HU209941B - Method for identifying and using biosynthetic or regulatory genes for enhanced production of beta-lactam antibiotics, these comprising vector, and transformation of fungi cells - Google Patents

Abstract

The present invention provides novel methods and compositions for the production of increased secondary metabolites. The methods allow for the identification of sequences which, when transformed into host cells that produce a secondary metabolite, enhance the production of the secondary metabolite. The procedure is illustrated by the example of penicillin. Thus, the invention also relates to the isolation and sequence analysis of the acyltransferase gene of P. chrysogenum, for incorporation of the isolated gene into a vector, and for vector transformation of the fungal host cell. EN 209 941 Description range: 26 pages (including 12 sheets)

Description

FIELD OF THE INVENTION The present invention relates to the isolation, cloning, insertion into a vector of genes for enhanced production of β-lactam antibiotics and transformation of fungal host cells producing said antibiotic.

Over the past four decades, classical strain development techniques have resulted in an enormous increase in penicillin production. These classical strain development techniques were based primarily on the systemic treatment of Penicillium chysogenum mutant mutation followed by the selection of multiple penicillin producing mutants. However, advances in cloning techniques have provided a potentially new tool for further improving penicillin production in this fungus.

Penicillin is produced by the filamentous fungus P. chrysogenum during some enzymatic steps [e.g., E. Alvarez et al., Antimicrob. Agents Chemother., 31, 1675-1682 (1987)]. These steps are illustrated in Figure 1. Throughout the patent, genes that are directly involved in the biosynthetic process are described, that is, genes that encode active enzymes in a multi-step process leading to the formation of a secondary metabolite. That is, the G or V production of penicillin are genes encoding the enzymes shown in Figure 1. The first reaction is the formation of the o- (1-α-aminoadipyl) L-cysteinyl-D-valine tripeptide from α-aminoadipic acid, cysteine and valine. The enzyme responsible for the reaction is ACV synthetase (hereinafter ACVS), a large-molecule, multifunctional enzyme. The cyclic formation of the tripeptide is accomplished by isopenicillin N synthetase (hereinafter IPNS) or cyclase. The reaction product is isopenicillin N. It is a compound containing the typical cyclic β-lactam structure and has antibacterial activity. In the final step of penicillin formation, a more hydrophobic side chain of the N-α-aminoadipic acid side chain of isopenicillin is replaced. In industrial production, the commonly used hydrophobic side chain or phenylacetic acid resulting in penicillin G or phenoxyacetic acid resulting in penicillin V. It has been suggested that the side chain exchange reaction is catalyzed by a single enzyme [A. L. Demain and N.A. Solomon (editors):

Antibiotics containing the β-lactam structure I. Springer Verlag, Berlin (1983); A. L. Demain Publication 189-228. Site]. However, there is also the possibility of a two-step reaction, the product of which is 6-APA (E. Alvarez et al., Supra). The enzyme involved in the final reaction was found to be acylCoA: 6-APA acyltransferase (hereinafter AT), which was purified to homogeneity (E. Alvarez et al., Supra). The involvement of a second enzyme that catalyzes the IPN-6APA reaction has not been confirmed or ruled out so far.

It is unclear whether one or more enzymatic reactions limit the rate of penicillin biosynthesis and, if so, which enzymatic process is involved.

Because the process of penicillin biosynthesis begins with three amino acids, each of which is otherwise part of other metabolic pathways, these regulatory steps of these processes may also influence penicillin biosynthesis. On the other hand, penicillin production is subject to a complex mechanism of carbon catabolism repression and nitrogen source control [H. Kleinkauf, H. von Döhren, H. Donnauer, and G. Nesemann (editors): Reguládon of secondary metabolite formád. VCH Verlaggesellschaft, Weinheim (1985); J.F. Martin et al., Pp. 41-75. Site]. Regulatory proteins are also involved in this type of regulation. These regulatory proteins and the proteins they regulate have been found to indirectly participate in the biosynthetic pathway of a secondary metabolite, in this case penicillin.

To date, only one of the enzymes involved in penicillin G biosynthesis has been cloned and sequenced, the isopenicillin N synthase (IPNS) or cyclic [L. G. Carr et al., Gene, 48: 257-266 (1986)] using the appropriate Acremonium chrysogenum gene [S. M. Samson et al., Natur. 318, 191-194 (1985)]. The latter gene was cloned and identified by purifying the IPNS protein, determining the amino-terminal amino acid sequence, and generating a synthetic oligode1 based on this sequence. FLOW CHART

L-cysteine

L-α-amino adipic acid.

ACV synthetase (ACVS)

L-valine δ - (L-α-aminoadipyl) -L-cysteinyl-D-valine isopenicillin N (IPN)

Isopenicillin N synthetase (IPNS; cyclase) acyl-CoA: IPN acyltransferase (AT) penicillin -G or -V

6-Aminopenicillanic acid (6-APA) phenylacetic acid or phenoxyacetic acid

A set of zoxynucleotides and a cosmid genomic library were probed with a mixture of these oligodeoxynucleotides (S. M. Samson, supra).

Attempts to improve the strain using the cloned Penicillium chrysogenum isopenicillin N synthetase genes resulted in increased enzyme activity in Penicillium chrysogenum but neither improved penicillin production nor stimulated penicillin synthesis [P. L. Skatrud et al., Poster Presentation at the 1987 Meeting of the Society of Industrial Microbiology, (Baltimore, August 1987), excerpted by SIM News (Vol. 37, p. 77, 1987)].

Biosynthesis of β-lactam antibiotics has been shown to undergo glucose repression [J. F. Martin and P. Lir, TIBS, 3, 39-4 (1985). Glucose repression is undoubtedly in the interest of ACVS tripeptide formation and IPNS activity (Revilla et al., J. Bact., 168, 947952 (1986)). On the other hand, as far as acyltransferase is concerned, the data are less convincing. Revilla et al., Supra, have reported that AT is not subject to glucose repression, but other data suggest that AT does not have, or at least reduced, its activity in the presence of glucose [B. Spencer and T. Maung, Proc. Biochem. Soc., 29-30 (1970)].

It is not known which glucose repression exerts its effect on expression; this effect may occur at either the level of transcription or translation. If the foregoing is valid, differences in mRNA levels between producing and non-producing cultures could be used to isolate said gene.

There are other uncertainties in penicillin producing cells regarding the levels of mRNA encoding different enzymes.

Subtraction isolation techniques are used to identify genes involved in the production of secondary metabolites in microorganisms. The procedure is illustrated by the example of penicillin produced by P. chrysogenum.

The figures are briefly described below.

Figure 1 is a physical map of lambda clones isolated by the process of the invention. The lambda G2 and lambda B21 clones contain the cyclase and acyltransferase gene cluster. Lambda clones B9, G5 and L5 contain the critical Y gene. Other lambda clones contain other cryptic genes. E = EcoRI; B BamHI; H = Hindiyl; K = KpnI; S = Scarf; Sa = SacI; Sp = Sphl; P = PstI; X = Xhol; Kb = Xbal; Hp = Hpal; N = Ncol; Bg = BglII.

right arm of bacteriophage lambda EMBL3 (9 kb);

—— left arm of bacteriophage lambda EMBL3! _____ (20.3 kb);

l ---- ^{* 1} hybridization region with pen ⁺ cDNA;

unexplored section of the map;

() restriction site position / occurrence unclear;

la and ea are the left and right arms of bacteriophage lambda.

Figure 2 shows the nucleotide sequence of the acyltransferase gene of P. chrysogenum and the deduced amino acid sequence thereof.

Figure 3 shows the nucleotide sequence of the pyrG gene of P. chrysogenum and the deduced amino acid sequence thereof. This sequence forms the major part of the 2.4 kb EcoRI fragment which acts as a transformation stimulating sequence.

Figure 4 shows the nucleotide sequence of the protomer of the phosphoglycerate kinase gene of P. chrysogenum.

Figure 5 shows a restriction site and functional map of pUC13: pyrG.

Figure 6 shows a restriction site and functional map of pPS54.

Figure 7 shows a restriction site and functional map of pRHO5.

Figure 8 depicts a restriction site and functional map of pGJ101 A and B.

Figure 9 shows a restriction site and functional map of pPS47.

According to the invention, DNA fragments containing mono- or polycysteric sequences are identified. The sequences encoded by the gene (s) are translated into enzymes that are involved in the production of secondary metabolites or other commercially important products. The desired sequences are identified by comparing RNA sequences isolated from a competent microorganism producing a secondary metabolite, one in which the gene is actively expressed and one in which the expression is silent. In this way, DNA fragments encoding one or more genes which are expressed differently and which are involved in the production of a commercially important product are obtained. The term "differential expression" is used in the application to express the gene (s) in question when the expression is only explicitly active under certain specific conditions but other, well-defined conditions (i.e., as used herein, it is understood that expression) low level, such as 5% or less relative to the active gear). Lack of expression may be caused by repression or lack of induction of gene expression, or by mutation or other event that results in silent transcription of the gene (s) in question. The isolated DNA is obtained by screening a collection of genes. The collection may be a genomic or cDNA collection containing, for example, a lambda or cosmid cloning vector or an expression vector. Using a cDNA probe rich in sequences expressed during secondary metabolite biosynthesis, positive hybrids can be identified in the collection for further manipulation to construct expression constructs for the enzyme (s) involved in secondary metabolite production. Therefore, a gene collection from a microorganism was narrowed using two cDNA probes, one of which is derived from an active transcriptional stage3.

It is rich in sequences and the second derives from a transcription-silent situation. Only clones containing a gene (s) that are actively expressed under defined active conditions can be isolated by comparison and subtraction.

This procedure is exemplified by the isolation of genes involved in the biosynthesis of a secondary metabolite, namely, penicillin, using two cDNA probes derived from lactose growing (producing) and glucose growing (non-producing) mycelium.

The DNA sequences identified contain at least one gene that encodes at least one antibiotic biosynthetic enzyme and / or, throughout the biosynthetic process, encodes a regulated protein or, more generally, any protein that participates in the biosynthesis of said antibiotic in any way, either positive or negative. .

When properly introduced into a suitable host microorganism, the positively-acting constructs, in particular in β-lactam-producing microorganisms, will greatly enhance the efficiency of the biosynthetic process through increased gene expression or higher levels of gene expression. On the other hand, structures which have a negative effect on the production of the antibiotic (for example by-products) can be isolated. These constructs are used to inactivate the negatively working gene by gene replacement or other equally effective means. In both cases, the yield of the desired antibiotic will be higher during industrial production. This procedure is shown on β-lactam producing microorganisms. The process is of particular use for the production of β-lactam producing microorganisms for the production of antibiotics, mainly penicillins. Preferably, the expression cassette contains genes encoding enzymes that catalyze rate-limiting steps or genes encoding transcription-inducing regulatory proteins or other genes.

The method of the invention also provides sequences for which, although the product encoded by them is not known, it has been found to result in increased production of the desired product. These sequences are referred to as "cryptic genes", i.e., those sequences obtainable by the isolation procedures described herein, and which contain genes for which no known function has yet been attributed. These genes are characterized by higher levels of expression and / or expression in the transformed microorganism compared to the untransformed host cell. In addition to the "cryptic genes", the process provides other genes, such as EPNS and acyltransferase. We have found that a cryptic gene called "Y" induces an enhanced biosynthesis of penicillin.

The microorganisms used in the present invention may also be prokaryotes and eukaryotes, for example bacteria belonging to the taxonomic group Actinomycetes or Flavobacteria or fungi including yeasts belonging to the species Aspergillus, Acremonium or Penicillium.

Depending on the origin of the fragment, different expression cassettes, whether of genomic or cDNA origin, or of prokaryotic or eukaryotic origin, may be constructed. For bacterial genomic DNA, the coding sequence for mono or polycistron may include its own transcriptional initiation regulatory region, as well as a transcriptional termination region, and a transcriptional termination region and appropriate translation signals, such as the Shine-Dalgarno sequence and stop codons. When genomic DNA is of fungal origin, usually only one gene will be linked to a transcriptional regulatory region, so each gene will have its own independent transcriptional regulatory region. When using cDNA, we must provide for a proper transcriptional regulatory region, depending on the host used for subsequent expression.

The genes of interest to them may be obtained by screening the DNA collection obtained from said microorganism by probes obtained from mRNA or DNA from a first culture of said microorganisms producing said secondary metabolites; and then filtering said DNA collection by probes obtained from mRNA or DNA from a second culture of said microorganism or a mutant which does not produce said secondary metabolite; then identifying fragments containing genes that are transcribed in said first culture and which are substantially non-transcribed in said second culture.

Particularly valuable genes include those expressed specifically during antibiotic biosynthesis, such as genes encoding β-lactam biosynthetic enzymes known in the art, such as tripeptide synthetase (ACVS), cyclase (IPNS), acyltransferase (AT), epimerase, expandase, hydroxylase, transcarbomoylase, methoxylase, transacetylase. Preferably, the gene encoding the IPN: 6-APA acyltransferase or the cryptic "Y" gene is present or expressed in greater amounts, and this desired antibiotic is present or expressed and results in a higher yield of the desired antibiotic in the transformed fungus.

Those skilled in the art will appreciate that a β-lactam-producing host cell carries the sequence of the gene (s) to be expressed or its native promoter recognized by the host cell RNA polymerase or linked to any other suitable promoter, such as another β-lactam biosynthetic gene or glycolytic gene such as phosphoglycerate kinase, glyceraldehyde phosphate dehydrogenase, triose phosphate isomerase promoter, or EF to translational factor promoter and so on.

Such a promoter can be used to:

20 control the expression of one or more genes that produce these enzymes. This leads to an increase in antibiotic production after transformation, since penicillin production is now possible under conditions that do not allow penicillin production in the untransformed host strain. For example, glycolytic enzymes are also expressed in the presence of glucose, while penicillin production is repressed by glucose (J.F. Martin, supra). By inducing expression of the penicillin biosynthetic genes under the control of a promoter of a glycolytic gene, the genes may also be expressed in the presence of glucose, and thus penicillin may be produced early in the fermentation when high concentrations of glucose are required to form mycelium. The selectable marker may also be placed under the control of such a promoter.

The invention exemplifies the promoter of the phosphoglycerate kinase gene of P. chrysogenum as a promoter capable of overcoming glucose repression of penicillin biosynthesis. The promoter was isolated from a genomic collection of P. chrysogenum using standard methods described in R. A. Hitzeman, Nucl. 10: 7791-7808 (1982)] using oligodeoxyribonucleotide probes. The nucleotide sequence of the phosphoglycerate kinase promoter is shown in Figure 4.

For the transformation of Penicillium, constructs are used which contain at least one selection marker for the transformed cells and preferably enhance the survival of the integrated DNA. Therefore, it is preferred that the vector contains a DNA sequence that is known to enhance the efficiency of the transformation. An example of such a DNA is the "ans" element isolated from Aspergillus nidulans (Ballance and Turner, Gene, 1985, 36, 321-331). The present invention provides a DNA sequence isolated from the genome of P. chrysogenum which has been found to have a similar effect to the "ans" sequence. Because this sequence is the natural sequence of P. chrysogenum, it can be used to increase the efficiency of transformation in P. chrysogenum. The DNA sequence contains the pyrG gene of P. chrysogenum, which can be used alone or in combination with the host pyrG, in which case the DNA sequence in question has both a selection of transformed cells and an effect enhancing transformation [v, i. EPA-260,762] provides or may be used in combination with another selection marker, for example a biocide in combination with such a selection marker, for example a gene encoding resistance to a biocide such as phleomycin. In the latter case, the selection of the transformed cells and the transformation enhancer are provided by two separate DNA sequences and the only function of the pyrG element is to increase the frequency of the transformation.

Markers for the selection of transformed clones may be homologous or heterologous biosynthetic genes that can complement a host cell's auxotrophic requirement caused by a defect in the metabolic pathway to an amino acid such as arginine or a nucleotide precursor such as uracil.

The structural gene providing the marker for selection may be either the wild-type Penicillium host cell or a heterologous structural gene operable in the host cell. For example, a structural gene encoding an enzyme involved in the metabolism process may be derived from Penicillium or other filamentous fungi such as Aspergillus, Neurospora or yeast if their structural gene is functional in the Penicillium host cell and converts autotrophy into prototrophy.

The complementary structural gene may be derived from a metabolic process such as the synthesis of purines or pyrimidines (nucleosides) or amino acids. Of particular interest are the structural genes encoding the pyrimidine metabolism enzymes, such as the gene encoding the orotidine 5'-phosphate decarboxylase enzyme (pyrG or pyr4). Other important genes are amino acid biosynthetic genes, such as ornithine carbomoyl transferase (argB) and arginine succinate lyase (arg4). The selection markers mentioned above are discussed in European Patent Application EP-A-260762.

Fermentation markers may be used instead of auxotrophic markers. For example, when amides are used as the sole source of carbon and nitrogen, or when different sugars such as galactose or the like are required for growth.

In addition, genes encoding biocidal resistance may be used. Examples of such biocides are hygromycin, gentamicin, phleomycin, plyphosate, bialaphos, and the like.

The present invention provides constructs that contain the desired sequence but may also have other functions, such as replication systems for one or more host cells, such as a target host cell for expression of a cloning host cell and / or a secondary metabolite; may contain one or more selectable markers for one or more host cells as mentioned above; genes that enhance transformation efficiency; or other special features.

The construct comprises at least one gene isolated by the method of the invention. The construct can be obtained in a conventional manner, such as isolating the other desired genes from the corresponding host cells, synthesizing all or some of the genes, or a combination of these methods. Similarly, regulatory signals, transcriptional and translational start and stop regions can be isolated from a natural source, synthesized, or combined. The various fragments may be treated by digestion with endonucleases (restriction), ligation, sequencing, in vitro mutagenesis, primer repair, or the like. Various manipulations are well known in the art and can be used for specific purposes.

HU 209 941 Β

The various fragments can be combined, cloned, isolated and sequenced according to conventional methods. After each manipulation, the DNA fragment or combination of fragments is inserted into a cloning vector, transformed with the vector, a cloning host cell, such as an E. coli cell, cultured, lysed, plasmid isolated, and the fragment by restriction analysis, sequencing, or the like. examined. E. coli cells can also be used as host cells to express the desired genes to produce large amounts of protein.

Various vectors may be used during the construction of the construct and to transform the host cell. These vectors may be cloning vectors, expression vectors, and vectors capable of integration into the host, or may use naked DNA for transformation and integration.

The cloning vector is typically characterized by containing a marker for the selection of a host cell containing the cloning vector and optionally a transformation enhancing sequence. It may also contain one or more polylinkers or complementary sequences for insertion, selection, manipulation, facilitation of sequencing, excision, and so on.

Expression vectors usually have a construct for insertion that includes a transcriptional and translational initiation and terminator regions and, on the other hand, may lack one or both regulatory regions, as these expression vectors may provide upon insertion of the protein product coding sequence.

DNA encoding the desired enzyme (s) may be introduced into a Penicillium host cell, essentially according to the method described in European Patent Application EPA-260762.

Effective transformation of Penicillium can result in the formation of transformed cells in which one or more expressing genes are separately integrated into the host genome (integrated genes). DNA constructs are provided that allow selection of transformed host cells. Transformation uses conditions that result in high-frequency transformation, while ensuring selection and isolation of transformed host cells expressing the desired structural gene (s). The resulting transformed cells allow constant integration and expression of the integrated DNA. The value of the transformed host of the present invention is that it can be used as a parent strain in strain development methods other than DNA-mediated transformation, such as protoplast fusion, mass mating, and mutation. The resulting strains are considered to be part of the invention.

The desired genes that we wish to introduce by transformation may be an integral part of the transforming vector, but it is often much more advantageous to introduce these genes into the transformation mixture via a separate vector, thereby co-transforming these genes with the selective vector. effective method for fungi [P. J. Punt et al., Gene, 56: 117-124 (1987); K. Wamars et al., Mol. Gene. Genet., 209, ΊΧ-ΊΊ (1987); I. E. Mattem et al., Mol. Gene. Genet. 270: 460-461 (1987).

The result of the transformation will be to produce at least one copy of the desired gene (s), often two or more, in most cases no more than ten. Their number depends on whether an integrated form is used or stable espeissomal maintenance is used. The number of integrated copies depends on whether the structures in question are multiplied and so on.

The present invention provides, by way of example, a method for isolating a gene that is involved in penicillin biosynthesis from a collection of genes derived from the producing microorganism, i.e., P. chrysogenum, using specific cDNA probes. This procedure serves as an example to identify the cryptic genes responsible for the increased production of secondary metabolites. This method, which seeks to isolate a DNA construct encoding one or more genes involved in the biosynthesis of secondary metabolites, involves screening the gene collection with a cDNA probe rich in sequences that are specifically expressed during biosynthesis. By "specific expression" we mean that the expression of these genes is silent or very low (e.g., less than 5% of the level of production) in the absence of penicillin production, but high in penicillin production. Finally, radioactive or otherwise labeled cDNA probes are synthesized from mRNA templates isolated from P. chrysogenum mycelia during the penicillin production phase.

The probes are then enriched for sequences that specifically hybridize to the desired genes, removing cDNA sequences that can hybridize to PenRillium-derived mRNA when conditions do not allow penicillin production. Such is the case with high glucose levels. Using this enriched probe, clones are selected from a collection of P. chrysogenum genes that do not hybridize to a probe from non-producing mycelia. A large number of clones isolated in this way have been found to encode the isopenicillin N synthetase (IPNS or cyclase) penicillin biosynthetic enzyme.

In addition, it has been found that there are several copies of a gene encoding a side chain exchange enzyme (acyltransferase) among selected clones. This was demonstrated by experiments using a DNA probe based on the amino terminal peptide sequence of the purified enzyme.

The identity of these clones has been biochemically and biologically verified. The nucleotide sequence of the acyltransferase gene and the deduced amino acid sequence thereof are shown in Figure 2. Surprisingly, both the gene encoding isopenicillin N synthetase and acyltransferase are present on the same DNA fragment. That's it

We demonstrated that the P. chrysogenum genome collection in the EMBL3 lambda vector was hybridized with probes specific for the two genes. The same clone is hydrated with both probes separately.

In addition, after generating a physical map of one genomic lambda clone and hybridizing the restriction digestions of the lambda clone with probes specifically designed for the two genes, it was found that the genomic structure corresponds to that shown in Figure 1 (clones B21 and G2). The location of the two genes on the same large DNA fragment allows the construction of strains of P. chrysogenum in which both the isopenicillin N synthetase and acyltransferase genes are present in larger numbers without disturbing the related structure or balanced expression of the two genes. In addition, by incorporating multiple copies of this large DNA fragment, it is possible for both genes on said DNA fragment to be expressed naturally in upstream and downstream sequences.

Both the balanced expression and the natural environment have a good effect on the efficiency of gene expression and thus on penicillin production. The technique of isolating the isopenicillin N synthetase plus acyltransferase gene group could be advantageously used to isolate other penicillin biosynthetic genes, and the penicillin biosynthetic genes could be grouped by chromosome walking techniques.

In the following, several genes have been isolated which have not yet been assigned any function, but which appear to be involved in the β-lactam biosynthesis. The physical map of the cryptic genes of the invention is shown in Figure 1 (clones B9-B23).

By transforming an appropriate host cell from the "cryptic" gene with the gene Y contained in clones B9, L5 and G5 on a 3.0 kb Sphl + BamHI alpha fragment, it stimulates penicillin production to 26% of that of the untransformed host cell. kal woman. It follows that the production of the Y gene is involved in the biosynthesis of penicillin. In addition, it follows that transformation using genes isolated by the method of the present invention without knowing the function or identity of the genes can be useful in repairing the P. chrysogenum strain.

It is a further object of the present invention to transform Penicillium chrysogenum cells with genes that are specifically expressed under conditions where the antibiotic is synthesized and which encode gene products that catalyze the biosynthetic reactions leading to the antibiotic in question.

One such enzyme, acyltransferase (hereinafter referred to as AT), catalyzes the final step of penicillin biosynthesis, whereby the amino-adipyl moiety of isopenicillin N is replaced by a hydrophobic acyl side chain precursor such as phenylacetic acid or phenoxyacetic acid, which is the penicillin Gt or Vt.

The acyltransferase gene of P. chrysogenum, including the nucleic acid sequence, is provided. The invention also relates to conservative mutations in which the sequence encodes the same amino acid sequence, but may differ from 30% of bases, but usually about 10% of bases. The invention also encompasses non-conservative mutations, with less than 10%, more often less than about 5%, and preferably no more than about 1% of the amino acids being substituted or deleted and when less than 5% amino acid, where the percentage refers to the number of naturally occurring amino acids. In addition, fragments of the nucleic acid encoding the enzyme can be used, usually at least about 9 codons, more often at least 15 codons - and expression products thereof as probes, for the production of antibodies, and so on. Probes can be used in other species to identify an enzyme by using nucleic acids for hybridization and antibodies to identify cross-reacting proteins.

Research on non-naturally occurring side chain precursors or other penicillins or cephalosporins as acyltransferase substrates can be complemented by research on mutant acyltransferase enzymes that accept other side chain precursors as their substrate, not only acid or phenyl, penicillins or cephalosporins other than isopenicillin N substrate. The present invention provides a starting material for mutant acyltransferase enzymes. E. coli is the best host for mutation cloning experiments; however, since E. coli lacks the splicing mechanism required to remove introns in the acyltransferase gene, a cDNA clone of the acyltransferase gene is the sequence of choice for expression of the enzyme in E. coli. This cDNA sequence can be easily mutated by methods well known in the art, such as treatment with irradiation (X-ray or UV) or chemical mutagens (ethyl methanesulfonate, nitrosoguanidine or methyl methanesulfonate), or site-specific mutagenesis, to obtain mutant enzymes which recognize the substrate exchange of non-natural side chain precursors and catalyze the formation of unnatural penicillins from isopenicillin N and penicillins or cephalosporins other than isopenicillin N.

Isolation of AT, Y, and other penicillin biosynthetic genes allows the identification of regulatory elements of individual genes such as a promoter, an upstream activating sequence (UAS), a terminator, and the like. This can be achieved by comparing genes with one another and by comparing sequences obtained with the isopenicillin N synthetase biosynthetic gene and other related genes. This latter comparison may also reveal the specific nature of the regulation of gene expression in the group of penicillin biosynthetic genes.

HU 209 941 Β

Identification of such a "penicillin biosynthetic regulatory element" leads to the identification of specific regulatory proteins by standard techniques such as gel retardation, cross-linking, DNA footprint analysis and the like. After isolation of the specific regulatory proteins by affinity chromatography, the gene encoding the respective protein is cloned and then manipulated in a suitable host cell.

Using the cloned AT gene, the Y gene and other penicillin biosynthetic genes, modified enzymes can be designed and synthesized. These modifications result in changes in the characteristics of the enzymes, such as changes in their pH and temperature optimum, changes in their stability, or changes in substrate specificity. Host strains transformed with genes encoding these modified enzymes can be programmed to perform antibiotic synthesis under different conditions or to synthesize another antibiotic, such as ampicillin instead of penicillin.

In another aspect of the invention, cloned genes can be used to transform host strains which naturally do not contain these enzymes. It is known that Streptomyces and Acremonium do not have the AT enzyme, whereas Penicillium, on the other hand, lacks the gene for the cephalosporin and cephamycin biosynthetic enzymes. The introduction of these genes into a host cell results in the synthesis of Penicillium cephalosporin or cephamycin and acremonium penicillin or cephalosporins with a hydrophobic side chain.

From the following results, it is clear that the production of secondary metabolites can be greatly enhanced by screening procedures that allow identification of DNA sequences associated with the production of secondary metabolites. Extraction methods used to identify specific sequences involved in the production of secondary metabolites can isolate mRNA and cDNA which, once identified, can be used as probes. Thus, it has been found that fragments containing genes of unknown function greatly enhance the production of secondary metabolites and these fragments can be transformed into host cells for production of the secondary metabolite. This procedure is illustrated by the example of penicillin.

We also disclose an acyltransferase gene that can be used in a variety of ways: as an enzyme for modifying β-lactam compounds, as a marker, as an antigen source for producing acyltransferase antibodies, as a promoter sequence source, as a source for expressing large amounts of protein for crystallization, in vitro mutagenesis to produce an enzyme with modified characteristics and so on.

All publications and patent applications mentioned in this patent are incorporated by reference as if each notice or patent application were to be specifically and individually used as reference.

While the invention has been described in some detail, and for purposes of clarity, illustrations and examples are provided, it will be apparent to those of ordinary skill in the art in light of the disclosure that certain changes and modifications are made without departing from the spirit or scope of the appended claims. .

The following examples are provided by way of illustration, but are not intended to limit the scope of the invention.

Example 1

Construction of a genomic library from Penicillium chrysogenum

Penicillium chrysogenum (ATCC 28089) is essentially according to methods known in the art [T. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. USA (1982)]. The chromosomal DNA is extracted from Penicillium chrysogenum by converting mycelia to protoplasts as previously described in EP-A-260762.

The protoplasts were then lysed by diluting the isotonic (0.7 M KCl) suspension with four volumes of TES buffer (0.05 M Tris · HCl, pH 8.0, 0.1 M EDTA, 0.15 M NaCl). Then, 1% sodium lauryl sulfate was added to the lysate and this mixture was incubated at 55 ° C for 30 minutes. After extraction with phenol and two chloroform, the DNA was precipitated with ethanol, dried and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The DNA solution was then treated with 100 µg / ml RNase at 37 ° C for 1 hour, followed by 200 µg / ml proteinase K at 42 ° C for 1 hour. The solution was extracted once with phenol and twice with chloroform. An equal volume of isopropanol is layered over the aqueous phase and DNA is collected from the interface by winding it onto a glass rod. After drying, the DNA is dissolved in TE buffer. The DNA composition thus obtained has a molecular weight of about 10 ⁸ . The DNA was partially digested with Sau3A, then ligated to BamHI-cleaved dephosphorylated EMBL3 arms (Promega Biotec, Madison, WI., USA) and packaged in lambda bacteriophage capsids using the Promega Biotec Packagene System. All reactions were carried out according to the manufacturer's instructions, except that the packing reaction was carried out at 22 ° C for 2-3 hours. The collections were grown by placing the packaged phages on a plate, incubating the plates for 7 to 8 hours at 37 ° C, and then adding the phages in 4 ml of SM buffer (0.1 M NaCl, 0.01 M MgSO ₄ , 0.01 M per Petri dish). , 05 M trisHCl, pH 7.5, 0.01% gelatin).

Example 2

Isolation of genes specifically expressed during penicillin biosynthesis by differential screening

Genes specifically and predominantly expressed during penicillin biosynthesis were identified by deleting the genomic library of Example 1

We digested with labeled cDNA probes synthesized from mRNA templates extracted from productive (lactose-growing) and non-producing (glucose-growing) mycelia and selected clones that predominantly gave a positive (+) probe.

Long-range RNAs were isolated from day 3 or 6 cultures, one time from the production medium (see Example 3) (+ production) and second from the same medium when lactose was replaced with glucose (- production). Mycelia were collected by filtration, frozen in liquid nitrogen, homogenized, and mRNA isolated by the guanidium isothiocyanate method described by T. Maniatis et al., Supra.

Against both mRNA populations, cDNA was synthesized and labeled with (α- ³² P) dATP at high specific activity in a 30 μΐ reaction mixture of the following composition:

12.5 mM MgCl ₂ 50 mM tris HCl; pH = 8.3 100 mM KC1 125 mM DTT 2 unit / μΐ RNase 500 μΜ dGTP 500 μΜ dCTP 500 μΜ TTP 25 μΜ dATP 0.1 μ _δ / μ1 BSA 100-200 pg / ml poly A ⁺ RNA 50-60 μ ^ πιΐ oligo ₁₂ _ ₁₈ 1.2 unit / μΐ reverse transcriptase 1.67 μΟί / μ! (a- ³² P) dATP

Poly A ⁺ RNA and oligo-dT were mixed separately, heated at 100 ° C for 1 minute, and cooled in ice water before being added to the reaction mixture. The reaction mixture was incubated for 1.5 hours at 42 ° C, then 5 μΐ of 1 mM dATP was added and incubated for another 30 minutes. The reaction mixture was then adjusted to 20 mM DETA, 20 mM NaOH (final volume 100 μΐ) and brought to 65 ° C. After incubation for one hour, 5 μΐ of 1 M tris was added. HCl (pH 8.3), 40 μΐ 0.1 M HCl, 7 μg bovine tuna DNA, 100 μΐ TES buffer (10 mM Tris, 1 mM EDTA, 1% SDS, pH 7, 5) and 200 μΐ of 5 M ammonium acetate and the DNA was precipitated with 800 μΐ of ethanol at -20 ° C for 16 hours.

The precipitate was collected by centrifugation, washed with 70% ethanol, dried and finally dissolved in 32.5 μΐ TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The (+) cDNA preparation is then enriched in sequences that are specifically expressed during penicillin biosynthesis by hybridization with a (-) mRNA composition in two successive hybridization steps (cascade) in a 75 μΐ reaction mixture as follows:

32.5 μΐ (+) cDNA 10 μΐ (-) mRNA (1 pg / μΐ) 30 μΐ 1 M sodium phosphate pH 6.8 1.5 μΐ 10% SDS ΐμΐ 0.5 M EDTA.

The reaction mixture was incubated for 16 hours at 68 ° C, 102 μΐ of water (final phosphate concentration 170 mM) was added, and the mixture was placed on a hydroxylapatite column previously equilibrated with 170 mM phosphate solution at 68 ° C. Under these conditions, the double-stranded nucleic acids are bound on the column while the single-stranded nucleic acids are dissolved. The eluate was collected, dialyzed against TE buffer for 1.5 hours, and precipitated with ethanol after addition of 4 μg of carrier (calf thymus) DNA. The procedure was repeated and the final unbound cDNA was used directly as a probe to screen the Penicillium genomic collection as follows:

A sample of the duplicate collection of Example 1 was plated on 5 Petri dishes to form approximately 1500 plaques per plate. Plaques were transferred to two Gene Screen Plus filters (New England Nuclear) according to the manufacturer's instructions. One filter group is probed with the labeled enriched (+) cDNA composition and the second filter group is probed with the labeled (-) cDNA as a control.

Positive plaques are purified and subjected to another filtration. By this procedure, 96 plaques that predominantly gave a positive signal with the (+) cDNA probe were selected.

Recombinant phage DNAs that gave a strong or medium signal at first screening were labeled with ³² P and used as probes to screen Northern blocks of Penicillium RNAs isolated from producing and non-producing mycelia to determine under both conditions. expression level. Accordingly, the recombinant clones were divided into three groups.

Group 1 contains genes that are expressed at high levels during penicillin biosynthesis.

Examples include the following clones:

G2 and B21

B9, L5 and G5

L12

K9

Group 2 genes have moderate expression. Examples include the following clones:

C12

P3 and Kll

B13

B20

Group 3 genes have poor expression. Examples include the following clones:

G3

G1 K16

L10 B23

The physical map of these recombinant phages is shown in Figure 2. Clones G2 and B12 gave a positive hybridization signal when tested with an isopenicillin N synthetase specific probe (S. M. Samson et al., Supra). Surprisingly, the same clone appeared to hybridize to an acyltransferase-specific probe (see Example 5).

HU 209 941 Β

Example 3

Purification of acyltransferase

Penicillium chrysogenum strain (ATCC 28089) was inoculated with 2 x 10 ⁶ conidium / ml) into a complex seeding medium consisting of: corn jam (20 g / l); alcohol production residue (20 g / l); sucrose (20 g / l); CaCO ₃ (5 g / L). The pH of the medium before sterilization is 5.7. The inoculum medium is incubated for 36 hours at 25 ° C and 250 rpm, and the resulting culture is used to inoculate 20 times the volume of complex medium. The production medium is 35 g / l dried jam, 25 g / l lactose, 2.5 g / l potassium phenylacetate, 3 g / l MgSO ₄ -7H ₂ O, 7 g / l KH ₂ PO ₄ , 2, 5 g of corn oil and 10 g / 1 CaC0 ₃ respectively. The incubation was continued for 48 hours and the mycelia were collected by filtration and the filter cake washed four times with cold 0.15 M NaCl.

200 g (wet weight) of mycelium are suspended in 700 ml of 0.05 M Tris-HCl buffer (pH 8, TD buffer) containing 5 mM dithiothreitol (DTT). The suspension is disintegrated in a ethanol / dry ice bath at 15 second intervals using Ballotini glass particles (Sigma, type V, diameter: 450-500 μπι) in Braun disintegrator (Braun, Melsunge, Germany). The extract is then centrifuged at 20,000 g for 30 minutes. This operation and each subsequent step are performed at 4 ° C. To 640 ml of the extract is slowly added 27 ml of a 10% (mixed) solution of protamine sulphate in 0.5 M Tris HCl, pH 8.0. After stirring for 45 minutes, the nucleic acid precipitate was removed by centrifugation at 20,000 g and the supernatant fractionated by precipitation with ammonium sulfate while maintaining the pH of the solution at 8.0 during the addition of ammonium sulfate. The fraction precipitated between 40-55% saturation was dissolved in 1M ammonium sulfate containing TD buffer and applied to a CL-4B column (1.8 x 16 cm) previously equilibrated with the same buffer. The column was washed with TF buffer at a flow rate of 5 ml / min until unbound protein was removed from the column.

The acyltransferase is then dissolved from the column with 40% ethylene glycol in 0.05M Tris HCl, pH 8.0.

The dissolved fractions were assayed for acyltransferase activity by adding 0.2 mM phenylacetyl coenzyme A, 0.2 mM 6-aminopenicillanic acid, 5 mM DTT, 0.1 M tris HCl (pH). = 8.0) and the reaction mixture containing the enzyme extract to a final volume of 200 μΐ was incubated at 25 ° C. After 10 minutes, quench the reaction with 200 μ 200 of methanol. The samples are centrifuged at 5,000 g and the penicillin G in the supernatant is determined by conventional microbiological or chromatographic methods.

The active fractions from the phenyl-Sepharose column were pooled and applied to a DEAE-Sephacel column (1.5 x 20 cm) previously equilibrated with TD buffer and the acyltransferase activity was solubilized with a linear TD gradient of NaCl (0-0.25 M). At a flow rate of 0.25 mL / min. The combined active fractions were precipitated with 70% ammonium sulfate, dissolved in 3 mL of TD buffer and applied to a TD-equilibrated Sephadex G-75 (coarse) column (2.6 x 70 cm). The acyltransferase was dissolved in TD buffer at a flow rate of 9 ml / h, and the subsequent fraction fractions were collected where the protein peak was symmetric with the corresponding acyltransferase activity and the final purity was 258 times.

Example 4

Determination of the amino terminal amino acid sequence of acyltransferase and design of appropriate oligonucleotide probe mixtures The enzyme preparation obtained in Example 3 was run on SDS-PAGE gel (UK Laemmli, Natura, 227, 680 (1970)) (13% acrylamide; 50 mA). A 29 kD band (approximately 10 μg of protein) was excised from the SDS gel and the protein was electrophoretically transferred onto a PCGM-2 membrane (polybrene impregnated glass fiber, Janssen, Beerse, Belgium) in a Multiphor II Nova biotype (LKB; 0.8). mA / cm ² ; 90 min; electrode buffer: 5 mM sodium borate, pH 8.0). After blotting, the PCGm membrane was washed four times with 25 mM sodium chloride and 10 mM sodium borate (pH 8.0) and dried.

The N-terminal amino acid sequence analysis of the protein thus adsorbed to PCGM was performed with a gas phase sequencer (Applied Biosystems, model 470A). The assay resulted in the following sequence:

thr-thr-ala-tyr-gln-cys-leu-pro-asn-gly-leu-ala-gln-asn glngly-asp-trp

Based on the underlined portion of the above amino acid sequence, the following oligodeoxynucleotide set was synthesized:

T

A C A T T

5 'CA GG CA AA TGGGA 3'

G A G C C

G

The amino terminal amino acid sequence of the 10 kD band that is sometimes present in the composition was also determined, but was not used to construct an oligodeoxyribonucleotide probe. The resulting sequence is as follows: Met-LeuHis-Ile-Leu-X-Gln-Gly-Thr-Pro-Phe-Glu-Ile-Gly-TyrGlu-His-Gly-Ser-Ala-Ala-Lys-Ala-Val- Ile-Ala.

Example 5

Identification of the acyltransferase gene

Some of the lambda clone DNA of Example 2 was digested with SalI restriction endonuclease, and the fragments were separated on a 0.7% agarose gel, transferred to a Genescreen Plus membrane, and hybridized with a mixture of ³² P-labeled oligonucleotides of Example 4. A positive signal-producing clone is map mapped using standard methods. Representative physical maps of two recombinant phages, lambda B21 and lambda G2, are shown in Figure 1. The oligodeoxyribonucleotide mixture was specifically hybridized to the EcoRI / HindIII subunit on the map.

HU 209 941 Β fragment. This and adjacent fragments were re-cloned in pTZ 18/19 (United States Biochemical Corporation) and subjected to nucleotide sequence analysis. The determined sequence and the deduced amino acid sequence are shown in Figure 2.

The amino-terminal amino acid sequence of the 10 kD band also present in the composition was determined and found to correspond to a DNA sequence upstream of the 29 kD sequence. Thus, it is likely that AT is synthesized as a 40 kD protein. This view is supported by the length of the AT messenger, which has been shown to be 1500 bases long (similar to isopenicillin N synthetase mRNA, which encodes a 38 kD protein), allowing for the presence of 100 bases of 3 'and 5' translated segments. .

The 29 kD protein amino acid sequence (extended to thr-Thr-Ala-Tyr-Cys-Gln-Pro-Asp-Gly-AlaLeu-Gln-Gly-Gln-Asn-Trp-Asp-Phe-Phe-Ser-Ala) -TrhLys-Gln-Ala sequence) and the 10 kD protein amino acid sequence indicates the presence of two introns. The existence of a third intron can also be assumed based on the gross amino acid composition of the 10 kD protein (groups 97) and the generally accepted sequence of its boundaries [D. J. Ballance, Yeast 2: 229-236 (1986)]. The presence of this third intron was confirmed by Norther biot hybridization with primary extension and using oligonucleotide probes from the coding and non-coding regions.

Example 6

Editing pPS47

The phosphoglycerate kinase (pgk) gene was isolated from the Penicillium genome collection by standard methods (Maniatis; Example 1), using the appropriate yeast gene as a hormone hybridization probe (Hitzeman et al., Supra). The pgk promoter region is described in part by the sequence shown in Figure 4, which is upstream of the pgk coding region.

The P. chrysogenum pgk promoter, as a 1.5 kb HindIII fragment, is cloned into pTZ18R and a clone with the correct orientation is selected.

The phleomycin resistance gene, isolated in the form of the 1.0 kb BamHI plus BglII fragment from pUT702 (Cayla, Toulose Cedex, France), was then cloned into this clone at the BamHI site of the polylinker. The pgk promoter is fused within the framework of the phleomycin resistance gene by unblocking the sequence to be deleted using the following oligonucleotide sequence:

5 '- GGA ACG GCA CTG GTC AAC TTG GCC ATG GTG GGT AGT TAA TGG TAT G - 3'

This oligonucleotide also introduces an NcoI site at the ATG site (underlined).

Example 7

Construction of a Transformation Vector with High Transformation Efficiency (pPS54)

In order to obtain a transformation frequency for P. chrysogenum that is high enough to allow the incorporation of genes by transformation or cotransformation to complement or amplify the non-selectable genes involved in β-lactam biosynthesis, it is desirable that the transformation vector introducing a transformation enhancer sequence [ans sequence in Aspergillus; D. J. Ballance and G. Turner, Gene, 36: 321-331 (1985). Surprisingly, the transformation stimulating sequence in P. chrysogenum is located on the DNA sequence containing the pyrG gene of P. chrysogenum. Part of this 2.4 kb Eco RI fragment is described in the nucleotide sequence shown in Figure 3. This DNA fragment is part of the 4 kb Sau3A partial fragment cloned into the BamHI site of plasmid pUC13 [J. Brass, Meth. Enzymol, 101, 20 (Acad. Press, 1983). The plasmid is designated pUC13 :: pyrG (see European Patent Application EP-A-260762 and Figure 5).

The 2.4 kb EcoRI fragment was inserted into the plasmid containing the Streptoalloteichus hindustanus phleomycin resistance gene (pPS47) under the control of the promoter of the phospholicerate kinase (pgk) gene from P. chrysogenum. The resulting structure is called pPS54.

The stimulatory effect of the pyrG fragment on the frequency of transformation is shown in Table 1 below.

Table 1

plasmid Transformed cell / pg DNA pPS 47 (phleo ^R ) 37 pPS 54 (phleo ^R , pyrg) 186

Example 8

Biological and biochemical verification of the identity of the AT clone

The identity of the AT clone is biologically confirmed by the addition of a P. chrysogenum Wis 54-1255 npe 8 acyltransferase negative mutant.

From a uracil-dependent derivative of Wis 54-1255 npe 8 strain Wis 54-1255 npe 8 pyrG (CBS 512.88) cells, 2 10 ⁷ protoplasts were made. The protoplasts were cotransformed with a mixture of 5 pg of pUC13 :: pyrG selective plasmid and 15 pg of lambda B21 DNA as described previously (EP-A-260762).

Hundreds of transformed cells are obtained from which the conidia are harvested and plated on plates containing the complex production medium of Example 1 at a density of 1-10 colonies per Perti dish. The plates are incubated for 3 days at 25 ° C and a spore suspension of penicillin sensitive Bacillus subtilis indicator strain is applied. Plates are incubated overnight at 30 ° C and the extent of inhibition zones on the bacterial lawn is determined.

Most of the transformed cells (75%) have a very small ring, similar to the penicillin-producing npe 8 pyrG recipient strain. The remaining 25% exhibited a high inhibition zone similar to that found in the wild-type strain Wis 54-1255.

HU 209 941 Β

From this it was concluded that the former group received only the pUC13 :: pyrG selective plasmid, while the latter group also received pUC13 :: pyrG and lambda B21, which restores penicillin production.

Cellular extracts of several transformed clones from both groups were determined for AT activity. After two days of growth, mycelia were harvested, washed as described in Example 3, frozen in liquid nitrogen and powdered. For each determination, 2.5 g of ground mycelium are suspended in 50 mM potassium phosphate buffer, pH 8.9 containing 5 mM DTT and 5 mM EDTA - final volume is 12.5 ml and the suspension is stirred for 25 minutes. The cell-free extract was obtained by centrifugation of the suspension at 1000 g for 5 minutes.

When determining AT activity, 2 ml of cell-free extract with 0.1 ml DTT (10 mg / ml), 0.2 ml 6-amino2. spreadsheet

Tribe Transforming vector Inhibition zone Penicillin G unit '/ mg protein formed Number of T copies Determined by Southern hybridization After 15 minutes After 30 minutes (1) Wis 54-1255 hpe 8 pyrG pUC 13 :: pyrG - examining 0.9 1 ' (2) u. the pUC13 :: pyrG plus lambda B21 - 1.7 1.1 1 ** (3) u. the u. the 11.9 9.5 > 1 (4) u. the u. the 10.8 7.0 > 1 (5) Wis 54-1255 not transformed 4.5 2.7 1

relative AT activity in the extract is inactive due to mutation **

Example 9

Increased Penicillin Production in a Host Strain Transformed with a Cryptic Y Gene In order to illustrate the effect of genes found to be involved in penicillin production, we increased the amount of such a gene in the Penicillium host strain. The "Y" gene contained in clones B9, L5 and G5 was cloned as 3 _; 0 kb BamHI plus SphI fragment to pPS47. The resulting pRH05 construct was transformed into P. chrysogenum Wis 54-1255 (ATCC 28089) cells and isolated phleomycin resistant clones. Several clones were tested for penicillin production in shaken bottles.

The results obtained with an isolated transformed cell are shown in Table 3.

Table 3

Tribe Relative penicillin production Wis 54-1255 100 Wis 54-1255 :: pRH05 122

Southern biot analysis showed that the transformed cell contains a higher amount of the Y gene than the untransformed host cell. Thus, it is incubated with Leppenicillanic acid (10 mg / ml) and 0.2 ml of phenylacetyl-coenzyme A solution (20 mg / ml) at 25 ° C.

After 15 and 30 minutes, the reaction was quenched with an equal volume of methanol and the sample was centrifuged (20 minutes; 5000 g). The supernatant is assayed for penicillin G content by known chromatography (HPLC) methods. The results of a typical experiment are summarized in Table 2. The data show that the transformed strains - (3) and (4) - AT have a 10 to 2 to 3-fold higher activity than the wild-type strain (5) AT, which is consistent with the increased gene content.

The IPNS + AT group was cloned into pPS54 to give vector pGJ101 A and B. A 5 kb Sal I fragment was blunt-ended with T4 DNA poly15 merase and, after treatment with T4 DNA polymerase, was ligated into a single HindIII site of pPS54 (ligase).

The Y gene significantly increased penicillin production.

The size of the transcript of the Y gene was determined by Northern biot hybridization: the transcribed sequence proved to be approximately 1.0 kb.

Claims (17)

PATENT CLAIMS CLAIMS 1. A method of isolating and optionally constructing a DNA library containing genes expressing β-lactam antibiotic-producing gom45bats which are directly or indirectly relevant for the production of a β-lactam antibiotic in such a fungus, characterizing that i) screening a collection of DNA prepared from such a fungus by probes derived from mRNA or DNA from a first culture of said fungus capable of producing said β-lactam antibiotic; ii) screening said DNA collection with probes derived from mRNA or DNA from a second culture of said fungus incapable of producing said β-lactam antibiotic; then 60 ii) identified a fragment containing such genes12 EN 209 941 Β fun, which are transcribed in the first culture but are not transcribed in the second culture; and iv) isolating these DNA fragments and optionally creating a bacteriophage library. 2. The method of claim 1, wherein said DNA collection is obtained from a first Penicillum culture capable of producing a β-lactam antibiotic and a second inability to produce said antibiotic. 3. A method for producing a recombinant transforming vector comprising the step of providing a suitable promoter, transcriptional sequence, translational activating sequence, regulatory sequence and DNA fragments according to claim 1, optionally a selectable marker gene and optionally a transformation efficiency. an enhancer sequence is ligated into a suitable vector. 4. The method of claim 3, wherein the DNA fragment is an acyltransferase gene, a cryptic Y gene or the bacteriophages L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16 and B23 in whole or in part. cryptic genes are ligated. The method of claim 3, wherein the promoter is a promoter of the Penicillium chrysogenum phosphoglycerate kinase gene. 6. The method of claim 3, wherein the gene encoding phleomycin resistance is inserted as a selection marker sequence. 7. The method of claim 3, wherein the promoter and the translational activator sequence of the acyltransferase gene of Penicillium chrysogenum are ligated as a promoter and a translational activating sequence. 8. The method of claim 3, wherein the regulatory sequence is a 3'-regulatory sequence of the acyltransferase gene of Penicillium chrysogenum. 9. The method of claim 3, wherein the DNA encoding the acyltransferase of the sequence shown in Figure 2 is inserted as a DNA fragment. 10. The method of claim 3 wherein the pyr G gene of Penicillium chrysogenum having the sequence shown in Figure 3 is ligated as a transformation efficiency enhancing sequence. 11. A method of producing a recombinant vector involved in enhanced production of a β-lactam antibiotic, comprising: i) digesting a suitable starting vector with a suitable restriction enzyme; ii) incorporating into the digested vector an acyltransferase gene, the cryptic Y gene or L12, K9, C12, P3, K11, B13, B20, G3, Gl, L10 directly or indirectly involved in the β-lactam antibiotic biosynthesis. , K16 and B23, one or more of the cryptic genes found in whole or in part. optionally subcloning, after digestion, a marker gene suitable for selection of said β-lactam antibiotic producing strain, and optionally, after digestion, a sequence that enhances the efficiency of transformation of the host cell with the vector and the vector; and iii) isolating the recombinant vector. 12. The method of claim 11 for the construction of plasmid pRH05 as a recombinant vector, comprising: 1) digesting pTZ18R with HindIII; ii) ligating the digested pTZ18R with a 1.5 kb HindIII fragment containing the pgk primer derived from P. chrysogenum; digesting the product with BamHI; ligating the pgk promoter fragment with the 1.0 kb BamHI / BglII fragment from pUT702 into the same reading frame as the phleo ^R (phleomycin resistance) gene; digesting the product with BamHI; and ligating with a 3.0 kb BamHI / SphI fragment containing the cryptic Y gene. 13. The method of claim 11 for the construction of plasmid pGJ101A and B as a recombinant vector comprising: i) digesting the ligation product obtained in steps i) to ii) of claim 12 with EcoRI; ii) ligating the product with a 2.4 kb Eco RI fragment containing the pyrG gene; digesting the ligation product with HindIII; ligating the product with a 5 kb SalI fragment containing the IPNS + AT group; transforming the ligation product with a suitable host cell; and selecting clones containing the IPNS + AT group in both orientations. 14. A process for the production of a transformed fungal host cell that produces an increased production of a β-lactam antibiotic, characterized in that i) transforming a suitable fungal host cell with a transforming vector produced according to claim 3; ii) cloning the resulting transformed cells; and iii) selecting clones that produce said β-lactam antibiotic in greater amounts than the parent host cell. 15. The method of claim 14, wherein the suitable fungal host cell is an acyltransferase gene, the cryptic Y gene or L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16 and B23 are transformed with a transforming vector comprising a gene selected from one or more of the cryptic genes found in bacteriophages. 16. The method of claim 14, wherein the Penicillium host cell is transformed. 17. The method of claim 14, wherein the host cell of the species Penicillium chrysogenum is transformed.