The present invention relates to a method of transforming a fungus belonging to the class Zygomycetes with a recombinant expression vector by means of recombinant DNA technology.

Most recombinant DNA work has been performed in bacteria, such as Eschericia coli, and therefore most efforts to transfer the knowledge developed by this basic research to areas of industrial utility have been centered around attempts to achieve industrial production of gene products expressed in E. coli or other bacteria such as Bacillus subtilis and, in some cases, in yeasts, principally Saccharomyces cerevisiae. However, the use of these organisms has certain draw¬ backs, principally because of difficulties in achieving expression of eucaryotic genes in these organisms, of maintaining the vector plas- mids in the cells, and/or because of the problem of recovering the expressed gene product from the culture.

It has now been found that certain fungi, more specifically filamen¬ tous fungi belonging to the class Zygomycetes, possess certain valu¬ able properties which may contribute to solving one or more of the problems outlined above.

A . Several species belonging to the class Zygomycetes, in particular those belonging to the genus Mucor, have been utilized industrially for a number of years, principally to produce proteolytic enzymes used as mil k coagulants in the production of a variety of cheeses . Large-scale production of these organisms which are grown in sub- merged culture or on a semisolid medium, is therefore well known with respect to optimal growth conditions such as the composition of the nutrient medium in which the culture is grown .

B . Fungi of the class Zygomycetes used in industrial production are non-pathogenic and therefore do not constitute an environmental hazard .

C. It is well known that at least certain products such as the proteo- lytic enzymes mentioned above, are excreted by the organisms into the culture medium. It is contemplated that the mechanism by which the fungi excrete these proteins may be utilized in various ways so as to obtain extracellular production of the gene products of the inserted genes which would greatly facilitate recovery procedures and there¬ fore reduce the cost of the end product.

D . It is known that certain genes (from these or other fungi) are not expressed in bacteria such as E. coli or yeasts such as Saccharomy- ces cerevisiae, possibly due to existence of introns in the DNA se¬ quence or the presence of alternative transcription, messenger RNA processing (e.g . exon splicing) or translation signals . It is contem¬ plated that such genes might be more easily expressed when inserted in fungi of the class Zygomycetes, such as Mucor species . It is also anticipated that certain eucaryotic genes which are difficult or impos¬ sible to express in bacteria or yeasts for these or other reasons may more easily be expressed in zygomycetous fungi .

It has hitherto not been possible to achieve transformation of Zygomy¬ cetes species, i . e. the direct incorporation of selected genes into the host organism by means of a plasmid vector. It has even been diffi¬ cult to obtain an adequate genetic analysis of Zygomycetes species by recombination studies due to the difficulties in utilizing their sexual cycle. Mating of ( ⁺ ) and (-) strains does not always result in the formation of zygospores, and in some species the numbers produced are low, while in others their development remains incomplete (cf . M. A. A . Schipper, Studies in Mycology 17, 1978, 1 -52) . Moreover, the successful germination of zygospores under laboratory conditions is rare and is preceded by a considerable period of dormancy (cf. W. Gauger, Mycoiogia 57 , 1965, pp. 634-641 ) .

The present invention has overcome these difficulties by providing a method by which genetic material may be introduced into a host orga¬ nism belonging to the class Zygomycetes.

Accordingly, the present invention relates to a method of transforming a fungus of the class Zygomycetes, in which sporangiospores or germ-

lings of a fungus of the class Zygomycetes are transformed with a re¬ combinant expression vector which is replicated in the fungal species .

I n the present context, the term "sporangiospores" denotes vegetati¬ ve, asexual spores of Zygomycetes species, and the term "germlings" denotes germinating spores .

It has surprisingly been found that, by suitable treatment, as descri¬ bed in fu rther detail below, sporangiospores and germlings may be brought to a condition in which they are capable of receiving external genetic material . This means, that by the method of the invention, it has become possible to obtain direct cloning of specific genes in fungi of the class Zygomycetes by the transformation methods usually em¬ ployed in recombinant DNA technology, including the use of a recom¬ binant expression vector.

The fungus employed for transformation pu rposes is preferably a fungus of the genus Mucor as these fungi have been used extensively for industrial purposes and, therefore, the conditions under which they should be cultivated in large-scale production are well known . The Mucor strain selected for transformation may be a mesophilic Mucor species such as M. circinelloides, M. racemosus or M. rouxii , or a thermophilic Mucor species such as M . miehei or M. pusilius .

One of the principal difficulties in obtaining transformation of fungi of the formation of protoplasts requires the removal of the cell wall by digestion with one or more lytic enzymes . It has for instance been found that the vegetative spores (sporangiospores) of Mucor may be a less suitable source of protoplasts due to the extreme resistance of the cell wall to lytic digestion . The high content of melanin (10% by weight of dry matter) is implicated in this resistance as it is a known inhibitor of cell wall lytic enzymes (A. T. Bull, Arch. Biochem. Bio- phys. 137, 1970, pp. 345-356) . I n accordance with the present inven- tion , however, a method has been developed whereby the transforma¬ tion is effected by treating germinating sporangiospores at a unique developmental stage with one or more suitable lytic enzymes, trans¬ forming the resulting protoplasts with the appropriate vector, and regenerating the cell wall .

The treatment of the germinating sporangiospores according to this method has become possible because of the finding that, during germination, a vegetative wall is formed de novo underneath the sporangiospore wall, later emerging to become the germ tube wall . The lack of continuity between spore and vegetative walls is correla¬ ted with marked differences in their composition , including the ab¬ sence of melanin in the latter. I nstead, chitin and chitosan have become the main components; in one Mucor species, M. rouxii, for example, they constitute 9.4% and 32.7% of the hyphal cell wall, respectively. It has further been found that to degrade this cell wall, lytic enzymes such as chitosanase may be employed. Chitosanases are not yet commercially available but are produced as extracellular en¬ zymes by a variety of microorganisms . The culture fluids from some of these microorganisms, or the chitosanases purified therefrom, can digest the hyphal cell walls of for instance Mucor and enable the formation of protoplasts from Mucor and the related genus Phycomy- ces. Chitinase which is commercially available may also be employed, but not as the sole lytic enzyme as it is unable or only partially able to hydrolyze chitosan .

One culture fluid which has been found useful for the present inven¬ tion is the culture fluid, optionally in concentrated form, of Strepto- myces sp. No. 6, also terme- ^** "streptozyme" which contains chitosan¬ ase in unpurified form and possibly also other lytic enzymes . It is also possible to add an enzyme which in itself has little or no lytic effect, but which increases the effect of chitosanase. I n experiments it has been found that when certain commercially available polysaccha- rases are employed together with chitosanase or the unpurified strep¬ tozyme, this results in increased yields of protoplasts, even when used in combination with a low concentration of the lytic enzyme proper, although none of these commercially available enzymes in themselves have any visible lytic effect.

It has been found that an increase in the concentration of the lytic enzyme .employed to digest the cell walls, increases the rate of proto¬ plast formation and the final yield of protoplasts obtained . When com- paring different batches of, e. g . , streptozyme, their efficacy in pro-

ducing protoplasts in Mucor species is directly proportional to the total chitosanase activity present. Thus, 0.5 mg/ml of a batch with a specific chitosanase activity of 2.3 produces equivalent results to 1 .0 mg/ml of a batch with a specific chitosanase activity of 1 .2.

It has also been found by experimentation that the formation of pro¬ toplasts from germinating sporangiospores is to some extent dependent on the stage of germination as the resistance to lytic enzyme seems to be reacqui red, though to a lesser extent, as the hyphae age and grow in length . I n fact, the yields of protoplasts obtained tend to be highest when the germlings had a germ tube length of 35-50 υm. Below this size, digestion of the cell wall at the growing hyphal tip still occurs, but the amount of material extruded decreases, with a consequent reduction in the number of protoplasts formed . When the germ tube length exceeds approximately 60 μm, the amount of hyphal structure visibly resistant to lytic enzyme action increases, the pro¬ toplast yield decreases, and the homogeneity of the protoplasts ob¬ tained also decreases (as evidenced by a greater variation in size, refractility and apparent membrane integrity) . Therefore, it is pre¬ ferred that, in order to obtain a high yield of morphologically homo- geneous protoplasts, the germlings to be treated with the lytic en¬ zyme be relatively homogeneous with respect to extent of germination (i . e. germ tube length) . Synchronous germination can be induced in the genus Phycomyces by various treatments such as heat shock; in another genus, Rhizopus, synch ronous germination may be induced by means of a phosphate buffer, pH 6.5, containing proline. However, although a specific induction method for Mucor has yet to be deve¬ loped, there are certain indications that a more synchronous germina¬ tion may also be obtained in Mucor, for instance by germinating freshly harvested sporangiospores in complete media instead of using frozen spores or minimal media, and by incubating the sporangio¬ spores at a temperature which approaches the optimal growth tempe¬ rature of the species in question, this latter effect having been observed for thermophilic species in particular.

I n some cases, it is necessary to employ osmotic stabilizers du ring protoplast formation . It is important that the osmotic stabilizer em-

ployed does not inhibit the activity of the lytic enzyme preparation to the effect that no cell wall degradation takes place. Solutions of sugar alcohols such as sorbitol and mannitol tend to be the most effective osmotic stabilizers, while the use of salt solutions, e. g . KCI, MgC (NH ₄ )-SO ₄ or MgSO ₄ seems to result in no protoplast formation at all . When employing sorbitol as the osmotic stabilizer when forming proto¬ plasts from Mucor germlings, the optimal concentration was found to be 0.35-0,5 M. At concentrations of 0.65 M or higher, no cell wall degradation is observed, and at concentrations of 0.3 M or lower, the action of the lytic enzyme tends to be accompanied by extensive cell lysis rather than protoplast formation .

The pH of the medium used during incubation should preferably be in the range of 4.5-7.5, so as not to interfere with the activity of the lytic enzyme employed .

Regeneration of the cell wall after transformation may be effected by washing the protoplasts twice in a suitable buffer or growth medium containing a suitable osmotic stabilizer such as sorbitol, and then plating the protoplasts on a solid agar medium also containing the osmotic stabilizer. The plating of the protoplast suspension may be performed by spreading the protoplasts directly on the surface of the solid agar medium, or preferably by mixing them into an agar overlay containing the osmotic stabilizer which is held in a molten condition and then poured onto the surface of the solid agar medium. Embed¬ ding the protoplasts in agar permits cell wall regeneration to occur reproducibly and at high frequencies .

I n accordance with the invention, the transformation procedure should preferably include a method of selecting the transformed cells . Accor¬ dingly, the fungal strain of the class Zygomycetes may conveniently be a suitable auxotrophic mutant strain (i . e. a strain which requires one or more growth factors not needed by the wild type strain) or a mutant strain which is sensitive to a certain antibiotic which strain , when transformed, is complemented by an expression vector carrying the counter-selectible prototrophic or antibiotic resistance gene.

Auxotrophic mutants have been found to be particularly useful in the method of the present invention . Such mutant strains may be produced by conventional mutagenization methods, including ultraviolet radia¬ tion , ionizing radiation or treatment with a chemical mutagen . The de- sired auxotrophic mutants are then isolated by selective elimination of the prototrophic survivors of the mutagenic treatment. Most such iso¬ lation methods are based on the fact that metabolically active cells are more sensitive to antibiotics and physical agents than inactive cells, such as ungerminated spores . Therefore, a preferential killing of pro- totrophs is the result when mutagenized cultures grown in minimal medium, i . e. a medium which does not contain the growth factors needed for the growth of auxotrophs, are exposed to a counter-selec¬ tive agent. Other methods include differential freeze-killing of germi¬ nating versus non-germinating spores, or differential heat sensitivity of germinated prototrophic spores as opposed to ungerminated auxo¬ trophic spores . The most advantageous method in the present context has been found to be an approach based on the use of an antibiotic which induces considerable changes in permeability by interaction with the sterols in the cell walls of metabolically active cells . A particularly advantageous antibiotic is a polyene antibiotic such as N-glycosyl-poli- fungin (NG P) which has the added advantage of being water-soluble unli ke most other polyene antibiotics such as nystatin .

Utilizing the enrichment method described above, it is contemplated that successive treatments with antibiotic may lead to a fu rther in- crease in the frequency of mutants obtained . Such additional cycles of counter-selection may be advisable when specific and rare phenotypes of mutants are needed . A further selection of auxotrophic mutants may be obtained by growing colonies which have survived the anti¬ biotic treatment and selecting those colonies which fail to grow on a medium which does not contain specific growth factors, but which grow on medium containing these growth factors .

Although the use of auxotrophic mutants is preferred, in particular where Mucor species are concerned, as Mucor shows a low intrinsic sensitivity to most of the antibiotics for which genes conferring resis- tance from other organisms are available, it is, however, also possible

to employ an antibiotic sensitive mutant strain . I n both cases, the expression vector used to transform the fungal cells should carry a gene which complements this property of the host organism, i .e. a gene which either mediates resistance to the antibiotic to which the strain is sensitive or which mediates prototrophy to the auxotrophic mutant so that it reverts to the wild type nutrient requirement.

In accordance with the invention, the expression vector used to transform the host cells may either be self-replicating, i . e. it may exist as an extrachromosomal entity the replication of which is inde- pendent of chromosome replication (i .e. a plasmid) , or the vector may be integrated stably into one or more of the host chromosomes by insertion, for instance by homologous recombination (the vector is so constructed that at least a portion of it corresponds to - is homolo¬ gous to - a portion of the chromosome; this results in a "joining" of the vector portion to the chromosome) and replicated concurrently with these. When the vector is an autonomously replicating plasmid, it carries an origin of replication which is capable of functioning in the host cell so that the plasmid is replicated in such a way that it is maintained in the cell .

I n some cases, it is preferred that the vector is a shuttle vector which can be replicated in a fungal species of the class Zygomycetes, in E. coli and/or in a yeast such as Saccharomyces cerevisiae. A shuttle vector may be advantageous as it facilitates the tranfer of genes already cloned in E. coli or yeast vectors and the construction, multiplication and analysis of vectors containing new or additional genes . Specific examples of such vectors are pMCL 1302 which is prepared according to the invention and, in Example 8 below, has been introduced in Mucor circinelloides, leucine auxotroph R7B . The strain carrying pMCL 1302 is deposited in the Centraal Bureau voor Schimmelkulturen under the Accession No. CBS 754.84. Another vector, produced in similar manner, is pMCL 1647 which has been introduced in the same Mucor strain . The strain of Mucor circinel¬ loides R7B carrying pMCL 1647 is also deposited in the Centraal Bureau voor Schimmelkulturen under the Accession No. CBS 755.84.

I n another aspect, the present invention relates to an expression vector for transformation of fungi of the class Zygomycetes, which carries a DNA sequence of zygomycetous DNA, a DNA sequence coding for a desired gene product, and a marker for the identification and/or selection of cells transformed with the vector, and which is replicated in fungi of the class Zygomycetes . In the present context, the term "DNA sequence" denotes a sequence of basepaired nucleic acids which include one or more structural genes as well as the appropriate transcriptional and translational start and stop signals, promoters, convenient restriction sites, etc. As described above, the selective marker may be a gene mediating antibiotic resistance to an antibiotic sensitive fungal strain or a gene mediating prototrophy to an auxotrophic fungal strain . I n the latter case, the gene may be one which, in its absence or in modified form, causes the organism to require the presence of a specific amino acid or several amino acids, one or more nucleotides or vitamins in the growth medium, and when this gene is complemented by the introduction of the vector into the host organism, the compound or compounds in question are no longer required for growth .

_* The invention further relates to an expression vector for transforma¬ tion of a fungus of the class Zygomycetes . The vector comprises a sequence of zygemycetous DNA, a marker for the identification and/or selection of cells transformed with the vector, and at least one restric¬ tion site for the insertion of a DNA sequence coding for a desired gene product. The vector is replicated in fungi of the class Zygomy¬ cetes . The restriction site should preferably be located on the vector at a site where the insertion of the DNA sequence coding for the desired gene product will not interfere with the replication of the vector and/or selection of the host organism to which the vector is to be transferred, that is, the restriction site should be located outside the origin of replication and the DNA sequence coding for the selec¬ tive marker. Examples of vectors which satisfy these requirements are pMCL 1302 and pMCL 1647 which are further described and characte¬ rized in the Examples and which are shown in Fig . 2.

The present invention also relates to a method of preparing an ex¬ pression vector as described above, which comprises inserting, in a

plasmid which is replicated in a fungus of the class Zygomycetes, a DNA sequence for a marker for the identification and/or selection of cells transformed with the vector, providing at least one restriction site for the insertion of the DNA sequence coding for a desired gene product at a location where the DNA sequence does not interfere with replication and/or selection, and inserting the DNA sequence for the desired gene product at this site. If a convenient restriction site is not available, it may be possible to provide it in the form of a linker inserted at a suitable site. The procedures employed to produce the expression vector of the invention may be those conventionally em¬ ployed for this purpose in the field of recombinant DNA technology.

The present invention additionally relates to a fungus of the class Zygomycetes which carries a recombinant expression vector which is replicated in the organism. Genetic transformation , i . e. the incorpora- tion of isolated genes in the form of isolated DNA, has previously only been achieved in filamentous fungi belonging to the class Asco- mycetes, namely Neurospora crassa (Case et al . , Proc. Natl . Acad . Sci . USA 76, 1979, p. 5259) , Podospora anserina (Stahl et al . , Proc. Natl. Acad. Sci. 70, 1982, p. 3641 ) and Aspergillus nidulans (Bal- lance et al . , Biochem. Biophys. Res. Commun. 112, 1983, p. 284) .

Finally, the present invention relates to a method of producing a gene product from a fungus of the class Zygomycetes, in which sporangio¬ spores or germlings of a fungus of the class Zygomycetes are trans¬ formed with a recombinant expression vector which is replicated in the fungal species and which carries a DNA sequence coding for a desi red gene product, the transformed sporangiospores or germlings are grown in a suitable culture medium to express said DNA sequen¬ ce, and the resulting product is harvested.

The transformed sporangiospores or germlings are grown in a suitable culture medium to express the DNA sequence. The cultivation is suit¬ ably performed using conventional techniques well known for large- scale production of Zygomycetes species, in particular Mucor species, such as the techniques described by, e. g. , K. Aunstrup, " Proteina- ses", in A. H . Rose, ed. , Microbial Enzymes and Byconversions;

Economic Microbiology, Vol . 5, Academic Press, New York, 1980, pp . 50-114, including the use of conventional nutrient media which are known to be suitable or optimal for the fungal species in question . For instance, Mucor species may be grown in submerged culture or on a semisolid medium. For submerged cultivation , it is preferred that the nutrient medium is relatively concentrated (with a dry matter content of up to about 10-15%) and that it has a high content of protein . The medium may be supplied with carbohydrates such as starch, starch derivatives or sugars, preferably in such a way that the carbohydrate concentration is kept low at all times (a so-called fed-batch process) . For semisolid fermentation, a medium such as bran (for instance wheat bran) may be moistened to a dry matter content of about 50%, inoculated with spores of the organism producing the desired product and left under aeration . The growth conditions may further include an adaptation to the optimal growth temperature of the fungus as well as certain specific nutrients such as mineral salts or trace minerals .

The harvesting of the resulting gene product may be performed by conventional methods in the manner most convenient to the production process employed, dependent, inter alia, on the type of product being produced, the end use of this product and, most importantly, whether the product is excreted into the medium or not. Most usu¬ ally, when dealing with excreted products which are found in soluble form in the medium, the solids (including residues of the medium components and host organisms) are removed by filtration and centri- fugation (cf. for instance K. Aunstrup, "Production , Isolation and Economics of Extracellular Enzymes", in L. B . Wingard et al . (eds . ) , Applied Biochemistry and Bioengineering, Vol . 2, Academic Press, New York, 1979, pp . 28-69.

When the fungus employed for the production of the gene product is a fungus of the genus Mucor, the growth conditions optimal for the cultivation of Mucor species include the presence of proteinaceous nu¬ trients such as soy protein , casein , whey powder, brewer's yeast or yeast extract, carbohydrate-containing nutrients such as ground grain, e. g . corn or barley, starch, e.g . corn or potato starch, or

starch hydrolysates, or sugars such as glucose, lactose or sucrose, and salts such as phosphates, magnesium salts, carbonates, sulpha¬ tes, ammonium salts or nitrates, and trace minerals such as Zn , Fe or Cu . The specific composition of the medium depends, to some extent, on the product to be produced. Specific media suitable for the pro¬ duction of, e. g. enzymes, are described in, e. g . , US Patent No. 3,988,207. Other factors influencing the growth of the organisms include the growth temperature.

As mentioned above, one of the valuable properties of fungi of the class Zygomycetes, especially of the genus Mucor, is their ability to excrete certain of their products, such as certain metabolites and enzymes, into the culture medium which, for instance,- is the case with certain enzymes such as acid proteases and other enzymes . I n a preferred embodiment of the present invention, this process is uti- lized so that the product of the DNA sequence inserted in the fungal cells is excreted into the medium by the natural route of these orga¬ nisms and recovered therefrom by established industrial procedures .

Thus, in order to obtain excretion of the product, it may be contem¬ plated to integrate the expression vector into the host chromosome at the site of a gene the product of which is known to be exported by the organism into the culture medium. The integration may be directed, for instance by homologous recombination, to occur in such a manner that the expression of the introduced gene utilizes the signals of the endogenous gene which it has replaced, including those responsible for the eventual export of the gene product into the culture medium.

One of the advantageous features of the method of the present inven¬ tion resides in the fact that products exported by the host cells into the medium may be recovered therefrom by simply filtering or centri- fugating off the host organisms together with residues of the medium components, without further purification being necessary. This, of course, greatly reduces the cost of the product in question .

For some special applications requiring a high degree of purity, how¬ ever, the product may be subjected to various purification procedures

according to need, for instance further filtration, precipitation in a manner known per se (usually with water-soluble organic solvents such as acetone or certain alcohols, or with inorganic salts such as ammonium sulphate or sodium sulphate) . If a further purification is needed, the partially purified product may be redissolved and sub¬ jected to various known affinity chromatographic methods such as gel filtration or ion exchange chromatography. The purification process may be accelerated by employing high performance liquid chromato¬ graphy. The mode and degree of purification depends on the product produced; thus, if the product is one which is to be used industri¬ ally, it may not be necessary to perform further purification than the basic recovery procedure by filtering off the host organisms.

The gene product to be produced by the method of the invention from the Zygomycetes species may be any product conveniently produced by recombinant DNA technology. Thus, the product may be a poly- peptide protein or fragment thereof, an enzyme or a non-proteinace- ous product of reactions of enzymes with a compound in the culture medium, or a low molecular weight product such as hormones or nucleic acids . Products of particular interest are products which are either known or expected to be expressed in or exported by other organisms such as bacteria or yeasts with great difficulty, if at all . Such products may comprise products from other fungal genera, the industrial fermentation of which is less well developed than that of fungi of the class Zygomycetes, in particular Mucor species, or genes of higher eucaryotes . I nteresting gene products are enzymes, inclu¬ ding lipase, amyloglucosidase, α-amylase, β-galactosidase, ceilulase or proteases such as chymosin . Some of these gene products may be produced by certain Zygomycetes species (e. g . Mucor species) , but may be produced in insufficient amounts, or may have undesirable properties such as excessive thermostability or thermolability, thus requiring a modification procedure to be industrially useful; it may also be desirable to confer a different specificity to the gene products by genetic alteration . The DNA sequence for the modified gene pro¬ duct may be inserted into a vector and introduced and expressed in a host organism, for instance in such a way that it replaces the inhe¬ rent DNA sequence for the natural product, in accordance with the method of the invention .

It should be noted that the gene encoding chymosin (calf rennet) which is extensively used as a milk coagulating enzyme in the pro¬ duction of cheese, has been introduced and expressed inside cells of Escherichia coli (cf. Nishimori et al . , Cene 29, 1984, p. 41 ; Emtage et al . , Proc. Natl. Acad. Sci. USA 80, 1983, pp. 3671 -3675) and in¬ side cells of the yeast Saccharomyces cerevisiae (Mellor et al . , Cene 24, 1983, p. 1 ; Goff et al . , Cene 27, 1984, p. 35) . Even when ex¬ pression is obtained in these organisms, it is therefore necessary to disrupt the cells in order to obtain the product after which the cell debris must be removed. Such a purification procedure is not neces¬ sary when introducing the same gene into species of the class Zygo¬ mycetes in such a way that the gene product is excreted, as outlined above.

The method of the present invention of producing a gene product from a fungus of the class Zygomycetes by transforming zygomycetous sporangiospores or germlings with an expression vector of the inven¬ tion , is distinguished from the known genetic transformation methods used in connection with filamentous fungi in the following way:

It allows di rect cloning of genes by complementation of suitable muta- tions in the recipient strain with plasmid DNA containing inserts from a genomic library of the wild-type strain . The plasmid may carry other genes to be expressed in the recipient strain . This is opposed to the known methods employed with filamentous fungi for the cloning of specific genes which use indirect means such as screening genomic libraries for the complementation of mutants in E. coli or S. cerevi¬ siae. This is not applicable to Zygomycetes species as zygomycetous genes cannot be functionally expressed in these two hosts generally employed for recombinant DNA work. The in vitro techniques of complementary DNA synthesis from messenger RNA and hybridization to synthetic oligonucleotide probes employed with the ascomycetous fungi Aspergiflus nidulans and Neurospora crassa and which might also be applicable to zygomycetous fungi are more cumbersome than the method of the present invention . The present invention is in fact believed to constitute the first instance of genetic transformation of fungi of the class Zygomycetes. The expression vector incorporated in

the fungus may be one of those described above, and the fungus is preferably a fungus of the genus Mucor, such as an auxotrophic mutant strain as described above.

The insertion of a DNA sequence coding for a desired gene product into the expression vector may be carried out in the following way: The expression vector is digested with one or more restriction en¬ zymes so that the circular molecule is opened at one position . The DNA sequence to be inserted is also digested with one or more re¬ striction enzymes to produce ends which may be ligated to the ends of the expression vector, using the enzyme T ₄ ~DNA ligase, either directly (in the case of complementary "cohesive" ends) or after the creation of "blunt" ends by treatment with DNA Polymerase 1 (Klenow fragment) or SI nuclease. The site of insertion of the DNA sequence coding for the desired gene product is so selected that it follows a promoter sequence known to function in Mucor. An example of the latter is the promoter sequence responsible for the expression of the Leu ⁺ gene of Mucor circinelloides which is known to be found in the 2. 1 kb Ava\ -Kpn\ fragment of the Mucor DNA insert of plasmid pMCL 1302.

The invention is fu rther described with reference to the drawings in which

Fig . 1 shows a restriction map of the parent plasmid YRp17 used for constructing expression vectors of the present invention . I n the Fig . 1 , the dotted areas denote DNA from E. coli, and the filled-in areas denote DNA from S. cerevisiae. The arrows denote the direction of transcription .

Fig. 2 shows restriction maps of two expression vectors according to the invention , pMCL 1302 and pMCL 1647 (not drawn to the same scale) . The thick lines denote YRp17 DNA (hatched areas denote DNA from E. coli and the filled-in areas denote DNA from S. cerevisiae) , and the thin lines denote the Mucor DNA inserts . The dotted lines indicate the region of homology between the two inserts .

Fig. 3 shows restriction maps of pMCL 002 and pMCL009 which are derivatives of pMCL 1302, in which the hatched areas denote E. coli DNA, and the thin lines denote Mucor DNA.

The invention is further described with reference to the following Examples.

MATERIALS AND METHODS

Strains

Mucor circinelloides f. lusitanicus CBS 277.49 (syn. Mucor racemosus

ATCC 1216b), Studies in Mycology 12, 1976, pp. 1-40, Mucor miehei CBS 370.65, Studies in Mycology 17, 1978, pp. 53-71,

Mucor miehei CBS 182.67, Studies in Mycology 17, 1978, pp. 53-71,

Mucor racemosus No. 50, Nippon Nogeikagaku Kaishi 55, 1981, pp.

561-571,

Mucor rouxii CBS 416.77, Biochem. Biophys. Ada 58, 1962, pp. 102-119,

Mucor pusillus IMI 96211, Appl . Microbiol . 16, 1968, pp. 1727-1733,

M. circinelloides leu-2A, was obtained as described in J. Cen . Micro- biol. 105, 1978, pp. 77-81,

Streptomyces sp. No. 6, J. Microbiol. & Serol . 84, 1968, pp. 173-182, and J. Cen. Microbiol. 72, 1972, pp. 281-290,

Escherichia coli HB101, J. Mol . Biol . 41, p. 459.

Media and growth conditions

Complete medium: YPG (3g of yeast extract, lOg of peptone and 20g of glucose per litre of distilled water) .

Minimal medium: YNB (0.5g of Difco yeast nitrogen base without amino acids and ammo¬ nium sulphate, 1.5g of ammonium sulphate and 1.5g of glutamic acid

per litre of distilled water with 1% of glucose and lyg/ml thiamine and niacin added after sterilization) .

For growth of auxotrophs, the minimal medium was supplemented with different amino acids at a final concentration of lOOyg/ml or with cas- amino acids at a concentration of 2mg/ml . To produce restricted colony growth, the media were adjusted to pH 3.0 with 1M HCI (added after sterilization) . Otherwise, the media were adjusted to pH 4.5 as described above. For solid media, pH 4.5, 20g/ml agar was included . I n the case of media with a pH of 3.0, double strength solutions of agar and the other media components were autoclaved separately to avoid acid hydrolysis of the agar.

Cultures of M. circinelloides, racemosus and rouxii were incubated at 28°C, and cultures of M. miehei and pusillus were grown at 40°C.

E. coli was grown at 37°C in LB medium (Virology 1, 1955, pp. 190- 206) containing 50μg/ml ampicillin or 25μg/ml tetracycline as required.

Chemicals

N-glycosyl-polifungin was prepared as described in German Patent No. 2239891 . Chitin and chitosan were of practical grade prepared from crab shells . Restriction enzymes, calf alkaline phosphatase, T4 DNA ligase and E. coli DNA polymerase I were obtained from Boehrin- ger Mannheim, Germany) . Novozym 234 (prepared from Trichoderma harzianum, mainly containing α-1 ,3-glucanase activity was obtained from Novo I ndustries A/S, Denmark) , helicase (I' l ndustrie Biologique, France) , laminarinase (β-1 ,3-glucanase ex mollusca, Calbiochem, USA) , β-glucanase isolated from Penicillium emersonii (BDH Biochemi- cals , England) and chitinase (Sigma, activity 3.0 units/mg ) . All enzymes were used according to the conditions specifified by the manufacturers . Polyethylene glycol (PEG) 4000 was from Fluka AG (Switzerland) . Heparin, bovine serum albumin (essentially free from fatty acids) , 3- (N-morpholino)propane sulphonic acid (MOPS) , and RNase A were from Sigma, USA, Sephadex® G50 was obtained from Pharmacia, Sweden, nitrocellulose filters ( BA 85) were from Schleicher

18

32 8- Schuell, Federal Republic of Germany, o p-dATP (>600 Ci/mmol) was from New England Nuclear, USA.

Isolation and assay of Streptozyme

Streptomyces sp. No. 6 was grown for 7-8 days at room temperature in the medium according to Skujins et al . , Arch. Bioche . Biophys. 111, 1965, pp. 358-364, with 0.5% (w/v) chitin and 0.1% (w/v) repre- cipitated chitosan (chitosan dissolved in 1% of acetic acid, neutralized with NaOH, then washed extensively with H~0) as the sole carbon sources. Cells were removed by centrifugation (5000 x g for 30 minu- tes) , and the supernatant brought to 95% saturation with ammonium sulphate at 4°C and left overnight with stirring . The precipitate was collected by centrifugation at 5000 x g for 60 minutes, resuspended in a minimal volume of cold 0.02 M sodium phosphate buffer at pH 6.5, and dialyzed extensively against the same buffer at 4°C. After cen- trifugation to remove insoluble material, the preparation was lyophili- zed and stored at -20°C. Protein was determined as described in Anal. Biochem. 72, 1976, pp. 248-254, using a Bio-Rad protein assay kit I with hen egg albumin as the standard . Chitosanase activity was determined as described in J. Bacteriof . 124, 1975, pp. 1574-1585. Chitosan was dissolved in 0.05 M maleic acid to 2 mg/ml, diluted 2-fold with H ₂ 0 and the pH adjusted to 6.0 with KOH . After preincu- bation of 375 μl aliquots at 30°C for 15 minutes, the reaction was started by addition of 125 μl enzyme in 0.01 M sodium phosphate buffer, pH 6.5, giving a final volume of 500 μl containing 0.5 mg/ml chitosan . After a further 15 minutes at 30°C, the reaction was stop¬ ped by adding 100 μl 1 M KOH, and the chitosan precipitated by incubation on ice for 30 minutes . The supernatant obtained after centrifugation in an Eppendorf minifuge for 10 minutes was assayed for hexosamine content by the indole method described in Methods of Biochemical Analysis, ed . D. Glick, I nterscience Publishers I nc. , New York, 1955, vol . 2, pp. 313-358. One unit of chitosanase activity was defined as the activity required to produce 1 μmol of hexosamine equivalent (mono- or oligosaccharide) per minute.

Isolation of DNA

Preparation of plasmid DNA from small cultures of E. coli were obtained by the rapid boiling method described in Anal. Biochem. 114, 1981 , pp. 193-197. They were treated with RNase A (1 mg/ml, preheated at 90°C for 10 minutes) for one hou r at 37°C prior to analysis by re¬ striction enzyme digestion and agarose gel electrophoresis . The pre¬ paration of plasmids from larger cultures of E. coli followed their amplification overnight in the presence of 150 μg/ml chloramphenicoi and was performed according to the method described in Biochim. Biophys. Ada 299, 1973, pp . 516-520, with minor modifications . The supernatant obtained after high speed centrifugation of the cell lysate was used directly for pu rification of the DNA by CsCI gradient cen¬ trifugation (p = 1 .55, containing 0.75 mg/ml ethidium bromide) .

Transformation of E. coli

E. coli HB101 was transformed to ampicillin resistance after induction of cell competence by the CaC method described in J. Mol . Biol . 53, 1970, pp . 150-162.

Hybridization analysis

DNA fragments generated after restriction endonuclease treatment were electrophoretically separated on agarose gels, then transferred to nitrocellulose filters according to the method described in J. Mol.

Biol. 98, 1975, pp. 503-517. Plasmid DNA was labelled in vitro with

32 o P-dATP by nick translation according to the method described i inn ,7

J. Mol. Biol. 113, 1977, pp. 237-251 . Specific radioactivities of 3-4x10

32 cpm per μg DNA were obtained . Hybridization of P-labelled plasmid

DNA to the DNA immobilized on nitrocellulose was performed at 68°C in 6 x SSC according to the method described in Molecular Cloning, a

Laboratory Manual, Cold Spring Harbor, New York, 1982. Autoradio- graphy was carried out using Kodak X-Omat RP X-ray film with Kodak X-Omatic regular intensifying screens at -80°C .

EXAMPLE 1

Isolation of auxotrophic mutants of Mucor circinelloides f. lusitanicus CBS 277.49 o

About 10 spores from the wild type strain CBS 277.49 were plated on YNB medium supplemented with 2 mg/ml casamino acids . The spores were irradiated with a dose of ultraviolet light to give a survival of 0.5-2% and incubated to allow them to complete a full vegetative cycle. For restricted colony growth the medium was adjusted to pH 3.0 with 1 M HCI , and 20 g/l of agar was added. After seven days at 28°C, the next generation of spores produced on each plate were indepen¬ dently collected and kept frozen at -20°C until further use. Survival titres were determined from colony counts of diluted samples before and after the mutagenic treatment.

Cultures inoculated with 10 ^s spores were grown in 25 ml of liquid mi- nimal medium, and incubated with shaking at 28°C for several hours until about 90% of the spore population had started to germinate (de¬ termined by visualisation under the microscope) . The germlings were collected by centrifugation, resuspended in an equal volume of YNB without ammonium sulphate, and dispensed into tubes before adding different aliquots of an aqueous solution of N-glycosyl-polifungin . After incubation in the presence of the antibiotic for th ree hours, the germlings were washed three times with H2O, plated on YNB medium at a dilution yielding 50-100 colonies per plate and incubated for three to five days for colony counting and replica plating .

The colonies surviving the antibiotic treatment were allowed to sporu- late and were replicated on plates containing YNB as well as on YN B plates containing casamino acids, to detect auxotrophic mutants . Those colonies which failed to grow on selective minimal medium (YN B) but capable of growing in the presence of casamino acids were classi- fied as auxotrophic mutants and their phenotype was further charac¬ terized . These auxotrophs were transferred to various supplemented minimal media, each one of them containing a combination of four or five different amino acids and one including them all (R. Holliday,

Nature 178, 1956, pp. 987) . The nutritional requirement of a particu¬ lar mutant was identified by detection of growth on one or more of the above-defined combinations .

Mutagen Concentration Number of Number of α Ό of N-glycosyl colonies auxotrophs Mutants -polifungin screened obtained units/ml

Ultraviolet light 0 952 0 <0, 10 1000 478 21 4,39

Types of auxotrophs obtained were: 2 requiring leucine, 9 requiring isoleucine, 2 requiring methionine, 1 requiring proline, 1 requiring cysteine or methionine.

The leucine auxotroph R7B, a derivative of M. circinelloides CBS 277.49 is deposited at the CBS under the Accession number CBS 753.84.

EXAMPLE 2

Isolation of auxotrophic mutants of Mucor miehei CBS 370.65.

The same procedure as under Example 1 was used except that the cultures were incubated at 40°C.

Mutagen Concentration Number of Number of g. Ό of N-glycosyl colonies auxotrophs Mutants -polifungin screened obtained units/ml

None 1000 2521 0 <0,04

Ultraviolet 0 1428 2 0, 14

50 867 0 <0, 11

100 1736 0 <0,06

1000 2726 10 0,36

The 12 auxotrophs obtained required lysine.

EXAMPLE 3

Formation of Protoplasts from M . circinelloides f .lusitanicus

Sporangiospores from cultu res of M. circinelloides f. lusitanicus grown for 4-6 days at room temperature on YPG , pH 4.5, agar plates were harvested by gently scraping with a glass rod into distilled water. After one wash in distilled water, they were resuspended in YPG, pH 4.5, at lOVml and germinated at 28°C with shaking. Ungerminated spores were removed by filtration through nylon cloth, mesh size 16 μm, and the germlings were washed twice with 0.5 M sorbitol . After resuspension in 0.5 M sorbitol and 0.01 M sodium phosphate buffer, pH 6.5, the germlings were added to an equal volume of streptozyme (cf . MATERIALS AND METHODS) dissolved in the same buffer to give final concentrations of lOVml germlings and 0.5-1 .0 mg/ml streptozyme (corresponding to approximately 1 .2 units/ml chitosanase) . After in¬ cubation at 23°C for up to 4 hours with gentle intermittent mixing, the number of protoplasts produced per ml was calculated by counting a minimum of two samples in a haemocytometer using phase contrast microscopy. When necessary, any undigested hyphae were removed by filtration of the protoplast suspension th rough nylon cloth , mesh size 10 μm.

Treatment with streptozyme causes digestion of the cell wall of the germlings, the cell membrane bulges out, is pinched off, and spheri¬ cal, osmotically sensitive protoplasts with an average size of 7 μm are released. The formation of protoplasts preferentially occurs at the hyphal apex which is the major site of cell wall synthesis . The total number of protoplasts formed varied with the conditions used, but it could be three times the total number of germlings treated . Mucor is coenocytic (i . e. has nonseptate hyphae) , and the formation of more than one protoplast per germling thus indicates an effective resealing of the cell membrane after a protoplast has been released .

The thus formed protoplasts of M. circinelloides f . lusitanicus were stable and regenerated at frequencies of up to 40% even after over¬ night incubation at 4°C in 0.01 M sodium phosphate buffer, pH 6.5, 0.5 M sorbitol . A comparison of different plating conditions for the regeneration of these protoplasts on solid media (2% (w/v) agar) showed that a 1% agar overlay consistently gave better results .

EXAMPLE 4

Formation of Protoplasts from Mucor miehei

The experiment was performed as described in Example 3 with the exception that the M. miehei cultures were grown at 40°C, and the resulting sporangiospores germinated at 25°C with a 2 hour prein- cubation at 40°C.

EXAMPLE 5

Construction of a genomic library of Mucor circinelloides CBS 277.49

The wild-type Mucor DNA used for construction of the genomic libra¬ ry was prepared as follows :

Sporangiospores were inoculated into one litre of YPG, pH 4.5, at a density of 10 /ml and grown for 16 hours at 28°C with shaking . The mycelium was harvested by filtration through nylon cloth (Monodur, mesh size 22 μm) , washed with cold distilled water, frozen in liquid nitrogen and ground using a mortar and pestle. The frozen powder was warmed to 0°C, an equal volume of TE buffer (10mM-Tris, ImM-EDTA) , pH 8.2, was added, and the slurry was filtered through nylon cloth (Monodur, mesh size 10 μm) . The solid residue was refro- zen , reground, warmed to 0°C and combined with the filtrate. The final slurry was brought to 1 .5% (w/v) SDS (sodium dodecyl sulphate) and combined with an equal volume of a mixture of phenol (containing 0.1% (w/v) 8-hydroxy-quinoline and saturated with TE buffer, pH

8.2) , chloroform and isoamyl alcohol (in the ratio 25: 24: 1 ) . After shaking at room temperature for five hours, the aqueous phase was obtained by centrifugation (12000 x g, for 10 minutes at room tempe¬ rature) , extracted twice with an equal volume of chloroform and iso- amyl alcohol (24: 1 ) , and then purified by CsCI gradient centrifugation .

The plasmid used was the yeast-E. coli shuttle vector YRp17 (Fig . 1 ) (D. Botstein, and R .W. Davis, In the Molecular Biology of the Yeast Saccharomyces cerevisiae, Cold Spring Harbor, New York, 1982, Vol . 1 I B, pp. 607) . It carries the TRP1 and URA3 genes for selection in yeast, and genes conferring resistance to ampicillin and tetracycline in E. coli. YRp17 was linearised with Bam \ , treated with calf alka¬ line phosphatase to prevent recircularisation, and then mixed with an equal amount of Mucor DNA that had been partially digested with Mbo\ to produce an average fragment size of lOkb. After treatment with T4 DNA ligase for 16 hours at 12°C, the resulting ligation mix¬ ture was used to transform E. coli H B101 to ampicillin resistance (M. Mandel and A. Higa, J. Mol. Biol. 53, 1970, pp. 159) . Of the trans- formants obtained, 93% were tetracycline sensitive indicating that their plasmids contained Mucor DNA inserts . A total of 60.000 transformants were divided into groups containing approximately 2.000 colonies each , amplified overnight by growth in one litre media, and used to prepare 30 independent pools of recombinant plasmid DNA . Aliquots of the plasmid pools obtained were digested with SσmH I and electrophor- esed in agarose gels (0,7% (w/v) ) in order to assess the size of the Mucor DNA inserts .

EXAMPLE 6

Transformation of Mucor circinelloides leucine auxotroph R7B = CBS 753.84

Sporangiospores of M. circinelloides R7B were suspended in 150 ml YPG , pH 4.5, at a concentration of lOVml and germinated for 3-5 hours at 28°C with shaking . The germlings were harvested by filtra¬ tion th rough nylon cloth (Monodur, mesh size 22 μm) , washed exten-

sively with 0,01 M sodium phosphate buffer, pH 6.5, and incubated in the same buffer containing 0.5 M sorbitol, 1 .5 mg/ml Novozym 234 and 0.5 mg/ml streptozyme (Skujins et al . , Arch . Biochem. Biophys. 111, 1965, pp. 358) for 2-3 hours at 23°C as described in Example 3. The protoplasts and any undigested cells remaining were pelleted by cen¬ trifugation (400 x g for 5 minutes at room temperature) , washed twice with 0.5 M sorbitol, once with 0.5 M sorbitol in MOPS buffer (10 mM 3- (N-Morpholino) propane sulphonic acid, pH 6.3, 50 mM CaCL) , then resuspended in the same solution to give a final volume of 2 ml .

Aliquots of 0.2 ml were added to 20 μl of the plasmid DNA obtained in Example 5 (0.1 -50 μg, pretreated with 1 mg heparin in 0.5 M sorbitol, MOPS buffer for 20 minutes on ice) plus 20 μl 40% (w/v) polyethylene glycol (PEG) 4000 in MOPS buffer. After incubation on ice for 30 minutes, 2.5 ml 40% (w/v) PEG 4000 in MOPS buffer was added and the incubation continued at room temperature for 25 min . The suspen¬ sions were diluted with 20 ml 0.5 M sorbitol in MOPS buffer, centri- fuged (400 x g, 5 minutes at room temperature) and the pellet resus¬ pended in 5 ml YPG pH 4.5, 0.5 M sorbitol . After 30 minutes incuba¬ tion at room temperature, the cells were centrifuged (400 x g for 5 minutes at room temperature) , and then resuspended in 5 ml of YNB, pH 4.5, 0.5 M sorbitol, and 1 ml aliquots were plated in soft agar overlays (9 ml of YNB, pH 3.0, 0.5 M sorbitol, 1% (w/v) agar) onto YNB pH 3.0, 0.5 M sorbitol agar plates . Assessment of the number of colony forming units was made by plating diluted aliquots onto YPG, pH 3.0, 0.5 M sorbitol . Plates were incubated at room temperature for 2-3 days before colonies were counted .

A total of approximately 2x10 ^s viable protoplasts of strain R7B were incubated with 10-50 μg of recombinant plasmid DNA in the presence of PEG and CaCL as described above, and then plated onto minimal media . After two days at room temperature prototrophic colonies appeared which were capable of sporulation and which could be trans¬ ferred to fresh minimal media with continued growth . No prototrophic colonies were obtained in parallel transformation experiments where either the DNA was omitted, or replaced by 25 μg of YRp17. Out of the 10 different recombinant plasmid pools tested, a total of fou r gave rise to Leu ⁺ colonies . The number of colonies obtained varied from

pool to pool but was in the range of 1 -38. The fact that the Leu ⁺ phenotype was due to plasmid-mediated complementation of the original mutation and not to spontaneous reversion was shown by the recovery of recombinant plasmids from the DNA of Mucor transformants as described in the following Example.

EXAMPLE 7

Transformation of E. coli with recombinant plasmids obtained from Mucor transformants

DNA from small cultures of Mucor for the analysis of transformants was obtained substantially as described in Yelton et al . , Proc. Natl. Acad. Sci. USA 84, 1984, pp . 1470. Cultures were grown from a spore inoculum in 100 ml of YNB, pH 4.5, for 20 hours at 28°C, after which the mycelium was harvested and ground as described above in Example 5. The ground cells were suspended in 10 ml 50 mM EDTA, pH 8.5, 0.2% (w/v) SDS, and mixed for one minute at room tempera¬ ture. The lysate was heated at 68°C for 15 minutes, cooled to room temperature and centrifuged (12000 x g for 15 minutes at room tempe¬ ratu re) . The supernatant (10 ml) was cooled on ice, 0.6 ml of 8.0 M potassium acetate, pH 4.2, was added, and the mixture incubated on ice for 60 minutes . After centrifugation (25000 x g, 15 minutes at 4°C) , the nucleic acids present in the supernatant were precipitated at room temperature by the addition of an equal volume of isopropanol and collected by centrifugation (12000 x g for 30 minutes at room temperature) . The pellet was resuspended in 6.0 ml of TE buffer, pH 7.6, and treated with 60 μl RNase A (10 mg/ml, preheated at 90°C for 10 minutes) for 1 hour at 37°C . After extraction with an equal volume of phenol, nucleic acids were precipitated at -20°C after the addition of 0.1 volumes 3.0 M sodium acetate, pH 4.5, and 2.5 volumes etha- nol . The pellet obtained after centrifugation (12000 x g for 30 minutes at 4°C) was resuspended in 500 μl TE buffer, pH 8.0, and the DNA content estimated by agarose gel eiectrophoresis .

Total DNA from 19 different Leu ⁺ Mucor transformants (cf . Example 6) grown in minimal medium was treated with Kpn\ , self-ligated and used to transform E. coli HB101 (cf . MATERIALS AND METHODS) . One ampicillin resistant colony was obtained . The plasmid recovered from this transformant was designated pMCL1302. The strain of E. coli HB101 pMCL1302 is deposited in the Laboratory of Microbiology at Delft (LMD) under the Accession No. LMD 84.94.

I n order to increase the number of bacterial transformants obtained, the Mucor DNA was purified by CsCI gradient centrifugation . DNA + from five Leu transformants was then successfully digested with

Sail , self-ligated and used to transform E. coli. I n this way, fou r ampicillin resistant colonies were obtained from two different DNA preparations . The plasmid recovered from one of these was designated pMCL1647. The strain of E. coli HB101 pMCL1647 is deposited in the LMD under the Accession No. LMD 84.95. Plasmid DNA from small cultures of E. coli as well as larger cultures of E. coli was prepared as described in MATERIALS AND METHODS .

Physical maps of plasmids pMCL1302 and pMCL1647 were obtained by single and double digestions with seven restriction enzymes ( Fig . 2) . They consist of YRp17 with Mucor DNA inserts of 10.5 kb and 4.0 kb respectively . The inserts in the two plasmids have a homologous region of 3.7 kb delimited by two Aval sites, but placed in the oppo¬ site orientation relative to the YRpl7 sequences . They originate from Mucor colonies transformed to Leu ⁺ with recombinant plasmid DNA from pools No. 13 and No. 16, respectively.

Plasmid pMCL1647 is smaller than the original transforming plasmid from pool No. 16, the insert of which can be estimated to be at least 9.9 kb.

EXAMPLE 8

Transformation of M. circinelloides R7B

The transformation was performed as described in Example 6.

The frequency of transformation of strain R7B to Leu ⁺ using the plasmid pMCL1302 is shown in the following table:

Experiment DNA No. viable No. Leu ⁺ transformation freq luency

ug ceils plated colonies per μg DNA per ^■ ✓ia ible cell

1 0 2.9 X 10 ⁷ 0 0 0

0.1 2.9 X 10 ⁷ 72 720 2.5 X 10 ^{" ε}

0.5 2.9 X 10 ⁷ 120 240 4. 1 X 10 ^{" ε}

1 .0 2.9 X 10 ⁷ 216 216 7.4 X 10 ^{" s}

2 0 3.2 X 10 ^ε 0 0 0

1 .0 3 ^* .2 X 10 ^s 600 600 1 .9 X 10 ^{" **}

3 0 5 X 10 ^s 0 0 0

1 .0 5 X 10 ^s 193 193 3.9 X 10 ^" --

5.0 5 X 10 ^s 234 47 4.7 X 10 ^" *

25.0 5 X 10 ^s 473 19 9.5 X 10 ^{" *} *

Both the concentration of DNA and the density of cells plated were found to have an influence on the number of Leu ⁺ colonies obtained. Up to 720 transformants per μg DNA were obtained using 0.1 -1 .0 μg plasmid DNA. An increase in the amount of DNA used gave an increa¬ se in the number of transformants but the frequency of transformati¬ on (per μg DNA) progressively decreased . This indicates that the DNA concentration was not the sole limiting factor. I n terms of the number of viable cells plated the transformation frequency varied from

-6 -4 2.5 x 10 to 8 x 10 . The relatively low frequency obtained at the

highest plating density probably reflects the inhibition of growth of the transformed cells by the non-transformed cells .

Treatment of strain R7B with pMCL 1647 also gave rise to Leu* colo¬ nies but the frequency of transformation was much lower than that obtained using pMCL 1302. I n an experiment conducted concurrently with experiment 1 , above, 1 .0 μg and 5.0 μg pMCL 1647 gave rise to 6 and 17 stable Leu ⁺ colonies, respectively.

These results demonstrate that the two plasmids pMCL 1302 and pMCL 1647 can serve as Mucor- yeast- E. coli shuttle vectors and can be used to obtain high frequency genetic transformation of Mucor with recombinant DNA.

Both vectors have a unique Sail restriction site (Fig. 2) which may be used for inserting a DNA sequence coding for a desired gene product. Cloning into this site does not affect the DNA sequences required for the expression of the yeast genes TRPl and URA3 and for autonomous replication of the vector (ARS) . Nor does cloning of a DNA sequence for the desi red product into this site interfere with the Mucor gene coding for leucine prototrophy and the E. coli ampicillin resistance gene, thus permitting maintenance and selection in all three hosts .

M. circinelloides R7B-1302 carrying the plasmid pMCL 1302 is deposi¬ ted in the CBS under the Accession number CBS 754.84.

M. circinelloides R7B-1647 carrying the plasmid pMCL 1647 is deposi¬ ted in the CBS under the Accession number CBS 755.84.

EXAMPLE 9

Hybridization analysis of Leu+ Mucor transformants

Final confirmation that the Leu ⁺ colonies obtained in Example 6 were transformants was provided by the detection of YRp17 plasmid se-

quences within their DNA. Total DNA was isolated from a number of transformants and purified by CsCI gradient centrifugation . The fragments obtained after digestion with BamH I or Pst\ were separated by agarose gel (0.7% w/v) electrophoresis, transferred to nitrocellu¬ lose filters (E. M. Southern, J. Mol. Biol. 98, 1975, pp. 503) and hybridized to ³² P-labelled (P .W.J . Rigby et al . , J. Mol. Biol. 113, 1977, p . 237) YRp17. Hybridization was performed at 68°C in 6 x SSC (0. 15 M NaCI, 0.015 M trisodium citrate, pH 7.0) according to Mania- tis et al . (Cold Spring Harbor, New York, 1982) . Autoradiography was carried out using Kodak X-Omat RP X-ray film with Kodak X-O- matic regular intensifying screens at -80°C.

All 22 transformants examined showed specific hybridization to YRp17, while DNA isolated from the recipient strain R7B contained no se¬ quences complementary to this probe. The hybridization patterns obtained were of two types and are presented in the following table.

To labelled YRp17 hybridise:

DNA fragments YRp17 R7B transformant transformant in kilobases from R7B-1302 R7B-1611 after digesti¬ BamH I Pst\ BamH \ Pst\ SσmH I Pst\ BamH \ Pst on with :

7 3.2 - 12 4.7 11 7.0

2.3 3.2 5.2 3.2

2.0

1.5 1.5 1.5

The patterns given for transformant 1302 are representative of all five transformants examined which originate from plasmid pool No. 13. Similarly, those given for 1611 are representative of all 14 transfor¬ mants examined which originate from plasmid pool No. 16. While the 3.2 kb and 1 .5 kb PstI fragments of YRp17 are present in all trans¬ formants, the 2.3 kb Pst\ fragment which contained the SσmHI cloning site has been replaced by two, more weakly hybridising fragments,

indicating the presence of an insert with at least one internal Pst\ site. After BamH \ digestion a single hybridising fragment is obtained in all transformants . These fragments are larger than linear YRp17 indicating the presence of inserts within the plasmid DNA.

32 5 The hybridisation of P-labelled plasmid pMCL 1302 to BamH I and

Pst\ digests of DNA from the untransformed strain R7B and from the

Leu ⁺ transformant 1302 reveals that the plasmid contains inserts of

Mucor DNA:

0 To labelled pMCL 1302 hy¬ bridize after digestion :

'with βσmH I with Pst\

DNA fragments in kilobases from:

5 trans¬ trans¬ formant formant

PMCL1302 R7B R7B1302 PMCL1302 R7B R7B1302

12 16 12 2x4.4 8.2 8.2

2.9 14 5.8 3.2 5.2 5.2

:0 2.1 5.8 3.3 2.2 4.4 3x 4.4

3.3 2.9 1 .6 3.2

2.0 2.2 0.95 2.2 0.7 1 .6 0.95

15 0.7

Since the DNA from strain R7B shares no homology with YRp17, the fragments hybridising from R7B represent Mucor genomic DNA present only in the insert of pMCL 1302. The hybridisation of pMCL 1302 to 0 DNA from the Leu ⁺ transformant 1302 reveals hybridisation to both sequences derived from the YRpl7 plasmid and the genomic sequences present in strain R7B . The homologous fragments have been italicized in the Tables shown above.

This proves that the plasmids pMCL 1302 and, analogously, pMCL 5 1647 are plasmids which contain Mucor, E. coli and yeast genes .

EXAMPLE 10

Autonomous replication in Mucor

In order to detect the presence of free plasmid molecules in Mucor transformants, DNA isolated from the transformants was electrophore- tically separated on 0.4% (w/v) agarose gels, with or without prior treatment with restriction endonucleases and then transferred to nitrocellulose filters according to the method described by Southern .

To the labelled 3.7 kb Aval fragment of the pMCL1302 plasmid hybridize: with undigested DNA after digestion with Hpa , fragments Rf in mm in kilobases and plasmids of rela¬ tive mobility in Rf transformant transformant

R7B R7B 1302 PMCL1302 R7B R7B i 1302 smear 6 mm 7 mm -25 kb 6 mm typical 14 mm 14 mm 14 mm for chrc ι- chromosomal ! mosomal band -25 kb

DNA 29 mm 21 mm 29 mm 36 mm 36 mm

46 mm 46 mm 46 mm

When the total DNA isolated from Mucor transformants is electropho-

32 resed in a 0.4% (w/v) agarose gel and hybridised to the P-labelled

3.7 kb Aval fragment of pMCL 1302, unique hybridization bands can be seen in the undigested DNA of the transformant but not in that of the Leu- strain as shown in the table above. The electrophoretic mo¬ bility of these bands is inversely proportional to the size of the transforming plasmid and the lowest band comigrates with covalently closed plasmid circles isolated from £. coli. I n order to confirm that the hybridization bands represent free plasmid molecules, the DNA was treated with the restriction endonuclease Hpal for which there are cleavage sites in the genomic DNA, but not in pMCL 1302. Any sequences integrated into the genomic DNA should have altered the electrophoretic mobility after Hpal digestion , while free plasmid mole¬ cules should remain unaffected. Digestion of the genomic DNA of

strain R7B results in the appearance of a unique band (-25 kb) of hybridization to the 3.9 kb Aval probe. Digestion of the transformant DNA does not otherwise alter the hybridization pattern obtained . Equivalent results were obtained with strain R7B transformed with the plasmid pMCL 1647.

I n order to further verify the presence of autonomously replicating plasmids, these were recovered from isolated uncut Mucor DNA. After transformation of M. circinelloides R7B to Leu ⁺ with plasmids pMCL 1302 or pMCL 1647, DNA was isolated from transformants as described in Example 7 and then used directly to transform E. coli HB101 . The number of amp colonies obtained ranged from 0-4 per μg DNA, with up to 200 totally from the DNA extracted from 100 ml of culture. Restriction fragment analysis of the plasmids isolated showed 35 to be identical to the original transforming plasmids .

The mitotic stability of the autonomously replicating plasmids was tested as follows:

M. circinelloides R7B was transformed to Leu ⁺ with pMCL 1302, and then three transformants were transferred to minimal and complete medium and allowed to complete the full vegetative growth cycle cul- minating in sporangiospore formation . The percentage of viable spo¬ rangiospores retaining the Leu ⁺ phenotype was estimated by plating sporangiospore suspensions onto complete medium followed by replica plating of colonies onto minimal medium and/or parallel plating of dilutions onto complete and minimal medium. Only 5% of viable spo- rangiospores were Leu ⁺ after the first transfer to complete medium compared with 45% when growth was continued under selective condi¬ tions . A further two successive transfers to complete medium reduced the percentage of Leu ⁺ sporangiospores to 0.8% and 0.056%, respec¬ tively.

To estimate plasmid copy number in strain R7B transformed with pMCL 1302, total Mucor DNA from strains R7B and R7B 1302 was. di¬ gested with Sail , denatured in 0.4 M NaOH for 10 minutes and chilled and diluted with an equal volume of cold 2 M ammonium acetate. Two-

fold serial dilutions were applied in 100 μl aliquots to a nitrocellulose filter using a Schleicher & Schuell filtration manifold . The filter was washed in 1 M ammonium acetate, then 2 x SSC, then 2 Denhardt's solution before baking at 80°C for two hours .

Hybridization of the DNA on this filter to the 3.7 kb Aval fragment of Mucor DNA indicates that the plasmid molecules are present in the transformants at 4-5 copies per haploid genome.

The data presented above is proof of the autonomous replication in Mucor of hybrid plasmids constructed from fragments of Mucor DNA inserted in YRp17. These plasmids transform Mucor at very high frequencies and can be recovered in an unmodified form from the transformant DNA. They are efficient shuttle vectors for the transfer of genetic material between Mucor, E. coli and 5. cerevisiae and enable an easy recovery of Mucor genes cloned by direct complemen- tation from a genomic library.

EXAMPLE 11

Identification of an autonomous replication sequence of Mucor

Plasmid pMCL 1302 (Fig . 2) contains the sequences denoted ARS and ORI which are responsible for the maintenance of the plasmid as an autonomously replicating unit in Saccharomyces cerevisiae and Esche- richia coli, respectively. I n order to determine their role, if any, in the autonomous replication of pMCL 1302 within Mucor, subclones lack¬ ing these sequences were prepared and used to transform M. circi¬ nelloides R7B to Leu ⁺ .

Plasmid pMCL 002 (Fig. 3) consists of the Mucor DNA insert of pMCL 1302 cloned into pBR322. It is devoid of all yeast sequences present in pMCL 1302, but is still found to exist as a free plasmid within Mucor transformants .

Plasmid pMCL 009 (Fig. 3) was prepared by digestion of pMCL 002 with Bgll which cleaves at three positions within the pBR322 sequen¬ ce, but not at all within the insert of Mucor DNA . The largest of the resulting restriction fragments contains the entire Mucor DNA insert plus 1 .8 kb of pBR322 and lacks the E. coli ORI plus at least 1 kb of pBR322 DNA on either side of this region . After isolation by agarose gel electrophoresis, this fragment was treated with T4-DNA ligase, and then the ligation mix was used directly to transform M. circinel¬ loides R7B to Leu ⁺ . DNA was isolated from four transformants and

32 analyzed by Southern hybridization to P-labelled pMCL 002. The uncut isolated DNA of these transformants gave hybridization patterns indicating the presence of free plasmid molecules. Digestion with Aval results in a pattern of hybridization consistent with the presence of pMCL 009, not pMCL 002, within the transformants . The absence of the E. coli ORI region is indicated by the replacement of the 4.1 and

4.7 kb Aval fragments of pMCL 002 by a single 6.5 kb fragment.

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