WO1992001046A1 - Laccase production by recombinant organisms - Google Patents

Laccase production by recombinant organisms Download PDF

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Publication number
WO1992001046A1
WO1992001046A1 PCT/FI1991/000214 FI9100214W WO9201046A1 WO 1992001046 A1 WO1992001046 A1 WO 1992001046A1 FI 9100214 W FI9100214 W FI 9100214W WO 9201046 A1 WO9201046 A1 WO 9201046A1
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Prior art keywords
laccase
according
enzyme
recombinant
trichoderma
Prior art date
Application number
PCT/FI1991/000214
Other languages
French (fr)
Inventor
Markku Saloheimo
Marja-Leena Niku-Paavola
Merja Penttilä
Jonathan Knowles
Anne Kantelinen
Original Assignee
Valtion Teknillinen Tutkimuskeskus
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Priority to FI903443 priority Critical
Priority to FI903443A priority patent/FI903443A/en
Application filed by Valtion Teknillinen Tutkimuskeskus filed Critical Valtion Teknillinen Tutkimuskeskus
Publication of WO1992001046A1 publication Critical patent/WO1992001046A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y110/00Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002Laccase (1.10.3.2)
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0055Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061Laccase (1.10.3.2)

Abstract

This invention relates to production of a ligninolytic oxidative enzyme, laccase, by recombinant DNA technology in suitable hosts. For that purpose the laccase gene is isolated from a suitable donor organism, the gene is transformed to a suitable host, the recombinant organism obtained is cultivated in conditions allowing expression of laccase and the laccase so produced is isolated. Laccase can be used to improve for instance the pulping process due to the ability of the enzyme to degrade and modify lignin. A suitable donor organism for the invention is Phlebia. A preferable host for laccase expression for the purposes of the invention is the species Trichoderma. According to the invention also enzyme preparates can be produced by Trichoderma which besides laccase include also other enzymes useful in for instance pulp and paper processing.

Description

Laccase production by recombinant organisms

Background of the invention

A. Field of the invention

The present invention relates to the gene encoding a ligninolytic oxidative enzyme, laccase, recombinant DNA molecules, containing said gene and microorganism hosts transformed with said recombinant DNA molecules. The invention also provides a process for production of lacca¬ se enzyme by recombinant DNA technology. Laccase and enzy¬ me preparations displaying laccase activity can be used for instance in the processes involved in pulp and paper manufacture and in treatment of waste waters.

B. Background art

Biotechnology as low-energy-demanding, non-corrosive and non-polluting is a repeatedly stated alternative for che¬ mical technology. The techniques applied presently in pulp and paper industry are due to the chemicals used hazardous for the environment.

In the wood tissue cellulose fibrils are held together by a matrix of hemicellulose and lignin. The conventional pulping process includes an energy demanding grinding step and the chemical steps where the cellulosic fibers are li¬ berated from their encasing matrix so that they can asso¬ ciate with one another, yielding strength in the final product.

Cellulose comprises 35-45 % of the dry weight of wood and is a linear polymer of glucose monomers coupled by β-1,4 bonds. The hemicelluloses are branched polymers composed of pentose (5-carbon) monomers, normally xylose and arabi- nose; and hexose (6-carbon) monomers, consisting of gluco¬ se, galactose, mannose and substituted uronic acid. Lignin is an extremely complex polymer formed of substituted phe- nylpropane units combined by a variety of bonds, of which the β-0-4 inter-unit linkage is the most prevalent. Lignin constitutes 15-30 % of dry wood weight.

The chemicals commonly used in pulping consist of sodium sulfide and sodium hydroxide (sulfate, Kraft process) or sulfur dioxide and calcium oxide (sulfite process). After this chemical treatment e.g. Kraft pulp still contains 5-8 % of residual modified lignin. To obtain a pulp of high brightness and brightness stability the residual lignin is removed by using strong oxidizing agents, e.g. chlorine, hydrogen peroxide, oxygen and ozone. The bleach¬ ing effluents contain as reaction products chlorinated phenolics which are toxic, carcinogenic and mutagenic.

Wood-rotting fungi can be classified according to their mode of action in wood decay. Soft-rot fungi attack wood under conditions of high humidity and cause softening of wood tissue and significant weight loss (Buswell and Odier, 1987). Wood polysaccharides are attacked preferen¬ tially although slow lignin degradation has also been re¬ ported. Brown-rot fungi colonize wood by advancing longitu¬ dinally through the lumina, removing mainly polysacchari¬ des and leaving behind a brown, modified lignin residue. Compared with white-rot fungi, brown-rot types are less capable of lignin degradation, the main modification being demethylation. White-rot fungi form a heterogeneous group consisting mainly of basidiomycetes. They are able to de¬ grade all the major components of wood and in particular lignin. Among white-rot fungi several strains have been isolated that degrade lignin very efficiently.

Lignin is highly resistant to biological attack; only hig¬ her order fungi degrade it completely (reviewed by Kirk & Farrell, 1987). The major degraders of "fully lignified" tissues (lignin > 20 %) are the Basidiomycetes that cause the white-rot type of wood decay. The lignin degradation system of white-rot fungi is induced during secondary metabolism. In laboratory cultivations the system is switched on under carbon, nitrogen or sulphur starvation of the fungus (Kirk and Shimada, 1985).

Lignin degradation mediated by white-rot fungi is thought to be a non-specific oxidative process (Kirk and Farrell, 1987). The initial step in this process is the removal of an electron from a subunit of lignin by a ligninolytic enzyme. The fungal enzyme can be either peroxidases that use H202 as their electron acceptor, or oxidases (laccases) that use 02 for this purpose. The ability of laccases to oxidize lignin model compounds with phenolic hydroxyl groups suggests that they are indeed likely to have a role in lignin degradation (Ishihara, 1980).

The most extensively investigated white-rot fungus is Phanerochaete chrysosporium Burds (Tien & Kirk, 1983). P. chrysosporium produces multiple extracellular heme- containing peroxidases (eg. Kirk et al. , 1986a; Leisola et al. , 1985). Using H202 as an electron acceptor, these enzymes oxidize phenolic lignin subunits by capturing one electron to create cation radicals. These radicals serve as intermediates leading to the later steps of lignin degradation (Kersten et al. , 1985; Hammel et al. , 1986).

The purified separate enzymes of P. chrysosporium are able to modify monomeric and dimeric structural components of lignin. Modifications include cα-cβ cleavage and cleavage of the β-0-4 ether bond, the basis of the so-called "depolymerizing" reactions. Other characteristic reactions include aromatic ring opening, demethoxylation, hydroxyla- tion, decarboxylation and phenol coupling reactions (Palmer et al. , 1987; Kirk and Farrell, 1987; Buswell and Odier, 1987; Tien, 1987).

As 80 % of intersubunit bonds involve linkages to cα or cβ carbons and up to 48 % of linkages in soft wood lignin are β-0-4-type, a tremendous interest arose towards the above mentioned ligninolytic enzymes. Patent applications (PCT W087/00550; PCT WO87/00564) have been filed introducing the application of separate purified ligninolytic enzymes and their mixtures for bleaching of Kraft pulp, for deco- lorization of pulp effluents, and for enchancing the strength properties and brightness stability of mechanical pulps. The disadvantage was the low amount of ligninoly¬ tic enzymes in the extracellular fluid (0.01 g/1) produced by the fungus. Better production strains of Phanerochaete, have been screened by conventional mutagenesis (Kirk et al. , 1986a, b) and also recombinant DNA technology has been applied (Farrell, 1987; Tien & Tu, 1987; Zhang et al. , 1986). The mutants obtained produced less than ten fold more enzyme than the parent strain. Recombinant peroxidase has until now been expressed only in bacteria in small amounts in denatured form, and an active form could be obtained only be refolding in the presence of heme (Farrell, 1987). Thus, the amounts of ligninolytic enzymes produced are still too low for practical applica¬ tions.

Recent information of the inability of the individual ext¬ racellular enzymes of Phanerochaete to depolymerize macro- molecular lignin, in the form as it is present in wood or pulps (Schoemaker & Leisola, 1990) has resulted in search for new enzymes and screening of other white-rot fungi. Early screening experiments in Finland (Hatakka & Uusi- Rauva, 1983) showed that the white-rot fungus Phlebia ra- diata degraded under certain circumstances wood lignin even more effectively than some of the Phanerochaete strains. Phlebia produces ligninolytic enzymes which are quite simi¬ lar to those of Phanerochaete (Niku-Paavola et al. , 1988; Saloheimo et al. , 1989; Karhunen et al. , 1990a,b). Unlike by Phlebia, however, laccase is not reported to be produ¬ ced by Phanerochaete. Phlebia radiata produces lignin peroxidase and oxidase activities to its culture fluids in conditions described by Kantelineή et al. (1989). Three lignin peroxidases (MW 42-45 kDa) and one Mn-dependent peroxidase (MW 49 kDa) of unspecific peroxidase type (EC 1.11.1) and one oxidase (MW 64 kDa) of laccase type (EC 1.10.3.2) have been purified from Phlebia culture filtrates and characterized (Niku-Paavola et al. , 1988; Karhunen et al. , 1990a,b) .

The characterization of the Phlebia laccase showed that it differs from the fungal laccases described so far in con¬ taining a novel combination of electron carriers as its prosthetic groups. The EPR spectrum exhibits features of type 1 and type 2 copper atoms (Karhunen et al. , 1990b). Most of fungal laccases e.g. Coriolus laccase have four copper atoms including also the type 3 binuclear copper pair (Malkin and Malmstrδm 1970). Moreover, this Phlebia enzyme is the first fungal laccase for which the presence of the cofactor PQQ (pyrroloquinoline quinone) has been proposed. In the enzymatic reaction four molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. Similar reaction is catalysed also by the Coriolus laccase (Sarkanen 1971) but the reaction products, dilignols, have different structu¬ res compared to those produced by the Phlebia laccase (Karhunen et al. , 1990b).

As mentioned above, the development of strains producing ligninolytic enzymes has not been successful using genetic engineering. Production of Phanerochaete peroxidase has not been obtained in yeast or in other filamentous fungi. The production levels obtained in bacteria (EP-patent application No. 87810516.2) are low because the enzyme is produced in inactive form and has to be renatured to re¬ gain it's activity. It is also noteworthy that the produc¬ tion of lignin peroxidase LIII of Phlebia radiata in the filamentous fungus Trichoderma reesei has been unsuccess¬ ful although LIII specific RNA was clearly detected (Saloheimo et al. , 1989). It thus seems that lignin per¬ oxidases cannot be expressed in other fungi. The only gene encoding a fungal laccase which has been reported to have " been isolated so far is the Neurospora crassa, a non-wood rotting fungus, laccase (German and Lerch, 1986; German et al. , 1988) but this gene has not been expressed in any other host.

Production levels of homologous enzymes can usually be in¬ creased by genetic engineering as shown for instance for the fungal enzymes α-amylase (EP-patent application No. 87103806.3) and for cellulases (Harkki et al. , 1990). This is possible if the corresponding genes have been isolated and techniques of genetic engineering have been developed for the organism. Using in principle the same techniques as for the production of homologous proteins it is also possible to express heterologous genes, such as those en¬ coding ligninolytic enzymes in a heterologous host provi¬ ded that the enzyme would be produced in enzymatically ac¬ tive form, would not be degraded by host cell proteases and preferably also in the case of an extracellular enzyme such as the ligninolytic enzymes, would be secreted by the heterologous host. These properties depend on the type of a protein expressed and has not been shown to be possible for lignin peroxidases.

Production in a heterologous host would be beneficial as the problem of contamination with other enzymes, for in¬ stance other ligninolytic enzymes produced by the fungus is avoided, when that is not desired. Furthermore, the production levels of ligninolytic enzymes are extremely low in white-rot fungi such as Phlebia and their expres¬ sion is regulated. Using a heterologous host for produc¬ tion, it could be possible to increase the production le¬ vels, if the heterologous promoter used is stronger than the natural promoter of the gene encoding a ligninolytic enzyme. The promoter can be also differently regulated than the endogenous one. This makes possible production of ligninolytic enzymes in completely novel conditions. It is also possible to use as hosts strains which already produ¬ ce useful enzymes and thus to produce an enzyme mixture, including also a ligninolytic enzyme, suited for the app¬ lication in question. Furthermore, as a host can be used a strain in which the expression of possible harmful pro¬ teins has been inactivated. This all would be possible if the ligninolytic enzyme in question can be expressed in the heterologous host in enzy atically active form.

Summary of the invention

This invention relates to DNA sequences encoding laccase enzyme and the production of laccase in suitable hosts. In the process the DNA sequences coding for laccase enzyme are isolated from a suitable donor organism; the DNA se¬ quences coding for laccase enzyme, as such or coupled to regulatory sequences, are transformed to a suitable host; the recombinant organism obtained is cultivated in growth conditions allowing expression of laccase; and the laccase enzyme is isolated from the culture broth or the crude en¬ zyme preparate produced by the organism is recovered.

Laccase can be used for instance to improve the processes involved in pulp and paper manufacture or in waste water treatment due to the ability of the enzyme to degrade and modify lignin and lignin-derived compounds. Suitable donor organisms for the purposes of this inven¬ tion are e.g. the Phlebia, Polyporus, Pleurotus, Phelli- nus, Coriolus, Panus, Lentinus, Bjerkandera, Agaricus and Schizophyllum species.

Fungi to be used as hosts for the purposes of the inven¬ tion are e.g. the fungi from genera such as Agaricus, Coriolus, Phanerochaete, Phlebia and Schizophyllum and other such as Aspergillus, Neurospora, Saccharomyces and Trichoderma. Especially preferred is Trichoderma.

Trichoderma reesei mutants have been described which lack some or all of the cellulases the fungus normally produ¬ ces. When these mutants are used as hosts according to this invention recombinant fungi are obtained which besi¬ des laccase do not produce unwanted cellulolytic enzymes but produce e.g. hemicellulases. So enzyme preparations useful in pulp and paper processing are obtained.

The present invention thus describes the isolation of the laccase gene from Phlebia radiata, characterization of the gene, as well as the transfer and expression of the gene in Trichoderma reesei. Recombinant DNA molecules, specifi¬ cally fungal vectors, comprising the said laccase gene and being suitable for transformation into the Trichoderma host, as well as the recombinant fungal hosts obtained are also described.

Thus the special objectives of this invention are to pro¬ vide

(1) the specific laccase gene of Phlebia radiata;

(2) specific cloning vectors for Trichoderma containing the laccase gene;

(3) filamentous fungal strains transformed with said vectors, which strains thus are able to produce lacca¬ se or laccase and other useful enzymes; and (4) a process for producing said laccase.

(5) a method for treating soluble waste lignin with said laccase.

Brief description of the drawings

Figs, la and lb describe the vectors pMS27 and pMS30 used in expression of laccase in Trichoderma.

Fig. 2 describes the laccase activity produced by several Trichoderma cotransformants assayed on plates. Negative control strains are shown on the left.

Fig. 3. Absorption spectra of soluble fractions of Kraft lignin treated with laccase. Control Kraft lignin ( ), lignin incubated with 7 nkat ( ) and 15 nkat (- « . ) of purified laccase produced by Trichoderma.

Detailed description of the invention

This invention relates to the cloning of genes coding for laccase.

Laccases (EC 1.10.3.2) are enzymes which catalyse the re¬ moval of electrons from phenolic compounds using 02 as an electron acceptor. Genes encoding laccase can be isolated from any laccase producing organism, such as from the wood-rotting filamentous fungi Coriolus, Polyporus, Pleurotus, Phellinus, Panus, Lentinus, Bjerkandera, Agaricus, Schizophyllum, and Phlebia. Preferable donor or¬ ganisms are Phlebia species, most preferably Phlebia radiata.

Genetic sequences which are capable of encoding a poly- peptide displaying laccase activity are derived from a variety of sources, such as from gene libraries prepared by techniques known in the art. These sources can include genomic DNA, cDNA, synthetic DNA, and combinations there¬ of.

Libraries containing clones encoding a laccase protein may be screened and a clone to the desired protein identified by any means which specifically selects for that protein's DNA such as, for example, a) by hybridization with an app¬ ropriate nucleic acid probe(s) containing a sequence spe¬ cific for the DNA of this protein, or b) by hybridization- selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the clones genetic sequences are themselves ca¬ pable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone.

Oligonucleotide probes specific for the laccase proteins which can be used to identify clones to such protein can be designed from knowledge of the amino acid sequence of the protein. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D. , In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357). Using the "codon usage rules" of Lathe, a single oligonucleotide sequence, or a set of oligonucleotide se¬ quences, that contain a theoretical "most probable" nuc- leotide sequence capable of encoding the protein's sequen¬ ce can be identified.

It is also noteworthy that the DNA sequence or parts of the DNA sequence encoding laccase, such as the sequence of the Phlebia laccase presented in this invention can be used to isolate laccase genes from other organisms by hybridization in non-stringent conditions using methods known in the art. In an alternative way of cloning a gene, a library is pre¬ pared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing a desired protein, into an expression vector. The library is then screened for members which express the protein, for example, by screening the library with antibodies to the protein as described in this invention.

Genes encoding laccase activity can be highly variable in nucleotide sequence as the regions in the protein respon¬ sible for the enzymatic reaction and for instance for coupling of the copper ions needed in activity, or the co- factors such as PQQ, are formed by only a few amino acid residues in the protein. Thus, the overall nucleotide sequence of the gene and consequently the amino acid sequence of a polypeptide can be different but still display laccase activity and be able to bind the cofac- tors. Due to the degeneracy of the genetic code even the laccases with the same aminoacid sequence can be encoded by genes having different nucleotide sequence. These can occur naturally or can be synthetized using methods known in the art. Furthermore, the gene and consequently the polypeptide displaying laccase activity can be accomp¬ lished by using only a part of the naturally occurring sequence or by combining only the parts responsible for activity, e.g. functional parts, from one or from several different enzymes.

The cloned DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in as¬ sociation with the native 5' promoter region of the DNA genetic sequences and/or with the 3 ' transcriptional ter¬ mination region. To the extent that the heterologous host such as Trichoderma can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5' and/or 3' non-transcribed regions of the native gene, and/or, the 5' and/or 3' non-translated regions of the mRNA, may be retained and employed for transcriptional and transla- tional regulation.

The precise nature of the regulatory regions needed for gene expression vary between species or cell types and production conditions used, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of tran¬ scription and translation respectively. A wide variety of transcriptional and translational regulatory sequences can be employed, since for instance filamentous fungi general¬ ly recognize transcriptional control sequences, for examp¬ le, those of other filamentous fungi.

Transcriptional initiation regulatory signals can be se¬ lected which allow for repression or activation, so that expression of the operably linked genes can be modulated.

Preferably, such regulatory sequences are homologous to the host such as to Trichoderma. A regulatory region, and especially a promoter, may be modified to contain only those sequence elements needed for expression and/or to retain a region which is responsible for high expression levels. Enhancer sequences may be introduced concurrently with the gene of interest as a separate DNA element but operably-linked to the gene. According to this invention, the genetic constructs which encode laccase enzymes which are desirable for pulp and paper processing purposes may be introduced into the genome of Trichoderma and expres¬ sion can be achieved by using strong promoters such as cbhl and, if desired, additional or modified regulatory regions such as, for example, enhancer sequences.

Transcriptional regulatory elements of other genes may be used where it is desired not to use the cbhl elements. For example a vector construction comprising the 3-phosphoglycerate kinase gene (pgk) (Vanhanen et al. , 1989) transcriptional regulatory regions may be used as 3-phosphoglycerate kinase, a key enzyme for ATP generation by glycolysis, is expressed in the presence of glucose un¬ der which conditions the synthesis of cellulases is re¬ pressed. Also other promoters functional in glucose medium can be used.

In a preferred embodiment, genetically stable transfor- mants of Trichoderma are constructed whereby a laccase protein's DNA is integrated into the host chromosome. The coding sequence for the desired protein may be from any source. Such integration may occur de novo within the cell or, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, DNA elements which promote integration of DNA sequences into a certain locus in chromosomes.

Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide resistance, towards phleo- mycin (Durand et al. , 1988), or allow transformants to be selected on acetamide (PenttilS et al.. , 1987). Complemen¬ tation of ArgB-auxotrophic strains can also be accomp¬ lished (PenttilS et al. , 1987) as well as TrpC- or PyrG- strains with the corresponding Asperqillus genes (our unpublished results; Gruber et al. , 1989; Berges et al. , 1989). The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or in¬ troduced into the same cell by cotransformation.

The DNA constructions prepared according to this inventi¬ on can be used to transform any Trichoderma strain. Such strains include, for example, T. reesei strains QM9414 (ATCC 26921), RUT-C-30 (ATCC 56765), and highly productive mutants like VTT-D-79125, which is a descendant of QM9414 (Nevalainen 1985, Technical Research Centre of Finland Publications 26, (1985), Espoo, Finland). The transforma¬ tion of Trichoderma may be performed by any technique known in the art and especially by the technique taught in EP-patent application 244 233.

The effectiveness of the expression of the desired gene can be dependent both on the number of copies of the desired gene integrated to the genome of Trichoderma and on the location of integration of the gene in the genome. The use of a linear DNA helps in directing the integration into a homologous locus. In a preferred embodiment, the integration of a desired gene is directed into the Trichoderma cbhl locus.

It is also possible to enrich Trichoderma hosts for an en¬ zyme such as laccase, whose activity is desirable for pulp and paper processing purposes by inactivating or eliminat¬ ing at least one cellulase enzyme by genetic engineering. Since the majority of the secreted proteins of Trichoderma may be the cellulase activity encoded by the gene cbhl, (the cellobiohydrolase, CBHI, protein), by constructing Trichoderma hosts in which the cbhl gene is mutated to an inactive form, the relative percent of the remaining pro¬ teins secreted by Trichoderma in the culture medium may be increased. Such cbhl~strains have been described (Harkki et al. , 1990) and can be used as host strains. The laccase gene is inserted preferably into the cbhl locus such that expression of the desired gene is operably linked to the strong cbhl promoter. A cassette comprising the laccase gene already operably linked to the homologous cbhl promo¬ ter and the cbhl terminator can also be inserted into the cbhl locus so that in integration the cbhl coding region is replaced by the laccase gene. Increased amounts of the desired heterologous enzyme are also achieved when Trichoderma's cellulase producing capacity is lowered in general, even if the heterologous gene is not inserted in¬ to the cbhl locus.

In addition, when it is desired to reduce residual lignin which is present in unbleached cellulose pulp, an enzyme preparation of the invention which contains high levels of hemicellulases and lignin degrading enzymes and low levels of cellulolytic enzymes is useful.

In the hosts of the invention, any one, some, or all of the cellulolytic enzymes can be eliminated, reduced, inac¬ tivated, or repressed by methods known in the art so as to result in the host's partial or complete inability to degrade cellulose to glucose.

For example, inactivation of genes coding for cellulolytic activities may be performed as described in European Pa¬ tent Applications EP 137,280 and EP 244,234 or by Harkki et al. (1990) .

Homologous genes which it is desirable to inactivate or delete include, for example, the cellulase genes cbhl, cbh2, egll, eg!2 (which encode the proteins cellobiohydro- lase I, cellobiohydrolase II, endoglucanase I and endoglu- canase II) or combinations of these genes. Cloning of these cellulase genes has been described (Shoemaker et al. , (1983)); (PenttilS, M. , et al. , (1986)); (Van Arsdel, J.N.V. , et al. , (1987)); (Teeri et al. , 1987a)); (Chen, CM., et al. , (1987)); (Saloheimo, M., et al. , (1988)).

Eliminating the activity of any of these genes will result in a host which is partially or completely deficient in its ability to degrade cellulose to glucose. The fungal strain can be also mutagenized by conventional means, by radiation or by chemicals, to inactivate the expression of e.g. cellulase genes as described by Neva- lainen and Palva (1978) and these strains can be used as hosts to express the laccase gene. It is also possible, by conventional mutagenesis and screening methods, to obtain strains with increased ability to produce laccase.

Examples of other desired pulp and paper processing enzy¬ mes which the fungal host can produce together with lacca¬ se include, for example, hemicellulases and pectin- degrading enzymes which several fungi such as Trichoderma, are inherently capable of producing.

Trichoderma is advantageous as a host because it naturally produces a wide spectrum of enzymes useful in treatment of lignocellulosic material, the proportions of which can be manipulated by genetic engineering for different applica¬ tions to provide enzyme preparations tailored for those purposes.

Trichoderma is an especially useful and practical host for the synthesis of the enzyme preparations of the invention because Trichoderma is capable of secreting protein at large amounts, for example, concentrations as much as 40 g/1 culture fluid have been reported; the homologous Trichoderma cbhl promoter provides a very convenient pro¬ moter for expression of genes-of-interest because it is a strong, single copy promoter which normally directs the synthesis of up to 60 % of the secreted protein from the Trichoderma host; the transformation system is highly ver¬ satile and can be adapted for any gene of interest; the Trichoderma host provides an "animal cell type" high man- nose glycosylation pattern; and culture of Trichoderma is supported by previous extensive experience in industrial scale fermentation techniques. It is noteworthy that once the laccase gene has been iso¬ lated it can be reintroduced to the original host and thus obtain recombinant strains possibly producing increased levels of laccase. Improvement of homologous gene expres¬ sion has been shown for α-amylase (application 87103806.3) and for cellulases (Harkki et al. , 1990), for instance. The gene can be also introduced to other Basiodiomycete fungi, to the white-rot fungi already producing ligninoly¬ tic enzymes such as to Phanerochaete using the methods known in the art, such as the transformation method de¬ scribed for Phanerochaete (Alic et aJL. , 1989).

It is clear that the laccase enzyme can be produced in ot¬ her fungi already capable of producing laccases such as Coriolus, Agaricus and Neurospora. Furthermore, we show in this invention, that unlike peroxidases, laccases can be produced in fungi belonging to completely other fami¬ lies, such as in Trichoderma which belongs to Fungi Imperfect! and not naturally classified to fungi producing ligninolytic enzymes. Trichoderma has been reported to possess a poorly defined sexual stage, the fungus Hypocrea which belongs to Ascomycetes, a taxu clearly different to Basiodiomycetes which most of the fungi producing lignino¬ lytic enzymes belong to, such as the species Phlebia. Thus, taxonomically unrelated fungi are capable of produc¬ ing active laccase enzyme and secrete it to the fungal culture medium and obviously also incorporate the cofac- tors needed for activity into the enzyme, as shown here by secretion of active Phlebia laccase by Trichoderma.

In a preferred embodiment, laccase is secreted into the surrounding medium due to the presence of a homologous secretion signal sequence. If a desired protein does not possess its own signal sequence, or if such signal sequen¬ ce does not function well in Trichoderma or in the organism of question, the desired coding sequence may be linked to

Figure imgf000020_0001

18

any signal sequence which will allow secretion of the pro¬ tein from a Trichoderma or other host, for example, the signal sequence of the Trichoderma cellobiohydrolase I protein. Such signal sequences may be designed with speci¬ fic protease sites such that the signal peptide sequence is amenable to subsequent removal.

The host cells, such as Trichoderma, may be cultivated and the desired enzymes produced by cultivating the host strain having the desired properties under any conditions which allow expression of the desired enzymes. The crude enzyme preparate produced by the organism is recovered or if preferred it can be partially purified. For certain applications pure laccase is preferred and can be obtained using the methods described (Niku-Paavola et al. , 1988).

The enzyme preparates obtained may be used in treatment of lignocellulosic material in paper and pulp industry. In pulp and paper industry laccase could facilitate the pro¬ duction of mechanical and chemical pulp. Laccase modifies lignin by making it more hydrophilic. Thus the separation of cellulose fiber from lignin could be easier after laccase treatment in mechanical pulping. Further the solu¬ bility of lignin would be increased by laccase to benefit chemical pulping either before or after cooking. Laccase enzyme has been shown to be effective in degradation of native-type wood-powder lignin and polymerization of so¬ luble black-liquor lignin. Laccase also improved the bleachability of pine sulphate pulp when used together with hemicellulases (Kantelinen, 1989). Decolorization of pulp mill, cotton mill hydroxide and cotton mill sulfide effluents by laccase has been demonstrated (Davis and Burns, 1990).

Laccase catalyses the polymerization of compounds contai¬ ning free phenolic hydroxyl group. In pulp and paper in- dustry laccase could facilitate the elimination of pheno¬ lic oligomers liberated from pulp by other ligninolytic enzymes, as precipitating catalyst. Laccase could decrease the toxicity of bleaching effluents and other pollutants by precipitation and facilitate their separation. Laccase has been shown to directly dechlorinate chlorophenolic compounds present in bleaching waste waters (Roy-Arcand and Archibald, 1991).

Example l

Isolation of the laccase gene of Phlebia radiata

Phlebia radiata strain ATCC 64658 was grown as described (Saloheimo et al. , 1989) and RNA was isolated according to Chirgwin et al. (1979). Poly A+ RNA was isolated from the total RNA by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972) and cDNA synthesis was carried out according to Teeri e_t a_l. , (1987b). The cDNA was cloned into gtll vector (Young and Davies, 1983) at the EcoRI site using E. coli Y1090 (Young and Davies, 1983) as a propagation host. The cDNA bank was screened with laccase specific antibodies (Niku-Paavola et al. , 1988) as described earlier (Saloheimo et al. , 1989). A positively reacting clone of 800 bp was isolated which was further used as a probe in isolation of a longer cDNA clone of 1.5 kb. This cDNA was cloned into Bluescribe Ml3+ (Vector Cloning Sys¬ tems, San Diego, USA) yielding the plasmid pMS20.

On the basis of the protein data it was known that this sequence was too short to code for the corresponding lac¬ case enzyme. Therefore a chromosomal DNA bank was con¬ structed. Chromosomal DNA of Phlebia was isolated accord¬ ing to Raeder and Broda (1985). This total DNA was cut with Mbol and ligated to the EMBL3 vector (Kaiser and Murray, 1985) cut with BamHI and EcoRI. The laccase cDNA clone was used as a probe to isolate the chromosomal copy of the gene. The chromosomal gene was transferred into a Bluescribe Ml3+ vector as a 1.6kb Sad (plasmid pMS25) and as a 2.0kb Asp718 (plasmid pMS24) fragment and the 5' re¬ gion of the gene was sequenced. Based on this sequence two primers 5 ' CCTCTCCAGTCTCCAAGCA, 5 'GCGAAGACCGTCACAGT (SEQ ID NO. 1 and SEQ ID NO. 2) were designed and used in a PCR reaction to isolate the missing 5' end of the cDNA from cDNA prepared from mRNA induced for laccase expres¬ sion. This fragment was cloned into Bluescribe Ml3+ vector (yielding plasmid pMS28). The sequence of the laccase cDNA and the chromosomal gene were determined from these plas¬ mid clones using standard methods (SEQ ID NO. 3).

Example 2

Construction of vectors for expression of laccase in

Trichoderma

The 5' end of the chromosomal laccase gene in pMS25 was transferred to pUClδ (Norrander et al. , 1983) plasmid as an Asp718-BamHI fragment. This fragment was released from the pUC18 vector by digesting with Asp718 and ligated with pMS24 vector digested with Asp718. This ligation yielded plasmid pMS26 that has the whole chromosomal laccase gene from the BamHI site 24 bp upstream from the initiation co¬ don to the Asp718 site about 250 bp downstream from the termination codon. Plasmid pMS26 was digested with EcoRI and Sphl and a 2.6 kb fragment containing the laccase gene was isolated from an agarose gel and treated with Klenow DNA polymerase to yield blunt ends. The expression vector pAMHHO (Saloheimo et al. , 1989) was digested with SacII and Ndel and treated with SI nuclease, Klenow DNA polyme¬ rase and calf intestinal alkaline phosphatase. The DNA fragment containing the chromosomal laccase gene was liga¬ ted with the prepared expression vector, resulting in the plasmid pMS27 (Fig. la). The 5' end of the laccase cDNA was cloned from pMS28 into pMS20 as an Asp718 fragment. The resulting plasmid pMS29 contains the full-length laccase cDNA. This plasmid was digested with EcoRI and Sphl and the cDNA fragment was isolated from an agarose gel and treated with the Klenow polymerase. The expression vector used for the laccase cDNA was pAPH120. It is essentially pAMHHO (Saloheimo et al. , 1989) with the phleomycin resistance cassette from the plasmid pAN8-l (Mattern et al. , 1987) inserted to the EcoRI site as an EcoRI-Ndel fragment. pAPH120 was digested with SacII and Ndel and treated with SI nuclease, Klenow DNA polymerase and calf intestinal alkaline phosphatase. The fragment containing the full-length laccase cDNA was ligated with the prepared vector pAPH120 yielding the plasmid pMS30 (Fig. lb).

Example 3

Transformation of Trichoderma, purification of the laccase producing clones and their analysis.

Trichoderma reesei strain RUT-C-30 (Montenecourt and Eve- leigh, 1979) was transformed essentially as described (PenttilS et al. , 1987) using 5 yg of the plasmid p3SR2 (Hynes et al. , 1983) together with 15 μg of the plasmid pMS27 or pMS30. Before the transformations the expression cassettes were released from pMS27 and pMS30 by digesting with EcoRI and Sphl. The Amd+ transformants obtained were streaked twice onto asetamide plates and thereafter their laccase production was tested by a plate assay: the trans¬ formants were inoculated as patches on minimal medium pla¬ tes (PenttilS et aj.. , 1987) containing 2% cellobiose as carbon source, sophorose solution (20 mg/ml) was pipetted onto the fungal inoculum, and the plates were incubated at 28°C for two days whereafter ABTS-solution (10 mg 2,2'- azinodi-[3-ethylbenzthiazoline sulfonate]/ml) was pipetted onto the colonies. Laccase producing colonies could be distinguished by their greenish colour (Fig. 2). Spore suspensions were made from the clones showing laccase ac¬ tivity and the spores were plated onto Potato Dextrose agar (Difco) to obtain single spore cultures. These were tested for laccase activity by a plate assay as described above and spore cultures were prepared from these purified active clones.

DNA was isolated from some of the laccase producing clones as described earlier (Raeder and Broda, 1985), digested with appropriate restriction enzymes and analysed by Southern blotting using techniques known in the art to ve¬ rify the presence of the laccase gene in the transformants and its possible location in the cbhl locus. Total RNA was isolated from active clones grown as described below (example 4) with methods described in example l and the expression of the laccase gene in Trichoderma was analysed by Northern blotting with conventional methods using a laccase specific probe.

Example 4

Laccase activity produced by Trichoderma

Several of the laccase producing purified clones were grown in shake flasks to study the production of Phlebia laccase in Trichoderma. 50 ml of whey spent grain medium (Uusitalo et al. , 1990) was inoculated with 107 spores/ml and grown at 28°C with shaking (180 rpm) for four days. CuS04 (1.4 μg/1) was added to the culture medium in some experiments. The culture supernatant was separated from the mycelium by filtration. Enzyme activity test was car¬ ried out as described (Niku-Paavola et al., 1988) by using 100 μl of the culture supernatant. The clones showing po¬ sitive reaction on a plate assay (Example 3) secreted ac¬ tive laccase enzyme as shown by the activity assay. Table 1

STRAIN ACTIVITY nkat/ml

Rut-C-30 (nontransformed) 0 pMS27-3-l 1.06 pMS27-5-3 0.76 pMS27-10-3 0.84 pMS27-21-2 0.85

PMS30-4-2 0.99 pMS30-10-2 1.08

PMS30-13-3 1.23

PMS30-17-2 1.11

The secretion of laccase by Trichoderma was also verified by Western blotting from the clones showing highest acti¬ vity by conventional methods running culture supernatants on SDS-PAGE and treating the blotted filter with polyclo- nal laccase specific antibodies. Some of the transformants were fermented in a Chemap CF-3000 bioreactor (10 1) in a Solka floe cellulose/spent grain medium (Uusitalo et a_l. , 1990). The temperature of the cultivation was 33°C for the first 48 h and 29°C after that, pH was controlled to >4.5 by NH40H addition and pθ2 was adjusted to >30% by agita¬ tion speed with a constant aeration rate of 5 1/min. The cultivation was carried out for 100-150 h. The peak lacca¬ se activity levels (7-7.7 nkat/ml) secreted to the bio¬ reactor medium were 6 - 7 times higher than those from shake flask cultivations.

The recombinant enzyme was purified essentially according to (Niku-Paavola e_t al. , 1988) and shown to have similar molecular mass (64 kDa), antigenic properties and specific activity (395 nkat/mg) to those of laccase produced by Phlebia. All of the enzyme was secreted as active accord¬ ing to the enzymatic and immunological quantitation. The heterologous laccase was secreted in three isoelectric forms (pi 4.00, 3.88, 3.86), in addition to the one (pi 3.94) form produced by Phlebia.

Example 5

The use of laccase activity produced by Trichoderma for polymerization of soluble waste lignin

Kraft pine lignin (Indulin; AT West Wago, Covington, VA, U.S.A.) was treated in a mixture of 7-15 nkat of purified laccase enzyme produced by Trichoderma and 100 mg of sub¬ strate in 10 ml of 50 mM Na-acetate buffer, pH 5.5. After incubation for 20 h at room temperature the mixtures were centrifuged and the supernatants were subjected to an ab¬ sorption spectral analysis HPLC anlysis (Niku-Paavola et al. , 1988) and to GLC-MS (gas-liquid chromatography-mass spectroscopy) . For GLC-MS, the reaction products from the supernatant were extracted to chloroform at pH 2.0 and analysed as their trimethylsilyl derivatives. The solid residue of Kraft lignin was washed with distilled water and the dry weight was estimated.

Due to the action of the laccase produced by Trichoderma, the absorption spectrum of the buffer-soluble fraction of Kraft lignin changed (Fig. 3). HPLC analysis showed a complete elimination of the monomeric lignin-related com¬ pounds. GLC-MS analysis did not reveal any ring cleavage products created from these compounds. The enzyme appears to polymerize the lignin monomers thus affecting the ab¬ sorption spectrum as seen in Figure 3. Deposited microorganism

The following strain was deposited according to the Buda¬ pest Treaty at the Centraalbureau voor Schimmelcultures (CBS), Oosterstraat 1, NL-3740 AG Baarn, Netherlands.

Strain Deposition number Deposition date

Trichoderma reesei CBS 409.91

PMS27-21-2 July 2, 1991

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Sequence Listing

SEQ ID NO: 1

SEQUENCE TYPE: Nucleotide

SEQUENCE LENGTH: 20 bases

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: genomic DNA

ORIGINAL SOURCE ORGANISM: Phlebia radiata ATCC 64658

IMMEDIATE EXPERIMENTAL SOURCE: synthetic

PROPERTIES: laccase gene, 5' flanking area

5' TCC TCT CCA GTC TCC AAG CA

SEQ ID NO: 2

SEQUENCE TYPE: Nucleotide

SEQUENCE LENGTH: 17 bases

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: genomic DNA

ORIGINAL SOURCE ORGANISM: Phlebia radiata ATCC 64658

IMMEDIATE EXPERIMENTAL SOURCE: synthetic

PROPERTIES: laccase gene, corresponding to amino acids 137 - 142, lower strand

5* GCG AAG ACC GTC ACA GT SEQ ID NO : 3

SEQUENCE TYPE: Nucleotide with corresponding protein

SEQUENCE LENGTH: 2122 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: CDNA to mRNA, genomic RNA

ORIGINAL SOURCE ORGANISM: Phlebia radiata ATCC 64658

IMMEDIATE EXPERIMENTAL SOURCE: genomic library, CDNA library, PCR-product from cDNA

FEATURES: Exons 1-183, 234-302, 358-478, 536-649, 700-763,

811-909, 962-1118, 1178-1378, 1434-1490, 1544-2122

PROPERTIES: laccase (E.C 1.10.3.2) activity of the product

ATG CAC ACT TTC CTT CGC TCC ACG GCA CTC GTT GTG GCA GGC CTG 45 Met His Thr Phe Leu Arg Ser Thr Ala Leu Val Val Ala Gly Leu 1 5 10 15

TCT GCC CGC GCC CTT GCC AGC ATT GGG CCC GTT ACC GAC TTT CAC 90 Ser Ala Arg Ala Leu Ala Ser lie Gly Pro Val Thr Asp Phe His 20 25 30

ATC GTC AAC GCC GCC GTC TCT CCC GAT GGT TTC TCT CGC CAG GCT 135 lie Val Asn Ala Ala Val Ser Pro Asp Gly Phe Ser Arg Gin Ala 35 40 45

GTC CTG GCT GAG GGT GTC TTC CCT GGC CCG CTC ATC GCC GGC AAC 180 Val Leu Ala Glu Gly Val Phe Pro Gly Pro Leu lie Ala Gly Asn 50 55 60

AAG GTACTCCATT TCTCTCTCAT CCCCGGGATA TGCGCTGACG GT 225

Lys

CGGCACAG GGC GAC AAT TTC CAG ATC AAT GTC ATT GAC GAA TTG A 270

Gly Asp Asn Phe Gin lie Asn Val lie Asp Glu Leu T

65 70

CC AAC GCA ACT ATG TTG AAG ACT ACC ACT ATC GTCAGTTCAC TTG 315 hr Asn Ala Thr Met Leu Lys Thr Thr Thr lie 75 80

CCACTCC CCGTGTTCTC ACTCTGAATT AATCGTCTTT GGTAG CAC 360

His 85

TGG CAT GGC TTC TTC CAG CAC GGT ACA AAC TGG GCT GAT GGA CCG 405 Trp His Gly Phe Phe Gin His Gly Thr Asn Trp Ala Asp Gly Pro 90 95 100

GCA TTT ATC AAT CAA TGC CCC ATT GCG TCT GGT GAC TCG TTC CTG 450 Ala Phe lie Asn Gin Cys Pro lie Ala Ser Gly Asp Ser Phe Leu 105 110 115

TAC AAC TTC CAG GTG CCC GAC CAA GCT G GTATGTCCCA GTTTCCT 4S5 Tyr Asn Phe Gin Val Pro Asp Gin Ala 120

GTT GTTGGATAAT ACCAGCATCT TAATTATTAT GATACAG GT ACC 540

Gly Thr 125

TTC TGG TAT CAC AGC CAT CTC TCC ACG CAG TAC TGT GAC GGT CTT 585 Phe Trp Tyr His Ser His Leu Ser Thr Gin Tyr Cys Asp Gly Leu 130 135 140

CGC GGA CCT TTC GTA GTG TAC GAT CCT GCT GAC CCG TAC CTT GAC 630 Arg Gly Pro Phe Val Val Tyr Asp Pro Ala Asp Pro Tyr Leu Asp 145 150 155

CAG TAC GAC GTC GAT GAT G GTACGCAAGA GGCATTTATT GTTTTC 675

Gin Tyr Asp Val Asp Asp 160

GCTT ATATATTGAA CCCTGCTCAG AC AGC ACT GTT ATC ACT TTG G 720

Asp Ser Thr Val lie Thr Leu A 165 1 CG GAC TGG TAT CAC ACC GCC GCG AGA TTG GGG AGC CCT TTC CC GT 765 la Asp Trp Tyr His Thr Ala Ala Arg Leu Gly Ser Pro Phe Pro 70 175 180

AAGTACTC AGAGCTTGTC CATTGTCAAA TGCTCATTCC GCTGCAG 810

C GCT GCG GAT ACC ACC TTG ATC AAC GGC CTT GGA CGG TGC GGT GA 855 Ala Ala Asp Thr Thr Leu lie Asn Gly Leu Gly Arg Cys Gly Gl 185 190 195

A GCC GGC TGC CCG GTC TCT GAT CTT GCT GTC ATC TCG GTT ACT AA 900 u Ala Gly Cys Pro Val Ser Asp Leu Ala Val lie Ser Val Thr Ly 200 205 210

A GGC AAA CG GTATGTCGAT CGTTCAGTAG CATGTTAGTA TCTAAC 945 s Gly Lys Arg 215

ATTT TCTGCCTTCC AG C TAC CGT TTC CGC CTG GTT TCC ATC TCT T 990

Tyr Arg Phe Arg Leu Val Ser lie Ser C 220 225

GC GAT TCA TTC TTC ACA TTT AGC ATC GAT GGG CAC AGT CTG AAC G 1035 ys Asp Ser Phe Phe Thr Phe Ser lie Asp Gly His Ser Leu Asn V 230 235 240

TC ATT GAG GTT GAT GCT ACC AAC CAC CAG CCG TTG ACC GTC GAC G 1080 al lie Glu Val Asp Ala Thr Asn His Gin Pro Leu Thr Val Asp G 245 250 255

AG CTC ACT ATT TAT GCA GGC CAG CGC TAC TCC TTC ATC GTAAGTT 1125 lu Leu Thr lie Tyr Ala Gly Gin Arg Tyr Ser Phe lie 260 265

TCA AGTGTAACGT GACTCAGACT GAAGGTTATT GATGACATTT GT 1170

TACTTAG CTC ACG GCC GAC CAA GAC GTC GAT AAC TAC TGG ATC CG 1215 Leu Thr Ala Asp Gin Asp Val Asp Asn Tyr Trp lie Ar 270 275 280

A GCA AAC CCC GGT ATT GGC ATC ACC ACA GGC TTT GCG GGC GGT AT 1260 g Ala Asn Pro Gly He Gly He Thr Thr Gly Phe Ala Gly Gly II 285 290 295

C AAC TCT GCT ATT CTT CGT TAT GAT GGT GCC GAC GTG GTC GAG CC 1305 e Asn Ser Ala He Leu Arg Tyr Asp Gly Ala Asp Val Val Glu Pr 300 305 * 310

T ACA ACT ACT CAG GCG ACA AGT CCC GTT GTG CTG AGC GAG TCG AA 1350 o Thr Thr Thr Gin Ala Thr Ser Pro Val Val Leu Ser Glu Ser As 315 320 325

C TTG GCC CCA CTG ACC AAC GCT GCT GCT GTCAGTCTTA TCTTTTG 1395 n Leu Ala Pro Leu Thr Asn Ala Ala Ala 330 335

TGT ATTGCTTTGC T^GCGTCTAA CCGGTAGATC TTTAG CCT GGT C 1440

Pro Gly L TC CCG GAG GTT GGC GGT GTC GAC CTT GCC CTC AAC TTC AAC CTG A 1485 eu Pro Glu Val Gly Gly Val Asp Leu Ala Leu Asn Phe Asn Leu T 340 345 350

CC TTC GTGAGTACCC TGGCTGAATC GAGCGGATAT TTGTTGAACG 1530 hr Phe 355

CAGGGTTGTG CAG GAT GGC CCC TCC CTA AAA TTC CAA ATC AAC GG 1575 Asp Gly Pro Ser Leu Lys Phe Gin He Asn Gl 360 365

A GTC ACC TTC GTT CCG CCC ACC GTG CCC GTT CTT CTC CAA ATC CT 1620 y Val Thr Phe Val Pro Pro Thr Val Pro Val Leu Leu Gin He Le 370 375 380

C AGT GGT GCC CAG TCG GCT GCA GAC CTA CTG CCA TCC GGA AGC GT 1665 u Ser Gly Ala Gin Ser Ala Ala Asp Leu Leu Pro Ser Gly Ser Va 385 390 395

G TAC GCG CTG CCT TCG AAC GCG ACC ATC GAG CTG AGT CTG CCC GC 1710 1 Tyr Ala Leu Pro Ser Asn Ala Thr He Glu Leu Ser Leu Pro Al 400 405 410

C GGC GCA CTG GGC GGC CCG CAC CCC TTC CAC TTG CAC GGC CAC AC 1755 a Gly Ala Leu Gly Gly Pro His Pro Phe His Leu His Gly His Th 415 420 425

C TTC AGC GTC GTC CGT CCC GCT GGC TCC ACG ACG TAC AAC TAT GT 1800 r Phe Ser Val Val Arg Pro Ala Gly Ser Thr Thr Tyr Asn Tyr Va 430 435 440

C AAC CCC GTC CAG CGT GAC GTC GTG AGC ATT GGA AAC ACC GGC GA 1845 1 Asn Pro Val Gin Arg Asp Val Val Ser He Gly Asn Thr Gly As 445 450 455

C AAC GTC ACA ATC CGC TTC GAT ACT AAC AAC CCG GGC CCG TGG TT 1890 p Asn Val Thr He Arg Phe Asp Thr Asn Asn Pro Gly Pro Trp Ph 460 465 470

C CTC CAT TGC CAC ATT GAC TGG CAT CTC GAG GCT GCT TTG CCG TT 1935 e Leu His Cys His He Asp Trp His Leu Glu Ala Ala Leu Pro Le 475 480 485

G TCT TCG CTG AGG ACA TCC CTG ACG TTG CGT CCA TTA ACC CTG TC 1980 u Ser Ser Leu Arg Thr Ser Leu Thr Leu Arg Pro Leu Thr Leu Se 490 495 500

C CCC AGG ACT GGT CCA ACC TGT GCC CTA TCT ACA ACG CTC TGG AC 2025 r Pro Arg Thr Gly Pro Thr Cys Ala Leu Ser Thr Thr Leu Trp Th 505 510 515

G CAT CTG ATC ACT AGC GGA TTC GCA TCA ATC ATA CAG TGG ATG AT 2070 r His Leu He Thr Ser Gly Phe Ala Ser He He Gin Trp Met Me 520 525 530

G GGT GGT AAC GGA. CTA TTT GCA CCA CAT GCT CTT TCA TTT CTC GG "115 t Gly Gly Asn Gly Leu Phe Ala Pro His Ala Leu Ser Phe Leu Gl 535 540 545

G TCG CAG 2122 y Ser Gin 548

Claims

We claim :
1. A DNA sequence coding for laccase enzyme capable of re¬ moving electrons from phenolic compounds using 02 as the electron acceptor.
2. A DNA sequence according to claim 1 originating from any wood rotting fungus.
3. A DNA sequence according to claim 1 coding for a poly¬ peptide comprising essentially the following amino acid sequence
MetHisThrPheLeuArgSerThrAlaLeuValValAlaGlyLeuSerAlaArgAlaLeu AlaSerlleGlyProValThrAspPheHisIleValAsnAlaAlaValSerProAspGly PheSerArgGlnAlaValLeuAlaGluGlyValPheProGlyProLeuIleAlaGlyAsn LysGlyAspAsnPheGlnlleAsnVallleAspGluLeuThrAsnAlaThrMetLeuLys ThrThrThrlleHisTrpHisGlyPhePheGlnHisGlyThrAsnTrpAlaAspGlyPro AlaPhelleAsnGlnCysProIleAlaSerGlyAspSerPheLeuTyrAsnPheGlnVal ProAspGlnAlaGlyThrPheTrpTyrHisSerHisLeuSerThrGlnTyrCysAspGly LeuArgGlyProPheValValTyrAspProAlaAspProTyrLeuAspGlnTyrAspVal AspAspAspSerThrVal11eThrLeuAlaAspTrpTyrHisThrAlaAlaArgLeuGly SerProPheProAlaAlaAspThrThrLeuIleAsnGlyLeuGlyArgCysGlyGluAla GlyCysProValSerAspLeuAlaVallleSerValThrLysGlyLysArgTyrArgPhe ArgLeuValSerlleSerCysAspSerPhePheThrPheSerlleAspGlyHisSerLeu AsnVallleGluValAspAlaThrAsnHisGlnProLeuThrValAspGluLeuThrlle TyrAlaGlyGlnArgTyrSerPhelleLeuThrAlaAspGlnAspValAspAsnTyrTrp IleArgAlaAsnProGlylleGlylleThrThrGlyPheAlaGlyGlylleAsnSerAla IleLeuArgTyrAspGlyAlaAspValValGluProThrThrThrGlnAlaThrSerPro ValValLeuSerGluSerAsnLeuAlaProLeuThrAsnAlaAlaAlaProGlyLeuPro GluValGlyGlyValAspLeuAlaLeuAsnPheAsnLeuThrPheAspGlyProSerLeu LysPheGlnlleAsnGlyValThrPheValProProThrValProValLeuLeuGlnlle LeuSerGlyAlaGlnSerAlaAlaAspLeuLeuProSerGlySerValTyrAlaLeuPro SerAsnAlaThrlleGluLeuSerLeuProAlaGlyAlaLeuGlyGlyProHisProPhe HisLeuHisGlyHisThrPheSerValValArgProAlaGlySerThrThrTyrAsnTyr ValAsnProValGlnArgAspValValSerlleGlyAsnThrGlyAspAsnValThrlle ArgPheAspThrAsnAsnProGlyProTrpPheLeuHisCysHisIleAspTrpHisLeu GluAlaAlaLeuProLeuSerSerLeuArgThrSerLeuThrLeuArgProLeuThrLeu SerProArgThrGlyProThrCysAlaLeuSerThrThrLeuTrpThrHisLeuIleThr SerGlyPheAlaSerllelleGlnTrpMetMetGlyGlyAsnGlyLeuPheAlaProHis AlaLeuSerPheLeuGlySerGln or a fragment thereof wherein the said polypeptide or fragment displays laccase activity.
4. A DNA sequence according to claim 1 coding for a laccase having two copper ions.
5. A DNA sequence according to claim 1 coding for laccase originated from a group of white-rot fungi comprising Agaricus, Coriolus, Phanerochaete, Pleurotus, Polyporus, Phellinus, Panus, Lentinus, Schizophyllum, Bjerkandera, preferably Phlebia.
6. A DNA molecule comprising a DNA sequence according to any of the claims 1-5.
7. A DNA molecule according to claim 6, wherein the said DNA sequence is expressed under fungal regulatory regions.
8. A recombinant DNA molecule according to claim 7, wherein said fungal regulatory regions are selected from the group comprising the promoter regions and optionally also the terminators of the fungal glucoamylase, β- glucosidase, cellobiohydrolase, endoglucanase and hemi- cellulase genes and promoters functional in glucose medium, or functional parts thereof.
9. A recombinant DNA molecule according to claim 7, where¬ in regulatory regions are selected from the group compri¬ sing the promoters and optionally also the terminators of the cellobiohydrolase genes cbhl and cbh2 and the endoglu¬ canase genes egll and eg!2 of Trichoderma.
10. A recombinant DNA molecule according to claim 7 being the plasmid pMS27.
11. A recombinant DNA molecule according to claim 7 being the plasmid pMS30.
12. A recombinant organism transformed with the DNA sequence according to claim 1 and expressing laccase activity.
13. A recombinant organism according to claim 12 which is a fungus.
14. A recombinant organism according to claim 13 being a filamentous fungus selected from the group comprising Coriolus, Neurospora, Phlebia, Phanerochaete, Pleurotus, Polyporus, Phellinus, Panus, Lentinus, Schizophyllum, Bjerkandera, Aspergillus and Trichoderma species.
15. A recombinant filamentous fungus according to claim 14 being Trichoderma.
16. A recombinant filamentous fungus according to claim 15 being selected from the group comprising T. reesei strains pMS27-3-l, pMS27-5-3, pMS27-10-3, pMS27-21-2, pMS30-4-2, pMS30-10-2, pMS30-13-3 and pMS30-17-2.
17. A recombinant filamentous fungus according to claim 16 being T. reesei pMS27-21-2.
18. A recombinant filamentous fungus according to claim 15, being transformed with the DNA molecules according to any of the claims 6-11.
19. A process for the production of laccase enzyme in re¬ combinant hosts, which process comprises:
(a) isolating the DNA sequences coding for laccase en¬ zyme from a suitable donor organism;
(b) transforming the DNA sequences coding for laccase enzyme, as such or coupled to regulatory sequences, to a suitable host; and
(c) cultivating the recombinant organism in growth conditions allowing expression of laccase.
20. A process according to claim 19, wherein the donor or¬ ganism is Phlebia.
21. A process according to claim 19, wherein the host to be transformed is of genus Trichoderma.
22. A process for recovering the enzyme preparate secreted by the organism according to claim 12 and optionally also purifying the laccase enzyme.
23. Enzyme preparates or mixtures displaying laccase ac¬ tivity as produced by a recombinant organism according to claim 12.
24. Laccase as produced by a recombinant organism accord¬ ing to claim 12.
25. A method of treating waste waters containing phenolic compounds wherein said method comprises addition of the enzyme preparates of claims 23-24 to waste waters.
PCT/FI1991/000214 1990-07-06 1991-07-08 Laccase production by recombinant organisms WO1992001046A1 (en)

Priority Applications (2)

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FI903443 1990-07-06
FI903443A FI903443A (en) 1990-07-06 1990-07-06 Framstaellning of lackas through rekombinantorganismer.

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