MXPA99009238A - Gene conversion as a tool for the construction of recombinant industrial filamentous fungi - Google Patents

Abstract

The present invention relates to filamentous fungi that comprise in their genomes at last two substantially homologous DNA domains which are suitable for integration of one or more copies of a recombinant DNA molecule and wherein at least two of these DNA domains comprise an integrated copy of a recombinant DNA molecule. The invention also relates to methods for preparing such filamentous fungi and for further multiplying the DNA domains with integrated recombinant DNA molecules through gene conversion or amplification.

Description

CONVERSION OF GENES AS A TOOL FOR THE CONSTRUCTION OF INDUSTRIAL ORGANISMS RECOMBINANTS Field of the invention The present invention relates to the genetic engineering of microorganisms used in industrial fermentation processes. BACKGROUND OF THE INVENTION An ever increasing number of products is produced by microbial fermentation on an industrial scale. Such processes vary from primary and secondary metabolites, such as, for example, citric acid and antibiotics, respectively, to proteins, enzymes and even to whole microorganisms. , v gr, in the form of yeast for bakery or biomass. Traditionally, the microorganisms in question have undergone programs of classical strains environments consisting of successive cycles of mutagenesis and subsequent selection of mutants with improved performance. More recently, also genetic engineering, is say, recombinant DNA technology, has been applied to industrial microorganisms This technology, not only allowed the improvement of production levels of products made naturally by the microorganism in question, but also allowed the development of totally new products and / or processes w, '1 •Y markers in order to promote the amplification of the genetic material located between them, in particular to obtain the strains with greater production of penicillin by random mutagenesis with nitrosoguanidine in order to increase the number of copies of the vector once it has been integrated in the genome of the microorganism. However, ES 2 094 088 does not describe whether the approach described above could be extended successfully. In addition, the method described in ES 2 094 088 suffers from several drawbacks. For example, the amplification of complex DNA structures is present only at a low frequency. In addition, the use of mutagen nitrosoguanidine results in unwanted spontaneous mutations in the microorganism's genome. In addition, the mutagenic treatment can result in the deletion of the sequences of the amplified region present in the vector and, therefore, the deletion of the gene of interest located between them; see also Fierro, Proc. Nati Acad. Sci. USA (1995), 6200-6204. Also, as described above, the random integration of the vector into the genome of the microorganism will result in unpredictable genotypes of the transformant. Therefore, the technical problem described in the present invention is to provide a generally applicable approach for the construction of recombinant strains of filamentous fungi that contain multiple copies of a recombinant DNA molecule integrated in predetermined target sites, * defined in its genome and whose system does not depend on the use of a particular type of selectable marker for transformation. BRIEF DESCRIPTION OF THE DRAWINGS Abbreviations used in the figures: 5 Restriction enzymes and restriction sites: A = Apal; Ba = Ba HI; B = BglII; Bs = SssHII; E = EcoRI; H = Hindlll; K = Kpnl; N =? / Del; No = Notl; Ps = Psíl; P = Pvull; Sa = I left; Se = Seal; S = Smal; Sn = SpaBI; Spe = Spel; Sp = Sphl; Ss = Sstll; Xb = Xbal; X = Xhol; Nr = Nrul; Hp = Hpal; Sf = Sfil; Ns =? / S / I; 10 Bst = BstXl. LEGENDS OF THE FIGURES Figure 1: Schematic view of three AglaA sites in A. niger glacial DNA amplicons CBS 646.97 (deposited on April 11, 1997 in Centraal Bureau voor Schimmelcultures, 15 Baarn, The Netherlands) transformant (ISO-505 ), each marked by a different restriction site (Sa HI, came out or Bglll). The truncation of each glaA site differs by approximately 20 or 60 bp, to visualize each truncated glacial site by PCR-based DNA label testing. 20 ~ Figure 2: Physical maps of the glaA site in parental A. niger CBS 646.97 (top) and three "X-labeled" glaA sites truncated in the A. niger ISO-505 recombinant strain (X serves for the 9 restriction sites FiamHIHI, Sali or BglII). 7 Figure 3: Construction route of the intermediate vector f? pGBGLA16 Figure 4: Schematic presentation of a fusion PCR to destroy the Kpnl * in the 3'-glaA DNA sequence and to add cloning sites on the shore for the appropriate cloning in pGBG AId resulting in the intermediate vector pGBGL / 418. Figure 5: Schematic presentation of a fusion PCR to include a SamHI restriction site between the 5 'and 3'-glaA sequences and to add the cloning sites on the shore for appropriate cloning in pGBGLA18 resulting in the pGBDELd gene replacement vector. Figure 6: Schematic presentation of a fusion PCR to include a restriction site I left between the 5 'and 3'-glaA sequences and 15 to add the cloning sites on the shore for proper cloning into pGBGL418 resulting in the replacement vector of genes pGBDEL9. Figure 7: Schematic presentation of a fusion PCR to include a BglII restriction site between the 5 'and 3'-glaA sequences and 20 to add the cloning sites on the shore for cloning r appropriate in pGBGLA18 resulting in the vector of # gene replacement pGBDEL11. Figure 8: Schematic presentation of hybridization patterns observed after digestion with Kpnl or ßamHl of chromosomal DNA isolated from the parent strain of A. niger CBS I 646.97 probed with the DNA fragment 5'-glaU HindlU / Xhol (a) DNA fragment of 3'-glaA from sall / Sall (b). Figure 9: Schematic presentation of 5 hybridization patterns observed after Knpl or SamHI digestion of chromosomal DNA isolated from A. niger transformants CBS 646.97, where the linearized pGBDELd vector was integrated into the glaA white site via a double crossing, probed with the Hindlll / Xhol 5'-glaA DNA fragment (a) the 3'-glaA DNA fragment from sall / Sall (b). In the genome of individual transformants the original Kpnl site located within the 3'-glaA sequence of the host can be destroyed (K *) but not necessarily, as a consequence of the double-crossing recombination event. Figure 10: Schematic presentation of patterns of Hybridization observed after digestion with "pnl or SamHI" * of chromosomal DNA isolated from the A. niger transformant GBA-201 free of the amdS selection marker gene, comprising the AglaA DNA amplicon labeled Baml and two remaining intact glacial amplicons, probed with the 5'-glaA DNA fragment.

Hindlll / Xhol (a) the 3'-glaA DNA fragment from sall / Sall (b). Figure 11: Schematic presentation of hybridization patterns observed after digestion with Kpnl or SamHI of chromosomal DNA isolated from the transformants of A. niger GBA-201, where the vector linearized pGDEL9 has integrated a double crossing, probed with the fragment of DNA 5'-g / aA of Hmdlll / Xhol (a) the 3'-glaA DNA fragment of sall / Sall (b) In the individual transformant genome the original Kpnl site located within the glaA sequence of the host was can destroy (K *), but not necessarily as a consequence of the recombination event of "double crossing" Figure 12: Schematic presentation of hybridization patterns observed after digestion with pnl or SamHI of isolated chromosomal DNA transformant A niger GBA-201 free of the amdS selection marker gene, comprising two amplicons of "glaA" DNA labeled "BamHl and Sali" and a remaining intact glaA amphcon, probed with the Hindlll / Xhol 5'-glaA DNA fragment (a) the fragment of 3'-glaA DNA from Sall / Sall (b) l Figure 13: Schematic presentation of patterns of Hybridization observed after digestion with Kpnl or BamHl of chromosomal DNA isolated from the transformants A niger GBA-201, !? where the vector pGBDEL11 hneapzado has integrated via a double course, it has been probed with the DNA fragment '-glaA of Hindlll / Xhol (a) the 3'-glaA DNA fragment of sall / Sall (b) In the individual transformant genome in the original localized Kpnl site of the host 3'-glaA sequence could be destroyed (K *) but not necessarily as a consequence of a double cross-recombination event e% Figure 14: Schematic presentation of hybridization patterns observed after Kpnl or SamHI digestion of chromosomal DNA isolated from the A. niger transformant GBA-201 free of the amdS selection marker gene, comprising three amplicons of? s / aA DNA "labeled with ßamHI and Salí", probed with the Hindlll / Xhol d'glaze DNA fragment (a) the sall / Sall 3'-glaA DNA fragment (b). Figure 15: Physical map of acetamide selection marker and pGBAAS-1 vector targeting glaA. Figure 16: Physical map of acetamide selection marker and pGBTOPFYT-1 vector targeting glaA. Figure 17: Schematic illustration of the target PCR test to determine whether the phyA expression cassette (a) or the amdS cassette (b) is directed adjacent to one of the white 3'-3"-glaA site in the ISO transformants- 505 of A. niger, the address to one of these cassettes results in both cases in amplification of a DNA fragment of adequate size of 4.2 kb Figure 18: Schematic illustration of the three domains of AglaA in ISO-505 of A. niger, the hybridization pattern is shown from an Ss / II digestion, sonded with a DNA fragment of 3"-glaA Sall / Xhol 2.2 kb. Figure 19: Schematic illustration of the domains of AglaA (each marked by B *, Sa * and Ba *) in transformants ISO-505 of A. niger pGBAAS-1 / pGBTOPFYT-1, in which the multiple cassettes of amdS were they run in one of three AglaA domains of the ISO-505 A. niger guest. The hybridization pattern is shown from a BgIII digestion probed with a DNA fragment of 3"-glaA Sall / Xhol 2.2 kb.Note that the presence and intensity of the BgIII hybridization fragment of 7.5 kb depends on the number (n) of the integrated amdS cassettes Figure 20: Schematic illustration of the three domains of AglaA (each marked by B *, Sa * and Ba *) in transformants ISO-505 of A. niger pGBAAS-1 / pGBTOPFYT-1, in the which multiple phyA have been integrated adjacent to the white sequence 3'-3"-AglaA from one to three domains of AglaA from host lSO-505 of A. niger and with the downstream addition of a multiple cassette of amdS. The hybridization pattern is shown from a BgIII digestion probed with a DNA fragment of 3"-g / aA Sall / Xhol of 2.2 kb Note that the intensity of the BgIII hybridization fragment of 7.5 kb depends on the number (n) of the integrated phyA and amdS cassettes Figure 21: Schematic illustration of the AglaA domains (each marked by B *, Sa * and Ba *) in A. niger transformants of A. niger pGBAAS-1 / pGBTOPFYT-1, in which multiple amdS cassettes adjacent to the 3'-3"-glaA target sequence are integrated from one to three AglaA domains of the ISO-505 host of A. niger with the downstream addition of one to multiple phyA cassettes . The hybridization pattern is shown from a BglII digestion probed with the DNA fragment of 3"-glaA Sall / Xhol of 2.2 kb.Note that the intensity of the BgIII hybridization fragment of 7.5 kb depends on the number (n) of the integrated phyA and amdS cassettes Figure 22: Schematic illustration of the three domains of AglaA (each marked by B *, Sa * and Ba *) in transformants ISO-505 of A. piger pGBAAS-1 / pGBTOPFYT-1, in which multiple cassettes of phyA are integrated adjacent to the white sequence of 3'- 3"-glaA from one to three AglaA domains of the ISO-505 host of A. niger and with the downstream addition of one to multiple cassettes of phyA and amdS.The hybridization pattern is shown from a digestion of Bglll probed with the DNA fragment of 3"-g / aA of Sall / Xhol of 2.2 kb. Note that the intensity of this 7.5 kb BglII hybridization fragment depends on the number (n) of the integrated phyA and amdS cassettes. Figure 23: Schematic illustration of the three domains of AglaA (each marked by B *, Sa * and Ba *) in A. niger transformants ISO-505 pGBAAS-1 / pGBTOPFYT-1, in which multiple amdS cassettes are integrated adjacent to the 3'-3"-glaA target sequence from one to three AglaA domains of the host ISO-505 of A. niger and in addition to one or multiple amdS cassettes downstream.The hybridization pattern is shown from a digestion of Bglll probed with the 3"DNA fragment -glaA from Sall / Xhol of 2.2 kb.

Figure 24: Physical map of ISO-505 transformants of A. niger free of the amdS gene containing one copy of phyA (a), two copies of phyA (b) and three copies of the gene p? YA (c) directed at the site AglaA "marked BamHl". A schematic hybridization pattern is shown from the BamHl and Bglll digestions, probed with the 3"-g / aA probe Figure 25: Schematic illustration of the three glaA domains in the transformant ISO-505-2 of A. niger, in which two phyA cassettes are directed to the AglaA amplicon "labeled SamHII" showing the genotype of "DNA label" ßa / 77HI + / Sa / I + / ßg / H + (A), ßamHI2 + / Sa / I7ßg ir (B) and ßamHI2 + / Sa / I + / ßg / ir (C) The hybridization standards are shown from a SamHI digestion probed with the DNA fragment 3"-glaA from Sall / Xhol of 2.2 kb. Note that the intensity of both fragments of BamHI hybridization depends on the presence and number of different amplicons. Figure 26: Schematic illustration of three glaA domains in an ISO-505-2 transformant of A. niger, in which two phyA cassettes are directed in the AglaA amplicon "labeled ßamHH" showing the "DNA label" genotype SamH / Sa / I + / ßs / II + (A), ßamHI2 + / Sa / r / ß / H + (B) and ßaA77HI2 + / Sa / r / ßg / II "(C) The hybridization pattern is shown from a BamHl digestion probed with the Hindll / Xholl 1.5 kb S'-glaA DNA fragment Note that the intensity of the BamHl hybridization fragments depends on the presence and number of different amplicons.

Figure 27: Schematic illustration of the glaA domains in the transformant ISO-505-2 of A. niger, in which two cassettes of phyA are directed in the amplicon AglaA "marked ßamHII" having the genotype of "DNA label" ßamHI + / Sa / r / ßg / II + (A), ßa7? HI2 + / Sa / r IBglll + (B) and ßa / 77HI2 + / Sa / I + / ßg / ir (C). The hybridization pattern is shown from the digestion of ßamHI probed with the DNA fragment 5'-g / aA of Hindlll / Xhol of 2.2 kb. Note that the intensity of both fragments of BamHl hybridization depends on the presence and number of different amplicons. Figure 28: Schematic illustration of the glaA domains in the transformant ISO-505-2 of A. niger, in which two cassettes of phyA are directed in the amplicon AglaA "marked ßamHII" having the genotype of "DNA label" ßa7 ? HI + / Sa / / ßg / H + (A), ßamHI2 + / Sa / F l Bglll * (B) and ßamHI2 + / Sa / I + / ßg / IF (C). The hybridization pattern is shown from the digestion of SamHI probed with the DNA fragment 3"-g / aA of Sall / Xhol of 2.2 kb Note that the intensity of both fragments of BamHl hybridization depends on the presence and number of different amplicons Figure 29: PEN amplicons in P. chrysogenum Relative positions of penicillin biosynthetic genes, HEL and HELF, within a single amplicon (in brackets) Multiple PEN amplicons are present as direct repeats (n). the 3 'end of HELF extends into the adjacent PEN amplicon.

Figure 30: Quantification of PEN amplicons by Southern analysis. The relative positions of the ßsfXI sites, probes and sizes of the hybridization fragments expected from the niaü site (A) and PEN amplicon (B) the amount of DNA that is hybridized to the HELE probe depends on the number of PEN amplicons (n). Figure 31: Quantification of PEN amplicons by CHEF. Relative positions on the Notl sites and the HEL probe are indicated. The sites of Notl are located outside the PEN amplicon. The sizes Expected% fragments of hybridization depend on the number of st 10 PEN amplicons (30 + n x 57) kb. Figure 32: Directed integration. (A) Expression vectors containing the gene of interest, regulated by a suitable promoter (P) and terminator (T). The cassette is flanked by the 5"and 3" regions homologous to the target site ("and 'to distinguish the sequence of the vector of the genomic sequence, respectively). The cassette and the flanks are cloned into an E. coli vector for the propagation of I expression vector. Prior to transformation, the expression vector is targeted with the restriction enzyme R to create a linear fragment and remove the E. coli sequence. (B) The integration is presented in the homologous region in the genome with the free 5 'and 3' ends of the transformation fragment as hot spots for recombination. (C) The resulting transformant contains 1 or multiple copies (n) of the transformation fragment (between - - brackets) integrated in the white site.

Figure 33: Expression Vector pHELE-A1. The transformation fragment, comprising the expression cassette amdS flanked by the 50 'and 3' regions of HELE, was isolated as a Sfil fragment. These sites were introduced via the oligo used for CPR of the corresponding flanks Figure 34: Expression Vector pHELF-A1. The fragment of The transformation, comprising the expression cassette amdS flanked by the 5 'and 3' regions of HELF, was isolated as a Sfil fragment. These sites were introduced via the oligo used to CPR of the corresponding flanks Figure 35: Expression Vector pHELF-A1. The transformation fragment, comprising the cefE expression cassette flanked by the 5 'and 3' regions of HELF, was isolated as a Sfil fragment. 15 Figure 36: Expression Vector pHELF-A1. The transformation fragment, comprising the cefF expression cassette flanked by the 5 'and 3' regions of HELF, was isolated as a Sfil fragment. Figure 37: loss of amdS through recombination. (A) 20 direct repeats of the 5 'and 3' flanks arise by the random integration of expression cassettes. (B) The 5 'and 3' flanks are equal and the recombination occurs through a single crossing. (C) The region between the crossing site is lost. Note that any number of cassettes (including the genes of interest) can be lost, since all are flanked by direct repeats. Figure 38: CefE integration in HELE. Physical map of cefE cassette (A) alone or (B) multiple in HELE. The relative positions of the Nrul sites, cefE probe and expected sizes of hybridization fragments are indicated. The 6.0 kb fragment is presented by multiple integrations of the cassette and its intensity depends on the cassette number present (n). Figure 39: CefE integration in HELE determined by TAFE. Relative positions of the Hpal sites, probes and expected sizes of hybridization fragments are indicated. The size of the hybridization fragment depends on the number (n) of the integrated cassette (5.9 + n x 6.0) kb. Figure 40: Casete and address CPR. The schematic presentation of specific pattern and oligo combinations. Essential for the address PCR is that the oligo is located upstream of the flank 50. Therefore, although several cassettes may be present, only the 3 'cassette is oligo of a PCR product. Relevant domains, oligos, and expected sizes of CPR products are indicated. (A) Cepheus cassette CPR, (B) CepE headset CPR in HELE, (C) amdS address CPR in HELE (D) amdS cassette CPR, (E) cefE- combination direction PCR amdS, (F) AmdS-cefE combination direction PCR, (G) AmdS-amdS combination address (S) NiaD sine (S) cassette CPR (cefF) cassette CPR (Steering RCP) of cefF integrated in HELF (K) RCP address of amdS in HELF. Figure 41: Integration of amdS in HELE. Relative positions of the Hpal sites, amdS probe and expected sizes of the hybridization fragments are indicated. Figure 42: Probes: isolated DNA fragments for the preparation of probes: (A) cefE probe: Ndel-Nsil fragment of pHEL-E1. (B) cefF probe: Ndel-Nsil fragment of pHEL-F1. (C) amdS probe: Notl fragment of pHELE-A1. (D) HEL probe: SpLl-Notl fragment of pHEL-A1. (E) niaD probe: PCR product of oligo 28 and 29 with chromosomal P. chrysogenum DNA as standard. Figure 43: Gene conversion. Gene conversion and resulting duplication of cefE through the substitution of amdS by CefE in another PEN amplicon (in brackets). Figure 44: Integration of (cefE + CefF) in an amplicon. The relative positions of Notl sites, HELE probe and expected sizes of specific hybridization fragments for the integration of CefE and cefF in HELF on the same amplicon are indicated. Note that the Notl site is incorporated via the CefE and cefF cassette integration. Figure 45: CefE integration in HELF. Physical map of cefE cassettes (A) alone or (B) multiple in HELF. The relative positions of the Nrul sites, cefE probe and expected sizes of hybridization fragments are indicated. The 5.7 kb fragment is presented by multiple integrations of the cassettes and its intensity depends on the cassette number present (n). Figure 46: Quantification of cefE in gene converters (cefE + cefF). The relative positions of the Nrul sites, probes and expected sizes of hybridization fragments for cefE (A) cassettes alone or (B) multiple in HELE. The 6.0 kb fragment present by multiple integrations of the cassette and its intensity depend on the cassette number present (n). (C) niaD site. The gene preservers will have a hybridization pattern identical to the mother strain but an increased intensity relative to the r? / AD signal. Figure 47: Quantification of cefE in gene converters (cefE + cefF). The relative positions of the Nrul sites, probes and expected sizes of hybridization fragments for cefE (A) cassettes alone or (B) multiple in HELF. The 5.7 kb fragment present by multiple integrations of the cassette and its intensity depend on the number of cassette present (n). (C) niaD site. The gene preservers will have a hybridization pattern identical to the mother strain but an increased intensity in relation to the niaD signal. Figure 48: Quantification of CefE gene converters. The relative positions of the Nrul sites, probes and expected sizes of hybridization fragments for cefE (A) cassettes alone or (B) multiple in HELE. The 6.0 kb fragment present by multiple integrations of the cassette and its intensity depend on the cassette number present (n). (C) niaD site. The gene preservers will have a hybridization pattern identical to the mother strain but an increased intensity relative to the niaD signal. DESCRIPTION OF THE INVENTION In a first aspect the present invention relates to a filamentous fungus which has an integrated DNA molecule recombining in at least two DNA domains substantially homologous to its chromosomes, wherein the DNA domains are not the repetitions of ribosomal DNA. The filamentous fungi of the invention are prepared by transforming a filamentous fungus with at least two of said DNA domains into its genome with a recombinant DNA molecule and identifying a transformant with at least one recombinant DNA molecule integrated into it. less one of the DNA domains and by the subsequent identification of strains in the progeny of the transformant, whose DNA domain comprising the recombinant DNA molecule has been multiplied through the conversion of genes with the other versions of the DNA domain or through the amplification of DNA domain comprising the recombinant DNA molecule. In other words, the filamentous fungus of the invention has incorporated at least one recombinant DNA molecule in at least one of its endogenous domains of DNA in its genome. The filamentous fungi of the present invention are eukaryotic microorganisms including all filamentous forms of the Eumicota division of the fungal kingdom (see for example Lasure and Bannett, 1985, Fungal Taxonomy, in: gene manipulations in Fungi, pp. 531-535, Academic Press, Inc.). The filamentous fungi of the present invention are morphologically, physiologically and genetically different from yeasts: in contrast to yeasts such as S. cerevisiae, the vegetative development of filamentous fungi is by elongation of the hyphal and the metabolism of carbon is necessarily aerobic . In addition, yeasts such as S. cerevisiae have a prominent stable diploid phase, whereas in filamentous fungi, eg, diploides Aspergillus nidulans and Neurospora crassa only exist briefly before meiosis. In addition, many industrially important filamentous fungi belong to the subdivision of Deuteromictona, also known as the Imperfect Fungus, an artificial group of fungi that are distinguished by the absence of any known sexual form. Preferred filamentous fungi of the invention belong to the genus of Aspergillus, Trichoderma, Penicilllum, Cephalosporium, Acremonium, Fusarium, Mucor, Rhizophus, Phanerochaete, Neurospora, Humicola, Claviceps, Sordaria, Ustilago, Schizophyllum, Blakeslea, Mortierella, Phycomyces and Tolypocladum. The even more preferred filamentous fungi of the invention are fungi belonging to the Aspergillus niger group as defined by Raper and Fennell (1965, In: The genus Aspergillus, The Williams &; Wilkins Company, Baltimore), e.g., A. niger, the Aspergillus flavus group as defined by Raper and Fennell (supra), e.g., A. oryzae, as well as the fungus Trichoderma reesei and Penicillium chrysogenum. The invention was exemplified by the filamentous fungi A. niger and P. chrysogenum. The filamentous fungi according to the invention comprise in their genome at least two substantially homologous versions of the DNA domain suitable for the integration of one or more copies of a recombinant DNA molecule, wherein the DNA domain is not ribosomal DNA. In the examples herein, the amplified glucoamylase site (glaA) of A. niger and the amplified penicillin set of P. chrysogenum are used as DNA domains for the integration of the recombinant DNA molecules. Both the A. niger glacial sites and the P. chrysogenum penicillin complex, comprising the penicillin biosynthetic genes pcbAB, pcbC and penDE, are presented in a single copy in the wild-type genomes of the A. niger strains. and P. chrysogenum, respectively. Strains containing multiple copies of the DNA domains, as used in the present examples, can be obtained in classical strain programs by selecting strains with improved production of glucoamylase and penicillin, respectively. Frequently, said improvements in production are the result of the amplification of a DNA domain in the selected strains. Said amplified DNA domains referred to as amplicons hereinafter. Although the present invention preferably uses said amplicons as DNA domains for the integration of the recombinant DNA molecules, the invention does not expect to be limited to the same. In fact, any DNA domain from which two or more substantially homologous versions occur in the genome of a filamentous fungus can be used as long as the two functional criteria are met: 1) that the DNA domain must be adequate to accept the integration of a recombinant DNA molecule; 2) that the DNA domain should be capable of recombination with the other substantially homologous versions of the domain in the fungal genome in order to achieve multiplication of the integrated recombinant DNA molecule through gene conversion. In order to meet the first criteria, a DNA domain must be of sufficient length in order to allow it to target the recombinant DNA molecule in the domain through homologous recombination. For this purpose a DNA domain comprises at least 100 bp, preferably at least 1 kb and more preferably at least 2 kb. The suitability of a DNA domain for the integration of the present one of a recombinant DNA molecule is therefore determined by the requirement that integration into the DNA domain does not alter the functions that are essential for the viability of filamentous fungi in question.

For the second functional criterion, that is, the capacity for recombination with the other substantially homologous versions of the domain in the fungal genome, it is required to allow gene conversions between different versions of the DNA domain. The minimum requirement for this purpose is that each version of the domain is flanked at either end of the domain by DNA sequences that are sufficiently homologous to the corresponding flanking sequences of the other regions of the DNA domains so as to allow homologous recombination between the flanking sequences. The result of this homologous recombination is a gene conversion where one of the versions of the DNA domain is replaced by a duplicated version of another DNA domain containing the integrated recombinant DNA molecule. The minimum requirements with respect to length and degree of homology of the flanking sequences that still allow the conversion of genes is not exactly known and may vary depending on the organisms in question. Probably a minimum length of 100 bp with a global homology of at least 60% would still allow the conversion of genes. Obviously the frequency of gene conversion will increase with the increasing length and homology of the sequences that flank the DNA domain. Preferably the different domains are flanked by the sequences of at least 1 kb that share at least 80% homology. The fungal genome can contain one or different types of DNA domains as defined above.

Examples of different types of domains that are not perfect copies with each other are allelic variants, gene families and / or genes encoding isoenzymes. Most preferred are domains that are exact copies with one another, differing most in the presence of the integrated recombinant DNA molecule. Examples of said identical domains are amplicons. Therefore, in a preferred embodiment of the invention, said DNA domains suitable for the integration of one or more copies of a recombinant DNA molecule are amplicons. The overall length of the DNA domains is not important and can vary from less than 1 kb to several hundred kb, e.g., in the present examples the length of the DNA domains vary from approximately 57 kb per unit for the penicillin set amplified to more than 80 kb per unit for the glazed amplified site. The recombinant DNA molecule comprises any combination of genetic elements required to introduce a desired genetic modification into the filamentous fungi of the invention. The recombinant DNA molecule comprising any genetic element, parts thereof or combinations thereof, such as a gene (coding part or complete site), a cDNA, a promoter, a terminator, an intron, a signal sequence, a regulatory DNA sequence or a recognition sequence of DNA binding proteins. Genetic elements may also include DNA sequences that are modified, ie, that contain one or more nucleotide alterations (e.g., insertions, deletions, substitutions). The desired genetic modifications include any modification, i.e., insertions, deletions and / or substitutions of DNA sequences in a selected filamentous fungus that are the result of the introduction of one or more genetic elements mentioned above into the fungus by transformation or co-transformation. -transformation of the recombinant DNA molecule. It will be understood that several of said genetic modifications can be introduced in independent transformation cycles. According to one embodiment of the invention, the recombinant DNA molecule integrated into the DNA domain of filamentous fungi contains one or more expression cassettes for the expression of one or more desired genes. An expression cassette is understood herein as a DNA fragment comprising a desired gene that will be expressed and which is operably linked to the appropriate expression elements that are capable of effecting and regulating the expression of the gene. Said expression elements include promoters, signal sequences, terminators and the like. If the filamentous fungus is indicated for the production of proteins or enzymes whether meteorological or homologous, the desired gene preferably encodes a secreted protein or enzyme and therefore comprises a signal sequence that affects the secretion of the enzyme. This embodiment is, for example, exemplified by the integration of the expression cassettes comprising the A. niger phytase gene in the A. niger glacial amplicons. However, it will be clear to the skilled person that the invention can be applied to any protein or enzyme of interest. Alternatively, if the filamentous fungi are intended for the production of primary or secondary metabolites, the desired genes usually encode one or more intracellular enzymes that are involved in the biosynthesis of these metabolites. In a preferred embodiment according to this aspect of the invention, the filamentous fungus comprises one or more recombinant DNA molecules integrated in the DNA domain, whereby the recombinant DNA molecules comprise one or more expression cassettes for the intracellular enzymes. that are part of a metabolic pathway that is not native to the filamentous fungus. Examples of said filamentous fungi include, for example, P. chrysogenum strains that have integrated in their cassettes expression of penicillin amplicons in a deacetoxycephalosporin C synthetase (expandase) and a desacetylcephalosporin C synthase (hydroxylase), which allows these strains synthesize adipoyl-7-aminodesacetoxycephalosporanic acid and adipoyl-aminodesacetylcephalosporanic acid, respectively. The mechanism by which the recombinant DNA molecule is integrated into the DNA domain is not important to the invention and may depend on the application of the present invention. The integration of the recombinant DNA molecule into the DNA domain can occur by random integration but more preferably integration can occur by homologous recombination, either by a single junction recombination event, i.e., resulting in an insertion of the recombinant DNA molecule or by a double-crossing recombination event, resulting in a replacement by the recombinant DNA molecule, replacing part of the original sequences of the DNA domain. In order to promote integration through homologous recombination the recombinant DNA preferably contains sequences that are homologous to the target sequences for integration into the DNA domain. Preferably said target sequences in the DNA molecule are identical to the target sequence in the DNA domain. In a preferred embodiment of the invention each version of the substantially homologous DNA domain present in the filamentous fungus comprises an integrated copy of the recombinant DNA molecule. Complete occupation with integrated recombinant DNA molecules from the entire DNA domain present in the fungus produces the highest possible copy number of the recombinant DNA molecule and provides a more stable situation because said fungus does not contain DNA domains. " vacuums "that could function as donors of gene conversions with full DNA domains, thus reducing the number of copies of the integrated recombinant DNA molecule. Any such versions of the same type of DNA domains can be occupied with recombinant DNA molecules or each version of the different types of DNA domains. Of occurrence, each version of all types of the substantially homologous DNA domains present in the filamentous fungus genome comprises an integrated copy of the recombinant DNA molecule. In an advantageous aspect of the invention, the DNA domain used for the integration of the recombinant DNA molecule is a domain which in its native state comprises an endogenous gene capable of expression at high levels. Usually it is known that the level of expression of an integrated recombinant gene can vary greatly depending on the genomic site where said gene is integrated. The advantage of using highly expressed domains for the integration of recombinant genes that will be expressed is that these domains are at least capable of supporting the expression of higher levels of the androgen gene. Therefore, it is likely that such domains also support expression at high levels of a recombinant gene. We have also found that the integration in the A. niger glaU domain and the joint integration of P. chrysogenum penicillin as described in the examples herein provides higher expression levels for gene copy compared to integration in some genomic sites. In this context it will be understood that a gene capable of expression at high levels is defined as a gene which, when expressed at the maximum level, it produces an mRNA that constitutes at least 0.1% of the total mRNA population, preferably at least 0.5% of the total mRNA and more preferably at least 1% of the total mRNA. Examples of such highly expressible endogenous genes of whose domains in which they are contained are particularly suitable for the integration of the recombinant DNA molecule of the invention are genes encoding glycolytic enzymes, amylolytic enzymes, celuiolytic enzymes and / or antibiotics. Even more preferred are domains comprising genes involved in industrial processes and known to be expressed at high levels such as glucoamylase genes, TAKA amylase genes, cellobiohydrolase genes and penicillin biosynthetic genes. In a further aspect of the invention, the highly expressed endogenous gene is inactivated in each copy of the DNA domain in the filamentous fungus in cases where expression of the endogenous gene is not required. In these cases, the inactivation of the expression of high levels of the endogenous gene makes available the energy and resources that also carry out the expression of the gene of interest. Furthermore, in the case that both the desired enzyme that will be produced by the integration of the recombinant DNA molecule and the enzyme encoded by the endogenous gene are secreted enzymes, the inactivation of the endogenous enzyme will result in more pure preparations of the enzyme desired. Preferably the endogenous gene is inactivated by an irreversible suppression of at least part of the endogenous gene in order to exclude the reversal of inactivation. More preferably the inactivation of the endogenous gene is affected by an irreversible deletion comprising at least part of the promoter and the upstream inactivation sequences. This is particularly advantageous where the cases where the expression of a desired gene encoding an enzyme that will be produced by the integration of the recombinant DNA molecule is driven by a promoter derived from the endogenous gene, because it eliminated competition to limit potentially the transcription factors required for the expression of the desired gene. In a further embodiment of the invention, each version of the DNA domains is distinguished from the other versions of the domains in the filamentous fungus by means of a single sequence tag. Said unique sequence labels allow to monitor the gene conversions between the different domains that facilitate the screening and / or selection of the converters with a desired genotype. Any form of sequence tags can be used while allowing the detection of different versions of the domain: eg, varying from the restriction sites that are detected in Southern blot analysis to complete the selectable marker genes providing an easily analysable phenotype. A particularly useful embodiment of the sequence tag is exemplified herein and allows each of the domains to be detected in a single PCR using a pair of oligo nucleotides to initiate PCR. The domains are modified in such a way that in the RCP each version of the domains will produce a fragment of RCP with a single length. The length and intensity of the PCR fragments obtained indicate the presence and number of copies of each of the domains, respectively. This form of sequence tag, referred to as "DNA markers", allows the genotype of large numbers of converting colonies to be easily analyzed, in order to obtain a converter with the desired genotype. The present invention also relates to methods for preparing the filamentous fungi of the invention. These methods comprise the step of transforming a filamentous fungus comprising one or more of its chromosomes into at least two substantially homologous DNA domains suitable for the integration of one or more copies of a recombinant DNA molecule and wherein the DNA domains are not the ribosomal DNA repeats, with a recombinant DNA molecule. The transformation of filamentous fungi is currently routine for the skilled person and a variety of transformation protocols suitable for filamentous fungi are available. The transformation of the filamentous fungi with the recombinant DNA molecule requires the use of a selectable marker gene which allows to distinguish the fungal cells that have absorbed the transforming DNA to form the non-transformed cells. A variety of selectable marker genes is available for use in the transformation of filamentous fungi. Suitable labels include auxotrophic marker genes involved in the metabolism of amino acids or nucleotides, such as for example genes encoding ornithine-transcarbamylases (argB), orotidine-5'-decarboxylases (pyrG) or glutamine-amido-transferase indoleglycerol-phosphate synthase phosphoribosyl-anthranilate isomerases (trpC) or that are involved in the metabolism of carbon or nitrogen, such as, for example, niaD or facA and antibiotic resistance markers such as genes that provide resistance against phleomycin, bleomycin or neomycin (G418). Preferably, bidirectional selection markers are used for which it is possible for positive and negative genetic selection. Examples of such bidirectional markers are the pyrG, facA and amdS genes. Due to their bidirectional ability these markers can be deleted from the transformed filamentous fungus while leaving the introduced recombinant DNA molecule in place, in order to obtain filamentous fungi that do not contain selectable markers. This essence of this MARKER GENE FREE ™ transformation technology is described in EP-A-0 635 574, which is incorporated herein by reference. Of these selectable markers, the use of dominant and directional selectable markers such as acetamidase genes similar to the amdS genes of A. nidulans, A. niger and P. chrysogenum is most preferred. In addition to their bidirectional capacity these markers provide the advantage that they are dominant selectable markers, the use of which does not require mutant strains (autotrophic), but can be used directly in wild-type strains. A further embodiment therefore relates to filamentous fungi according to the invention, wherein the recombinant DNA molecule lacks a selectable marker, or more preferably filamentous fungi according to the invention which collectively lack a selectable marker. The selectable markers used to transform the filamentous fungi of the invention with the recombinant DNA molecule can be physically bound to the recombinant DNA molecule to be transformed or they can be in a separate DNA molecule that is co-transformed with the recombinant DNA molecule. desired. Cotransformation is routinely used by those skilled in the art because it occurs at a relatively high frequency in filamentous fungi. A next step in the methods for preparing the filamentous fungi of the invention comprises selecting a transformant with at least one recombinant DNA molecule integrated in at least one of the DNA domains of the filamentous fungi. A number of routine techniques are available to the experts in order to determine which transformants obtained have an integration of a recombinant DNA molecule in one of their DNA domains. In a further step, the selected transformant is propagated and from its progeny a strain is selected in which at least two of the DNA domains comprise the integrated recombinant DNA molecule. This means that the strain is selected in which the DNA domain comprising the integrated recombinant DNA molecule is multiplied, either through the conversion of genes with an "empty" DNA domain or through amplification. These events of conversion and / or amplification of genes occur spontaneously at a low frequency. The exact frequency at which these events occur, it can depend on a number of variables including the fungi in question and the number, length and degree of homology of the DNA domains. However, we have found that these frequencies are high enough to allow the strains in which these events have been sieved to be screened and selected using currently available analysis techniques. Strains in which the DNA domain comprising the integrated recombinant DNA molecule is multiplied can be identified, eg, by simply screening the strains with higher production levels of the product, which are synthesized by the recombinant DNA molecule, or alternatively said strains can be identified by analyzing their genotype eg, by the "DNA label" test as described above. A method according to the invention may comprise additional steps in which one of the strains in which the multiplication of the DNA domain has been presented comprises the integrated recombinant DNA molecule propagating and where it forms its progeny strains are selected in which additional copies of the DNA domains comprise the integrated recombinant DNA molecule. These strains then again can be subjected to this procedure until a strain is obtained in which each of the DNA domains comprises the integrated recombinant DNA molecule. As described herein, such strains provide the advantages of a high copy number of the recombinant DNA molecule and improved stability. In a further aspect of the method for preparing the filamentous fungi of the invention, the recombinant DNA molecule comprises sequences that are substantially homologous to the DNA domains. The presence of said substantially homologous sequences in the recombinant DNA molecule will significantly increase the frequency at which the recombinant DNA molecule is integrated into the DNA domain. The minimum requirement for substantially homologous sequence in the recombinant DNA molecule is not known but in practice frequencies are obtained that target the reasonable target with homologous target sequences of at least 1 kb, preferably at least 2 kb. As mentioned before one aspect of the invention relates to the use of bidirectional selectable markers for transforming filamentous fungi with the recombinant DNA molecule. Once said transformant has been obtained it is advantageous to subject them to selection against selection for the absence of bidirectional marker. Such unlabeled transformants can then undergo further transformations or they can be propagated to screen their progeny strains with conversions and / or gene amplifications of the DNA domain comprising the recombinant DNA molecules. In an alternative method for preparing a filamentous fungus according to the invention a filamentous fungus is transformed with a recombinant DNA molecule and a transformant is selected with at least one recombinant DNA molecule integrated in a predetermined genomic target sequence. This transformant is subsequently propagated and strains with at least two DNA domains comprising an integrated recombinant DNA molecule are selected from its progeny. In this case the recipient filamentous fungus that will be transformed with the recombinant DNA molecule does not necessarily contain multiple copies of the DNA domain. The DNA domain is instead amplified after the recombinant DNA molecule is integrated therein. The present invention allows the preparation of recombinant filamentous fungi comprising on one or more of its chromosomes at least two DNA domains substantially homologous for the integration of one or more copies of a recombinant DNA molecule, wherein the DNA domains do not are the ribosomal DNA repeats and wherein at least two domains of DNA comprise an integrated copy of a recombinant DNA molecule. These recombinant fungi can be used in the processes for the production of a product of interest. Said process will only include the steps of culturing the recombinant cells in a medium that leads to the production of the product of interest and the recovery of the product of interest from the culture medium and / or the fungus. The products of interest can be proteins, such as an enzyme and / or primary metabolites, such as CO2, alcohol or organic acids, and / or secondary metabolites, such as antibiotics or carotenoids. The product of interest can also be the recombinant fungi by themselves, that is, the biomass obtained in the process. Examples of enzymes and proteins that can be produced in the filamentous fungi of the invention include lipase, phospholipases, phosphatases, phytases, proteases, pullulanases, esterases, glycosidases, amylases, glucoamylases, catalases, glucose oxidases, β-gluocosidases, arabinofuranosidases, rhamnosidases, apiosidases, cimosin, lactoferrin, cell wall degradation enzymes such as cellulases, hemicellulases, xylanases, mannases, pectinases, rhamnogalacturonase and the like. Some of the advantages of the filamentous fungi of the invention and methods for their preparation are summarized below: The present invention provides greater versatility compared to the systems available for recombinant DNA molecules integrating non-random multiple copies into filamentous fungi. Greater versatility because the present invention is not confined to the use of selectable marker genes deficient for transformation, and also because the present invention is not confined solely to the use of ribosomal DNA sequences as a target sequence for integration. The filamentous fungi of the invention provide greater genetic stability of the multiple integrated copies of the recombinant DNA molecule compared to the recombinant filamentous fungi in which the recombinant DNA molecules are randomly integrated into dispositions dispositions one after the other. In particular, filamentous fungi with a copy number of randomly integrated recombinant DNA molecules will usually have large numbers of recombinant DNA molecules repeated randomly integrated into only a few genomic sites. This configuration is inherently less stable than a contiguity according to the invention, wherein only a few repeating copies of the recombinant DNA molecule (preferably no more than five copies) are integrated into different genomic sites such as the domains of DNA substantially homologous. For example, a filamentous fungus according to the invention may have a copy number of 15, whereby the fungus may have three copies repeated one after the other of a recombinant DNA molecule integrated into each of five DNA domains substantially homologous In contrast, a conventional recombinant filamentous fungus with a copy number of 15 can have 10 copies integrated in an undefined genomic site and 5 copies in another undefined genomic site. In accordance with the present invention it has been found that the latter configuration is less stable with respect to the loss of the recombinant DNA molecules compared to a fungus according to the invention having the same number of copies. The filamentous fungi of the invention are expected to provide higher expression levels by gene copy compared to filamentous fungi with integrations in the ribosomal DNA, because the latter did not evolve to support the transcription of RNA polymerase II from high level RNA. the genes that encode proteins. The filamentous fungi of the invention are strains of multiple copies of which the genotype can be completely defined at least insofar as it refers to the integrated recombinant DNA molecules. This will facilitate the obtaining of regulatory approval for processes and products in which these fungi are involved. For similar reasons the filamentous fungal phenotype will be more predictable compared to conventional recombinant filamentous fungi in which recombinant DNA molecules are randomly integrated because random integration can integrate the expression of unknown genes that reside in, or are close to, of the integration sites. Methods for the preparation of the filamentous fungi of the invention provide the advantage of synergy with early classic strain improvement programs. As the preferred embodiment of the invention was desilted before, industrial filamentous fungi are used which have been obtained in ways of improving normal classical strains. The use of said fungi is not only advantageous because they will often comprise amplicons that are suitable for the integration and subsequent multiplication of the recombinant DNA molecules according to the invention, but also because the industrial filamentous fungi will have accumulated ( a large number of) mutations that are advantageous not only for the production of the product in which the program has been promoted to improve the strain (the "previous" product) but also for other new products. By modifying these improved industrial filamentous fungi, the genes were involved in the production of the "previous" product in a way that allows the fungus to produce a new product, the advantageous mutations will now contribute to the efficient production of the new product. In this form the present invention can have significant use for the development of the strains of the new production.

The present invention allows the "design and construction" of recombinant filamentous fungi. The term "design and construction" means a single level of copies of a design that is made of all the desired genetic modifications that will be introduced in a DNA domain. V.gr., in the case of the production of a desired protein one or more expression cassettes for the protein are integrated in the DNA domain and in the same resident domain they inactivate endogenous genes that could negatively influence the expression of the desired protein . In the case of a mixture of proteins, the design will be produced and will include adjusting the expression levels of the different proteins to the desired relationships. The latter may be particularly advantageous in the case of engineering metabolic pathways where a number of new metabolic activities will be introduced, forming part of a new metabolic pathway, in a predetermined relationship. Once these designs of the desired genetic modification have been established in the DNA domain at a single level of copies, the construction process can begin, which means that the domain of unique copies designed is multiplied by comparison and / or Gene amplification of the domain comprising the desired genetic modifications until the desired production level is reached.

EXAMPLES Nomenclature A. niger Aspergillus niger P. chrysogenum Penicillium chrysogenum S. clavuligerus Streptomyces clavuligerus A. nidulans Aspergillus nidulans PhyA Gen. phyA from A. niger, which codes for amdS phytase AmdS gene from A. nidulands, which codes for acetamidase (Corrick et al., 1987 Gene 53: 63-71) cefE CefE gene of S. clavuligerus, which codes for deacetoxycephalosphsin C synthetase. (Kovacevic et al., 1989 J. Bacteriol 171: 398-400) cefF CefF gene from S. clavuligerus, which encodes deacetylcephalosporin C synthetase. (Kovacevic, S. and Miller, J.R. 1991 J. Bacteriol. 173: 398-400) .. niaD Genia niaD of P. chrysogenum encoding nitrate reductase. (Haas et al., 1996 Biochem Biophys, Acta 1309: 81-84) Glaciated A. niger glacial gene encoding glucoamylase gpdA gdpA gene from A. nidulans, which encodes glyceraldehyde 3-phosphate dehydrogenase. (Punt et al., 1988 Gene 69: 49-57) pcbC GencocC from P. chrysogenum, which encodes isopenicillin N synthase (IPNS) (Carr et al., 1986 Gene 48: 257-266) PgpdA promoter gpdA PgiaA promoter glaA PpcbC promoter pCOC TpenDE terminator penDE Tamds terminator amdS TgiaA terminator glaA GLA glucoamylase protein from A. niger CHEF abbreviations Homogeneous Electric Fields Subjects (electrophoresis) TAFE Alternative Field Electrophoresis Inverse Kb kilo base Pb base pairs ADCA aminodesacetoxycephalosporanic acid ADAC aminodesacetylcephalosporanic acid Oligo oligonucleotide RCP Polymerase Chain Reaction Oiigonucleotides: 1. 5 '-gta gct gcg gcc gcc tec gtc ttc act tet tcg ecc gca ct-3' 2. 5 '-cag agg gca tgc ggc cgt ate ggc cgg tga caa here tea tcc aac gcc-3' 3. 5 '-atg ttt aag ctt ggc cga tac ggc caa aac acc ttt gat ttc-3 '4. 5' -cata gtt gcg gcc gct ect cae taa cga gcc age aga tat cga tgg-3 '5. 5' -aag ctt atg cgg ccg cga att cga gct ctg tac agt gac 3 '6. 5' -cgg tac gtg cgg ccg cte gta cea tgg gtt gag tgg tat g-3 * 7. 5 '-ata tgt gcg gcc gct tta cat ggt caa tgc aat tag atg gtg g-3 '8. 5'- ata act cta gag gcc cta ccg gcc ttt gca aat ata ctg taa gaa cc-3r 9. 5 '-gta tat tet gca ggg ccg gta ggg cea here gtt tec gca ggt g-3' 10. 5 '-gta tgg gcg gcc gta tta cata cta gaa tat ggg aac ctg tgg g-3' 11. 5 '-etc gag tgc sgc cgc aaa gct age ttg ata tcg aat tec tta tac tgg gcc tgc tgc att g-3 '12. 5' -gtc cat atg ggt gtc tag aaa aat aat ggt gaa aac ttg aag gcg-3 ' 13. 5 '-cat atg gcg gac acg ccc gta ccg ate ttc-3' 14. 5 '-atg cat tgg etc gtc atg aag age cta tea tec ggc ctg ccggg etc gtt ctt cgc-3' 15. 5 '-cag cta ccc cgc ttg age aga cat c-3 '16. 5' -gtc agg gaa gaa falls gag ggc gca g-3 ' 17. 5 '-ccc tet ctt cgL cgt tgt cea cgc c-3' 18. 5 '-atg tec ttg gcc gac ttc age tcg g-3' 19. 5 '-gac gag cea atg cat ctt ttg tat g-3 '20. 5' -cgg gta etc gct cta ect act tcg g-3 '- 21. 5' -gcc cag tat aag gaa ttc gat ate aag-3 '22. 5' -agg gzc gac act agt tet aga gcg g -3 '23. 5' -gac gtt ate gga cgg aga etc agt g-3 '24. 5' -gcc tac tet gtt ctg gag age gc-3 '25. 5' -ccc cea tec cgg tea cgc act cgc g -3 '26. 5' -cae aga gaa tgt gcc gtt tet ttg g-31 27. 5 '-tea cat ate ccc tac tec cga gcc g-3' 28. 5 '-gtc gcg tat ccc agg-3' 29 5 '-gtc aaa gga tat gca tac-3' 30. 5r -age tta tgc ggc cgc gaa ttc agg tac cgt ate tcg aga- 3 '31. 5' -aat ttc tcg aga tac ggt acc tga att cgc ggc cge ata- 3 '32. 5' -gtg cga ggt acc ate ate aat cea ttt cgc-3 '33. 5' -atg gtt caa gaa etc ggt age ctt ttc ctt gat tct-3 ' 34. 5 '-aga ate aag gaa aag gct acc gag ttc ttg aac cat-3' . 5 '-ate aat cag aag ctt tet etc gag acg ggc ate gga gtc CC: g-3 '36. 5' -gac cat gat tac gcc aag ctt-3 '37. 5' -gga tec tta act agt taa gtg ggg gcc tgc gca aag-3 ' 38. 5 * -tta act agt taa gga tec here ate aat ceattt cgc-3 ' 39. 5 '-gct cta gag cgg ccg cga att cat ccg gag ate c-3' 40. 5 '-ctt tgc gca ggc ccc cac-3' 41. 5 '-tgc agg gta aat cag gga-3' 42. 5 ' -tec gct aaa ggt ggt cgc g-3 '43. 5' -ccc cag cat cat tac acc tc-31 44. 5 '-aaa gga ccc gag ate cgt ac-3' 45. 5 '-tet cga tac ca ggt falls falls ggg c-3 '46. 5' -gca tec ate ggc falls cgt cat tgg a-3 '47. 5' -ate cag acc age here ggc age ttc g-3 '48. 5' -tec gca tgc cag aaa gag tea ccg g-3 '49. 5' -gtc gac tta act agt taa ggc ttc aga cgc age gag-3 ' 50. 5 '-tta act agt taa gtc gac here ate aat cea ttt cgc-3' 51. 5 '-aga tet tta act agt taa gtg gcc tga here gtg ccg-3' 52. 5 '-tta act agt taa aga tet here ate aat cea ttt cgc-3' Materials and Methods General Procedures The normal molecular cloning techniques such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, Southern analysis, transformation of E. coli ea, where it was carried out as described by Sambrook and others (1989) "Molecular Cloning: a manual laboratori", Cold Spring Harbor Laboratories, Cold Spring Harbor, New York and Innis and others (1990) "PCR protocols, a guide to methods and applications" Academic Press, San Diego. The synthetic oligo-deoxynucleotides were obtained from ISOGEN Bioscience (Maarssen, The Netherlands). DNA sequence analyzes were carried out on an Applied Biosystems 373A DNA sequencer, according to the manufacturer's instructions. Transformation of Aspergillus niger. The transformation of A. niger was carried out according to the method described by Tilburn J. et al. (1993) Gene 26, 205-221 and Kelly, J. & Heynes, M. (1985) EMBO J., 4, 475-479 with the following modifications: The spores were grown for 16 hours at 30 ° C on a rotary shaker at 300 rpm in Aspergillus minimal medium. The minimum medium of Aspergillus contains per liter: 6 g of NaNO3; 0.52 g of KCl; 1.52 g of KH2PO4; '1.12 ml of KOH 4 m; 0.52 g of MgSO4.7H2O; 10 g of glucose, 1 g of casamino acids; 22 mg of ZnSO4.7H2O; 11 mg of H3BO3; 5 mg FeSO4.7H2O; 1.7 mg of CoCl2 .6H2O; 1.6 mg of CuSO4 .5H2O; 5 mg of MnCl2 .2H2O; 1.5 mg Na2MoO .2H2O; 50 mg EDTA; 2 mg of riboflavin; 2 mg thiamine-HCl; 2 mg of nicotinamide; 1 mg of pyridoxine-HCL; 0.2 mg of pentatotenic acid; 4 μg biotin; 10 ml of Penicillin (50 lU / ml) Streptomycin (5000 UG / ml) solution (Gibco). - Novozym 234 (Novo Industries) was used instead of helicase for the protoplast preparations; After the formation of protoplasts (60-90 minutes), KC buffer solution (0.8 M KCl, 9.5 mM citric acid, pH 6.2) was added to a final volume of 45 ml, the protoplast suspension was centrifuged for 10 minutes. minutes at 3000 rpm at 4 ° C in a rolling bowl rotor. The protoplasts were resuspended in 20 ml of KC buffer and subsequently 25 ml of STC buffer solution (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2) was added. The protoplast suspension was centrifuged for 10 minutes at 3000 rpm at 4 ° C in a roll cuvette rotor, washed in STC buffer and resuspended in STC buffer at a protoplast concentration of 108 / ml. to 200 μl of the protoplast suspension the DNA fragment dissolved in 10 μl in the TH buffer solution (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA), was added 100 μl of a PEG buffer (20 μL). % of PEG 4000 (Merck), 0.8 M of sorbitol, 10 mM of Tris-HCl pH 7.5, 50 mM of CaCl 2); after incubation of the DNA protoplast suspension for 10 minutes at room temperature 1.5 ml of the PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2) was added slowly, with repeated mixing of the tubes. After incubation for 20 minutes at room temperature, the suspensions were diluted with 5 ml of 1.2 M sorbitol, mixed by inversion and centrifuged for 10 minutes at 4000 rpm at room temperature. The protoplasts were gently resuspended in 1 ml of 1.2 M sorbitol and plated in selective regeneration medium consisting of minimal Aspergillus medium without riboflavin, thiamine. HCL, nicotinamide, pyridoxine, pantothenic acid, biotin, casamino acids and glucose, supplemented with 10 mM acetamide as the sole source of nitrogen, 1 M sucrose, solidified in 2% bacteriological agar # 1 (Oxoid, Eng / and). After incubation for 6-10 days at 30 ° C, the plates were plated as replicate in selective acetamide plates consisting of Aspergillus minimal medium with 2% glucose instead of sucrose and 1.5% agarose instead of agar. . Unique transformants were isolated after 5-10 days of growth at 30 ° C.

Transformation of Penicillium chrysogenum. The protoplast transformation procedure mediated by Ca-PEG was used. The preparation of protoplasts and transformation of P. chrysogenum was carried out according to the method described by Gouka et al., Journal of Biotechnology 20. (1991), 189-200 with the following modifications: after transformation, the protoplasts were seeded on selective regeneration media plates consisting of minimal Aspergillus medium osmotically stabilized with 1.2 M sucrose, containing 0.1% acetamide as the only source of nitrogen and solidified with 1.5% bacteriological agar # 1 (Oxoid, Eng / and). After 5-8 days of incubation at 25 ° C the transformants were paired. TAFE DNA fragments ranging from 20 to 50 kb were separated in 1% agarase gels by TAFE, using a Beckman Geneline ™ II device (Beckman) according to supplier instructions. The electrophoresis parameters: 4 sec. A, 4 pulse times in second B to 300 mA for approximately 18 hours at 14 ° C. CHEF DNA fragments larger than approximately 50 kb were separated on 1% agarose gels by CHEF electrophoresis, using an HCER-DRII module (BioRad), equipped with a 760 Pulse Wave Exchanger, Model 200 / supply 2.0 power and CHEF electrophoresis cell. The electrophoresis parameters: initial time 50 sec, final time 90 sec., Starting ratio 1.0, 200 V for 20 to 24 hours at 14 ° C. Quantification of PCR products and hybridization signals The PCR products and hybridization signals were quantified from photographs of agarose gels stained with ethidium bromide or autoradiograms, respectively, according to the ImageQuaNT ™ software (Molecular Dynamics). DNA Marking and Hybridizations DNA labeling and DNA hybridizations were in accordance with ECL ™ direct nucleic acid labeling and detection systems (Amersham LIFE SCIENCE, Little Chalfont, Eng / and). Specific PCR procedure for cassette and DNA labels in A. niger colonies A. niger spores were plated on PDA plates (Potato Dextrose Agar, Oxoid, prepared according to the supplier's instructions). After growth for 48 hours at 30 ° C, a? -a- parts of mycelium from a single colony were transferred to 50 μl of Novozym (5 mg of Novozym 234 (Novo Industries) per ml of KC (KCl 0.8 M , 9.4 mM citric acid, pH 6.2)) and incubated for 1 hour at 37 ° C. Subsequently, 300 μl of DNA dilution buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 1 mM EDTA) was added and the suspension was boiled for 5 minutes at 100 ° C, followed by centrifugation vigorous to break the mycelium intact. 5 μl of these mixtures were primed as a standard in 50 μl PCR reactions containing 4 μl 10 x Super Taq PCR buffer (HT Biotechnology Ltd.), 8 μl dNTP (1.25 mM each), 20-80 ng of each oligo nucleotide and 1U Super Taq (HT Biotechnology Ltd. Cambridge, UK). The optimal amount of oligos was determined experimentally for each batch purchased. Twenty-five cycles of amplification, (Each: 1 minute 94 ° C, 1 minute 55 ° C and 1.5 minutes 72 ° C) and each final extension step of 7 minutes 72 ° C, were carried out in a DNA amplifier (e.g., Perkin-Elmer, Hybaid). For the DNA label test, as the first oligo 40/41 group was used, for the phyA cassette CPR test of the oligo group 42/43 and for the amdS cassette test the oligo group 15/16. Subsequently, to analyze the PCR products, 20 μl of the PCR mixture was analyzed by agarose gel electrophoresis on a 2% agarose gel in TBE buffer (0.09M Tris, 0.09M H3BO3, 2mM EDTA, pH 8.3). Specific CPR procedure for target for Aspergillus niger. The preparation of the DNA standards and PCR reaction conditions were used as described for the white CPR test for Penicillium. Oligo 46/48 was used as primers to determine if the amdS cassette is directed adjacent to the glaU white site and the set of oligos 46/47 to determine if the phyA cassette is directed adjacent to the glaU white site. The CPR conditions used were as described for the white CPR test for Penicillium. White PCR of transformants of P. chrysogenum Approximately one third of colonies of 4 days of age was incubated for 2 hours at 37 ° C in 50 μl of KC buffer (60 g / l KCl, 2 g / l of citric acid, pH 6.2), supplemented with 5 mg / ml of Novozym ™ 234. Subsequently 10 μl of 10 mM Tris, 50 mM EDTA, 150 mM NaCl, 1% SDS, pH 8 and 400 μl of QIAquick ™ PB buffer (Quiagen Inc., Chatsworth, USA) were added. The extracts were gently resuspended and loaded onto a rotating QIAquick ™ column. The columns were centrifuged for 1 minute in microfuge and washed once with 500 μl of QIAquick ™ PE buffer. Traces of ethanol were removed by a final rapid spin. Chromosomal DNA (PCR pattern) was eluted from the column by the addition of 50 μl of H2O and subsequent centrifugation for 1 minute at 130000 rpm. The PCR reactions contained 10 μL of buffer solution eLONGase ™ (Life Technologies, Breda, The Netherlands), 14 μL of dNTP (1.25 mM each), 1 μL of enzyme mixture eLONGase ™, 1 μL of standard and 30- 50 ng of each oligo, in a final volume of 50 μl. The optimal amount of the oligo was determined experimentally for each batch purchased. On average, 30 to 50 ng were used. The reactions were carried out under the following cycle conditions: 1x (2 minutes 94 ° C), 10x (15 seconds 94 ° C, 30 seconds 55 ° C, 4 minutes 68 ° C), 20x (15 seconds 94 ° C) , 30 seconds 55 ° C, 4 minutes of start with inclination of 20 seconds per cycle, 68 ° C), 1x (10 minutes 68 ° C). The 8μl samples were loaded on agarose gels for analysis of PCR products. Catheter PCR of P. chrysogenum transformants Approximately one third of 4-day-old colonies were incubated for 2 hours at 37 ° C in 200 μl of DVB buffer solution (10 mM Tris-10 mM NaCl, 1 mM EDTA , pH 7.5), supplemented with 5 mg / ml Novozym ™ 234. The mixture was boiled for 8 minutes and subsequently vigorously rotated to break the intact mycelium. Five μl of these mixtures were used as a standard in 50 μl of PCR reactions containing 5 μl of RCP 10 x Super Taq buffer solution (HT Biotechnology Ltd., Cambridge, UK), 8 μl of dNTP (1.25 mM each), 1 U Super Taq and 20 -80 ng of each oligo. The optimal amount of the oligos was determined experimentally for each batch purchased. On average, 20 to 80 ng were used. The reactions were carried out with the following cycle conditions: 25 x (1 minutes 94 ° C, 1 minutes 55 ° C, 1.5 minutes 72 ° C), 1 x (8 minutes 72 ° C). The 15 μl samples were loaded on agarose gels for analysis of PCR products. Isolation of chromosomal DNA The spores of A. niger and P. chrysogenum were inoculated in 20 ml of minimal Aspergillus medium (see transformation of A. niger) in a 100 ml conical flask and grown for 20-24 hours at 30 ° C. on a rotary shaker at 300 rpm. 5-10 ml of a well-developed culture were inoculated in a fresh minimum medium of Aspergillus and were grown for another 20-24 hours at 30 ° C in a rotary shaker at 300 rpm. After growth, the mycelium was recovered by filtration on a Miracloth filter (Cal Biochem) and washed with 15 ml of KC buffer (0.8 m KCl, 9.5 mM citric acid, pH 6.2). per gram of washed mycelium (3-5 g of a 100 ml culture) were added 4 ml of KC buffer and 0.25 ml of Novozym (50 mg Novozym 234 (Novo Industries) / ml of KC buffer) and the Protoplast formation was allowed to take place at 30 ° C under moderate agitation. After the protoplast formation, the KC buffer solution was added to a volume of 45 ml and the protoplast suspension was centrifuged at 3000 rpm at 4 ° C for 5 minutes in a rolling cup rotor. The protoplasts were resuspended in 20 ml of KC buffer. Then 25 ml of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2) were added and then mixing the suspension was centrifuged subsequently at 3000 rpm at 4 ° C for 5 minutes in A rolling-bowl rotor was resuspended in 50 ml of STC buffer and again centrifuged for 5 minutes at 2500 xg at 4 ° C in a rocking-pan rotor. To 0.3-0.6 ml of the protoplast pellet was added 60 μl-K (20 mg / ml) and 1 ml of melting point agarose (0.8% in 1 M Sorbitol (0.45 M EDTA) were added. to mix by twist, the suspension was transferred to wells of a preformed flexifold mold and fixed on ice for approximately 15 minutes. Then, the agarose plugs were transferred to 2.5 ml of sarcosyl solution (1% Na-lauroyl sarcosine in 0.5 M EDTA). 60 μl of Proteinase-K (20 μg / ml) was added and after incubation for 16-20 hours at 50 ° C the sarcosyl solution was replaced by 10 ml of 60 mM EDTA solution. After 2 hours at 50 ° C, the EDTA solution was replaced by 10 ml of 50 mM fresh EDTA and incubated for another 2 hours at 50 ° C. Finally, the EDTA solution was replaced by 10 ml of 50 mM fresh EDTA solution and the agarose plugs stored at 4 ° C. Restriction enzyme digestions of chromosomal DNA agarose plugs Part of DNA agarose plugs (prepared as described above), containing 1-3 μg of chromosomal DNA, was incubated in 1 ml of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) for 1 hour at 37 ° C. 1 ml of TE buffer was refreshed and the incubation continued for another hour at 37 ° C. Then the TE buffer solution was replaced by 200 μl of an appropriate regulatory solution for each restriction enzyme as recommended by the supplier. After an incubation at 37 ° C for one hour, the restriction enzyme buffer was cooled (50 μl) and incubated for 10 minutes at 65 ° C to dissolve the agarose. Finally, 20-40 Units of the appropriate restriction enzyme was added and the digestion mixture was incubated for 16 hours at a temperature as recommended by the supplier for each restriction enzyme. Anti-amdS selection procedure In most of the derived vectors the amdS selection marker gene was placed between the DNA repeats. Therefore, in transformants made with these vectors, the removal of the amdS marker gene can be achieved either by internal recombination on flanking DNA repeats within the cassette or by homologous recombination on repeats that are created by the integration of a single crossing event. The selection of cells that have lost the amdS selection marker is achieved by growing on plates containing fluoroacetamide. The cells that host the amdS gene metabolize fluoroacetamide to ammonium and fluoroacetate that is toxic to the cell. Consequently, only cells that have lost the amdS gene are able to grow on plates containing fluoroacetamide. In the case of the removal of the amdS marker from the Aspergillus transformants, the spores of these transformants were seeded in selective regeneration medium (described above) containing 5 mM of fluoroacetamide and 5 mM of urea instead of 10 mM of acetamide, 1.1 % glucose instead of 1M sucrose and 1.1% instead of 2% bacteriological agar # 1 (Oxoid, Eng / and). After 7-10 days of growth at 30 ° C the colonies alone were recovered and plated on 0.4% potato dextrose agar (Oxoid, Eng / and). In the case of the removal of the amdS marker from Penincillium transformants, the spores of these transformants were plated in minimal Aspergillus medium containing 10 mM fluoroacetamide and 5% glucose, solidified with 1.5% bacteriological agar # 1 ( Oxoid, Eng / and). After 5-10 days of growth at 25 ° C, resistant colonies appeared. Bioanalysis of E. coli The transformants were grown on YPD agar medium for 5 days. E. coli ESS2231 was used as an indicator bacterium in an agar layer, which also contained Bacto penase that may be able to discriminate between the production of penicillin and cephalosporin, according to methods well known in the art (Guttiérez et al. Mol. Gen. Genet, 1991 225: 56-64). Transformants expressing cefE were identified by a clarification of the agar layer around the colony. Improvement of industrial strain of P. chrysogenum Normal mutagenic techniques and sieving procedure well known to persons skilled in the art (Rowlands, 1984 Enzyme Microb. Technol. 6: 3-10) were used to isolate the strain of P. chrysogenum CBS 6.49.97 (deposited on April 11, 1997 in Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) of Wis54-1255 (ATCC 28089). Fermentations of adipoyl-7-ADCA and adipoyl-7-ADAC The fermentative productions and the quantification of adipoyl-7-ADCA and adipoyl-7-ADAC were essentially as described for the production of 2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propionyl-7-ADCA (WO 95/04148) with the exception of 1u was added 3 g / l of adipic acid to the culture medium in place of 3'-carboxymethylthiopropionic acid. Adipoyl-7-ADCA and synthetic adipoyl-7ADAC were used as reference substrates. Aspergillus niger shake flask fermentations From the recombinant strains the A. niger control was generated a large batch of spores by sowing spores or mycelia on PDA plates (Potato Dextrose Agar, Oxoid), prepared according to the instruction of the maker. After growth for 3-7 days at 30 ° C the spores were recovered after the addition of 0.01% Triton X-100 to the plates. Then approximately 107 spores of selected transformants were washed with sterile water and control strains were inoculated into shake flasks, containing 10 ml of liquid pre-culture medium contained per liter: 30 g maltose H2O; 5 g of yeast extract; 10 g of hydrolyzed casein; 1 g of KH2PO4; 0.5 g MgSO4.7H2O; 0.03 g ZnCl2; 0.02 g CaCl2; 0.01 g of MnSO4 '4H2O; 0.3 g of FeSO4 7H2O; 3 g of Tween 80; 10 ml of penicillin (500 IU / ml) / Streptomycin (500 UG / ml); pH 5.5. These cultures were grown at 34 ° C for 20-24 hours. 10 ml of this culture were inoculated in 100 ml of fermentation medium A. niger containing per liter: 70 g maltodextrins; 25 g of hydrolyzed casein; 12.5 g of yeast extract; 1 g of KH2PO4; 2 g of K2SO4; 0.5 g of MgSO4.7H2O; 0.03 g of ZnCl2; 0.02 g of CaCl2; 0.01 g of MnSO4.4H2O; 0.3 g of FeSO4.7H2O; 10 ml of penicillin (5000 IU / ml) / Streptomycin (5000 UG / ml); adjusted to pH 5.6 with H2SO. These cultures were grown at 34 ° C for 6 days. The samples taken from the fermentation broth were centrifuged (10 ', 5,000 rpm in a rolling-cup centrifuge) and the supernatants were recovered. Glucoamylase or phytase activity analyzes (see below) were carried out on these supernatants. Glucoamylase Activity Analysis The activity of glucoamylase was determined by incubating 10 μl of a six-fold diluted sample of the culture supernatant in 0.032 M NaAc / HAc pH 4.05 with 115 μl of 0.2% (w / v) p-nitrophenyl aD-glucopyranoside (Sigma) in 0.032 M NaAc / HAc pH 4.05. After a 30 minute incubation at room temperature, 50 μl of 0.3 M Na 2 CO 3 was added and the absorbance at a wavelength of 405 nm was measured. The A405nm is a measure for the production of GLA. Analysis of phytase activity 100 μl of supernatant (diluted when necessary) of fermentations of Aspergillus niger shake flask or micro-titration (as reference 100 μl of demineralized water) was added to a 900 μl mixture, containing acetate buffer sodium 0.25 M pH 5.5, 1 mM phytic acid (sodium salt, Sigma P-3168) and incubated for 30 minutes at 37 ° C. The reaction was stopped by the addition of 1 ml of 10% TCA (tricholoracetic acid). After the reaction was finished, 2 ml of reagent (3.66 g of FeSO4.7H2O in 50 ml of ammonium molybdate solution (2.5 g of (NH4) 6MO7O24.4H2O and 8 ml of H2SO4 diluted to 250 ml with water were added. demineralized) was added The absorbance of the blue color was measured spectrophotometrically at 690 nm Measurements indicate the amount of phosphate released in relation to the phosphate calibration curve on the scale of 0-1 mMoles / L. Cultivation of microorganisms The strain of S. clavuligerus ATCC 27064 was cultured at 27 ° C in Tryptic soy broth (Difco). The strains of P. chrysogenum were cultured at 25 ° C in complete YPD medium (1% yeast extract, 2% peptone , 2% glucose.) For solid media, 2% Bacto-agar was added Transformants were purified by repeated culture on YPD agar Stable single colonies were used to prepare agar slants for the production of spores. A. niger strains were grown at 30 ° C in plates that They contain PDAs The transformants were purified by repeated culture on PDA plates. The unique stable colonies were used to prepare newly inclined media for the production of spores. The E. coli strains were cultured according to normal procedures (Sambrook) EXAMPLES OF A. NIGER 1.1 Selection and characterization of Aspergillus niger host. 1.1.a Rational. The presence of the repeated DNA domains in the genome is essential to use gene conversion as a tool to amplify the inserted recombinant expression cassettes. For example, the glaA gene of A. niger strains that produce high glucoamylase content may be present in the amplified DNA domains. In order to identify suitable hosts for the production of industrial enzymes, we screened A. niger strains with improved production of glucoamylase. Here we describe the procedure of how to select and characterize said strains of A. niger containing the required DNA amplicons. 1.1. b Selection of mutants of A. niger. From A. niger CBS 513.88 (deposited on October 10, 1988) we selected several mutant strains that show an improved production of glucoamylase on the level of shake flask. Mutagenesis was carried out on the spores of A. niger CBS 513.88 by UV treatment. Surviving spores (approximately 1%) were tested for their ability to produce glucoamylase in shake flask fermentations as described in Materials and Methods. After 6 days of growth the activities of the glucoamylase enzyme were determined as described in Materials and Methods. . Several mutant strains of A. niger showing improved glucoamylase production levels even up to 600 U glucoamylase / ml. The A. niger CBS strain 513.88 reaches a level of approximately 200 U / ml glucoamylase. The best production of the A. niger mutant strain was deposited as A. niger CBS 646.97 on April 11, 1997 in Centraal Bureau voor Schimmecultures, Baarn, The Netherlands. 1.1. c Genetic characterization of A. niger CBS 646.97. To determine whether those described above increased glucoamylase production is the consequence of an amplification of the glaA sites, chromosomal DNA was isolated from the mutant strain A. niger CBS 646.97 and its A. niger CBS 513.88 stem. Southern analyzes were carried out on an isolated DNA, digested with EcoRI and SalI and probed with a DNA fragment of glucoamylase / phytase (fusion of glaA / phyA: an Eco RI fragment / mH I fragment of pFYT3, which it was described in Patent Application EP 0420358A1)). The autoradiographs clearly show that the improved mutant glucoamylase mutant strain contains multiple copies of (3-4) glaA genes. 1.1. d. Size of glacial amplicons in the mutant strain of A. niger. TAFE analyzes revealed that the DNA fragments were amplified in the selected mutant strain of A. niger. The chromosomal DNA was derived from the original A. niger CBS 513.88 strain as well as from the A. niger CBS 646.97 strain as described in materials and Methods. Digestions with Hindlll and the eight restriction enzymes Notl, .Swal, Ascl and Pací were performed and subsequently delivered for TAFE analysis using several probes specific for glaA sites derived from pAB6-1 (see Patent Application EP 0357127A1 ). No differences could be observed with respect to the size of the glaA hybridization DNA fragments with the restriction enzymes mentioned above between the mother strain and the mutant. The largest hybridization DNA fragment could be detected in all cases with the restriction enzyme SwaI: approximately 80 kb. The only difference observed is the intensity of the DNA band of glaA hybridization. Although the intensity of the hybridization glaze tables are difficult to quantify accurately, TAFE autoradiographs clearly show that the A. niger CBS 646.97 mutant strain contains additional copies of the observed glaA amplicon. In addition, we can not find another restriction enzyme that is cut out of the detected glacial amplicon. This means that the glauc amplicon spans at least 80 kb in size or larger. 1. 2 Modifications of the glai site on several amplicons of A. niger strain CBS 646.97. 1.2.a. Rational. As previously described (Patent Application EP 0635574A1) a genomic target gene can be deleted approximately in A. niger using the "MARKER GENE FREE" approach. In this patent application, an example of how to suppress the specific DNA sequences for glaA in the genome of A. niger CBS 513.88 was extensively described. The described replacement vector is integrated into the genomic sequence of A. niger glaA via double homologous homologous recombination. The deletion vector comprises regions of DNA homologous to the target site of glaA and the selectable marker gene of amdS driven by the gpdA promoter sequence. In addition, in this vector the amdS selectable marker gene is flanked at both sites by glaA sequences as direct DNA repeats to subsequently facilitate the appropriate elimination of the amdS marker gene. These vectors direct the replacement of the glaA gene by the amdS marker gene. Subsequently, by carrying out the counter selection of fluoroacetamide on these transformants, the amdS marker gene can be appropriately suppressed by an internal recombination event between the direct repeats of 3'-gla-A DNA. This resulted in a strain of A. niger CBS 513.88 recombinant from AglaA FREE MARKER GENE, which finally has no foreign DNA sequences. It is obvious that this "MARKER-FREE" approach is especially useful for modifying and deleting the genomic sequences of a host. This example describes the proper deletion of all the promoter and coding sequences of the glaA gene in A. niger CBS 646.97, in three successive transformation cycles using a set of three "special designated" pGBDEL replacement vectors for these three deletion sites a set of more or less three pGBDEL vectors of equal glaA replacement were constructed and used. The reason for including in each pGBDEL vector a given sequence in the single restriction enzyme recognition, is to recognize and visualize in the recombinant AglaA A. niger strain derived each truncated glau site by Southern analysis (described in the example). 1.2.e). The unique restriction site in each pGBDEL vector is placed at the 5 'end of the glaA site by trickling. However, in each pGBDEL vector, the glaA sequences inclined to address the target of the gene replacement vector at one of the glaA sites are slightly different. The consequence is that each glacial site truncated in the A. niger host is slightly different as well. The reason for including this slight difference is to visualize each glaucous site truncated by a rapid PCR test on the colonies: the so-called "DNA label" test (Figure 1). Although not essential, this aspect of incorporated DNA is especially useful for monitoring, with the help of the phenomenon described in this application for gene conversion events, the events between glacial amplicons in A. niger, the "construction" process for amplify the expression cassettes (phytase), which refer to one of the truncated glacial sites of host A. niger. the details of this "construction" process for deriving strains of rDNA for enzyme production, using the conversion events between the glaA amplicons in A. niger are described below (examples 1.5 and 1.6). 1.2.b Description of pGBDEL vectors for glaze replacement. All pGBDEL replacement vectors comprise: - a white 5'-glaA sequence with a size of 2 kb from the Hindlll site up to 200 bp in pGBDEL-5, or 180 bp in pGBDEL-9 and 140 bp in pGBDEL-11, upstream from the unique site of Xhol within the glacial promoter sequence. - a white sequence of 3'-g / aA with a size of 2 kb from the glate retention codon to the Salí site; - the amdS gene as a selectable marker under the control of the gpdA promoter; - two direct repeats of DNA of 3'-g / aA of size of 1 kb to facilitate the removal of the last of the selectable marker of amdS and, - an additional restriction site (ßamHI in pGBDEL-5; I left in pGBDEL-9 and Bglll in pGBDEL-11) placed in a glacial truncation.

Using these pGBDEL replacement vectors, the 4.3 kb glaA sequences in the A. niger host that will be deleted will include the 2 kb glacial promoter sequences (ranging from 200, 180 bp or 140 bp in pGBDEL-5, pGBDEL-9 and pGBDEL-11, respectively, upstream of Xhol) up to the start codon of ATG and the entire glaA coding sequence. For a schematic view, refer to figure 2). 1.2.C. Construction of intermediate vectors of pGBGLA. For the construction of the intermediate vector pGBGZ., 416 two oligo nucleotides 830/31 were synthesized, containing the restriction sites of enzymes: Notl, Ecol, Kpnl and Xhol. The oligo adapted with a Hindlll restriction site at the 5 'end and with an EcoRI restriction site at the 3' end. By the insertion of this nucleotide oligo into pTX18R the EcoRI site will be destroyed. Both oligo nucleotides 30 and 31 were cloned into the EcoRI and HindIII sites of plasmid pTZ18R. The control digests were carried out to ensure that the desired enzyme restriction sites for kpnl, Xhol, Notl and Hindlll were properly incorporated and the EcoRI site was destroyed. The derived plasmid was designated pTM1 (see figure 3). From pTM1, the DNA fragment of EcoRI and Kpnl was isolated and purified by gel electrophoresis after digestion with both restriction enzymes, to insert the EcoRl / Kpnl fragment of pGBDEL4L comprising the sequence PgdpA / a / 77dS. The construction of pGBDEL4L was described extensively in our Previous Patent Application (EPA 0635574A1). PGBDEL4L was digested with EcoRl / Kpnl and Xhol (the latter to avoid molecular cloning of another DNA fragment of pGBDEL4L having the same size). The correct EcoRl / Kpnl DNA fragment, comprising the PgpdA / amdS sequence, was purified by gel electrophoresis and cloned into the EcoRl / Kpnl sites of pTM1. This intermediate vector was designated pGBGLA16 (see figure 3). To properly insert the 3'-g / aA sequence with a size of 2.2 kb in pGBGL \ 16, the fragment needs some modifications. First a fusion PCR with pAB6-1 was carried out as a standard and two groups of oligo nucleotides as primers (32/33 and 34/35) to destroy the Kpnl restriction site within the sequence of 3'-g / aA 2.2 kb and to create the appropriate cloning sites at the 5 'junction (Kpnl) and the 3' junction (Xhol, Hindlll). The pAB6-1 standard comprises a 16 kb long Hindlll fragment in pUC19, containing the glaA genomic site (molecular cloning was extensively described in EPA 0357127A1). In the first PCR, oligo nucleotides 32 and 33 were used as an initiator set to amplify a part (1 kb) of the sequence 3'-g / aA. In the second PCR the oligo nucleotides 34 and 35 were used to amplify the remaining sequence of 1.2 kb flanking 3'-g / aA. After purification by gel electrophoresis both amplified fragments were used as a standard in a fusion PCR with oligo nucleotides 32 and 35 as the primer set. A schematic presentation of the PCR fusion is presented in Figure 4. The 2.2 kb 3'-glaA fusion PCR fragment obtained was purified by gel electrophoresis, digested with KpnI and Xhol and inserted by molecular cloning into the Kpnl / Xhol sites of pGBGLA16, giving pGBG A18 (see figure 4). 1.2.d. Construction of glacial replacement PGBDEL vectors. To obtain the three final vectors of pGBDEL, the white sequence '-glaA, a single restriction site for each vector and the 3'-glaA sequence of 1 kb as a direct repeat has to be inserted into the Hindlll / Notl sites of pGBG A18. To this end, three separate fusion CPRs were carried out to construct pGBDELd, -9 and -11. Construction of pGBDELd. A first PCR was carried out to modify the 5'-g / aA target sequence at the 3 'end with two synthetic oligo nucleotides: 36, containing a part of the nucleotide sequence of pUC19, the HindIII site of the sequence 5'. -g / aA and 37 equaling approximately 200 bp upstream of the Xhol site in the glaA promoter, which comprises a BamH1 site, check codons in all reading frames and the first 18 nucleotides (5 'end) of the direct repeat 3'-g / aA of 1 kb. The second PCR was carried out to modify the direct repeat 3'-g / aA at the 5 'end with the oligo nucleotides 38, which is the inverse of 7 and the oligo nucleotide 29, which is equal to the sequences around the site EcoPI of the direct repeat of 3'-glaA, added with the restriction sites X? »Al and Notl for the appropriate cloning in pTZ18R and pGBGLA18, respectively. In both PCRs, the amplification pAB6-1 was used as a standard. The amplified DNA fragments for both PCRs were separated by gel electrophoresis and used as templates in the fusion PCR with 36 and 39 as primers. The DNA fragment fragment with a size of 3 kb was purified by gel electrophoresis, directed with Híndlll and Notl and subsequently molecularly cloned into the appropriate sites in pGBG A18, giving the first pGBDELd gene replacement vector (see figure 5). Construction of pGBDELT The first PCR was carried out the synthetic oligo nucleotides: 36 and 49 (as described above) pairing at approximately 180 bp upstream of the Xhol site in the glaA promoter, comprising an additional SalI site, the stop codons in all reading frames and the first 18 nucleotides (5 'end) of the 3'-glaA direct repetition of 1 kb. The second PCR was carried out to modify the direct repeat of 3'-g / aA at the 5 'end with the oligo nucleotides 50, which is the inverse of 49 and the oligo nucleotide 39. In both PCR amplifications pAB6 was used -1 as a pattern. The amplified DNA fragments from both PCRs were separated by gel electrophoresis and used as templates in PCR fusion with oligo nucleotides 36 and 39 as primers. The DNA fusion fragment with a size of 3 kb obtained was purified by gel electrophoresis, digested with HindIII and NotI and subsequently molecularly cloned into the appropriate sites in pGBG A18, giving the second pGBDEL9 gene replacement vector (see figure 6). Construction of pGBDEL11 The first PCR was carried out the synthetic oligo nucleotides: 36 and 51 pairing at approximately 140 bp upstream of the Xhol site in the glaA promoter, which comprises an additional Bgll site, the codons of challenge in all reading frames and the first 18 nucleotides (5 'end) of the 3'-glaA direct repeat of 1 kb. The second PCR was carried out to modify the direct repeat of 3'-g / aA at the 5 'end with the oligo nucleotides 52, which is the inverse of 52 and the oligo nucleotide 39. In both PCR amplifications pAB6 was used -1 as a pattern. The amplified DNA fragments from both PCRs were separated by gel electrophoresis and used as templates in PCR fusion with oligo nucleotides 36 and 39 as primers. The DNA fusion fragment with a size of 3 kb obtained was purified by gel electrophoresis, digested with HindIII and NotI and subsequently molecularly cloned into the appropriate sites in pGBGL / 418, giving the second gene replacement vector pGBDEL11 (see figure 7). 1.2.e. Deletion of the glaA promoter and coding sequences and incorporation of specific "DNA tags" in A. niger. In this example the successful suppression of the three glaA genes present in A. niger 649.97 was described using the pGBDEL vectors in the "MARKERS GENES FREE" technology in three successive transformation cycles. By liberalizing the pGBDEL vectors with Xhol and Hindlll and the fact that the rDNA cassettes were flanked by the DNA sequences homologous to the glaA target site of the host genome, the pGBDEL vectors were integrated by a double crossover event at one of the sites of the host's glaucoma, resulting in a replacement of the glaA sequence by the glaA site by trickling pGBDEL vectors. However, the frequency of the target direction of a pGBDEL vector by a double crossover event is limited and depends on the number of white glacial genomic sites of the host. Thus, although linearized vectors are used comprising homologous regions of glaze of size 2 kb on both sides, still most of the generated transformants contain randomly integrated vectors. However, the transformant having the desired genetic aspect can be easily selected and verified by application a) the PCR-based DNA label test, b) the specific PCR procedure for the blank and c) by detailed Southern analyzes as indicated in Materials and Methods. Finally, once the appropriate transformant was genetically verified the amdS marker gene was removed by applying the anti-fluoroacetamide selection procedure. Due to the incorporated 3'-g / aA DNA repeats, flanking the cassette of the PgpdA / amdS marker gene, after each cycle of the amdS selection marker was eliminated by an internal recombination event. The modified A. niger strains thus obtained were subjected to successive transformation sites to eliminate and modify the glaA sites. Modification of the first glacial amplicon in CBS 646.97 of A. niger with pGBDELd. A. niger CBS 646.97 was transformed with 5 μg of liberalized pGBDEL5 DNA (H /? DIII / X / 7? I). The transformants were selected on selective plates containing acetamides and spores prepared after repeated growth on selective medium.

A restricted number of transformants was selected and the isolated chromosomal DNA was extensively analyzed by Southern analysis using the Kpnl and SamHI and Hindlll / Xhol digestions of 5'-g / aA and the Sali fragments of 3'-g / aA of 2.2 kb as probes. See figure 8 to compare the hybridization patterns of SamH1 from the AglaA site of the host strain and the two remaining AglaA sites of the transformant.

Transformants showing separate hybridization patterns as indicated in Figure 9 were selected. Subsequently, in these the counter selection procedure was carried out. The spores were planted in medium containing fluoroacetamide (for details see Materials and Methods). On average 1-2% of the spores planted were able to grow under these selective conditions. PCR analyzes performed directly on the mycelium of these developing colonies with amdS-specific oligos 15 and 16 as primers, revealed that all growing cells are also recombinant cells that have lost the amdS marker gene. Surprisingly, however, Southern analyzes detailed in many recombinant cells showed that in most cases (frequency of approximately 90%) the hybridization patterns were observed which are characteristic only for the parental strain 636.97, instead of the standard surpassed for a truncated glacial site and the remaining two intact glacial sites. In addition, the intensities of the DNA hybridization fragments indicated that the number of glacial amplicons did not decrease in these amdS-negative strains. Approximately 10% of the cells show the expected hybridization pattern as indicated in Figure 10. Therefore, in these cases the amdS marker gene was also deleted via the internal recombination event on the direct repeats of 3'-g / aA of flinching, as expected.

This unexpected recombination event in most of the negative strains of amdS can be explained only by the presentation of a genetic phenomenon, called gene conversions. An aspect that is used later (see example 1.5 and 1.6) to amplify the enzyme encoding expression cassettes directed to one of the truncated glaUic amplicons of host CBS 646.97 from A. niger. A negative strain for amdS, showing the hybridization patterns for a truncated glacial site and two intact glacial sites as presented in figure 10, was designated A. niger GBA-201 and subjected to a second transformation with pGBDEL- 9. Modification of the second glacial amplicon in CBS 646.97 of A. niger with pGBDELT. A. niger CBS 646.97 was transformed with 5 μg of liberalized pGBDELT DNA (Hindlll / Xhol). Again the transformants were selected on selective plates containing acetamides and spores prepared after repeated growth on selective medium. Because the bank direction of pGBDEL9 can also occur at the truncated glaU sites marked with previous SamHI from the host, only the transformants were selected and analyzed by Southern analysis, still showing the presence of the "SamHl" glaze site by trickling . For this purpose the first tests of "DNA tag" bases on CPR were carried out in mycelia of these transformants. Only these transformants showing the 200 bp DNA fragment characteristic of the glaA site trickling from "phylaHI" were analyzed by the PCR methods, followed by extensive Southern analysis as indicated above. Subsequently, the spores of the transformant showing the correct hybridization pattern (Figure 10) were seeded in medium containing fluoroacetamide (for details see Materials and Methods). Again on average 1-2% of the spores planted were able to grow under these selective conditions. The others appeared to have lost the amdS gene as revealed after the application of the cassette CPR test on the mycelium of these colonies. As a second selection criterion the "DNA label" test was carried out. Only these transformants were subjected to the detailed Southern analysis as described above, showing the desired DNA labeling pattern of 82 bands of 200 and 220 bp) referring to the presence of the truncated glaucin amplicon of "SamHl" and the glacier amplicons. truncated from "I left", respectively. A negative strain for amdS, showing the hybridization patterns for the two truncated glacial amplicons and an intact glacial amplicon (Figure 12), was designated A. niger GBA-202 and subjected to a third transformation with pGBDEL- eleven. Modification of the third glacial amplicon in CBS 646.97 of A. niger with pGBDEL11.

A. niger GBA-202 was transformed with 5 μg of liberalized pGBDELT DNA (Hindlll / Xhol). Again the transformants were selected on selective plates containing acetamides and spores prepared after repeated growth on selective medium. Also in this case the bank direction of pGBDEL11 can also occur in the previous glacial truncated sites of the host, only the transformants were selected and analyzed by Southern analysis, still showing the presence of fragments of 200 and 229 previously representing the glacier amplicons. of "BamHI" and "Salí". The transformant showing the correct hybridization pattern as indicated in Figure 13 was subjected to the counterselection procedure to obtain free recombinants of marker genes. Again, on average, 1-2% of the spores sown were able to grow under these selective conditions. The others appeared to have lost the amdS gene as revealed after the application of the cassette CPR test on the mycelium of these colonies. As a second selection criterion the "DNA label" test was carried out. Only these transformants were subjected to detailed Southern analysis as described above, showing the desired DNA labeling pattern of 82 bands of 200 and 220 and 300 bp) referring to the presence of the truncated glacial amplicon from "Sali" and "SamHI". "glaA, respectively.

A negative strain for amdS, showing the hybridization patterns (Figure 14) for the three glacial amplicons was designated A. niger ISO-505. 1.3. Description and construction of expression vectors of pGBAAS and pGBTOPFYT-1 1.3.a. Rational. The pGBTOP vectors, comprising the expression cassette for an enzyme, are introduced into the A. niger host strains by co-transformation with the amdS selectable marker gene that contains the vector, which are designated pGBAAS-1. Both vectors comprise two DNA domains homologous to the glaA sites of the A. niger host strain to direct the target of the liberalized plasmid to one of the glauca sites by trickling from A. niger ISO-505. These domains, each approximately 2 kb in size, are homologous to the glaA sequence without downstream coding and are specified as 3'- and 3"-glaA domains, including a single restriction site (Xhol). in pGBAAS-1 and Hindlll in pGBTOPFYT-1) to obtain, after removal of the cloning vector pTZ18R from E. coli before transformation by digestion.The restriction terminal of the DNA enzyme that is almost complementary to the sites Host glacial white In pGBAAS-1 the amdS gene is driven by the strong gpdA promoter Using said promoter most of the transformants obtained will have only one integrated selection marker cassette.

These transformants being crucial to carry out the next step, selected by the recombined strains FREE OF MARKERS GENES applying the technology FREE OF MARKERS GENES described previously. The presence of multiple amdS genes will certainly affect the frequency to remove them in the "one-step selection procedure" or they will be impossible. In pGBTOPFYT-1 phyA is driven by Pg / aA- As indicated above, the composition of this vector is designated in such a way that the liberalized expression cassette is integrated into a glaA bank site in A. niger ISO-505. To obtain the transformants that have multiple copies of the expression cassette against a single copy of the co-vector (necessary to finally obtain the recombinant strains FREE OF MARKERS GENES) in only one transformation cycle and both directed to the same target site of the genome host, the ratio of liberalized DNA fragments from both cassettes is crucial. 3.1.b. Construction of the integration vector pGBAAS-1. All the details relating to the construction of pGBAAS-1 can be found in one of the previous patent applications EPA 06335574A1. In this patent application the construction of the pGBG vector A-50 of the amdS selection marker gene is extensively described. For naming reasons only, then in this vector it is renamed as pGBAAS-1 (Aspergillus A_mdS Shuttle). See figure 15 for the physical map. 3. 1 C. Construction of the integration vector pGBTOPFYT-1.

Also the construction of pGBTOPFYT-1 can be found in the same patent applications as mentioned above. In this patent application the construction of the phytase expression vector pGBGLA-53 is described extensively. Again for nomenclature reasons only, then in this vector they are renamed pGBTOPFYT-1 (JHerra lies for Envelope Proteins). See figure 16 for the physical map. 1.4 Development of a FREE MARKER GENE phytase, which produces A. niger strain, containing one or multiple phytase expression cassettes all directed to one of the glazed amplicons of A. niger ISO-505. 1.4.a. Rational. The help of this example is to show that the expression cassettes (only one or even multiple copies) can be perfectly targeted to a predefined target site in the genome of a host cell by co-transformation with a vector having a selectable marker gene. The second part shows how to remove the selectable marker gene cassette by an internal recombination event without losing the targeted (multiple) enzyme expression cassettes. The target sites of choice in this case is the 4 kb glaze sequence located just downstream of the glacial stop codon. The integration of both linearized plasmids of pGBTOPFYT-1 and pGBAAS-1 is presented via a single cross-event in one of the truncated and marked glacial sites of the A. niger ISO-505 strain. To finally obtain the recombinants FREE OF GENES MARKERS selection, only these transformants are selected for the subsequent fluoroacetamide selection procedure, both plasmids having an appropriate form in the same site of glaze labeled from the host genome. 1.4.b. Co-transformation of A. niger ISO-505 with pGBAAS-1 DNA and liberalized pGBTQPFYT-1. Both plasmids, pGBAAS-1 and pGBTOPFYT-1, were linearized by digestion with Xhol and Hindlll, repetitively. The 2.8 kb E. coli cloning sequence was removed before transformation by gel electrophoresis. A ratio of 1 to 5 μg linearized DNA from pGBAAS-1 and pGBTOPFYT-1, respectively, was used to transform A. niger ISO-505 as described in Materials and Methods. Transformants were selected on acetamide plates. The spores of these transformants were isolated and plated, the individual colonies on the PDA plates. Approximately 500 transformants were subjected to the cassette CPR test. 1.4.c. Selection for co-transformants. Transformants having the amdS marker gene as well as the phyA expression cassette were identified by the cassette PCR test using oligo nucleotides specific for phytase 42/43 as primers. Positive transformants containing one or multiple phyA expression cassettes will show a specific DNA band of 482 bp in size. The co-transformation frequencies varied between 10 to 50%. 1.4.d. Selection of directed amdS * and phyA * co-transformants. On the identified co-transformants, directed PCR tests were carried out using two groups of oligo nucleotides 46/57 (primer set 1) and 46/48 (primer set 2). Co-transformants that show a positive result (amplification of a 4.2 kb DNA fragment, see figure 17) with one of these two primer sets were selected. A negative result indicates that all the cassettes are randomly integrated, a positive result with the initiator set 1 or 2, implies phyA or amdS is located adjacent to the glai white site, respectively. Between 5-50% of the co-transformants seemed positive. An identified number of identified strains was selected for extensive Southern analyzes. 1.4.e. Genetic analysis of co-transformants directed from amdS and phyA directed to the target by Southern analysis. Chromosomal DNA was isolated and Southern analyzes were carried out using BglII digestion hybridized with amdS and DNA fragment 3"-glaA from Sall / Xhol of 2.2 kb as probe.S schematic drawings of six DNA hybridization patterns observed for the most part they are shown in Figures 18 to 23. The co-transformants that show the characteristic hybridization pattern by having a single copy of arhdS and a cassette or multiple phyA cassettes (Figures 20-23), were subjected to the procedure of anti-selection of fluoroacetamide to finally remove the amdS selection marker gene 1.4.f.Selection of free phytase-producing strains of the amdS gene free gene. {amdS) The recombination on the direct repeats created the integration directed to the target of the amdS expression cassette can be selected in the fluoroacetamide medium .. In the present, we describe the free amdS recombinant strains, which still contain the integrated (multiple) phyA expression cassettes using the anti-fluoroacetamide selection procedure. The spores of selected amdS + / phyA + co-transformants targeted to the target were seeded on fluoroacetamide plates. Initially, the developing colonies were analyzed by several tests based on successive CPR. First, all progenies were probed for the genotype, amdS. "The selection for amdS 'genotypes was performed by carrying out the PCR cassette test on the colonies using the amdS-specific primers as described in Materials and Methods. however, surprisingly, in the form of "DNA tag" based on subsequent CPR, it appeared that in most of the progenies a complete glauc amplicon was also lost. In addition, most of the amdS 'recombinants were also lost to target phyA cassettes, as appeared in the third PCR test using the primer set specific for phyA. These results can be explained only by the presentation of a phenomenon, which is known as gene conversion. In several cases, a large DNA fragment or even the size of the complete glauc amplicon (>; _80kb) was replaced by another glacial amplicon. In most progenies (more than 90%), however, it appeared that a complete amplicon of glaucoma was suppressed. Therefore, to fully cover the observed results, terms such as conversion and amplicon suppression are more appropriate. The observed phenomenon was used to determine if all the cassettes of amdS and phyA in the co-transformants were directed to the site in the same place of truncated glaucous and in which one of them. To determine this, only one or two colonies resistant to flouroacetamide from each co-transformant was tested, which will result in the genotype: amdS7p 7yA7g / aA-amplicon2 + carrying out a mixed PCR test using phyA and primer sets of oligo nucleotides of "DNA label". The results showed that transformants can be made by having the amdS and phyA cassettes integrated into one of the three glacial amplicons. Subsequently, from said identified co-transformants, the additional progenies were tested by applying a PCR test mixed with the sets of phyA primers and "DNA tag". The presentation of the desired amdS '/ phyA' / g / aA-amplicon3 + genotype varied strongly (from 1 to 20%) between the progenies of the individual co-transformants. This frequency lost to a large extent depending on the genetic composition of the amdS and phyA cassettes within the glacial amplicon of the preselected co-transformants. In this case, in a cotransformant, multiple cassettes have been directed to the target in a glade site and many repetitions have been created. The consequence of the fluoroacetamide counter-selection procedure is that the amdS cassette loss may occur differentially, resulting in amdS progenies having a different number of phyA cassettes.For this reason, from each pre-selected cotransformant, they were selected several recombinants of progenies (all showing the desired genotype of aA77dS7p / 7yA7g / aA-amplicon3 +) but containing a different number of the remaining phyA cassettes The pre-selection of said progenies strains could easily be made by comparing the difference in intensity of the specific bands of phyA DNA with the DNA bands representing the three glaA amplicons in the PCR test described above Finally, to confirm the genetic composition of selected amdS progenies, Southern analysis was performed on the chromosomal DNA digested with SamHI and BglW, hybridized with a 3-g / aA DNA fragment from Sal \ IXho \ 2.2 kb as a specific probe. Strains showing the hybridization pattern as indicated in Figure 24 by having 1, 2 or 3 phyA cassettes targeted at one of the glaA amplicons marked with SamHI, were selected and designated A. Niger NP505-1, -2 and -3, respectively. These strains, together with those having the phytase cassettes in one of the other two glaA amplicons, were subsequently tested for their ability to produce phytase. The shake flask fermentations were carried out as indicated in Materials and Methods. Measurement of phytase activity in the supernatant revealed that all strains produce the same amount of phytase (100 U / ml) per copy of the phyA gene and appeared to be independent of the glaA amplicon in which the phyA genes were located. 1.5 Selection of converters comprising an increase in the phytase cassette from 1 to 2. 1.5.a Rational. The number of copies of an expression cassette targeting a DNA amplicon can be increased through gene conversion. Consequently, the production of the enzyme encoded by the gene can be increased. We have described the selection and characterization of phyA converters, showing an increased production of phytase. 1.5.b Selection of putative phyA converters. As an example, strain A. Niger NP505-2 was chosen. Recombinant strains containing less than one or more phyA cassettes targeted in another glacial amplicon may also be used. Spores of the strain of A. Niger NP505.2, which contain two phyA cassettes directed to the glaA amplicon marked with ßamHI were plated in the plates containing PDA medium and after 2-3 days the PCR test was carried out in the individual colonies using the specific primers of the DNA label of p? yA (see Materials and Methods). Using this method, a genetic exchange can be visualized very quickly (conversion / amplification or deletion) between the three labeled glacial amplicons. Although the frequencies of the genotypes obtained vary greatly, on average we observed that more than 95% of the colonies tested showed no change in the three glacier amplicons. 5% of the progenies showed either a deletion of one or two glaA amplicons, or an amplification of one of the glaA amplicons and the desired converter was also detected, as a result of a conversion between the glaA site "labeled with ßamHI" and one of the other two glacial amplicons (one example of each is presented in figure 25). 1.5.c Southern analysis of phyA converters. To determine whether the number of phyA copies in these converters are also double, several of these converters (BamHI2 + / Sai / Bglir or BamHI2 + / Sair / BglH + genotype) were subjected to extensive Southern analysis using Sa HI and Sg / ll digestions. probed with the 5'g / aA DNA fragment of Hind \ / Xho \ specific for GlaA and the fragment of adN 3"-glaA of Sal \ / Xho \, respectively.

The hybridization patterns of SamHI digests of the parenteral strain and selected converters were shown in Figure 25. As expected and compared to the strain of N. niger NP505-2, the PCR identified BamH \ 2 + genotypes also showed in the SamHI digestions inverse intensities for hybridization DNA fragments of 2.5 kb and 4.2 kb. In the SamHI2 + genotype, the 2.5 kb fragment represents two amplicons of glaA labeled with SamHI and the 4.2 kb fragment of the remaining SalI glia amplicon or ßg / ll, depending on the genotype of selected BamH \ 2+ converters. The observed results clearly indicate that a predefined amplicon can be multiplied by "gene conversion" at the expense of another related amplicon. The hybridization patterns of the Sg / ll digestions clearly showed that in the selected SamHI2 + converters, the number of phyA gene copies is also doubled. As indicated in Figure 26, a digestion of Sg / ll of chromosomal DNA from the parental strain shows 5 fragments of DNA hybridization using the 3"-g / aA fragment as a probe.The 5 rows of 14.9, 11.7, 7.5 , 5.7 and 5.6 represent, respectively, the 5 'flanking sequence, the Sa / I labeled amplicon, the phyA-phyA transition, the 3' flanking flange sequence and the glaA amplicon marked with 11. In the parental strain, all these bands have more or less the same intensity of hybridization, as also shown in Figure 26, in the selected converters with the genotype BamHl2 * / Sal * / Bglir or BamHl2 * / Sal * / BglII *, the DNA fragment of 5.6 kb or 11.7 kb, respectively, is absent while the intensity of the hybridization fragments of 14.9, 7.5 and 5.7 kb is doubled.

This result indicates that the 2 phyA cassettes located within the glaA amplicon labeled with ßamHI of parental strain is also doubled by the conversion event in converters of SamHI2 + selected. 1.5.d Analysis of phyA converters for the production of phytase. In order to determine if the selected phyA converters also show an increased production of phytase, fermentations of shake flasks were carried out as described in Materials and Methods. On average, phytase expression of parental strain NP505-2 (which has 2 copies of phyA genes) was measured at a level of approximately 200 U / ml. As expected for both selected converters with the SamHI2 + / Sa / or ßamHI2 + / Sg / i genotype, phytase levels up to 400 could be detected U / ml. Both strains, designated A. Niger NP505-4 and -5, were used in Example 1.6 to further increase the number of phyA gene copies. 1.6 Selection of converters comprising multiple modified DNA amplicons. 1. 6.a. Rational. The expression cassettes targeted in one of the DNA amplicons present in a host strain can be multiplied only by screening progenies of the parent strain and selecting the recombinant strains in which the desired conversion has occurred. Although to a low degree, as described above, conversions between these DNA amplicons occur spontaneously. This gene conversion process can be used repeatedly, resulting in new converters having an improved number of modified DNA amplicons (phyA). Using this approach, we have identified a converter that finally has 6 copies of phyA genes, all equally divided over the three modified glaA amplicons of the A. niger ISO-505 host. The degree of "enrichment of the enzyme expression cassette" depends on the number of DNA amplicons originally present in the host strain. However, applying this PCR-based DNA label test also seemed possible to isolate progenies that have a DNA label profile that could be explained only by a spontaneous amplification of one of three amplicons of glaA DNA. By repeatedly using these recombinant strains, one could finally select, having additional g / aA-DNA amplicons compared to the host strain, each containing 2 phyA expression cassettes. 1.6.b. Selection of putative converters comprising 6 copies of phyA genes. The spores of A. niger strain NP505-4, described in example 1.5 (d), containing 4 phyA expression cassettes in total (two phyA cassettes in each amplicon of glaA marked with ßamHI) were seeded of PDA and after 2-3 days the DNA label PCR test was carried out on the individual progenies using the specific label-AD primers. As stated above, again approximately 95% of the colonies tested show no change in the genotype of glaucous amplifications. Approximately 5% of the progenies showed a DNA labeling pattern that targets both a deletion of one of the amplicons labeled with ßamHI or Sa / I or even a spontaneous amplification of one of the glaA amplicons marked with ßamHI. In addition to these recombinant strains, the converters having the desired DNA label standard also identified the ßamHI DNA label band with a size of 200 bp with a slightly increased intensity. All strains of the progeny showing the genotype of "ßamHI3 +" in the "ßan7HI3 + / Sa / l +" converter were subjected to Southern analysis. To increase the phyA gene in the "BamH \ 3 * / Sal \ +" converter to 8, spores were seeded in the PDA plates and after 2-3 days of growth, the PCR test was carried out. DNA tag in the individual progenies using specific DNA tag primers. At a rather low frequency (1 in 1000) a "SamHI4 +" converter showing a DNA tag pattern with an increased intensity of the 200 bp ßamHI tag DNA fragment and the subsequent loss of the DNA fragment of the Sa / I tag with the remaining 220pb size (figure 27). This converter strain was also subjected to Southern analysis. 1-6-c Genetic characterization of converters by Southern analysis. The number of phyA gene copies of the above-identified converters (PCR-based genotypes "ßamHI3 +", "ßamHI3 + / Sa / l +" and "ßamHI4 +" was determined by Southern analysis using digestions of ßamHi and ßg / ll, hybridized respectively with DNA fragments specific for 5'-g / aA of Hindi / Xho \ and 3"-g / aA of Sal \ IXho \, as probes The expected hybridization patterns of the ßamHI digestions of the parental converters (genotype) ßamHI2 + / Sa / l + / ßg / ir or ßa / 77HI2 + / Sa / r / ßg / H +) and the selected converters thereof are shown in figures 25 and 27. The digestions of ßamHI also show a single fragment Hybridization of 2.5 kb for the "BamH \ 3+" converter. For "ßa / r7hl3 + / Sa /" an additional band of 4.2 kb was detected, indicating that in this converter the amplicon labeled with Sa / I is still present as Predicts in the result obtained from the DNA label pattern The "ßamHI4 +" converter shows the same pattern No hybridizing the converter "ßamHI3 +". By comparing, the hybridization patterns of the original parental strain, A. Niger NP505-2 and the two parental converter strains ßamH | I2 + /, Sa / I IBglW and ßa / 77HI3 + / Sa / r / ßg / H +, see previous figures. The expected hybridization patterns of a ßg / ll digestion in DNA isolated from the strains mentioned above are indicated in figure 28. For the "BamH \ 3+" as well as for the "BamH \ A +" converter, three bands were detected of hybridization (14.5, 7.5 and 5.7), all with equal intensities, indicating that in addition, the 5 'and 3' flanking sequences of the phyA gene copies were increased up to 6 and 8, respectively. For the "ßamHI3 + 7Sa / l +" converter, the expected pattern was observed, including the band with a size of 11.7 kb (characteristic for the presence of the Sali amplicon). The intensity of this band is of course three times smaller. These results clearly indicate that the converters can be selected, containing three and up to four amplicons of glaA labeled with BamHI, all containing 2 cassettes of phyA. 1.6.d Analysis of phytase production by fermentations in shake flask. To determine if the converters "ßamHI3 +" and "BamH \ 4+" also show an increased production of phytase, the fermentations in shake flasks were carried out as described in Materials and Methods. On average, phytase expression of parental strain NP505-2 (which has 2 copies of phyA genes) was measured at a level of approximately 200 U / ml. As predicted for these three selected converters, phytase levels of approximately 600 U were measured for the "ßamHI3 + / Sa / l +" genotypes and approximately 800 U / ml for the "ßamHI4 +" converting strain. Then, these strains were designated A. niger NP505-6, -7 and -8, respectively. 1.7 Direct selection of recombinants that have improved phytase production levels. 1.7.a Rational. Due to the fact that the amplicons of glaA DNA were marked by so-called DNA labels, the converters could be selected only by carrying out PCR-based DNA label testing. Applying this genetically based screening approach, we were able to show and prove that the phyA expression cassettes multiplied as a result of conversions between the amplicon of glaA marked with BamHl modified by phyA and the other two amplicons. As stated earlier, we observed a linear relationship between phyA gene copy numbers and phytase production levels indicating that selection may also be based on expression. In this example, we show the isolation of converters that have increased phyA gene copy numbers by screening progenies of a recombinant strain of phyA targeting the initial BamH? -la blank for increased phytase expression levels in microwell plates. 1.7.b. Screening for increased phytase expression levels. The spores of single colonies of the A strain.

Niger NP505-2 were inoculated into 96-well plates, containing 200 μl of A. niger fermentation medium as described in Materials and Methods. After 7 days of growth at 34 ° C, 100% humidity and light agitation, the activity of the phytase enzymes in the supernatant of each well was determined. Enzyme expression levels of approximately one thousand progenies were tested. Cells exhibiting increased phytase activity were recovered, the spores were collected and applied to an additional microtiter sieve cycle. Again, cells with increasing activity of the phytase enzyme were found. In the subsequent fermentations in shake flasks, said identified strains show phytase expression levels up to 600 U / ml. 1.7.c Genetic characterization of identified recombinant strains. The improvements in phytase production observed from the strains described above appeared to be the result of an increase in the copy number of phyA genes, either by amplification or conversion of the glaA site marked with ßamHI containing phyA. First, the genotype of these selected strains was determined by applying the DNA label test and secondly by Southern analysis. The DNA label test clearly indicated that all classes of "DNA label" genotypes were among the selected strains. These strains showed the same genotypes that were previously isolated either by carrying out the DNA labeling test as a selection criterion similar to: ßamHI2 + / Sa / l + / ßg / ll ", ßamHI2 + / Sa / + / ßg / ir, ßamHI3 + / Sa / l + / ßg / ll ", ßamHI2 + / Sa / 7ßg / ll +, ßamHI3 + / Sa / l7ßg / ir. All these genotypes were confirmed by Southern analysis as described in previous examples. EXAMPLES OF PENINCILLUIM 2.1 Selection and characterization of the host of P. chrysogenum 2.1.a Rational. The amplification of genes inserted through the conversion of genes requires the presence of multiple homologous DNA domains in the genome. These domains have been described for P. c? Rysogenum (in this report, they are called PEN amplicons: Fierro et al., 1995, Proc. Nati, Acad. Sci. USA 92: 6200-6204). The penicillin pool, a 15 kb domain spanning penicillin biosynthetic genes pcbAB, pcbC and penDE, is located in these PEN amplicons (Fig. 29) (Ten et al., 1990 J. Biol. Chem. 265: 16358 -16365; Smith et al., 1990 EMBO J. 9: 741-747). We have described the selection and characterization of the host strain of P. c / 7rysogenum CBS 649.97, which contains multiple copies of the PEN amplicon. 2.1.b Quantification of PEN amplicons. The strain of P. chrysogenum CBS 649.97 was obtained by classical improvement of strains of strain WÍ54-1255 (ATCC 28089), which contains a PEN amplicon alone (Fierro et al., 1995 Proc. Nati. Acad. Sci.

USA 92: 6200-6404). The number of PEN amplicons in strain CBS 649.97 was determined by Southern analysis. To this end, the chromosomal DNA was digested with ßsfXI and hybridized to HELE and niaD probes. A 7 fold higher ratio of HELE / niaD hybridization was detected for CBS 649.97 compared to Wis54-1255 (Fig. 30,42). Similarly, the DNA digested with Not? - by CHEF, was hybridized to the HELE probe (FIG. 42). A major hybridization signal, approximately 420 kb larger, was detected in CBS 649.97 than in Wis54-1544 (Figure 31). These results indicated the presence of approximately 7 PEN amplicons in the CBS strain 649.97. 2.2 Construction of expression vectors. 2.2.a Rational. The targeted (target) insertion of rDNA molecules into the genome occurs through homologous recombination. Therefore, rDNA cassettes should be flanked by DNA fragments homologous to the target site in the genome (Fig. 32). Two target domains within the penicillin array, defined as HELE and HELF, were chosen for the direct insertion of the expression cassettes in the PEN amplicons (Fig. 29). Here we describe the design and construction of the expression vectors of amdS, cefE and cefF used in this report. 2.2.b Basic design of expression vectors. Linear DNA molecules are crucial for targeted integration in the genome. In addition, both 5 'and 3' ends (flanks) must consist of DNA homologous to the desired integration site. The transformation fragments, therefore, comprise the expression cassette (the gene of interest regulated by a suitable promoter and terminator) flanked by the 5 'and 3' target domains. These fragments were cloned into an E. coli vector for propagation of the plasmid. The resulting expression vectors were designed so that the E. coli sequences were removed during lienarization and isolation of the transformation fragment (Fig. 32). Therefore, the recombinant strains will be free of E. coli DNA (E. P. 0635 574 A1). 2.2.c Construction of the expression vector pHELE-A1. The target domain of the HELE 5 'target was amplified by PCR of the chromosomal DNA of the P. chrysogenum strain CBS 649.97, using oligo 1 and 2. The resulting product was cloned as a? / Oyl-SpHI fragment in pZErO ™ -1 (Invitrogen, Carlsbad, USA), giving the pHELE 5 'plasmid. Similarly, the target domain of the HELE 3 'target was amplified with PCR using oligo 3 and 4 and cloned as a Hind-Not fragment in pHELEd', giving the plasmid pHELF53. The amdS expression cassette, comprising amdS regulated by PgpdA and amdS, was amplified by PCR from plasmid pGBDEL4L (EP 0 635 547 A1), using oligo 5 and 6. The resulting product was cloned as a fragment of Not \ in pHELE 53, giving the expression vector pHELE-A1 (Fig. 33). 2.2.d Construction of the expression vector pHELF-A1. The direction domain to the 5 'HELF target was amplified by PCR of the chromosomal DNA of P. chrysogenum strain CBS 649.97, using oligo 7 and 8. The resulting product was cloned as a fragment of Not \ -Kba \ in pZErO ™ -1 (Invitrogen, Carlsbad, USA), giving the phelfd 'plasmid. Similarly, flanking 3 'HELF was amplified by PCR, using oligo 9 and 10 and cloned as a Pst \ -Not \ fragment in pHELFd', giving the plasmid pHELF53. The cassette of d amdS expression, encompassing amdS regulated by PgpdA and amds, was amplified by PCR from plasmid pGBDEL4L (EP 0 63d d47 A1), using the oligo dy 6. The resulting product was cloned as a fragment of Notl in pHELE d3 , giving the expression vector pHELE-AI (Fig. 34). 0 2.2.e Construction of the expression vector pHELE-E1. The PpCbc was amplified by PCR of the genomic DNA of the strain of P. c /? Rysogenum CBS 649.97, using oligo 11 and 12. The resulting product was cloned as a fragment of Xho \ -Nde \ in pGSEWA (WO 9d / 04148 ), giving pISEWA-N. Finally, the cefE expression cassette, d regulated by Ppobc and TpenDE, was cloned as a fragment of Not \ from pISEWA-N in pHELE53, giving the expression vector pHELE-E1 (Fig. 35). 2.2.f Construction of the expression vector pHELF-F1. The cefF gene was amplified by PCR of the genomic DNA of S. clavuligerus ATCC 0 27064, using oligo 13 and 14, according to the Long-Scale Extended PCR System ™ (Boehringer Mannheim). Cycle conditions: 30x (1min 98 ° C, 5min 70 ° C), 1x (7min 72 ° C). The resulting product was cloned as a Nde \ -Nsi fragment in pISEWA-N (replacing cefE), yielding pISFWA. Finally, the cefF expression cassette, regulated by Pp0bc and TpenDE, was cloned as a NotI fragment of pISFWA in pHELF63, giving the expression vector pHELF-F1 (Fig. 36). 2.3 Modification of PEN amplicons with CEFE 2.3.a Rational. The efficient selection of transformants requires a selectable marker. Mushroom co-transformation is a well-known procedure by which the host was simultaneously transformed by two different rDNA molecules; one containing the gene of interest and the other containing the selection marker. The use of the bidirectional amdS marker allows the repeated transformation of the host of P. c. Rysogenum (E.P. 0 63d 647 A1). Recombination over direct repeats created by targeted integration will result in the loss of the amdS cassette and can be selected in the Fluoroacetamide medium (Fig. 37). Here we describe the selection of amdS-free recombinants containing the CEFE expression cassette integrated in one of the PEN amplicons. 2.3.b Selection of co-transformants of amdS, cefE directed to the target. The strain of p. cbrysogenum CBS 649.97 was cotransformed with fragments of HELE-A1 and HELE-E1 lined with Sfil (Fig. 33, 36). Transformants containing amdS were selected on acetamide plates and tested for CEFE by cassette PCR, using oligo 17 18 (Fig. 40a). The co-transformants with amdS or cefE integrated in HELE, were identified by PCR that targets the target, using oligo 19, 20 and 21 (Fig. 40.b., c). These strains were used to isolate the amdS recombinants. "2.3.c Selection of cefE recombinants (marker-free) from amdS The spores of the amdS co-transformants, cefE, described in 2.3.b, were seeded on plates in the medium of fluoroacetamide The recombinants that retain cefE were identified by the bioassay of E. coli The loss of amdS but the presence of at least one copy of cefE was confirmed by the cassette PCR, using the oligo 15, 16 , 17 and 18 (Fig. 40. a, d) .These strains were selected for Southern analysis 2.3.d Southern analysis of amdS 'recombinants.The integration of cefE in HELE and the number of copies of cefE in the recombinants of amdS ', cefE * was analyzed by Southern and TAFE To this end, the chromosomal DNA of recombinants was digested with Nrul and hybridized to the cefE probe (Fig. 38, 42). DNA digested with Hpal, separated by TAFE (Fig. 39). In a hybridization pattern expected for the integration of cefE into HELE, they were selected for further experiments. 2.4 Modification of a second amplicon with amdS 2.4.a Rational. The conversion of Genes essentially replaces the conversion of the genetic information of an acceptor DNA strand with a donor DNA strand. Both the donor and acceptor threads can be marked to view this event. The strains, as described in 2.3, already have an amplicon marked by cefE (in this case, the donor thread). We describe the modification of an acceptor thread by inserting a cassette of amdS alone into a PEN amplicon different from cefE (Fig. 43). 2.4. Direction to the amdS target to a second PEN amplicon. The strains, as described in 2.3.d, containing cefE in an amplicon, were transformed with the fragment HELE-A1 linearized with Sfil (Fig. 33). The integration of amdS and the direction to the HELE target was tested by the cassette and target address CPR using oligos 16, 16 and 19, 20, respectively (Fig. 40c, d). The integrations of amdS adjacent to cefE or multiple copies of amdS at a single HELE site were identified by target-directed PCR using the oligo 20, 21, 22, and 23 (Fig. 40e, f, g). The remaining strains, therefore, containing a single cassette of amdS integrated in a second amplicon, were selected for Southern analysis. 2.4.c Southern analysis of amdD transformants. The chromosomal DNA of the transformants described in 2.4. b were digested with Hpal and hybridized to an amdS probe (Fig. 41, 42). Strains with a hybridization pattern expected for the correct integration of amdS into HELE were selected for gene conversion experiments. 2.5 Production increase by gene conversion. 2.5.a Rational. The number of copies of an integrated gene is increased through the conversion of genes (Fig. 43). Consequently, the production of the enzyme encoded by the gene or its catalytic activity can be increased. Recombinant strains of P. chrysogenum expressing cefE, produce adipoyl-7-ADCA when fermented under the appropriate conditions (Crawford et al., 1995 BIO / Technol.13: 58-62). Here we describe the selection and characterization of cefE gene converters and the resulting increase in production of adipoyl-7-ADCA. 2.5. b Selection of CefE gene converters. The spores of strains described in 2.5.a, containing cefE and amdS in different PEN amplicons, were seeded in plates in fluoroacetamide medium to select amdS 'recombinants. The genotype of amdS ', cefE * of these recombinants was confirmed by cassette PCR using oligos 15, 16, 17, 18, 26, and 27 (Fig. 40. a, d, h). Oligo 26 and 27 were included to amplify a fragment of the unique niaD site, which served as an internal reference for the relative quantification of the PCR products. Gene converters, identified as strains that lose the amdS marker but obtain copies of cefE, as judged by the increased cefE / niaD ratio to the parental strain, were selected for Southern analysis. 2.5.C Southern analysis of the cefE gene converters. The chromosomal DNA of the strains selected in 2.5.b was digested with Nrul and hybridized to cefE and niaD probes. Strains with a hybridization pattern identical to the parental strain, but increasing ratio of cefE / niaD hybridization signals, were selected for fermentations of adipoyl-7-ADCA (Fig. 48). 2. 5.d Production of adipoyl-7-ADCA gene converters of cefE. The production of Adipoyl-7-ADCA from the gene converters selected in 2.7.b was determined by fermentations in shake flasks. All the selected strains produced significantly more adipoyl-7-ADCA than the corresponding parental strains. 2.6. Simultaneous increase of different expression cassettes by gene conversion. 2.6.a Rational. Domains that span large segments of the amplicons, or that extend the edges of the amplicons, can participate in the conversion of genes. Therefore, the number of copies of different genes adjacent to each other can be increased simultaneously by the conversion of genes. Recombinant strains of P. chrysogenum expressing cefE and cefF produce adipoyl-7-ADAC, when fermented under the appropriate conditions (Crawford et al., 1995 BIO / Technol.13: 58-62). Here, we describe the simultaneous conversion of genes from expression vectors of cefE and cefF, which are directed to the site in different locations of the same PEN amplicon (cefE + cefF), and the resulting increase in the production of adipoyl-7-ADAC. 2.6.b Selection of transformants of (cefE + cefF). The strains, as described in 2.3.d, containing cefE integrated in HELE, were co-transformed with fragments of HELE-F1 and HELE-A1 linearized with Sfil (Fig. 34,36). Transformants were selected on acetamide plates and tested by cassette PCR for cefF and amdS, using oligos 15, 16, 17 and 25 (Fig. 40d, j). The integration of amdS or cefE in HELF was determined by targeting PCR to the blank using oligo 20, 21 and 24 (Fig. 40 j, k). The chromosomal DNA of these strains was digested with Notl, graphed and hybridized to the HELE probe (Fig. 44). Strains with a hybridization fragment corresponding to the integration of cefF or amdS in HELF in the PEN amplicon that already contains cefE, were used for the selection of marker-free recombinants (amdS ~). 2.6.C Selection of recombinants (cefE + cefF) MARKER FREE. The spores of strains described in 2.6. b were seeded onto plates of fluoroacetamide medium for the selection of amdS 'recombinants. Recombinants that lost amdS but that retained cefE and cefF, were identified by cassette PCR, using oligos 15, 16, 17, 18 and 25 (Fig. 40a, d, j). The chromosomal DNA of these strains was digested with Nrul and hybridized to the cefF probe (Fig. 42, 45). The strains with correct integration of cefF in HELF were used for the selection of gene converters. 2.6.d Selection of gene converters (cefE + cefF). The spores of the strains described in 2.6.c above, were grown to single colonies and analyzed for the amount of cefE, cefF and niaD by cassette PCR, using oligos 17, 18, 26, 26 and 27 (Fig. 40a , h, 1). The gene converters were identified by the simultaneously increasing relationship of cefE / niaD and cefF / niaD, compared with parenteral strains. The increased copy numbers of cefE and cefF was confirmed by Southern analysis. To this end, the chromosomal DNA was digested with Nrul and hybridized to the cefE and niaD probes (Fig. 46). The spots were divided and subsequently hybridized to the cefF and niaD probes (Fig. 47). Strains with increased cefE ratios (niaD and cefF / niaD compared to the stock strains were selected for adipoyl-7-ADAC 2.6.e Production of adipoyl-7-ADAC gene converters (cefE + cefF). of adipoyl-7-ADAC of the gene converters selected in 2.6.d, was determined by shake flask fermentations.All the selected strains produced increasing amounts of adipoyl-7-ADAC purchased with the parenteral strains.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT (A) NAME: Gist-Brocades B.V. (B) STREET: Wateringseweg 1 (C) CITY: Delft (D) STATE: none (E) COUNTRY: The Netherlands (F) ZIP CODE (ZIP): 2600 MA (G) TELEPHONE: +31 (0) 15-2799111 (H) TELEFAX: 31 (0) 15-2793957 (ii) TITLE OF THE INVENTION: Conversion of genes as a tool for the construction of recombinant industrial organisms (iii) SEQUENCE NUMBER: 52 (iv) COMPUTER LEADABLE FORM: ( A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: GTAGCTGCGG CCGCCTCCGT CTTCACTTCT TCGCCCGCAC T 41 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (ii?) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: CAAAGGGCAT GCGGCCGTAT CGGCCGGTGA CAAACATCAT TCAACGCC 48 ( 2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: line al (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: ATGTTTAAGC TTGGCCGATA CGGCCAAAAC ACCTTTGATT TC 42 (2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : another nucleic acid (A) DESCRIPTION / desc = "oligonucleotides" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: CAAGTTGCGG CCGCTCCTCA CTAACGAGCC AGCAGATATC GATGG 45 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH 39 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE MOLECULES: another nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: AAGCTTATGC GGCCGCGAAT TCGAGCTCTG TACAGTGAC 39 (2) INFORMATION FOR SEQ ID NO : 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: CGGTACGTGC GGCCGCTCGT ACCATGGGTT GAGTGGTATG 40 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: ATATGTGCGG CCGCTTTACA TGGTCAATGC AATTAGATGG TGG 43 2) INFORMATION FOR SEQ ID NO: 8: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: ATAACTCTAG AGGCCCTACC GGCCTTTGCA AATATACTGT AAGAAC 47 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETICAL: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: GTATATTCTG CAGGGCCGGT AG.GGCCAACA GTTTCCGCAG GTG 43 2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) ) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (ii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: GTATGGGCGG CCGCTTTACA ACTAGAATAT GGGAACCTGT GGG 43 2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11: CT03AGTOCGGCO3CAM (-CTAGCnT ^ 60 G 61 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: GTCCATATGG GTGTCTAGAA AAATAATGGT GAAAACTTGA AGGCG 45 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: CATATGGCG ACACGCCCGT ACCGATCTTC 30 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: ATGCATTGGC TCGTCATGAA GAGCCTATCA TCCGGCCTGC GGCTCGTCT TCGC 54 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: CAGCTACCCC GCTTGAGCAG ACATC 25 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡i) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16: GTCAGGGAAG AACACGAGGG CGCAG 25 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17: CCCTCTCTTC GTCGTTGTCC ACGCC 25 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 18: ATGTCCTTGG CCGACTTCAG CTCGG 25 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (ni) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 19: GACGAGCCAA TGCATCTTTT GTATG 25 2) INFORMATION FOR SEQ ID NO : 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: CGGGTACTCG CTCTACCTAC TTCGG 25 2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: other nucleotide acid (A) DES CRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 21: GCCCAGTATA AGGAATTCGA TATCAAG 27 2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: ( A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 22: AGGGTCGACA CTAGTTCTAG AGCGG 25 2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡i) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: SI ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: GACGTTATCG GACGGAGACT CAGTG 25 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: GCCTACTCTG TTCTGGAGAG CTGC 24 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (ii?) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 25: CCCCCATCCC GGTCACGCAC TCGCG 25 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (B) DESCRIPTION / desc = "oligonucleotide" (ii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 26: CACAGAGAAT GTGCCGTTTC TTTGG 25 (2) INFORMATION FOR SEQ ID NO: 27 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (B) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: TCACATATCC CCTACTCCCG AGCCG 25 (2) INFORMATION FOR SEQ ID NO : 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (B) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 28: GTCGCGTATC CCAGG 15 2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs ( B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (B) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: GTCAAAGGAT ATGCATAC 18 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) ) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (B) DESCRIPTION: / desc = "oligonucleotide" (ni) HYPOTHETIC: YES (x) DESCRIPTION OF SEQUENCE: SEQ ID NO: 30: AGCTTATGCG GCCGCGAATT CAGGTACCGT ATCTCGAGA 39 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) FOR MA OF THREAD: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (B) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 31: AATTTCTCGA GATACGGTAC CTGAATTCGC GGCCGCATA 39 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 32: GTGCGAGGTA CCACAATCAA TCCATTTCGC 30 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 33: ATGGTTCAAG AACTCGGTAG CCTTTTTCCTT GATTCT 36 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 34: AGAATCAAGG AAAAGGCTAC CGAGTTCTTG AACCAT 36 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 35: ATCAATCAGA AGCTTTCTCT CGAGACGGGC ATCGGAGTCC CG 42 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE : other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 36: GACCATGATT ACGCCAAGCT T 21 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iíi) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 37: GGATCCTTAA CTAGTTAAGT GGGGGCCTGC GCAAAG 36 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 38: TTAACTAGTT AAGGATCCAC AATCAATCCA TTTCGC 36 2) INFORMATION FOR SEQ ID NO: 39: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (??) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 39: GCTCTAGAGC GGCCGCGAAT ACATCCGGAG ATCC 34 2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleotide acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 40: CTTTGCGCAG GCCCCCAC 18 2) INFORMATION FOR SEQ ID NO: 41 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡i) TYPE OF MOLECULE: another nucleic acid ( A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 41: TGCAGGGTAA ATCAGGGA 18 2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / d esc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 42: TCCGCTAAAG GTGGTCGCG 19 (2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 43: CCCCAGCATC ATTACACCTC 20 (2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (ii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 44: AAAGGACCCG AGATCCGTAC 20 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION / desc = "oligonucleotide" (ii) HYPOTHETICAL: YES (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 45: TCTCGATACC AAGGTCACCA CGC 25 (2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION / desc = "oligonucleotides" (i? i) HYPOTHETIC: YES (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 46: GCATCCATCG GCCACCGTCA TTGGA 25 (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡) i) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 47: ATCCAGACCA GCACAGGCAG CTTCG 25 (2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 48: TCCGCATGCC AGAAAGAGTC ACCGG 26 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (C) DESCRIPTION / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 49: GTCGACTTAA CTAGTTAAGG CTTCAGACGC AGCGAG 36 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (C) DESCRPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 50: TTAACTAGTT AAGTCGACAC AATCAATCCA TTTCGC 36 2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 51: AGATCTTTAA CTAGTTAAGT GGCCTGAACA GTGCCG 36 2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETIC: YES (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 52: TTAACTAGTT AAAGATCTAC AATCAATCCA TTTCGC 36

Claims (29)

1. CLAIMS 1. A filamentous fungus that has a recombinant DNA molecule integrated into at least two domains of DNA capable substance homologues of its chromosomes, where the DNA domains are not the ribosomal DNA repeats.
2. 2. A filamentous fungus according to claim 1, wherein a DNA molecule is integrated into each of its substantially homologous DNA domains.
3. 3. The filamentous fungus of claims 1 or 2, wherein the DNA domains are amplicons.
4. 4. The filamentous fungus of any of claims 1 to 3, wherein the DNA domain is a domain which in its native state comprises an endogenous gene capable of high level expression.
5. 5. The filamentous fungus of claim 4, wherein the endogenous gene is selected from the group consisting of genes encoding glycolytic enzymes, amylolytic enzymes, cellulolytic enzymes and antibiotic biosynthetic enzymes.
6. 6. The filamentous fungus of claim 5, wherein the endogenous is selected from the group consisting of a glycoamylase gene, a TAKA amylase gene, a cellobiohydrolase gene, and penicillin biosynthetic genes.
7. 7. The filamentous fungus of claims 4 to 6, wherein the endogenous gene is inactivated in each copy of said DNA domain.
8. 8. The filamentous fungus of claim 7, wherein the endogenous gene is inactivated by irreversible suppression of at least part thereof.
9. 9. The filamentous fungus of claim 8, wherein the irreversible deletion comprises at least part of the promoter and upstream activation sequences.
10. The filamentous fungus of claim 1 to 9, wherein each version of the DNA domains are distinguished from the other versions of the domains by means of the unique sequence tag.
11. 11. The filamentous fungus of claim 10, wherein the sequence tag is a restriction site.
12. 12. The filamentous fungus of any of claims 1 to 11, wherein the DNA molecule contains an expression cassette for the expression of a desired gene.
13. 13. The filamentous fungus of claim 12, wherein the desired gene encodes the secreted enzyme.
14. 14. The filamentous fungus of claim 12, wherein the desired gene encodes the intracellular enzyme.
15. 15. The filamentous fungus of claim 14, wherein wherein one or more recombinant DNA molecules comprise one or more expression cassettes of the intracellular enzymes are integrated into the DNA domain and wherein the intracellular enzymes are part of a pathway. metabolic that is not native to the filamentous fungus.
16. 16. The filamentous fungus of claim 15, wherein the intracellular enzymes are selected from the group comprising a deacetoxycephalosporin C tapese (expandase) and a desacetylcephalosporin C tapese (hydroxylase).
17. 17. The filamentous fungus of any of claims 1 to 16, wherein the DNA molecule lacks a selectable marker gene.
18. 18. The filamentous fungus of claim 17, which lacks a selectable marker gene.
19. 19. The filamentous fungus of any of claims 1 to 18, which belongs to a genus selected from the group consisting of Aspergillus, Trichoderma, Penicillium, Cephalosporium, Acremonium, Fusarium, Mucor, Rhizophus, Phanerochaete, Neurospora, Humicola, Claviceeps, Sordaria , Ustilago, Schlzophyflum, Blakeslea, Mortierella, Phycomyces and Tolypocladium.
20. 20. The filamentous fungus of claim 19, which is selected from the group consisting of members of the Aspergillus niger group, Aspergillus orizae, Trichoderma reesei and Penicillium chrysogenum.
21. 21. A method for preparing a filamentous fungus according to any of claims 1 to 20, wherein the method comprises the steps of: (a) transforming a filamentous fungus comprising one or more of its chromosomes into at least two domains of substantially homologous DNAs suitable for the integration of one or more copies of a recombinant DNA molecule and wherein the DNA domains are not repetitions of ribosomal DNA, with a recombinant DNA molecule; recombinant DNA molecule; (b) selecting a transformant with at least one recombinant DNA molecule integrated in at least one of the DNA domains; (c) propagating the transformant obtained in (b) and selecting its progeny in a strain in which at least two domains of DNA comprise the integrated recombinant DNA molecule.
22. The method of claim 21, further comprising steps (a), (b) and (c), the steps of: (d) propagating the strain in which at least two of the DNA domains comprise a DNA molecule and selecting from its progeny a strain in which additional copies of the DNA domains comprise an integrated recombinant DNA molecule; (e) repeating step (d) until a strain is obtained in which each of the DNA domains comprises the integrated recombinant DNA molecule.
23. The method of claims 21 or 22, wherein the recombinant DNA molecule comprises sequences that are substantially homologous to the DNA domains.
24. 24. The method of any of claims 21 to 23, wherein a bidirectional selectable marker is used for the transformation of the filamentous fungi in step (a), and wherein, before step (c), the transformants are selected in the absence of the bidirectional marker.
25. 25. The method of claim 24, wherein the bidirectional marker is a dominant marker.
26. 26. A method for the production of a protein of interest, comprises the steps of: (a) cultivating a filamentous fungus according to any of claims 1 to 20 under conductive conditions for the expression of the protein of interest; and (b) recovering the protein of interest.
27. 27. The method of claim 26, wherein the protein of interest is a secreted protein.
28. 28. The method for the production of a metabolite of interest comprising the steps of: (a) cultivating a filamentous fungus of any of claims 1 to 20 under conducting conduits for the production of the metabolite of interest; and (b) recovering the metabolite of interest.
29. 29. The method of claim 28, wherein the metabolite of interest is a secondary metabolite.