WO2016016805A1

WO2016016805A1 - Gene construct for the transformation of yeast strains

Info

Publication number: WO2016016805A1
Application number: PCT/IB2015/055692
Authority: WO
Inventors: Daniele Bianchi; Giuliana Franzosi; Silvia Galafassi; Concetta COMPAGNO
Original assignee: Eni SpA; Versalis SpA
Current assignee: Eni SpA; Versalis SpA
Priority date: 2014-08-01
Filing date: 2015-07-28
Publication date: 2016-02-04
Anticipated expiration: 2017-02-01

Abstract

The present invention relates to a gene construct based on auxotrophy for uracil suitable for the transformation of yeasts of the species Rhodosporidium azoricum. The invention also relates to a vector containing said gene construct, a yeast of the species Rhodosporidium azoricum transformed with said gene construct and a transformation method.

Description

GENE CONSTRUCT FOR THE TRANSFORMATION OF YEAST STRAINS

The present invention relates to a gene construct based on auxotrophy for uracil suitable for the transformation of yeasts of the species Rhodosporidium azoricum.

The invention also relates to a vector containing said gene construct, a yeast of the species Rhodosporidium azoricum transformed with said gene construct and a transformation method.

The development of biotechnology has led to the identification of expression systems optimal for the production and purification of a metabolite of interest. These technologies therefore allow the availability of "engineered" organisms characterized by an ever-increasing yield, safety and rapidity of the production process and quality of the product obtained.

In particular, among the most advantageous eukaryotic expression systems, yeasts belonging to the group of ascomycetes have been widely studied and characterized.

The stable integration of DNA segments in the genome of yeasts through transformation and recombination is well known (Hinnen A., Hicks J.B., Fink G.R. Trasformation of Yeast. Proc. Natl. Acad. Sci. U.S.A. 1978 75 ( 4 ) : 1929-33 ) . Furthermore, the construction of auxotrophic mutants represents a simple system for having available selective markers in DNA transformation with genes of interest. These systems are generally composed of two elements: the gene encoding an enzymatic activity essential for the biosynthetic pathway of a particular compound (generally an amino acid or a nitrogenous base) , including the promoter and terminator sequences that guarantee its correct expression and the corresponding auxotrophic strain for that particular compound (Gene 263 : 159-169 (2001) ) .

The enzyme orotidine-5' -monophosphate (OMP) decarboxylase normally encoded by the gene URA3 converts OMP to uridine-5'- monophosphate (UMP) in the last passage of the biosynthetic pathway of pyrimidine, essential for the synthesis of DNA and RNA. In strains missing the gene URA3 , growth can be possible by uracil addition in the culture medium. This same mutation, on the other hand, entails ability to grow in media containing 5' -fluoroorotic acid (5'-FOA), which conversely are toxic for strains having this enzymatic activity (gene URA3) , as they are capable of transforming 5'-FOA into 5-fluorouridine monophosphate, i.e. into a compound harmful for the cell. For these reasons, mutations in this gene can be easily detected, numerous protocols have therefore been defined that envisage the use of this system in both S. cerevisiae

(Alani E, Cao L, Kleckner N. 1987. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116: 541-545) and in other yeasts

(Alani E, Cao L, Kleckner N. 1987. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116: 541-545; Ngan WY, Nga BH, Pridmore D, et al . 2000. Transformation of Endomyces fibuliger based on its gene for orotidine-5' -phosphate decarboxylase. Gene 254: 97-103) and fungi (Fierro F, Laich F, Garcia-Rico RO, Martin JF. 2004. High efficiency transformation of Penicillium nalgiovense with integrative and autonomously replicating plasmids. Int J Food Microbiol 90: 237-248; Goosen T, Bloemheuvel G, Gysler C, et al . 1987. Transformation of Aspergillus niger using the homologous orotidine-5' -phosphate- decarboxylase gene. Curr Genet 11: 499-503) .

In Basidiomycetae yeasts, such as, for example, Cryptococcus neoformans, Cryptococcus gattii,

Phanerochaete chrysosporium, Rhodotorula graminis _r Rhodotorula sloofiae and Sporobolomyces sp. , unlike the Ascomycetae yeasts such as Saccharomyces cerevisiae_r from selection on plates with 5'-FOA, both clones with mutations in the gene URA3 and in the gene URA5 can be obtained (Kwon-Chung KJ, Varma A, Edman JC, Bennett JE . 1992. Selection of ura5 and ura3 mutants from the two varieties of Cryptococcus neoformans on 5-fluoroorotic acid medium. Journal of Medical and Veterinary Mycology 30(1) : 61-69) . Furthermore, in some cases, only URA5 mutants are obtained. With respect to the species belonging to the Rhodosporidium genus, URA5 mutants have only been described for the species R. kratochvilovae (in Abbott EP Ianiri G, Castoria R, Idnurm A. 2013. Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts. Applied Microbiology and Biotechnology 97(1) :283-295) .

For induction of mutations in the genes URA3 and URA5, 5'-FOA is employed, which is a compound used for the selection of uracil auxotroph strains, as it generates mutations in the URA3 and URA5 genes in yeasts commonly studied such as S. cerevisiae and in other yeasts and fungi (see articles cited above) . International patent application WO2009126890 describes recombinant oleaginous fungi for the biological production of carotenoids and/or retinoic acid, whereas US patent application US20100305341 describes recombinant oleaginous fungi for the biological production of sterols.

International patent application WO2012097091 describes microorganisms, among which also yeasts belonging to the Rhodosporidium genus, which have an improved fermentative activity. Recombinant systems also comprising the URA5 gene are also described, but only for the microorganism S. pombe, a fungus belonging to the ascomycetes group.

As is known, among Basidiomycetae yeasts, those belonging to the Rhodosporidium species are of particular interest as they are known oleaginous capable of accumulating lipids even naturally, so that, when cultivated under particular favourable conditions, they can increase the conversion of carbon sources into fatty acids and triglycerides (as described in literature in M. Khot, S. Kamat, S. Zinjarde, A. Pant, B. Chopade and A. RaviKumar. Single cell oil of oleaginous fungi from the tropical mangrove wetlands as a potential for biodiesel. Microbial Cell Factories 2012, 11:71. and G. Katre, C. Joshi, M. Khot, S. Zinjarde, A. Ravikumar. Evaluation of single cell oil (SCO) from a tropical marine yeast Yarrowia lipolytica NCIM 3589 as a potential feedstock for biodiesel. AMB Express 2012, 2:36) . In this case, the yeasts belonging to the Rhodosporidium genus are the best producers of lipids, specifically over 60% of the dry weight of their biomass when cultivated under nitrogen-, phosphate- or sulfate-limited conditions (Li YH, Zhao ZB, Bai FW, et al. 2007. High density cultivation of the oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microbiol Technol 41:312-317; Wu SG, Hu C, Jin G, et al . 2010. Phosphate-limitation mediated lipid production. Bioresour Technol 101: 6124- 6129; Wu SG, Zhao X, Shen HW, et al . 2011. Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions. Bioresour Technol 102: 1803-1807) .

The aspects of interest of yeasts of the Rhodosporidium genus can be improved by acting on specific biosynthetic pathways. This can be obtained by genetic engineering, wherein fragments of exogenous DNA encoding, for example, enzymatic or regulatory activities of interest, are inserted in the genome of the target microorganism by gene recombination.

The phylogenetic distance between different genera and/or species, however, limits the use of molecular tools already known, for example, for Ascomycetous yeasts such as S. cerevisiae in Basidiomycetous yeasts, consequently making them unsuitable for use in Rhodosporidium species.

Even among the species belonging to the same genus, therefore apparently close from a phylogenetic point of view, DNA fragments of one species may not be functional in another nearby species, thus indicating the need for specific molecular tools. See for example Applied Microbiology and Biotechnology 97 ( 1 ) : 283-295 (mentioned above) , in which DNA fragments of Basidiomycetous Rhodotorula sloffiae have proved not to be functional in the phylogenetically close yeast Rhodotorula glutinis .

In this respect, the Applicants have experimentally observed that URA3 mutants cannot be obtained by the exposure of yeasts of the species Rhodosporidium azoricum to 5'-FOA.

In order to overcome this problem, a molecular marker has been developed, containing the sequence of the marker gene URA5 encoding the orotidine monop osp ate phosphorylase enzyme (OMPPase) and including the promoter and terminator sequences, and an uracil auxotroph strain wherein the URA5 gene has a mutation that makes the enzyme inactive, and which therefore makes the microorganism unable to autonomously reproduce itself without uracil.

Nowadays, in fact, no description can be found, either in scientific literature or in filed patents, of protocols, genes or strains able of envisaging or expressing selection molecular tools based on the URA5 gene, and through which genetic modifications in uracil auxotroph species belonging to the species Rhodosporidium azoricum can be effected. For these reasons, the molecular tools known in literature cannot be used to modify yeasts of the Rhodosporidium azoricum species at a genetic level.

In the light of what is indicated above, there is an evident need for having molecular tools suitable for transforming yeasts of the Rhodosporidium azoricum species capable of overcoming the drawbacks of the known art .

The Applicants have now found a gene construct based on auxotrophy for uracil suitable for the transformation of yeasts of the species Rhodosporidium azoricum auxotrophic for uracil.

The specific attention with respect to this particular yeasts species is due to the fact that the yeasts belonging to the species Rhodosporidium azoricum are not only oleaginous but also have other advantages.

In fact, they showed a good resistance to the toxic compounds usually present in media deriving from the hydrolysis of lignocellulosic materials. Furthermore, these yeasts are capable of utilizing sugars with five carbon atoms and fermentative set-ups have been advantageously developed that allow high concentrations of biomass production.

A first aspect of the present invention therefore relates to a gene construct having the characteristics according to claim 1.

In a second aspect, the invention relates to a method for transforming yeast cells of the species Rhodosporidium azoricum auxotrophic for uracil, wherein said method is effected according to claim 14.

In a third aspect, the invention relates to a transformation vector as described in claim 15.

In a fourth aspect, the invention relates to a yeast cell of Rhodosporidium azoricum transformed as described in claim 16.

In a fifth aspect, the invention relates to a yeast cell of Rhodosporidium azoricum as described in claim 17.

In the present invention, the expression gene construct refers to a polynucleotide which contains information necessary for the transformation and expression of at least one desired characteristic in the target organism. In particular, said polynucleotide is a DNA fragment. This definition also comprises, moreover, the expressions "expression cassette" and "transformation cassette".

Further characteristics and advantages of the present invention will be apparent from the following detailed description.

An object of the present invention relates to a gene construct comprising a sequence encoding the URA5 gene for the transformation of yeast strains of the species Rhodosporidium azoricum auxotrophic for uracil.

The gene construct of the present invention comprises the sequence of the URA5 gene of 696 base pairs with identification number 1 (SEQ ID NO: 1), which was obtained from the genome of Rhodosporidium azoricum by direct sequencing, following amplification of the corresponding fragment by PCR and identified thanks to the construction of degenerated promoters (primers) on the sequences of the URA5 genes of similar yeasts present in public databases.

Upstream of the gene, there is the promoter sequence. Said sequence of the promoter is preferably the sequence with identification number 2 (SEQ ID NO: 2), of 995 base pairs. Downstream of the gene, there is the terminator sequence. Said terminator sequence is preferably the sequence with identification number 3 (SEQ ID NO: 3) of 415 base pairs. The promoter and terminator sequences were also amplified from the regions adjacent to the gene URA5 in the genome of Rhodosporidium azoricum.

According to a preferred aspect, the gene construct of the present invention comprises the sequence with identification number 4 (SEQ ID NO: 4), wherein the sequence of the URA 5 gene has 770 base pairs, as it also comprises the intron of SEQ ID NO: 5 (see also the explanation of figure 2) .

According to a preferred aspect of the present invention, the sequence of the URA5 gene of the gene construct encodes a polypeptide with an amino acid sequence with identification number 6 (SEQ ID NO: 6) .

Alternatively, any promoter/terminator pair of constitutive (autologous) genes of Rhodosporidium can be used: according to a preferred aspect, the promoter of the (autologous) phosphoglycerate kinase (PGK) gene with the sequence having identification number 7 (SEQ ID NO: 7) , is used.

As an alternative to the promoters with SEQ ID NO: 2 or SEQ ID NO: 7, the promoter of the (heterologous ) TEF gene of Ashbya gossypii (Ji, L., Z.- D. Jiang, Y. Liu, C. M. J. Koh and L.-H. Zhang. 2010 A simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells. Fungal Genetics and Biology, 47:279-287.) with the sequence having identification number 8 (SEQ ID NO: 8) can be used.

The terminator of the (autologous) gene of phosphoglycerate kinase (PGK) with the sequence having identification number 9 (SEQ ID NO: 9) or the terminator of the (heterologous) TEF gene of Ashbya gossypii with identification number 10 (SEQ ID NO: 10), can be used as terminators.

Particularly preferred promoter, URA 5 gene and terminator constructs are those having identification number SEQ ID NO: 11, wherein the promoter and the terminator respectively have SEQ ID NO: 7 and SEQ ID NO: 9 (autologous), or having identification number SEQ ID NO: 12, wherein the promoter and the terminator respectively have SEQ ID NO: 8 and SEQ ID NO: 10

(heterologous) .

The combination of promoters and terminators of an autologous or heterologous origin alongside the URA5 gene should not be ruled out.

The term gene construct of the present invention

(promoter- URA5 gene sequence-terminator) therefore represents a molecular tool which, if inserted in a transformation vector, allows the transfer and expression of exogenous DNA in a target organism auxotrophic for uracil, relying on a selective system capable of verifying its correct integration, i.e. a selection marker, in this case URA5, whose gene sequence has been obtained from the genome of Rhodosporidium azoricum by means of PCR amplification.

According to a preferred aspect of the present invention, said organisms are yeasts of the genus Rhodosporidium, preferably Rhodosporidium azoricum. In this case, this gene expression construct can be used for the transformation of a strain of Rhodosporidium azoricum, or more generally a strain of the genus Rhodosporidium, which is auxotrophic for uracil, i.e. having the URA5 gene inactivated.

According to a preferred aspect, the gene construct of the present invention is included in an expression vector, preferably a plasmid.

Various types of vectors can be used for effecting the transformation with the gene construct of the invention. Preferably, said construct can be inserted into different types of bacterial plasmids well-known in the art, such as, for example, the commercially available plasmids TOPO, pUC18, pJET1.2, pSP72 and those of the pGEM family, so as to be able to easily manipulate the construct and obtain a sufficient quantity for the transformation of yeast cells. The cassette can then be excised from the plasmid or the plasmid can be simply linearized to facilitate its recombination in the genome of the target yeast.

A further object of the present invention relates to a method for the transformation of yeast cells of the species Rhodosporidium azoricum auxotrophic for uracil and selection of the transformants comprising: - transforming yeast cells with the gene construct as defined in the present invention;

selecting transformants by culture on a minimal medium, containing no uracil.

According to a preferred aspect, the minimal medium mainly used for the culture of yeasts comprises glucose from 5 to 50 g/1, preferably 20 g/1, YNB (Yeast Nitrogen Base) W/O amino acid from 3.35 to 13.4 g/1, preferably 6.7 g/1.

For the solid version, the minimal medium preferably envisages the addition of agar 20 g/1 (from 15 to 30 g/1) .

A further object relates to a transformation vector comprising the gene construct as defined in the present invention .

Another object of the present invention relates to a yeast transformed with the gene construct as defined above, said yeast belonging to the species Rhodosporidium azoricum and being auxotrophic for uracil. The strain belonging to the species Rhodosporidium azoricum uracil auxotroph was deposited under the Budapest Treaty at the Leibniz-Institut DSMZ

Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), InhoffenstraBe 7 B 38124 Braunschweig (Germany), on May 6, 2014, accession number DSM 28738.

According to a preferred aspect, said gene construct also comprises at least one nucleotide sequence and/or gene of interest to be transferred into the genome of target yeasts.

According to a further preferred aspect, the at least one nucleotide sequence and/or gene of interest to be transferred into the genome of target yeasts through the construct of the invention encodes resistance to antibiotics.

Description of the figures

The present description will now be described, for illustrative and non-limiting purposes, according to its preferred embodiments, with particular reference to the enclosed figures, in which:

figure 1 shows:

figure 1A: gene construct of the invention comprising a gene of interest to be transferred into the genome of target yeasts;

figure IB: gene construct of figure 1A inserted in a bacterial plasmid;

figure 1C: gene construct of the invention comprising two genes of interest to be transferred into the genome of target yeasts;

figure 2 shows the nucleotide sequence of the URA5 gene of Rhodosporidium azoricum. The start (ATG) and stop (TAG) codons of the transcript (dark grey) are shown, whereas an intron (non-encoding area situated inside the gene, with sequence SEQ ID NO: 5) , is shown in light grey. The URA5 gene inserted in the gene construct of the invention corresponds to the underlined nucleotide sequence (SEQ ID NO: 1, without intron) . The region upstream of the underlined part is the promoter with SEQ ID NO: 2 and the region downstream of the underlined part is the terminator with SEQ ID NO: 3. The whole nucleotide sequence comprising promoter, URA5 gene

(including intron) and terminator has SEQ ID NO: 4 ; figure 3 shows examples of mutations in the URA5 gene that can lead to uracil auxotrophy Nucleotide sequences of the URA5 gene of Rhodosporidium azoricum are shown: the wild-type

(wt) strain (SEQ ID NO: 13) is that from which the mutants are obtained (therefore its sequence encodes a functional enzyme) , whereas the sequences called "mutant U24" (SEQ ID NO: 14) and "mutant U27" (SEQ ID NO: 15) are those relating to the gene not functioning in different mutants indicated as an example (in U24 see residue nr. 420; in U27 see residue nr. 614) .

The mutant U27 is that used in the strain deposited with number DSM 28738 on May 6, 2014, at the Leibniz- Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), InhoffenstraBe 7 B 38124 Braunschweig (Germany) .

The sequence called "consensus" is automatically generated by the program used for the alignment (MultAlin software, "Multiple sequence alignment with hierarchical clustering" F. CORPET, 1988, Nucl . Acids Res., 16 (22), 10881-10890) : in fact it creates this sequence "consensus" which indicates the "common" base in all the aligned sequences, whereas it inserts a dot where there are modifications.

EXAMPLE S

Example 1

An example is provided hereunder of the method for obtaining the gene construct object of the invention and the transformation of a strain of Rhodosporidium azoricum auxotrophic for uracil.

The first step of the strategy used for the sequencing of the gene URA5 of Rhodosporidium azoricum consisted in the search for regions having a high homology among the protein sequences of URA5 deriving from yeasts, close from a phylogenetic point of view {Malassezia globosa, Cryptococcus gattii, Melampsora larici-populina) , whose sequences were present in public databases. The conserved regions among the different sequences compared were thus identified and degenerated primers were then constructed (following the method proposed by Rose, T.M., E.R. Schultz, J.G. Henikoff, S. Pietrokovski , CM. McCallum and S. Henikoff. 1998. Consensus-degenerate hybrid oligonucleotide primer for amplification of distantly- related sequences. Nucleic Acids Research, 26(7) :1628- 1635) .

From the sequencing of the first fragment obtained, the sequences upstream and downstream were also sequenced, thanks to Genome Walking techniques (in this case the commercial kit "DNA Walking SpeedUp" of the firm SeeGene, cat. num. K1501 was used) .

The complete sequence obtained is shown in Figure 2 (SEQ ID NO: 4) . The construct was then amplified from the genome of Rhodosporidium azoricum by means of PCR using the primers URA5_f 5' -CAAATCGGAGGAGGAAACG-3' and URA5_r 5' -ATGTGCTAGAGAGAGAGGAC-3' . In order to obtain sufficient quantities of material for the transformation of the yeast, the cassette was inserted in a plasmid capable of autonomously replicating in E. coli, using the commercial kit TOPO TA (Invitrogen) . The transformants obtained were verified through a PCR effected directly on the colonies with the primers contained directly in the kit. A clone containing URA5 was then grown for a night on a liquid medium with ampicillin and the plasmid was extracted the day after by means of a column having a high affinity for DNA

(Pure Yield plasmid MiniPrep system, Promega) . The plasmid was then cut with the restriction enzyme BamHI, which linearizes it, to allow the integration in the receiving genome.

In parallel, mutants were obtained with the URA5 gene non-functional. To do this, the yeast Rhodosporidium azoricum was grown in a rich medium

(glucose 20 g/1, yeast extract 20 g/1, peptone 20 g/1) and 10⁷ cells were then plated on a minimal agar medium containing 5' -fluoroorotic acid (glucose 20 g/1, Yeast Nitrogen Base w/o amino acids 6.7 g/1, agar 20 g/1, 5- fluoroorotic acid 1 g/1, uracil 50 mg/1) . The plates were then incubated at 30°C until colonies appeared large enough to be transferred onto other plates. The actual mutation in the URA5 gene into the mutants thus obtained was verified by amplifying the URA5 gene and by sequencing it. Figure 3 shows the nucleotide sequences of two strains obtained as an example (mutants U24 and U27) .

In order to verify the functionality of the cassette, the uracil auxotroph mutants were transformed with the constructed cassette. The protocol envisaged the collection of a culture of exponentially growing cells at about 1-4 x 10⁷ cells ml^-1 in YPD medium (100 ml), by centrifuging for 5 minutes at 4, 000 rpm. The pellet was washed with 10 ml of buffer LiAc/TE (0.1 M Lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 8) and gently re-suspended in LiAc/TE at a final concentration of 1-4 x 10⁹ cells ml^-1. 100 μΐ of cell suspension were prepared in a sterile eppendorf test-tube for each transformation treatment, to which the DNA was added in a volume of 10 μΐ . The test-tube was gently shaken and incubated at room temperature for 5 minutes. 280 μΐ of PEG 4000 50% in LiAc/TE were added to each test-tube

(eppendorf) . The test-tubes were inverted 5-6 times to guarantee a complete mixing and then incubated at 30 °C for 45 min.; 1/10 of the volume of dimethylsulfoxide

(DMSO) , i.e. about 43 μΐ, were added to each aliquot. The cells were subjected to thermal "shock" at 42°C for 5 minutes and then immediately re-immersed in ice, in order to stimulate the DNA insertion. The cells were then washed with water 3-5 times to eliminate the residues, subjecting the cells to centrifuges of 15 seconds at 13,000 rpm, to isolate the pellet. The cells were then plated on a minimal medium, with no uracil, so as to only allow the growth of the cells transformed and which had therefore acquired a functional copy of the URA5 gene .

The clones of Rhodosporidium azoricum,. which, after transformation, showed a growth capability on plates of minimal medium, were analyzed to verify the real insertion of the wild-type URA5 gene in the genome and that it was not a reversion of the phenotype, an event that can occur spontaneously, even if rarely.

A PCR reaction was then carried out, using a pair of primers capable of revealing the presence of URA5 fused to the plasmid TOPO TA used as cloning vector. This PCR revealed the presence of the hexogen gene in all the clones obtained from the transformation of two different ura^~ strains, thus demonstrating the functioning of the gene construct object of the invention .

Example 2

An example is provided hereunder of the use of the gene construct of the invention for the expression of a gene for resistance to an antibiotic in Rhodosporidium azoricum.

In order to verify the functionality of the gene construct object of the invention and of the transformation and selection system connected to it, the expression of the gene for resistance to the antibiotic geneticin (commonly known as G418, from the name of the commercial product) was chosen, already successfully used in basidiomycete yeasts (Hua, J., J. D. Meyer and J. K. Lodge. 2000. Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans. Clinical and Diagnostic Laboratory Immunology, 7: 125-128) and to which Rhodosporidium azoricum is sensitive.

In this case, it was decided to use the module KANMX4 (SEQ ID NO: 16, promoter, gene and terminator) present in the plasmid pFA6a (SEQ ID NO: 17) normally maintained in E. coli (Wach, A., A. Brachat, R. Poehlmann and P. Philippsen. 1994. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793-1808) which contains the gene encoding an amyloglycoside phosphotransferase isolated from the bacterial transposon Tn903_r flanked by the promoter and terminator sequences of the gene TEE of Ashbya gossypii (SEQ ID NO: 8 and SEQ ID NO: 10, respectively), also already successfully tested in other basidiomycete yeasts (Ji, L., Z.-D. Jiang, Y. Liu, C. M. J. Koh and L.-H. Zhang. 2010 A simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells. Fungal Genetics and Biology, 47:279-287) . The KANMX4 module was amplified by PCR using the primers KANf 5'-ATTGGATCCGATATCAAGCTTGCCTCG-3' and KANr 5'-ATAGGATCCCACTGGATGGCGGCGTTA-3', which, in addition to the sequence homologous to the module, contain restriction sites recognized by the enzyme BamHI . The fragment obtained was purified from enzymes and salts present in the PCR reaction, then digested with the restriction enzyme BamHI, so as to generate, at the ends, single strand sequences. The plasmid TOPO TA was also digested with the same enzyme, in which the gene URA5 of Rhodosporidium azoricum had been previously inserted (see Example 1) and the two fragments were then ligated to obtain a plasmid containing, in sequence, the gene URA5 and the module KANMX5, and the ligation mixture was transformed into E. coli, made competent for the transformation. The transformants obtained were verified by means of a PCR carried out directly on the colonies, with the primers KANf and KANr . A clone containing the module ΚΆΝΜΧ4 correctly inserted was then grown overnight in a liquid medium with ampicillin and the plasmid was extracted the next day by using a column having a high affinity for DNA (Pure Yield plasmid MiniPrep system, Promega) . The plasmid was then cut with the restriction enzyme Spel (which linearizes the same plasmid) to allow its integration in the receiving genome.

The uracil auxotroph mutants were then transformed with the gene construct thus constructed. The protocol envisaged the collection of a culture of exponentially growing cells at about 1-4 x 10⁷ cells ml^-1 in YPD medium (100 ml), by centrifuging for 5 minutes at 4,000 rpm. The pellet was washed with 10 ml of buffer LiAc/TE (0.1 M Lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 8) and gently re-suspended in LiAc/TE to a final concentration of 1-4 x 10⁹ cells ml^-1. 100 μΐ of cell suspension were prepared in a sterile eppendorf test-tube for each transformation treatment, to which the DNA was added in a volume of 10 μΐ . The test-tube was gently shaken and incubated at room temperature for 5 minutes. 280 μΐ of PEG 4000 50% in LiAc/TE were added to each test-tube (eppendorf) . The test-tubes were inverted 5-6 times to guarantee a complete mixing and then incubated at 30°C for 45 min.; 1/10 of the volume of dimethylsulfoxide (DMSO) , i.e. about 43 μΐ, were added to each aliquot. The cells were subjected to thermal "shock" at 42 °C for 5 minutes and then immediately re-immersed in ice, in order to stimulate the DNA insertion. The cells were then washed with water 3-5 times to eliminate the residues, subjecting the cells to centrifuges of 15 seconds at 13, 000 rpm, to isolate the pellet. The cells were then plated on a minimal medium, containing no uracil, so as to only allow the growth of the cells transformed and which had therefore acquired a functional copy of the URA5 gene.

The clones of Rhodosporidium azoricum,. which, after transformation, showed a growth capability on plates of minimal medium, were analyzed to verify the real insertion of the wild-type URA5 gene and KANMX4 in the genome by means of a PCR with the pairs of primers URA5_f and URA5_r and KANf and KA r .

The clones obtained were parallely plated on plates containing the antibiotic geneticin (glucose 20 g/1, yeast extract 10 g/1, peptone 20 g/1, agarose 20 g/1, G418 100 mg/1) to verify the effective expression of the gene of interest inserted in the gene construct, demonstrating the correct functioning of the gene construct object of the invention.

Claims

Gene construct comprising a sequence encoding the URA5 gene for the transformation of yeast strains of the genus Rhodosporidium azoricum auxotrophic for uracil.

Gene construct according to claim 1, wherein said sequence is coding for a polypeptide with the amino acid sequence SEQ ID NO: 6.

Gene construct according to any one of the previous claims, wherein said sequence encoding the URA5 gene is the sequence SEQ ID NO: 1.

Gene construct according to any one of the previous claims, wherein said construct further comprises a promoter sequence of SEQ ID NO: 2, SEQ ID NO: 7 or SEQ ID NO: 8, preferably of SEQ ID NO: 2.

Gene construct according to any one of the previous claims, wherein said construct further comprises a terminator sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 10, preferably of SEQ ID No. 3.

Gene construct according to claim 1, wherein said construct further comprises a promoter sequence of SEQ ID NO: 2 and a terminator sequence SEQ ID NO: 3.

Gene construct according to claim 1, wherein said construct further comprises a promoter sequence of SEQ ID NO: 7 and a terminator sequence SEQ ID NO: 9.

Gene construct according to claim 1, wherein said construct further comprises a promoter sequence of SEQ ID NO: 8 and a terminator sequence SEQ ID NO: 10.

Gene construct according to claim 3, having the sequence SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 12.

10) Gene construct according to any one of the previous claims further comprising at least one nucleotide sequence and/or gene of interest to be transferred into the genome of target yeasts.

11) Gene construct according to claim 10, wherein at least one nucleotide sequence and/or gene of interest to be transferred into the genome of target yeasts are included in at least one independent gene construct which comprises a promoter and a terminator.

12) Gene construct according to any one of claims 10- 11, wherein said at least one nucleotide sequence and/or gene of interest encode resistance to antibiotics .

13) Gene construct according to any one of the previous claims, wherein said construct is comprised in an expression vector, preferably a plasmid.

14) Method of transformation of yeast cells of the species Rhodosporidium azoricum auxotrophic for uracil and selection of the transformants comprising :

transforming yeast cells with the genetic construct as defined in any one of claims 1-13;

- selecting transformants by culture over a minimal medium, containing no uracil.

15) Transformation vector, preferably a plasmid, comprising the gene construct as defined in any one of claims 1-13.

16) Yeast of the species Rhodosporidium azoricum transformed with the gene construct as defined in any one of claims 1-13.

17) Yeast deposited on May 6, 2014 under the Budapest Treaty at the Leibniz Institute DSMZ, DSM accession code 28738.