and map of pFS2 are shown in Figure 4. Example 2 Vectors With The R388-Dihydropteroate Synthetase Gene Under The Control of the P.chrysogenum trpC Promoter and Terminator. Sequence of the plasmid R388 dihydropteroate synthetase gene, and a DNA fragment containing the same. Plasmid R388 contains the dihydropteroate synthetase gene in a BamHI fragment of 1.7 Kb (Ward, J.M. et al "Physical and genetic analysis of the Inc-W group plasmids R388, Sa and R7K." Plasmid 7: 239-250 (1982)). Analysis by subcloning of various subfragments expressing or not expressing sulphonamide resistance in E. coli , together with partial nucleotide sequence, indicate that the putative structural gene is located between a NcoI and an AvaI restriction site (Figure 5). The larger NcoI-BamHI fragment of 1.15 Kb was used in subsequent constructs as the structural dihydropteroate synthetase gene. P.chrysogenum trpC promoter and terminator. -pPctrpC1 contains a single NcoI restriction site (CCATGG) overlapping the initial ATG of the trpC gene see Figure 3, (Sánchez. F. et al "The complete nucleotide sequence of the trpC gene from P.chrysogenum " Nucl. Acids Res. 15 1874 (1987)). It also contains a unique NsiI site 16 bp downstream from the stop codon of the gene. Thus digestion of pPctrpC1 with NsiI (and converting the sticky ends to blunt ends using T4 DNA polymerase) followed by digestion with NcoI and purification of the larger fragment (3.35 Kb) resulted in the original vector pUC13 being linked to the promoter and terminator of the trpC gene. Ligation of promoter, terminator and gene: pML1. The fragment thus obtained was ligated to the NcoI-BamHI fragment of the gene (where the BamHI sticky end was converted to a blunt end by Klenow polymerase). E. coli DH1 competent cells were transformed with the mixture and clones containing the construct were selected. Plasmid from one of these clones was purified and named pML1. The construction of pML1 and its map are shown in Figure 6. A vector containing the trpC and Su r markers: pML2. A smaI-SalI fragment containing the sequence trpC promoter -Su r gen- trpC terminator. The SalI sticky end was converted to a blunt end and the fragment was ligated to SmaI-digested pPctrpCl. E. coli DH1 competent cells were transformed with the mixture and clones containing the desired construct were selected. All the clones analyzed contained the Su r gene with the same direction of transcription as the trpC gene. The plasmid from one of these clones was named pML2. Figure 6 shows the construction and map of pML2. Example 3 Transformation of P.chrysogenum trp2 to MTX Resistance. P.chrysogenum trp2 has been deposited at the Commonwealth Mycological Institute under deposit number CMICC 3027092. 50 ml of semidefined medium supplemented with tryptophan (250 µg/ml) and indole (50 µg/ml) were inoculated with 10⁵ spores/ml and incubated at 28°C for 44 h (shaking, 300 rpm). Mycelium was recovered by filtration, washed thoroughly with distilled water and resuspended in 1.2M KCl (10 ml per gram of wet weight). Protoplasts were formed by incubation of this suspension with Novozyme 234 (Novo Enzyme Industries, 20 mg per g of mycelium) at 28°C for 4-5 hours with gentle shaking. Protoplasts were separated from mycelial debris by filtration through glass-wool, pelleted by low-speed centrifugation (10 min, 800g), washed with 1.2M KCl and finally resuspended in 0.5 ml of 1.2M KCl per gram of initial wet weight. At this point protoplasts were counted under the microscope and appropriate dilutions plated in MM supplemented with tryptophan (250 µg/ml) and indole (50 µ/ml) and in the same medium containing the osmotic stabilizer (1.2M KCl). For transformation, protoplasts were pelleted by low-speed centrifugation, resuspended (10⁹ protoplasts/ml) in 10 mM Tris-HCl, 10 mM CaCl₂, 1.2 M KCl, pH 8.0 and incubated for 10 minutes at 30°C. 0.1 ml samples were mixed with plasmid DNA and 2 ml of 30% PEG 6000, 10 mM CaCl₂, 1.2 M KCl, 10 mM Tris-HCl, pH 8.0 were added and the mixture incubated for 5 minutes at room temperature. After recovery by low-speed centrifugation, the protoplasts were resuspended in 1 ml of 1.2M KCl. The DNA used in these transformation experiments were plasmids pFS1 and pFS2. For plating, samples of treated protoplasts were mixed with 5 ml of osmotically stabilized agar medium (see below) maintained at 50°C, and Petri dishes containing 30 ml of the medium were overlaid with the mixture. When selection was for methotrexate resistance, the drug (2625 µg) was present only in the overlay medium, the amount being calculated for a final concentration of 75 µg/ml in the total volume of medium. Protoplasts transformed with pFS2 were plated on minimal medium containing the osmotic stabilizer (selection for trp⁺ transformants), on minimal medium containing tryptophan, indole, the osmotic stabilizer and Mtx (selection for Mtx r transformants) and on minimal medium containing the osmotic stabilizer and Mtx (selection for double transformants trp⁺, Mtx r ). Trp⁺ transformants appeared at the usual frequency of 50 per µg, but, when these clones were assayed for the expression of Mtx resistance (by streaking on minimal medium containing 75 µg/ml of Mtx) only 4% expressed resistance. Mtx resistant transformants or double transformants appeared at a frequency of 0.6-6 per µg, consistent with the above result. All the Mtx resistant transformants were concomitantly trp⁺. Protoplasts transformed with pFS1 (lacking portions of the trpC gene) were only selected for Mtx resistance. They appeared at a frequency of 0.6 per µg. Southern blot analysis of total DNA from transformants showed that those clones expressing Mtx resistance contained several copies of the transformant plasmid, integrated in a tandem fashion. Thus pFS1 and pFS2 readily transform P.chrysogenum trp2 to methotrexate resistance. The fact that selection for methothrexate results in several copies of the vector being integrated into the genome, although decreasing the frequency of transformation, means that any sequence inserted in this vector will be present in multiple copies in tranformed clones. pFS1 and pFS2 contain single restriction sites for the enzymes EcoRI, PstI, SmaI and XbaI not affecting any of the markers, making them available as cloning sites. Example 4 Transformation of P.chrysogenum ATCC 10003 to MTX Resistance . Protoplasts of the strain P.chrysogenum ATCC 10003 were prepared as described for the P.chrysogenum trp2 strain (Example 3) except that indole and tryptophan were omitted from the incubation medium. The DNA used in transformation experiments were pFS1 and pFS2. Selection for Mtx resistance was carried out by plating protoplasts on minimal medium containing the osmotic stabilizer and Mtx to give a final concentration of 75 µg/ml. Mtx resistant transformants appeared at a frequency of 1 per µg. Thus vectors pFS1 and pFS2, in addition to the advantages indicated in Example 3, are able to transform a wild type strain of P.chrysogenum to Mtx resistance and, accordingly any P.chrysogenum strain sensitive to Mtx. Example 5 Transformation of P.chrysogenum trp2 to SU Resistance . Protoplasts were prepared as described in Example 3, and transformed with pML2. For plating, samples of treated protoplasts were mixed with 5 ml of osmotically stabilized agar medium (see below) maintained at 50°C, and Petri dishes containing 30 ml of the medium were overlaid with the mixture. When selection was for sulfamethoxazole resistance, the drug (70 mg) was present only in the overlay medium, at a final concentration of 2mg/ml of total volume. Selection was for trp⁺ phenotype (by plating on minimal medium plus the osmotic stabilizer), for sulfamethoxazole resistance (by plating on minimal medium containing indole, tryptophan, the osmotic stabilizer and sulfamethoxazole) and for trp⁺ and sulfamethoxazole resistance (by plating on minimal medium containing the osmotic stabilizer and sulfamethoxazole). Trp⁺ clones appeared at the usual frequency of 50 per µg. When these clones were assayed for the expression of sulfamethoxazole resistance (by streaking on minimal medium containing sulfamethoxazole at a concentration of 2mg/ml) about 35% expressed the resistance. Su resistant transformants, or double transformants, appeared at a frequency of 10-15 per µg, consistent with the above result. All the sulfamethoxazole resistant transformants were concomitantly trp⁺. Protoplasts transformed with plasmid pML1 (lacking portions of the trpC gene) were only selected for Su resistance. They appeared at a frequency of 5 per µg. Southern blot analysis of total DNA transformants showed that those clones expressing sulfamethoxazole resistance contained the tranformant plasmid. Thus pML1 and pML2 readily transform P.chrysogenum trp2 to sulfamethoxazole resistance. pML1 and pM12 contain single restriction sites for the enzymes EcoRI and SmaI that do not affect any of the markers, leaving them available as cloning sites. Example 6 Transformation of P. Chrysogenum ATCC 10003 to SU Resistance . Protoplasts were prepared as described in Example 4. The DNA used in transformation experiments were pML1 and pML2. Selection for sulfamethoxazole resistance was carried out by plating on minimal medium containing the osmotic stabilizer plus sulfamethoxazole at a concentration of 2 mg/ml. Clones expressing the resistance gene appeared at a frequency of 0.5 per µg. Thus pM11 and pML2, in addition to the advantages indicated in Example 5, are able to transform a wild type strain of P.chrysogenum to sulfamethoxazole resistance and accordingly, any P.chrysogenum strain sensitive to sulfamethoxazole. The plasmids were deposited at the National Collection of Industrial Bacteria in strains of E.coli DH1 on 23 March 1987 as follows:- Strain AM949 containing p FS1 : NCIB 12436 Strain AM950 containing p FS2 : NCIB 12437 Strain AM951 containing p ML1 : NCIB 12438 Strain AM952 containing p ML2 : NCIB 12439.

Claims: A drug resistant strain of Penicilium chrysogenum wherein the said drug resistance is conferred by a segment of heterologous DNA capable of conferring resistance to sulfonamide, trimethoprim or methotrexate. A strain according to claim 1 wherein the said DNA is a heterologous gene, or genes, operably linked to a Penicilium chrysogenum promoter region. A strain according to claim 2 wherein the said promoter region has identifying characteristics of promoter regions controlling the expression of a trpC gene. A strain according to claim 3 wherein the trpC gene is a trpC gene in pPctrpC1 (NCIB-12222) or pPctrpC6 (NCIB-12223). A strain according to any of claims 1 to 4 wherein the resistance gene is derived from plasmid R388. A strain according to any of claims 1 to 4 wherein drug resistance is conferred by more than one segment of heterologous DNA each conferring resistance to any one of the drugs sulfonamide, trimethoprim, or methotrexate. A vector capable of conferring resistance to Penicilloum chrysogenum comprising at least one methotrexate, trimethoprim or sulfonamide resistance gene operably linked to a trpC promoter region. A plasmid selected from the group consisting of: pFS1, NCLB 12346 pFS2, NCLB 12347 pML1, NCLB 12348, and pML2, NCLB 12349. A strain of Penicillium chrysogenum transformed with any of the vectors of claims 7 or 8. A method of conferring drug resistance to Penicillium chrysogenum comprising conferring resistance to the drug sulfonamide, trimethoprim or methotrexate to Penicillium chrysogenum , where the drug resistance is conferred through transformation with a heterologous segment of DNA encoding the drug resistance as a dominant marker.

Inventors: CARRAMOLINO-FITERA, LAURA, ORTEGA, AGUSTIN PEREZ ARANDA, SANCHEZ, FLORENTINA SANCHEZ, SUSAN, VICTOR RUBIO
Applicants: ANTIBIOTICOS, S.A.
Application year: 1987
Main classification: C12N15
IPC codes: C12N9, C12N15, C12N1, C12R1