DD296699A5 - NEW EXPRESSION SYSTEM - Google Patents

Abstract

Recombinant DNA molecules encoding pectinase expression systems (PL expression systems) and their derivatives, such as the structural genes of PLA, PLB, PLC, PLE and PLF, and the corresponding regulatory sequences, e.g. As promoter, signal and terminator sequences, and hybrid vectors, which consist of corresponding DNA, including hybrid vectors with a DNA coding for homologous or heterologous polypeptides, hosts, especially filamentous fungi, z. Aspergillus hosts transformed by these hosts, methods for the production of the recombinant DNA molecules and the hosts, and the use of the recombinant DNA molecules for the production of new expression systems. Another object is the production of polypeptides by this DNA and these hosts.

Description

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Field of application of the invention

The invention relates to the field of genetic engineering and describes new DNA molecules which are formed from DNA sequences which code for various pectin lyases of Aspergillus niger and / or their promoter, signal and terminator sequences. The novel DNA molecules are useful for the overproduction of pectin lyases in Aspergillus and / or for the construction of hybrid vectors that express foreign genes in filamentous and other fungi. It is now possible to produce a single pectin lyase that is not contaminated with other pectin lyases.

Initial situation of the invention

Although numerous polypeptide expression systems for prokaryotic and eukaryotic hosts are already known in the field of genetic engineering, there continues to be a need for novel systems which have advantages over the known systems.

Very widely used are the prokaryotic host Escherichia coli and the eukaryotic yeast host, e.g. B. Saccharomyces cerevisiae, for which a large number of different hybrid expression vectors, especially plasmids, has been developed. The disadvantages of E. coli hosts are that they can not glycosylate the polypeptide formed and, due to lack of secretion, the foreign peptide can accumulate within the host cell and prevent further growth. The yeast hosts glycosylate, but like E. coli, they do not secrete the polypeptides, except for very small ones, into the nutrient medium. Yeasts secrete only in the periplasmic space. Higher eukaryotic hosts are mammalian cancer cells capable of glycosylating and secreting into the nutrient medium, but their cultivation is very slow and very expensive, and there is a risk of isolating oncogenic nucleic acids along with the desired peptide from which the peptide can not be released.

In the search for other hosts, filamentous fungi, for example Neurospora crassa, Aspergillus nidulans and Aspergillus niger, were also investigated. Such fungi are already widely used for industrial purposes, but their use in genetic engineering has lagged behind, mainly due to the lack of appropriate transformation systems. In contrast to Saccharomyces cerevisiae, filamentous fungi do not contain any plasmids which could be used for the introduction of foreign genes and for phenotype selection. However, it is possible to transform filamentous fungi with foreign plasmids containing a selectable marker gene. All vectors previously described for filamentous fungi do not replicate autonomously, as is the case with yeasts, but are integrated into the fungal chromosome. This happens only with a very low frequency. On the other hand, it is advantageous that the integrative transformation makes the transformants mitotically very stable, even under non-selective conditions. The stable integration of more than one hundred copies has been reported. The first described vector for filamentous fungi contained as selectable marker the qa-2 gene of Neurospora crassa. This gene encodes the enzyme catabolic dehydroquinase and can be used as a functional complication of N. crassa aro mutants (Case, ME, Schweizer, M., Kushner, SR, and Giles, NH [1979], Proc. Natl. Acad USA 76,5259-5263). Aro mutants can not grow on minimal medium without an aromatic amino acid supplement. Transformation of N.crassa by the qa-2 vector was accomplished by integration of a single copy of the plasmid into the chromosome. The integrated qa-2 gene was still bound to the bacterial plasmid sequences in 30% of the stable Aro "4ntegranten (Case, ME, [1982] in Genetic Engineering of Microorganism for Chemicals [genetic engineering of microorganisms for chemicals] [Hollander, Α., DeMoss, D., Kaplan, S., Konisky, J., Savage, D., and Wolfe, RS, eds.), Pp. 87-100, plenum.) This observation coalesced the cotransformation of nonselective DNA sequences Selective to feasible tasks In Aspergillus nidulans, which has a sexual cycle suitable for classical genetic manipulation, both negative and positive selection systems have been identified, using heterologous DNA from N. crassa or homologous DNA, the functional complementation of A. nidulans pyrG mutants obtained by transformation with plasmids containing the pyrG gene (Bailance et al., BBRC 112,284 [1983]; Tilburn et al., Gene 26,205,1983)

In other systems, mutations were functionally complemented at the trpC or argB locus by transformation with the appropriate plasmids. (Yelton, et al., PNAS 81, 1470, 1984; Yelton et Timberlake, J. Cell Biochem., Suppl., 9C, 173, 1985; Johnstone et al., EMBOJ., 4, 1307, 1983).

A dominant positive selection system has also been developed using the amdS gene isolated from A. nidulans and allowing A.niger transformed with it to grow on acetamide as the sole nitrogen source (Tilburn et al., Gene 26: 205, 1983; Wernars et al , Curr. Genet., 9,361, 1985; Kelly, JM, et al., EMBOJ, 4,475, 1985).

In comparison to N. crassa or A. nidulans A. niger is by far the more important organism. It is widely used in the industrial production of enzymes, e.g. B. for use in the food industry. A. niger differs from A. nidulans in its secretory capacity in that it secretes a variety of hydrolytic enzymes, e.g. B.

Glucoamylase, α-amylase, pectinase, cellulase, β-glucanase, β-galactosidase, naringinase, pentosanase, acid protease and lignase, with the glucoamylase and pectinase complexes being the most important.

A. niger has no known sexual cycle. Therefore, mutations can not be introduced via meiotic recombination. However, by classical mutation and selection methods, extensive strain improvements have been achieved in the secretion of hydrolytic enzymes.

Of the genes of the A. niger enzymes, only those of glucoamylase (Boel et al., EMBO J. 3,1581,1984) and alcohol and aldehyde dehydrogenase (WO 86/06097) were characterized together with their promoter and signal sequences and in transformation experiments with A nidulans and A. niger respectively. Selection markers for A. niger were the heterogeneous amds gene (Kelly and Hynes, EMBO J. 4,475,1985) and the argB gene (Buxton et al., Gene 37,207,1985, EP 184438, WO 86/06097), both of A. nidulans were used.

A. niger is the most important organism for the industrial production of pectin-degrading enzymes. Pectins are high molecular weight (20,000 to 40,000D) polygalacturonides comprised of α-1,4-glycosidically linked D-galacturonic acid polymers which are naturally occurring as constituents of higher plant cells, where they are attached to cellulose molecules and where they are predominantly present in the cellulosic molecule Primary cell wall and the middle lamella occur. The richest pectin sources include lemon and orange peel containing about 30% of this polysaccharide. Pectin enzymes degrade the carbohydrate polymer substrate either by hydrolysis of the α-1,4-glycoside bond (polygalacturonase) or by transelimination of the α-4,5-unsaturated galacturonic residue of the pectin molecule (different pectin lyases). The system name of pectin lyase is pectin transeliminase (EC 4.2.2.10).

In A. niger, the proteins of the pectin complex are not constitutively expressed. Expressed under inducing conditions

A. niger when using pectin or its degradation products, the above enzymes, including PLI, when other carbon sources, such as glucose or sucrose are limiting. In surface cultures, pectin enzymes tend to be associated with the outer cell wall. An increase in the pectin and Ca ²⁺ concentration in the medium leads to complete secretion.

Pectinases, such as PLI, are used by the food industry primarily for clarifying fruit juices.

A.niger purified two different pectin lyases, PLI and PLII, and partially purified from F.E. A. Van Houndenhoven (22). PLI contains four mannose residues, while PLII contains two mannose and glucose residues. The enzymes have different molecular weights (PLI: 37.5 kD, PLII: 36 kD). The amino acid sequence of PLI was determined and disclosed in EP 88101 397.3. This invention was based on a partial structure determination of pectin lyase I (PLI), which enabled the synthesis of DNA probes encoding relevant parts of the protein. With the help of the DNA probes, it was possible to screen for DNA coding for PLI and isolate it, possibly together with its precursors and sequences, from a gene library of A. niger.

By hybridizing parts of the PLI gene to an A. niger genomic library, further PL genes could be discovered that are the subject of the present invention. The PLI structural gene with the N-end flanking region generated by

A. niger N 756 is identical at the N-terminus with the PLD gene obtained from A. niger N400. Accordingly, the latter is not part of the present invention. The present PLA appears to be part of the previously purified PL mix called PLII.

Hereinafter, PLI is also referred to as PLD, and the PLI structural gene has the name pel D.

Objectives of the invention

The aim of the invention are recombinant DNA molecules consisting of DNA sequences encoding novel pectin lyase expression systems and their derivatives, such as the structural genes of these pectin lyases and corresponding regulatory sequences, e.g. Promoter, signal and terminator sequences, and hybrid vectors consisting of corresponding DNA, including hybrid vectors having a DNA coding for homologous or heterologous polypeptides, hosts, especially filamentous fungi, e.g. Aspergillus hosts that have been transformed by these vectors, methods for making these recombinant DNA molecules and these hosts, and the use of the recombinant DNA molecules for the production of new expression systems. Another object is the production of polypeptides by means of this DNA and these hosts. The novel pectin lyase expression systems consist of DNA sequences encoding the promoters, the signal sequences, the structural genes and the terminators of these novel pectin lyase genes. The novel pectin lyases are referred to as PLA, PLB, PLC, PLE and PLF.

In particular, it is an object of the present invention to construct hybrid vectors consisting of DNA sequences encoding the promoters of PLA, PLB, PLC, PLE and PLF, optionally for the signal sequences of these proteins and optionally for their terminators. These hybrid vectors are used for the inclusion of genes encoding certain proteins and for the transformation of filamentous fungi such as Aspergillus, Penicillium and Cephalosporium. The co-transfusion of A. niger with two different plasmids can be carried out with one of the plasmids selected from the group of novel hybrid vectors of the invention and the other being a specially designed selection plasmid which becomes active in conjunction with a mutant strain of a filamentous fungus.

The present invention also relates to the overproduction of these novel pectin lyases in the Aspergillus species and the production of individual PL uncontaminated by other PLs or their specified synthetic mixtures. In addition, the present invention relates to the production of any protein whose structural gene can be expressed in the presence or under the control of the present recombinant PL DNA. The various objects of the invention will be apparent from the following detailed description of the invention.

Detailed description of the invention

The recombinant DNA molecules

In particular, the present invention relates to a recombinant DNA molecule consisting of a DNA sequence encoding the expression systems of the pectin lyases PLA, PLB, PLC, PLE or PLF or their derivatives.

By the term "expression system" is meant a DNA sequence capable of expressing a polypeptide consisting of the promoter, the signal sequence, the structural gene and the terminator.

When the term "derivative" is used in conjunction with the novel DNA sequences, it is intended to include larger derivatives or derivatives with flanking sequences, fragments of said DNA sequence, mutants, especially naturally occurring mutants, and DNA sequences that correspond to the genetic Code are degenerate.

Larger derivatives of the novel recombinant DNA molecules are those which can be excised from the A. niger genome and consist of the DNA sequences shown in Figures 1 to 3.5 and 6, such as in a genomic Library of A.niger N 400 can be found, obtained by fragmentation of the nucleic acids, treatment of the fragments with a suitable restriction enzyme, eg EcoRI, BamHI or HindIII, ligation into a suitable vector, e.g. the lambda phage and plasmid pBR322, cloning, e.g. B. in E. coli, and re-cutting with the same restriction enzyme. Such derivatives are z. For example, the inserts of plasmids pGW820, pGW830, pGW840, pGW850, pGW860 and pGW880 shown in Figures 1,2,3,4,5 and 6 are shown.

Fragments of the DNA sequence of Formula I (Figure 9) are those located between two restriction sites of these plasmids and retain promoter, signaling, structural or terminator functions.

Preferred fragments are those containing the promoter sequence, the signal frequency, the structural gene of a novel PL or the terminator sequence or any combination of these elements.

The fragments of the DNA sequence may contain linkers that allow successful binding to other DNA molecules.

The PL promoter sequence is present in the DNA region in front of the structural gene and consists of up to 2000, preferably up to about 1000 to 1400 nucleotides. In pGW820, the promoter is located on the sequence between Sail (1240) and PstI (2420) as restriction sites. For the promoter sequence, the TATAA boxes are important as the RNA polymerase recognition sites. On PeIA, the TATAA box is located at position 1221 (Fig. 10), at pelB at position 951 (Fig. 1) and at pelC at position 1261 (Fig. 12). The short promoters from, for example, the TATAA boxes to the beginning of the signal sequences are of particular interest for uninduced expression of the polypeptides. If pectin-induced expression is required, the sequences above the TATAA boxes are required for the regulation of the promoter's strength. Suitable fragments can be added to these fragments.

The promoter of the PLI of A. nlger is inducible, i. h., the expression of the structural gene attached thereto, z. For example, the structural gene coding for a PL or foreign gene is induced by the addition of pectin or pectin degradation products to the medium. If pectin is lacking and there is sufficient glucose, the promoter will not be effective. If the upstream regulatory sequences are absent, the promoter becomes constitutive.

The DNA coding for the signal sequence extends between the end of the promoter and the beginning of the sequence coding for the mature protein. In PLA and PLB the signal sequence contains about 20 amino acids, in pelC it is about 18 amino acids. The corresponding coding region at pelA extends from the ATG codon at position 1361 down to the PstI cut location at position 1420, at pelB from position 1134 to at least position 1191 and at pelC from position 1368 to position 1422.

As can be seen from formulas I, II and III, the structural genes of PLA, PLB and PLC contain several introns. The structural genes may also contain suitable linkers.

The terminators begin with the stop codons, e.g. TAA, and may extend to one of the restriction sites within these sequences. The terminator fragment contains at least 300bρ and may contain suitable linkers.

Suitable linkers for the above fragments have a DNA sequence that fits into the restriction site of the DNA to which the fragment is bound. They may contain a fixed restriction site.

Fragments of the DNA sequence of the invention are also those composed of smaller fragments, e.g. For example, those containing the promoter, the promoter and the signal or structural sequence, the signal and the structural sequence or the structural gene without the introns and the like.

Mutants of the DNA sequence of the invention are, for example, naturally occurring mutants. The invention also includes natural or synthetic mutants of the signal sequence having a similar or identical hydrophobicity profile, e.g. For example, when codons for the polar amino acids lysine (K ⁸⁺ ), tyrosine (Y * ") and arginine (R ⁶⁺ ) are exchanged by codons for other amino acids with similar charges and the hydrophobic amino acids alanine (A), leucine ( For example, the codon for the positively charged lysine may be replaced by a codon for arginine, and vice versa, the codon for the negatively charged tyrosine may be replaced by a codon for glutamate or aspartate and or the codon for the non-polar, hydrophobic alanine by one of the codons for threonine, proline, valine, isoleucine, leucine, methionine or phenylalanine, etc. Other mutations are "silent mutations" in which one or a few nucleotides, e.g. Up to about 30, by one or more other nucleotides, whereby the new codons have the coding for the same amino acid (s).

DNA sequences of the invention that are degraded, like the silent mutations, are those that are degraded within the meaning of the genetic code such that an infinite number of nucleotides are replaced by other nucleotides without altering the amino acid sequence for which they code , Such degenerate DNA sequences may be useful because of their different restriction sites.

Recombinant DNA molecules containing a DNA sequence of the invention or its derivative are, above all, recombinant vectors, also referred to as hybrid vectors, which contain these DNA sequences as inserts. They can be used for cloning in hosts such as bacteria, fungi or animal cells. Such hybrid vectors are derived from any vector used in the field of genetic engineering, such as phages, cosmids, plasmids or chromosomal DNA, such as the derivatives of phage λ, ζ. NM 989, or Phag M13, e.g. B. M 13mp8 phage DNA (Ut 15) linearized by BamHI digestion, bacterial plasmids, e.g. pBR322, pUN 121, pUC 18, or yeast plasmids, e.g. Yeast 2u plasmid, or chromosomal DNA derived e.g. of Aspergillus, e.g. A.niger, e.g. the phage disclosed by EP 184438 or defective plasmids or defective plasmids in the presence of a helper phage or a helper plasmid, which allow the replication of these defective phage or plasmids, z. B. M13 (+) KS vector in the presence of z. B. the helper phage M13 K07. A hybrid vector according to the invention contains, in addition to the DNA sequences according to the invention or its derivative, a replication site and optionally, depending on the type of DNA derivative, an expression control sequence, such as an extension sequence, above the activation site, a promoter and signal sequence and / or a structural gene other than that present in A. niger-derived sequences. Such an extension sequence may be derived from the extra chromosomal ribosomal DNA of Physarum polycephalum (PCT / EP 8500278), or it may be the upstream activating site of the acid phosphatase PH05 gene (EP Appl. No. 86111820.6) or PH 05, trp PH 05 -G APDH hybrid (EP Appl. No. 86111820.6) or the similar promoter.

Structural genes ligated to the present promoter, signal and / or terminator sequences are, in addition to those encoding pectinase PIA Pl-B, PiC, PLE and PLF with or without inirons, other homologous pectinase genes, г. В. the PLD (or PLI) gene, or other Aspergillus genes and heterologous structural genes derived from genomic DNA or from aDNA that can be produced via the mRNA route or chemically synthesized, with coding for a wide variety of useful peptides, including glycosylated polypeptides, especially of higher eukaryotic, especially mammalian, such as animal or especially human, origin, such as enzymes that can be used, for example, for the production of food and for carrying out enzymatic reactions in chemistry, or polypeptides which are useful and valuable for the treatment of or prevention of disease in humans and animals, for example, hormones, polypeptides having immunomodulatory, antiviral and antitumor properties, antibodies, viral antigens, vaccines, coagulation reactors, foods and the like.

Examples of such heterologous structural genes are, for. For example, those encoding hormones such as secretin, thymosin, relaxin, calcitonin, luteinizing hormones, parathyroid hormone, adrenocorticotropin, melanocyte stimulating hormone, β-liptropin, urogastrone or insulin, growth factors such as epidermal growth factor, insulin-like growth factor (IGF), e.g. IGF-I and IGF-II, mast cell growth factor, nerve growth factor, glial derived nerve cell growth factor or transforming growth factor (TGF) such as TGFβ, growth hormones such as human or bovine growth hormone, interleukin such as interleukin-1 or-2, human macrophage migration inhibitory factor (MIF), interferons such as human α-interferon, for example interferon-aA, aB, aD or aF, β-interferon, γ-interferon or a hybrid interferon, for example an aA-aD or aB-aD hybrid interferon, especially the hybrid interferon BDBB, proteinase inhibitors, such as a _r antitrypsin, SLPI and the like, hepatitis virus antigens, such as hepatitis B virus surface or -Kernantigen or hepatitis A virus antigen, or hepatitis nonA-NonB antigen, plasminogen activators such as tissue plasminogen activator or urokinase, tumor necrosis factor, somatostatin, renin , β-endorphin, immunoglobulins such as the light and / or heavy chains of immunoglobulin D, E or G or human Mouse hybrid immunoglobulins, immunoglobulin binding factors such as immunoglobulin E binding factor, calcitonin, human calzitonin related peptide, blood coagulation factors such as Factors IX or VIIIc, erythropoietin, eglin such as eglin C, hirudin, desulfatorhirudin such as desulfatorhirudin variant HV1, HV2 or PA, human superoxide dismutase, virenthymidine kinase , β-lactamase, glucose isomerase. Preferred genes are those coding for a human alpha interferon or hybrid interferon, human tissue plasminogen activator (t-PA), hepatitis B virus surface antigen (HBVsAG), insulin-like growth factor I and II, eglin C and desulphatohirudin, eg variant HV1. In the hybrid vectors of the present invention, the present promoter and / or signal sequence are operably linked to the polypeptide coding region so as to ensure effective expression of the polypeptide.

The DNA molecules of the present invention may contain selective markers, depending on the host to be transformed, selected and cloned. Any marker gene can be used which facilitates the selection of transformants due to the phenotypic expression of the marker. Suitable markers are, above all, those which express antibiotic resistance, eg. Against tetracycline or ampicillin, or, in auxotrophic yeast mutants, genes that complement host lesions. Corresponding genes, for example, confer resistance to the antibiotic cycloheximide or mediate prototrophy in an auxotrophic yeast mutant, for example the genes ura3, leu2, his3 or trpi. It is also possible to use as structural markers structural genes associated with an autonomously replicating segment, provided that the host to be transformed is auxotrophic for the product expressed by the marker. Of particular importance are marker genes which complement A. niger host lesions, encoding the argB gene encoding ornithine carbomoyltransferase, e.g. Derived from A. niger or A. nidulans (EP 184438) or A. nidulans DNA fragments homologous to the N.crassa pyr4 gene (26).

Preferred embodiments of the present invention are hybrid vectors in which the structural gene, in particular the heterologous structural gene, is operatively linked to the promoter and signal sequences of the present invention. Such a preferred hybrid vector is z. B, pUC 19 / pel; IFN AM 119. Another preferred hybrid vector is e.g. M13 (+) KS / pelA-IFNAM119.

In another preferred embodiment of the present invention, the structural gene, in particular the heterologous structural gene, is operatively linked directly to the promoter sequences of the present invention. Such a preferred hybrid vector is z. M13 (+) KS / pelA-ss-IFN AM 119.

The DNA engine kit of the invention and its derivatives, including fragments, can be used to screen DNA gene banks or mRNA for other similar DNA or mRNA.

Process for the preparation of the recombinant DNA molecules

The invention also relates to a method for the production of a recombinant DNA molecule coding for the expression system of pectin lyase PLA, PLB, PLC, PLE or PLF or its derivative, which consists in cultivating a host linked to a DNA molecule which contains a DNA sequence coding for the pectin lyase expression system or its derivative, and to isolate the desired recombinant DNA molecule or its derivative or to produce this by in vitro synthesis.

The breeding of the hosts is carried out in a conventional nutrient medium, which can be supplemented or freed from chemical compounds which allow a positive or negative selection of the transformants, d. that is, those hosts that lack the desired DNA molecule along with a selection marker from the non-transformants, i.e., those hosts lacking the desired DNA molecule.

All hosts applicable in this field can be used, e.g. B. bacteria such as E. coli, fungi such as Saccharomyces cerevisiae or especially filamentous fungi such as Aspergillus, z. A preferred host is A. niger An 8, a novel mutant lacking the pyrA gene, as described below. The transformation of the hosts takes place according to conventional methods.

The DNA sequence coding for the PL expression system is obtained from filamentous fungi containing such a system, especially from their genomic library, or also via the mRNA.

Hereinafter, the preparation of the present PL expression systems will be described in more detail. A genomic library can be prepared e.g. By partial digestion of A. niger strain genomic DNA, e.g. N756 or N400, with e.g. Sow 3Al or Mbol and cloning the high molecular weight DNA fragments into a suitable host vector, e.g. the E. coli plasmid pUN121, or a lambda vector, e.g. B. EMBL4. Any other A. niger strain that produces the desired PL can also serve as a source for the genomic library, and any other suitable vector can also be used as a recipient for the fragments. In order to successfully screen the genomic library for DNA sequences encoding the PL, a hybridizing DNA probe is needed. This may be a synthetic DNA probe if the sequence of the desired PL is known, or it may be a known PL gene or parts thereof. The first method was used to find the PLI gene, as described in EP 88101397.3. The second method was used in the present invention. Since the entire DNA sequence of the PLI gene has become available, either DNA sequences containing the entire gene, or any part thereof with at least 14 bp, can now be used for screening, as can screening for mRNA encoding the PL gene. System includes.

For screening purposes the DNA probes are labeled 5'-radioactively by the known methods using y ^ P-ATP and T4 kinase. Host microorganisms carrying the nucleic acids of the present invention as an insert are identified by hybridization with the labeled DNA probe on filter copies of the gene library. Clones that hybridize to one or more DNA probes are isolated and propagated. The hybridization conditions used may be more or less strict, eg, simply by choosing different temperatures, and in combination with the use of different DNA probes derived from the PLI (or PLD) gene, e.g. Example by measuring the various hybridization reactions on the complete 1, бкЬр-ВатНІ / Pstl fragment, the 649bp BamHI / Xhol fragment (the N-end fragment) and the 244 bp Xhol / Pstl fragment (the C-terminal fragment) of pCG 3 B11 or pGW840 (Figure 4), the clones can be classified into different classes based on their degree of homology (Tables II or IV). Five λ vectors (λ-PL 113, λ-PL 122, λ-PL 109, λ-PL 102 and λ-PL 116) were finally identified, restricted and their pel genes were produced in pBR322 subclones resulting in plasmids pGW820 , pGW830, pGW850, pGW860 and pGW880 (Figures 1 to 3.5 and 6). The five genes of these clones are referred to as pelA, pelB, pelC, pelE and pelF, respectively.

The genes can be sequenced. Complete sequences of pelA, рѳІВ and pelC are represented by formulas I, II and III (Figures 10, 11 and 12).

A computational search for consensus sequences of exon / intron splice junctions as well as intron interior sequences (Boel, E., et al. [24] and Mount SM [25]) leads to the presence of four introns in pelA and pelB, as in pelD (Figures 10 and 11) and of three introns in pelC (Figure 12).

Plasmids pGW820, pGW830, pGW850, pGW860 and pGW880 are used to make other recombinant DNA molecules of the invention. These other DNA molecules are prepared in a conventional manner using conventional restriction enzymes, linkers, ligation, propagation and isolation processes. For example, plasmid pGW820 is restricted with HindIII. The 3.9kbp HindIII fragment is subcloned into the HindIII site of pBR322. The 3.3 kbp BamHI-HindIII fragment of the recovered plasmid pGW822 with the pelA gene is cloned into the BamHI and HindIII sites of the vector pUC 19 to give a vector designated pUC 19 / pelA. The structural gene of pelA is excised by digestion with Sail and Psti. Into the remaining fragment containing the pelA promoter, signal and terminator sequences, a gene encoding the interferon hybrid BDBB is ligated between the signal and terminator sequences by a suitable linker. The recovered plasmid is designated pUC19 / pelA-IFN AM 119 (Figure 11) and contains the promoter and signal sequence of pelA, the IFN-BDBB gene and the pelA terminator attached to the HindIII-BamHI fragment of pUC 19 is attached. This plasmid is cotransformed with pCG 59D7 into the uridine auxotrophic A. niger mutants An 8 (DSM 3917). Transformants are selected in minimal medium containing arginine and Bacto agar and analyzed for interferon expression.

In another embodiment of the invention, the signal sequence of pelA can be deleted. The recovered plasmid is designated M13 (+) KS / pelMss-IFN AM 119 and contains the promoter sequence of pelA, the IFN-BDBB gene and the pelA terminator attached to the HindIII-BamHI fragment of the Bluescript M 13 (FIG. +) KS vector is attached. This plasmid is cotransformed with pCG59D7 into the uridine-auxotrophic A.niger mutant An8 (DSM3917). Transformants are selected in minimal medium containing arginine and bacto-agar and analyzed for interferon expression. Mutants containing novel restriction sites can be prepared, for example, in vitro by site-directed mutagenesis by conventional methods (see review article by MJ Zoller and M. Smith, Methods Enzymol., 100, 488 [1983]; D.Botstein and D.Shortle, Science 229, 1193 [ 1985] or K. Norris et al., Nucl. Acids Res. 11, 5103 [1983]). For higher expression levels, any eukaryotic terminator sequence can be ligated to the end of the structural gene.

Bacteria are transformed by a conventional method, and the transformants are characterized by their resistance, e.g. B. against tetracycline identified.

Specifically, the described expression vectors are propagated in suitable E. coli host strains, such as HB101, transformed and selected according to conventional methods in the art. The increased plasmid DNA is isolated from the bacteria by conventional methods, as described above all by Birnboim and DoIy (23). Similarly, other plasmids can be constructed with other homologous or heterologous genes. The DNA molecules of the present invention can also be prepared by conventional methods by in vitro synthesis. The in vitro synthesis can be used especially for the preparation of smaller fragments of the PL expression system, z. As DNA sequences with the coding for the promoter or the signal sequence of PL or their mutants.

DNA molecules according to the present invention which contain a PL promoter and optionally a PL signal sequence, a PL structural gene, any other heterologous structural gene and / or a PL terminator may also be used for the transformation of filamentous fungi such as Aspergillus, Penicillium or Cephalosporium, z. A. nidulans, A. oryzae, A. carbonarius, A. awamori and especially A. niger.

To facilitate the selection of the transformed from the untransformed fungi, the DNA molecules of the invention carry a selection marker or, alternatively, the fungi are cotransformed with a second vector containing such a marker. As in other systems, such a selection marker is an expressible structural gene whose expressed polypeptide (an enzyme) confers resistance to compounds that are toxic to the transformant or that completes the enzyme system of a mutant lacking such an essential polypeptide. Such marker genes are, for example, the known qa-2, pyrG, pyr4, trpC, amdS or argB genes.

As described in EP 88101397.3, A. niger genomic library has been used to isolate a novel marker gene named pyrA, which is related to pyrg from A. nidulans and pyr4 from N. crassa and has similar function, namely the production of the enzyme orotidine-S'-phosphatdekarboxylase. This enzyme catalyzes the decarboxylation of orotidine 5'-phosphate in uridylic acid (uridine-5'-phosphate) and also of fluoroorotic acid into the toxic fluorouridine. An E. coli clone containing the pyrA gene was identified by hybridization to the 1.1 kb Hind III fragment of pDJB2 (24), which contains part of the pyr4 gene, but may also be DNA from any other руг gene encoding orotidine 5'-phosphate decarboxylase. From a positive clone named E. coli B75183 / pCG59 D7, the plasmid pCG59 D7 with the pyrA gene was isolated and used for the co-transformation of an A. niger-pyrA 'mutant. Such a pyrA ~ mutant lacks the orotidine 5'-phosphate decarboxylase gene, and therefore is incapable of producing the corresponding enzyme. Such a mutant was prepared by treating conidiospores of A. niger N756 under a UV-irradiation mutation, and the colonies surviving in the presence of fluoroorotic acid and uridine were selected. Colonies surviving in the presence of fluoroorotic acid and absence of uridine were eliminated. The remaining uridine-requiring mutants belong, according to their ability to be transformable, to two complementation groups, pyrA and pyrB, which are represented by the mutants An8 and An10, respectively. They are treated in the form of their protoplasts under transforming conditions with the pyrA-containing plasmid pCG59D7. It was found that only the An 8 colonies were transformed and contained the pyrA gene, as indicated by the hybridization ability of digested DNA with DNA from pUN 121.

The invention also relates to hosts transformed with the hybrid vectors of the invention and methods for their preparation. Such transformants are, for example, bacteria such as E. coli or filamentous fungi such as Aspergillus, Penicillium or Cephalosporium and in particular A. nidulans, A.oryzae, A. carbonarius, A.awamori and preferably A. niger, z. B. A. niger An8. The invention also relates to a method for the production of such transformants, which includes the treatment of a host under transforming conditions with a recombinant DNA molecule, in particular a hybrid vector, according to the invention, optionally together with a selection marker gene, and the selection of the transformants. The invention also relates to the use of the recombinant DNA or its derivatives for the production of hybrid vectors which express useful homologous or heterologous polypeptides. Examples of genes encoding such useful polypeptides are given above.

The invention also relates to a method for the production of polypeptides which is characterized in that a hybrid vector according to the invention is expressed in a suitable host. If necessary, the polypeptide is isolated in a conventional manner. Depending on the construction of the vector, the products are either expressed or, if a signal sequence is present, expressed and secreted.

This method involves the production of useful homologous or heterologous proteins in a suitable host, e.g. As the Aspergillus species, by culturing a transformed with an expression hybrid vector host. Examples of genes encoding such proteins are given above.

It is now also possible to produce individual polypeptides PLA, PLB, PLC, PLD, PLE or PLF, that is, in pure form and not contaminated by other PL, whereby various methods can be used. For example, a method for the production of a single polypeptide selected from the group consisting of PLA, PLB, PLC, PLD, PLE and PLF is characterized in that a host incapable of expressing a pectin lyase PL is transformed with a DNA expression vector expressing the product of PLA, PLB, PLC, PLD, PLE or PLF. A host incapable of expressing a pectin lyase PL is either a microorganism lacking a corresponding gene, e.g. A PL'-Aspergillus strain, another fungus not belonging to the Aspergillus or any other eukaryotic or prokaryotic microorganism or an Aspergillus strain whose production of PL in a suitably conditioned growth medium is suppressed. For example, a single PL gene can be expressed under the control of the glucoamylase promoter in A. niger or A. awamori, under the control of the PH05 promoter in S. cerevisiae, or under the control of a PL promoter in a PL ^- -A. niger strain. The invention is illustrated by the attached drawings, in which

Figure 1 shows a restriction map of pGW820 with the pelA gene; Figure 2 shows the restriction map of pGW830 with the pelB gene;

Figure 3 shows the restriction map of pGW850 with the pelC gene; Figure 4 shows the restriction map of pGW840 with the pelD gene; Figure 5 shows the restriction map of pGW880 with the pelE gene; Figure 6 shows the restriction map of pGW860 with the pelF gene; Figure 7 shows the sequencing strategy for the pelA gene; Figure 8 shows the sequencing strategy for the pelB gene; Figure 9 shows the sequencing strategy for the pelC gene; Fig. 10 shows the sequence of a DNA molecule pelA of formula I containing the gene coding for PLA; Fig. 11 shows the sequence of the DNA molecule pelB of formula II containing the gene coding for PLB; Fig. 12 shows the sequence of the DNA molecule pelC having the formula IM, which contains the gene coding for PLC; Fig. 13 shows the homology at the amino acid sequence level between PLD, PLA and PLC; Fig. 14 shows the construction of the vector pUC 10 / pelA-IFN AM 119 with the gene coding for the hybrid interferon BDBB; Figure 15 shows the non-coding strand of a portion of the pelA-IFN fusion on plasmid pelA-IFN AM 119 with signal sequence extracted aligned with the oligonucleotide starter used for deletion mutagenesis of the pelA signal sequence.

The following examples serve to illustrate the invention but are not to be considered as limiting in any way.

The abbreviations have the following meaning:

Amp ampicillin

ATP adenosine triphosphate

BSA bovine serum albumin

CIP calf intestinal alkaline phosphatase

DNA deoxyribonucleic acid

DTT 1,4-dithiothreitol

EDTA ethylenediaminetetraacetic acid disodium salt

EGTA Bis (aminoethyl) glycol ether) N, N, N ', N'-tetraacetic acid

kbp kilobase pair

LMP Low melting point

mOsm Milliosmole

PEG polyethylene glycol

RNA ribonucleic acid

rpm revolutions / min

SDS sodium dodecyl sulfate

Tc tetracycline

Tris tris (hydroxymethyl) aminomethane

U units

V volts

Buffers, media, reagents:

HHA B / g 10-fold restriction enzyme buffer useful for BamHI, BglII, HalllI, Mbol, PstI and Xhol digestions

and 60 mM Tris-HCl (pH 7.4), 6 mM β-mercaptoethanol, 60 mM MgCl ₂ , 500 mM NaCl,

0.1% BSA, 0.1% gelatin contains EcoRI buffer 5-fold restriction enzyme buffer used for EcoRI digestions and 50 mM Tris-HCl

(pH 7.2), 25 mM MgCl ₂ , 250 mM NaCl, 0.05% BSA, 0.05% gelatin contains IPTG 100 mM isopropyl-β-thio-galactopyranoside (23.8 mg / ml) in H ₂ o

LC medium 1% trypticase peptone (BBL), 0.5% yeast extract (BBL), 0.8% NaCl, 1 ml Tris-HCl, pH 7.5, per liter

Minimal medium 1.05% K ₂ HPO ₄ , 0.45 KH ₂ PO ₄ , 0.1% (NH ₄ ) ₂ SO ₄ , 0.05% for E. coli sodium citrate.2H ₂ 0.1 mM MgSO ₄ , 1 mM thiamine -HCl, 0.2% glucose

2 XTY medium per liter 16 g tryptase peptone (BBL), 10 g yeast extract, 5 g NaCl TBE buffer 1 liter contains 10.8 g Tris, 5.5 g boric acid, 4 ml 0.5 M EDTA (pH 8.0 )

TE buffer 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)

low 10mM Tris HCl (pH 8.0), 0.1mM EDTA (pH 8.0)

TE buffer

X-gal 2% (S-bromo-1-chloro-S-indolyl-β-galactoside) in dimethylformamide

SSC 0.15 M NaCl, 0.015 M sodium citrate

Minimal medium 1 liter contains 1.5 g KH ₂ PO _4, 0.5 g KCI, 0.5 g MgSO ₄ 7H ₂ 0,4,0 g NH ₄ Cl, 10.0 g glucose, traces of FeSO _4, for A. niger MnSO ₄ , ZnCl ₂ , adjusted to a pH of 6.5 with NaOH

Complete medium minimal medium plus 0.2% trypticase peptone for (BBL) 0.1% casamino acids (Difco), 0.1% yeast extract for A. niger (BBL), 0.05% sodium ribonucleic acid salt (ICN, Cleveland, USA), 2 ml of vitamin solution per liter

Vitamin solution per 100 ml 10 mg thiamine, 100 mg riboflavin, 10 mg pantothenic acid, 2 mg biotin, 10 mg p-aminobenzoic acid, 100 mg nicotinamide, 50 mg pyridoxine-HCl

For plates, all media are solidified by the addition of 1.5% agar (BBL), for Topagar (ose) 0.7% agar (BBL) or agarose (SEAkem) are used.

PBS per liter of O, 37 g of NaH ₂ PO ₄ , 2.7 g of Na ₂ HPO ₄ , 8.5 g of NaCl PSB 10 mM Tris-HCl (pH 7.6), 100 mM NaCI, 10 mM MgCl ₂ , 0.05% gelatin LDB 10 mM Tris-HCl (pH 7.6), 10 mM NaCl, 10 mM MgCl ₂ The following strains were used: A. niger wild-type N 400 A. niger N 593 cspA, pyrA A. niger N 756 selected for the high production of pectinase complex enzymes A. niger To 8 DSM 3917, uridine auxotrophic mutant from A. Niger N 756 E. coli NM 539 metB, supE, hsdM ⁺ , hsdFT, supF, (P 2cox 3) E. coli MH 1 araD 139, лІасХ 74, galU, galK, hsr ', hsrtT, strA E. coli JM 103 ліас-рго, thi, strA, supE, endA, sbcB, hsdR 4 F \ traD36, proAB, lacIq, ΖΔΜ15 E. coli J M109 Д (Іас-ргоАВ), recAI, endAI, gyrA 96, thi, hsdR 17, supE 44, relAI, λ ", (F ', traD 36, proAB, lacIq, ΖΔΜ15] E. coli CJ 236 (dut-1, ung-1, thi-1, relA-1, pCJ 105 (Cm ^r ); BIO-RAD muta genes M13 in vitro kit)

E. coli MV1190 [A (lac-proAB), thi, supE, A (srl-recA) 360: Tn 10 (tet ') (F': traD36, proAB, Iac 1 "ΖΔΜ 15)] Handbook for the BIO-RAD MUTA-GENE M 13 in vitro mutagenesis described.

The following vectors are used:

EMBL4

EMBL4 is a lambda exchange vector with a cloning capacity of 9-23kbp (Frischauf et al., Lit.2). It contains a multiple cloning region between the lambda arms and the non-essential fill region. This allows multiple restriction enzyme digests to be performed in a manner that reduces the relegation of the filler to the vector arms by introducing foreign DNA of interest. The vector also uses the Spi phenotype to allow direct selection for recombinants (Zissler et al., Ref. 20).

pCG3B11

This plasmid has been described in European Patent Application 88101397.3, which contains the pelD (PLI) gene of A. niger N 756 cloned in pBR322.

pPL29-5

This plasmid has been described in European Patent Application 88101397.3, which contains the N-terminal EcoRI fragment of the pelD (PLI) gene.

pPL35-5

This plasmid has been described in European Patent Application 88101397.3, which contains the C-terminal EcoRI fragment of the pelD (PLI) gene.

pBR322

Plasmid pBR322 carries genes for resistance to the antibiotics ampicillin (amp ¹ ") and tetracycline (tet®) .The plasmid has several distinct cloning sites, some of which are either in the amp or tet gene, so that the insertion of cloned DNA leads to antibioticsensitive bacteria.

pEMBL18undpEMBL19

These plasmids were described by Dente et al. (Refs. 7,8).

pGW613

This plasmid was developed by Goosen et al. a. (Ref 12).

PhagM13mp

The vectors M13mp 18 and M 13mp 19 (Norrander et al., Ref. 21) are derivatives of the single-stranded DNA bacteriophage M13 and have been designed to facilitate DNA sequencing because they involve the cloning of DNA fragments at a versatile polylinker site and the cloning thereof Allow restriction fragments in both possible orientations. Sequences which are cloned into these vectors can readily be used as templates for sequencing reactions or for the production of single-stranded probes using a standard oligodeoxyribonucleotide starter and the Klenow fragment of E. coli DNA polymerase I. The vector DNA carries the E.coli lac operon promoter and the genetic information of the first 145 amino acids of β-galactosidase. The polylinker sequences containing the multiple restriction sites are inserted into the sequence lacZ. The polylinker receives the lacZ reading frame and the vector gives the allelic complementation of a LacZa host strain, resulting in blue plaques on the plates containing IPTG and X-gal. Recombinant phages containing inserts that disrupt the reading frame or otherwise interfere with the expression of the lacZa peptide appear as colorless plaques.

pCG59D7

This plasmid is described in EP 88101397.3 and can be obtained from Escherichia coli BJ5183 / pCG59D7 (DSM3968). The plasmid contains the pyrA gene and is used for cotransformation of A.niger pyrA 'mutants.

M13 (+) KS (Bluescript) (STRATEGENE, San Diego, CA, USA) dsDNA vector comprising the replication origin of phag M13. In the presence of a helper phage, z. For example, M13KO7 (ref 29) or R408 (ref 9), the (+) strand of the vector is replicated, packaged and released into the medium.

M 13KO 7

Helper M 13 phage for M 13 (+) KS. From Mead et al. (Ref 29).

Helper M 13 phage for M 13 (+) KS. From Russell et al. (Lit.9).

example 1

Construction of genomic library of Aspergillus niger

Example 1.1

Isolation of high molecular weight DNA from A. niger

Conidiospores of Aspergillus niger strain N400 are inoculated in 200 ml minimal medium to a final spore density of 10 ⁶ spores / ml and shaken in an Erlenmeyer flask of 11 capacity for 24 hours at 28 ⁰ C with 300U / min using a rotary shaker New Brunswick , The mycelium is harvested by filtration using a Myracloth Books funnel, washed with cold, sterile saline, frozen in liquid nitrogen, and either stored at -60 ° C or used directly. The method used to isolate DNA for genomic library production is based on the method described by Yelton et al. (Ref. 1). The DNA yield in this method is about 50-100,000 g / g mycelium.

For the construction of the library, 10 g of mycelium are ground in liquid nitrogen in 1 g portions in a brown microdosage brewer. The ground mycelium is placed in an 11-liter sterile Erlenmeyer flask containing 200 ml of extraction buffer (50 mM EDTA, pH 8.5, 2% SDS) and 200 μM diethylpyrocarbonate. The mixture is slowly warmed to room temperature and then heated at 68 ° C for 20 minutes with occasional shaking. The suspension is cooled to room temperature and centrifuged at 12,000xg for 15 minutes. Derobenaufschwimmenden layer are ¹ / i6 volume of an 8 M Kaliumazetatlösung, pH 4.2, was added and the mixture is allowed for one hour on ice. The precipitate is removed by centrifugation (20 min, 16000xg, 4 ° C). The nucleic acids are precipitated from the supernatant layer by incubation with 0.6 volumes of isopropanol on ice for 15 min. The pellet is collected by centrifugation (10 min, 6000xg, 4 ° C), washed with 70% ethanol and dried briefly. The pellet is resuspended in 10 ml TE containing 20 цд / ті RNase A (Boehringer Mannheim), and incubated at 37 ⁰ C for 15 minutes. The DNA is with nukleasefreier pronase (1 mg / ml final concentration) (Koch Light, Coinbrook), duration 1 hour at 37 ⁰ C. The Pronasevorratslösung in TE buffer contains 20 mg / ml enzyme was incubated at 37 ° C for one hour to digest the nucleases.

In 9 ml DNA solution, 8.5 g of CsCl are dissolved, 0.2 ml of 10 mg / ml ethidium bromide are added, and this solution is centrifuged either in a Beckman SW41 rotor at 33,000 rpm for 60 hours or in a Beckman 50Ti rotor Quick-release tubes for 40 hours at 45000 rpm. The DNA tape is picked up by piercing the tube. Ethidium bromide is removed by multiple extraction with water and NaCl-saturated isopropanol. 5 volumes of TE are added, the DNA is extracted with TE-saturated phenol, phenol / chloroform / isoamyl alcohol, 25: 24: 1, and chloroform / isoamyl alcohol, 24: 1. The DNA is precipitated by the addition of 0.1 volume of 3M sodium acetate, pH 5.2.2.5 volumes of ethanol and incubation at -20 ° C overnight. The precipitate is collected by centrifuging (1 h; 4 ° C), washed with 70% ethanol, dried and dissolved in 400μΙ lower TE buffer.

Example 1.2

Partial Digestion of A. niger DNA with Mbo! and Isolation of the Fragments To determine the Mbol concentration which gives the largest amount of fragments between 13.6 and 23kbp, portions of 1 μg of A. niger N 400 DNA in a suitable buffer with decreasing amounts of Mbol (FIG. 0.5-0.001 units) for a period of one hour at 37 ° C in a volume of 10μΙ digested. The reaction is stopped by the addition of 1 μM 0.25 M EDTA and the samples are placed on a 0.6% agarose gel in TBE containing 1 μg / ml ethidium bromide. Suitable labels are a mixture of lambda DNA and BgIIII-digested lambda DNA giving bands of 49,22,8,13,6,9,8, 2,3kbp and a non-visible fragment of 0,45kbp. The required Mbol concentration at which a high yield of the desired fragments of 13.6-23 kbp is achieved is 0.02 units of DNA. Thus, 200μg DNA is digested in a total volume of 2ml and divided into 20 equally sized portions immediately after addition of the enzyme. After one hour at 37 ° C, these portions are placed on ice and a 1 μg sample is run on a gel to test for proper digestion. When the result is positive, EDTA is added to a final concentration of 25 mM to stop the reaction, the enzyme is heat-inactivated at 65 ° C. for 10 minutes, the samples are pooled and the DNA is precipitated, washed, dried and seeded in 400 μM TE. Buffer resolved.

The fragmented DNA is separated overnight on a 0.4% preparative agarose gel (mean well 120 χ 1.5 mm). Lambda DNA digested with Bgl II is used as a marker to determine the size of the partially digested DNA fragments after electrophoresis at 4 ° C and 40 V (3 V / cm). The gel area containing the fragments of the correct size is cut out of the gel and the DNA is electroeluted from the gel in a sterile dialysis tube in 2 ml of TBE buffer for 2 to 3 hours at 100V. The current is reversed for 30 seconds and the buffer containing the DNA is collected. Then the fragments are concentrated by ethanol precipitation and dissolved in 100 μΙ low TE buffer.

Example 1.3

Preparation of Vector DNA and Cloning of A. niger High Molecular Weight DNA Fragments in EMBL4 The genomic library of A. niger strain N400 is constructed into the lambda vector EMBL4. The vector having a cloning capacity of 9 to 23 kbp is used by Frischauf et al. (Ref. 2) and by Kam et al. (Ref. 3) and was published by Promega Biotechn. Inc. related. In order to avoid double inserts originating in different parts of the genome, cloning was carried out with a minimum fragment length of 13.6 kbp.

Lambda EMBL4 DNA is completely digested with 50 units of BamHI in a suitable buffer in a volume of 10μΙ for 2 hours at 37 ° C. The enzyme is inactivated for 10 min at 65 ⁰ C. The NaCl concentration is increased to 15 mM, and 50 units of Sail are added, followed by incubation at 37 ° C. for a further 2 hours.

After addition of EDTA to 25 mM and inactivation of the enzyme (10 min, 65 ° C), the solution is extracted with equal amounts of phenol (TE-saturated), phenol / chloroform / isoamyl alcohol, 25: 24: 1, and chloroform / isoamyl alcohol , To weed out the small BamHI / Sall polylinker fragments, the DNA is precipitated with 0.6 volumes of isopropanol after 0.1 volume of 3M sodium acetate, pH 5.2, was added. After 15 min on ice and 15 min centrifugation at 12000xg and 4 ° C, the precipitate is washed thoroughly with 70% ethanol, dried and dissolved in 40μΙ low TE buffer.

Example 1.4

Ligation and In Vitro Packaging of A. niger Genomic DNA Fragments It is essential that the cos sites of the vector prepared according to Example 1.3 be hybridized prior to the ligation reaction. The vector in 10OmM Tris-HCl, pH 7.5 and 1OmM MgCl ₂ for 10 min at 65 ⁰ C. and then for one hour hybridized at 42 ° C. From probing, a ratio of vector to fragments of about 1: 1 (weight basis) is determined which yields a maximum of recombinants. Ligation is carried out in 50 mM Tris-HCl, pH 7.5, 10 mMMgCl ₂ , 10 mM DTT and 1 mM ATP using 9.5 g of vector and 10 μg of DNA fragments in a total volume of 100 μM. Then, DNA ligase (BRL) at a concentration of 0.5 units / pg of DNA is added and the ligation mixture is incubated overnight at 14 ^° C. To test for ligation, a sample of the ligated DNA is assayed on an agarose gel. In addition, an amount of 0.5 cc of vector is ligated without the addition of fragments in a volume of 5 μΙ.

The large volume ligation mixture is concentrated by ethanol precipitation and dissolved in 20 μΙ low TE buffer prior to in vitro packaging. In vitro packaging is carried out with Promega Packagene extracts according to the manufacturer's instructions using ΙΟ-μΙ portions to package 1 μς of the DNA. Of the high molecular weight lambda-cl857-Sam7 supplied with the extracts, 1 μ s are separately packaged to provide control. After packaging 500μΙ phage solution buffer (PSB) and 5μΙ chloroform are added. The recombinant phag stocks can be stored at 4 ° C. The library obtained was constructed from two separate ligation experiments.

Example 1.5

Titration and Amplification of Genomic Library of A. nlger Strain N 400 Cells of E. coli NM 539 are incubated on an LC medium containing 0.2% maltose, 10 mM MgSO ₄ and 1 mM CaCl ₂ to an optimum density of 1.0 (600 nm) pulled. Then, aliquots of this culture are added to 0.2 ml of 0.1 ml of a phag dilution series in PSB. After adsorption, the phage over 20 min at 37 ⁰ C 3 mL 0.6% sodium LC top agar is added to 45 ° C, the mixture is plated on LC agar plates and these are incubated at 37 ⁰ C overnight. The titration results, expressed as the number of plaque forming units (pfu) of jemlPhag suspension, are 12 χ 10 ⁵ and 4.2 χ 10 ^s pfu / ml for the two phag stock solutions prepared according to Example 1.4. After subtracting the background calculated from controls with no fragments (17% and 40%, respectively), the absolute number of recombinants is 6 x 10 ⁵ , which is more than 200 times the genome length.

In order to expand the library, portions 80 are used to μΙ by both phage stock solutions to infect E. coli NM539 cells which are plated in top agarose on LC-LC-agarose plates and then incubated at 37 ⁰ C overnight. The phage are eluted from the agarose by gently shaking the plates with 5ml PSB per plate for one hour at room temperature. PSB is collected, centrifuged to remove the bacteria (10 min at 6000xg) and chloroform added (final concentration 0.5%). Both phag stock solutions, which are expanded approximately to the same extent, are then mixed (40 ml stock), titrated (8 x 10 ⁹ pfu / ml) and stored at 4 ° C.

Example 2

Screening of the genomic library of A. niger N400 for the pectin lyase D gene (pelD) and isolation of the gene

Example 2.1

Screening the library

To isolate the pelD gene from the lambda genomic library, which was obtained as described in Example 1, 2.5 χ 10 ⁴ phage are mixed with liquefied LC top agarose and plated on 5 LC Ag Árp latte η. The plates are incubated overnight at 37 ⁰ C for 2 hours and cooled at 4 ° C. On each plate, two replicates are made according to Benton and Davis (ref. 4) and their plaque hybridization method. The first filter (Schleicher and Schuell, BA85, or Millipore, HATF

085) is placed on top of the plate for one minute, the second replica for 2 minutes, and the position of the replicates is marked using pink ink.

After removal of the filters, they are placed in a dish containing 100 ml of a denaturing solution of M NaCl, 0.5 M NaOH), where they remain for 1 min., Then they are also added for one minute in 100 ml renaturation solution (0.5 M Tris / HCl, pH 7.5, 1.5M NaCl). The filters are then placed in a dish containing 3x SSC (SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.0). The filters are gently scrubbed with a gloved hand to remove bacterial debris, placed in a fresh dish with 3x SSC, and gently cautious for 20 min at room temperature

shaken. The filters are transferred to Whatman 3MM paper and dried at room temperature for 10 minutes. The filters are baked on Whatman 3 MM paper in an oven at 80 ⁰ C for a period of 2 hours. They are then washed 0.5h in 6x SSC at room temperature and then transferred to 50 ml of prewarmed (56Ό prehybridization mixture prepared from 50mMTHs-HCl, pH 7.5.1mM EDTA, 1M NaCl, 0.5% SDS, 0.1 % Sodium pyrophosphate, 10X Denhardt's solution (5% Denhard's solution = 1% BSA, Boehringer V fraction; 1% polyvinylpyrrolidone -40; 1% Ficoll 400) Freshly added to the prehybridization mixture: 0.1 mg / ml denatured salmon sperm DNA ( Maniatis et al., P.327, Lit.5) and 0.01 mg / ml poly-rA Prehybridization is carried out with gentle shaking at 56 ° C. for 4 hours .The probe for hybridization is the single-strand-break-transfused 3.5 kbp EcoRI fragment of pPL35-5 containing the C-terminal region of the pelD gene of A. niger strain N756 (available from E. coli BJ 5183 / pCG 3B11, DSM 3916, EP 88101397.3) isolated from a low-melting agarose gel, and 0.3 ug of the fragment are einzelstra ng-break-translated (Maniatis et al., pp. 109-112; Ref. 5). The single strand break-translated DNA is denatured in a boiling water bath for 10 minutes, cooled on ice, and added to 50 ml of prewarmed prehybridization mixture. The prehybridized filters are transferred one by one to the hybridization mixture. The hybridization is carried out with gentle shaking overnight at 56 ⁰ C. The filters are washed at 56 ° C: 2x 30min with 0.514x SSC, 0.1% SDS and 2x 30min with 2x SSC, 0.1% SDS. The filters are transferred to 3 MM paper and dried for one hour by air. After being glued onto 3MM paper and marked with radiolabeled ink, the filters are covered with Saran overlay material and autoradiographed overnight at -70 ° C including Konica X-ray films and Kodak X-ray chromatographic cassettes and normal intensification screens In this way 44 positive signals are obtained from the 5 screened plaques Positive plaques are picked up with a sterile Pasteur pipette by carefully positioning the plates on the autoradiogram in the correct manner and using the ink markings. containing the positive plaques are dispersed in 1 ml of PSB The phage are allowed to diffuse from the agar for one hour at room temperature with occasional vortexing The agar and bacterial cell debris are removed by centrifugation for 5 minutes, 10μl of chloroform are added and the phag Stock solutions at 4 ° C The positive clones are designated X-PL1 to X-PL4A. Since phage are plated at high density, the positive plaques must be cleaned twice, by plating them at low density (± 100Phag per plate, 0.1 ml of a 10 ³ dilution of the phag stock solution) and the entire replicate plating procedure, hybridization and recording the positive plaques are repeated.

Example 2.2 Isolation of lambda DNA

To isolate DNA from the recombinant clones, first amplify the phage by infecting E. coli NM 539 with 0.1 ml of the phag stock solutions derived from purified clones as in Example 1.5 and then incubate the cells in LC top agarose plated on LC agar plates. This gives plates with confluent lysis of the indicator bacteria. 5ml of PSB are placed over the plates and the phage are eluted by gentle shaking over a period of two hours. The eluted phage are harvested, cell debris is removed by centrifugation, and chloroform is added to 1%. The resulting plate lysate is stored at 4 ° C. It usually has a titer of about 10 ¹¹ pfu / ml.

Since small-scale isolation procedures from plate lysate stock solutions and small-scale liquid cultures (Maniatis, et al., Pp. 65-68, reference 5) do not always yield digestible DNA, phages are isolated from 250 ml lysates. These are prepared by growing the host, E. coli NM 539, to an ODeO of 0.3 in LC + 0.2% maltose, 10 mM MgSO ₄ , 1 mM CaCl ₂ and then adding about 10 ¹⁰ pfu of plate lysate. After further growth, the bacteria dissolve within 4 hours.

RNase and DNase are added to the lysates to a final concentration of 2Kd / L, and the lysate is allowed to stand at room temperature for one hour. Cell debris is removed by centrifugation (20min, room temperature, ЮОООхд). In the supernatent layer, 14.8 g NaCl and 25 g PEG 6000 are dissolved to give final concentrations of 1 M NaCl and 25% PEG. The phage are allowed to precipitate for one hour on ice or overnight at 4 ° C and harvested by centrifugation (20min; ЮОООхд; 4 ° C). The pellets are resuspended in 3 ml of lambda dilution buffer (LDB = 1OmM Tris / HCl, pH Wert7,5,2OmM MgCl _2, 10OmM NaCl) suspergiert.ln4ml derPhagsuspension are dissolved 2.5 g of CsCI, and the mixture with the pipette in 5- ml Beckman snap tubes, which are then filled to the same CsCl concentration in LDB. The tubes are sealed and centrifuged overnight in a Beckman Rotor VTi65.2 at 50000 rpm and 4 ^: C. The cloudy phag band, visible under a normal lamp, is dotted from the side of the rack. CsCl is removed by dialysis in sterile tubes using 211 mM Tris / HCl, pH 7.5, 19 mM MgCl ₂ .5 mM NaCl at 4 ° C. over a period of 4 hours. The buffer is changed once. Phage are collected in sterile Greiner tubes, the volume is adjusted to 1 ml with the dialysis buffer, 50 μL 10% SDS, 50 μM 0.5 M EDTA, pH 8.0, and 25 μM of 20 mg / ml nuclease-free Pronase added and the tubes incubated for 1 hour at 37 ° C. The volume is increased to 3 ml with TE and this solution is extracted with equal volumes of TE-saturated phenol, phenol / chloroform / isoamyl alcohol, 25: 24: 1, and chloroform / isoamyl alcohol, 24: 1. After 0.1 volume of 3M sodium acetate, pH 5.2, and 2.5 volumes of ethanol are added, the DNA is precipitated at -20 ⁰ C overnight and collected by centrifugation (1 h, 20000X g at 4 ⁰ C ). The pellet is washed with 70% ethanol, dried and dissolved in 200 μΙ low TE buffer. The yield of phag-DNA is about 200μg / 250ml lysate.

Example 2.3

Restriction analysis of pelD clones

Of the positive phages, 10 were arbitrarily selected for further analysis. It is digested lyg phage DNA with 10 units of Eco RI or Bgl II in a volume of 20 μΙ for a period of 2 hours at 37 ⁰ C in the buffers recommended by the manufacturer. The samples are separated on a 0.6% agarose gel using 1 μg Lambda el 857-Sam 7 DNA digested with Hind III (Lambda χ Hind III) as marker, and EMBL4 DNA and pCG 3B11 -DNA, digested with

EcoRI, serves as a control. The gel is photographed on a UV transilluminator using Polaroid 667 films (4s, aperture 8). The DNA is transferred to nitrocellulose filters from Schleicher and Schüll, BA85, according to the method of Southern (1975) as described by Maniatis, pp. 382-386 (ref. 5). The filters are labeled with a mixture of ³² P-labeled (single strand break-translated) fragments of the pelD gene that span the region of the entire gene. These fragments are the 2.9 kbp EcoRI fragment of pPL29-5 and the 3.5 kbp EcoRI fragment of pPL35-5. The hybridization and washing conditions are identical to those described in Example 2.1.

The tape lengths are calculated from the photographs and autoradiograms by graphical comparison on plain logarithmic paper with the known marker bands. Restriction maps of the clones are given in Fig. 1. Clones λ-ΡΙ.4, -14, and -40 contain both the 2.9 and 3.5 kbp EcoRI hybridization fragments as found in pCG3B11. λ-ΡΙ_5, -6, -10 and -33 contain the 3.5 kp EcoRI fragment together with another hybridizing EcoRI fragment. The clones X-PL26 and -28 contain the 2.9 kbp EcoRI fragment together with another hybridizing fragment, while A-PL9 contains only a small hybridizing 1.2 kbp EcoRI fragment. The complete 5.0 kbp Bgl II fragment of pCG 3 B11, which contains the entire pelD structural gene, is contained only in clones A-PL4, -14 and -40. The 3.7 kbp hybridization band present in clones A-PL5, -6, -10, -14, -33 and -40, but not in pCG3 B11, is located downstream of the pelD gene in FIG , 0-kbp fragment. Some hybridizing BglII fragments still contain 1.2 kbp of the right arm of the vector. All clones also contain non-hybridizing fragments that are identical. Therefore, since it seems obvious that the restriction patterns of both the structural gene and the gene in the environment are identical in all clones, it is concluded that they originate in the same gene, the pelD gene of A. niger strain N400 , From the number of cloned clones and the assumed genome length of 3 × 10 ⁷ bp, it is deduced that for 25,000 clones with an average insert length of 15 kbp at least 12 pelD clones are found. Since only 2 of the 10 clones analyzed are identical, the complexity of the library is even higher than expected (see Example 3).

Example 2.4

Subcloning of pelD fragments

The 5.0 kbp BglII fragments of \ -PL4 and pCG3B11, which contain the pelD genes of A. niger N400 and N 756, respectively, are subcloned into the BamHI site of plasmid pBR322 (see cloning vectors). In 20μΙ 4цд X-PL4 are 2цд and digested with 10 units of BglII pCG3B11 over two hours at 37 ⁰ C until Kompletion. The fragments are separated on a 1% low melting point agarose gel (BRL) in TBE + 1 cc ethidium bromide at 50V. The 5.0-kbp fragment is cut out from the gel, diluted with 0.4 ml of TE, it is added to an equal volume of phenol, and the mixture is heated to 65 ⁰ C 10 min to melt the agarose. After centrifuging at 12,000 rpm for 5 minutes in an Eppendorf 5414S bench top centrifuge, the aqueous phase is extracted with phenol / chloroform and chloroform, precipitated with ethanol, washed, dried and finally dissolved in 10 μΙ low TE buffer.

1 цd of pBR322 DNA prepared by the large scale plasmid production method (Maniatis, et al., P.86; lit.5) and purified by CsCl gradient centrifugation was added with 5 units of BamHI for 2 hours 37 ° C digested in a volume of 20μΙ. 2 μΙ of 10 χ CIP buffer and 0.02 units of CIP are added and incubated for a further 30 minutes. EDTA is added to a final concentration of 25 mM and the mixture is heated to 65 ° C for 10 min. The solution is diluted to 200μΙ with TE and extracted three times with TE-saturated phenol, once with phenol / chloroform and once with chloroform. After ethanol precipitation, the DNA is dissolved in 50 μM low TE buffer. A DNA fragment of 100 ng is ligated with 20 ng of the vector DNA in a volume of 20 μΙ under the conditions described in Example 1.4. Half of the ligation mixture is used for the transformation of E. coli MH 1-competent cells prepared by the CaCl ₂ method (Maniatis, et al., P 250, ref 5). The cells are plated on LC agar plates containing 50 μg / ml ampicillin and incubated overnight at 37 ° C. The transformants are tested for tetracycline sensitivity by staining in colonies first on LC plates containing 20 pg / ml Tc and then on LC + amp plates. The transformants are drawn overnight at 37 ⁰ C in 5 ml of LC + amp and the DNA using the alkaline lysis miniprep method (Maniatis, inter alia, S.368; ref 5) isolated from 1.5 ml of culture. The Miniprer DNA is digested with BamHI and PstI and the fragments are analyzed on a 1% agarose gel. Two plasmids with their origin of A-PL 4 containing the 5.0 kbp Bgl II fragment in opposite orientations are designated pGW840 and pGW841. Other plasmids derived from pCG3B11 are designated pGW870 and pGW871. The MH-1 cells which they receive which are maintained at -70 ⁰ C to glycerol. Plasmid DNA from 0.5 L cultures of these clones is isolated on a large scale (Maniatis et al., P.86; lit.5), purified by CsCl centrifugation, phenolated, ethanol precipitated and dissolved in 400 μM low TE buffer , The yield is about 500μg. The plasmids are subjected to restriction mapping. The map for plasmid pGW840 is shown in Fig. 2 and is indistinguishable from that for pGW870. Also, plasmids pGW841 and pGW871, which contain the fragment in the opposite orientation, are indistinguishable, indicating the identity of the pelD genes from A. niger strains N400 and N756.

Example 3

Screen after. Isolation and characterization of pelO-related genes

Example 3.1

Generation of Hybridization Conditions by Analysis of Genomic Transfers of A. niger N400 DNA In a volume of 20μΙ at 37 "C for 2 hours, 2 ^ g DNA samples of the A. niger strain N400, as described in Example 1.1, using BamHI (BRL) or Hind III (BRL) digested in HHA BOg buffer The digested samples were electrophoresed on 0.6% agarose gels at 40 V for 18 hours is transferred to nitrocellulose filters and baked as in Example 2.3 The (pre-) hybridization solutions are identical to those described in Example 2.1.

The temperature used in prehybridization is always the same as the hybridization temperature. The single-strand break-translated BamHI / PstI fragment of pGW840, which contains the coding region of the pelD gene, was used as a probe. The hybridization is carried out for 16 hours and for 40 hours at different temperatures (52 ° C, 60 ° C and 68 ⁰ C). At the three different temperatures, the following two washing conditions are used: 2x 30min 5x SSC, 0.1% SDS followed by 2x30min3x SSC, 0.1% SDS compared to 4x 30min 5x SSC, 0.1% SDS. At 68%, a wash is also included twice with 2x SSC, 0.1% SDS, and twice with 0.2x SSC, 0.1% SDS. At 68 ° C and using the 0.2x SSC wash, only homologous hybridization occurs. When higher salt concentrations are applied, some other weak signals appear. At 52 ^° C, the background is strong and the hybridization is weak. The best conditions for "heterologous" hybridization are achieved at 60 ° C over a period of 40 hours using the 5 χ SSC and 3 χ SSC wash combination, which can be further optimized by using 4x SSC in conjunction with 2x SSC instead of 5χ SSC in conjunction with 3x SSC The signals seen in these genomic transfers of A. niger N400 when probed with the 1.6 kbp BamHI: PstI fragment of pGW840 are summarized in Table I.

Table 1

Hybridization signals in A. niger N 400 genomic DNA probed with the 1.6 kb BamHI / PstI fragment of pGW840

BamHI Hind III

7.5 kbp strongly homologous; pelD 21.0 kbp strongly homologous; PELD

4.3 kbp strong 3.9 kbp strong

4.1 kbp weak 7.1 kbp weaker

18.0 kbp weak 4.4 kbp weak

8.4 kbp very weak 4.6 kbp weak

1.5 kbp very weak

Example 3.2

Screen the library

On 5 plates, 2.5 × 10 ⁴ phages of the N400 lambda library are plated and 3 nitrite cell replicates are made from each plate, as described in Example 2.1. The (pre-) hybridization buffer is also described in Example 2.1. The first replica of each plate is hybridized with the 1.6 kbp BamHI / PstI fragment of gGW840 at 60 ⁰ C for 40 hours and washed twice at that temperature for about 30 minutes with 4x SSC, 0.1% SDS, and twice Washed for 30 min with 2x SSC, 0.1% SDS, as described in Example 3.1. These conditions are called heterologous conditions.

The second replicate from each plate is hybridized at 68 ^° C. to the same fragment and washed twice with 30 μl of 2% SSC, 0.1% SDS, and twice for 30 min with 0.2% SSC, 0.1% SDS. These conditions are called homogeneous conditions. The third replicate from each plate is homologously hybridized to the 0.35 kbp BamHI fragment of pGW840 located in the promoter region of pelD immediately following the 1.6 kbp BamHI / PstI fragment. This gives 29 plaques which hybridize heterologously, but not (or very weakly) homologously. They are referred to as λ-PL 101 to λ-PL 129.

This gives 19 plaques that hybridize strongly homologous and heterologous with the BamHI / PstI probe. They are referred to as λ-PL 130 to X-PL148 and are considered as pelD clones. Of these, 2 hybridize only weakly to the 0.35 BamHI probe (λ-ΡΙ.145 and λ-ΡΙ_146) and therefore likely contain only part of the promoter region. The others hybridize strongly. Only one clone is obtained which only hybridizes with the 0.35 kbp BamHI probe (X-PL149).

All clones are picked and purified twice by plating and heterologous hybridization with the 1.6 kbp BamHI / PstI probe. In a second screening experiment, an additional 23 pelD clones and 25 other clones were detected (\ -PL151bisX-PL198).

Example 3.3

Characterization of lambda clones by plaque hybridization signal with different pelD-derived probes

In this experiment, a classification of the isolated clones using different parts of the coding region of pelD as a probe is described. Included are λ-PLI 01 to -130, while K-PlA is used as a positive control. Also included is λ-PL 145. The clones are plated and only one replica is made, which is divided into 6 parts. In this way, differences in the hybridization signal between replicas should be excluded. Separate portions of the replicates are hybridized with the following ³² P-labeled probes, both homologous and heterologous:

Figure 1: the complete 1.6 kbp BamHI / PstI fragment of pGW840

2: the 649 bp BamHI / Xhol fragment of gWG 840 (N-terminal coding region of pelD) 3: the 244 bp XhoI / PstI fragment of pGW840 (C-terminal coding region of pelD).

As expected for pelD clones, λ-Ρί4, -130, and -145 and λ-PLIOI strongly hybridize under homologous and heterologous conditions with these probes. The latter clone, λ-PL 101, is located at the edge of a filter in Example 3.2 and is therefore not listed as pelD in the first screening. The clones just named are classified as class I (pelD) clones. All other clones can be divided into two other classes: Class II not only hybridizes heterogeneously with the entire probe, but also with the N-terminal probe. Homologous hybridization is only weak with the entire pelD gene as a probe. Class III clones do not hybridize at all with the N-terminal probe and are not homologous with the entire probe. The C probe does not hybridize with class II and III clones. From these experiments, it was concluded that at least two related genes are in the isolated clones. Class II clones include: λ-Ρί104, -105, -109, -113, -114, -115, -119, -122, -124, -125, -128 and -129. The class III clones are: λ-Ρί102, -103, -106, -107, -108, -110, -111, -116, -117, -118, -120, -121, and-123.

Example 3.4 Restriction analysis of isolated lambda clones (class I, II)

Plate lysate stock solutions, 250 ml liquid lysates and large scale phag-DNA preparations from these lysates are prepared as in Example 2.2. This happens for 24 clones, of which 3 are pelD clones {λ-ΡΙ4, -130, -145). A clone DNA is lost during DNA isolation (λ-PL 124). The DNA isolated from these clones is digested in 1 pg samples in a total of 20 μΙ with 10 units of enzyme. All clones are digested with EcoRI, BamHI, HindIII, EcoRI / BamHI, BamHI / HindIII, EcoRI / HindIII and EcoRI / BamHI / HindIII. The fragments are separated on 0.6% agarose gel and transferred to nitrocellulose filter as in Example 2.3. Transmissions are heterologously hybridized to the 1.6 kbp BamHI / PstI fragment of pGW840 as described in Example 3.1.

Two clones (λ-PL 112 and λ-PL 129) do not hybridize. The tape lengths are calculated from both the photographs and the autoradiograms. Apart from clone λ-PL 113, all clones contain too many fragments to derive a complete map.

However, it is possible to derive a map of the hybridization region and, by combining data to other non-hybridizing bands, assign which clones are derived from the same gene. It is clear that of the remaining 18 analyzed clones of classes II and III, there are 5 genes. They are referred to as pelA, pelB, pelC, pelE and pelF. The assignment of clones to these genes is summarized in Table II.

Table II

Assignment of isolated clones to different pel genes

(part means that the hybridization bands are only partially present in these clones)

class gene clones I. PELD PL 4, PL 130, PL 145 (part) II. PelA Peleb PelC PL 113, PL 104, PL 115, PL 125 (part) PL 119, PL 122, PL 128, PL 105 (part) PL 109

pelE PL 116, PL 107 (part)

pelF PL 102, PL 103, PL 110, PL 120, PL 121 (part), PL123 (part)

Table III Comparison of hybridization bands in chromosomal transfers and isolated genes

The tape length is specified in kbp.

EcoRI BamHI Hindill

chromosomally 2.9 + 3.5 7.5 21.0 homologous signal pelD + larger 4.3 3.9 strongest heterologous bands signals 18.0 7.1 weaker signals 4.4 weak signals 8.4 4.6 weak signals 4.1 1.5 weak signals PELD 2.9 + 3.5 7.5 large pelA 7.5 4.3 3.9 pelB 8.7 large 7.1 pelC 5.0 large 4.4 pelE 6.2 8.4 4.6 pelF large 4.1 1.5 + 2.8 (very weak)

A comparison of the length of the hybridization fragment of the isolated genes and the length of the hybridization fragments of genomic DNA transfers is given in Table III. From this data, it is concluded that all bands that hybridize in the genomic transfers are present in the isolated clones and that no other related genes can be isolated under these hybridization conditions. The plaque hybridization results (Example 3.3) show that each gene, unless partial clones are considered, has a specific pattern of hybridization with the probes tested. This is summarized in Table IV.

Table IV

Comparison of signal strength of different genes in homologous and heterologous plaque hybridization with different petD probes after the experiment in Example 3.3. The degree of hybridization is expressed by the number of + symbols. The homologous pelD gene hybridizes with at least 10+ characters; + very poor hybridization, - no hybridization

Heterologous hybridization Homologous hybridization

Probe whole N-term. C-term. Whole N term. C-term.

Gene gene

pelF ++++ + ± ± n - -

Example 3.5 Subcloning of pelD-related genes into pBR322

Subclones of the hybridization fragments, which are obtained from the λ clones obtained in Example 3.3, are prepared in the vector pBR322, which is digested by EcoRI, BamHI or Hind III and subsequently dephosphorylated. Fragment isolation from LMP agarose gels, vector production, ligation, transformation of E. coli MH1, miniprep DNA isolation, and large-scale plasmid isolation are performed according to the standard procedures described in Example 2.4.

The fragments are ligated into the appropriate vector as described in Example 2.4. Transformed E. coli MH 1 cells are plated to LC + 50pg / ml amp. The transformants are tested for tetracycline sensitivity. The EcoRI clones are all resistant. Then, miniprep DNA is digested with appropriate enzymes to test for the correct inserts and to determine the orientation of the fragments. The selected plasmids are summarized in Table V. Cells that have taken up are stored at -70 ° C on glycerol.

Table V Subclones of pel genes in pBR322

Plasmid gene fragment origin orientation

pGW820 pel A 7.5 EcoRI X-PL113 2

pGW821 4.3 BamHI 2

PGW822 3.9 Hindill. 2

pGW823 7.5 EcoRI 1

pGW824 4.3 BamHI 1

pGW825 3.9% 1

pGW830 pelB 7.1 Hindill λ-ΡΙ.122 1 pGW850 pGW851 pelC 5.0 EcoRI 5.0 EcoRI X PL109 1 2 pGW860 pelF 4.1 BamHI X PL102 1

pGW880 pelE 4,6Hindlll X-PL109 1

pGW881 4,6Hindlll 2

Plasmids pGW820, -830, -860 and -880 were isolated on a large scale and subjected to restriction mapping.

Maps of this plasm ide are given in Figures 1 through 6.

To determine gene location and orientation, Southern transfers of plasmid digests are heterologously hybridized to the 1.6 kbp BamHI / PstI fragment of pGW840, and identical transfers are heterologously hybridized to an N-terminal fragment of the same plasmid encoding the 649 -bp-BamHI / Xhol fragment for the mapping of pGW820 and -830 and the 766-bp BamHI / EcoRI fragment for pGW850 and -860.

The plasmids pGW820, pGW830, pGW850, pGW860 and pGW880 were cloned in E. coli HB101 and deposited on February 1, 1988 at the Deutsche Sammlung für Mikroorganismen, Grisebachstr.8.3400 Göttingen. They received DSM Nos. 4,388, 4389,4390,4391 and 4392, respectively.

Example 4 Sequence determination of the genes pelD, pelA, pelB and pelC Example 4.1

Partial sequence of the pelD gene of the A. niger strain N400 and its identity with pel I of A. niger strain N After the LMP agarose gel electrophoresis as described in Example 2.4, suitable restriction fragments of pGW840 are isolated. These are cloned into the M13mp18RF and M13mp 19RF vectors according to the BRL-M 13 cloning / dideoxy sequencing handbook (p.26, 27; Lit6). Transformation of E. coli JM103, plating to minimal X-gal.

Indicator plates and the isolation of single-stranded DNA from recombinant phage are performed according to the instructions in the same manual (pages 29-34). Some clones are sequenced according to the Sanger dideoxy chain termination method described on pages 38-74 of the BRL Handbook

The sequences determined between nucleotides -457 and +100 are identical to the corresponding sequence of the PU gene derived from A. niger N756 and present in plasmid pCG3B11 (EP88101 397 3). The sequence derived from BamHI from position 457 to position +100, consists of 457 nucleotides of the promoter sequence, 57 nucleotides encoding the signal sequence, and 43 nucleotides encoding the first ISN-terminal ammosaurs of the mature PLD proteme.

Because of the identical restriction maps of the N400 pELD gene in pGW840 and the N756 pelD gene in pCG3B11 and the completely identical data of the N-terminal sequence, it is assumed that the two genes are identical. Thus, it is believed that the PLD protein is linked to the identical to earlier PLI protein

Example 4 2

Sequence determination of the pelA, pelB and pelC gene

Fragments of pGW820, pGW830 and pGW850 are subcloned into M13mp 18RF and Μ13mp 19RF (see Example 4.1) or into the plasmids pEMBL 18 and pEMBL19 (Dente et al., Lit.7, 8) Single-stranded DNA from the plasmid clones is obtained by infection with the helper -Phag R408 (Russell et al., Lit.9).

Exonuclease III deletion clones were prepared from pGW850 according to the method of Hemkoff (Lit.28). The 3.0kb SmaI-BglII fragment from pGW850, which has the pelC gene, is subcloned into pEMBL19. With Saml and Sacl 50 цд of this clone are digested and digested after extraction with phenol / chloroform and ethanol precipitation with exonuclease III at different time intervals samples are taken, the exonuclease III is inactivated by the addition of NaCl and EDTA, and it is 10 min incubated at 70 ⁰ C. Supernatant ssDNA ends are removed by S1 nuclease, and the cohesive ends are flattened with T4 DNA polymerase. Following self-ligation and transformation of E. coli JM103 with these deletion clones, single-stranded DNA is obtained by infection with helper phage R408. pGW820 is sequenced from the Pvull site at position 1000 to the Clal site at position 4175 (see Figure 1). For pG W 830, the 2 774 bp Xhol fragment is sequenced (see FIG. 2). pGW850 is sequenced from the EcoRI site in position 0 to position 3168 (see Fig.3)

Sequence strategies for these genes are shown in Figures 7, 8 and 9. Several oligonucleotides are synthesized using the phosphoramidite method of Caruthers (Ref. 10) using an Applied Biosystems oligonucleotide synthesizer (Model 380B). They are used as sequencing initiators, their position is shown in Figures 7 and 8.

The entire sequence of the pelA gene is shown in Figure 9 in the 5 '- »3' direction. The 3175bp sequence starts with the first nucleotide of the Pvull site at position 1000 of pGW820 and ends at the last nucleotide of the ClaI site at position 4175 in pGW820.

The pelA sequence consists of 1360 nucleotides of the promoter region, 1355 residues of the structural part and 360 nucleotides of the terminator region.

The amino acid sequence of PLA (see Example 6.3) is preceded by a signal sequence of 20 amino acids, as can be deduced from the nucleotide sequence.

MET-LYS-TYR-SER-THR-ILE-PHE-SER-ALA-ALA-ALA-ALA-VAL-PHE-ALA-GLY-SER-ALA-ALA-ALA

The homology with the signal sequence of the pelD gene is indicated by an asterisk. The first 5 residues of the mature protein also have the same amino acid sequence in pelA and pelD.

The entire sequence of the pelB gene is given in Fig. 10, also in the 5 '-> 3' direction. The 2,774bp sequence begins with the first nucleotide of the XhoI site in pGW830 and terminates at the last nucleotide of the second XhoI site.

The pelB sequence comprises 1133 nucleotides of the promoter region, 1373 residues of the structural part and 268 of the terminator region. The pel B gene encodes a signal sequence of at least 20 amino acids.

The pelC sequence consists of 1367 nucleotides of the promoter region, 1340 residues of the structural part and 461 of the terminator region. The pelC gene encodes a signal sequence of 18 amino acids

pelC MET-LYS-VAL-PROX-X-PHE-LEU-GLN-LEU-LEU-CYS-LEU

pelB * *

pelA * * *

pelD * * * *

pelC ASN-ALA-ALA pelB *

pelA *

pel D *

PelC LEU-ALA-SER-ALA pelB * *

pelA * *

pelD * * *

The homology with the signal sequence of the pelA, pelB and pelD gene is indicated at the individual positions with an asterisk. The X's are included to align the sequences

The search for consensus sequences for fungal exon / intron splice junctions in intron interior sequences (Baliance, Ref. 11) leads to the postulation of the presence of four introns in the genes pelA, B and C. The positions at which the introns are located within the coding regions of these pel genes are conserved in the sense that there are potentially five intron sites but each gene lacks another intron. However, only three introns are postulated for the pelC gene. Of these, only one is in a position corresponding to a position of an intron in pelD and pelA (intron 5). The positions of these introns and their corresponding length are summarized in Table Vla.

Table VIa

Positions of introns in the sequences of pelD, pelA, pelB and pelC

The positions refer to the coding sequences in Figures 10, 11 and 12 and to the sequence of pelD

gene pel D pelA pelB pelC intron Position length, bp Position length, bp Position length, bp Position length, bp 1 202-267 66 absence 1337-139862 absence 2 410-47162 1708-175952 1543-159856 absence 3 598-66063 1886-193348 1725-178157 absence 4 absence absence absence 1853-193078 5 1446-150257 2031-209464 absence 1998-206265 6 absence absence absence 2232-229463 7 absence 2329-238254 2113-2169 57 absence

The length of the exons between the introns in these cases is the same for the four pel genes. Figure 13 shows the aligned, deduced amino acid sequences of PLA, PLB, PLC and PLD. In the N-terminal part of the coding regions of pelA, pelB and pelD, homology of about 80% is observed based on nucleotide sequence homology, and more than 80% on the basis of amino acid sequence homology. This percentage is much lower in pelC. In the C-terminal part of pelA, pelB and pelD, beyond the remainder 285 of the mature protein (which corresponds to residue 305 in Fig. 13), homology largely disappears and returns only near the C-terminus. Apart from the consensus sequences of the splice signal and the 5 'and З' splice sites (Table VI b), no homology is detected in the introns.

Table VIb

Comparison of intron consensus sequence

intron

Donor exoni

lasso

intron

Acceptor intron

exon 2

pelD pelB 1 1 AAGAC AGAAC GTGAGTTT. GTAGGTCG. .32 .32. GGCTGACC. .GCCTAACA. .12. , .8th. .TGCCAG .ATCCAG TTTCG CTTCG pe ID pe IA pelB 2 2 2 ACCTA GAATA ACTTA GTAAGTTG. GTATGTCC. GTATGTTG. .37. .29. .31. .TGCTGACA. .ATCTAACT. .TGCTGATA. ..3. ..1. ..3. .GGATAG .GGATAG .GTATAG CAACA CTACA TGATA pelD pelA pelB 3 3 3 ATCCA ATCCA ATCCA GTATGCAT. GTAGGTTA. GTGAGTGC. .32. .25. .33. .AACTAACC. .CTCTAACG. .TGCTAATA. ..9. ..1. ..2. .CCACAG .AATCAG .GGCCAG GAACA GAACA GAACA pelD pe IA pe 1С 5 5 5 TTACT TTACT GCGAG GTAAGTCG. GTACGTCT. GTAAGACA. .31. .40. .39. .CACTAATG. .AGTTAACA. .CGTTGACT. ..4. ..2. ..4. .CTTCAG .TGACAG .GATTAG ACTGC ACCGC ACCTC pelC 6 CCAGA GTACGTGT. .30. .AACTAACA. .11. .TCACAG ACAAC Pelea Peleb 7 7 ACTGT ACGCC GTAAGTTG. GTATGTCG. .24. .28. .ACCTGACT. .TACTGACA. ..8th. , .7. .TTGCAG .CTACAG GTCAA GTGAA CONSENSUS GTA GT--. g с a .24. 40 .-CTAAC-. t G t ..1. 12 , CAG T

The deduced amino acid sequences for the proteins PLA, PLB, PLC and PLD indicate a total of 359 residues for the first two proteins, 360 for POC and 354 for PLD. In the context of N-glycosylation, all four proteins have a potential glycosylation site at position 109 (in the mature protein) (in the PLC, this corresponds to position 105 in the unaligned sequence). PLB has a second potential site at position 232, while PLD has two other potential glycosylation sites at residues 255 and 329.

Example 5

Co-transformation of A. niger using the A. niger pyrA gene as a selection marker and of various A. niger pel genes as co-transforming plasmids

Example 5.1

Reproduction and Purification of Plasmids Used for the Transformation of A. niger All plasmids are propagated in E. coli strain MH1 and the plasmid DNA is prepared from 500 ml cultures grown overnight by the method by Maniatis et al. (pp. 90-91, ref. 5). To 2.5 ml of DNA solution in TE containing up to 1 mg of DNA was added 2.2 g of CsCl and 1 ml of ethidium bromide (10 mg / ml in water). The solution is placed in quick-release tubes which are filled and sealed as recommended by the manufacturer (Beckman). The centrifugation is performed in a VTi 65.2 rotor at 20 ⁰ C over a period of 16 hours at 45 000 rev / min. Of the two fluorescent bands visible under ultraviolet light, the lower one, in which the plasmid DNA is located, is isolated by lateral puncturing of the tube. The DNA solution (about 1 ml) is extracted five times with water-saturated butanol to remove the ethidium bromide. Then, the DNA is precipitated by ethanol, brought back to TE in suspension, extracted once with phenol / chloroform and chloroform, reprecipitated and stored at -20 ⁰ C.

Plasmid pGW613 carrying the OMP decarboxylase gene (pyrA) on a 5 kbp A. niger DNA fragment is used to select for transformants (Goosen et al., Ref 12).

Plasmids pGW820, pGW830, pGW840, pGW850, pGW860 and pGW880, described in Example 3.5, are used as transforming plasmids.

Example 5.2

Preparation of protoplasts and transformation of the uridine auxotrophic mutant A.niger strain N593 The A.niger strain N593 (cspA, pyrA), which is a derivative of the parent strain N400, was prepared by positive selection on the toxic, 5-fluoroorotinic acid analog in yeast (Boeke Ref. 13) in the presence of uridine, such as that of Goosen and others (Ref 12).

Liquid minimal medium supplemented with 0.5% yeast extract, 0.2 Kacasaminosäuren, 1OmM uridine (Janssen Chimica) and 50 mM glucose, inokultiert with 10 ⁶ conidiospores of A. niger N593 and 20 hours at 30 ⁰ C in an orbital shaker New Brunswick incubated. Mycelium is harvested by filtration through Miracloth, washed with isoosmotic minimal medium (STS STC) with the following composition: 1.33 M sorbitol, 10 mM Tris-HCl, pH 7.5, 5 mM CaCl ₂ , adjusted to 1.7 mM. An amount of 1 g of mycelium is re-suspended in 20 ml of STC. Within 2 hours, 234 (Novo Industries, Denmark) and incubating the mixture at 30 ⁰ C in a shaker with 95 U / min protoplasts were liberated from the mycelium by the addition of 250 mg of filter-sterilized Novozym. The protoplasts are separated from residual mycelium by filtering using a funnel with a glass wool plug and cleaned by centrifugation (10 min 2 500 rpm), pelleted and re-suspended in STC.

For transformation 5 χ 10 ⁶ protoplasts in 200μΙ be given (less than 20 μΙ volume) are then incubated together with 1 \ ig pGW631 and Юцд one of the plasmids pGW820, pGW830, pGW850, pGW860 or PGW 880th For pGW 840, a ratio of 1: 3 is used using бцд pGW613. After addition of plasmid DNA to the protoplasts, 50 μM PCT (10 mM Tris-HCl, pH 7.5.5 mM CaCl ₂ 25% PEG 6000) is added and the incubation mixture is kept on ice for 20 min.

Then another 2 ml of PCT are added and the mixture is incubated for a further 5 min at room temperature. Finally 4ml STC are added and mixed. Aliquots of 1 ml of this final transformation solution are mixed with 4 ml of liquefied isoosmotic MM top agar stabilized by 0.95 M sucrose (adjusted to 1.7 mMsm). The protoplast mixture is immediately plated on agar plates containing the same isoosmotic minimal medium and these are incubated at 30 ^° C. Corresponding control experiments involve protoplasts treated as well, but without plasmid DNA, and on non-stabilized minimal medium with 2.5 mM uridine. After 3 days of growth at 30 ⁰ C well growing transformants appear that form spores (50-150 transformants per \ ig pGW613). In addition, a large number of presumably abortive transformants appear. The spores of individual transformants are then taken and plated separately on 50 mM minimal glucose medium to obtain single colonies which are used to avoid the spores for further analysis of the transformants. In any event, at least ten transformants are grown on minimal liquid medium, the DNA is extracted and analyzed for the presence of co-transforming plasmid DNA. Fungus DNA is isolated by a method that is slightly modified from the method for isolating plant RNA (Slater, Ref 14). The mycelium is washed with saline, frozen in liquid nitrogen and 0.5 g is broken using a microdismembrator (Braun). The mycelium powder is extracted with freshly prepared extraction buffer. The extraction buffer is prepared as follows: 1 ml triisopropylnaphthalenesulfonic acid (TNS), 20 mg / ml, is added with 1 ml p-aminosalicylic acid (PAS), 120 mg / ml, and 0.5 ml 5x RNB buffer (5x RNB buffer contains 121.1 g Tris, 73.04g NaCl and 95.1gTA in 11, pH 8.5). After the addition of 1.5 ml of phenol, the extraction buffer is equilibrated for 10 min at 55 ° C. The warm buffer is then added to the mycelial powder and the suspension is thoroughly mixed for one minute with a vortex mixer. Then one milliliter of chloroform is added and mixed for 1 minute.

The aqueous phase is separated off by centrifuging (10 min, 10,000 g) and extracted once more with an equal volume of phenol / chloroform (1: 1) and then twice with chloroform.

The aqueous phase contains both RNA and DNA. The DNA is precipitated at room temperature with 2 volumes of ethanol and collected by centrifugation (10 min, 10000 g), washed twice by dissolution in distilled, sterile water and reprecipitation with ethanol. The RNA is removed by adding RNase A (20 цд / ті) to the final solution. The method yields high molecular weight DNA (100 to 200 kbp) in a yield of about ЗООдd DNA / g mycelium which is further purified by CsCl / ethidium bromide density gradient centrifugation as described essentially by Maniatis et al., P.93; Ref 5, has been described.

Digestions of chromosomal DNA (2Kd) are transferred by incubation at 37 ° C with EchoRI for pelA (pGW820) and pelC (pGW 850) transformants, HindIII for pelB (pGW 830) transformants, and EcoRI or BglII for pelD transformants the duration of 2 hours. In all cases, initially 20 units of restriction enzyme are used, then the incubation is prolonged by the addition of another 20 units of restriction enzymes for a further 2 hours. The digestions are carried out in the appropriate reaction buffers supplied by BRL.

Then the DNA is fractionated to 0.6% agarose, transferred to nitrocellulose and hybridized essentially as described by Maniatis et al. (Lit.5), pp. 382-386, for which the corresponding ³² P) -labeled Probes are used.

The following cotransformation frequencies are found: pGW820 (pelA) 70%; pGW830 (pelB) 70%; pGW840 (pelD) 15%; pGW850 (pelC) 60%.

The mass ratio of pGW 613 to the transforming plasmid pGW820, pGW830, pGW950 (1:10) and pGW840 (1: 3) leads in the majority of the cases observed to the multiple copy of the chromosomal integration of cotransforming plasmids.

Example 5.3

Genomic analysis of the pelA-transfected A. niger strain A15

A number of the multicopy transformants described in Example 5.2 were analyzed and analysis of the pelA transformant A15 is given as a detailed example.

DNA of the A. niger strain N 593 and the multi-copy transformed pelA strain Al 5 are isolated and analyzed by hybridization of Southern transfers, as described in Example 3.1. The pelA probe used is the 7.5 kbp EcoRI fragment (see FIG. 3 a). In genomic transmission, an intense band of 7.4 kbp is found in the transformed strain A15 resulting from type I and / or type II integration events (see Hinnen et al., Ref 14a). There are also several smaller bands of varying lengths found that represent gene fragments of integration events type II of conversions.

The optical densities of the 7.4kbp EcoRI bands in the A. niger pelA transformants A15 and the A. niger N593 autoradiograms are scanned using an Ultroscan XL (LKB) instrument. Values are corrected for small differences in DNA concentration between N593 and transformant A15 by scanning the detected densities with a second probe that hybridizes with the pyruvate kinase gene present as a single copy in both strains. From these data it is calculated that there are about 19 intact copies of the pelA gene in the pelA transformant of A15.

Using the HindIII insert of pGW830 (Figure 3b) as a probe, analysis of Southern transfer of transformed strain A15 shows that integration of the pelA sequences did not occur at the pelB site. Similarly, analysis of transformed pelB strain B13, pelC strain C15 and pelD strain D12 with appropriate probes yields copy numbers of about 15.50 and 10, respectively.

Example 5.4

Northern transfer analysis of induced and uninduced mycelia of the рѳІА-transformed A. niger strain A15 and A. niger N400

Minimal medium (250ml), the 5OmM glucose or 1% apple pectin (Brown Ribbon, de 72.8%, Obipektin AG, Bischofszeil), is inoculated with a spore density of 10 ^е spores / ml with spores of transformants A15 or spores of the wild-type strain N400 , and after 30 hours of growth at 30 ° C is harvested. There also be included samples of the culture medium (2ml), based on 211OmM sodium phosphate buffer, pH 7.0, at 4 ⁰ C dialyzed and stored at -20 ° C. Nucleic acids are isolated from the mycelium, as described in Example 5.2. The RNA is precipitated from the aqueous phase after chloroform extraction by the addition of Vs volume of 8 M LiCl. The RNA is collected by centrifugation (300 U / min, 30 min) using a centrifuge MSE collected Spuer-Mlnor and then once with cold (-20 C ⁰⁾ 2 M LiCl, and once with cold (-20 C ^0), 96% washed ethanol. Finally, the RNA is dissolved in distilled water at a concentration of about 1 mg / ml. The preparations are essentially free of DNA and give a yield of 1-2 mg of RNA per gram of mycelium. Quantities of about 50 μg of RNA are treated according to Maniatis et al., Pp. 202-203 (ref. 5) on denatured formaldehyde 1% agarose gel using HindIII-digested lambda DNA or the RNA ladder of BRL as molecular weight marker. The gels are transferred and baked to Nytran (Schleicher and Schuell), as recommended by the manufacturer, and hybridized for 16 hours at 68 ⁰ C with the 7.4 kbp EcoRI-pelA-DN Α probe. The hybridization and washing procedures are the same as for DNA hybridization under homologous conditions according to Example 3.2. Both A. niger strains produce a pectin-inducible mRNA of about 1.6 kbp in length. The scanning of the optical density of the two autoradiograms has a 15 to 20 fold difference in intensity between the A15 transformant and the wild-type strain, which correlates with the increase in copy number calculated from the Southern transfer analysis.

Example 5.5

Pectinylase production by the transformed A. niger strain D12

The A. niger-pelD transformant D12 obtained according to Example 5.2 is grown in a two step culturing procedure similar to the method described by Hermersdörfer et al. (27) for the production of polygalacturonase. On top of a liquid culture, a mycelial layer is formed by modulation of 10 ⁶ spores per milliliter of

Precultivation medium (PCM) consisting of sugar beet pulp (4%) and NH ₄ NO ₃ (1%), pH 4.5. After a growth period of 5 days at 30 ⁰ C the solid in 30ml medium mycelium is washed with brine and 50ml Hauptkultivationsmedium (MCM) transmitted. This culture is grown at 30 ° C in an orbital shaker at 100 rpm. The main culture medium per liter of medium is composed of glucose (5%), pectin (de 72.8%, 0.1%), NH ₄ NO ₃ (0.75%), KH ₂ PO ₄ (0.5%), FeSO ₄ .7H ₂ O (0.03%), MgSO ₄ .7H ₂ O (0.03%), CaCO ₃ (0.03%), NaNO ₃ (0.03%), pH 4 ; 5. Within 24 hours, samples are taken at different time intervals and analyzed by SDS-polyacrylamide gel electrophoresis. The expression of pelD is maximal as early as 7 hours after the start of cultivation. The protein migrates identically to PLI, which was used as a reference.

Example 5.6

Production of pectin lyase A by the transformed A. niger strain A15 and by A. niger N400 The dialyzed culture filtrates (Example 5.4) are lyophilized, resuspended in 0.2 ml distilled water and resuspended on SDS-polyacrylamide gel electrophoresis (10% gel) Standard methods (Laemmli, ref 15). The separated proteins are transferred to nitrocellulose (Towbin et al., Ref 16). The transfers are saturated for five hours at room temperature with 1% BSA solution in 10 mM Tris-HCl buffer, pH 7.5, with 0.35 M NaCl. This is followed by a sixteen-hour incubation at room temperature with polyclonal antibody (0.1%), drawn from two pectinylases purified according to van Houdehoven (1975) in 50 ml of the same buffer containing 15 mM NaCl, 0.5% BSam 1 % Triton X100,0.5% deoxycholate and 0.1% SDS. The transfers are then rinsed five times with 200ml of PBS before incubation with 30μΙ goat anti-rabbit IgG-HRP (Sigma) as the second antibody per 50ml, then washed 5 times with PBS again. Finally, the transfers are stained using 4-chloro-1-naphthol as the substrate. 40 mg of the substrate are dissolved in 0.5 ml of 96% ethanol and mixed with 100 ml of 50 mM Tris-HCl, pH 7.5, and 30 μl of 30% hydrogen peroxide and then added to the transfers.

Example 5.7

Screening of the pelA-transformed strains by halo formation on pectin-containing solid media Conidia of pelA-transformed strains are inoculated with a spore density of about 40 colonies per petri dish on sterile filter paper (Schleicher and Schuell) applied to top of 1% agarically solidified minimal medium. which contains 0.01% Triton-X 100 and apple pectin (1%, De 72.8%, Obipektin) as carbon source. The incubation period is 72 hours at 30 ° C and gives single colonies (2-4mm). The filter paper is transferred to another sterile Petri dish, and the medium in this dish is stained with at least 5 ml of a ruthenium red solution (0.1%) in distilled water, incubated for 2 hours and then washed several times with distilled water to obtain to remove the unadsorbed dye, followed essentially by the procedure described by Ried and Collmer (ref 17) for polygalacturonate to characterize the bacterial pectate lyase enzyme activity.

Transformants with an overproduction of PL Α protein are detected by an increase in the diameter of the halo around the individual colonies compared to the parent strain N400.

Example 6

Isolation, purification and characterization of pectin lyase A (PLA) from A. niger pelA transformant A15

Example 6.1

Culture conditions for the production of pectin lyase A

An A. niger pelA transformant A15, as described in Example 5, is drawn on complete medium in Petri dishes for a period of 3 days at 28 ⁰ C to produce conidia. The conidia are harvested from these plates by suspending the spores in 5 ml of sterile saline containing 0.005% Tween 80. The suspension is stirred vigorously on a Griffin shaker for 20 minutes. Minimal medium (ref 21) containing 1% apple pectin (Brown Ribbon, de 72.8%; Obipektin AG, Bischofzeil) is spiked with a spore density of 10 ^e spores / ml using 250 ml of medium in 11-conical flasks are inoculated. The mycelium is drawn for 40 hours at 30 ⁰ C on a Gallenkamp Oribtalschüttelapparat at 200U / min. After culturing, the mycelium is removed by filtering over Miracloth using a Buchs funnel. The culture filtrate (ref 21) is diluted with distilled water (ref 21), the pH of the filtrate (pH 3.7) is brought to 6.0 using 1N NaOH, then again with filter paper Whatman 1 filtered.

Example 6.2 Cleaning the PLA

The diluted culture filtrate (4I) is placed in a DEAE-Sepharose (Pharmacia) rapid-flow column (10 cm x 2.6 cm) which has been preequilibrated with 20 mM sodium phosphate buffer, pH 6.0. The column is eluted with the same buffer until the absorbance at 280 nm reaches a value of 0.1 OD. The pectinase activity is then eluted from the column using a linear gradient composed of 20 mM sodium phosphate buffer (150 ml) and 20 mM sodium phosphate buffer with 1 M NaCl (150 ml).

The fractions are checked for the presence of active pectinylase according to the procedure described by van Houdenhoven (ref 18, p. 11), using pectin from Brown Ribbon (i.e., 72.8%) as a substrate. The activity appears around a concentration of 0.43M NaCl in the salt gradient. The active fractions are then pooled and dialysed twice with 112 mM of piperazine-HCl buffer, pH 5.5 (sample size 45.5 ml). In the next step, the dialysed sample is subdivided into 3 equal sized portions which are subjected to anionic exchange chromatography in three separate runs using a standard MONO Q column from Pharmacia in conjunction with the Pharmacia FPLC system. The column is pre-equilibrated with 20 mM piperazine-HCl buffer, pH 5.5. After introduction of the enzyme sample, the column is eluted with the same buffer at a flow rate of 1 ml / min until baseline absorbency is restored. Then a linear salt gradient is applied. A final concentration of 0.6M NaCl is achieved in the context of 22 ml of the elution buffer supplied to the columns.

The active fractions (4.4 ml) are collected, diluted to 25 ml using the equilibration buffer, and the same chromatographic procedure repeated. Two fractions containing the active enzyme (total volume 3 ml) are then dialysed from 25 mM piperazine buffer, pH 5.0, and injected in 1 ml portions onto a MONO P (Pharmacia) column, which is co-injected with the same Buffer was pre-equilibrated. Following injections, a pH gradient is established using a 5% solution of Polypuffer TM 74 (in distilled water brought to pH 3.0 using 4N HCl). The active enzyme (3.2 ml) occurs at a pH of 3.2. The drug is extensively based on 5OmM sodium phosphate buffer, pH 6.0, dialyzed and at -20 ⁰ C. Purification results are summarized in Table VIIA and compared to data from a similar purification of pectin lyase produced by A. niger strain N400 (Table VII b).

Table VII a and VIIb

Purification scheme for pectin lyase A from the A. niger pelA transformant N 593 and pectin lyase from the A. niger strain

Vila step Volume ml Total Activity (I.U.) Specific pectin lyase activity (I.U./mg protein) DEAE-Sepharose MONOQ Chromatography (2x) MONOP Chromatogr. VIIb wild-type step 45.5 2.3 3.2 ml ml 39.8 19.9 17.4 Overall activity (I.U.) 4.9 18.4 28.6 Specific pectin lyase activity (I.U./mg protein) DEAE-Sepharose MONOQ Chromatography (2x) MONO P-Chromatogr. 43.0 1.1 1.4 10.1 2.5 1.7 0.9 6.3 10.0

Protein concentrations were determined using the BCA protein pro-reagent (Pierce) according to the manufacturer's instructions.

The total activity of A. niger N 593 transformed with pGW820 has increased approximately tenfold compared to the total activity of the wild type after purification, and the specific activity about threefold.

Example 6.3

Determination of the Amino Acid Sequences of the N-terminal Part of Pectin Lyase A 500 μg of pectin lyase (PLA), which were purified according to Example 6.2, were dialyzed three times from 11 distilled water filtered through Millipore and lyophilized. The amino acid sequences are determined on an Applied Biosystems Model 470A protein sequencer attached directly to a 120 A PTH analyzer according to the method described by Hunkapiller (ref 17)

 For the enzyme, the following N-terminal amino acid sequence is determined:

VAL-GLY-VAL-SER-GLY-SER-ALA-GLU-GLY-PHE-ALA-GLU-T-VAL-THR-GLY-GLY-GLY-ASP-ALA.

The amino acid sequence thus determined corresponds exactly to that based on the nucleotide sequence of the pelA gene (see Example 4.2).

Example 6.4

Properties of pectin lyase A

The A. niger strain N400 and the pelA transformant A15 are cultured as in Example 6.1, and the enzyme is purified from the culture filtrate according to the procedure in Example 6.2. The two enzyme preparations thus obtained are compared with pectin lyase II, which was purified according to van Houdenhoven (ref. 18).

SDS-polyacrylamide gel electrophoresis using 10% gel yields a single band of identical molecular weight in all three cases when 2 to 5K protein are used in each case.

Using standard techniques (see, e.g., Instruction Sheet by Pharmacia, Isoelectric Focussing, 1982), isoelectric focusing was performed. An instrument FSBE-3000 and the corresponding power supply ECPS 3000/150 (Pharmacia) were used. The recovered PLA is homogeneous after isoelectric focusing and has a lower isoelectric point (O, 2pH units) than PLII, which is given as 3.75 by van Houdenhoven (Ref. 18). The PLII purified from the N400 filtrate is heterogeneous after isoelectric focusing. The major band is in position consistent with PLA protein. Two smaller bands are slightly displaced to the cathode. Consequently, the A. niger multicopy transformant A15 lacks the smaller enzyme forms that are visible at N 400.

A direct comparison of the kinetic parameters of PLA and PLH determined in the latter case by van Houdenhoven (ref 18) using 95% esterified apple pectin as substrate reveals identical K _n , and V _max values for both enzymes.

Example 7 Isolation, purification and characterization of pectin lyase D (PLO) from the A. niger-pelD transformant 012 Example 7.1 Culture conditions for the production of pectin lyase O.

The A. niger-pelD transformant D12, obtained according to example 5.2, is initially drawn into aliquots of 300 ml according to example 5.5. After a growth period of 5 days in PCM at 30 ⁰ C the mycelial mat on 20 mM sodium phosphate buffer (pH 6.0) containing 0.1 M sodium chloride, is transferred. By shaking the mycelium in 150 ml on an orbital shaker at 200 rpm for 2 hours, most of the pectin lyase is released into the medium.

Example 7.2 Purification of PLD

After removal of the mycelium by filtration, the enzyme solution is delivered to a DEAE-Sepharose rapid flow column (Pharmacia) (25 cm x 1.25 cm) pre-equilibrated with 20 mM sodium phosphate buffer (pH 6.0) containing 0.2 M sodium chloride was. The column is eluted with the same buffer until the absorbance at 280 nm reaches a value of <0.1. The pectin lyase activity is eluted from the column using a linear sodium chloride gradient (0.2-0.7M) in 20 mM sodium phosphate buffer (pH 6.0) (800 ml total volume). The fractions are screened for the presence of pectin lyase D by SDS-polyacrylamide gel electrophoresis and Western transfer (Towbin et al., Ref 16), as described in Example 5.6. The fractions containing pectin lyase D are pooled and dialyzed from 20 mM sodium phosphate buffer (pH 6.0) containing 0.15M sodium chloride and anion exchange chromatography using a standard Pharmacia ΜΟΝΟ Q column in conjunction with FPLC System subjected. After loading, the column is washed with the same buffer before applying a linear gradient of 50 mM Narium chloride in 20 mM sodium phosphate buffer (pH 6.0). The active fractions are collected and aliquots of 1 ml are added to a 100 ml Superose 12 gel permeation column equilibrated in 20 mM sodium phosphate (pH 6.0) containing 0.2M sodium chloride and with connected to the FPLC system. Active enzyme fractions (1 ml each) recovered from the GPC column are analyzed by SDS-polyacrylamide gel electrophoresis to check the purity of the recovered pectin lyase.

Table VIII Purification scheme of pectin lyase D from the A. niger-pelD transformant D12

Step Volume Activity Specific pectin

(ml) (I.U.) lyase activity

(I.U./mg protein)

Culture filtrate 1950 109 0.096

DEAE Sepharose FF 103 32.2 5.65

MONO Q and GPC 14.5 10.4 7.5

Example 7.3 Determination of the amino acid sequence of the N-terminal part of pectin lyase D

500 μl of pectin lyase D, purified according to example 6.2, are purified three times by means of distilled water, 11 filtered through Millipore, and lyophilized. The amino acid sequences are determined on an Applied Biosystems model 470A protein sequencer that has been directly coupled to an analyzer 120A PTH using the method described by Hunkapillar (Ref. 19)

 For the enzyme, the following N-terminal amino acid sequence is determined:

VAL-GLY-VAL-SER-GLY-THR-PRO-VAL-GLY-PHE-ALA-SER-SER-ALA-THR-GLY-GLY-GLY-ASP-ALA-THR

The particular amino acid sequence is exactly the same as that based on the nucleotide sequence of the pelD gene.

Example 8 Isolation, purification and characterization of pectin lyase B (PLB) from A. niger trartformant B13 Example 8.1 Culture conditions for the production of pectin lyase B

The A. niger pelB transformant B13, which was obtained according to Example 5.2, is analyzed according to Example 5.3 for its multiple copy character. The Northern transfer analysis according to the method described in Example 5.4 shows that a pectin-inducible mRNA with a length of about 1.6 kb is produced. After growth for 35 hours at 30 ° C in a minimal medium containing 1% citrus pectin (de 72.8%) and 1% wheat bran, the enzyme is produced as in Example 5.4. The enzyme is also produced using a medium consisting of 4% sugar beet pulp and 1% NH ₄ HO. ₃

Example 8.2 Cleaning the PLB

The culture filtrate is diluted twice with distilled water, and the pH is brought to 6.0 with 1N NaOH, then filtered again with Whatman 1 filter paper. The dilute filtrate (21) is then added to a DEAE-Sepharose fast flow column (Pharmacia) (9cm x 3.2cm) which is loaded with 50 mM sodium acetate buffer, pH 5.0,

was pre-equilibrated. Part of the pectin lyase activity is present in the eluate, as can be demonstrated by the method described by van Houdenhoven (ref 18, p. 11), whereby Brown Ribbon pectin (de 72.8%) or strongly esterified Pectin (de 94%) used. The majority of the pectin lyase activity can be eluted by the addition of a salt gradient. This activity appears in the salt gradient and is found to be identical to PLA on the basis of the elution behavior and the apparent molecular weight in SDS-polyacrylamide gel electrophoresis, as described in Example 6.

The eluate from the DEAE high-speed column is dialyzed using distilled water and fed to a MONO S (Pharmacia) column previously equilibrated with a 20 mM sodium acetate buffer, pH 4.5. The enzyme is eluted from the column by using a sodium chloride salt gradient of 0-1.0M in the same buffer. The enzyme is almost immediately eluted from the column. The active fractions are pooled and then diluted approximately fourfold with distilled water. Rechromatography of the enzyme solution yields fractions containing the pure PLB protein based on SDS-polyacrylamide gel electrophoresis.

Example 8.3

Properties of pectin lyase B

The properties of the enzyme purified from pelB transformant B13 according to example 8.2 are determined and compared with those of pectin lyase A and D.

SDS-polyacrylamide gel electrophoresis using 10% gels results in a single band with an apparent molecular mass of 39.7 kDa. Under the same conditions, the apparent molecular mass of PLA and PLD is 49.1 kDa and 52.3 kDa, respectively.

Isoelectric focusing is carried out as described in Example 6.4. The isoelectric point of PLB is 6.0 when working with a pH gradient between pH's 3 and 7. The sample is fed approximately in a position corresponding to a pI of 5.0.

The purified enzyme PLB is also reactive with polyclonal antibodies prepared from PLI and PLII as tested by Western transfer analysis. PLD, A and B can thus easily be distinguished by the values of their apparent molecular mass.

Example 8.4

Kinetic properties of PLB

The purified according to Example 8.2 enzyme is characterized kinetically. The enzyme catalyzes a pectin lyase reaction and is active over a wide range of pH's (pH 5-9.5) as tested in a Mcllvaine buffer (μ = 0.5) at different pH's (van Houdenhoven Ref 18). The pH optimum is broad (pH 7.5-8.5). At a pH of 6.0, the activity is about 55%; at a pH of 9.5 it still accounts for 85% of the activity at a pH of 8.0. Both strongly esterified pectin (de = degree of esterification) = 94%) and pectins with a lower degree of esterification, such as brown ribbon apple pectin (de 72.8%; Obipektin AG, Bischoffzeil) can be used as a substrate. However, the highest activity is achieved with strongly esterified pectin. The enzyme does not react with polygalacturomat. The pectin lyase reaction catalyzed by PLB can be inhibited by the addition of EDTA to the reaction mixture (operating at a final concentration of 3 mM); The enzyme is reactivated by the addition of an excess of Ca ²⁺ ions. So pelB encodes a Ca ²⁺ -dependent pectin lyase. At 25 ° C, the enzyme has an approximate turnover rate of 6500 and a K _m value of 2.5 mM when heavily esterified pectin is used as the substrate. These values are determined by the method of van Houdenhoven (ref 18) using a Mcllvaine buffer, pH 8.0 (μ = 0.5).

Example 9 Expression and secretion of foreign genes under the control of the pelA promoter

The 3.9kb HindIII fragment of plasmid pGW 820 is subcloned into the HindIII site of pBR 322. The plasmid DNA of ampicillin-resistant transformants is analyzed by HindIII and SalI restriction digests. A clone containing the pelA insert in a clockwise orientation is called pGW822. The inducible promoter of pelA is used to express foreign genes in A. niger. On plasmid pGW822, the complete pelA gene is present. The promoter and the pelA signal sequence are located on a 1 kb BamHI-PstI fragment. The PstI site at nucleotide position 1420 (Figure 10) coincides with the З 'end of the signal sequence and can be used for frame fusion to the coding sequence of a foreign gene. The transcriptional termination signals of pelA are on a 1.3 kb SalI HindIII fragment.

Example 9.1

Subcloning the pelA gene into the vector pUC19

The 3.3 kb BamHI-HindIII fragment of plasmid pGW322 contains the pelA gene. The fragment is cloned into the vector pUC 19 (Pharmacia), which was cut with BamHI and HindIII. The purified fragments are ligated. An aliquot of the ligation mixture is used to treat Ca ² ~ treated, competent JM 109 cells. The successful cloning of the 3.3 kb BamHI-HindIII fragment into pUC19 is carried out by selection on ampicillin plates in the presence of X-GaI and IPTG (T. Maniatis et al., In "Molecular Cloning, A Laboratory Manual"). Cold Spring Harbor Laboratory, 1982, p.52) White ampicillin-resistant transformants are picked out The plasmid DNA of these colonies is analyzed by BamHI / HindIII double digestion A transformant with a correct insertion is designated pUC19 / pelA An analogous construction with pUC 18 gives pUC18 / pelA.

Example 9.2

Expression of Human Hybrid Interferon BDBB Under the Control of the pelA Promoter 9.2.1: Construction of the plasmid pUC 19 / pelA-IFN AM 119 (see Fig. 14)

Plasmid риСІЭ / реІА is digested with Sail. The cohesive ends of the linear fragment are reacted in a reaction with Klenow DNA polymerase I (BRL) in the presence of each 0.1 mM dCTP, dGTP, dATP. dTTP, 60 mM Tris-HCl, pH 7.5, and 10 mM MgCl _{2 were} filled at room temperature over a period of 30 minutes. The reaction is stopped by the addition of EDTA to a final concentration of 12.5 mM. The DNA is precipitated with ethanol. The linear fragment is digested with PstI. After phenol / chloroform extraction, the DNA is precipitated with ethanol.

An oligodeoxynucleotide linker of the formula

Pstl ^DdeI

(I) 5'-TGTGATCTGCC -3 '

(II) З'-ACGTACACTAGACGGAGT -5 '

is ligated into the PstI site of the linearized plasmid. The linker fills in the 3'-end of the PstI site that coincides with the end of the pELA signal sequence and assures in frame fusion to the coding sequence of interferon-BDBB. (I) represents the 5 'terminal nucleotide sequence of the interferon BDBB gene up to the Ddel restriction site.

In each case 20OpMoI of the oligonucleotides (I) and (II) are phosphorylated and hybridized. The PstI cut pUC 19 / PeIA plasmid and a one hundred fold molar excess of the double stranded linker DNA are dissolved in 60 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM ATP, 5 mM DTT and 400 units T4 DNA. Ligase ligated for 16 hours at 15 ° C. The DNA ligase is inactivated for 10 min at 85 ° C. The excess linkers are removed by precipitation in the presence of 10 mM EDTA, 30 mM sodium acetate, pH 6.0, and 0.54 volumes of isopropanol.

The large 5 kb fragment is isolated on a 0.6% preparative agarose gel. The fragment contains the pelA promoter and the signal sequence (BamHI [PstI] / linker) and the pelA terminator (Salllglatt-HindIII) in the vector pUC19.

The plasmid pJDB207 / PH05-IFN AM 119 (EP 205404) contains the gene for hybrid a-interferon BDBB under the control of the regulated promoter of the yeast acid phosphatase (PHO5). The plasmid DNA is digested with BamHI and HindIII. The 1.3kb BamHI-HindIII fragment is isolated which contains the PHO 5 promoter, the IFN BDBB coding sequence and the PHO 5 transcription termination sequences. The fragment is purified by DE 52 chromatography and ethanol precipitation and further digested with Taql. The cohesive ends of the TaqI restriction fragments are reacted in a reaction with Klenow DNA polymerase I (BRL) in the presence of 0.1 mM each of dCTP and dGTP, 60 mM Tris-HCl, pH 7.5.1 mM MgCl ₂ the duration of 30 min at room temperature. The reaction is stopped by the addition of EDTA to a final concentration of 12.5 mM. The DNA fragments are precipitated with ethanol and further digested with DdeI. The DNA fragments are separated on a 0.8% preparative agarose gel. The 549bp Ddel [Taql] smooth fragment is electroeluted from the gel, purified by DE 52 ion exchange chromatography and precipitated with ethanol. The DNA fragment is brought back into suspension in H ₂ O at a concentration of about 0.1 pmol / μΙ.

0.2 pmol of the 549 bp Ddel [Taql] -shad fragment and 0.1 pmol of the 5kb vector fragment are dissolved in 10 μl of 60 mM Tris-HCl, pH 7.5.1 mM MgCl ₂ , 3.5 mM ATP, 5 mM DTT and 200 units of T4 DNA ligase (Biolabs) ligated for 16 hours at 15 ° C. An amount of 1 μΙ of the ligation mixture is used to transform Ca ²⁺ -treated, competent HB 101 cells.

Plasmid DNA is prepared from 12 ampicillin-resistant, transformed colonies and analyzed by EcoRI, Pvull, CIaI and EcoRI / HindIII digestion. A clone with the expected restriction pattern is selected. The correct in-frame fusion of the peA signal sequence and the mature coding sequence of interferon-BDBB is confirmed by DNA sequencing. The plasmid DNA of the selected clone is designated pUC 19 / pelA-IFN AM 119.

An analogous construction with pUC18 / pelA gives the plasmid pUC18 / pelA-IFN AM 119.

Example 9.2.2

Cotransformation of the Aspergillus niger Mutant to 8 mitpCG59D7 and риСІЭ / pelA-IFN AM 119 The uridine auxotrophic mutant AN 8 {й DSM 3917, disclosed in EP88101397.3) is transformed with plasmid pCG 59 D7 to give uridine prototrophs. Together with the transforming plasmid, the nonselective plasmid pUC 19 / pelA-IFN AM 119 is added to give random cointegrates upon co-transformation.

Congenial spores of A. niger An 8 are grown for 4 days at 28 ° C until complete sporulation in complete medium. 2 × 10 ⁸ conidiospores are used to inoculate 200 ml minimal medium supplemented with 1 g / l arginine and uridine.

After 20 hours of growth at 28 ⁰ C and 180U / min, the mycelium is harvested by filtration through Miracloth, washed twice with 10 ml 0.8M KCl, 5OmM _CaCl2 washed and brought back to the suspension in 20 ml 0.8 M KCl, 5OmM _CaCl2, 0 , 5 mg / ml Novozym 234 (Novo Industries). The mixture is incubated in a Rüttet-Wasserbad (30 ⁰ C, 50U / min) until microscopically a maximum protoplast release can be detected (90-120min). The protoplast suspension is filtered through a glass wool plug in a funnel to remove mycelial debris. The protoplasts are pelleted by gentle centrifugation (10 min, 2000 rpm) at room temperature and washed twice with 10 ml 0.8 M KCl, 50 mM CaCl ₂ . Finally, the protoplasts in 200 to 500μΙ 0.8M KCI, 5OmM CaCl _{2 brought} back to the suspension, with a concentration of 1 χ 10 ⁸ / ml is achieved. For transformation, an aliquot of 200 μΙ of the protoplast dispersion is incubated with 5MgpCG59D7 and 10 μg of PUC19 / pelA-IFN AM 119 DNA, 50 μM PCT (10 mM Tris-HCl, pH 7.5, 5 mM CaCl ₂ , 25% PEG 6000). The incubation mixture is kept on ice for 20 min, an additional 2 ml of PCT are added, and the mixture is incubated at room temperature for a further 5 min. 4 ml of 0.8 M KCl, 5 mM CaCl _{2 are} added and aliquots of 1 ml of the final transformation solution are mixed with minimal agar lignified medium (minimal medium + 1 g / l arginine + 10 g / l Bacto agar [Difco]), stabilized with 0.8M KCl, mixed. The mixture is immediately poured onto agar plates with the same medium and incubated at 28 ° C

After two to three days of growth at 28 ° C, stable transformants appear as vigorously growing and sporulating colonies on a background growth of many hundreds of small, presumably abortive, transformants.

Example 9.2.3

Expression of the Hybrid Interferon BDBB Gene Under the Control of the pelA Promoter 37 transformants of the cotransformation experiment (Example 9.2.2) are removed and analyzed for interferon expression. Interferon activity is determined by the method of Armstrong {J.A. Armstrong, Appl. Microbio. 21,732 [1971]) using human CCL-23 cells and vesicular stomatitis virus (VSV) as the challenge virus.

Conidial spores of transformants are individually pre-cultured in 50 ml of a preculture medium (Pectin Slow Set I [Unipectin, SA, Redon, France], 3 g / l, NH ₄ Cl, ₂ g / l, KH ₂ PO ₄ , 0.5 g / l, NaCl, 0.5 g / l, Mg ₂ SO ₄ .7H ₂ O, 0.5 g / l, Ca ₂ SO ₄ .2H ₂ .5 g / l, pH 7.0). The preculture is incubated for 72 hours at 250 rpm and 28 ° C. 10% of the preculture is used to inoculate 50 ml of the main culture medium (soybean meal, 20 g / l, pectin slow set, 5 g / l). The culture is grown for 72 to 96 hours at 250 rpm and 28 ° C.

Samples are taken at various times (every 20 hours), the cells are pelleted by centrifugation and disrupted by sonication disintegration. The supernatant layer and cell extracts are both assayed for interferon activity as described (see above). It is noted that the mass of interferon activity is secreted into the medium.

Example 10

Construction of plasmid M13 (+) KS / pelA _A ss-IFN AM 119

The 2.8 kb expression cassette comprising of plasmid M13 (+) KS / pelA _u ss-IFN AM 119 contains the inducible pelA promoter, the coding sequence of hybrid interferon BDBB and the pelA transcriptional terminator on the Bluescript M13 ( +) KS vector. Hybrid interferon BDBB is expressed and located in the host cell. Plasmid M13 (+) KS / pelA- _A ss-IFN AM 119 (see Example 9.2.1) derived from pUC18 / pelA-IFN AM 119th The pelA signal sequence (ss) is switched off by site-directed mutagenesis (see Fig. 15). It is expected that hybrid interferon, which was expressed without signal sequence from the construction, is located in the cytosol.

Example 10.1

Subcloning the pelA-IFN AM 119 cassette into the Bluescript M 13 (+) KS vector Plasmid pUC 18 / pelA-IFN AM 119 (see Example 9.2.1) is digested with BamHI and HindIII. The 2.8kb BamHI-HindIII fragment contains the pelA promoter, the signal sequence, the IFN-BDBB coding sequence and the pelA terminator. The BamHI-HindIII fragment is isolated and ligated to the Bluescript M13 (+) KS vector (Stratagene) cut with BamHI and HindIII. An aliquot of the ligation mixture is used to transform Ca ²⁺ -treated, competent JM 109 cells. The successful cloning of the 2.8 kb BamHI HindIII fragment into M13 (+) KS is selected for ampicillin plates in the presence of X-GaI and IPTG. Twelve white, ampicillin-resistant colonies are recorded. The plasmid DNA of these colonies is analyzed by BamHI / BglII double digestion. A transformant with the correct insert is designated M 13 (+) KS / pelA-IFN AM 119.

Example 10.2

Recovery of single-stranded DNA from cells containing the Bluescript M 13 (+) KS plasmid. Plasmid M13 (+) KS / pelA-IFN AM 119 is used to transform the competent E. coli strain CJ 236 (dut-1 , ung-1, thi-1, relA-1, pCJ105 (Cm '), BIO-RAD Muta-Gene M13 in vitro mutagenesis set). This strain allows the inclusion of uracil in the DNA. CJ 236 is drawn into 10 ml LB medium containing 100 mg / l ampicillin. At ODeoo of 0.5, the cells are over-infected with Phag M13KO7 (Mead et al., Lit 29) at a multiplicity of infection of 50. After one hour at 37 ⁰ C is added kanamycin (50mg / l), and the culture is incubated at 37 ° C 5 to 6 hours on a shaker. The non-coding strand relative to the pelA-IFN AM 119 insert of plasmid M 13 (+) KS / pelA-IFN AM 119 is synthesized, packaged and released to the medium. From the supernatant layer of the culture, single-stranded DNA is prepared according to Kunkel et al. (Ref. 30).

Example 10.3

Lateral oligonucleotide mutagenesis on the single-stranded DNA template Lateral deletion mutagenesis is performed on the non-coding, single-stranded pelA-IFN AM 119 template to resolve the pelA signal sequence (Figure 15).

The mutagenic starter consists of a portion of the pelA promoter sequence, including ATG (nucleotide positions 1343 to 1363 in Fig. 10), and 21 nucleotides encoding the amino acids 1 to 7 of the mature IFN-BDBB. For mutagenesis 20OpMoI of the mutagenic starter are phosphoylated in 20μΙ of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 5 mM DTT and 0.5 mM ATP using 8 units of T ₄ polynucleotide kinase (Boehringer). After one hour at 37 ° C, the reaction is stopped by heating to 65 ° C for 10min.

There are 0.2 pmoles of single-stranded template with 10pMoI of phosphorylated mutagenic Oligodeoxyribonukleotidstarters and 1OpMoI the universal sequencing starter M13 in 30μΙ 2OmM Tris · HCl, pH 7,5,1OmM MgCl _2, 5OmM NaCl, 1 mM DTT at 80 ° C incubated for 5 min. The solution is allowed to cool slowly to room temperature over a period of 30 minutes; 10 μΙ of the enzyme-dNTP- (-dATP-, -dGTP-, -dCTP-, -dTTP-) solution are added to the hybridized mixture and the 1 μM buffer (0.2 M Tris HCl, pH 7.5.0 , 1 M MgCl ₂ ), 5 μM ₂ mM dNTP mixture, 0.5 μM ATM ₂ μM, 0.2 μM DTT, 0.5 μM T ₄ DNA ligase (Biolabs, 400 U / μΙ) and 1.2 μM Klenow DNA Polymerase (BRL, 5 units / μΙ). The mixture is incubated at 15 ^° C. for 16 hours. The reaction is stopped by incubation at 65 ^° C. for 10 minutes. 1 μΙ and 3μΙ of the ligation mixture are used to transform 0.2 ml of the competent cells of the repair minus strain E. coli MV 1190 UMIac-proAB), thi, supE, A (sr1-recA) 306:: Tn10 (tef) (F ': traD36, proAB, Iac Ι ⁴ ΖΔΜ15)]. Strain MV1190 is described in the manual for the in vitro mutagen set BIO-RAD MUTA-GENE M13. Twelve ampicillin-resistant colonies are recorded. Plasmid DNA is prepared and analyzed by seal digestion. Successful triggering of the pelA signal sequence removes a seal site. Correct plasmids are at the seal site in

Dep.-Nr. Disposal date DSM 4388 February 1, 1988 DSM 4389 February 1, 1988 DSM 4390 February 1, 1988 DSM 4391 February 1, 1988 DSM 4392 February 1, 1988

Bluescript vector linearized. A plasmid is further analyzed. The correct association between the pelA promoter and the coding sequence for hybrid IFM BDBB with ATG is confirmed by DNA sequencing. Proper construction is referred to as M 13 (+) KS / pelAAss-IFN AM 119.

Example 10.4

Cotransformation of A. niger and expression of hybrid interferon

The plasmids M 13 (+) KS / pelAAss-IFN AM 119 and pCG59D7 are used for the cotransformation of the A. niger mutant An8 according to Example 8.2.2. The transformants are cultured as in Example 8.2.3. Samples are taken at various times (every 20 hours), the cells are pelleted by centrifugation and disrupted by sonication disintegration. The cell extracts are tested for interferon activity (see above). The mass of interferon activity is found intracellularly.

Deposit of microorganisms

The following microorganisms were deposited under the Budapest Treaty at the German Collection of Microorganisms, Grisebachstr. 8, D-3400 Göttingen:

Microorganisms Escherichia coli HB101 / pGW 820 Escherichia coli HB 101 / pGW 830 Escherichia coli HB101 / pGW 850 Escherichia coli HB 101 / pGW860 Escherichia coli HB101 / pGW 880

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Claims (6)

A method for producing a polypeptide, characterized in that a hybrid vector having a DNA sequence encoding the expression system of pectin lyases PLA, PLB, PLC, PLE or PLF or its derivative is expressed in a suitable host. 2. The method according to claim 1, characterized in that a hybrid vector with a structural gene which is heterologous to Aspergillus niger, is expressed in a suitable host. 3. The method of claim 1 for the overproduction of pectin lyases PLA, PLB, PLC, PLE or PLF in Aspergillus species, characterized by the cultivation of an Aspergillus host, which has been transformed with an expression hybrid vector for the expression of these pectin lyases. A method according to claim 2 for the production of hybrid interferon-BDBB in Aspergillus species, characterized by the cultivation of an Aspergillus host transformed with an expression hybrid vector for the expression of said hybrid interferon. 5. Process for the preparation of pectin lyases PLA, PLB, PLC, PLE or PLF in pure form. 6. Pectin lyases PLA, PLB, PLC, PLE or PLF in pure form.