MXPA98010517A - Process for the inactivation of genes which code for enzymes for the catabolism of phenyl acetate, plasmids involved in such process and strains transformed therewith - Google Patents
Process for the inactivation of genes which code for enzymes for the catabolism of phenyl acetate, plasmids involved in such process and strains transformed therewithInfo
- Publication number
- MXPA98010517A MXPA98010517A MXPA/A/1998/010517A MX9810517A MXPA98010517A MX PA98010517 A MXPA98010517 A MX PA98010517A MX 9810517 A MX9810517 A MX 9810517A MX PA98010517 A MXPA98010517 A MX PA98010517A
- Authority
- MX
- Mexico
- Prior art keywords
- gene
- nidulans
- leu
- thr
- inactivated
- Prior art date
Links
- WLJVXDMOQOGPHL-UHFFFAOYSA-M phenylacetate Chemical compound [O-]C(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-M 0.000 title claims abstract description 60
- 229940049953 phenylacetate Drugs 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 20
- 230000015556 catabolic process Effects 0.000 title claims abstract description 11
- 230000035569 catabolism Effects 0.000 title claims abstract description 11
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- 229960000626 benzylpenicillin Drugs 0.000 claims abstract description 37
- 229940049954 Penicillin Drugs 0.000 claims abstract description 36
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- HSMNQINEKMPTIC-UHFFFAOYSA-N N-(4-aminobenzoyl)glycine Chemical compound NC1=CC=C(C(=O)NCC(O)=O)C=C1 HSMNQINEKMPTIC-UHFFFAOYSA-N 0.000 claims abstract description 23
- 241000712503 Sicyos pachycarpus Species 0.000 claims abstract description 22
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Abstract
Process for inactivating genes which code for enzymes of the phenyl acetate catabolism, plasmids containing them and strains transformed therewith. The process applies preferably to the gene pahcA of A.nidulans and to the gene pahA of P. chrysogenum, genes which code respectively for competitor enzymes for this substrate with the biosynthetic enzymes of penicillin. The non expression of said enzymes favors the synthesis of this antibiotic, increasing its production, with a smaller demand of phenyl acetate per culture unit.
Description
PROCEDURE OF INACTIVATION OF GENES THAT CODIFY FOR ENZYMES OF CATABOLISM OF PHENYLACETATE, PLASMIDES THAT INTERVENE AND STRAINS TRANSFORMED WITH THEM.
Field of the invention
The present invention is based on the characterization of new DNA compounds and recombinant DNA molecules encoding the enzyme phenylacetate 2-hydroxylase obtained from the filamentous fungi Aspersillus nidulans and Penicillium chrysogenum. The transformation of penicillin-producing strains with the aforementioned DNA compounds allows to increase the production of this antibiotic.
State of the art
The last step of the biosynthetic pathway of benzylpenicillin (penicillin G) in certain filamentous fungi consists in the conversion of isopenicillin N into penicillin G. This involves a transacylation reaction, in which the L-aminoadipa side chain or the Isopenicillin N is replaced by another of phenylacetic acid. The enzyme that catalyzes this reaction (acyl-CoA: isopenicillin N acyltransferase) uses activated phenylacetic acid in the form of a thioester of coenzyme A (CoA) as one of its substrates. Penicillium chrysogenum, the fungus used for the industrial production of penicillin, as5. As Aspergillus niduians, they are unable to synthesize phenylacetate. Therefore, in order to favor the biosynthesis of penicillin G, it is necessary to add an excess of phenylacetate to the industrial cultures of P. c. Rysogrenun. This excess, which prevents the undesired biosynthesis of other penicillins with
REF. 29075
aliphatic side chains, increases the final costs of the fermentation process. A part of the phenylacetate added to the penicillin production cultures can be metabolized. It is a well-established fact that a considerable amount of 2-hydroxyphenylacetate accumulates in the fermentations of P. chrysogenum. This precursor fraction of the side chain obviously does not contribute to the biosynthesis of penicillin G. Both Aspergillus nidulans and P. chrysogenum synthesize penicillin from three precursor amino acids through the same enzymatic steps. Also, like P. chrysogenum, A. nidulans converts a fraction of phenylacetate to 2-hydroxyphenylacetate when the first excess compound is added to the cultures. In addition to converting phenylacetate to 2-hydroxyphenylacetate, A. nidulans can also catabolize the latter compound and, in fact, is able to use phenylacetate as the sole carbon source. The catabolic route in A. nidulans is described in the following scheme:
Phenylacetate * phenylacetate hydroxylase 2-hydroxyphenylacetate "2-hydroxyphenylacetate hydroxylase Ho ogentisate * homogentisate dioxygenase Maleilacetoacetate maleyacetoaceto-isomerase Fumarylacetoacetate ** fumarylacetoacetate hydrolase Fumarate + Acetoacetate
- - The ortho-hydroxylation of phenylacetate is the first step of this route. A second hydroxyphenyl reaction converts 2-hydroxyphenylacetate to 2,5-dihydroxyphenylacetate (= homogentisate). The homogentisate is catabolized through fumarylacetoacetate in fumarate and acetoacetate, which are incorporated in the Krebs cycle (see Fernández Cañón and Peñalva, Proc.Nat.Acid Sci USA 92: 9132-9136, 1995). The ability of the said filamentous fungi to partially or completely catabolize phenylacetate is a detrimental characteristic in the production of penicillin. Therefore, the total or partial elimination of this characteristic would result in strains with improved production capacity of penicillin G. The elimination by classical mutagenesis of this deleterious characteristic (ie, the ability to partially or fully catabolize phenylacetate) in the penicillin-producing fungi would require enormous selection efforts among a large number of strains. On the other hand, said mutagenesis often causes second mutations that could eliminate useful characteristics of the parental strains., generating, for example, auxotrophic mutations or mutations that decrease the vigor of growth or conidiation critical for industrial fermentation. Therefore, it is appropriate to use genetic engineering techniques, lacking the aforementioned limitations, to eliminate the ability to completely or partially catabolize phenylacetate. An essential requirement for performing directed genetic engineering is the cloning and characterization of the gene (s) that mediate the aforementioned harmful characteristic of the penicillin-producing fungi.
Detailed description of the invention
The object of the present invention aims to solve the existing problems of the State of the Art previously listed. It consists of characterizing a fungal gene
which codes for a phenylacetate 2-hydroxylase activity, an enzyme that catalyzes the first enzymatic step of the catabolism of phenylacetate in Aspergillus and Penicillium and its use to eliminate the gene present in the genome of the penicillin-producing fungi by recombinant DNA technology. The present patent describes how this gene is inactivated in a strain constructed by genetic engineering. This strain, which is unable to catabolize phenylacetate, produces higher levels of penicillin than the parental strain. In addition, the maximum penicillin production of this recombinant strain requires a lower excess of phenylacetate than that required by the parental strain. Inactivation of the catabolism of phenylacetate using the above (s) DNA compound (s) results in a significant improvement of penicillin production. The sequence of a 1,986 base pair (bp) genomic DNA fragment of A. nidulans, which includes the new gene used for the present invention is shown in SEQ ID NO: 1. The gene was named phacA (phac by the use of phenylacetate (phenylacetate)) and codes for an enzyme that ortho-hydroxylated phenylacetate (said reaction being the first step of the phenylacetate catabolism in A. nidulans). The nucleotide sequence of the complementary DNA clones (cDNA) covering the entire coding region and its subsequent alignment with the genomic DNA sequence revealed the following characteristics of this gene: The gene codes for a 518 amino acid polypeptide. The first methionine is encoded by an ATG triplet in position 82, while the end codon (TAG) is located in position 1810. These positions correspond to the nucleotide sequence shown in SEQ ID NO: 1 - The coding region is interrupted by three introns of 65, 56 and 53 nucleotides in length (SEQ ID NO: 1).
The deduced polypeptide of 518 residues is shown in the three letter amino acid code, below its corresponding exons in SEQ ID NO: 1 and also, in an isolated context, in SEQ ID NO: 2. The molecular weight of the protein deduced is 58,495 g / mol. A search using the public access server BLAST of the National Center for Biotechnology Information (NCBI, USA) in the databases of protein sequences (for example SwissProt and PIR) and in the conceptual translation of sequence databases DNA (for example the GenBank and? MBL databases) in the six possible reading frames, revealed similarity of the amino acid sequence with members of the cytochrome P450 family (hemo-thiolated protein). These proteins usually intervene in oxidative processes. In fact, the sequences between residues 431 to 439 correspond to the peptide containing the amino acid cysteine Gly-X-Gly-XXX-Cys-X-Gly (where X denotes any amino acid) involved in the binding of the heme group, which it is characteristic of these hemotylated proteins. The new DNA compound, whose structure is described in detail in SEQ ID NO: 1 and which was isolated from a natural microorganism, can be fully synthesized using automatic DNA synthesizers. In addition, due to the degenerate nature of the genetic code, the protein encoded by the new DNA compound could be encoded by alternative DNA sequences. These are included in the present invention. On the other hand, any natural genetic variant derived from the new DNA compound described above is considered equivalent thereto. These genetic variants include homologous genes in organisms closely related to A. nidulans in evolutionary terms, such as P. chrysogenum. These genes can be easily identified using the DNA compound of A. nidulans as a hybridization probe as will be described below.
The Gen. phacA of A. nidulans can be used as a molecular probe for the search of functional homologous genes in genomic or cDNA libraries of other fungal species by hybridization. For example, in the patent pre-sentate its use is described to trace a library of P. chrysogenum, demonstrating that the gene homologous to phacA of A. nidulans can be isolated from industrial strains of P. chrysogenum. The sequence of a 2.558 bp genomic DNA fragment of P. chrysogenum isolated by hybridization with a phacA gene probe is shown in SEQ ID N0: 3. The gene of P. chrysogenum homologous to the phacA of A. nidulans was designated pahA (for phenylacetate h droxilation). The paiifl gene of P. chrysogenum codes for a polypeptide of 516 amino acids that shows 84% identity with the amino acid sequence of PhacA, the protein product of the phacA gene of A. nidulans. The sequence of the pahA gene product of P. chrysogepu-ri is shown in SEQ ID N0: 4. As indicated above, this new DNA compound isolated from P. chrysogenum is also included in the present invention, as well as the other homologous genes that can be isolated from other fungal species by similar procedures. Cloned genes can be used to generate loss-of-function mutations by reverse genetics. For this, a new recombinant DNA molecule can be constructed in an Escherichia coli vector, carrying a truncated version of the fungal gene, which can be used to inactivate the endogenous gene by transformation. For example, this recombinant plasmid can contain the 5 'region of the phacA gene of A. nidulans, followed by a modification in the coding region of the phacA gene, consisting of the 289 bp Nael -Kpnl fragment being replaced by a fragment Xbal of 3.2 kb containing the argB * gene of
TO . nidulans (see Fig. 1). Next, the 3 'region of the phacA gene is arranged. Expression of this mutant p? AcA gene would result in a PhacA protein truncated at residue 297 and, therefore, lacking 221 carboxy-terminal residues. These latter residues, absent in the mutant protein, comprise the aforementioned peptide that includes the Cys residue, which is involved in the binding of heme groups and is essential for the activity of this type of proteins. A linear fragment of DNA containing the aforementioned regions can be separated from the vector sequences by means of suitable restriction enzymes and then purified by standard techniques. This linear fragment can be used for the transformation of an argB strain "from A. nidulans into prototroph for arginine.
As the DNA used in the transformation lacks the sequences required for its autonomous replication, the prototrophic transformants result from the integration of the DNA fragment into the genome. One of the possible modes of integration that would originate an argrB 'phenotype would occur through a double crossing between the linear DNA molecule and the phacA locus residing in the genome of the fungus, as outlined in Figure 1. This double event Cross-linking results in the substitution of the original phacA gene for the truncated gene generated in vitro, resulting in the loss of the phacA function. The transformants carrying said substitution
(ie, with the loss-of-function mutation) can be recognized by their hybridization pattern when their genomic DNA is digested with the appropriate restriction enzymes and analyzed by hybridization using the * Southern technique, using the phacA or phacA genes as probes. argB radioactively labeled with 32P. In this way, the hybridization patterns of other types of transformants can be easily distinguished from those corresponding to a gene substitution. The transformants that are lacking
The function provided by the phacA gene can be purified for subsequent assays using standard techniques. In contrast to the wild type strain, the trans-formants of A. nidulans generated by reverse genetics that lack the function of the phacA gene can not grow in phenylacetate as the sole carbon source. However, they grow in 2-hydroxyphenylacetate or 2,5-dihydroxyphenylacetate. This demonstrates that the new DNA compound (the phacA gene from A. nidulans) codes for an enzymatic activity required for the orthohydroxylation of phenylacetate, an essential step for the use of phenylacetat in A. nidulans. P. chrysogrepum does not use phenylacetate as the sole carbon source but, as previously shown, contains a gene that codes for an enzyme homologue
PhacA. Therefore, the new DNA compound provides a method to eliminate lateral metabolic conversions that can decrease the accumulation of phenylacetate available for penicillin biosynthesis. In P. chrysogenum a methodology similar to that described can be applied to inactivate the pahA gene, using the new DNA compound of P. chrysogenum included in this invention and any of the transformation markers already available for P. chrysogenum, for example the trpC gene. It will also be appreciated that other transformation markers other than argB (in A. nidulans) or trpC (in P. chrysogenum) could be used to interrupt the coding region of the new DNA compound. These include other auxotrophic markers (for example pyrG or riJoB) and antibiotic resistance genes (for example, genes that confer resistance to phleomycin or hygromycin B in fungi). Such methods, basically equivalent to those used here, are also included in the present invention. Figure 2 shows one such method, the
-which can be used to alter the pañA gene of P. chrysogenum. In this case, in the inactivation construct, an internal region of the pahA gene has been replaced by a chimeric gene where the gdh gene promoter of P. chrysogenum (gene encoding the glutamate dehydrogenase enzyme activity) controls the expression of the bacterial gene The plasmid that includes the inactivation construct is called pALP696.Therefore, transformants can be selected for their ability to grow in media containing phleomycin (Kolar, M., Punt, PJ). , van del Hondel, CAMJJ and Schwab, H. Gene 62: 127-134, 1988) Additionally, other strategies can be used to generate, by inverse genetics, loss-of-function mutations in a gene that has been cloned and characterized. example, the transformation with a circular plasmid carrying an internal fragment of the target gene originates, after the homologous integration of a single copy of the transforming DNA, two co incomplete pias of the gene lacking functionality if the fragment included in the plasmid is properly chosen. Methods that use these or other different strategies to generate a mutation of loss of function in the desired gene by means of reverse genetics are also included in this invention. Fungal strains in which the enzymes involved in the modification or catabolism of phenylacetate have been removed show improved penicillin production properties. However, its capacity for growth in the means of production of penicillin is indistinguishable from that of the parental strain. For example, the transformants of A. nidulans with the phacA gene replacement described above (ie, lacking phenylacetate 2-hydroxylase activity) produce 3 to 8 times more penicillin than the parental strain and are less dependent on the amount of added phenylacetate (cf. Figure 3). In this way, while reducing phenylacetate from the
- - 0.125% (w / v) at 0.0625% (w / v) results in a significant decrease in penicillin production levels in the wild strain, transformants lacking the function of the phacA gene produce levels of elevated penicillin, with both concentrations of phenylacetate. Finally, this invention not only concerns the phacA gene and its homologs. We use here the gene coding for phenylacetate 2-hydroxylase, because this enzyme catalyses the first step of the catabolic route of phenylacetate and the production strains of P. chrysogenum secrete significant amounts of 2-hydroxyphenylacetate. Once characterized, other genes involved in the metabolism of phenylacetate could be inactivated using a method similar to that described here. This invention, ie, that the penicillin productivity of fungal organisms can be improved by inactivating individual genes of phenylacetate metabolism by the use of reverse genetics, also covers these alternative possibilities.
Examples
EXAMPLE 1. CLONING AND CHARACTERIZATION OF THE phacA GENE OF A. nidulans.
The cDNA clones corresponding to the phacA gene were isolated by differential selection of a cDNA library, following the steps described below:
a) Obtaining a population of cDNA corresponding to gene transcripts that are preferably expressed when culturing the fungus in the presence of phenylacetate (for use as a "plus" probe in the screening of the library). The A. nidulans A26 strain from the Fungal Genetics Stock Center collection (Department of Microbiology, University of Kansas Medical Center) was grown at 37 ° C
in an adequately supplemented minimum medium containing (in g / 1) KP0, H /, (13.6), (NH,) aSO ,, 2.0; MgSO 4 x 7H., 0, 0.25 and Fe x 7H0, 0.0005, with 0.3% (w / v) glucose as a carbon source. The moment in which the glucose was consumed was determined by analyzing the glucose concentration in the culture medium (using an enzymatic "kit"). This generally occurred at 18 hours of incubation with constant agitation. Two hours later phenylacetate was added to the culture, to a final concentration of 10 mM and the culture was shaken at 37 ° C for 1 hour more in order to induce the expression of the mentioned genes. After this time, the mycelium was collected by filtration, washed with water, frozen in liquid nitrogen, lyophilized and used to isolate total RNA. This total RNA preparation was used to isolate poly (A *) mRNA by affinity chromatography on oligo-dT cellulose. Single-stranded cDNA was prepared in the presence of the reverse transcriptase of the avian myeloblastosis virus (of commercial origin), using as a template 2 μg of said mRNA isolated from mycelium induced with phenylacetate and oligo-dT (15 -mer) as an initiator. The reaction was incubated for 1 hour at 42 ° C in a buffer containing 10 M Tris-HCl pH 8.8 (at 25 ° C), 50 mM KCl, 0.1% Triton X-100, 5 mM MgCl ,, 10 mM of each dNTP and 0.5 units of siRNA. After the synthesis of the first strand, the template RNA was removed by incubation for 1 hour at 60 ° C with 3M NaOH. After neutralization (with acetic acid), the single-stranded DNA was recovered by centrifugation by precipitation with 2.5 volumes of absolute ethanol for 2 hours at -80 ° C. This cDNA was subtracted with a 30-fold excess of poly (A *) RNA isolated from the mycelium collected at the time of glucose depletion, following the procedure deferred by Sargent, T.D., Methods Enzymol. 152: 423-432, 1587. The hybrid cDNA-RNA molecules were separated by hydroxyapatite chromatography at 68 ° C and discarded. The remaining cDNA was hybridized as mentioned, with an excess of poly (A ') RNA from mycelium grown in the absence of phenylacetate. Again the motives were discarded
cDNA-RNA hybrid cells as indicated and the resulting single-chain cDNA population was collected, which represented transcripts of genes expressed preferentially in the presence of phenylacetate. This cDNA was uniformly marked with [a "JP] dCTP and excess of all other dNTPs, in the presence of the Klenow fragment of DNA polymerase and random sequence hexanucleotides as" primers. "The population obtained from 3aP-labeled cDNAs (specific activity > 10 'cpm / mg) was used as a probe to screen a cDNA library constructed as described in section c).
b) Obtaining a cDNA probe corresponding to genes transcribed in the absence of phenylacetate ("less" probe). Single-stranded cDNA was synthesized and marked with "P as described in section a), but using mRNA from cultured mycelium until the glucose was depleted from the culture medium and incubated for an additional 1 hour at 37 ° C in the absence of phenylacetate. .
c) Construction of a cDNA library in the vector gTlO, using cDNA obtained from mycelium cultivated in the presence of 10 mM phenylacetate as the sole carbon source. To construct this library, the synthesis reaction of the first cDNA chain was carried out as described above. The obtained cDNA-RNA hybrid was converted to double-stranded cDNA by introducing random breaks in the RNA chain with RNAse H, which were used as starter points by DNA polymerase I of Escherichia coli. To add EcoRI ends to this blunt cDNA preparation, synthetic adapters were incubated which had the blunt phosphorylated end and the EcoRI overhang with the double stranded cDNA in the presence of T4 DNA ligase. Next, the EcoRI end cDNAs were purified and phosphorylated with T4 polynucleotide kinase and ATP. The phosphorylated cDNA was mixed with the arms of the vector?
previously digested with EcoRI and dephosphorylated and the mixture was incubated with T4 DNA ligase. Recombinant DNA molecules were packaged in vitro, using commercial packaging extracts. Recombinant phages in which a cDNA insert had inactivated the cl gene (in which the EcoRI site is present) were selected for their lytic phenotype in a strain of E. coli hfl (F-, thi-1, thr-1). , leuB6, lacYl, tonASl, supE44, hflA150, [chr-.:Tnl0].
In this way, a total of 107 recombinant clones were obtained.
d) Scanning of the library. The cDNA library was plated using E. coli strain C600 hfl 'and the lysis plates obtained were transferred in duplicate to nitrocellulose filters. One of the replicates was hybridized with the "plus" cDNA probe (described in a)) and the second replica was hybridized with the "minus" cDNA probe (described in b)). The clones that hybridized with the "plus" probe and did not hybridize with the "minus" probe were then selected and purified.
and; Identification of the cDNA corresponding to the phacA gene. The cDNA inserts were excised from the vector by digestion with the Notl endonuclease, using the Notl cut sites included in the EcoRl adapter. The inserts of the selected clones were subcloned in plasmid pBluescript SK (+) digested with Notl and then sequenced by standard procedures. The sequences of the inserts of cAD? were translated into the six possible reading frames and compared using the BLAST algorithm with the conceptual translation in the six reading frames of the AD sequence databases? of public access GenBank and EMBL or with the protein sequence databases SwissProt and PIR. One of the sequences of the cAD inserts? generated an extensive framework
open reading, not interrupted by termination codons. The deduced amino acid sequence showed a significant identity with amino acid sequences of the proteins of the cytochrome P450 family, which are generally involved in oxidation reactions. As will be demonstrated below (see Example 3), the open reading frame encodes a phenylacetate 2-hydroxylase. This cDNA insert was used to hybridize new cDNA clones corresponding to this gene. These clones were sequenced by both strands and the nucleotide sequence obtained showed a complete open reading frame of 1,554 bp in length (without the translation stop codon) coding for a 518 amino acid polypeptide (SEQ ID NO: 2) . The identity of this sequence with members of the cytochrome P450 family established unambiguously that this deduced protein represents a new member of this family.
f) Cloning of the phacA gene. A genomic DNA library of A. nidulans constructed in the vector? EMBL4 was screened with a cDNA probe labeled with J? P corresponding to the almost complete transcript. The positive clones were purified and their inserts were characterized by digestion with restriction enzymes and hybridization with the aforementioned probe. In this way, the hybridization zone of two contiguous Ba HI fragments of 2.4 and 1.9 kb in length was mapped. In Fig. 4 a restriction map of the genomic region containing these fragments is shown. Said BamHI fragments were subcloned into pBluescript SK (+). The recombinant plasmid containing the 2.4 kb Ba-nf-T fragment was designated pBS-FG4A, while the plasmid carrying the 1.9 kb BamHI fragment was designated pBS-FG4B.
- -EJEMPLO 2. CLONING AND CHARACTERIZATION OF THE PAHA GENE OF P. chrysogepuip, FUNCTIONAL HOMOLOGOUS OF THE PhacA GENE OF A. nidulans.
a) Cloning and determination of the nucleotide sequence of the pahA gene of P. chrysogenum. A genomic library of P. chryssgrenu.7. Wisconsin 54-1255, constructed with cloned Sau3A partial fragments at the BamHI site of? EMBL4, was screened with a radioactively labeled probe (with J2P) consisting of an almost complete length cDNA of the phacA gene. About 12,000 clones were transferred to cellulose filters by standard techniques. They were then hybridized for 24 h at 37 ° C in a buffer containing 50% formamide, 5 x Denhart's solution, 5 x SSC, 0.1% SDS, 50 μg / ml of single-stranded herring sperm DNA and 50 ng of the labeled probe. The final wash of the hybridized filters was carried out in 0.2 x SSC, 0.1% SDS at 37 ° C. Through this procedure, 10 clones were purified and their DNA was isolated. Next, the inserts of said clones were characterized by digestion with restriction enzymes and hybridization with the above-mentioned probe. The hybridization region was located on a 2.58 kb XhoJ fragment, whose restriction map is shown in Fig. 5. This DNA fragment was subcloned into the plasmid pBluescript SK (+) giving rise to the plasmid designated pALP520. The nucleotide sequence of the Xhol fragment including the pahA gene (Fig. 5) was determined by the dideoxynucleotide method (Sanger, F., Nicklen, = 5. and Coulson, AR (1977) Proc. Nati. Acad. Sci. USA 74, 5463-5467).
b) Construction of a cDNA library of P. chrysogenum from purified RNA under conditions of penicillin production. Characterization of the cDNA corresponding to the pahA gene.
- - The mycelium from fermentor cultures of an industrial strain of P. chrysogrenuzn, grown under conditions of production of penicillin G, was collected at 100, 124 and 148 hours of incubation and was used to obtain poly (A *) mRNA. with the "Poly (A) Quick mRNA Purification Kit" (Stratagene). This poly (A +) mRNA was used as a template for the construction of a cDNA library using the "ZAP-cDNA Synthesis Kit" (Stratagene) according to the manufacturer's recommendations. The double-stranded cDNA was inserted between the EcoRI and Xhol restriction siof the phage vector? ZAP, placing the 5 'region of the transcripts next to the EcoRl site. The library was packaged with packaging extracts "Gigapack II Gold" (Stratagene) generating around 10"independent clones. The screening of this cDNA library was carried out according to standard procedures (Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2 * ed., Cold Spring Harbor Laboratory Press, New York ), using as probe an EcoRV fragment of 1174 bp internal to the pahA gene (Figure 5). In this way, twelve positive recombinant clones were isolated (denominated from # 1 to # 12) and the clone with the largest insert (approximately 1600 bp) was chosen for its characterization. Next, the complete nucleotide sequence of this clone was determined, observing that it included an open reading frame (ORF) of 1548 bp, which codes for a polypeptide of 516 amino acids with a molecular weight of 58,112 Da and an identity of 84. % with the amino acid sequence of the protein encoded by the phacA gene of A. nidulans. As was the case with its homologous gene of A. nidulans, the "investigation of the databases showed a considerable identity of the PahA protein with members of the P450 protein family.The PahA protein contains the sequence Gly-X-Gly-XXX- Cys-X-Gly (residues 430-438, SEQ ID NO: 4) characterizing this type of protein (see Descrip-
- Detailed Description of this invention). From these results it is concluded that the pahA gene of P. chrysogenum is the homolog of the phacA gene of A. nidulans.
EXAMPLE 3. INACTIVATION OF THE PhacA GENE OF A. nidulans THROUGH REVERSE GENETICS.
The wild allele of the phacA gene from A. nidulans was replaced by a mutated allele, in which part of its coding region, presumably essential for its function (residues 298 to 392 in SEQ ID NO: 1), had been deleted. replaced by the argB 'gene. Therefore, this genetic manipulation truncathe phacA gene in codon 297 generating a null mutated allele. On the other hand, a Kpnl-EcoRI fragment was purified from
1.7 kb, from pBS-FG4B (Figure 4) and subcloned into pUC18 digested with these two enzymes, originating the pUCB plasmid. Next, a Nael fragment was purified from
1.9 kb from pBS-FG4A (Figure 4) and was cloned into pBluescript SK (+) digested with Smal. A plasmid was selected in which the coding strand of the incomplete phacA gene (starting at the Nael site) was in the same orientation as the gene coding for β-galactosidase in the vector. This plasmid was called pBSA. A DNA fragment of Xbal-HindIII was purified from pBSA and inserted into pUCB digested with Xbal-HindIII, originating the pUCA-B plasmid. Finally, a 3.2 kb DNA fragment, which contained the argB 'gene of A, was inserted. nidulans in the unique Xbal site of pUCA-B, to give pPh'acA:: argB '. Starting from the lacZ promoter of pUC18, pPhacA :: argB includes a 0.94 kb fragment of the 5 'region of the phacA gene, followed sequentially by the first 297 codons of its genomic sequence, the 3.2 kb fragment that contains the argB gene, the genomic sequence
- -of the phacA gene corresponding to codons 393-518 and 1, 2 kb from the 3 'region of the phacA gene. A linear fragment containing all these regions can be purified from the plasmid by EcoRI digestion (Figure 6). Likewise, a strain of A. nidulans that includes a disruption-deletion mutation in the phacA gene can be constructed by transformation using this linear fragment of EcoRI and following the strategy summarized in the scheme described above. For this, protoplasts of the A strain were transformed. nidulans biAl, methGl, argB2, with 2 μg of said DNA fragment, using the protocol of Tilburn, J., Scazzocchio, C, Taylor, G.G., Zabicky-Zissman, J.H., Lockington, R.A. and Davies, R.W. (1983) Gene 26: 205-211. The transformants were selected by arginine prototrophy using an appropriate selective medium and then purified. DNA was isolated from the mycelium corresponding to a series of transformants, digested with PstI and analyzed by Southern hybridization, using probes specific for the phacA and argB genes. Several transformants showed the expected hybridization pattern for the double crossover event shown in Figure 1. For example, using the 0.9 kb PstJ fragment of the phacA gene as a probe (see Fig. 4), found that the 0.9 kb PstI individual hybridization band of the recipient strain had been replaced by a 3.8 kb band in the transformants exhibiting the type of integration shown in Figure 1. This band also hybridized with the argB probe. Two of these transformants were chosen for further characterization and were designated AphacA # 1 and AphacA # 2. These transformed strains had the capacity to grow in culture media with phenylalanine, 2-hydroxy-phenylacetate or 2,5-dihydroxyphenylacetate as the sole carbon source, but on the contrary, they did not grow in culture media with phenylacetate as the sole carbon source. This
supports the conclusion that the cytochrome P450 enzyme encoded by the phacA gene of A. nidulans (and by extension the one encoded by the pahA gene of P. chrysogenu-p) possesses an activity that hydroxylates phenylacetate to 2-hydroxyphenylacetate, catalysing this activity the first step of the phenylacetate pathway in A. nidulans (see State of
Technique) . The strain of A. nidulans AphacA # 1, which was phenotypically indistinguishable from the AphacA # 2 strain, has been deposited in the Spanish Type Culture Collection (CECT), University of Valencia, Research Building, Burjasot Campus, 46100, Valencia, dated June 19, 1996, as A. nidulans biAl veAl methGl argB2 phacA :: [pPhacA :: argB "] with the accession number CECT 20195. Obtaining transformants of P. chrysogenum with the inactivated pahA gene is very evident to any expert in the art. the pahA gene of P. chrysogenum by hybridization with the phacA gene of A. nidulans present in the transformant CECT 20195 or alternatively by hybridization with synthetic oligonucleotides based on SEQ ID No: 1. Subsequently and using the plasmid pALfleo7 (deposited in the Spanish Type Culture Collection (CECT), University of Valencia, Research Building, Burjasot Campus, 46100 Valencia, dated February 20, 1997, as CECT 4849) the plasmid pALP696 would be constructed as indicated in figure 7 of the present patent.
EXAMPLE 4. TEST OF THE INACTIVATION OF THE FUNCTION OF THE PhacA GENE AND OF THE INCREASE IN THE PRODUCTION OF PENICILLIN.
To check if eliminating the phenylacetate 2-hydroxylase activity encoded by the phacA gene improved penicillin production was achieved, experiments were performed to measure the levels of penicillin produced by the
strain? phacA compared to a phacA strain. Both strains were compared by fermentation in a flask in a minimum medium containing 2.5% (w / v) lactose as the main carbon source and 2.5% (w / v) solid corn steep, with different amounts of phenylacetate sodium (in w / v) as indicated. The cultures were inoculated in all cases with an equal number of viable conidiospores (2 x loVml) and incubated at 37 ° C with orbital shaking (250 r.p.m.). Samples were taken at different times, filtered through Miracloth and the supernatants were used to measure the penicillin produced by bioassay. For the bioassay, 1 ml of a Microcccus luteus culture grown to a DO * 00 »6 was mixed with 1 1 of the Antibiotic No. 1 medium (Difco) at 50 ° C. This mixture was poured into 13.6 cm Petri dishes (65 ml / plate). In wells of 8 mm in diameter, 100 μl samples were placed corresponding to the supernatants of the cultures (suitably diluted in 10 mM sodium phosphate buffer, pH 6.8). The plates were incubated for 2 hours at 4 ° C and then for another 22 h at 37 ° C. Subsequently the diameter of the bacterial growth inhibition halos originated by the penicillin present in the samples was measured and the amount of antibiotic was estimated comparing with a calibration line made with different amounts of potassium penicillin G. Figure 3 shows that the highest levels of penicillin corresponding to the control strain phacA 'were obtained at 24 hours using 0.125% phenylacetate in the production medium. The level of production reached at this time was 1.8 μg / ml penicillin. The reduction to half the concentration of phenylacetate reduced the maximum levels of penicillin produced by this strain to
1.1 μg / ml. Figure 3 also shows that, in marked contrast, the highest levels of penicillin in a culture of the
- - AppacAcepa reached 5.6 μg / ml, that is, levels 3.1 times higher than those of its parental strain. In addition, as a result of the inactivation of the phacA gene, the increase in penicillin production was not affected by the reduction of the initial concentration of phenylacetate at 0, 0625%. It is concluded that a disruption-deletion mutation of the phacA gene encoding a phenylacetate 2-hydroxylase activity, performed by means of reverse genetics techniques as outlined in Figure 1, originates at least two advantages in the production of penicillin by A. nidulans: (i) penicillin levels rise significantly and (ii) penicillin production depends to a lesser degree on the external supply of phenylacetate.
Detailed description of the figures.
Figure 1: Strategy used to generate a disruption-deletion mutation in the phacA gene of A. nidulans by homologous recombination. The supposed crossings are shown with large crosses in bold. Figure 2: Strategy used to generate a disruption-deletion mutation in the pahA gene of P. chrysogenum by homologous recombination. The supposed cross-links are shown with large discontinuous crosses. Figure 3. Production of penicillin from a transformed strain phacA :: argB of A. nidulans compared to a wild-type strain (phacA *). Abscissa: time in hours (h); Ordered: production of penicillin. { μg / ml). Continuous line: Phenylacetate concentration of 0.125%; Dashed line: Phenylacetate concentration of 0.0625%. Figure 4: Restriction map of the genomic region that includes the phacA gene of A. nidulans, showing the fragments that were subcloned to generate pFG4A and pFG4B. In dark outline the 3 introns.
- - Figure 5: Restriction map of the genomic region that includes the pahA gene of P. chrysogenu-p, showing the 2.55 kb Xhol fragment that was subcloned to generate pALP520. In darker stroke the 3 introns. Figure 6: Restriction map and relevant characteristics of the inactivation construction pPhacA:: argB *. In dark outline the sequencing region that includes the phacA gene;
In white line the flanking areas anterior and posterior to the phacA gene; In striped line the region argB *. Figure 7: Restriction map and relevant characteristics of the inactivation construction pALP696. In dark outline the sequencing region that includes the pahA gene (1745 bp); In white line the flanking areas before and after the pahA gene (4.143 bp); In line marked the region of resistance to bleomycin, gen ble * (2048 bp).
LIST OF SEQUENCES
GENERAL INFORMATION:
APPLICANT: NAME: ANTIBIOTICS, S.A.U. STREET: Avda. de Burgos, 8-A CITY: Madrid STATE OR PROVINCE: Madrid COUNTRY.- Spain POSTAL CODE: 28036 TELEPHONE: 91-3841200 FACSIMILE: 91-3841220
TITLE: "PROCEDURE OF INACTIVATION OF GENES THAT CODIFY FOR ENZYMES OF CATABOLISM OF PHENYLACETATE, PLASMIDES THAT INTERVENE AND STRAINS TRANSFORMED WITH THEM".
NUMBER OF SEQUENCES: 4
ADDRESS FOR CORRESPONDENCE: RECIPIENT: ANTIBIOTICS, S.A.U. STREET: Avda. De Burgos, 8-A CITY: Madrid STATE OR PROVINCE: Madrid COUNTRY: Spain POSTAL CODE: 28036
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INFORMATION ABOUT THE LAWYER / AGENT: NAME: ALBERTO DE ELZABURU REGISTRATION NUMBER: 232/1 REFERENCE NUMBER / REGISTRATION: PCT-42
INFORMATION ABOUT TELECOMMUNICATIONS: TELEPHONE: 91 7009400 FACSIMILE: 91 3193810 TELEX OR EMAIL: elzaburu@elzaburu.es
INFORMATION FOR SEQ ID NO .: 1
CHARACTERISTICS OF THE SEQUENCE: LENGTH: 1986 base pairs TYPE: nucleotides NUMBER OF HEBRAS: 2 CONFIGURATION: linear TYPE OF MOLECULE: genomic DNA HYPOTHETICAL: NO ANTI-SENSE: NO SOURCE OF ORIGIN: Aspergillus nidulans IMMEDIATE SOURCE: plasmid pPhacA:: argB * POSITION IN THE GENOME: unknown CHARACTERISTICS: NAME / KEY: coding sequence SITUATION: 82..1810 OTHER INFORMATION: phacA gene
DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1
C CAGCCAGATA TAAAGGCCGT 21 CCTTAGACGA CTTGATCTTA CTCTGGTTTC TAAAAGTTCA CTGATTATCG CAAGGTATCC 81 ATG TCT CTT CAA ACA ATC GGG ATC GCC GCT GTC GCG GTG GTC TAT TTT 129 Met Ser Leu Gln Thr lie Gly He Ala Ala Ala Ala Val Val Tyr Phe 5 10 15
CTC ATC CGC TAC TTC AAC CGC ACA GAC ATC CCA AAG ATC AAG GGT CTC 177 Leu He Arg Tyr Phe Asn Arg Thr Asp He Pro Lys He Lys Gly Leu 20 25 30 CCT GAA GTT CCA GGC GTA CCG ATC TTT GGC AAC CTC ATC CAG CTC GGT 225 Pro Glu Val Pro Gly Val Pro He Phe Gly Asn Leu He Gln Leu Gly 35 40 45 GAC CAG CAC GCG ACC GTA GCG CAG AAA TGG GCG AAG AAA TTT GGA CCT 273 Asp Gln His Wing Thr Val Wing Gln Lys Trp Wing Lys Lys Phe Gly Pro 50 55 60 GTT TTC CAG GTT CGC ATG GGG AAT AAA GTGAGTTCAG TGTCTGCATT TGTAAC 326 Val Phe Gln Val Arg Met Gly Asn Lys 55 70 AGAC GACAATATTG CGAGAATAAT TCTGACCTAA CACAG CGC GTT GTC TTC GCA 380 Arg Val Val Phe Wing 75 AAC ACC TTC GAC TCT GTC CGT CAG CTA TGG ATC AAA GAT CAG TCC GCG 428 Asn Thr Phe Asp Ser Val Arg Gln Leu Trp He Lys Asp Gln Ser Wing 80 85 90 CTC ATC TCC CGG CCG ACC TTT CAC ACC TTC CAC AGT GTA GTT TCC AGC 476 Leu He Ser Arg Pro Thr Phe His Thr Phe His Ser Val Val Ser Ser 95 100 105 110 TCT CAG GGA TTC ACC ATC GGA ACG TCG CCG TGG GAC GAG TCG TGC AAG 524 Ser Gln Gly Phe Thr He Gly Thr Ser Pro Trp Asp Glu Ser Cys Lys 115 120 125 CGT CGT CGG AAG GCT GCA GCC ACA GCC TTG AAC CGC CCG GCT ACC CAG 572 Arg Arg Arg Lys Ala Ala Ala Thr Ala Leu Asn Arg Pro Ala Thr Gln 130 135 140 TCG TAT ATG CCT ATT ATC GAT CTT GAG TCG ATG TCG AGT ATC CGG GAA 620 Ser Tyr Met Pro He He Asp Leu Glu Ser Met Ser Ser He Arg Glu 145 150 155 TTG CTC AGG GAT AGC GCG AAT GGA ACA ATG GAT ATC AAC CCG ACA GCG 668 Leu Leu Arg Asp Ser Wing Asn Gly Thr Met Asp He Asn Pro Thr Wing 460 165 170 TAC TTC CAG CGG TTC GCG TTG AAC ACG AGC TTA ACA TTG AAC TAT GGA 716 Tyr Phe Gln Arg Phe Ala Leu Asn Thr Ser Leu Thr Leu Asn Tyr Gly 175 180 185 190
- -
ATC CGA ATC GAG GGC AAT GTG AAC GAT GAG CTT TTG CGC GAA ATT GTC 764 He Arg He Glu Gly Asn Val Asn Aap Glu Leu Aru Glu He Val 195 200 205 GAT GTC GAG CGC GGG GTG TCG AAC TTC CGG AGT ACC AGC AAC CAG TGG 812 Asp Val Glu Arg Gly Val Ser Asn Phe Arg Ser Thr Ser Asn Gln Trp 210 215 220 CAG GAC TAT ATC CCG CTC CTG AGA ATC TTC CCG AAG ATG AAC CGC GAG 860 Gln Asp Tyr He Pro Leu Leu Arg He Phe Pro Lya Met Asn Arg Glu 225 230 235 GCT GAG GAG TTC CGG GTG CGG AGA GAC AAG TAT CTT ACC TAT CTT TTG 908 Wing Glu Glu Phe Arg Val Arg Arg Asp Lys Tyr Leu Thr Tyr Leu Leu 240 245 250 GAT GTT CTC AAG GAT CGC ATT GCA AAG GGA ACC GAC AAG CCC TGT ATT 956 Asp Val Leu Lys Asp Arg He Wing Lys Gly Thr Asp Lys Pro Cys He 255 260 265 270 ACT GGA AAC ATC CTC AAA GAC CCT GAG GCT AAG CTC AAT GAT G GTATG 1004 Thr Gly Asn He Leu Lys Asp Pro Glu Wing Lys Leu Asn Asp 275 280 CCGTC CGGTCCTGTC CGCsCTTGAA GGAAAATAAA OATTAACAGA GTCTAG CC GAG 1060 Wing Glu 285 ATT AAA TCG ATC TGC TTG ACC ATG GTC TCC GCC GGC CTC GAT ACT GTT 1108 He Lys Ser He Cys Leu Thr Met Val Be Wing Gly Leu Asp Thr Val 290 295 300 CCG GGC AAC TTG ATC ATG GGC ATC GCG TAC CTC GCC TCC GAA GAC GGC 1156 Pro Gly Asn Leu He Met Gly He Ala Tyr Leu Wing Ser Glu Asp Gly 305 310 315 CAA AGG ATC CAG AAG CGC GCC CAC GAC GAG ATC ATG AAA GTC TAC CCG 1204 Gln Arg He Gln Lys Arg Wing His Asp Glu He Met Lys Val Tyr Pro 320 325 330 GAC GGC GAT GCA TGG GAG AAA TGC CTG CTC GAA GAG AAA GTC CCC TAC 1252 Asp Gly Asp Wing Trp Glu Lys Cys Leu Leu Glu Glu Lys Val Pro Tyr 335 340 345 350 GTC ACA GCT TTG GTC AAA GAA ACC CTC CGC TTC TGG ACT GTC ATT CCC 1300 Val Thr Ala Leu Val Lys Glu Thr Leu Arg Phe Trp Thr Val He Pro 355 360 365
- ATC TGT CTG CCT AGA GAA AAC ACC AAG GAT ATT GTC TGG AAC GGA GCC 1348 He Cys Leu Pro Arg Glu Asn Thr Lys Asp He Val Trp Asn Gly Wing 370 375 380 GTT ATC CCT AAG GGA ACG ACC TTT TTC ATG AAC GCC TAC GCT GAC GAC 1396 Val He Pro Lys Gly Thr Thr Phe Met Asn Ala Tyr Ala Ala Aßp 385 390 395 TAC GAC GAA ACA CAC TTC ACC AAT CCA CAC GCC TTT GAA CCA GAA CGC 1444 Tyr Asp Glu Thr His Phe Thr Asn Pro His Wing Phe Glu Pro Glu Arg 400 405 410 TAC CTC ACC GCC TCA TCT GAC GGC TCC GGC ACT CCA CAC TAC GGC TAC 1492 Tyr Leu Thr Wing Being Ser Asp Gly Ser Gly Thr Pro His Tyr Gly Tyr 415 420 425 430 GGC GCG GGC TCG CGC ATG GTACCTCCCC CACAACCCAC ATGTTATATA GACAAT 1546 Gly Wing Gly Ser Arg Met 435 ACTG ATTTATTATG AAG TGC GCT GGC TCG CAT CTT GCG AAC CGC GAG CTC 1596 Cys Wing Gly Ser His Leu Wing Asn Arg Glu Leu 440 445 TTC ACC GCT TAC GTC CGT • CTG ATC ACA GCA TTT ACC ATG CAT CCA GCT 1644 Phe Thr Wing Tyr Val Arg Leu He Thr Wing Phe Thr Met His Pro Wing 450 455 460 AAG AGG GCT GAG GAT CGG CCG ATC CTC G AT GCG ATT GAG TGT AAT GCG 1692 Lys Arg Wing Glu Asp Arg Pro He Leu Asp Wing He Glu Cys Asn Wing 465 470 475 ATC CCG ACC GCT TTG ACG ACG GAG CCG AAG CCC TTC AAG GTT GGG TTT 1740 He Pro Thr Ala Leu Thr Thr Glu Pro Lys Pro Phe Lys Val Gly Phe 480 485 490 495 AAA CCG AGA GAT CCT GTC TTG GTA AGG AAA TGG ATT GCG GAG AGT GAG 1788 Lys Pro Arg Asp Pro Val Leu Val Arg Lys Trp He Wing Glu Ser Glu 500 505 510 GAG AGG ACG AAG CAC CTG AAT TAGTTTTTCT TTTCTTTCTT GCGTGCAAGT TAC 1842 Glu Arg Thr Lys His Leu Asn 515 AGCGCAATTT ATACTTAGCT GGGACATGGT CGATTGGAGT ATACTGATTT CAGCAACAGC 1902 GCAAATAAAA ATAAGGCAAC CAATAGAAAT GCAACAATAT GCAATCTCCC AAGACCATTA 1962 AATGGTTGAT CAGATGATCC CAAG 1986
INFORMATION FOR THE. SEQ ID NO. : 2
CHARACTERISTICS OF THE SEQUENCE: LENGTH: 518 amino acids TYPE: amino acids NUMBER OF HEBRAS: 1 CONFIGURATION: linear TYPE OF MOLECULE: peptide SOURCE OF ORIGIN: Aspergillus nidulans CHARACTERISTICS: OTHER INFORMATION: amino acid sequence of the enzyme phenylacetate 2 hydroxylase CHARACTERISTIC: OTHER INFORMATION: Weight molecular 58495 Da.
DESCRIPTION OF THE SEQUENCE SEQ ID NO: 2
Met Ser Leu Gln Thr He Gly He Wing Wing Val Wing Val Val Tyr 5 10 15 Phe Leu He Arg Tyr Phe Asn Arg Thr Asp He Pro Lys He Lys 20 25 30 Gly Leu Pro Glu Val Pro Gly Val Pro He Phe Gly Asn Leu He 35 40 45 Gln Leu Gl and Asp Gln His Wing Thr Val Wing Gln Lys Trp Wing Lys 50 55 60 Lys Phe Gly Pro Val Phe Gln Val Arg Met Gly Asn Lys Arg Val 65 70 75 Val Phe Wing Asn Thr Phe Asp Ser Val Arg Gln Leu Trp He Lys 80 85 90 Asp Gln Ser Wing Leu He Ser Arg Pro Thr Phe His Thr Phe His 95 100 105 Val Val Ser Ser Ser Gln Gly Phe Thr He Gly Thr Ser Pro 110 115 120 Trp Asp Glu Be Cys Lys Arg Arg Arg Lys Wing Wing Wing Thr Wing 125 130 135 Leu Asn Arg Pro Wing Thr Gln Ser Tyr Met Pro He He Asp Leu 140 145 150 Glu Ser Met Ser Ser He Arg Glu Leu Leu Arg Asp Ser Ala Asn 155 160 165 Gly Thr Met Asp He Asn Pro Thr Wing Tyr Phe Gln Arg Phe Wing 170 175 180 Leu Asn Thr Ser Leu Thr Leu Asn Tyr Gly He Arg He Glu Gly 185 190 195 Aan Val Asn Asp Glu Leu Leu Arg Glu He Val Asp Val Glu Arg 200 205 210
- Gly Val Ser Asn Phe Arg Ser Thr Ser Asn Gln Trp Gln Asp Tyr 215 220 225
He Pro Leu Leu Arg He Phe Pro Lys Met Asn Arg Glu Wing Glu 2- > J 235 240
Glu Phe Arg Val Arg Arg Asp Lys Tyr Leu Thr Tyr Leu Leu Asp 245 250 255
Val Leu Lys Asp Arg He Wing Lys Gly Thr Asp Lys Pro Cys He 260 265 270
Thr Gly Asn He Leu Lys Asp Pro Glu Wing Lys Leu Asn Asp Wing 275 280 285
Glu He Lys Ser He Cys Leu Thr Met Val Ser Wing Gly Leu Aap 290 295 300
Thr Val Pro Gly Asn Leu He Met Gly He Wing Tyr Leu Wing Ser 305 310 315
Glu Asp Gly Gln Arg He Gln Lys Arg Ala His Asp Glu He Met 320 325 330
Lys Val Tyr Pro Asp Gly Asp Wing Trp Glu Lys Cys Leu Leu Glu 335 340 345
Glu Lys Val Pro Tyr Val Thr Ala Leu Val Ly3 Glu Thr Leu Arg 350 355 360
Phe Trp Thr Val He Pro He Cys Leu Pro Arg Glu Asn Thr Lys 365 370 375
Asp He Val Trp Asn Gly Wing Val He Pro Lys Gly Thr Thr Phe 380 385 390
Phe Met Asn Ala Tyr Ala Ala Asp Tyr Asp Glu Thr His Phe Thr 395 400 405
Asn Pro His Wing Phe Glu Pro Glu Arg Tyr Leu Thr Wing Ser Ser 410 415 420
Asp -Gly Ser Gly Thr Pro His Tyr Gly Tyr Gly Wing Gly Ser Arg 425 430 435
Met Cys Ala Gly Ser His Leu Ala Asn Arg Glu Leu Phe Thr Ala 440 445 450
Tyr Val Arg Leu lie Thr Ala Phe Thr Met His Pro Ala Lys Arg 455 460 465
Wing Glu Asp Arg Pro He Leu Asp Wing He Glu Cys Asn Wing He 470 475 480
Pro Thr Ala Leu Thr Thr Glu Pro Lys Pro Phe Lys Val Gly Phe 485 490 495
Lys Pro Arg Asp Pro Val Leu Val Arg Lys Trp He Ala Glu Ser 500 505 510
Glu Glu Arg Thr Lys His Leu Asn 515
INFORMATION FOR SEQ ID NO .: 3
CHARACTERISTICS OF THE SEQUENCE: * LENGTH: 2558 base pairs TYPE: nucleotides NUMBER OF HEBRAS: 2 CONFIGURATION: linear TYPE OF MOLECULE: Genomic DNA
HYPOTHETICAL: NO ANTI-SENSE: NO SOURCE OF ORIGIN: Penicillium chrysogenum IMMEDIATE SOURCE: plasmid pALP520 POSITION IN THE GENOME: unknown CHARACTERISTICS: NAME / KEY: coding sequence SITUATION: 371. .2094 CHARACTERISTICS: OTHER INFORMATION: gene pahA
DESCRIPTION OF THE SEQUENCE SEQ ID NO: 3
CTCGAGAAGG TCTGTTGACA CGAGTCCACA AGATGTTGCG AAGTATATAA TATGAGAAAT 60 GGATATACAG CGGAAAATTG AAGTTTCATT CCGCGGGGGC GsGGAACAAG AGGAAGCGCG 120 GAATGAACAT TCCGTCTATG CGCGGTGGAG AGATCTTCGA ACTGGGGACT TGTGGACTTG 180 GACCTATGAG AGAGCTGCCT TTACTGATTC TGGGACTTGT GGCCAATGAA ACATGTCATG 240 GATGTTACAG CTTTCTTTAT GCGACTCTCT AAACTCGGAA ATATGCCGTA GCCGAAATGC 300 AGGAAAGGCT GGGCTATAAG AGATGCATGC TCCCACCGAA CGTCTGCAAT TCCTTGTTTC March 60 ACTCTATATC ATG GCC ATC CAA ACA CTC GCC GTC GCC GTG ATC ACG GTG GTC 412 Met Al to H e G ln Thr Leu Ala Val Wing l H e Thr Val Val 5 10 TAT TTC GTC ATT CGA TAC TTC AAC CGC ACT GAT ATC CCT AAG ATT AAA 460
Tyr Phe Val He Arg Tyr Phe Asn Arg Thr Asp He Pro Lys He Lys 15 20 25 30 GGC CTC CCG GAG ATT CCT GGT ATA CCC ATA TTT GGC AAT CTA TTG CAG 508
Gly Leu Pro Glu He Pro Gly He Pro He Phe Gly Asn Leu Leu Gln 35 40 45 CTA GGA GAT CAA CAT GCC ACA GTC ACG GGG AAA TGG GCA AAG AAA TTT 556
Leu Gly Asp Gln His Wing Thr Val Thr Gly Lys Trp Wing Lys Lys Phe 50 55 60 GGC * CCA GTT TTC CAA GTG CGC ATG GGA AAC AAG GTAAGTAATC AAATAATCTT 609 Gl and Pro Val Phe Gln Val Arg Met Gly Asn Lys 65 70
TTAAATCGGA CATTCTGATA ATCTAATGCC CTTTTAG CGC ATC GTG TTC GCC AAC 664 Arg He Val Phe Wing Asn 75 GGC TTT GAC TCC GTT CGT CAA TTG TGG ATT AAG GAC TCG TCG GCT CTG 712 Gly Phe Asp Ser Val Arg Gln Leu Trp He Lys Asp Ser Ser Ala Leu 80 85 90 95 ATC TCC CGC CCA ACT TTC CAC ACT TTC CAC AGC GTC GTC TCC AGT TCA 760 He Ser Arg Pro Thr Phe His Thr Phe His Ser Val Val Ser Ser 100 105 110 CAG GGC TTC ACG ATC GGA ACT TCC CCG TGG GAT GAT TCC TGT AAG AAA 808 Gln Gly Phe Thr He Gly Thr Ser Pro Trp Asp Asp Ser Cys Lys Lys 115 120 125 CGT CGC AAG GCC GCT GCC ACT GCG CTG AAT CGA CCG GCC GTG CAG TCG 856 Arg Arg Lys Ala Ala Ala Thr Ala Leu Asn Arg Pro Wing Val Gln Ser 130"135 140 TAT ATG CCC ATC ATC GAT CTT GAA TCC AAT TCC AGC ATC AAA GAA CTG 904 Tyr Met Pro He He Asp Leu Glu Ser Asn Ser Ser He Lys Glu Leu 145 150 155 TAC CGG GAC AGC CAA AAT GGC AAA CGT GAT GTG AAT CCC ACT GCA TAC 952 Tyr Arg Asp Ser Gln Asn Gly Lys Arg Asp Val Asn Pro Thr Wing Tyr 160 165 170 175 TTC CAG CGA TAC GCT TTC AAC ACC AGT TTG ACT TTG AAC TAT GGA TTC 1000 Phe Gln Arg Tyr Wing Phe Asn Thr Ser Leu Thr Leu Asn Tyr Gly Phe 180 185 190 CGC ATC GAG GGC AAT GTG GAT GAT ACG CTG CTG CAT GAG ATT GTG GAT 1048 Arg He Glu Gly Asn Val Asp Asp Thr Leu Leu His Glu He Val Asp 195 200 205 GTG GAG CGT GGT GTG TCC AAC TTC CGC AGC ACT TCG AAC AAC TGG CAG 1096 Val Glu Arg Gly Val Ser Asn Phe Arg Ser Thr Ser Asn Asn Trp Gln 210 215 220 GAC TAC ATT CCC CTA TTG CGC ATT TTC CCC AAG ATG AAC AAT GAG GCC 1144 Asp * Tvr He Pro Leu Leu Arg He Phe Pro Lys Met Asn Asn Glu Wing 225 230 235 GCT GCC TTC CGG GGT CGC GAT AAA TAT CTO ACC TAC TTG CTC GAT 1192 Wing Asp Phe Arg Gly Arg Arg Asp Lys Tyr Leu Thr Tyr Leu Leu Asp 240 245 250 255C AAG GAT CGA ATC GCC AAG GGA ACC GAT AAG CCT TGC ATC ACT 1240 Met Le Lyß Asp Arg He Wing Lys Gly Thr Asp Lys Pro Cys He Thr 260 265 270 GGT AAT ATC TTG AAG GAT CCC GAG GCT AAG CTG AAT GAT G GTGAGTTACA 1290 Gly Asn He Leu Lys Asp Pro Glu Wing Lys Leu Asn Asp 275 280 AAATCTCGCG AAATCTTTGT GAATGTTGGT ATTGAGTACT CATCTATCGT CTAG CC 1346 Wing 285 GAG GTG AAA TCG ATC TGT TTG ACT ATG GTG TCG GCT GGC CTT GAC ACC 1394 Glu Val Lys Ser He Cys Leu Thr Met Val Be Wing Gly Leu Asp Thr 290 295 300 GTT CCG GGC AAC TTG ATT ATG GGA ATT GCC TAC CTG GCA TCT GAA GAC 1442 Val Pro Gly Asn Leu He Met Gly He Wing Tyr Leu Wing Ser Glu Asp 305 310 315 GGC CAA AGA ATT CAG AAA AAG GCC TAT GAT GCA ATC ATG GAG GTA TAC 1490 Gly Gln Arg He Gln Lys Lys Wing Tyr Asp Wing He Met Glu Val Tyr 320 325 330 ccs GAC ssc GAT GCT TGG GAG AAA TGT TTG GTG GAG GAG AAG GTC CCT 1S38 Pro Asp Gly Asp Wing Trp Glu Lys Cys Leu Val Glu Glu Lys Val Pro 335 340 345 TAT GTG ACG GCA CTG GTC AAG TAG GTC TTG CGC TTC TGG ACG GT C ATT 1586 Tyr Val Thr Ala Leu Val Lys Glu Val Leu Arg Phe Trp Thr Val He 350 355 360 365 CCT ATT TGT CTG CCA CGC GAG AGC ACC AAG GAT ATC CAG TGG AAT GGA 1634 Pro He Cys Leu Pro Arg Glu Ser Thr Lys Asp He Gln Trp Asn Gly 370 375 380 GCT ACA ATC CCT GCC GGG ACG ACC TTT TTC ATG GTATGTTGCG CCTTGCTTCT 1687 Wing Thr He Pro Wing Gly Thr Thr Phe Phe Met 385 390 GTCCCTTTCG GGACGTTGCT AACAGGATCT GATAG AAT GTT TGG GCT GCC GAT 1740 Asn Val Txp Wing Wing Asp 395 TAC GAT GAA GAT CAC TTT AAG GAT GCC GAT AAA TTC ATC CCG GAG CGT 1788 Tyr Asp Glu Asp His Phe Lys Asp Wing Asp Lys Phe He Pro Glu Arg 400 405 410
- -
TAT TTG GAG GCC AGT GAG GGT GCA GGC ACT CCA CAC TAT GCA TAT GGA 1836
Tyr Leu Glu Wing Ser Glu Gly Wing Gly Thr Pro His Tyr Wing Tyr Gly 415 420 425 430 GCG GGA TCA CGT ATG TGT GCA GGC TCA CAT CTC GCC AAT CGC GAG CTG 1884
Wing Gly Ser Arg Met Cys Wing Gly Ser His Leu Wing Asn Arg Glu Leu 435 440 445 TTC ACT GCT TTT ATC CGC CTC GTC ACT GCT TTC AAC ATG CAC ACG GCG 1932 Phe Thr Wing Phe He Arg Leu Val Thr Wing Phe Asn Met His Thr Ala 450 455 460 AAG GAG ACG GCC GAC CGA CCG ATT CTG AAT GCA ATT GAG TGC AAT TTG 1980 Lys Glu Thr Wing Asp Arg Pro He Leu Asn Wing He Glu Cys Asn Leu 465 470 475 ATT CCG ACA GCC TTG ACA ACC GAG CCG AAG CCA TTC AAG GTT GGC TTT 2028 He Pro Thr Ala Leu Thr Thr Glu Pro Lys Pro Phe Lys Val Gly Phe "480 485 490 AGT GCA CGC GAC CCT AAG AAG CTT GAG CAG TGG ATT GCT GAG AGC GAT 2076 Ser Ala Arg Asp Pro Lys Lys Leu Glu Gln Trp He Ala Glu Ser Asp 495 500 505 510 GAA CGG ACC AAA GAT CTA TAAGAGGAAG TTTTATCTGA TATGATTCCC TTTTTTTTTT Glu Arg Thr Lys Asp Leu 515 TCGGAGGCTA TTTATGTATC TACTTGAATA TATGT AAAT AAAGGCAATG AAGTATAGAT 2194 ATAGACCTGC CCAGGTGAGA CCTACATGTA TGTAATCGAC GTCTCCCAGT ACCTATTTAG_2254_GAACCGAATA GAAATTTGAG TGATGAAGGG TOAAGAATAA CCTCAGAGAA CATAGGTCTA 2314 CTAAGATCTG CTAATTCTTA GACTCCTTTC CCTGAATTAT TGCCAATTTC CCCAGTTGTC 2374 TCTCTCCTCT ATGACTCCCG GCCTTGTCAC ACCGGCGGAA TCTCCCCGCA TTTTCCTCGA 2434 CCTCACCCTT TTCTTCCTCT TCACCCTCTC CCCATCCACT TGAAATGAAC CTCTGATCGC 2494 AATCAATTGC ATCCCGAAGT CATTTTGGAT GATGAATAAT CCGCAGATGT ACCCTGTCCT 2554 CGAG 2558
INFORMATION FOR SEQ ID NO. : 4
CHARACTERISTICS OF THE SEQUENCE! LENGTH: 516 base pairs TYPE: amino acids
- NUMBER OF HEBRAS: 1 CONFIGURATION: linear TYPE OF MOLECULE: peptide CHARACTERISTIC: OTHER INFORMATION: amino acid sequence of the enzyme phenylacetate 2 hydroxylase CHARACTERISTIC: OTHER INFORMATION: molecular weight 58112 Da.
DESCRIPTION OF THE SEQUENCE SEQ ID NO: 4
Met Wing He Gln Thr Leu Wing Val Wing Val He Thr Val Val Tyr Phe 1 5 10 15 Val He Arg Tyr Phe Asn Arg Thr Asp He Pro Lys He Lys Gly Leu 20 25 30 Pro Glu He Pro Gly He Pro He Phe Gly Asn Leu Leu Gln Leu Gly 35 40 45 Asp Gln Hi s Thr Wing Thr Val Thr Gly Lys Trp Wing Lys Lys Phe Gly Pro 50 55 60 Val Phe Gln Val Arg Met Gly Asn Lys Arg He Val Phe Wing Asn Gly 65 70 75 80 Phe Asp Ser Val Arg Gln Leu Trp He Lys Asp Ser Be Ala Leu He 85 90 95 Ser Arg Pro Thr Phe His Thr Phe His Ser Val Val Ser Ser Gln 100 105 110 Gly Phe Thr He Gly Thr Ser Pro Trp Asp Asp Ser Cys Lys Lys Arg 115 120 125 Arg Lys Ala Ala Ala Thr Ala Leu Asn Arg Pro Ala Val Gln Ser Tyr 130 135 140 Met Pro He He Asp Leu Glu Ser Asn Ser Ser He Lys Glu Leu Tyr 145 150 155 160
Arg Asp Ser Gln Asn Gly Lys Arg Asp Val Asn Pro Thr Wing Tyr Phe 165 170 175 Gln Arg Tyr Wing Phe Asn Thr Ser Leu Thr Leu Asn Tyr Gly Phe Arg 180 185 190
He Glu Gly Asn Val Asp Asp Thr Leu Leu His Glu He Val Asp Val
195 200 205 Glu Arg Gly Val Ser Asn Phe Arg Ser Thr Ser Asn Asn Trp Gln Asp
210 215 220 Tyr He Pro Leu Leu Arg He Phe Pro Lys Met Asn Asn Glu Ala Wing 225 230 235 240
Asp Phe Arg Gly Arg Arg Asp Lys Tyr Leu Thr Tyr Leu Leu Asp Met 245 250 255
Leu Lys Asp Arg He Wing Lys Gly Thr Asp Lys Pro Cys He Thr Gly
260 265 270 Asn lie Leu Lys Asp Pro Glu Al'a Lys Leu Asn Asp Ala Glu Val Lys
275 280 285 Be He Cys Leu Thr Met Val Ser Wing Gly Leu Asp Thr Val Pro Gly
290 295 300 Asn Leu He Met Gly He Wing Tyr Leu Wing Ser Glu Asp Gly Gln Arg 305 310 315 320
He Gln Lys Lys Wing Tyr Asp Wing He Met Glu Val Tyr Pro Asp Gly 325 330 335
Asp Wing Trp Glu Lys Cys Leu Val Glu Glu Lys Val Pro Tyr Val Thr
340 345 350 Wing Leu Val Lys Glu Val Leu Arg Phe Trp Thr Val He Pro He Cys
355 360 365 Leu Pro Arg Glu Be Thr Lys Asp He Gln Trp Asn Gly Wing Thr He
370 375 380 Pro Wing Gly Thr Thr Phe Phe Met Asn Val Trp Wing Wing Asp Tyr Asp 385 390 395 400
Glu Asp His Phe Lys Asp Wing Asp Lys Phe He Pro Glu Arg Tyr Leu 405 410 415
Glu Ala Ser Glu Gly Ala Gly Thr Pro His Tyr Ala Tyr Gly Ala Gly
420 425 430 Ser Arg Met Cys Wing Gly Ser His Leu Wing Asn Arg Glu Le. Phe Thr
435 440 445 Wing Phe He Arg Leu Val Thr Wing Phe Asn Met His Thr Wing Lys Glu
450 455 460 Thr Wing Asp Arg Pro He Leu Asn Wing He Glu Cys Asn Leu He Pro 465 470 475 480
Thr Ala Leu Thr Thr Glu Pro Lys Pro Phe Ly3 Val Gly Phß Ser Ala 485 490 495 Arg Asp Pro Lys Lys Leu Glu Gln Trp He Wing Glu Ser Asp Glu Arg 500 505 510 Thr Lys Asp Leu 515 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:
Claims (27)
1. A method of inactivation in microorganisms of genes coding for enzymes of the catabolism of phenylacetate and competing for this compound with the biosynthetic enzymes of penicillin, characterized in that it consists of an integrative transformation by homologous recombination between at least one exogenous DNA compound and minus one part of the sequence of the gene that is to be inactivated.
2. A method according to claim 1, characterized in that the transforming DNA compound is a circular molecule containing at least one expressible fragment of the gene to be inactivated.
3. A method according to claim 1, characterized in that the transforming DNA compound is a linear molecule containing at least one DNA fragment not included in the sequence of the gene to be inactivated, but containing at least one transformation marker that interrupts the coding sequence thereof.
4. A method according to the preceding claims, characterized in that the transformant DNA molecule has, totally or partially, a copy of the sequence of the gene to be inactivated with a mutation, preferably, of change of reading frame, meaningless or deletion, which results in a phenotype of loss of function of said gene.
5. A method according to any of the preceding claims, characterized in that the ... organism undergoing transformation in which the gene is inactivated is capable of producing penicillin G or V.
6. A method according to claim 5, characterized by i-croorgan-L-s product of penicillin G or V, is an ongo. - 7 A process according to claims 1 to 6, characterized in that the transformed crogarganism is Aspergillus nidulans. 8 A process according to claims 1 to 6, characterized in that the transformed pilocogganism is Penicillium chrysogrenuin. 9. A process according to claims 1 to 7, characterized in that the transforming DNA compound used is totally or partially in-clible in a vector, preferably in the plasmid pPhacA:: argB. 10 A process according to claims 1 to 6 and 8, characterized in that the transforming DNA compound used is included, totally or partially, in a vector, preferably in the plasmid pALP696. eleven . A method according to claims 1 to 7 and 9, characterized in that the gene that is inactivated is represented by SEQ ID NO: 1, its imitated gene sequences and / or homologous genes. 12 A method according to claim 11, characterized in that the gene that is inactivated encodes a polypeptide represented by SEQ ID NO: 2, or similar sequences that express P450 phenylacetate 2-hydroxylase activity. 13 A method according to claims 1 to 6, 8 and 10, characterized in that the gene that is inactivated is represented by SEQ ID NO: 3, its mutated gene sequences and / or homologous genes. 14 A method according to claim 13, characterized in that the gene that is inactivated encodes a polypeptide represented by SEQ ID NO: 4, or similar sequences exhibiting P450 phenylacetate 2-hydroxylase activity. fifteen . A transformed strain of Aspergillus nidulans and derivatives thereof incorporating at least one exogenous DNA co-position characterized in that it consists of a truncated, incomplete or inactive sequence of at least one gene coding for an enzyme of the catabolism of phenylacetate and - which inactivates the endogenous gene after its integration by homologous recombination. 16. A transformed strain of A. nidulans according to claim 15, characterized in that the gene to be inactivated codes for an enzyme that mediates the hydroxylation of phenylacetate. 1
7. A transformed strain of A. nidulans according to claims 15 and 16, characterized in that the gene that is inactivated is phacA, whose sequence and flanking regions are described in SEQ ID N0: 1, its mutated gene sequences and homologous genes. 1
8. A transformed strain of A. nidulans according to claim 17, characterized in that the gene that is inactivated is homologous to phacA, of origin other than A. nidulans and whose homology with the DNA sequence of the phacA gene of A. nidulans is sufficient to mediate homologous recombination with the endogenous locus of A. nidulans. 1
9. A transformed strain of A. nidulans according to claims 15 to 18, characterized in that the gene that is inactivated encodes a polypeptide represented by the protein sequence SEQ ID NO: 2, or similar sequences expressing P450 phenylacetate 2-hydroxylase activity. 20. A transformed strain according to claims 15 to 19, characterized in that it consists of a pure strain of A. nidulans CECT20195, its mutants and / or transformed derivatives. 21. A plasmid pPhacA:: argB of inactivation of the phcA gene of A. nidulans as shown in the restriction map of Figure 6 and consisting of the plasmid pUC18 containing an EcoRI insert of 6.8 kb which it includes the phacA gene of A. inactivated nidulans by inserting a 3.2 kb fragment which in turn contains the argB gene of A. nidulans. 22. A transformed strain of Penicillium chrysogenum and ilutants d - rivß ± -s of the? M ---- a char-E-rizad-- porq-e ccpti-f --- a DNA that consists of a truncated, incomplete or inactive sequence of a gene that codes for an enzyme of the catabolism of phenylacetate and that inactivates the endogenous gene after its integration by homologous recombination. 23. A transformed strain of P. chrysogenum according to claim 22, characterized in that the gene to be inactivated codes for an enzyme that mediates the hydroxylation of phenylacetate. 24. A transformed strain of P. ciirysogenup. according to claims 22 and 23, characterized in that the gene is phcA, whose sequence and that of its flanking regions are described in SEQ ID NO: 3, its mutated gene sequences and / or homologous genes. 25. A transformed strain of P. chrysogenum according to claims 22 to 24, characterized in that the gene that is inactivated encodes a polypeptide represented by the protein sequence SEQ ID N0: 4, or similar sequences expressing P450 defined phenylacetate 2-hydroxylase activity . 26. A transformed strain of P. chrysogenum according to reiviix-Lic-ation 24, characterized in that the gene is phacA, of A. nidulans or any other homologous DNA compound isolated from a source other than P. chrysogenum and whose homology with the The panA DNA sequence of P. chrysogenum is sufficient to mediate homologous recombination with the endogenous loco of P. chrys ogen um 27. A plasmid pALP696 inactivating the pahA gene of P. chrysogenum, as shown in the restriction map of Figure 7 and consisting of the plasmid pBC KS + containing a Sali insert of 7.9 kb which includes the pahA gene of P. chrysogenum inactivated by inserting a 2.0 kb fragment which in turn contains the blek gene of S. hindustanus expressed under the control of the gdh promoter of P. chrysogenum.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES9700833 | 1997-04-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA98010517A true MXPA98010517A (en) | 1999-09-20 |
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