PT91410B - PROCESS FOR IDENTIFYING AND USING BIOSAETIC OR REGULATING GENES FOR THE ENHANCED PRODUCTION OF SECONDARY METABOLITES - Google Patents

Abstract

The invention relates to a process for the isolation of DNA fragments which express during the metabolism of micro-organisms and which are directly or indirectly essential for the production of a secondary metabolite or indirectly essential for the production of a secondary metabolite in that micro-organism, preferably a bacteria or a fungus, which comprises the stages of: a) scrutinizing a DNA bank prepared from the said micro- organism with sensors obtained from mRNA and DNA derived from a first culture of the said micro-organism which produces the said secondary metabolite, b) scrutinizing the said DNA bank with sensors obtained from mRNA or DNA derived from a second culture of the said micro-organism or a mutant thereof which does not produce the said secondary metabolite, and c) identification of the genes in the said first culture which are not substantially transcribed in the said second culture.

Description

description

PROCESS FOR THE IDENTIFICATION AND USE OF BIOSYSTIC GENES OR REGULATION FOR IMPROVED GOAL PRODUCTION.

SECONDARY BULLETS

INTRODUCTION

Technical Domain domain of the present invention concerns the isolation and use of genes for the production of secondary metabolites.

Background and Literature of Interest

As a result of improvements in the classic strain, penicillin production has been largely absent for the past four decades * These improvements of the classic strain were initially based on mutagenic treatments of Penicillium chrysogenua and subsequent selection of the mutants that produce more penicillin. The development of cloning techniques, however, introduced a new, potentially more powerful tool for further improvement of penicillin production in this fungus.

Penicillin is produced by the filamentous fungus P. chrysogenua in various enzymatic phases (eg E. Alvarez et al., Antimlcrob. Agents Chemother. 31 (1987) pp. 1675-1682). These phases are shown in Figure

1. Throughout this specification, genes directly involved in the biosynthetic pathway are those genes that encode the active enzymes in various stages that lead to the production of a secondary metabolite. Thus, in the case of the production of penicillin G or V, the genes that code for the enzymes are shown in Figure 1. The first reaction is the formation of the tripeptide £ - (L-O ^ - amylnoadipil) —L-cis. teini1-D-vanila from amino-adipic acid, cysteline and valine. The enzyme that is responsible for this reaction is the synthesis of ACV (hereinafter referred to as ACVS), a large multifunctional enzyme. The tripeptide is cycled by the action of N-isopenethyl synthase (hereinafter referred to as IPNS) or cyclase. The reaction product is isopenylcine N, a compound which contains the typical O-lactam ring structure and which has antibacterial activity. The final phase in the formation of penicillin consists of the exchange of the side chain of the isopenyllline N Î ± -oainoadlopic acid for a more hydrophobic side chain. The hydrophobic side chains generally used in industrial production are either phenylacetic acid, resulting in penicillin G, or phenoxyacetic acid, resulting in penicillin

V. It was suggested that the side chain exchange was a reaction catalyzed by a single enzyme (AL Demain (1983) in: AL Demain and NA Solooon (ed), Àntibiotics containing theJ3-lactam structure I. Springer Verlag, Berlin; pp. 189-228). However, a two-phase reaction involving the

6-ÀPA as an intermediary (E. Alvarez et al., See above). The enzyme that has been identified as being involved in the final reaction is acylCoA; 6 — APA acyltransferase (hereinafter referred to as AT); this enzyme was purified to homogeneity (E. Alvarez et al .. see above). The involvement of a second enzyme, catalyzing the transformation of IPN into 6-APA, cannot yet be confirmed or excluded.

It is not clear whether one or more enzyme reactions limit the speed of the penicillin biosynthesis process and if so, which enzyme phases are involved.

Since the penicillin biosynthetic pathway begins with three amino acids, which in turn each participate in other metabolic pathways, the regulatory phases in these pathways will also influence the penicillin biosynthesis. On the other hand, penile production is subject to a complex mechanism of catabolic repression of carbon sources and regulation of the nitrogen source (JF Martin et al. In: H. Kleinkauf, H. von Dohren, R. Donnauer and G Neseman (eds), Regulation of secondary metbollte formation, VCH Verlaggesellschaft, Weinheim (1985), pp. 41-75). They may also be involved in these types of regulation regulatory proteins. These regulatory proteins and the proteins regulated by them, are defined as being indirectly involved in the biosynthetic pathway of a secondary metabolite, in this case penicillin.

Until recently, the gene for only one of the enzymes active in the biosynthetic pathway for penicillin G, isopeni synthase, had been cloned and sequenced. cilina N (IPNS) or cyclase (L.G. et al., Gene 48 (1986) pp. 257—266), using the corresponding Acremonium Chrysogenum gene (S. M. Samson et al. Nature 318 (1985) pp. 191-194). Es_

This gene was cloned and identified by purifying the IPNS protein, determining the amino acid amino acid sequence. nal, preparing a set of oligodeoxyribonucleotides in accordance with this sequence and probing a genomic bank of cojbids with these mixed oligodeoxyribonucleotides (S.M. Samson, see supra).

Strain improvement studies using the Penicillium chrysogenum isopenicillin N synthase genes cloned into Penicillium chrysogenum resulted in enhanced enzyme activity, but no improvement in penicillin production was found, nor was it verified. stimulated penicillin synthesis (P.L. Slcatrud et al., Poster presentation 1987 Annual meeting of Society of Industrial Microbiology, Baltimore, August 1987, abstract published in SIM News 37 (1987) pp. 77).

It has been reported that the biosynthesis of β-lactam antibiotics is subjected to glucose suppression (J.F. Martin and P. Liras, TIBS 3 ^ (1985), pp. 39-44). This glucose repression was unequivocally established for the formation of the tripeptide by ÀCVS and for the activity of IPNS (Revilla et al., J. Bact. 168 (1986), pp. 947-952). For acyltransferase, on the other hand, the data is less convincing. Revilla et al (see above) report that AT is not subjected to glucose repression, but other data suggest that AT activity is absent, or at least decreased, in the presence of glucose (B.Spencer and T. Maung, Proc. Biochem, Soc. 1970, pp. 29-30).

It is not known at what stage of the expression. healthy repression by glucose is exerted; this repression can take place both at the level of transcription and at the level of translation. If the first regulation applies, the differences in mRNA levels between the cultures they produce and those they do not produce can be used to isolate those genes.

In addition, there is uncertainty in the levels of RNÀm’s that code for the various enzymes in them.

squid that produce penicillin.

SUMMARY OF THE INVENTION

Subtraction isolation methods are used to identify genes associated with the production of secondary metabolites in microorganisms. The method is exemplified with the production of penicillin in P. chrysogenum.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1

The biosynthetic pathway for penicillin 6 or V in P. chrysogenum * is shown schematically.

Figure 2

Physical map of lambda clones isolated by the method of the present invention. The lambda G2 and lambda B21 clones contain the cluster of the cyclase and acyl transferase genes. Lambda clones B9, G5 and L5 contain the cryptic Y gene. Other lambda clones contain other cryptic genes.

E - EcoRI; B - BamHI; H «HindIII; K - Xpnl; Sa «Saci;

Sp - Sphl; P - PstI; X - Xhol; Xb · Xbal; Hp · Hpal;

N - Ncol and Bg - BglII

K \\\\\\\\\ 5NS5NI · right arm of the bacteriophage lambda EMBL3 (9 Xb) | | «Left arm of bacteriophage lambda EMBL3 (20.3 kg) _n region that hybridizes to pen ⁺ cDNA ------ · unclear region of the map () - position / occurrence of unclear restriction site la and ra are the left arm and the right arm respectively of the lambda bacteriophage.

Figure 3

Nucleotide sequence and amino acid sequence deduced from the acyltransphere gene.

if _P. chrysogenua.

Figure 4

Nucleotide sequence and amino acid sequence deduced from the pyrG gene of P_chrysogenua. Es. this sequence forms the major part of the 2.4 kb EcoRI fragment that acts as a transformation stimulus sequence.

Figure 5

Nucleotide sequence of the pronator of the P. chrysogenua phospho-lycerate kinase gene.

Figure 6

national in 7 pUC13i ypyrG. a place in restriction and a fun map a place in restriction and a fun map national in pPS54.

Figure 8

A restriction site and a functional map of pRR05.

Figure 9

A restriction site and a functional map of pGJOl A and B.

Figure 10

A restriction site and a functional map of pPS47.

DESCRIPTION OF THE SPECIFIC FORMS OF ACCOMPLISHMENT

According to the present invention, fragments of DNA are included which include sequences that are Donocistronic or polycistronic. The gene (s) encoded by the sequences are translated into enzymes related to the production of secondary metabolites or other products of commercial interest. These sequences of interest are identified by comparison with RNA sequences isolated from a competent organism to produce the second metabolite, in which the genes of interest are actively expressed, and a microorganism in which the expression is silent. In this way it is possible to obtain fragments of DNA that encode one or more genes that are differentially expressed and that are involved in the formation of a product of commercial interest.

differentially expressed term is used through its application to express the gene or genes of interest which are specifically active only under certain defined conditions and which are absent (which means in the present specification to be present at a low level of 5X or compared to the active phase) under other equally well-defined conditions. The absence of expression can be a result of repression or the lack of induction of gene expression, mutation or other events that result in the silence of the transcription of the gene (s) of interest. The DNA that is isolated may result from a selection from a gene bank, whether from a genomic bank or a bank containing cDNA contained for example in a lambda vector or a cosmid cloning vector or an expression vector. By employing a cDNA probe enriched for sequences expressed during the biosynthesis of secondary metabolites, positive hybrids can be identified in the bank for subsequent manipulation to allow the preparation of expression constructs for the enzyme (s) as. associated with the production of the secondary metabolite. Therefore—

Next, a gene bank of a microorganism is searched using two cDNA probes, one of which is enriched in transcriptional active state sequences and the other is derived from the transcriptional silence situation. By comparison and subtraction, it is possible to isolate clones that contain gene (s) that are actively expressed only under the defined active conditions.

The method is exemplified by the isolation of genes involved in the biosynthesis of a secondary metabolite, more specifically penicillin, using two eons of cDNA, from mycelium grown in lactose (producer) and mycelium grown in glucose (non-producer).

The identified DNA sequences contain at least one gene that codes for an antibiotic biosynthetic enzyme and / or a regulatory protein from the integral pathway or more generally any protein that is involved, whether positive or negative , in the biosynthesis of said antibiotic.

The constructs that act positively, when properly introduced into a suitable host microorganism, increase the efficiency of the biosynthetic and operative pathways in the production of microorganisms that produce - lactams by increasing the dose of the gene or by higher expression of the gene. On the other hand, constructions that have a negative effect on the production of antibiotics (e.g. formation of by-products) can be isolated. These constructs are used to inactivate the gene that acts negatively by replacing the gene or by other methods with a similar effect. Both uses result in higher yields of the desired antibiotic during industrial production. This method is exemplified by microorganisms that produce —lactam for the production of antibiotics, particularly penicillins, and finds particular application in these cases. Preferably, the integrated expression block (expression cassette) will include genes encoding for enzi.

but that catalyze speed-limiting phases or genes that code for regulatory proteins for the induction of transcription or any other.

The exposed method furthermore provides sequences for which the encoded product is not known, but it appears that the sequence provides an improvement in the yield of a desired product. These sequences are referred to as cryptic genes, which means sequences that can be obtained by the isolation methods described herein, sequences that encompass genes for which no known function is yet attributable. These genes are characterized by being dosed and / or expressed in higher amounts in transformed host microorganisms compared to their non-transformed hosts. In addition to the cryptic genes, other genes provided are the IPNS and aclltransferase genes. A cryptic gene called Y has been found to provide improved penicillin biosynthesis.

The microorganisms used in the present invention include both prokaryotes and eukaryotes, including bacteria, such as those belonging to the taxonomic group of Actinomycetes or Flavobacterium, or fungi including yeasts, belonging to the genera Aspergillua, Acre monium or Penicillium.

Depending on the origin of the fragmen. several integrated expression blocks, either genomic or cDNA, prokaryotic or eukaryotic, can be constructed. With genomic DNA from a bacterium, the fragment containing a monocistronic or polycistronic coding region can include its own transcription start regulation region as well as a suitable transcription termination region and translation signals, for example a sequence from Shine — Delgarno and stop codons. When genomic DNA is from a fungus, normally only one gene is associated with a transcription start regulation region, such that each gene has its own independent transcription start regulation region. When using cDNA,

it is necessary to provide an appropriate transcriptional start regulation region, depending on the host organism used for subsequent expression.

The genes of interest can be obtained by searching a DNA bank prepared from said microorganism with probes obtained from mRNA or DNA derived from one ₍ first culture of said microorganism that produces said secondary metabolite; by searching for said bank of DNA with probes obtained from mRNA or DNA derived from a second culture of said microorganism or its autant, which does not produce said secondary metabolite, and identification of fragments containing genes transcribed in said first culture that are not substantially transcribed in said second culture.

Especially valuable genes include genes that are expressed specifically during antibiotic biosynthesis, including genes encoding for β-lactam biosynthetic enzymes known in the art, for example tripeptide synthase (ACVS), cyclase (INPS), acyltransferase (AT) , epimerase, expandase, hydroxylase, transcarbamoylase, methoxylase and transacetylase. Preferably, the genes encoding for IPN: 6-APÀ-acyltransferase or the cryptic gene ϊ are dosed or expressed in higher quantities, which results in higher yields, of the desired antibiotic in the transformed fungus.

It will be of interest to specialists. It is important in the matter that the gene (s) to be expressed in a host that produces a lactam may contain their own native promoter sequence that is recognized by a host cell RNA polymerase, or may be linked to any other suitable promoter, eg a different lactam biosynthetic gene or a glycolytic gene such as phosphoglycerate kinase, glyceraldehyde phosphate dehydrogenase, trlose phosphate isomerase or translation elongation factor, Ef-Tu, or the like.

Such a promoter can be used to influence the regulation of the expression of one or more

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genes that code for said enzymes. This will lead to an increased production of the antibiotic after transformation, a way that penicillin production is now possible under conditions where in the untransformed host strain do not lead to the production of penicillin, for example, glycolated enzymes are expressed in the presence of glucose , while penicillin production, on the other hand, is repressed in the presence. glucose (J.F. Martin, see above). By subjecting the expression of the penicillin biosynthetic genes to the regulation of a promoter of a glycollic gene, the genes can also be expressed in the presence of glucose and thus penicillin can be produced earlier in fermentation, when a high concentration of glucose is required for the production of a sufficient amount of mycelium. The selection marker can also be kept under the regulation of this promoter.

The present invention exemplifies the promoter of the P. chrysogenun phosphoglyceratokinase gene as a promoter to be used to overcome glucose repression of penicillin blosynthesis. This promoter was isolated from the genomic bank of P, chrysogenum by normal methods, using ollgodeoxyribonucleotide probes from the yeast sequence published by R.A. Hitzman et al., Nucleic Acids Res. 10 (1982) pp. 7791—7808. The nucleotide sequence of a phosphoglyceratokinase promoter is specified in Figure 5.

For the transformation of Penicillium. constructions that include at least one pa marker are employed. for the selection of transformed cells and, preferably, to enhance the conservation of integrated DNA. However, the vector preferably includes a DNA sequence known to increase transformation efficiency. An example of a DNA sequence with this property is the element ans, isolated, from Aspergillus nidulans (cf. Ballance and Tutner, Gene 36 (1985) pp. 321-331). The present invention provides a DNA sequence, isolated from a P. chrysogenum genome. which has been identified as a sequence with an effect similar to the effect of the βοβ sequence. Once

this sequence is native to P. chrysogenua, can be used to increase transformation efficiencies in P. chrYsogenum. The DNA sequence comprises the PyrG gene of P. chrysogenua and can be used either alone, in combination with a pyrG host. this case in which the aforementioned DNA sequence provides both the selection of transformants and the effect of increasing the transformation (cf. EPA-260762), or in combination with another selection marker, for example a gene that codes for resistance to a biocide, such as ileum, mycine. In the latter case, the selection of transformants and the transformation enhancement effect are provided by two separate DNA sequences and the only function of the pyrG element is to enhance the transformation frequencies.

Useful markers for the selection of transformant clones can be homologous or heterologous biosynthetic genes capable of complementing an auxotriphic need of the host cell, caused by a defect in the metabolic pathway for an amino acid, for example arginine, for a nucleotide precursor, for example example uracil, and similar.

The structural gene providing the marker for selection may be native to the host, ro wild type Penicillium or a heterologous structural gene as soon as it is functional in the host. For example, structural genes that code for an enzyme in a metabolic pathway can be derived from Penicillium or other filamentous fungi, for example Aspergillus. Neurospora, Podospora. or yeast, in which the structural and functional gene in the host Penicillium and complements the auxotrophy for prototrophy.

Complementary structural gene can be derived from a metabolic pathway, such as the synthesis of purines or pyrimidines (nucleosides) or amino acids.

Of particular interest are the structural genes encoding enzymes in the pyrimidine pathway, for example the gene encoding the orotidine-5'-phosphate decarboxylase enzyme (pyrG or pyr4). Other genes of interest are the bios- 12 genes

synthetic amino acid compounds, for example, orinitine-carbamoyl transferase (arqB) and arginine suctionate lyase (arq4) The number of the aforementioned selection markers is provided by EP-A-260762.

Instead of auxotrophic markers, fermentation markers, such as the ability to use amides as the sole source of carbon or azo, to culture in various sugars, for example in galactose, or the like, can be used.

In addition, genes encoding biocide resistance, such as hygromycin, gentamicin, phleomycin, glyphosate, bialaphos, and the like, can be used.

Constructions are provided which contain the sequence of interest and which may include other functions, such as replication systems in one or more hoses. stonemasons, for example in cloning hosts and / or target hosts for expression of secondary oetabolite; one or more markers for selection on one or more hosts, as indicated above; genes that enhance the efficiency of transformation; or other specialized function.

The construct must contain at least one gene isolated by the method of the present invention. The construct can be prepared by conventional means by isolating other desired genes from a suitable host, synthesizing all or some of the genes or their combinations. Similar to the regulation signals, the regions of beginning of trans. and translation and termination regions can be iso. from a natural source, can be synthesized or obtained by combinations of these methods. The various fragmen. All of these can be subjected to endonuclease digestion (restriction), ligation, sequencing, in vitro mutagenesis. repair by initiators or similar technique. The various combinations are well known in the literature and can be used to achieve specific purposes.

The various fragments can be combined.

cloned, isolated and equated according to the techniques, “13 -

conventional techniques. After each manipulation the DNA fragment or combination of fragments can be inserted into the cloning vector, the vector can be transformed into a cloning host, for example in E. coli, that of the cloning host can be cultured and lysed, the plasmid can be isolated and the fragment can be analyzed by restriction analysis or by sequencing, their combinations or similar operations. E. coli can also be used as a host for the expression of genes of interest for the purpose of production. high amounts of protein.

Various vectors can be used during the development of the construction and transformation of the host cell. These vectors can include cloning vectors, expression vectors and vectors that provide integration into the host or the use of simple DNA for transformation and integration.

The cloning vector will be characterized, for the most part, by a marker for the selection of a host containing the cloning vector and optionally a transformation stimulation sequence, it may have one or more polylinkers, or additional sequences for insertion, if_ selection, manipulation, ease of sequencing, excision or similar.

The insertion of a construct that includes the transcription start and translation region and the termination regions can normally be achieved by means of expression vectors; alternatively the construction can be read. establish one or both of the regulatory regions, which will be provided by the expression of the vector in the insertion of the sequence coding for the protein product.

DNA encoding the enzyme (s) of interest can be introduced into a Penicillium host in a substantial manner according to the procedure described in EP-A-260762.

The efficient transformation of Penicil liun is provided to produce transformants that have one or more structural genes capable of expression, particularly

integrated into the host genome (members). DNA constructs are prepared that allow selection of transformed host cells. Conditions for transformation are used which result in a high frequency. transformation to thereby ensure the selection and isolation of transformed hosts that express the structural gene (s) of interest. Transformants provide stable maintenance and expression of integrated DNA. It is to be noted that the host transformed according to the present invention can be used as a starting strain in strain improvement processes instead of using a DNA mediating transformation, for example, protoplast fusion, mass matching and mutation. The resulting strains are considered. considered to be part of the present invention.

The genes with interest to be in. Transformation troducts can form an integral part of the transformation vector, but it will often be more convenient to provide these genes in a separate vector in the transformation mixture, thereby introducing said genes by cotransformation together with the selective vector, which is a process reasonably effective on filamentous, sos fungi (PJ Punt et al., Gene 56 (1987) pp. 117-124; X. Wernars et al, Mol. Gen. Genet: 209 (1987) pp. 71-77; IE Mattern et al., Mol. Gen, Genet. 210 (1987) pp. 460-461).

As a result of the have transformation. frog at least one copy of the gene (s) of interest, often two or more, usually not exceeding about 100, most usually not exceeding about 10. The number will depend above all on whether to employ stable episomal integration or maintenance, the number of integrated copies and the construction to be prepared to undergo amplification and similar factors.

The present invention exemplifies a method for isolating genes that involves the biosynthesis of penicillin from a gene bank of the producing organism, P .. chrysogenum, using specific cDNA probes, a method that is paradigmatic for the identification of cryptic genes

to improve the production of secondary metabolites. This method for the isolation of DNA constructs that encode one or more genes that take part in the biosynthesis of secondary metabolites, comprises searching a gene bank with a cDNA probe enriched in sequences specifically expressed during biosynthesis. By express specifically, it is meant that the expression of these genes is silent or at a very low level (for example less than 5% of the production level) in the absence of penicillin production and in contrast to high during penicillin production . To this end, radioisotope-labeled cDNA probes or otherwise are synthesized in mRNA templates isolated from P. chrysogenum mycelium during the penicillin production phase.

The probes are then enriched in sequences that specifically hybridize with the desired genes by eliminating those cDNA sequences that hybridize with mRNA derived from a Penicillium fermentation under conditions that do not allow the production of penicillin, for example by the high glucose concentration. Using this enriched probe, clones are selected from a chrysogenum gene bank that does not hybridize with a probe derived from a non-producing mycelium. It appears that a large number of clones thus isolated encode the biosynthetic enzyme of penicillin synthase isopenicillin N (IPNS or cyclase).

In addition, some copies of the gene encoding the side chain exchange enzyme (acyltransferase) are found among the selected clones. This presence is proven with experiments using a DNA probe, based on the amino acid terminal sequence of the purified enzyme. The entity of these clones is verified biochemically and biologically. The nucleotide sequence and the deduced amino acid sequence of the acyltransferase gene are specified in Figure 3. Surprise. Accordingly, the genes encoding the isopenicillin N synthase and acyltransferase enzymes are both present

in an ADR fragment. This presence was demonstrated by the hybridization of a genomic bank of chrysogenua in the ilium vector of EMBL3 with separate probes, specific for each of the two genes mentioned. Identical clones hybridize separately with the two probes.

In addition, after the construction of a physical map of a genomic lambda clone and the hybridization of digestion products of the lambda clone with sepa probes, used for both genes, the genomic organization is verified to be as outlined in Figure 2 (clones B21 and G2). The presence of the two genes of an extensive DNA fragment allows the construction of strains of P. chrysogenum with a higher dosage of the isopenicillin N synthase and acyltransferase genes, without disturbing the relative organization or balanced expression of the two genes. In addition, the introduction of multiple plots of said extensive DNA fragment allows the expression of the two genes of said fragment in its natural environment with sequences upstream and downstream that are identical to the normal situation.

Both balanced expression and the maintenance of the natural environment can be beneficial for the efficiency of the expression gene and thus for the production of penilin. The isolation techniques of the isopenicillin N synthetase and acyltransferase genes can be applied with advantage to the isolation of other bios genes. synthetic penicillin by chromosomal movement techniques, in which penicillin biosynthetic genes can be clustered.

In addition, several cryptic genes have been isolated, for which no function has yet been identified but which probably play a role in the biosynthesis of —lactams. Figure 2 shows a physical map of the cryptic genes of the present invention (clones B9 to B23).

Of these cryptic genes, the gene designated by Y, present in clones B9, L5 and G5, when used in the transformation (into a 3.0 kb Sphl + BamRI sub-fragment) of a suitable host, stimulates the production of penicilli17

in 26Z, in comparison with the untransformed host. This fact demonstrates the involvement of the Y gene product in penicillin biosynthesis. In addition, this observation demonstrates that the transformation using the genes that isolated Fr. The method of the present invention, without knowledge of its function or identity, can be applied successively to the improvement of the P. chrysogenum strain.

The present invention is further exemplified by the transformation of Penicillium chrysogenoa with genes that are specifically expressed under conditions in which the antibiotic is synthesized, and which code for gene products that catalyze biosynthetic reactions that go against said antibiotics.

One of these enzymes, acyltransferase (hereinafter referred to as AT), catalyzes the final phase in the biosynthesis of penicillin, that is, the exchange of the amlnoadipyl fraction of Isopenicillin N with a precursor of the la chain. hydrophobic acyl ester, for example phenylacetic acid or phenoxyacetic acid, thereby giving rise to penicillin G or penicillin V, respectively.

The acyltransf era gene is prized. if P_. chrysogenum, including the nucleic acid sequence. The present invention provides conservative mutations, in which the sequences encode the same amino acid sequences, but can have up to 30Z of different bases, or mutations that are not conservative, in which less than about 10Z, more usually less than about 5Z, and preferably no more than IX of the amino acids are substituted or eliminated, and there is less than 5Z of amino acids inserted, the percentage being based on the number of amino acids occurring. natural science. In addition, fragments of the two acids encoding the enzyme can be used as probes for the production of antibodies, usually at least about 9 codons, more usually at least about 15 codons, as well as their expression products or similar. The son. can be used to identify the enzyme in other species by using nucleic acids for hybridization or

antibodies for identification of cross-reactive proteins.

Searching for unnatural side chain precursors or other penicillins or cephalosporins that can serve as a substrate for acyltransferesis po. to be complemented by the search for mutant acyltransfer enzymes that can accept precursors of a side bitch other than phenylacetic acid or phenoxyacetic acid, or penicillins or cephalosporins other than isopenicillin N. The present invention provides the material press for such a search for a mutant acyltransferase enzyme. The best host for mute cloning experiments. is E. coli: since E. coll does not have the cutting apparatus for removing the ions present in the acyltransferase gene, a cDNA clone of the acyltransferase gene is the preferred sequence for the expression of the ee enzyme E. coll. This cDNA sequence can be easily mutated by processes well known in the art such as, for example, radiation treatment (X-rays or UV) or mutagenic chemicals such as ethyl methanesulfonate, nitrosoguanldine or methyl methanesulfonate) or specific mutagenicity for a place, to get mutant enzymes that put it there. recognize unnatural side chain precursors as a substrate and catalyze the formation of unnatural penicillins from isopenicillin N, or on the other hand catalyze the exchange reaction in penicillins and cephalosporins other than isopenicillin N.

isolation of AT and Y genes and other biosynthetic genes allows for the identification of regulatory elements of individual genes such as a promoter, an upstream activator sequence (SAM), a terminator and si. pillars. This can be accomplished by comparing the sequence of the genes among themselves and by comparing the sequence as obtained for the isope synthetase biosynthetic gene. nicillna N and other related genes. This last comparison, however, may present the specific nature of the regulation of gene expression in the group of penl biosynthetic genes

cilina.

The identification of such a bioasynthetic regulatory element of penicillin leads to the identification of specific regulatory proteins by means of standard techniques such as gel retardation, crosslinking, DNA tracing and the like. Isolation of specific regulatory protein by affinity chromatography results in the cloning of the gene encoding that protein and subsequent meningulation in a suitable host.

By using the AT gene, the Y gene and other cloned penicillin biosynthetic genes, modified enzymes can be designed and synthesized. These modifications will result in modification of the characteristics of the enzymes, such as a change in the optimum pH or temperature values, a change in the establishment or a change in the specificity of the substrate. The host strains, transformed with genes that code for these modified enzymes, can be programmed to perform antibiotic synthesis under different conditions or to synthesize alternative antibiotics, for example ampicillna instead of penicillin.

In another aspect of the present invention, the cloned genes can be used to transform host strains that do not naturally contain these enzymes. It is known that in Streptomyces and Acremonlum the AT enzyme does not occur, whereas in Penlcillium, on the other hand, the genes of the blosynthetic enzymes of cephalosporin and cephamycin do not occur. The introduction of such genes into hosts will result in blosynthesis of cephalosporin or cephamycin by Penlcillium or penicillin or cephalosporins with a hydrophobic side chain by Acremonlum.

It is evident from the following results that the production of secondary metabolites can be greatly increased using selection processes that allow the identification of DNA associated with the production of a secondary metabolite. Using subtraction methods on specific identification strings associated with the production—

secondary metabolites, it is possible to isolate and identify the mRNA and cDNA to be used as probes. Thus, it appears that fragments containing cryptic genes, which do not yet have a known function, have a greatly increased production of secondary metabolites and can be used to transform a host for the production of the secondary metabolite. This process is specifically exemplified for penicillin.

In addition, an acyltransferase gene according to the present invention can be used in several ways, as an enzyme for the modification of β-lactam compounds, as a brand, as a source of an antigen for the production of antibodies to acyltransferase , as a source for a promoter sequence, as a source for expressing high amounts of protein for crystallization, as a template for in vitro mutagenesis to obtain an enzyme with modified characteristics, etc.

All publications and patent applications referred to in this specification are hereby incorporated by reference as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Although the preceding invention has been described in full detail by means of illustrations and e. xemples for the sake of clarity and understanding, will be quickly. It is evident to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made without exceeding the spirit or scope of the claims in the appendix.

The following examples are intended to illustrate the invention without limiting its scope.

EXAMPLE 1

Construction of a genomic bank of Penicillium ctinsogenua

A genomic bank of Pe niclllium chrysogenum (ATCC 28089) was constructed substantially according to procedures known in the art (T. Maniatis et al., (1982), Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N. Y.). The chromosomal DNA was extracted from Penicillium chrysogenum by the formation of protoplasts from the mycelium as previously described in EP-A260762.

The protoplasts were then lysed by diluting the isotonic suspension (0.7 M XC1) with four volumes of TES buffer (0.05 M Tris-HCl pH 8.0, 0.1 M EDTA, 0.15 M NaCl). To the lysate, 1Z sodium lauryl sulfate was added and the mixture was incubated at 55 ° C for 30 minutes. After extraction with phenol and two extractions with chloroform, the DNA was precipitated with ethanol, dried and dissolved in a 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 and then with proteinase K at 200 µg / ml at 42 ° C for 1 hour. The solution was extracted once with phenol and twice with chloroform. An equal volume of isopropanol was poured over the aqueous phase and the DNA was collected at the interface by rotating the loop with a glass rod. After drying, the DNA was dissolved in TE buffer. The molecular weight of a β DNA preparation

thus obtained was about 10. The DNA was partially digested with Sau3A. linked to BamHI-cut dephosphorylated EMBL with 3 arms (Promega Biotec, Madison WI, USA), and packaged in lambda bacteriophage capsids using Packagene Sys. has to Promega Biotec. All reactions were carried out according to the manufacturer's recommendations except for the packaging reaction which was carried out at 22 ° C for 2 to 3 hours. The banks were amplified by inocula22

Plated phage plates, incubation for 7 to 8 hours at 37 ° C and phage elution using 4 ml SM buffer (0.1 M NaCl, 0.01 M MgS0 ₄ , HCl 0, 0.5 M pH 7, 5, gelatin 0.012) per Petri dish.

EXAMPLE 2

Isolation of genes specifically expressed during biosynthesis of penicillin using a different separation process.

The genes that are expressed specifically or predominantly during penicillin biosynthesis were identified by probing the genomic bank of Example 1 with labeled cDNA probes synthesized in mRNA templates extracted from producer (grown in lactose) and non-producer (grown in glucose) mycelium , and clones that predominantly give a positive signal with the previous (+) probe are selected.

Messenger RNAs were isolated from cultures with 3 or 6 days in the production medium (cf. Example 3) (preparation +) or in the same medium with the glucose-substituted lactose (preparation-). The mycelium was collected by filtration, frozen in liquid nitrogen and homogenized and the mRNA was isolated using the guanidinium isoriocyanate method as described by T. Maniatis et al. (see above).

CDNAs were synthesized and 3 2 labeled for high specific activity with Q * - pJ ~ dATP against the two mRNA populations in a 30 1 reaction mixture containing

12.5 mM MgCl ₂ 50 mM Tris-HCl pH 8.3 100 mM KC1 125 mM DTT 2 u / a1 RNasin 500 am dGTP

500 aM dCTP 500 the ^m dTTP 25 AM dATP 0.1 Ag / ml BSA 100-200 Ag / ml poly A ⁺ RNA 50-60 Ag / ml oligo dT ₁₂ _ ₁₈ 1.2 u / a! reverse tranacryptase 1.67 / Ai / 1 U? - ³² p] dATP

ηοβ which PoliA ⁺ RNA and oligo-dt were mixed separately, heated to 100 ° C for 1 minute and cooled in ice water before adding to the reaction mixture. After 1.5 hours of incubation at 42 ° C, 5 µl of 1 mM dATP was added and the incubation continued for 30 minutes. Subsequent. The reaction mixture was then brought to 20 mM in EDTA, 40 mM in NaOH (final volume 100 χΐ) and heated to 65 ° C. After 1 hour of incubation, 5 µl of 1 M Tris-HCl pH 8.3 was added,

1 of 0.01 N HCl, 7 g of calf thymus DNA, 100 1 of TES buffer (10 mM Tris, 1 mM EDTA, 1Z SDS pH 7.5) and 200 æl of 5 mM ammonium acetate and precipitated DNA with 800 l of ethanol for 16 hours at -20 ° C.

The precipitate was separated by centrifugation, washed with 702 ethanol, dried and dissolved in 32.5 L TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). The cDNA (+) preparation was then enriched in the sequences expressed specifically during penicillin biosynthesis by two successive cycles (cascades) of hybridization against a mRNA preparation (-) in a 75 µl.1 reaction mixture containing

32.5 /.(1 (+) CDNA 10 Al (-) mRNA (l ^ g / _A l) 30 ,"1 1M NaPO ^ pH 6.8 1.5 Al 102 SDS 1 0.5 M EDTA

After incubation for 16 hours at 68 ° C, 102 „1 of water (final concentration of 170 aM phosphate) was added and the mixture was passed through a balanced hydroxylapatite column and 170 mM phosphate at 68 ° C. Under these conditions, double-stranded nucleic acids bind to the column while single-stranded acids are eluted. The eluate was collected, diluted against TE buffer for

1.5 hours and precipitated with ethanol after the addition of 4 µg of vehicle DNA (calf thymus). This process was repeated and the final vehicle cDNA (calf thymus). This process was repeated and the final unbound cDNA was used directly as a probe to search the genomic bank for a Penicillium strain as follows:

A sample from the amplified bank of Example 1 was inoculated onto 5 Petrl capsules so that each capsule contained 1500 plates. The plates were transferred in duplicate to Genes Screen Plus filters (New England Nuclear) according to the manufacturer's recommendations. A set of filters was probed with the tagged enriched cDNA (+) preparation; the duplicate set was probed with the cDNA (-) for comparison.

Positive plates were purified and subjected to a second selection. For this purpose, 96 plaques were selected which gave a predominantly positive signal with the cDNA probe (+).

The DNAs of the recombinant phage that had given a strong or moderate signal in the initial selection were skin markets and were used as an opportunity to perform a Northern blot search of Penicillium RNAs isolated from a producing micelle. and non-producer, in order to establish levels of expression under both conditions. Hereby the recombinant clones were divided into three groups:

Class 1 contains highly expressed genes during penicillin biosynthesis and is exemplified by clones

* G2 * Β9, L5 and G5 * L12 * K9

Class 2 moderately expressed, exemplified by * C12 * P3 and 111 * B13 * B20

Class 3 weakly expressed, exemplified by * G3 * G1 * 116 * IOL * B23

The physical maps of these recombinant phages are shown in Figure 2. Clones G2 and B21 gave a positive hybridization signal when probed with a specific N isopenicillin synthase probe (S.M. Samson et al., See supra). Surprisingly, it appears that the same clones hybridize to a specific acyltransferase probe (see Example 5).

EXAMPLE 3

Purification of acyltransferase

The strain Penlcillium chrysogenum (ATCC 28089) was inoculated (at 2 x 10 ^ conids / ml) in a complex inoculation medium containing: corn maceration liquid (20 g / 1); distillery soluble (20 g / 1); sucrose (20 g / 1); CaCOg (5 g / 1) (pH before sterilization 5.7). After 36 hours of incubation at 25 ° C and 250 rpm, the culture obtained was used to inoculate twenty volumes of complex production medium containing: corn maceration solids (35 g / 1)? lacto, if (25 g / 1); potassium phenylacellate (2.5 g / 1); MgSO ^^ E ^ O

(3 g / 1); KH ₂ PO ₄ (g / 1) j Corn oil (2.5 g / 1); CaC (> ₃ (10 g / 1). After the incubation continued for another 48 hours, the mycelium was collected by filtration and the filter cake was washed four times with cold 0.15 M NaCl.

200 g (wet weight) of mycelium were suspended in 700 ml of 0.05 M Tris-HCl buffer (pH 8) containing 5 mM dltiotreltol (hereinafter referred to as TD buffer) and the mycelium was disintegrated in a Braun disintegrator (Braun, Meleungen, RFA) using Ballotini glass spheres (Sigma type V, 450 to 500 µm in diameter) for periods of 30 s at intervals with refrigeration in an ethanol / dry ice bath.

The extract was then centrifuged for 30 minutes at 20,000 x g. This and all the following steps were performed at 4 ° C. 640 µl of extract 27 µl of a solution of 10% w / v protaamine sulfate in 0.05 M Tris-HCl to pB 8 were added slowly after stirring for 45 minutes the precipitated nucleic acid was removed by centrifugation. at 20,000 xgeo supernatant was fractionated by precipitation with ammonium sulfate while maintaining the pH of the solution at 8.0 during ammonium sulfate additions. The fraction that precipitated between 40Z and 55Z of the saturation was dissolved in TD buffer containing 1M ammonium sulphate and applied to a CL-4B (1.8 x 16 cm) column of equilibrium with the same buffer. The column was washed with TD buffer at a speed of 5 ml / min until no more unbound proteins were released.

Then the acyltransferase was eluted from the column with ethylene-glycol 40Z in 0.05 M Tris-HCl at pH 8.0.

The eluted fractions were analyzed for the detection of acyltransferase activity by incubation at 25 ° C in a reaction mixture containing 0.2 mM phenylacetylcozyzase, 0.2 mM 6-aaminopenicillanic acid, dithiothrei! 5 mM tol, 0.1 M Tris-HCl at pH 8.0 and enzyme extract in a final volume of 200 µl. After 10 minutes the reaction was stopped by adding 200 µl of methanol. The samples were centrifuged at 5000 x g and penicillin G was analyzed in the—

brenadante by conventional microbiological methods.

The active fractions of the phenylsepharose column were pooled and applied to a Sephacel-DEAE column (1.5 x 20 cm) equilibrated with buffer and the acyltransferase activity eluted with a linear NaCl gradient (0 to 0.25 M) in buffer TD at a flow rate of 0.25 ml / mln. The combined active fractions were precipitated with 70% ammonium sulfate and the solid was dissolved in 3 ml of TD buffer and applied to a column of Sefadex G — 75 (coarse grain) (2.6 x 70 cm) equilibrated with TD buffer. The acyltransferase was eluted using TD buffer with a flow rate of 9 ml / h and was collected in the last part of the eluted fractions as a symmetrical peak of protein corresponding to the acyltransferase activity: The final purification was 258 times.

EXAMPLE 4

Determination of the amino acid terminal amino acid sequence and design of the corresponding mixtures of ollgonucleotide probes.

The enzymatic preparation, obtained as described in Example 3, was run on a SDS-PÀGE gel (U.K. Laemmli, Nature, 227 (1970) pp. 680 ff) (132, 50 A acrylamide). A 29 kD band (about 10 g protein) was separated from the SDS gel and the protein was electrophoretically transferred to a PCGM-2 membrane (impregnated glass fiber, polybene, Jansen, Beerse, Belgium) ), using a Nova Multlphor II stain unit (LXB; 0.8 mÀ / cm; 90 min; 5 mM sodium borate electrode plug at pH 8.0). After spotting, the PCGM membrane was flushed four times with a solution containing 25 mM NaCl and 10 mM sodium borate at pH 8.0 and was air-dried. The protein band absorbed by PCGM thus obtained was analyzed to determine the N-terminal amino acid sequence, using a gas phase sequencer (Applied Biosystems model 470 a). The following sequence was determined:

According to the underlined part of the amino acid sequence. the following sets of oligodeaoxirri were synthesized. bonucleotides:

T

A C A T T

5 * CA GG CA AA TGGGA 3 ’

G A G C C

The terminal sequence of amino acids from a 10 kD band present sometimes in the preparation was also determined, but was not used for the construction of an oligodeoxyribonucleotide probe. The obtained sequence is:

Met-Leu-His-Ile-Leu-X-Gln-Gly-Thr-Pro-Phe-Glu-Ile-Gly-Tyr-Gli £ -His-Gly-Ser-Ala — Ala-Lys-Ala-Val-Ile -Allah.

EXAMPLE 5

Identification of the acyltransferase gene

DNA from several clones of Example 2 was digested with the restriction endonuclease Sail, the fragmen. All of them were separated on a 0.7Z agarose gel, transferred to Genescreen Plus and hybridized with the oligonucleotide mixtures of Example 4 marked at the end by E ^ Pj. Clones that gave a positive signal were mapped by restriction analysis using normal methods. Two physical maps represent. Representatives derived from the recombinant phage, lambda B21 and lambda G2, are shown in Figure 2. The mixture of oligodeoxyribonucleotides specifically hybridizes with the subfragment EcoRI / HindIII indicated on the map. This and adjacent fragments were recloned in pTZ 18/19 (United States Biocheaical Corporation) and subjected to nucleotide sequence analysis.

taken. The determined sequence and the deduced amino acid sequence are shown in figure 3.

The amino-terminal amino acid sequence of a 10 kD band also present in the preparation was determined and found to correspond to a DNA sequence upstream of the 29 kD sequence. Therefore, AT is probably synthesized as a 40 kD protein. This notion is confirmed by the length of the AT messenger, which has been shown to be 1500 bases (similar to the isopenicillin N synthetase mRNA encoding a 38kD protein), thus taking into account the 3 'and 5 * untranslated regions 100 bases.

The amino acid sequences of the 29kD proteins (which have been extended to Thr-Thr-Ala-Tyr-Cys — Gln-Leu — Pro — Asp — Gly — Ala-Leu-Gln-Gly-Gln-Asn-Trp — Àsp- Phe— Ser-Ala — Thr-Lys-Gln-Ala) and 10 kD revealed the presence of two introns. A third intrao is postulated on the basis of crude composition and 10 kD protein amino acids (97 residues) and following consensus to their limits (D.J. Ballance, Yeast 2 (1986) pp. 229-236). The presence of the third intrao was confirmed by primer extinction and Northern blot hybridization using ollgonucleotide probes from coding and non-coding regions.

EXAMPLE 6

Construction of pPS47 phosphogiicerate kinase gene (pgk) was isolated from a Penicillium genomic bank by normal methods (Manlatis; Example 1), using the corresponding yeast gene (Hitzeman et al., See supra) as a hybridization probe . The pgk promoter region is paired. specified by the sequence shown in Figure 5, which is located directly upstream of the Pgk coding region.

pgk promoter from P .. chrisogenua

it was cloned into pTZ8R as a 1.5 kb HlndlII fragment and a clone having the desired orientation was selected.

Subsequently, the resistance gene. phleomycin was cloned into a BamHI site of the polylinker of this clone as a 1.0 kb BaaHI - BglII fragment, isolated from pUT702 (Cayla, Toulouse Cedex, France). The pgk promoter was fused to the phleomycin resistance gene in frame agreement by exterine closing the cycle of the sequence to be deleted using an oligonucleotide with the sequence: *

I

I

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

In addition, this oligonucleotide introduces an Ncol site at the ATG position (underlined).

EXAMPLE 8

Biological and biochemical verification

PA IDENTITY OF AT CLONES

The identity of the AT clones was verified by complementation of a negative mutant in relation to J? Acyltransferase. chrysoRenum Wis 54 - 1255, npe 8.

Co-transform 2 x 10? protoplasts of a derivative requiring uracil of strain Wis 54 - 1255 npe 8, Wis 54 - 1255 npe 8 pyrG (CBS 512.88) with a 5 µg mixture of the selective plasmid pDC 13 :: pyrG and 15 µg. g of B21 lambda DNA as previously described (EP-A-260762).

A few hundred transformants were obtained from which the conidia were separated and plated in the complex production medium of Example 1 at a density of one to ten colonies per Petri dish. After three days of incubation at 25θϋ, the plates were covered with a spore suspension of a penicillin sensitive Bacil las subtilis indicator strain and incubated overnight at

ο - -

C to determine the size of the zones of inhibition in the bacterial field.

Most of the transformants (75Z) had very small halos, similar in size to those of the non-peni producing pyrG npe 8 receptor strain. cilina. The remaining 25Z showed wide zones of inhibition comparable to those of the wild type strain, Vis 54-1255. It was concluded that the first class received only the plasmid se— lectlvo pUC 13:: pyrG. while the latter had received both pOC :: pyrG and lambda B21, which restores penicillin productivity.

For several transformant clones of the two groups the level of AT activity in isen extracts. Cell counts were determined as follows: mycelium was collected after two days of incubation as described in Example 3, washed, frozen in liquid nitrogen and sprayed. For each analysis, 2.5 µl of crushed micelle were suspended in 50mH potassium phosphate buffer (pH 8.0) containing dithiothrei. 5 mM tol and 5 mH EDTA (final volume 12.5 ml) and stirred for 25 minutes. The cell-free extract was obtained by centrifuging the suspension (5 minutes at 1000 x g).

AT activity was analyzed by incubating 2 ml of cell-free extract with 0.1 ml of dithio. treitol (10 mg / ml), 0.2 ml of 6-aminopenilclic acid (10 mg / ml) and 0.2 ml of phenylacetyl solution - coenzyme A (20 mg / ml) at 25 ° C.

After 15 or 30 minutes, the reaction was stopped by adding an equal volume of methanol and the sample was centrifuged (20 minutes at 5000 x g). The supernatant was then analyzed to determine the penicillin G formed by chromatographic methods known in the speclalide. (HPLC). The results of a typical test are shown in Table 2 below. These data show that in transformed strains (3) and (4) the level of AT activity is increased, 2 to 3 times beyond that of the wild type according to the increased dosage of the gene.

cluster of IPNS plus AT was sub.

cloned into pPS54, giving rise to pGJO1 A and B. A 5 kb fragment was neutralized by the action of a DNA T4 polymerase and ligated into the unique HindIII site of pPS54, after treatment of this vector with DNA T4 polymerase.

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EXAMPLE 9

Increased production of penicillin on the back of the host strain transformed with the cryptic Y gene

To show the effect of the aqnl genes identified as involved in the production of penicillin, the genetic load of one of these genes was increased in a host strain of Penicillium. For this purpose, the Y gene, contained in lambda B9, L5 and G5 clones, was subcloned as a 3.0 kb BamHI-Sphl fragment in pPS47. The resulting construct, pRH05, was used for £ transformation. chrysogenum Wis 54-1255 (ATCC 28089) and phleomycin resistant clones were isolated. Several clones were tested for penicillin production in shaken Erlenmeyer flasks.

The results obtained for an isolated transformant are shown in the following Table.

Table 3 strain relative production of penicillin

Wis 54-1255 100

Wis 54-1255 :: pRH05 122

The increased genetic dosage of the Y gene in the transformant, by comparison with the untransformed host, was confirmed by Southern blot analysis.

Consequently, the increased genetic dosage of the Y gene, a cryptic gene, isolated by the method of the present invention, results in a substantial increase in penicillin production.

The size of the transcription for the Y gene was determined by Northern blot hybridization: the transcription is about 1.0 kb in length.

Claims (1)

- 1 · Process for the isolation of DNA fragments which are expressed during the metabolism of organisms and which are directly and indirectly essential for the production of a secondary metabolite in this microorganism, preferably a bacterium or a fungus, characterized by understanding the phases in : (a) screening a bank of DNA prepared from said microorganism with probes obtained from mRNA or DNA derived from a first culture of said microorganism, which produces said secondary metabolite; (b) scrutinizing said DNA bank with probes obtained from mRNA or DNA derived from a second culture of said microorganism or a mutant that does not produce said secondary metabolite; and (c) identifying the gene containing fragments transcribed in said first culture that are not substantially transcribed in said second culture. - 2 · Process for the preparation of a DNA construct characterized by comprising the phases of: (a) isolation of fragments that are expressed during the production of a secondary metabolite according to claim 1; and (b) subcloning the Isolated fragments to form a DNA construct that contains the gene or genes. of interest and all the necessary transcription and translation elements and optionally a selection marker. - 36 3 (a) (b) (c) (d) 4 _ Process for obtaining or increasing the production of a secondary metabolite in a microbial host characterized by comprising the phases of: preparation of DNA constructs according to claim 2; transforming a host candidate with these DNA constructs; cloning of the resulting transformants; and identifying clones that produce said secondary metabolite at a higher level than! said host candidate. ί 4. A process according to claim 1, characterized in that said first and second cultures have the ability to produce βlactam antibiotics. - 5 * I Process according to claim 1, i characterized by the said first and second cultures i be Penicillium, Aspergillus, Acremonium, Flavobacterium or Actinomycetes cultures. j - ^{6 <} *! I Process for the preparation of a vector by incorporating a sequence encoding a gene directly or indirectly involved in the biosynthetic pathway leading to a secondary metabolite, optionally a marker for selection in a host that produces said secondary metabolite and optionally a sequence to improve the efi37 science transformation of said vector in said property ameniti ^ Deiro, wherein said gene is _R is selected from a group consisting of acyltransferase gene by cryptic Y gene and the genes cryptic whole contained ^ entity or partially in L12 phage. I9, C12, P3, K11, B13, B20, G3, Gl, IOL, 16 and B23. - 7 ^a process for the preparation of a vector characterized in that it comprises a sequence which encodes. stands for acyltransferase with a structure as shown in Figure 3. - 8 ^the method according to claim CaO 6 , characterized in that it comprises a sequence coding for acyltransferase which was modified by insertion mutagenesis, deletion of mutagéneee, utagéneses a combination of insertion and deletion mutagenesis , or specifi. to a location in the AON sequence specified in Figure 3. - 9 ^the method according to claim CaO 6 wherein said gene contains a promoter PA. transcription different from the endogenous promoter. - 10 A process for the preparation of a transformed host characterized in that in the formation of trans ^ a DNA construct prepared according to the process of claim 2. Process for the preparation of a transformed host in which a copy of a sequence containing a functional gene in that host is incorporated as a result and which codes for a protein directly or indirectly involved in the biosynthetic pathway leading to a secondary metabolite giving as the result is a higher production in said secondary metabolite characterized in that said gene is selected from a group consisting of the acyltransferase gene, by ge. ne cryptic Y and by the cryptic genes contained entirely or partially in the phage L12, 19, C12, P3, 111, B13, B20, G3, Gl, IOL, K16 and B23. 12. A process according to claim 10 or 11, characterized in that said host has the ability to produce antibodies to lactam. 13. A process according to claim 10 or 11, characterized in that said host is a Penicillium. Aspergillus, Acremonium, Fiavobacterlum or ActinomYcetes ♦ 14. A process according to claim 10 wherein said secondary metabolite is a β-lactam. 15. ^A process according to claim 10 characterized in that said β-lactam is penicillin. 16. ^A process according to claim 13, characterized in that said host is of the species Penicillium chrysogenum or Acremonium chrysogenum or E. coli. 17. ^A process as claimed in claim 13 wherein the start of transcription of said set gene is regulated by a transcription start regulation region!> I different from that of the wild type. h i; - 18 ^to - ί Process for the preparation of a DNA construct characterized by incorporating the promoter of the P. chrysogenum phosphoglycerate kinase gene and genes prepared according to claim 2 under the regulation of the transcription of said promoter. Process according to claim 2, characterized in that the selection marker is a phleomycin resistance gene which is expressed under the transcriptional regulation of the phosphoglycerate kinase gene promoter. i - 20 ^to ! í Process for the preparation of a recombinant DNA construct characterized by incorporating the promoter and the translation activation sequence of the Penlcillium chrvsogenum acyltransferase gene. - 21 · Process for the preparation of a recombinant DNA construct characterized by incorporating the 3 'regulatory sequences of the Penylcillium chrysogenum acyltransferase gene. - 22 · Process for the preparation of a DNA construct characterized by incorporating a transformation reinforcement sequence that contains the sequence of Figure 4 together with another sequence other than the jungle type. gem. 23. The method of claim 22 wherein said sequence other than that of the wild type contains a gene encoding an enzyme or Penlcillium activity. - 24 · Process to improve the yields of an antibiotic secondary metabolite characterized by understanding the phases of: (a) culture of a transformed host containing an extra set of a sequence containing a gene, Isolated according to the process of any of claims 1 or 2, which codes for a protein directly or indirectly involved in the biosynthetic pathway of a secondary metabolite . tlblotlco resulting in a higher (b) higher production of said antibiotic; and isolating the resulting antibiotic. 25. The method of claim 24 wherein said antibiotic is a loop. taaaa. 26. ^The method of claim 25 wherein said host is a Penicillum, Aspergillua, Acremonium, Flavobacterium or Actinomycetes. 21. The method of claim 26 wherein said host is Penicillium chrysogenum. 28. ^The method of claim 24 wherein said gene codes for Î ± -cyltransferase. I - 29 ^to process according to claim CaO 24 wherein said gene encodes a protein with the structure as shown in Flg. 3. - 30 ^the method according to the claims. section 24 characterized by said cryptic Y gene. The applicant claims priority. des of European patent applications filed 11 and A-like and a 3 of 1988 and March 1989 under Nos 88201714.8 and 89200522.4 ^fl s, respectivaoente Lisbon, August 9, 1989 THE LVA LS PROPERTY OFFICIAL AFIE5i2 RESUME PROCESS FOR THE IDENTIFICATION AND USE OF BIOSYNTHETIC GENES OR REGULATIONS FOR IMPROVED PRODUCTION OF SECONDARY METABOLITES The invention relates to a process for the isolation of DNA fragments that express the metabolism of microorganisms durably and which are directly or indirectly essential for the production of a secondary metabolite or indirectly for the production of a secondary metabolite in this microorganism , preferably a bacterium or fungus, comprising the phases of: (a) screening a DNA bank prepared from said microorganism with probes obtained from mRNA or DNA derived from a first culture of said microorganism, which produces said secondary metabolite; (b) scrutinizing said DNA bank with probes obtained from mRNA or DNA derived from a second culture of said microorganism or a mutant that does not produce said secondary metabolite: and (c) identification of genes in said first cul. that are not substantially transcribed in said second culture.