WO2004074490A2 - Method for transforming blakeslea strains - Google Patents

Abstract

The present invention relates to the production of a molecule of interest by a host cell. Provided is a method for producing at least one molecule of interest by a host cell, comprising providing a host cell with at least one isolated nucleic acid, culturing said host cell and allowing said host cell to produce said at least one molecule of interest, wherein said host cell comprises a fungus of the family Choanephoreaceae, for example Blakeslea. Also provided is a method for providing said host cell with said nucleic acid.

Description

Title: Method for transforming Blakeslea strains.

The present invention relates to the production of a molecule of interest by a host cell. More specifically, the invention relates to methods for producing valuable compounds, such as carotenoids, proteins, and other biomolecules, using a fungus as host ceU.

The Kingdom of the Fungi includes over 250,000 different species and contains members central to every ecosystem on our planet. Fungi are universally consumed as food and are used for the industrial manufacture of chemicals and enzymes, collectively representing industries that contribute ca. $35 billion to the US economy only each year. As a group, fungi have an enormous impact on the world economy : yeast is used extensively in the brewing industry, filamentous fungi are used both for the production of foodstuffs and industrial production of enzymes and chemicals, and Basidiomycetes are consumed as food all over the world.

Some fungi, or spontaneous mutants thereof, are natural hyperproducers of a molecule of interest. However, it is generally preferred to use a genetically engineered fungus as a host cell, because this allows to increase or otherwise modulate the production level of such a molecule of interest. Therefore, the construction of various fungal genetic systems, is of paramount importance for the development of inexpensive and flexible production systems for the production of various kinds of commercially relevant molecules. For example, a host cell is subjected to genetic engineering to obtain an increased production, or an altered production profile, of biomanufactured products

In general, the availability of a genetic transformation system is a prerequisite for metabolic engineering of an organism by recombinant DNA technology. This enables the analysis of gene function and regulation and allows the amplification of homologous nucleic acid sequences and the introduction of novel (heterologous) nucleic acid sequences. The first transformation procedures for filamentous fungi were reported for the ascomycetous species Neurospora crassa, Aspergillus nidulans and Podospora anserina. These protocols were based on the transformation of protoplasts by a combination of CaCta and polyethylene glycol (PEG). Using a method based on the transformation of protoplasts by electroporation the transformation efficiency was further improved and extended the group of potential hosts to basidiomycetous fungal species. Successful procedures for the transformation of the fungi of the class Zygomycetes were reported for Mucor circinelloides (van Heeswijck and Roncero, 1984), P rasitella simplex (Burmester 1992), Phycomyces blakesleeanus (Reυualta and Jayaram 1986), Rhizopus niveus (Takaya et al. 1994; 1996), Rhizomucor pusillus (Wada et al. 1996) and Absidia glauca (Wδstemeyer et al., 1987). In most studies, selection is based on the complementation of auxotrophic recipients because the transformation efficiency using a dominant marker is typically low. Transformation studies indicate that all studied zygomycetes tend to replicate introduced plasmids exclusively autonomously (Schilde et al., 2001). As a result, the mitotic stability of a transformed zygomycete is low. Only when using a strong selection pressure on marker stability after transformation and by using complementation assays, transformants could be isolated in which the introduced plasmids were integrated in the genome.

It stems from the above that the development of inexpensive and flexible biological production systems for the production of various kinds of commercially relevant molecules depends to a significant extent on the construction of a genetically engineered host cell wherein the production of such a molecule can be optimised.

The filamentous fungus Blakeslea trispora is an important industrial source for among others natural carotene, as it contains beta-carotene and precursors for beta- carotene synthesis. Unfortunately, no transformation methods are available for B. trispora to date.

The present invention provides a method for producing at least one molecule of interest by a host cell, comprising providing a host cell with at least one isolated or recombinant nucleic acid, culturing said host cell and allowing said host cell to produce said at least one molecule of interest, wherein said host cell comprises a fungus of the family Choanephoreaceae. In a preferred embodiment of the invention, said host cell comprises a Blakeslea spp., such as Blakeslea trispora.

Earlier studies have proven that B. trispora strain improvement through mutagenesis is difficult because all life stages of B. trispora are multinucleate. In those sporadic cases in which Blakeslea mutants were obtained which showed an altered carotene composition, they were found to sporulate very poorly or not at all (Metha and Cerda-Olmedo, 1995). Defective sporulation is a shortcoming for a mutagenic approach to increase or modulate the carotene content of Blakeslea. In addition, the increases in carotene production in Blakeslea mutants, as described in Hterature thus far, are modest (Finkelstein et al., 1995). For comparison, certain Phycomyces blakesleanus mutants accumulate nearly pure β-carotene at close to 1000 times the wild-type level (Cerda- Olmedo 1989). Nevertheless, from an economic point of view B. trispora is still the most preferred producer of carotenoids.

Despite many efforts, all attempts to generate Blakeslea spp. stable mutants in an efficient manner have failed thus far. As a consequence, B. trispora and other members of the family of Choanephoreaceae could not be fully exploited as a host cell for the production of valuable molecules, such as carotenoids. The invention now provides a solution for this problem by providing a method for producing a molecule of interest by a fungus of the family Choanephoreaceae, wherein said fungus is provided with at least one isolated or recombinant nucleic acid allowing production of said molecule of interest, for instance by modulating at least one component related to the production of said molecule.

In a preferred embodiment, modulating comprises stimulating or enhancing the production of a molecule of interest by a host cell by providing a host cell with a nucleic acid encoding a protein, such as an enzyme, to enhance a component related to the production of said molecule of interest. Some host cells are natural producers of one or more molecules of interest. In a specific aspect of the invention, the production of a desired molecule by a natural hyperproducer is further enhanced by providing said host cell with an isolated nucleic acid. In one embodiment, a nucleic acid is used which allows modulation of a component related to the production of said desired molecule. For example, a nucleic acid encoding a homologous or endogenous protein is used when practicing a method according to the invention, e.g. to achieve overexpression of said endogenous protein or polypeptide. In one aspect, an endogenous protein comprises a protein with enzymatic activity. Overexpression of an enzyme catalysing the production of a desired molecule or a precursor of said molecule typically leads to enhanced production of said molecule.

In yet another aspect of the invention, a molecule of interest is produced by a host cell by providing said host cell with at least one nucleic acid capable of modulating at least one endogenous component, such as an enzyme, related to the production of said molecule of interest. In one embodiment, said at least one endogenous component is inhibited. For example, the invention provides a method to reduce the activity of an enzyme involved in the conversion, e.g. degradation, of a molecule of interest to a less valuable molecule, thereby enhancing the accumulation of a desired molecule. Reduction or inhibition of an endogenous enzyme of a host cell can for instance be achieved by homologous recombination via controlled or site specific genomic integration. Inhibition by anti-sense RNA and preferably by double stranded anti-sense RNA is another method to obtain reduction or inhibition of endogenous enzyme activity in a host cell. (Bass BL., 2000; Tijsterman M., et al 2002).

In another embodiment of the invention, a host cell is provided with a nucleic acid encoding a heterologous protein or polypeptide. Said heterologous protein comprises a heterologous enzyme, which for example allows the production of a new product by a host cell via the conversion of one or more substrates, be it produced via an endogenous or via a heterologous pathway. Thus, according to a method provided it is possible to alter the profile o the molecules produced by a host cell. As is discussed below, expression of one or more heterologous enzymes in the fungus B. trispora is suitably used to change the profile of metabolites that is produced by said fungus. Expression of an enzyme can also give rise to the production of metabolites, such as carotenoids, that are normally not produced by said host cell. In another embodiment, a method for producing a molecule of interest by a host cell relates to co-regulated (over)expression of multiple enzymes.

In one embodiment of the invention, a molecule of interest comprises a metabolite. The term metabolite as used herein refers to any substance produced or used by a host cell during metabolism. In a preferred embodiment, a molecule of interest comprises a metabolite of the carotenoid pathway (see Fig. 1), such as β-carotene (also known as pro-vitamin A), lycopene, canthaxanthin or astaxanthin. Carotenoids are important compounds for human health and development, and are widely distributed in, amongst others, fruits and vegetables. Due to their antioxidant, pro-vitamin A activity (Weisburger 1991), inhibition of cancer cell proliferation (Zhang et al., 1992), immune system stimulation (Jyonouchi et al., 1995) and other health promoting properties, carotenoids have been the subject of numerous studies. The anti-tumor action of β- carotene has been extensively described and is presumed to arise from its provitamin A activity (Johnson, 2002). Besides its antioxidant property (singlet oxygen quenching and peroxyl radical scavenging), lycopene is also known for its role in growth control and induction of cell-cell communication (Stahl and Sies, 1996). Epiάemiological studies implicate lycopene in the prevention of cardiovascular diseases and some types of cancers, such as prostate cancer and gastrointestinal cancer (Clinton, 1998). Studies concerning the beneficial role of astaxanthin revealed preventive effects against cancer and several degenerative diseases (Jyonouchi et al., 1995).

Besides their use as "functional food" because of their health stimulating effects, carotenoids are also used as food supplements and colorants. The carotenoid beta- carotene is used as a natural vitamin, as an antioxidant, and as an orange/red pigment in food, feed, pharmaceuticals and cosmetics. Astaxanthin, for example, is used as the "natural" colorant in the aquaculture farming and in the poultry industry. For the farming of aquatic animals like salmon, trout, shrimp, lobster and other red or pink organisms, astaxanthin is typically used as a fish feed additive in order to colour their flesh. Since the colour of the meat of these expensive fish is important for the appreciation by the consumer, the use of astaxanthin as a fish feed additive is an absolute requirement for marketing. An increase in aquaculture farming and an increased awareness ofthe beneficial effects of carotenoids for human health contribute to an ever growing demand for carotenoids, be it obtained via synthetic or biological procedures. Efficient commercial chemical synthesis and extraction processes for beta- carotene have been used since the 1950s. However, due to Food and Drug Administration (FDA) regulations covering chemically synthesised products, food additives such as carotenoids are preferably obtained from biological sources.

A recently developed economic bioprocess is based on the culture of hypersaline green microalgae (Dunaniella spp) in salt ponds or lakes. However, this bioprocess can however only be used in countries in which suitable climatic conditions and pristine salt- lake environments prevail. As said, the Mucorales fungus Blakeslea trispora is a second important industrial source for natural carotene, containing β-carotene and precursors of its synthesis. Other Mucorales fungi which are natural beta-carotene producers include Choanephora, Mucor, Parasitella, Phycomyces, and Pilaria. However, these are lower producers compared to Blakeslea species.

With a method according to the invention, it is now possible to modulate Blakeslea spp. in such a manner that it produces a substantial level of one or more carotenoids, such as β-carotene (also known as pro- vitamin A), lycopene or canthaxanthin. For example, a host cell is provided with a nucleic acid encoding an enzyme involved in the carotenoid biosynthetic pathway, such as prenyltransferase, geranylgeranyl diphosphate (GGPP) synthase, phytoene synthase, phytoene desaturase or lycopene cyclase. As mentioned before, the production of a molecule of interest can also be enhanced by reducing the activity of an enzyme involved in the conversion, e.g. degradation, of a molecule of interest to a less valuable molecule, thereby enhancing the accumulation of a desired molecule. Reduction or inhibition of an endogenous enzyme involved in the carotenoid synthesis, achieved by genetic inactivation such as homologous recombination or gene silencing through anti-sense DNA technology, is advantageously used to achieve accumulation of an intermediate or precursor molecule of interest. For example, a method is provided for the functional inactivation of lycopene cyclase in a host cell, catalysing the conversion of lycopene to beta-carotene, which generally results in lycopene accumulation in said host cell.

In an other embodiment, a method is provided for producing a molecule of interest wherein said molecule comprises a metabolite of the mevalonate pathway or a metabolite of the isoprenoid pathway (see Figure 4). A host cell is for example provided with at least one gene encoding acetoacetyl-CoA thiolase (AACT); 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGS); HMG-CoA reductase (HMGR); mevalonate kinase (MK); 5-phosphomevalonate kinase (PMK); or 5-diphosphomevalonate decarboxylase (PMDC). Again, the invention furthermore provides inactivation of one of these enzymes in a host cell to obtain a host cell which produces or accumulates one or more mevalonate and/or isoprenoid metabolic intermediates. Besides inactivation, replacement of an endogenous enzyme by an enzyme that catalyses the same reaction, but with better characteristics with regard to catalytic properties and feedback inhibition, is also comprised by the invention.

In yet another embodiment of the invention, a method is provided for modulating the production of a polypeptide of interest by a host cell. To this end, a host cell is provided using a method according to the invention with a nucleic acid, such as a DNA sequence, encoding said polypeptide of interest. A polypeptide according to the invention comprises a homologous, or endogenous, polypeptide, encoded by a homologous nucleic acid sequence, as well as a heterologous peptide encoded by a heterologous nucleic acid sequence. A heterologous sequence according to the invention comprises any open reading frame coding for a protein of interest. Such a protein of interest typically comprises a valuable protein, such as human serum albumin, or a cytokine such as I -3, a protein hormone such as insulin, factor VIII, tPA, EPO, α-interferon, and the like. Also provided is a method for using Blakeslea spp. as a host cell for the production of industrial enzymes, detergent enzymes such as proteases and lipases and the like, cell wall degrading enzymes, such as xylanases, pectinases, cellulases, glucanases, polygalacturonases and the like, and other enzymes which may be useful as additives for food and feed (e.g. phytases and phospholipases).

It is likely that, in the near future, fungi and moulds will be frequently used for the production of various kinds of commercially important enzymes and proteins. The reason for it is the, in some cases, extensive ability to secrete enzymes and proteins, to fold protein in the correct secondary structure, due to the secretion system, and their ability to glycosylate proteins. Each fungus and yeast produce different structures of glycomoieties, which gives different recognition signals to immune systems. Both the correct secondary structure of a protein of interest as well as its glycosylation profile (oligosaccharide chains) are often indispensable for their biological activity and in some cases also for protein stability which for instance protects a protein against the attack of proteinases. As is exemplified in the detailed description, the invention now allows to provide a filamentous fungus of the family of Choanephoreaceae, such as Blakeslea spp., with an isolated or recombinant nucleic acid encoding a polypeptide of interest. Herewith, the invention provides a method for expressing a recombinant protein using Blakeslea spp. as a host cell, comprising culturing a host cell, said host cell provided with a nucleic acid encoding a protein of interest, and allowing expression of the protein of interest by said host cell.

A method is provided for producing at least one molecule of interest by a Blakeslea spp., comprising providing Blakeslea with at least one isolated or recombinant nucleic acid. In a specific embodiment of the invention, Blakeslea is provided with a nucleic acid by polyethyleneglycol (PEG)-mediated transformation of protoplasts. For protoplast transformation, the quality and the quantity of the protoplasts is essential. In our approach protoplasts ware made from germinating spores. The length of the germ tubes is a critical parameter.If the germ tubes are too short or too long, hardly any protoplast will be released. It was found that the optimal germination tube length for making protoplasts is 2-3 times the spore diameter. To obtain a substantial amount of germ tubes with a length of 2-3 times the spore diameter , synchronisation of the germination process is a prerequisite . Synchronisation of the germination of spores of B. trispora has not been described before. As is exemplified in Example 9, the invention provides a method to synchronise germination of spores of Blakeslea spp. This approach may also be applied to spores of other fungi.

According to the invention, germinating spores are preferably treated with one or more cell wall digesting enzymes (e.g. chitosanase) to obtain protoplasts. Subsequently, the protoplasts can be provided with a nucleic acid, for instance using PEG. However, protoplasts can also be transformed by other methods such as electroporation.

Herewith, a method is provided for providing a host cell with at least one isolated nucleic acid, wherein said host cell comprises a fungus of the family Choanephoreaceae. Thus, the invention provides a transformation procedure for a fungus of the family of Choanephoreaceae, which as said was not available before.

A transformation method of the invention can be used to provide a fungus with a nucleic acid of interest. In a preferred embodiment of the invention, a host cell is provided with an isolated or a recombinant nucleic acid, wherein said nucleic acid is further provided with a transcription promoter and a termination sequence. A transcriptional promoter typically promotes stable gene expression in a host cell. It is preferred that said promotor comprises a homologous expression signal of said host cell, for instance a region found upstream of a highly expressed gene of said host cell. A highly expressed gene is for example a gene encoding an enzyme of the glycolytic pathway, such as glyceraldehyde-3-phosphate dehydrogenase (gpd). As is exemplified herein, we cloned the promoter and termination regions of gpd of B. trispora. A preferred vector comprises a PgdpT-G418-TgpdT sequence (see example 6). Furthermore, it was found that an integrative vector comprising at least part of an rDNA sequence of B. trispora works particularly well in a method of the invention.

The invention further provides a host cell and a nucleic acid for use in a method according to the invention. For example, the invention provides recombinant DNA comprising a transcription promoter, downstream a termination sequence and in between a sequence to be expressed, in operable linkage therewith. A preferred transcription promoter according to the invention comprises a region found upstream of a highly expressed gene of Blakeslea trispora, in particular the glycolytic pathway gene coding for glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). A transcription terminator comprises for instance a region found downstream of the above-mentioned gene.

Use of a host cell for the production of an industrially relevant molecule of interest, or a precursor thereof, is also provided herein. According to a method provided, a wide range of different molecules can now be produced in a host cell of the family of Choanephoreaceae. Such molecules comprise both proteinaceous and non-proteinaceous substances, ranging from pharmaceutically relevant enzymes, hormones, cytokines to food or feed additives, such as carotenoids, vitamins, colorants, antioxidants, and precursors thereof.

DETAILED DESCRIPTION

Trisporic acids are a group of compounds, produced upon mating ofthe (+) and (-) mating type cultures of mucoraceous fungi. They are a group of oxygenated 18 carbon atom derivatives of β-carotene (Fig. 2). Trisporic acids are end products o the sex hormones (pheromones) that stimulate both carotenogenesis and inducing sexual reproduction (development of zygophores; van den Ende, 1978; Sutter, 1987). These sex- specific pheromones are, depending upon the species, between 100- and 1.10⁸ —fold less abundant than the end product, trisporic acids (Sutter et al., 1989). Combined mating type cultures of B. trispora accumulate around 2% trisporic acid- A, 15% trisporic acid-B, and 83% trisporic acid-C also called factor beta 1, beta 2, and beta 3 (due the fact that they stimulate the synthesis of β-carotene) (Caghoti et al., 1966). Figure 2 illustrates the relationship between the sex-specific pheromones and trisporic acids.

Trisporic acids obtained from mixed cultures of B. trispora can be used to derepress enzyme(s) of the carotenoid biosynthetic pathway thereby stimulating the production of β-carotene in cultures with separate strains. For example, addition of 21.8 units of trisporic acid increases by 422% the yield of β-carotene in the (-) strain and by 71% in the (+) strain of B. trispora. However, stimulation of he carotene biosynthesis per unit trisporic acid in cultures of the (-) strain was only 20-30% of the carotene biosynthesis in mixed cultures (Sutter and Rafelson, 1968).

If trisporic acid is not added to the growth medium, mating of the (+) and (-) mating type of B. trispora is from an industrial / commercial point of view a prerequisite for a profitable carotenoid production. The addition of trisporic acid is also needed under certain specific growth conditions. The pathway towards β-carotene has to be blocked in order to produce lycopene, a precursor molecule of β-carotene (Fig. 1). Yet this can be achieved either by blocking the conversion of lycopene into β-carotene with a specific inhibitor ofthe lycopene cyclase or by using a mutant strain in which the lycopene cyclase has been inactivated genetically. Trisporic acid is a degradation product of β- carotene (Fig 3). No β-carotene production means no trisporic acid production, which means no stimulation of the lycopene production.

Trisporic acid is not only a very intense stimulator of carotene synthesis, but it is a general stimulation of all isoprenoid biosynthesis, including sterols (especially the triterpene ergosterol). A schematic representation o the mevalonate and isoprenoid pathway is shown in Figure 4. Although the mechanism of increased flux in these pathways remains unknown the increase in sterols is still remarkable (Gooday, 1994). 3- Hydro-3-methylglutaryl CoA (HMG-CoA) reductase (HMGR) is considered to be a major point of regulation of the isoprenoid pathway (Hampton et al. 1996). The post- translational regulation of HMGR is mediated in two different ways. Firstly, HMGR is regulated by phosphorylation / dephosphorylation. The enzyme is inactivated upon phosphorylation, mediated by an AMP-activated protein kinase (Carling et al., 1994; Mitchenhill et al., 1994). Secondly, HMGR activity is controlled by (protein) degradation of the enzyme. The degradation is controlled by sterols (Nakanishi et al., 1988; McGee et al., 1996). One would expect that an increase in sterols in B. trispora should lead to a decrease of the HMGR activity and consequently a decrease in the carotenoid production. Nevertheless, in B. trispora a simultaneous increase of β-carotene and sterols takes place. It might be suggested that trisporic acid is also involved in the regulation of HMGR degradation by protecting the enzyme for degradation. The fungal metabolite lovastatin (also known as mevinohn) is a potential inhibitor of HMGR. It is beheved that micro-organisms which are resistant to lovastatin have a modified HMGR that is no longer inhibited by sterols. Finkelstein and co-workers (1995) showed that selection of B. trispora mutants on lovastatin yielded a mutant that (after mating with an opposite mating type) doubled its β-carotene production from 3.5 g 1 to 7 g 1 after 7 days of fermentation. This suggests that HMGR may have become less sensitive to sterols, and subsequent degradation, resulting in higher levels of carotenoids in B. trispora.

However, the maximum amount of β-carotene productivity in B. trispora appears to be limited since after several rounds of mutagenesis no further increase in β-carotene content was reported. This may partially be due to adverse mutations, which occur during random mutagenesis.

Recombinant DNA technology could probably overcome these bottlenecks in strain development. However, in order to be able to obtain recombinant strains one needs an effective transformation process and adequate expression regulating sequences. The transformation methods and new expression tools provided herein create a wealth of possibilities for increasing the carotenoid levels and producing new and valuable biomolecules in B. trispora.

Optimisation of carotenoid production in B. trispora with recombinant DNA technology

Identification of B. trispora cells or protoplasts that have taken up isolated or recombinant nucleic acid can be achieved by exploring dominant selection markers or marker genes which complement auxothrophic mutants. It is preferred that the marker genes are present in B. trispora in an expressible form. This can be achieved by the coupling the gene of interest to an upstream region (promoter) which promotes stable gene expression in B. trispora and a signal for the termination on transcription (terminator) in B. trispora. Examples of such promoters are the upstream region of cloned genes from other Zygomycetes e.g. the upstream regions of the genes encoding actin or the transcription elongation factor (ie/) from Absidia glauca (Burmester, 1995) or the upstream regions of the genes encoding 3-phosphoglycerate kinases of Rhizopus niveus (Takaya et al., 1994). Preferably, the upstream regions of genes from B. trispora are used. More preferably, promoters of highly expressed genes in B. trispora are used. Examples of such genes, of which the expression patterns have been identified in other yeasts and fungi, include the glycolytic pathway genes, phosphoglucoisomerase, phosphofructokinase, triosephosphoisomerase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, phosphoglucomutase, enolase, pyruvate kinase and alcohol dehydrogenase. Isolation of B. trispora genes and upstream regions from genomic DNA of Blakeslea is readily achieved using molecular biological techniques well known to the person skilled in the art.

A marker gene comprises an open reading frame coding for reduced sensitivity against a selective agent. Whereas the open reading frame coding for an enzyme giving G-418, kanamycin and/or neomycine resistance, such as an enzyme encoded by the nptll gene, was used satisfactorily in a method according to the invention, the invention is not limited to the use of this selection marker. Other useful dominant selection markers include, but are not limited to, phleomycin or hygromycin resistance genes.

In addition, marker genes that complement an auxotrophy of the host (auxotrophic selection marker) can be used. Suitable auxotrophic strains are e.g. mutants in the biosynthetic pathway of amino acids (e.g. tryptophane, leucine and histidine) and nucleosides (e.g. uridine-mono phosphate and uracil). Examples of such genes include the genes encoding α-isopropylmalate isomerase (leuA), orotidine-5'- phosphate decarboxylase (pyrG) and dihydroorotic acid dehydrogenase (ura). For this approach, isolated and characterised genes from closely related fungi, especially Zygomycetes, can be used (e.g. Diaz-Minguez et al., 1990; Benito et al., 1992; Roncero et al., 1989; Takaya et al., 1996). More preferably, the homologous genes from B. trispora are used. These genes can be isolated from B. trispora by e.g. heterologous hybridisation, heterologous complementation, amplification techniques or other known procedures to those skilled in the art.

Using a method for providing a host cell with a nucleic acid as herein disclosed, essentially any gene of interest can be expressed in B. trispora. For example, this includes genes involved in the carotenoid pathway with the objective to manipulate the carotenoid production in B. trispora. Various carotenoid biosynthetic genes have been isolated from bacteria, algae, fungi and plants (reviewed by Lee and Schmidt-Dannert, 2002), including the genes encoding geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase. For certain applications homologous genes like lycopene cyclase/phytoenesynthase (CARRP) and phytoene dehydrogenase (GARB) from B. trispora may be preferred. (De la Fuente Moreno J.L. et al 2003). Any one (or more) of these genes can be used to manipulate the enzymatic activity in B. trispora, by placing the gene of interest under control of a high level expression promoter. According to a method provided herein, increased expression of a gene of interest, be it a homologous or a heterologous gene, can change the carotenoid composition by changing the accumulation of intermediates and/ or end product. Expression of a protein of interest may also give rise to carotenoids not known to be produce in B. trispora naturally, such as canthaxanthin. An open reading frame that is suitably employed in a method provided includes but is not limited to the one encoding a ketolase, which converts β-carotene in canthaxantin obtained from Agrobacterium aurantiacum (Misawa et al., 1995). Also, co-regulated overexpression of multiple carotenogenic genes can be carried to produce interesting carotenoids. For example the carotenoids astaxanthin (by co expression of genes encoding a β-carotene ketolase and a β-carotene hydroxylase from e.g. A. aurantiacum) and lutein (by co-expression of genes encoding a lycopene-ε-cyclase (e.g. from Arabidopsis thaliana) and a β-carotene hydroxylase (from e.g. A. aurantiacum)).

From hterature it is known that the stability of HMGR of S. cerevisiae is controlled by protease attack upon the membrane-spanning domains (Hampton et al., 1996). Shimada and co-workers (1998) demonstrated that (over)expression of a truncated form of the S. cerevisiae HMGR in Candida utilis resulted in an increased carotenoid production. Even higher production performances were obtained when this modification was combined a specific inactivation of the squalene synthase encoding gene (ERG9). Expression of a recombinant HMGR, preferably the gene encoding HMGR from B. trispora in B. trispora, that has become insensitive for phosphorylation and degradation through genetic engineering is one of the approaches provided herein that allows an increased carotenoid production.

Roa and Modi (1977) showed that activation of carotenogenesis in B. trispora by trisporic acid molecule was by derepression of the enzyme(s) catalysing the conversion of 5-phosphomevalonate to dimethylallyl diphosphate (Fig. 4). In one embodiment ofthe invention, the 5-phosphomevalonate and 5-diphosphomevalonate decarboxylase encoding genes are isolated from B. trispora using hybridisation or amplification techniques know in the art. Introduction of deregulated gene copies, by placing the expression of a heterologous promoter, is an advantageous approach to overcome the derepression of the carotenogenic pathway in the absence of trisporic acid.

Of special interest according to the present invention, are nucleic acid sequences coding for carotenoid biosynthetic genes from B. trispora. Isolation of these genes is possible using known procedures to those skilled in the art (see review by Sandmann, 1994), such as heterologous hybridisation and/or complementation of carotenoid biosynthetic mutants of B. trispora or recombinant E. coli strains which has modified in order to produce carotenoids (Misawa et al., 1995).

Via site specific integration by homologous recombination specific carotenoid biosynthetic mutants can be generated. Once cells have been transformed according to a method provided, some transformed plasmid will integrate at the specific locus resulting in a non-functional gene copy.

The carotenoid pathway as present in B. trispora (Fig. 1) is profitably used as a source of intermediates like geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranygeranyl diphosphate (GGPP) wherein these intermediates are converted to respectively monoterpenes, sesquiterpenes, and diterpenes. Expression of a gene of interest related to the isoprenoid pathway can give rise to new and valuable terpenoids in B. trispora. An open reading frame suitable employed in such a method includes but is not limited to the ones encoding amorpha-4,ll-diene synthase obtained from Artemisia annua L (Wallaart et al., 2001) and taxadiene synthase obtained from Taxus brevifolia (Trapp and Croteau, 2001). The first enzyme converts farnesyl diphosphate into amorpha-4,ll-diene, which is the first step of the biosynthetic pathway of the antimalarial drug artemisinin. The latter one, which is a diterpene cyclase, catalysis the first step in taxol biosynthesis.

Experimental part

Strains: Escherichia coli DH5α: supE44ZαcU169 (80ZαcZM15) hsdRll recAl endKl gyr A96 thi-1 relAl

Blakeslea trispora DSM 2387, mating type + Blakeslea trispora DSM 1254

Plasmids: pPRlTN (Verdoes et al. 1999) pGEM® T-Easy (Promega)

Media:

LB (Luria Bertani): 10 g 1 bacto trypton, 10 g/1 yeast extract, 5 g/1 NaCl. PDB (Potato Dextrose Broth, Duchefa, Haarlem The Netherlands); 4 g/1 Potato extract, 20 g/1 glucose, thiamine hydrochloride 0.0002 % (w/v).

YpSs medium: yeast extract 4 g/1, soluble starch 15 g/1, K2HPO4 1 g/1, and

MgSO₄.7H₂O 0.5 g/1.

For plates bacto agar (15 g/1) was added to the media.

For YpSs selection medium plates: sorbitol 109 g/1 was added and after autoclaving 2 ml kanamycin (100 mg/ml) was added per litre medium.

GAY medium: glucose 70 g/1, L-asparagine 2.0 g/1, yeast extract 1.0 g/1, K2HPO4

1.5 g/1 and MgSO4.7H2O 0.5 g/1. The pH was adjusted to 5.5. The solution was sterilized by autoclaving. Thereafter, 30 microliter of a filter sterilized stock solution of thiamine hydrochloride was added.

V8-medium: V8 vegetable juice (CampbeU Foods N.V. Belgium) 200 mlΛ, CaCO₃ 3 g/1, Bacto agar 16 g/1. The solution was sterihsed by autoclaving.

MMY medium: Glucose monohydrate 20 g/1, MgSO4.7H₂O 0,5 g/1, L-asparagine monohydrate 1,5 g/1, yeast extract 2 g/1. (separately dissolved under heating):

- 1 ml/1 solution A: - 0.012 g thiamin dichloride

- Fill to 100 ml with distilled water (Every month fresh)

- 1 ml/1 solution B: - 0.5 g FeCL

- Fill to 100 ml with distilled water

- 1 ml 1 solution C: - 0.06 g HBO3

- 0.04 g (NH4)6Mo₇O24.4H₂O

- 0.2 g CuSO₄.5H₂O

- 2.0 g ZnSO₄.7H₂O

- Fill to 1 liter with distilled water

- 2.5 ml/1 solution D - 184 g KH₂PO₄

- Fill to 1 liter with distilled water

The solution was filled to 1 htre with distilled water and sterilised by autoclaving. Regeneration medium: yeast extract 5 g1, D-glucose 20 g/1, sorbitol 218 g/1. STC buffer: Sorbitol 218,6 g/1, CaCl₂ 1,5 g/1, 1M Tris-HCl (pH 7.5) lOml 1. 60% PEG 4000: Poly ethylene glycol 4000 600g/l, CaCl₂ 1.5 g/1, 1M Tris-HCl (pH7.5) 10 ml/1. When appropriate the following antibiotics were added: Ampicillin 50 μg/ml Kanamycin 100-200 μg/ml

Cell wall digesting enzyme solution:

Chitosanase RD (US Biologicals) 10 mg, NOVO SP 10 mg, both enzymes dissolved in 2 ml 0.6 M sorbitol and after 5 min. of centrifugation at 15000 g in a microfuge the supernatant was used for digesting the cell wall in order to obtain protoplasts.

Methods:

All molecular cloning techniques were essential carried out as described by Sambrook et al. (1989). Most ofthe enzyme incubations and DNA modifications were performed with enzymes purchased from MBI Fermentas (St Leon-Roth, Germany) following the instructions ofthe suppher. DNA was labelled with ³²P- -dCTP using a random hexanucleotide mix and large DNA polymerase fragment I (Klenow).

Isolation of chromosomal DNA from Blakeslea trispora was performed using the DNeasy® Plant mini kit (Qiagen). For the isolation of total RNA of 23. trispora the RNeasy® Plant mini kit (Qiagen) was used. Isolation of DNA f agments from agarose was performed using the Qiaex® II gel Extraction kit (Qiagen), and plasmid DNA from E. coli was isolated with GenElute™ Plasmid Miniprep kit (Sigma). Isolation of PCR fragments was carried out using PCR purification kit. For the cloning of blunt-ended DNA fragments generated during PCR amplification the A tailing procedure as described by the suppher of pGEM® T Easy (Promega) was used. For this procedure recombinant Taq purchased from MBI Fermentas was used.

All nucleotide sequence analysis was carried out by Baseclear (Leiden, The Netherlands)

Transformation of E. coli was performed according the SEM method described by Inoue et αZ.(1990). E. coli was cultivated in LB media with the appropriate selective agents. Seed cultures of B. trispora were prepared by inoculating a flask containing 50 ml GAY medium supplemented with 0.1 % SPAN with chunks of agar medium containing mycehum or 5 x 10⁷ spores. These cultures were incubated for 2 days at 29 °C on a rotary shaker set at 200 rev/min. Reverse transcriptase (RT) reaction were carried as follows. Different amounts of total RNA (0.5; 1; 2; and 4 ul) were mixed in a reaction tube with 0.5 μg oligo dT primer (SEQIDNO: 15) and H₂O was added until the final volume was 10 μl. The RNA and primer were denatured by heating the mixture at 70 °C for 10 min. and directly chilled in ice. Then the following components were added:

- 4 μl of a 5 x First strand buffer (provided by Life Technologies™)

- 0.4 μl DTT (100 mM)

- 2 μl dNTPs (10 mM)

- 1.6 μl H₂O the reaction mixtures were preheated (2 min. 42 °C) and finally, 2 μl (200 U/μl) of Superscript ™II RNase H-Reverse Transcriptase (Life Technologies™) were added. The first strand reaction was performed at 42 °C for 1 h and the reaction was stopped by incubating the reaction mixture for 10 min. at 70 °C.

Polymerase Chain Reactions (PCR) experiments were performed in mixtures having the following composition:

1 x Pfx Amplification buffer

10 ng plasmid DNA or 1 μg chromosomal DNA

0.3 mM of each primer

- 0.3 mM dNTP mix

- 1.0 mM MgSO₄

1.5 unit Platinum Pfx DNA polymerase (Life technologies®) autoclave, distilled water to 50 μl Reactions were carried out in an automated T gradient thermocycler (Biometra®). Conditions: 5 min. 95 °C (for DNA, primers, reaction buffer and water), then the reaction mixture (dNTPs, MgSO4 and polymerase) was added and PCR was continued by 35 repeated cycli; 30 sec. at 92 °C, 45 sec. at 50 °C, 1-2 min. at 68 °C ending with an additional elongation step of 10 min. at 68 °C. For the construction of the chimeric DNA fragments (so-called recombinant PCR) the reactions were performed as described above except that the two DNA fragments with compatible ends were added as a template in equimolar amounts.

Example 1

Synthesis of a specific probe for the glvceraldehvde-3-phosphate dehydrogenase encoding gene (gpd) o B. trisyora by PCR The polymerase chain reaction technique was used to synthesise a specific DNA probe for the glyceraldehyde-3-phosphate dehydrogenase (GPD) encoding gene of B. trispora. GPD is a highly expressed enzyme of the glycolytic pathway. In one of the public databases a partial sequence of a putative GPD encoding gene of a B. trispora strain (CBS130.59) was retrieved (EMBL accession number AJ278318). Based on the nucleotide sequence two primers (SEQIDNO: 7 and 8) were designed. These primers were used to synthesise a DNA fragment with chromosomal DNA of B. trispora (strains

DSM 2387) as template. A single DNA fragment of approximately 300 bp was synthesised under the applied conditions. The fragment was purified from the PCR mixture, ligated together with pGEM®-T Easy. The ligation mixture was transformed into E. coli DH5α and the cell/DNA mixture was plated on LB-agar plates containing ampicillin, IPTG (0.1 mM) and X-gal (20 mg/ml). Plasmid DNA was isolated from several white colonies. One plasmid, named pBtgpd#5, was selected for restriction analysis and nucleotide sequence determination (SEQIDNO: 1). Comparison of the nucleotide and deduced amino acid sequence in the public databases showed significant homology with

GPD encoding genes from other fungi.

Example 2

Isolation of the gpd gene of B. trispora

Chromosomal DNA isolated from B. trispora DSM 2387 was incubated with a selection of different restriction enzymes (Bglϊl, BamBl, EcόRl; HindlH, Kpnl, Sacϊ, Pstl, Xbal, Xhoϊ). The DNA fragments were separated on 0.8 % agarose gel, transferred to a Nytran N membrane and hybridised with the ³²P labelled- EcόRl fragment of 334 bp, isolated from of pBtgpd#5, as probe. The blot was hybridised for 16 h in a hybridisation solution (1 M NaCl; 1% SDS; 10 % PEG 6000) at 65 °C. Afterwards the blot was washed 3 times for a period of 15 min. at 65 °C with a wash solution (2 X SSC; 0.1 % SDS). From the autoradiogram it was concluded that a H dlll fragment of approximately 2.7 kb hybridised with the probe whereas all of the hybridising fragments, obtained after the incubation with other restriction enzymes, were larger than 8 kbp. The presence of a putative HinάlU restriction site in the insert of pBtgpd#5 suggest that this 2.7 kb fragment should, depending on the presence and size of introns, the coding region of the gpd gene and a significant part of the promoter region.

Cloning ofthe promoter region ofthe gpd gene of B. trisopora by inverse PCR To isolate the Hindlll fragment of 2.7 kb the method of inverse PCR was applied. In short, 5 μg of chromosomal DNA of B. trispora DSM 2387 was digested with Hindϊlϊ. After separation on a agarose gel DNA fragments with a length between 2.5 and 3.0 kb were isolated and purified. Finally the fragments were dissolved in 50 μl H₂O. The presence of the fragment of interest was tested by a PCR using the primers 5' gpd (SEQIDNO: 7) and 3'gpd (SEQIDNO: 8). Different amounts (2.5; 5; and 10 μl) of the fragments were self-ligated in a total volume of 50 μl containing 1 x ligase buffer and 2 Units of T4 DNA ligase. The hgation mixture was incubated at 14 °C for 16 h. Subsequently 5 μl of each hgation mixture was used in the PCR. For these PCRs both rec Taq and Hotstart Taq were tested. Only the combination consisting of HotStart and the primers 5' Bt gpd inv2 (SEQIDNO: 11) and 3'Btgpd2 (SEQIDNO: 9) resulted in the amplification of DNA fragment of approximately 2.5 kbp. The specificity of the amphfied product was tested by using two nested primers 5'Bt gpd3inv (SEQIDNO: 10) and 3'Btgpd3inv (SEQIDNO: 12). As this PCR resulted in the synthesis of a fragment of about 2.2 kbp it was decided to purify and clone the product in pGEM®-T Easy. The fragment was cloned in two orientations yielding the plasmids pBtinvPCR#2.1 and pBtinvPCR#3.7. By making use of standard sequence primers (M13rev and T7), internal deletion clones (Ncoϊ) of pBtinvPCR#2.1 and pBtinvPCR#3.7, and primers deduced from the data obtained after a first sequence run, e,g. Btgpd 4 (SEQIDNO: 13) and Btgpd 5 (SEQIDNO: 14) the total nucleotide sequence of the fragment was determined (SEQIDNO: 3). The translation start was found by sequence alignment of the deduced amino acid sequence with known GPD proteins. The fragment contains an upstream region (promoter) of 1.4 kb and the N-terminal part of the GPD (aa 1 - 309) protein. Within this fragment also three introns were present (SEQIDNO: 3).

Cloning of the terminator region of the gpd gene of B. trisoyora by RT- PCR

An approach based on reverse transcription (RT) and polymerase chain reaction (PCR) was set up to isolate the carboxy- terminal domain of the GDP protein of B. trispora and the region of transcription termination. Total RNA was isolated from mycehum of B. trispora DSM 2387. Therefor an Erlenmeyer flask of 500 ml with 150 ml PDB medium supplemented with thiamine.HCl (vitamin B) was inoculated with an agar plug of B. trispora DSM 2387. The flask was incubated on a shaking platform (180 rpm/min) at 28 ° for 48 h. Total RNA was treated with RNAse-free DNAse. Four μl of this total RNA were then used in a RT reaction according to the protocol of the suppher of the reverse transcriptase (Life Technologies). One μl (out of 20) was used as template in the following PCR using the primers 5'Btgpd (SEQIDNO: 7) and oligo-dT-PCR primer AS (SEQIDNO: 15). Analysis of the reaction mixture by DNA electrophoresis showed the amplification of a specific fragment with a length of approximately 650 bp. The DNA fragment was purified from the PCR mixture, A-tailed and cloned in pGEM®-T Easy. The nucleotide sequence of the insert in pCLIPPBt#20 was determined. The fragment encodes the C-terminal domain ofthe GPD protein (aa 234 -337) and a termination region of 182 bps (SEQIDNO: 2). The entire amino acid sequence of GPD of B. trispora DSM 2387, consisting of 337 amino acids, is shown in SEQIDNO: 4.

Example 3

Isolation of an autonomously replicating sequence for B. trispora To set up a transformation procedure it would be beneficial to design a replicative vector. Such a vector should contain an autonomously replicating sequence (ARS). Compared to integrative vectors it is expected that much higher transformation efficiency can be obtained. However the isolation of an ARS from B. trispora or an ARS functional in B. trispora has not been reported yet. But it has been shown that sequences from e.g. the yeast Saceharomyces cerevisiae, which are involved in autonomously replication, can be functional in Zygomycetes (van Heeswijck and Roncero, 1984). Furthermore fragments supporting autonomously replication, the so-called ARS, from zygomycetous fungi have been identified and isolated by functional analysis in S. cerevisiae (Revuelta and Jayaram, 1986; Burmester and Wostemeyer, 1987). And also ARS from Mucor circinelloides and Phycomyces blakesleeanus were used in Rhizopus niveus (Takaya et al., 1994 and 1996).

To construct an autonomously replicating plasmid for B. trispora the ARS element from Absidia glauca (Burmester and Wostemeyer, 1987) was amplified by PCR and cloned in pGEM-T Easy, yielding pGEM-ARS-4^.

Example 4

Isolation of rDNA fragments of B. trispora DSM 2387

In general autonomously replicating plasmids in mucoralean fungi are unstable (Schilde et al. 2001). For industrial applications it is preferred to construct recombinant strains in which the introduced sequence can be maintained without selection e.g. by (site specific) integration in the genome of the recipient strain. To facilitate the efficiency of integration of an introduced plasmid in B. trispora and to minimise the effect of the site of integration on gene expression we used the rDNA loci as integrative platform. As the rDNA cluster is present in multiple copies in the genome it will enhance the change for a stable integration event. Partial nucleotide sequence of Blakeslea trispora ribosomal RNA genes have been submitted to public databases (EMBL accession numbers; AJ278366; AF157124 and AF157178). By in silico restriction analysis the presence of unique restriction sites (CZαl and H dIII) could be established in the parts encoding the 18S rRNA and 5.8S rDNA. To isolate these DNA fragments four primers were designed (SEQIDNO: 16-19). After a PCR using chromosomal DNA as template the 5.8S and 18S ribosomal RNA genes were synthesised as a fragment of about 550 and 1700 bp, respectively. The fragments were isolated and cloned in pGEM® T-Easy yielding the plasmids pBt5.8S and pBtl8S. The complete nucleotide sequences of the inserts were determined (SEQIDNO: 5 and 6)

Example 5

Determination o the resistance level of B. trisyora against kanamycin One of the marker genes that can be used for the selection of putative transformants of B. trispora is the ap S'll/nptll gene (Beck et al., 1982) from transposon Tn5. Expression of this gene results in kanamycin resistance in fungi and bacteria and also G-418 resistance in fungi. A serial dilution of kanamycin was made in both YPD agar medium + vitamin Bl and YpSs agar medium. This resulted in plates with a kanamycin concentration ranging from 50 to 400 μg/ml. These plates were inoculated with a suspension of spores in water. The plates were incubated for 48 h at 28 °C. Although germination was observed on YpSs plates with a kanamycin concentration of 100 μg/ml, there was no continuation of growth of the mycehum. At a kanamycin concentration of 200 μg/ml no germination was observed on YpSs plates, which were inoculated with spores.

From these data it is concluded that kanamycin at a concentration of 200 μg/ml or higher can be used as selective agent for isolation of transformants of B. trispora.

Example 6

Construction of integrative vectors for B. trisyora

The nptll gene, also referred to as the G-418 or kanamycin/neomycin resistance gene, is placed under control of the homologous expression signals which are the promoter and terminator ofthe GPD encoding gene of B. trispora (example 2). The vector pPRlTN was used as starting material. This vector contains the nptll gene and the expression is controlled by the promoter and terminator of the gpd gene ofthe carotenoid producing yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma). The fragment that contains the 18S rDNA gene of 5. trispora contains a H dlll restriction site. This site is used to linearize the vector in the rDNA part prior to transformation. Therefore, the H dIII site in pPRlTN has to be removed. The vector pPRlTN was digested with H dlll and the site was filled by the polymerase activity of the Klenow fragment of E. coli DNA polymerase I. The treated fragment was ligated and the hgation mixture was introduced in E .coli. Plasmid DNA was isolated from several transformants and was analysed using various restriction enzymes. The new constructed vector was named pPRlTNΔHindlll (Figure 5).

To join two unrelated sequences (in this example G418 and ΥgpdBt) a approach of "recombinant PCR" can be used. The procedure will be illustrated hereafter. Two primary polymerase chain reactions (PCRs) were performed to amplify the 3'end of the nptll gene and the terminator region of the gpd gene. For the first the primer combination GC 06 (SEQIDNO: 28) and GC 07(SEQIDNO: 29) and for the latter the primer combination GC08 (SEQIDNO: 30) and GC09 (SEQIDNO: 31) were used. To the two "inside" primers (GC 07 and GC08) a sequence has been added to the 5'end ofthe primers. This implies for GC07 the 5' end of TgpdBt and for GC08 the 3'-end of nptll. In a secondary PCR the overlapping primary PCR products are mixed and denaturated. One of the two heteroduplexes, the one with the recessed 3'end, is extended by the DNA polymerase yielding a fragment that is the sum of the two overlapping primary products. This product is then amplified by using the only the most left- end right-most primers (GC06 and GC09). After the secondary PCR the expected fragment of about 600 bp was gel purified from the PCR mixture and cloned in pGEM®-T Easy, yielding the plasmid pG418-Tgpd. To prevent the introduction of unwanted amino acid substitutions, due to errors introduced by the Tαq DNA polymerase, the complete nucleotide sequence of the insert was checked. The 3' end of the nptll gene and the TgpdBT were re-isolated from the vector pG418-Tgpd as a Ncol-Notϊ fragment of 518 bps. This fragment was used to replace the terminator sequence of he gpd gene of X. dendrorhous (ΥgpdXd) that is present in the vectors pPRlTN and pPRlTNΔHindlll yielding the plasmids pCLIPP89 and pCLIPP89ΔHindIII, respectively. This first cloning steps are illustrated in figure 5.

The recombinant PCR technique, as described above, was used to join the promoter region of the gpd gene of B. trispora with the 5'end o the nptll gene. As the length of the promoter region is not estabhshed three different primary PCR were set up to amplify this region. The combination ofthe primers GC00/GC03 (SEQIDNO: 22 and 25) GC01/GC03 (SEQIDNO: 23 and 25) GC02/GC03 (SEQIDNO: 24 and 25) yielding a promoter region with a length of approximately 1400, 750 and 500 bps- The 5' -end of the nptll gene, a fragment of 213 bp, was amplified using the primers GC04 (SEQIDNO: 26) and GC05 (SEQIDNO: 27) and the plasmid pPRlTN as template. Three secondary PCR were set up with the most left- end right-most primers, GC00/GC05, GC01/GC05 and GC02/GC05). After the secondary PCRs the expected fragments of about 1635, 975 and 720 bp were gel purified from the PCR mixtures and cloned in pGEM®-T Easy, yielding respectively, the plasmids pPgpd(A)Bt-G418, pPgpd(B)Bt-G418 and pPgpd(C)Bt-G418 The promoter regions (PgpdBt) and the 5'end of the nptll gene were re-isolated from these plasmids as a Sαcl- Pstl fragment. These fragments were used to replace the Sacl- Pstl fragment (PgpdXd -G418) in pCLIPP89, yielding pCLIPP90A, pCLIPP90B and pCLLPP90C (see Figure 5). Subsequently, the EcoRI fragment of 550 bp, containing the 5.8S rRNA of B. trispora, was isolated from pBt5.8S and cloned in the EcoRI sites of pCLIPP90A, pCLIPP90B and pCLIPP90C. This yielded the final transformation vectors pCLIPP91A, pCLIPP91B and pCLIPP91C (Fig. 6).

The same cloning procedure was carried out for the vector pCLIPP89ΔHindIII. The outcome of these steps resulted in the plasmids pCLIPP90AΔHindIII, pCLIPP90BΔHindIII and pCLIPP90CΔHindIII (Fig. 7). In these vectors a part of the 18S rDNA of B. trispora was introduced. The 18S rDNA sequence was isolated from pBtl8S as a EcoRI fragment of 1.6 kR The fragment was cloned in the EcoRI site of pCLIPP90AΔHindIII, pCLIPP90BΔHindIII and pCLIPP90CΔHindIII yielding, depending on the orientation o the rDNA sequence, pCLIPP92A, pCLIPP92B and pCLIPP92C (Fig. 7).

In summary, the newly constructed vectors contain an expression cassette for B. trispora and other zygomycetes fungi consisting of ^~PgpdBt-G4l8-TgpdBt and a part of the rDNA sequence of B. trispora. These latter sequences contain unique restriction sites (CZαl and H dlll) and are included to facilitate stable integration of the vector in the genome after the introduction of the plasmid DNA in a cell.

Example 7

Construction of autonomously replicating vectors for B. trispora To construct an autonomously replicating plasmid for B. trispora the ARS element from Absidia glauca was reisolated from the plasmid pGEM-ARSAg (see example 3) as an EcoRI fragment and cloned in the unique EcoRI site of pCLIPP90A-C yielding pCLIPP97A-C, respectively (Fig. 8).

Example 8

Sporulation and harvesting of spores

A plug with a diameter of 0.5 cm of freshly grown mycehum of B. trispora strain DSM 2387 was placed in the centre of a 9 cm diameter, V8 plate with 50 μg/ml ampicillin. After incubation for about 2 days at 28 °C the whole plate was covered with mycehum. To induce sporulation 1 ml sterile water was added to the mycehal mat, which was then hghtly rubbed by using a glass rod, until the mycehal mat became transparent. Cultivation was continued overnight at 22 °C which resulted in abundant formation of spores. The spores were harvested by adding 5 ml water, containing 0.1% Tween-80^®, and subsequently rubbing the culture with a glass rod. The spore suspension was transferred to a sterile tube and the concentration of spores was determined by using a haemocytometer. A fully-grown plate will yield at least 50 x 10⁶ spores.

Example 9

Synchronous germination

50 x 10⁶ freshly harvested spores were transferred into a sterile 50 ml polypropylene screw-cap tube and centrifuged for 5 minutes at 1500g. The pelleted spores were resuspended in 9 ml MMY, followed by addition of 1 ml 1% Tween-80^®. The suspension was subsequently incubated on the rotary shaker (New Brunswick Scientific, G10 Gyrotory shaker) at 235 rev/min. at 28 °C for approximately 7 h. Under these conditions the spores will only sweU, but no germination tubes are formed. The screw cap tube with spores was put on ice. After a period of 16 h on ice (0 °C) the tube with the spore suspension was incubated for 2 h on the rotary shaker at 235 rev/min and 28 °C. At this point nearly 100% of the spores were swollen but no germination tubes were formed. Successively the spore suspension was transferred into a 250 ml Erlenmeyer containing 100 ml MMY supplemented with 50 μg/ml ampicillin and incubated on the rotary shaker at 235 rev/min and 28 °C. According to Van Heeswijck et al. 1984, the optimal germination tube length, for making protoplasts, is 2-3 times the spore diameter. The length ofthe germ tubes is a critical parameter; if the germ tubes are shorter or longer, hardly any protoplast will be released. Within 2 h of incubation this optimal germ tube length was obtained as monitored by 400x phase contrast microscopy.

Example 10

Protoplast formation

The germinated spore suspension was transferred to two 50ml sterile polypropylene screw-cap tubes and centrifuged for 5 minutes at 1500g. After resuspending the two peUets each in 10 ml 0.1% Tween-80 and dividing them evenly over four glass tubes the suspension was centrifuged for 5 min at 1500g. The germinated spore suspension was washed three times by repeated centrifugation for 5 min at 1500g and resuspension in fresh 0.6M sorbitol. The pellet from each tube was resuspended in 2 ml Cell wall digesting enzyme solution and transferred to a small glass petridish (diameter: 4.5 cm). The petridishes were put on the rotary shaker and incubated for 3 h at 100 rev/min and 28 °C. Protoplast formation was verified by visual inspection through a 400x magnifying phase contrast microscope. During this process, starting with the germination of about 50 x 10⁶ spores, about 37.5 x 10⁶ spores were lost due to handling steps and the high affinity of the highly hydrophobic spores with plastic. The remaining 12,5 x 10⁶ spores yielded about 16 x 10⁶ protoplasts.

Example 11

Protoplast transformation

A. Transfer of plasmid DNA to B. trisyora by Poly Ethylene Glvcol (PEG)

Protoplasts were freshly prepared according to the protocol of example 10. Transformation of the protoplasts was adapted from the protocol for Aspergillus nidulans as described by Yelton et al., 1984.

The protoplast suspension was evenly divided among 4 glass tubes (4 ml) and centrifuged for 5 minutes at 1500g. The pellet was washed 3 times in STC buffer through centrifugation and resuspension. Each pellet was gently resuspended in 100 μl STC buffer. To each tube 20 μl restriction mixture (4 μg DNA) was added by mixing the suspension gently by hand. Plasmid DNA was added in a linear form. The vectors pCLIPP91A and ρCLIPP92A were linearised with Clal and Hindlll, respectively. Restriction of these vectors with BamΗl also yielded good transformation results. The DNA concentration in the restriction mixture was 0.2 μg/μl .

After 25 minutes of incubation at room temperature 0.2 ml 60% PEG was added and mixed gently by hand, followed by a second addition of 0.2 ml and a third addition of 0.85 ml of 60% PEG solution. The mixture was incubated for 20 minutes at room temperature foUowed by centrifugation for 5 min. at 1500g. Each pellet was gently resuspended in 1ml regeneration medium and transferred into a glass 10 ml tube. After adding 1.5 ml regeneration medium the "protoplasts" were incubated Overnight at 18°C under standing condition. The following day the regenerated cells were spread (in portions of 200μl) on YpSs selection plates and incubated at 28°C. After approximately δ to 7 days individual colonies were transferred to fresh selection plates without sorbitol. Only the group of transformants yielded regenerated fungi growing on 200 μg/ml kanamycin containing YpSs plates. Although the control group had twice the size of that of the transformants, it did not yield a single regenerated fungus. The efficiency of the PEG-mediated transformation process was approximately 1.2 transformants/μg DNA/1 x 10⁶ protoplasts.

Spores of primary transformants were pooled and used to inoculate a serial dilution of kanamycin containing YpSs plates. This resulted in secondary transformants growing on kanamycin concentrations up to 800 μg/ml. Spores obtained after another vegetative cycles of growth, on 600 μg/ml kanamycin, were used to make protoplasts as described in example 9 and 10. The obtained protoplasts were plated out on YpSs selection plates in order to select uni-nucleate transformants. Subsequently single colonies were isolated on selective YpSS medium containing 600 μg/ml kanamycin. The presence of sequences derived from the transforming plasmid could be estabhshed by PCR on chromosomal DNA isolated from the recombinant strains (Fig. 12).

B. Transfer of plasmid DNA to B. trispora by electroporation.

Protoplasts, obtained as described in example 10, were centrifuged (δ min., 1000-2000 rp , 4 °C). The protoplasts were washed three times by carefully resuspending the pellets in 2δ ml ice-cold 0.6 M sorbitol, collecting the cells by centrifugation at 1000 rpm and aspirating the supernatant as much as possible. Finally, the protoplast pellet was resuspended in 1 ml of ice-cold sorbitol yielding a protoplast density of 1-2 x 10⁷-10⁸. This mixture was kept on ice until electroporation.

Depending on the protoplast density 80-400 μl of the protoplast suspension was mixed, by pipetting up and down several times, in a pre-chilled microcentrifuge tube with δ μl (1-2 μg) of plasmid DNA (in MQ). The plasmid DNA was added in either the circular or a linear form. The vectors pCLIPP91A-C and pCLIPP92A-C were linearised with Clal and Hindlll, respectively. The vector was purified and concentrated. The protoplast /DNA mixture was transferred into a pre-chilled electroporation cuvette with a 0.2 cm gap. Electroporation was carried out using a Multiporator (Eppendorf) the under the following settings: bacterial mode (time constant of 5 s) and the field strength was varied between 1.5 and 3.δ kV. The ideal electroporation conditions were 2.4 kV.

Following electroporation, 1 ml of sterile, ice-cold regeneration medium was added (1 M sorbitol, 2 % glucose, and 1 x Vogel's salts. The solutions were mixed with a Pasteur pipette and transferred to a microcentrifuge tube. The protoplasts were allowed to regenerate their cell wall during 24 - 36 h incubation at room temperature. Following the regeneration the cells were concentrated by centrifugation (4000 rpm, 5 min.) and resuspended in 100 μl PS-broth. The ceUs were plated out on selective medium (YpSs agar + vitamin Bl and 200 μg/ml kanamycin).

The presence of sequences derived from the transforming plasmid could be estabhshed by PCR and Southern blot analysis of chromosomal DNA isolated from the recombinant strains.

Example 12

Construction of a general cassette to express homologous and heterologous genes in B. trisyora

In example 6 we have used the method of recombinant PCR to fuse a gene of interest, in that specific example the nptll gene (conferring G-418/kanamycin resistance), with the promoter and terminator region ofthe gpd gene of B. trispora. Instead, it would be desirable to use a general expression cassette to prevent the necessity to carry out multiple amplification rounds for each gene.

To this end, the promoter region ofthe B. trispora gene was amplified using the primers GCOl (SEQIDNO: 23) and GC10 (SEQIDNO: 34) and _PBtinvPCR#2.2 as template. The PCR product of 0.8 kb was A-tailed and inserted in the cloning vector pGEM-T Easy, yielding pPgpdBtunivF/R (depending o the orientation o the fragment). The terminator region of the B. trispora gene was amplified using the primers GC11 (SEQIDNO: 35) and GC12 (SEQIDNO: 36) and pCLIPPBt#20 as template. The PCR product of 0.3 kb was A-tailed and inserted in the cloning vector pGEM-T Easy, yielding pTgpdBiunivF/R (depending of the orientation of the fragment). The promoter and terminator regions were reisolated as EcoRI-H dIII fragment of 0.8 and 0.3 kb from pPgpdBtunivF/R and pTgpdBtunivF/R, respectively. The fragments were cloned in the EcoRI digested vector pGΕM-T easyΔSpM yielding pCLIPP96 (Fig 9). The vector pGΕM- T easyΔSpM is a derivative of pGΕM-T Easy in which the Sphl site was removed by removing the protruding 3' termini of the Sphl site using T4 DNA polymerase. The expression cassette is flanked at both sites by the restriction sites Notl, EcoRI and Sαcl. Any gene of interest can be inserted in the Sphl site, treated with bacteriophage T4 DNA polymerase, and Pmel site of pCLIPP96. The restriction map and nucleotide sequence of the expression cassette is shown in figure 10.

Example 13

Heterologous expression of carotenogenic genes in Blakeslea trispora

With the exception of the first codon (ATG; Met) the coding sequence of the gene (carRA) encoding the bifunctional carotenoid biosynthetic enzyme phytoene synthase/lycopene cyclase of P. hlakesleeanus (Arrach et al., 2001) was amplified. This fragment was synthesised using the phosphorylated primers GC 13 (SEQIDNO: 37) and GC 14 (SEQIDNO: 38) and reverse transcribed mRNA of P. hlakesleeanus as template. The fragment was purified from the PCR mixture and cloned in the ρCLIPP96 (x Sphl/T4 DNA pol. x Pmel see example 12). The orientation of the insert was determined with restriction analysis. The complete expression cassette (PgpdBt-carRA-TgpdBt) was isolated as a Notl fragment of approximately 3.1 kb and was cloned in the corresponding site of pCLIPP9lAF yielding pCLIPP99F.

In order to develop B. trispora as a cell factory of carotenoids which are not naturally produced in B. trispora, a pilot study was set up in which to following heterologous carotenogenic genes were introduced in the expression cassette of B. trispora (see example 12).

With the exception of the first codon (ATG; Met) the coding sequence of the β- carotene hydroxylase encoding gene (crtZ) of Erwinia uredovora (Misawa et al., 1990) was amplified. This fragment was synthesised using the phosphorylated primers GC 15 (SEQIDNO: 39) and GC 16 (SEQIDNO: 40) and genomic DNA of E. uredovora as template. The fragment was purified from the PCR mixture and cloned in the pCLIPP96 (x Sphl/^rT4 DNA pol. x Pmel see example 12). The orientation of the insert was determined with restriction analysis. The complete expression cassette (PgpdBt-crtZ- TgpdBt) was isolated as a Noil fragment of approximately 1.6 kb and was cloned in the corresponding site of pCLlPP92AR yielding pCLIPPlOOF.

With the exception of the first codon (ATG; Met) the coding sequence ofthe β- carotene C(4) oxygenase encoding gene (crtW) of Agrobacterium aurantiacum (Misawa et al., 1995) was amplified. This fragment was synthesised using the phosphorylated primers GC 17 (SEQIDNO: 41) and GC 18 (SEQIDNO: 42) and genomic DNA of Agrobacterium aurantiacum as template. The fragment was purified from the PCR mixture and cloned in the pCLIPP96 (x SpM/T4 DNA pol. x Pmel see example 12). The orientation of the insert was determined with restriction analysis. The complete expression cassette (PgpdBt-crtW-TgpdBt) was isolated as a Noil fragment of approximately 1.8 kb and was cloned in the corresponding site of pCLIPP92AR yielding pCLIPPlOlF.

B. trispora strain DSM2387 (+) was transformed with pCLIPP99F, pCLIPPlOOF, pCLIPPlOlF and pCLIPP96 (control) and transformants were selected on YpSs agar plates containing kanamycin.

The different sets of transformants were cultivated in 50 ml GAY under selective conditions for 6 days at 28 °C. Flasks were inoculated with 2.5 ml seed culture. After the cultivation period the colour of the mycehum was determined (Table 1). The changes of colour indicate an increase of carotenoid production and/or a different carotenoid composition.

Table 1: Colour of genetically engineered B. trispora strains DSM2387

These data show the potentials of metabolic engineering of the carotenoid pathway of B. trispora. The examples are only used as an illustration. The deregulated overexpression of genes encoding for enzymes from the mevalonate pathway such as HMG-CoA reductase, phospho-mevalonate kinase and diphosphomevalonate decarboxylase, alone or in combination, may help to increase the metabolic flux through the pathway either by the amplification of the rate limiting reactions and/ or the elimination of regulatory mechanisms such as product inhibition and repression.

Metabolic engineering can be used to redirect the biosynthetic capacity of B. trispora from β-carotene towards mono- (Cio), sesqui- (C15), di- (C20), tri- (C30), tetra-(C4o), and polyterpenes (C45-i5o,ooo). The feasibility of this approach is illustrated in the next example, where the amorpha-4,ll-diene synthase encoding gene (ads) from. Artemisia annua L. is overexpressed in B. trispora.

Example 14

Heterologous expression of non-carotenogenic genes in Blakeslea trispora With the exception of the first codon (ATG; Met) the coding sequence of the gene (ads) encoding for the enzyme amorpha-4,ll-diene synthase of the plant Artemisia annua L. was amplified. This enzyme catalysis the cychsation of farnesyl diphposphate (FPP) into amorpha-4,ll-dieen, the first specific precursor of artemisinin. The sesquiterpenoid artemisine and its semi-synthetic derivatives represent a relatively new and very effective group of antimalarial drugs. The fragment was synthesised using the phosphorylated primers GC 19 (SEQIDNO: 43) and GC 20 (SEQIDNO: 44) and an ads cDNA clone as template (Wallaart et al., 2001). The fragment was purified from the PCR mixture and cloned in the pCLIPP96 (x Sphl/T4 DNA pol. x Pmel see example 12). The orientation of the insert was determined with restriction analysis. The complete expression cassette (PgpdBt-ads-TgpdBt) was isolated as a Noil fragment of approximately 2.7 kb and was cloned in the corresponding site of pCLIPP91AF yielding pCLIPP102F.

B. trispora strain DSM2387 (+) was transformed with pCLIPP102F and pCLIPP91AF (control) and transformants were selected on YpSs agar plates containing kanamycin. Transformants were cultivated in δO ml GAY under selective conditions for 6 days at 28 °C. Flasks were inoculated with 2.δ ml seed culture. The seed culture were prepared by inoculating a flask of selective GAY medium supplemented with 0.1 % SPAN with 1 x 10⁶ spores per ml. These cultures were incubated for 2 days at 29 °C on a rotary shaker set at 200 rev/min. From each culture 100-200 mg mycehum was homogenised using a mortar and pestle in δ ml pentane (p. a.). The pentane extract was decolourised (activated carbon) and centrifuged (δ min. 3,000x g). Then the extract was filtered over a column containing AI2O3 and anhydrous Na₂SO₄. The concentrated samples (under a stream of N₂) were analysed by GC-MS as described by Bouwmeester et al.(1999). Significant amounts of amorpha-4,ll-diene were detected in recombinant B. trispora strains transformed with pCLIPP 102. Amorpha-4,ll-diene was absent in the control strain, B. trispora DSM2387 [pCLIPP91AF].

Example 15

Gene silencing bv double stranded antisense RNA

To demonstrate that gene silencing in B. trispora by double stranded antisense RNA is effective a construct containing a Pyr-G cDNA clone was made. PCR with Proofstart DNA polymerase (Qiagen) was used to clone the promoter Ppyr-S cl (SEQIDNO: 49); Vyr-Xbal (SEQIDNO: 50) termination region Tpyr-^&αl (SEQIDNO: 51)); Tpyr-E HI (SEQIDNO: 52) o the Pyr-G gene with genomic DNA of B. trispora DSM 2387 as template. The cDNA clone of Pyr-G was cloned by using RT-PCR with the primer combination vjr-Xhol (SEQIDNO: 53) ; pyr-Xb l (SEQIDNO: 54) on cDNA obtained from total RNA isolated from mycehum of B. trispora DSM2387 as described in example 2. All PCR products were cloned in pGEM-Teasy. Yielding the vectors; pCLIPP AS1 (not shown) and pCLIPP AS2 for the two orientations of the Pyr-G termination region in pGEM-Teasy. pCLIPP AS3 (not shown) and pCLIPP AS4 for the two orientations of the Pyr-G promoter region in pGEM-Teasy and pCLIPP AS5 and pCLIPP AS6 (not shown) for the two orientations of the Pyr-G cDNA clone in pGEM-Teasy (Fig. 13) The orientation of pCLIPP ASl and pCLIPP AS2 was determent by using PCR with a standard sequencing primer present on pGEM-Teasy and one of the primers (SEQIDNO: 51) or (SEQIDNO: 52). The vector pCLIPP AS2 was cut with Xbάl and Sαcl and afterwards isolated from gel. Also pCLIPP AS4 was cut with these enzymes but this time the insert (Pyr-G promoter region) was isolated from gel. This insert was subsequently ligated into the Xbaϊ-Sacl cut vector pCLIPP AS2. The new constructed vector was named pCLIPP AS7. This construct was cut with Xbaϊ, subsequently dephosphorylated with Calf Intestine Alkaline Phosphatase (CIAP) to avoid self hgation, and purified through gel isolation. The cDNA clone of Pyr-G was cut out pCLIPP AS5 by digestion with Xbal-Xhoϊ and purification by gel isolation. A piece of "stuffer DNA" with at both ends a Xhol site was obtained from a human cDNA clone encoding the cytochrome P450 enzyme 3A4. This DNA fragment of δOO bp was also isolated from gel. In a so called four points hgation the Xhal-Xhόl cut cDNA clone of Pyr-G and the Xhol flanked stuffer DNA were ligated into the Xbal cut and CIAP treated pCLIPP AS7. This yielded pCLIPP AS8 and pCLIPP AS9 (not shown). The vector pCLIPP AS8 was cut with BamRΪ- Sad- Seal and the pyr-G construct was isolated from gel. The sticky ends of the restriction sites were filled by the polymerase activity of the Klenow fragment of E. coli DNA polymerase I. A δ' A overhang was introduced according to the instructions of the manufacturer of the pGEM-Teasy cloning kit. The fragment was cloned into pGEM- Teasy and yielding pCLIPP AS10 and pCLIPP ASH (not shown). The vector pCLIPP AS 10 was cut with Noil and Seal and the pyr-G construct, flanked with Noil sites was isolated from gel. PCLIPP92A was digested with Noil, subsequently dephosphorylated with CIAP, and purified through gel isolation. The Noil flanked pyr-G construct was ligated into this vector yielding pCLIPP AS 12 and pCLIPP AS 13 (not shown).

Transformation of B. trispora with these constructs (pCLIPP AS12 and pCLIPP AS 13) yielded transformants with the characteristics of a Pyr-G auxotrophic mutant. This implies that these transformants were able to grow on plates with lg 1 5-FOA (5- fluoro orotic acid). Growth on minimal medium was only possible if uracil was added. (Boeke et al., 1984; Benito, et al., 1992) This proves that gene silencing in B. trispora by means of double stranded antisense RΝA is very effective and may also be an effective approach for interfering in the isoprenoid/carotenoid pathway.

In analogy to this approach a construct was made in which the promoter and termination region of Pyr-G were replaced by the B. trispora gpd promoter and termination regions. Also this construct was very effective in gene silencing (results not shown).

LEGENDS

Figure 1. Biosynthetic pathway of β-carotene in Blakeslea trispora. Abbreviations:

IPP, isopentenyl diphosphate; DAMPP, dimethyl allyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate, GGPP, geranylgeranyl diphosphate. Figure 2. Structural formula for trisporic acids. X is an oxygen atom in the B form of trisporic acid and a hydroxyl group and hydrogen atom in the C forms. For both the C and D form Ri = R₂ = R3 = R4 = H. Adapted from Sutter et al.,

1996. Figure 3. Collaborative biosynthesis of trisporic acids by cross-feeding of intermediates between plus (+) and minus (-) mating types of Blakeslea trispora. Figure 4. A general representation of the isoprenoid pathway via mevalonate.

Abbreviations: IPP, isopentenyl diphosphate; DAMPP, dimethyl allyl diphosphate. Enzymes: AACT, acetoacetyl-CoA thiolase; HMGS, 3- hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK, δ-phosphomevalonate kinase;

PMDC, δ-diphosphomevalonate decarboxylase. Figure δ. Schematic representation of the construction of the marker cassette

(PgpdBt-G418-ΥgpdBt) for Blakeslea trispora. Figure 6. Schematic representation of the construction of integrative vectors for transformation of B. trispora; part 1. Figure 7. Schematic representation ofthe construction of integrative vectors for transformation of B. trispora; part 2. Figure 8. Schematic representation of the construction of autonomously replicating vectors for transformation of B. trispora. Figure 9. Schematic representation of the construction of a general cassette to express homologous and heterologous genes in B. trispora. Figure 10. The restriction map (A) and nucleotide sequence (B) of the expression cassette from pCLIPP96. Figure 11. Schematic representation of the various expression cassettes used to express heterologous genes in B. trispora

Figure 12. Analysis of PCR amplification products on agarose gel electrophoresis. A. PCR with the Pyr-G homologous primer combination Pyr-G S (SEQIDNO: 4δ):; Pyr-G AS (SEQIDNO: 46): and genomic DNA ofthe transformants (tr.) no. 1-7, pCHPP92A (con.), and, the wild type (WT) as template. H₂O was used as negative control. Pyr-G (EMBL database accession No. AJδ34694)is a housekeeping gene of B. trispora. (Quiles-Rosillo 2003) As expected an amplification product of 16δ4 bp was obtained only with the genomic DNA of the transformants and wild type as template. M; represents a DNA ladder ranging from 100 tot 1000 bp.

B. PCR with the primer NPT II homologous primer combination pRUG41 (SEQIDNO: 47):; pRUG940 (SEQIDNO: 48): and the same set of template DNA as used in A. Amplification products of the expected size of 412 bp were only obtained with the genomic DNA of the transformants and the positive control PC1IPP92A (con.).

Figure 13. Schematic representation of the construction of a double stranded antisense Pyr-G construct for expression in B. trispora.

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Claims

1. A method for producing at least one molecule of interest by a host cell, comprising providing a host cell with at least one isolated nucleic acid, culturing said host cell and allowing said host cell to produce said at least one molecule of interest, wherein said host cell comprises a fungus of the family Choanephoreaceae, preferably a Blakeslea spp., more preferably Blakeslea trispora.

2. A method according to claim 1, wherein said at least one nucleic acid encodes a homologous polypeptide or part thereof, preferably an enzyme involved in the production of said molecule of interest.

3. A method according to claim 1, wherein said nucleic acid encodes a heterologous polypeptide or part thereof, preferably an enzyme involved in the production of said molecule of interest.

4. A method according to claim 1, wherein said nucleic acid encodes said molecule of interest.

5. A method according to any one of claims 1-3, wherein said molecule of interest comprises a metabolite, preferably a metabohte of the carotenoid pathway, more preferably wherein said molecule is selected from the group consisting of β-carotene, lycopene, canthaxanthin or astaxanthin.

6. A method according to any one of claims 1-4, wherein said molecule of interest comprises a metabohte of the isoprenoid pathway or a precursor thereof.

7. A method according to any one of claims 1-6, wherein said nucleic acid is further provided with a transcription promoter, preferably a transcription promotor comprising a region found upstream of a highly expressed gene of said host cell.

8. A method according to claim 7, wherein said highly expressed gene is a gene encoding an enzyme of the glycolytic pathway, preferably glyceraldehydes-3-phosphate dehydrogenase.

9. A method according to any one of claims 1-8, wherein said host cell is provided with said nucleic acid using polyethyleneglycol (PEG).

10. A method for providing a host cell with at least one isolated nucleic acid, wherein said host cell comprises a fungus of the family Choanephoreaceae.

11. A method according to claim 10 wherein said host cell is provided with said nucleic acid using polyethyleneglycol.

12. A host cell provided with at least one nucleic acid, preferably a transgenic Blakeslea spp., obtainable by a method according to claim 10 or 11.

13. A nucleic acid for use in a method according to any one of claims 1 to 12.

14. Use of a host cell according to claim 12 or a nucleic acid according to claim 13 for the production of a pharmaceuticaUy relevant molecule, a food or feed additive.