WO1998014600A1 - Candida utilis transformation system - Google Patents

Abstract

The present invention discloses a transformation system useful to express heterologous proteins in the Candida utilis yeast, based on obtaining auxotrophic mutants of said species as well as the isolation of different genes, from a genomic library, which complement said auxotrophies. The transformation system uses as hosts new auxotrophic mutants obtained from the yeast NRRL Y-1084 of Candida utilis which are defective mainly in the biosynthetic ways of uracyl and histidine, which are transformed with plasmids containing as selection markers the genes URA3 and HIS3 of Candida utilis. Another aspect of the invention is the isolation of the gene coding for the enzyme sucrose invertase or β-fructofuranosidase of Candida utilis, as well as the identification of sequences for promoting, secretion signalling and termination of said gene INV1. These sequences are useful to obtain the expression of heterologous proteins in said yeast.

Description

 UTILIS CANDIDA TRANSFORMATION SYSTEM. Technical Sector

The present invention is related to the field of genetic engineering and biotechnology, and in particular to the development of a host-vector system for the genetic transformation of the yeast Candida or tilis, which allows the expression and secretion of heterologous proteins in this yeast, which can be subsequently used for various purposes. Previous Technique

Genetic engineering and biotechnologies in general have opened an unprecedented path in the production of compounds of interest both from a medical, nutritional or industrial point of view, which report great benefits to man.

Escherichia coli bacteria have been for some time the most widely used microorganism for these purposes by various biotechnology companies, due to the knowledge they have regarding their genetics, their easy handling and their large-scale culture systems.

However, the hopes of producing protein of interest in this microorganism are affected by various factors. In the first place, the presence of pyrogens and toxic compounds in the cell wall of Escherichia coli have created regulations that limit their use, when the products obtained are for medical use or for the food industry. Also the proteins that are overexpressed in E. coli generally appear in a more soluble form and cannot be secreted. On the other hand, the mechanisms of transcription, translation and post-translational processing differ from those present in eukaryotic organisms, so that the proteins produced differ somewhat from those obtained from natural sources.

The possibility of producing heterologous proteins in eucaπot systems, such as yeasts, has some advantages over prokaryotic systems. Among these we can point out the ability to grow at high cell densities and the possibility of adapting their culture to continuous systems. In addition, yeasts are able to secrete proteins to the culture medium in considerable greater amounts compared to E. coli, and the culture media used for yeast growth are cheaper than those used in bacteria (Lemome, Y., 1988. Heterologous expression ín yeast. 8th International Biotechnology Symposium, Paris, July 17-22). In addition, these systems can carry out other post-translational modifications such as glycosylation, which is absent in bacterial systems (Fiers, W., 1988. Engmeeπng Maximal Expression of Heterologous Gene m Microorganism. 8th International Biotechnology Symposium, Paris, July 17-22) In addition, these systems generally have some preference for the same use of codons used by the cells of higher organisms (Kigsman, SM et al., 1990. Heterologous Gene Expression m Saccharomyces cerevisiae, Biotechnology & Genetic Engmeeπng Reviews , 3, Ed. GE Russell).

All this has led to the development and dissemination of new transformation systems, and with particular interest in yeasts, such as those described first for species of the Saccharomyces genus, with special emphasis on Saccharomyces cerevisiae. However, protein expression in Saccharomyces has faced problems ranging from low levels of expression obtained using its homologous promoters until hyperglycosylation of the products secreted to the medium, that is why in recent years the search for unconventional yeasts for use in the expression of heterologous proteins has intensified.

The development of transformation techniques in other non-Saccharomyces yeasts, such as Hansenula polymorpha, Pichia pastoris, and yeasts of the genus Kluyveromyces (Sudbery, P., 1994. Yeast 10: 1707-1726) has allowed a rapid advance in knowledge and improvement of these systems, as well as their greater use, both for vaccination, diagnostic and industrial purposes.

Also within the Candida genus several transformation and expression systems have been reported, such as those reported for Candida tropical is, Candida boidiini, Candida glabra ta, Candida parapsilosis, Candida mal cough and Candida albicans, all of them with a marked medical interest, because many of these species cause opportunistic diseases in humans. Within the Candida genus, the Candida utilis yeast is particularly interesting because of its peculiar characteristics. First, it uses a wide spectrum of cheap carbon sources such as xylose, sucrose, maltose among others. Another interesting feature is that it is possible to efficiently produce a large number of cells in a continuous culture. In addition, Candida utilis, like Saccnaromyces cerevisiae and Kl uyveromyces lactis, have been authorized by the FDA (Food an Drug Administration) as safe sources of food additives. At the same time, Candida utilis has been used as a source for the industrial production of L-glutamma, ethyl acetate, mvertase, among other products. A preliminary transformation system in Candida utilis has been described by Ho, I. et. al., 1984, (Biotechnology and Bioengmeenng Symp. 14: 295-301). In the mentioned work there is no direct evidence of the presence of the antibiotic resistance marker, so the direct verification of the transformation is incomplete. Recently a new strategy for a transformation system for Candiαa utilis was reported by Kondo, K. et al., 1995, (J. Bacteriol. 177: 7171-7177). They obtained cycloheximide resistant transformants by using a marker gene that contains a mutated form of the πbosomal L41 gene that confers resistance. They also use ribosomal DNA fragments (rDNA) as a target of multiple integration, because the marker needs to be present in multiple copies to confer resistance.

However, until now, there is no transformation system in this yeast based on auxotrophy markers due to the absence of mutants for the development of said system.

If we take into account the knowledge obtained in its industrial exploitation and its novelty from the genetic point of view, Candida utilis could be an attractive organism for commercial use as an expression system for heterologous proteins.

Disclosure of the invention.

The objective of the present invention has been to provide a transformation system that allows to express heterologous proteins in Candida yeast or tilis, based on obtaining auxotrophic mutants of this species as well as in the isolation of genes, from a genomic library , which complement said auxotrophies. The transformation process described here provides means for the introduction of DNA fragments or sequences into a host strain of Candida utilis and allows this yeast to be used for protein expression and production. At the same time, the isolation of genes whose promoter and thermatoring sequences can serve for the expression of heterologous genes in said yeast has been a purpose of this invention. In addition, transformed yeast cells can be identified and selected by the methods described in the present invention.

New strains of Candida utilis, vectors and subclones are provided. The new strains are used as hosts for the introduction of recombinant DNA fragments.

The invention also relates to the stable transformation and maintenance of said DNA fragments in the host cells, where the marker is integrated by homologous recombination in the yeast genome. Specifically, the invention consists of a new transformation system for the yeast Canαida or tilis, which uses new auxotrophic mutants isolated from strain NRRL Y-1084 of said yeast as hosts. These mutants are deficient in the enzyme orotιdm-5 phosphate decarboxylase of the uracil biosmhesis pathway or in the enzyme imidazole glycerol phosphate dehydratase of the biosmtetic pathway of histidma, which were obtained by using classical mutagenesis caused by physical agents chemists known from the state of the art (Sherman, F. et al., 1986. Laboratory course: Manual for methods m yeast genetics. Cold Sprmg Harbor Laboratory Press, NY). These mutants showed a high stability (reversal frequency approximately 10) and can be efficiently transformed by the method described in the present invention. In turn, the URA3 gene, coding for the enzyme orotidin 5-monophosphate decarboxylase and the HIS3 gene coding for the enzyme Imidazol-glycerol phosphate dehydratase, were isolated as selection markers for the new Candida or tilis mutants, which were isolated at from a library of Candida utilis constructed in plasmid pUC19 and identified by complement of the pyrF and hιsb463 mutations of the E strains. coli MC1066 and KC8 respectively. Likewise, the URA3 gene of C. u til is complement the URA3 mutation of Sa ccnaro yces cerevisiae SEY 2202. The complete sequence of the isolated genes was determined and the predicted amino acid sequence showed a high similarity with that of the same gene from other yeasts and mushrooms .

The mtegrative vectors constructed to perform the transformation of this mutant were pURA5 and pUREC3. These plasmids contain the URA3 gene isolated from Candida utilis as a selection marker, in addition to presenting chromosomal DNA regions that are flanking it, thus favoring integration into the Candida utilis locus by homologous recombination. Another novel aspect of the present invention is the isolation of the gene that encodes the enzyme sucrose invertase or P-fructofuranosidase {INV1) of Candida utilis, as well as the identification of the promoter, thermator regions and the signal sequence of this gene, which can also be advantageously used in the protein expression of different origins in said yeast.

The present invention also provides a series of expression vectors based on the system described above, which are used for the transformation of mutants isolated from C. utilis with a view to obtaining heterologous proteins.

The transformation system of the present invention uses as new hosts auxotrophic mutants from strain NRRL Y-1084 of Candiaa or tilis, which are mainly defective in the biosynthetic pathway of uracil and histidma, within which they were selected by its characteristics the imitant CUT-35 for uracil, as well as for the histidm the TMN-3 mutant. EXAMPLES OF REALIZATION:

Example 1: Candi da utilis mutagenesis

In order to develop a transformation system in a microorganism, three elements are generally required: (1) a selection marker for the transformation, which may be an auxotrophy marker or a dominant marker,

(2) a suitable host mutant for said selection and

(3) a method to be able to transform this host and allow it to efficiently acquire foreign DNA.

With a view to achieving the second objective, a classic mutagenesis was carried out in the Candida utilis yeast. For this, cultures of the selected yeast strain (NRRL Y-1084), were inoculated in 100 ml of YPG medium (1% yeast extract, 2% peptone, 2% glucose) and incubated in a shack at 30 ° C of 10-20 hours 50 ml of culture were taken and centrifuged at 3000 rpm for 5 minutes. Later The cells were washed twice with sterile 0.1M citrate buffer (pH 5.5) and then resuspended in 50 ml of the same buffer. Next, 10 ml of this suspension is treated with a solution of NTG (Nitroso Guanidma) at a final concentration of 50 mg / ml. The suspension in the presence of the mutagene was incubated for 30 minutes at 30 ° C at rest. The mutagen is removed from the suspension by washing twice with distilled water. The cells were resuspended in 50 ml of YPG and then transferred to an erlenmeyer with 100 ml of YPG. This mutagenized cell culture was incubated at 30 ^" C for 48 hours. Enriqu czininiento with Nystatin.

Approximately 5 ml of the culture expressed for 48 hours in YPG was used to inoculate 100 ml of minimum medium. The minimum medium (YNB; Yeast Nitrogen Base) used for enrichment with the antibiotic was not supplemented with the metabolite produced by the biosynthetic pathway in which the defect is sought. For example, for the isolation of auxotrophic mutants to uracil, it is not added to the medium. Incubation was continued until the optical density (OD) of the culture reached 20-30 ^α of the initial OD. When the culture reached the desired OD, the cell suspension was treated with 25 units / ml of a nystatma solution. The solution treated with the antibiotic was incubated at 30 ° C for 30 minutes without stirring. The nystatma was removed from the medium by washing the cell suspension twice with distilled water and then the cells were resuspended in a suitable volume to obtain 150 to 200 colonies per plate. Landscape and Selection. Plates containing the mutagenized colonies according to the enrichment performed in the previous example were plated in YNB minimal medium with and without uracil. The colonies that did not grow in the absence of uracil were taken for further analysis.

Specifically to identify the presence of ura3 and ura5 mutants, the cells were grown in the presence of the toxic acid compound 5-fluorotok (5-FOA). Resistant colonies were selected as ura 3 or ura5 mutants. Example 2: Isolation of uz a3 mutants.

After enrichment with nystatin, the culture was washed twice with distilled H ₂ 0 and disseminated directly onto YNB plates containing 0.75 -q / ml of 5-FOA (5- fluoro-orotic acid; Fluka) and 40 Mg / ml of uracil . The plates were incubated for four days and the colonies that grew therein were analyzed to check the ura phenotype. Of the 4 x 10 "viable cells, after enrichment in nystatma, 79 colonies showed resistance to 5-FOA. These colonies could be URA3, ura5, or simply resistant to 5-FOA. To confirm auxotrophy to uracil, the mutant assumptions they were punctuated on YPG medium plates and incubated for 48 hours at 30 ° C. These YPG plates were replicated on YNB minimum medium with and without uracil, obtaining 67 mutants with ura phenotype.

These mutants were checked for the frequency of reversion, highlighting a group of about 23 mutants for presenting a reversal frequency of the order of 10 ^" , which gives them acceptable stability as hosts with a view to being transformed.

The ura auxotrophic mutants thus obtained were checked for ODCase activity (activity of the URA3 gene product), by the method described by Yoshimoto et. al., 1978 (Methods Enzymol. 51: 74-79), as well as 8 of them were determined Its growth parameters. The results of the reversal frequency, as well as these last determined characteristics, are shown in Table 1. Table 1. Summary of the characteristics of the most significant ura3 mutants. Name Frequency Activity Growth Reversal OMPDCase

CUT35 <5xl0 ^"7 +++ CUT43 <lxlO ^{" 7} +++ CUT61 <lxlO ^"8 +++ CUT65 <lxlO ^{" 8} ++ CUT70 <1x10 "+ CUT88 <7xl0 ^{" 7} +++ CUT93 lxlO ^"8 ++ + CUT166 6xl0 ^"8 +++

Example 3: Isolation of mutants other than the ura phenotype.

In order to have a variety of auxotrophic mutants other than uracil, the cell suspension obtained according to enrichment with nystatin was disseminated in YPG plates, which were incubated at 30 ° C for 50 hours. Subsequently, the colonies contained in the YPG plates were replicated on plates containing YNB minimum medium, and incubated at 30 ° C for 48 hours. Colonies that did not grow on the YNB plates were taken for later analysis.

As a result, about 2411 colonies were checked and a 2% appearance of auxotrophic mutants was obtained. These mutants were checked through the Holliday test and the Finchan test, from which it was obtained that 90% of the mutants obtained responded to the his phenotype, ^" 2% to the lys phenotype ^" , 1% to the leu phenotype ^" , 1% at phenotype meth, 1% to ade phenotype ^", and 5% did not show a simple auxotrophic phenotype (Naa).

The mutants obtained were checked for the frequency of reversal, thus selecting the most stable for presenting a frequency of reversal between 10 'and 10 (Table 2).

Table 2. Reversal frequency of the most significant different uracil mutants.

Ñ Name Phenotype Reversion Frequency

TMN31 his lxlO ^"8

TMN12 his 5xl0 ^"7

TMN13 his 2.5xl0 ^"7

TMN74 his 2x10 ^"

TMN45 lys 8x10 ^" °

TMN82 Naa 2x10 ^'

Example 4: Construction of a genomic bookstore of Candida utilis. The chromosomal DNA extracted from Candida or tilis NRRL Y-1084 was partially digested with the Sau3A enzyme and fragments of sizes between 6 and 9 kb were isolated by low melting agarose gel electrophoresis

(LGT) These fragments were ligated to the pUC19 vector previously digested with the restriction enzyme BamHI and treated with alkaline phosphatase. This bond was transformed into the Escherichia coli strain MC 1066 (F ', D Lac x74, hsr, hs, rpsl, galU, galK, trip C 9030F, leuB, pyrF :: tn5). Approximately 95% of recombinants were obtained in the genomic library.

Example 5: Isolation of the URA3 gene from Candida utilis.

In order to find Candiαa _^ ilis DNA fragments that complement the pyrF mutation of the αe Escneπcnia coli strain MC1066, aliquots of the previous transformation were plated in complete medium (LBA) and the grown colonies were replicated in selective medium with the objective of find recombinant individuals that complement this strain of Escheπcma coli, considering that the URA3 gene of Saccnaromyces zerevisiae complements this mutation in E. coli.

From the individuals that complemented, about 12 colonies were taken, the plasmid was purified and rechecked by transformation in this strain of Escneπchia coli MC1066 that the restoration of the URA3 phenotype is associated with the presence of the plasmid.

Parallel to these plasmids, a restriction analysis was carried out with the hindIII and EcoRI enzymes that allowed us to identify recombinant individuals with fragments of approximately 2.6 kb where the Candida utilis URA3 gene is contained. As a result of this analysis, two plasmids were chosen, called pURA2 and püRA5, to which a restriction map was made with a view to identifying the sites present in the cloned fragment. Figure 1 shows the map of the plasmid pURA5. This plasmid was subsequently used for subsequent complementation and sequence analysis. Example 6: Dimensioning and sequencing of the Candida utilis URA3 gene.

The plasmid pURA5 was digested with several restriction enzymes according to the sites present in the vector of the püC19 library. With a view to the boundary of the Candida URA 3 gene, you use the fragments corresponding to the digestions EcoRI (1.9 kb), HincII (1.5 kb), Sacl (l, lkb) were subcloned into pBluescπpt SK (+) giving rise to plasmids pUREc-3, püRHmc-1 and pURSac-4, respectively. Of these plasmids only pURSac-4 was not able to complement the pyrF mutation of Escherichia coli (Figure 2). The 1.9 kb EcoRI fragment (pUREc-3, Figure 3) containing the useful Candida URA 3 gene is double sequenced RAMIFICATION by the method of Sanger et. to the. (1977, Proc. Nati. Acad. Sci USA 74: 5463-5467).

For this, both universal oligos of the M13mp / pUC series were used, as well as internal oligos derived from the sequence. The complete 1179 bp sequence of the EcoRI fragment is shown in Figure 4 (No. Sec. Sec: 1, 2). This fragment contains an open reading frame of 800 bp (266 codons). The URA3 gene of Canαida or til is encodes for a theoretical molecular mass protein 29 436 Da. The nucleotide sequence flanking the ATG initiation codon (GAAAATG) corresponds well with the consensus reported in yeast (A / YAA / YAATG), by Cigan and Donehue, 1987 (Gene 59: 1-18). The 3'-untranslated region contains a possible TATAAAA polyadenylation signal (AATAAAA consensus) found in the 3 'terminal region of most eucaπotic genes (Guo, Z and Sherman, F., 1995. Mol. Cell. Biol. 15: 5983-5990). Example 7: Complementation analysis in Saccharomyces cerevisiae.

In order to verify that the cloned fragment corresponds to the URA3 gene of Candida utilis and not to a DNA fragment with suppressive activity, the 2.8 kb Kpnl / Xbal fragment of the purasmid pURA5 was cloned into a pBR322 derivative vector (pBSARTR -3) . The vector pBSARTR 3 contains a Autonomous replication sequence (ARS1) and the TRIP1 selection marker, both from Saccnaromyces cerevisiae. As a result, pUT64 vector was obtained for Saccharomyces cerevisiae (Figure 5) which was used to transform the Saccnaromyces cerevisiae strain SEY2202 (URA3-52-, leu2-111, hιs3-) using the Lithium Acetate method previously reported by Ito. et. al., 1983 (J. Bacteriol. 153: 163-168). The transformants were obtained 48 hours after the transformation was performed and the presence of the plasmid was checked by hybridization αe colonies and southern blot experiments.

The transformation frequency obtained (2-5 x 10 "transf / mg) corresponded to that reported in the literature for other auxotrophic markers obtained in yeasts.

Consequently, it was demonstrated by complementation that the URA3 gene of Candida or tilis is capable of complementing the URA3 mutation of Saccharomyces cerevisiae. Example 8: Transformation of Candida utilis NRRL Y-1084 CUT35 with the pure plasmids and by the lithium acetate method.

The ñútante ara 3 strain of Candiαa j til is CUT35 (Number of Pending Deposit) was transformed by the lithium acetate method reported by Ito. et. to the. (1983, J. Bacteπol. 153: 163-168) and using as a selection marker the Candida URA3 gene or previously isolated tilis. The transformation with plasmids pURA5 and p C URA3 carrying the URA3 gene of Candida utilis was carried out by integrating said gene by homologous recombination in the corresponding region of the Candida utii is genome. The plasmid pOC URA 3 was obtained by cloning the 1.9 kb EcoRI fragment of the URA3 gene from Candida utilis in the vector pUC19 (Figure 6). To do this, the plasmids were digested with the restriction enzyme Xhol which is located towards the end of the structural gene. The linearization of the plasmid favors the homologous integration in the genomic locus

The transformation procedure used, which is based on the treatment of intact yeast cells with alkali metal cations, is basically the same used for Saccnaromyces cerevisiae, with 50 mM Lithium Acetate instead of 100 mM. The selection of the transformants was carried out in a minimal medium YNB lacking uracil. The mitotic stability of these transformants is high, due to the homologous integration mechanism. The transformation frequency coincides with that reported for mtegrative plasmids of Saccnaromyces cerevisiae and other unconventional yeasts using integrative vectors (Table 3).

Ex mplo 9: Transformation of Candida utilis NRRL Y-1084 with plasmids pURA5 and pUCURA3 using the electroporation method.

The mutant strain ura3 of Candida utilis CUT35 was transformed by the electroporation method reported by Kondo, K. et al., 1995, (J. Bacteriol. 177: 7171-7177) and using the URA3 gene of Candida utilis as the selection marker previously isolated. The transformation with plasmids pURA5 and pxxC URA 3 carriers of the Candida utilis URA3 gene was carried out by means of α-integration of said gene by homologous recombination in the corresponding region of the Candida utilis genome. The transformation procedure used is based on the treatment of intact yeast cells with an electric field set to the following conditions 0.7 kV (3.5 kV / cm), a resistance of 800 Ω and _a capacitance of 25 F.

Prior to transformation, both plasmids were digested with the restriction enzyme Xhol which is toward the end of the structural gene which favors the homologous integration event.

The selection of the transformants was carried out in a minimum medium

YNB (Yeast Nitrogen Base) lacking uracil.

The transformation frequencies with both the plasmid pURA5 and the pO URA3 by this method, comparatively with that of lithium acetate are shown in Table 3.

Table 2. Transformation frequencies obtained in the transformation of the Candida utilis CUT35 strain by different methods. In all cases the plasmids were digested with Xhol since there were no transformants with circular plasmids.

Table 3

Transformation frequency Vector Concentration of (# transí. / ^ _Q)

DNA (l- ^l g)

LiAc Electroporation pUCURA-3 0. 1 - 70-90

0. 5 - 640

3 . 0 22 pure - 5 0. 1 - 40-50

0. 5 - 670 3 0 21 -

The mitotic stability of these plasmids is high, due to the transformation mechanism.

The transformation frequency coincided with that reported for mtegrative plasmids in Saccharomyces cerevisiae and other unconventional yeasts. Figure 7 shows the integration scheme of the possible integration events in the genome of Candida utilis as well as the Southern blot of some of the transformants obtained. Example 10: Isolation of the HIS3 gene from Candida utilis.

The HIS3 gene of Candida utilis was isolated and characterized from the library previously described in Example 4. DNA fragments, which contain the HIS3 gene of Candida utilis, were isolated from a genomic library for their ability to complement the mutation hιsB463 αe Escherichia col i KC8 (hsd, hιsB463, leuB6, pyrF :: Tn5 Km, trp (9830 (lact YA), stm, galU, gal), taking into consideration that the HIS3 gene of Saccharomyces cerevisiae complements the hιsb463 mutation of Escherichia coli, using fortuitous promoter sequences in Escherichia coli.

To isolate the gene encoding the enzyme Imidazole glycerol phosphate dehydratase from Candida or til is, approximately 10 ^" transforming cells were seeded in selective medium (M9 supplemented with uracil, leucma and tryptophan. Plasmid DNA was extracted from colonies capable of growing therein. medium and therefore capable of complementing the hιsb463 mutation of the Escnericma coli KC8 strain. The plasmids obtained were used to retransform the Escherichia coli KC8 strain. All plasmids capable of supplying the histidma requirement were called pHCU. To confirm that the colonies his ^" contained the HIS3 gene of Candida utilis and not a DNA fragment with suppressive activity, two of the plasmids obtained from his (pHCU37 and pHCU40) transformants were subjected to a PCR reaction. Two degenerate oligonucleotides were used for this , designed from two highly conserved regions in 5 sequences of IGPDases corresponding to yeasts and filamentous fungi and taking into account the use of codons of Candida utilis. The predicted peptide sequence, as well as the oligonucleotide sequence are shown in Figure 8. A PCR band of approximately 500 bp that Southerbott demonstrated that hybrid with the genomic DNA of Candida utilis, was cloned into T-Vector (pMOSBLUE, Amershan) and the predicted ammoacidic sequence of its nucleotide sequence was highly homologous to Hιs3p from other yeasts and fungi. Plasmid pHCU 37 (Figure 9), was used to determine the sequence of the HIS3 gene of Candida utilis. Example 11: Sequencing of the HIS3 gene of Candida utilis. The HIS3 gene of Candida or tilis was completely sequenced double RAMIFICATION by the method of Sanger et. to the. (1977). Universal oligonucleotides of the M13mp / pUC series were used. A total of 1190 bp of the plasmid pHCU37 were sequenced starting micially with oligos designed taking into account the previously cloned PCR fragment. For the sequence sequence of the gene, internal oligonucleotides designed from the micially obtained sequence were synthesized. The complete sequence of the gene is shown in Figure 10 (No. Sec. Sec.: 5, 6). The HIS3 gene of Candida utilis encodes a theoretical molecular mass protein of 24,518 Da. Example 12: Isolation of the INVl gene encoding the Candida utilis mvertase.

In order to isolate the INVl gene that encodes the Candida utilis mvertase, the fact that the amino acid sequence of this enzyme has highly conserved regions between the species was used, on this basis the sequences of the P-frctofuranosidases of yeasts They were aligned. Two degenerate oligonucleotides used in said PCR were designed according to the use of codons in Candida utilis. The peptide sequence as well as that of the degenerated oligos is shown in Figure 11. The PCR generated a 417 bp band that was subcloned into the T-Vector (pMOSBlue, Amersham). Said band was completely sequenced and the translation of said DNA fragment corroborates the presence of consensus regions in addition to high homology with the mvertases reported in the literature. This showed that the isolated fragment was belonging to the INVl gene that codes for a mvertase in Candida utilis. This fragment was used as sonαa to isolate the INVl gene from Candida or tilis. After researching the Candida utilis library, a total of 6 clones containing the INVl gene of Candida utilis were isolated. Two of the clones were selected for their size for the sequencing process (pCI-6 and pCI-12) using a PCR using the olges used in the isolation of the above fragment. These oligos were used to initiate the entire sequence of the gene through both chains of plasmid pCI-6.

Example 13: Sequencing of the INVl gene from Candida utilis. A total of 2607 bp of clone pCI-6 containing the INVl gene encoding the Candida utilis mvertase was completely sequenced double RAMIFICATION by the method of Sanger et. al., (1977), both universal oligos of the M13mp / pUC series as well as internal oligos derived from the sequence were used for this. The complete sequence of the 2607 bp fragment is shown in Figure 12 (No. Sec. Sec: 5, 6). This fragment contains an open frame αe reading αe 1602 bp (534 codons ¡. The Candida INVl gene utilis codes for a theoretical molecular mass protein 60 703 Da.

Considering that Candida utilis mvertase is a periplasmic enzyme, it must have a peptide signal at its N-thermal end. Analyzing the sequence towards the 5 end of the gene reveals two ATG codons (ATGi and ATG ₂ in Figure 12) giving rise to ORF coding for proteins that differ only in the size of their N-thermal ends. Applying von Heijne G.'s (1986, Nucí. Acids Res. 14: 4683-4690) algorithm to predict the cut-off site of the mature protein peptidase signal derived from both ATGs reveal that in both cases the cut-off site is located between the same residues (S39 and S40 for ATGi and S26 and S27 for ATG ₂ ), giving rise to peptide signals of 39 and 26 amino acids respectively. Taking into account the average size of the signal sequences in yeast (estimated around 20 residues), we can suggest that the initiation codon of the INVl gene is the second ATG. Eleven potential N-glycosylation sites according to the general NXT / S rule are found in the asparagraphs of positions 40, 88, 141, 187, 245, 277, 344, 348, 365, 373, 379 and 399 of the sequence of the mature protein. The 5-non-translated region shows two possible TATA boxes (TATAA consensus), in the -18 to -14 and -212 to -208 regions, in addition to several possible Migl repressor binding sites (SYGGRG consensus).

BRIEF DESCRIPTION OF THE FIGURES.

Figure 1. Plasmid pURA5, resulting from the Candida utilis library, where the Candida utilis DNA fragment that complements a pyrF mutation in Escneπchia with MC1066 and URA3 of Saccnaromyces cerevisiae SEY 2202 was identified. Figure 2. Location, restriction map and complement analysis of the URA 3 gene of Candida utlilis. Sequencing strategy of said gene.

Figure 3. Plasmid pUREC3, obtained from a digestion with the restriction enzyme EcoRI in plasmid pURA5 and capable of complementing the URA3 mutation of Saccharomyces cerevisiae.

Figure 4. DNA sequence corresponding to the fragment that contains the URA3 gene of Candida or til is. Figure 5. Plasmid pUT64 obtained for the complementation experiments of the ura3 mutation in Sa ccnaromyces cere ísiae.

Figure 6. The pOC URA3 plasmid map used in the Candida utilis yeast transformation experiments. Figure 7. (A) Scheme of possible integration events during transformation

(B) Southern blot of some of the transformants.

Figure 8. Peptide sequence and DNA corresponding to the oligonucleotides used in the PCR reaction for the isolation of the HIS3 αe Candida useful gene.

Figure 9. Plasmid pHCU37, resulting from the library of

Candida u tilis, which contains a fragment capable of complementing the hιsB463 mutation of Escheπchia coli KC8.

Figure 10. DNA sequence corresponding to the fragment that contains the HIS3 gene of Ca ndida or til is.

Figure 11. Peptide sequence and DNA corresponding to the oligonucleotides used in the PCR reaction for the isolation of the INVl gene from Candida utilis.

Figure 12. DNA sequence corresponding to the fragment containing the INVl gene of Candida or til is. LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) APPLICANT:

.A) NAME: CENTER OF GENETIC ENGINEERING AND BIOTECHNOLOGY

(B) ADDRESS: AVE. 31 BETWEEN 158 AND 190, CUBANACAN, BEACH.

(C) CITY: CITY OF HAVANA (D) STATE: CITY OF HAVANA

(E) COUNTRY: CUBA

(F) ZIP CODE: 12100

(G) TELEPHONE: 53 7 216013 (H) TELEFAX: 53 7 336008 di) TITLE OF THE INVENTION: TRANSFORMATION SYSTEM IN UTILIS CANDIDA. ím) NUMBER OF SEQUENCES: 6

! ιv) PRO MACHINE LEGIBLE FORM:

A) SUPPORT TYPE: Flexible disk, B) COMPUTER: IBM PC compatible 'OR OPERATING SYSTEM: PC-DOS / MS-DOS (D) LOGICAL SUPPORT: Patentln Relay # 1.0, Version # 1.30 (EPO)

(vi) PRIORITY REQUEST DATA:

(A) APPLICATION NUMBER: 82/96

(3) APPLICATION DATE: 03-OCT-1996

(2) INFORMATION FOR SEQ ID NO. one:

(i) SEQUENCE CHARACTERISTICS: Α) LENGTH: 1179 base pairs

3) TYPE: Z nucleic acid. RAMIFICATION: simple <Z, TOPOLOGY: linear di) TYPE OF MOLECULE: DNA (genomic)

(m) HYPOTHETICAL: NO dv) ANT I - SENT I DO: NO ívι) ORIGINAL SOURCE:

Αj ORGANISM: Candiαa utilis

(B) CEPA: NRRL Y-1084 (lx) FEATURES:

I?.) NAME / KEY: PEPTIDOO MADURO '3) LOCATION: 1..1179

Z t OTHER INFORMATION: / product = "Orotidm 5'-α-carboxylase monophosphate" / gene = "URA3" (xi) SEQUENCE DESCRIPTION SEQ ID NO. one:

CAAATAGCTC TCTACTTGCT TCTGCTCAAC AAGCTGCTGG AACTGCTGCT GCTCTTTTGG 60 GTTCAATTGG TCCATCCTTG CTACTTTTCC GCCTAGTTTC GATTCCGATT CTGATAGAGA 120

AGCCCAGCTA TGAATGGAAG AAATTTTTCA CTTTTGTATG TCCTTTTTTT CACGCTTCGT 180

TGCTTCGGAC AAAAAAATAG TGGAGGCACT CGGTGGAGGG AAGCTATCCT CGAGATGAAA 240

AATTTCAAGC TCATCTCATC GTCCAAGTGG GACAGCAAGC TGAGGCTTCT GAAGAGGTTG 300

AGGAAAATGG TCACCACGTT ATCGTACACA GAGAGGGCAT CGCACCCTTC GCCACTTGCT 360 AAGCGTCTGT TTTCGCTTAT GGAGTCCAAG AAGACGAACC TGTGTGCCAG TGTCGATGTT 420

CGTACCACAG AGGAGTTGCT CAAGCTCGTT GATACGCTTG GTCCTTATAT CTGTCTGTTG 480

AAGACGCATA TTGATATCAT TGATGACTTC TCTATGGAGT CTACTGTGGC TCCACTGTTG 540

GAGCTTTCAA AAGAGCACAA TTTCCTCATC TTTGAGGACC GTAAGTTTGC TGATATCGGC 600

AACACCGTCA AGGCACAGTA CGCCGGTGGT GCGTTCAAGA TTGCACAATG GGCAGACATC 660 ACCAACGCCC ACGGTGTCAC CGGTCGAGGT ATCGTCAAGG GGTTGAAGGA GGCTGCACAG 720

GAAACCACGG ATGAGCCAAG AGGGCTGTTG ATGCTTGCTG AGCTAAGCTC CAAGGGCTCC 780

TTCGCTCACG GGACATATAC CGAGGAGACC GTGGAGATTG CCAAAACTGA TAAGGACTTT 840

TGTATTGGAT TCATCGCACA GAGAGACATG GGTGGCAGAG AAGATGGGTT CGACTGGATC 900

ATCATGACAC CAGGCGT GG ACTCGACGAT AAGGGCGACT CCCTGGGCCA ACAGTACAGA 960 ACTGTCGATG AGGTTGTCAG TGGTGGCTGT GACATCATCA TCGTTGGTAG AGGCTTGTTT 1020

GGAAAGGGAA GAGATCCAAC AGTGGAAGGT GAGCGTTATA GAAAAGCAGG CTGGGATGCT 1080

TATCTCAAGA GATACTCAGC TCAATAAACG TTGAGCTCTG GCTTGTATAG GTTCACTTGT 1140

ATAAAATGTT CATTACTGTT TTCGGAAGTT GTAGATTGC 1179

(2) INFORMATION FOR SEQ ID NO. 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 266 amino acids

(3) TYPE: amino acid

(C) RAMIFICATION: simple

(0) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: PROTEIN

(iii) HYPOTHETICAL: NO (iv) ANT I - SENT I DO: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Candida utilis

(B) CEPA: NRRL Y-1084 (ix) FEATURES:

(A) NAME / KEY: PROTEIN

(B) LOCATION: 1,266

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2: Met Val Thr Thr Leu Ser Tyr Thr Glu Arg Ala Ser His Pro Ser Pro 1 5 10 15

Leu Ala Lys Arg Leu Phe Ser Leu Met Glu Ser Lys Lys Thr Asn Leu 20 25 30

Cys Ala Ser Val Asp Val Arg Thr Thr Glu Glu Leu Leu Lys Leu Val 35 40 45

Asp Thr Leu Gly Pro Tyr He Cys Leu Leu Lys Thi His He Asp He 50 55 60

He Asp Asp Pne Ser Met Glu Ser Thr Val Ala Pro Leu Leu Glu Leu 65 70 75 80

Ser Lys Glu His Asn Phe Leu He Phe Glu Asp Arg Lys Phe Ala Asp 85 90 95

: ie Gly Asn Thr Val Lys Wing Gln Tyr Wing Gly Gly Wing Phe Lys He 100 105 110

Gln Wing Trp Wing Asp He Thr Asn Wing His Gly Val Thr Gly Arg Gly 115 120 125

He Val Lys Gly Leu Lys Glu Ala Wing Gln Glu Thr Thr Asp Glu Pro 130 135 140

Arg Gly Leu Leu Met Leu Ala Glu Leu Ser Ser Lys Gly Ser Phe Ala 145 150 155 160 His Gly Thr Tyr Thr Glu Glu Thr Val Glu He Ala Lys Thr Asp Lys

165 170 175

Asp Phe Cys He Gly Phe He Ala Gln Arg Asp Met Gly Gly Arg Glu 180 185 190

Asp Gly Phe Asp Trp He He Met Thr Pro Gly Val Gly Leu Asp Asp 195 200 205

Lys Gly Asp Ser Leu Gly Gln Gln Tyr Arg Thr Val Asp Glu Val Val 210 215 220

Be Gly Gly Cys Asp He He He Val Gly Arg Gly Leu Phe Gly Lys

225 230 235 240 Gly Arg Asp Pro Thr Val Glu Gly Glu Arg Tyr Arg Lys Wing Gly Trp

245 250 255 Asp Wing Tyr Leu Lys Arg Tyr Ser Wing Gln 260 265

(2) INFORMATION FOR SEQ ID NO. 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1190 base pairs

(B) TYPE: nucleic acid

(C) RAMIFICATION: simple (D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic) (m) HYPOTHETICAL: NO (ív) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Candida utilis (B) CEPA: NRRL Y-1084

(íx) FEATURES:

(A) NAME / KEY: mature peptide

(B) LOCATION H.1190 (D) OTHER INFORMATION: / product = "Enzyme

Imidazol-glycerol phosphate dehydratase "/ gen =" HIS3 "

(xι) SEQUENCE DESCRIPTION: SEQ ID NO. 3:

ACCTCCCAAT CGCACAGGCA ACGATACAAA TTCAACGAGT ATTAACCATC TTGTGTGCTA 60

AAAAGAGTCG AAGAACAACA GTGCGCCAAA AAAAAAACTC CGGACCGCAC ACGACTCATC 120

GCTCTCGGAA TATCCCTCGG AATGCGCCAC TTCCGGGTGC GTGGCCATCG GAAGAGCGAA 180

GAGTCATCAC CATCGTACTT TAACGACTTA CTATTCTCAT TGAGTATTGA GAAGAAGGAT 240 AGAGAAATGG CTGAACGAAC GGTGAAACCC CAGAGAAGAG CTCTTGTGAA TCGTACAACA 300

AACGAAACGA AGATCCAGAT TTCCTTGAGT TTGGATGGTG GATACGTAAC GGTTCCGGAG 360

TCAATCTTCA AGGATAAGAA GTACGACGAT GCTACTCAAG TCACCTCTTC TCAGGTGATT 420

TCAATCAACA CGGGCGTTGG ATTCCTGGAC CACATGATCC ATGCTCTTGC GAAGCATGGT 480

GGGTGGAGTT TGATTGTGGA GTGTATTGGT GATTTGCACA TTGACGACCA CCACACCACC 540 GAGGACGTTG GTATTGCGCT GGGAGACGCC GTCAAGGAGG CCTTGGCATA TAGAGGTGTC 600

AAGAGATTTG GTAGCGGGTT TGCTCCATTG GACGAGGCTC TGAGCAGAGC CGTTGTTGAT 660

CTGAGTAACC GTCCGTTTGC CGTTGTTGAG CTGGGACTCA AGAGGGAAAA GATCGGTGAC 720

TTGTCATGTG AGATGATTCC TCACTTCTTG GAGAGTTTTG CCCAAGCAGC TCATATCACG 780 ATGCATGTTG ACTGTTTGAG AGGCTTCAAC GACCATCACA GAGCTGAATC CGCATTCAAG 840

GCCCTGGCAG TCGCCATTAA GGAATCCATC TCCAGTAACG GCACCAATGA TGTTCCCTCA 900

ACAAAGGGTG TTTTGTTCTA GATAGCAGTC TTTCTGTCTC TCTATTTATT CGATAAATAA 960

GAACTATGTA TATCTTTCTC TTTTAATTGT ATATGTACAT GCACAGCTGA CTTCATCAAC 1020

GGAAGATGTT ATTGAGTGCA GCCATTGTCT GACTGTCGTT ATCCTTCTTT GCGGATTTAC 1080

CAAGGACTCT ACGACCACTG GTGGCTTTGA TATGATTTCC GCCAGTACT TGTAAGAGGT 1140

GCAACGTCAA TGGAAACGGC ACCGTTAGCC TTGATGGTTG CACGGGTAGG 1190 (2) INFORMATION FOR SEQ ID NO. 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 210 amino acids

(B) TYPE: ammoacidic (C) RAMIFICATION: simple

(D) TOPOLOGY: linear

(n) TYPE OF MOLECLE: PROTEIN iin) HYPOTHETICAL: NO

(ív) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Candida utilis

(B) CEPA: NRRL Y-1084 ix) FEATURES:

(A) NAME / KEY: PROTEIN (B) LOCATION: 1,210

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4: Met Wing Glu Arg Thr Val Lys Pro Gln Arg Arg Wing Leu Val Asn Arg 1 5 10 15

Thr Thr Asn Glu Thr Lys He Gln He Ser Leu Ser Leu Asp Gly Gly 20 25 30

Tyr Val Thr Val Pro Glu Ser He Phe Lys Asp Lys Lys Tyr Asp Asp 35 40 45

Wing Thr Gln Val Thr Ser Ser Gln Val He Ser He Asn Thr Gly Val 50 55 60

Gly Phe Leu Asp His Met He His Ala Leu Ala Lys His Gly Gly Trp 65 70 75 80

Ser Leu He Val Glu Cys He Gly Asp Leu riis He Asp Asp His His 85 90 95 Thr Thr Glu Asp Val Gly He Ala Leu Gly Asp Ala Val Lys Glu Ala 100 105 110

Leu Ala Tyr Arg Gly Val Lys Arg Phe Gly Ser Gly Phe Ala Pro Leu 115 120 125

Asp Glu Ala Leu Ser Arg Ala Val Val Asp Leu Ser Asn Arg Pro Phe

130 135 140

Wing Val Val Glu Leu Gly Leu Lys Arg Glu Lys He Gly Asp Leu Ser

145 150 155 160

: ys GH Met He Pro His Phe Leu Glu Ser Phe Ala Gln Ala Ala His 165 170 175

He Thr Met His Val Asp Cys Leu Arg Gly Phe Asn Asp His His Arg 180 185 190

Wing Glu Ser Wing Phe Lys Wing Leu Wing Val Wing He Lys Glu Ser He 195 200 205

Being 210

(2) INFORMATION FOR SEQ ID NO. 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2607 base pairs

(B) TYPE: nucleic acid 'OR RAMIFICATION: simple (D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genus ico) (m) HYPOTHETICS: NO 'ιv) ANTI-SENSE: NO

'vi) ORIGINAL SOURCE:

(A) ORGANISM: Candida utilis (B) CEPA: NRRL Y-1084

(ix) FEATURES:

(A) NAME / KEY: mature peptide (3) LOCATION H.6060 (D) OTHER INFORMATION: / PRODUCT = "Mvertase enzyme

(β-fructofuranosidase) "/ gen =" INVl "

ixi) SEQUENCE DESCRIPTION: SEQ ID NO. 5:

ATCGGCACAG AAGCGACACT GATGTCCTCC GTCTAAAACT CATCGTTTAA TAACTTCTGC 60

ATTGGCAGCT CCGGAGCACA CTCAATTGGG ACTAAAAGAA GTAACATTTG TACTACAATG 120

AGTCGTATAG AGTCATGTAT AAGAAGAACA GCAAGAAAAG AAAATATTGG TGCAGAATTC 180 AACAGCTTCT GAGATCGTAA GAACAGCCAA TCATTTACCG GAATTCATTA TGATACCTAT 240

AGAAAGACAC AAATTGTTGG GTAAAACAAC AGAACATACC TGTATAGGGG TTTATACGAG 300

AATTTTCTTA GACGTCTCCC CCAGTGTCCG CCAAAGCAAC TTACATGTGG AGTTTGAATT 360

TGGATGCGCC TTTTCCTTTA AACGGTCACC TGAGGTCTGA ATCTCAATGC AAATATCATT 420 ACACCAATAA TAAAGGTGCA TATAACCCCA TAACCTGTAC ATAAAGAACG GCACATGATC 480

CAATTTATCG ACGTTATGCC TTGTCAGACC ATCGTCGTGA ACTTTTCTAA ACCGGATAAA 540

CTCTCGCACG GATTATAACG TGCGTCTGTG ATATGCACTC CGGAAAAAAC CCCCGTGGAG 600

AAGTGAAGCG GCCACCTGTG GAGCAGAAAT TTCGATCGAC GTTTCAAGTT CAAATGGTTT 660

CCTGTTGTCA AAGGGCTTGA GATTTACCAC TTGAGCATTT GTGCTCAGAA TTCGGAGAGC 720 ATTCCCATGA GTGGTGTCCA AAAACACTAT AAAAGCAGCA CAGGGATGTC GTTGACAAAA 780

GATGCCTCAG AGGACCAAGA AGACATCAAG AGTCTCACGA TGAACACTAG TTTAGTTGAT 840

TCCAGCATTT ACAGACCATT AGTCCATCTA ACGCCACCAG TGGGGTGGAT GAACGACCCT 900

AATGGTCTCT TCTACGATTC ATCTGAATCT ACTTACCATG TGTACTACCA ATACAACCCA 960

AACGATACGA TTTGGGGATT GCCTCTATAT TGGGGACATG CCACCTCTGA TGATTTGTTA 1020 ACGTGGGACC ACCATGCGCC TGCAATTGGA CCTGAGAATG ATGATGAGGG TATTTACTCT 1080

GGATCTATAG TCATAGACTA CGATAATACC TCAGGGTTCT TTGACGATTC AACAAGACCA 1140

GAACAGAGAA TCGTTGCCAT TTATACCAAT AACTTACCAG ATGTCGAGAC GCAAGACATT 1200

GCCTATTCCA CGGACGGTGG TTATACTTTC GAAAAGTATG AAAACAACCC AGTTATAGAC 1260

GTCAATTCGA CCCAATTTAG GGATCCGAAG GTGATTTGGT ATGAGGAAAC TGAACAATGG 1320 GTCATGACTG TGGCAAAGAG TCAAGAGTAC AAGATCCAGA TTTACACCTC TGACAATTTG 1380

AAAGACTGGA GTTTGGCCTC GAATTTCTCA ACCAAGGGTT ATGTTGGTTA TCAGTATGAA 1440

TGTCCAGGTC TATTCGAAGC CACTATTGAA AACCCAAAGA GTGGTGACCC AGAGAAGAAA 1500

TGGGTTATGG TCTTAGCAAT CAATCCAGGC TCACCTCTTG GTGGTTCCAT AAATGAATAC 1560

TTTGTTGGTG ATTTCAACGG TACTGAATTC ATTCCAGATG ATGACGCTAC AAGATTTATG 1620 GATACTGGTA AGGACTTCTA TGCCTTCCAA GCGTTCTTCA ATGCACCGGA GAATCGGTCA 1680

ATTGGAGTTG CCTGGTCATC GAACTGGCAG TATTCCAACC AGGTTCCGGA TCCTGATGGA 1740

TATAGAAGCT CCATGTCATC AATCAGAGAG TACACTCTGA GATATGTCAG TACGAATCCA 1800

GAATCTGAAC AGTTGATCCT TTGTCAAAAA CCATTCTTTG TGAACGAGAC AGACTTGAAG 1860 GTGGTTGAAG AGTACAAGGT TTCAAACAGT TCTTTGACCG TGGACCACAC GTTTGGAAGT 1920

AGCTTTGCAA ACTCCAACAC CACTGGACTG TTGGATTTCA ACATGACTTT CACGGTTAAC 1980 GGTACAACTG ACGTTACGCA GAAGGACTCC GTCACCTTTG AGCTCAGAAT CAAATCTAAC 2040

CAAAGCGACG AGGCAATTGC GCTTGGTTAC GATTACAACA ACGAGCAATT CTACATCAAC 2100

AGAGCCACAG AGAGCTACTT CCAGAGAACC AACCAGTTCT TCCAGGAGAG ATGGTCCACG 2160

TACGTTCAGC CTCTCACAAT CACCGAATCT GGTGATAAAC AGTACCAGCT CTACGGATTG 2220

GTTGATAACA ACATCCTTGA GTTGTACTTC AACGACGGGG CATTCACATC CACAAACACC 2280 TTCTTCTTGG AGAAGGGCAA GCCATCAAAC GTCGATATCG TGGCAAGCTC CTCCAAGGAG 2340

GCTTACCACC GTGGACCAGC TGACTGAGAC GTCTCACTGT TTGACGAATA CGCACGTGAA 2400

AGCTATATAA GGGATCACGT GGTCTAGCCA CCCCAGTCTA AAAGCTTCAG CAAACCGCCA 2460 CTATATAAAC AGACAGGTTT GTCACTTTTC AACAAAACAA ATATCTTCTT CTTTTACCCT 2520

TCAGAGTAGT TTGTACGAGT GCTTTTTTCA ATTATATATA CAACAACGTG AGCTGCCTTT 2580

GGATATGCAA TCAACAGCGC TCTCTTT 2607 (2) INFORMATION FOR SEQ ID NO. 6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 533 amino acids (B) TYPE: amino acid

C) RAMIFICATION: simple 'D) TOPOLOGY: linear

(ιι) MCLECULE TYPE: PROTEIN (íii) HYPOTHETICAL: NO (ív) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Candida utilis

(B) CEPA: NRRL Y-1084

(ix) FEATURES: (A) NAME / KEY: PROTEIN

'B) LOCATION H. 533

(xi) SEQUENCE DESCRIPTION: SΞC ID NO. 6: Met Ser Leu Thr Lys Asp Ala Ser Glu Asp Gln Glu Asp He Lys Ser 1 5 10 15

Leu Thr Met Asn Thr Ser Leu Val Asp Ser Ser He Tyr Arg Pro Leu 20 25 30

Val H s Leu Thr Pro Pro Val Gly Trp Met Asn Asp Pro Asn Gly Leu 35 40 45 Phe Tyr Asp Ser Ser Glu Ser Thr Tyr His Val Tyr Tyr Gln Tyr Asn

50 55 60

Pro Asn Asp Thr He Trp Gly Leu Pro Leu Tyr Trp Gly His Ala Thr 65 70 75 80

Ser Asp Asp Leu Leu Thr Trp Asp His His Ala Pro Ala He Gly Pro 85 90 95

Glu Asn Asp Asp Glu Gly He Tyr Ser Gly Ser He Val He Asp Tyr 100 105 110

Asp Asn Thr Ser Gly Phe Phe Asp Asp Ser Thr Arg Pro Glu Gln Arg

115 120 125

He Val Ala He Tyr Thr Asn Asn Leu Pro Asp Val Glu Thr Gln Asp 130 135 140

He Ala Tyr Ser Thr Asp Gly Gly Tyr Thr Phe Glu Lys Tyr Glu Asn 145 150 155 160

Asn Pro Val He Asp Val Asn Ser Thr Gln Phe Arg Asp Pro Lys Val 165 170 175

He Trp Tyr Glu Glu Thr Glu Gln Trp Val Met Thr Val Ala Lys Ser 180 185 190

Gln Glu Tyr Lys He Gln He Tyr Thr Ser Asp Asn Leu Lys Asp Trp 195 200 205

Be Leu Ala Be Asn Phe Ser Thr Lys Gly Tyr Val Gly Tyr Gln Tyr 210 215 220

Glu Cys Pro Gly Leu Phe Glu Ala Thr He Glu Asn Pro Lys Ser Gly 225 230 235 240

Asp Pro Glu Lys Lys Trp Val Met Val Leu Ala He Asn Pro Gly Ser 245 250 255 Pro Leu Gly Gly Ser He Asn Glu Tyr Phe Val Gly Asp Phe Asn Gly

260 265 270

Thr Glu Phe He Pro Asp Asp Asp Ala Thr Arg Phe Met Asp Thr Gly 275 280 285

Lys Asp Phe Tyr Wing Phe Gln Wing Phe Phe Asn Wing Pro Glu Asn Arg 290 295 300

Be He Gly Val Wing Trp Be Being Asn Trp Gln Tyr Be Asn Gln Val 305 310 315 320

Pro Asp Pro Asp Gly Tyr Arg Be Be Met Be Be He Arg Glu Tyr 325 330 335 Thr Leu Arg Tyr Val Ser Thr Asn Pro Glu Ser Glu Gln Leu He Leu

340 345 350 Cys Gln Lys Pro Phe Phe Val Asn Glu Thr Asp Leu Lys Val Val Glu 355 360 365

Glu Tyr Lys Val Ser Asn Ser Ser Leu Thr Val Asp His Thr Phe Gly 370 375 380

Be Be Phe Ala Asn Be Asn Thr Thr Gly Leu Leu Asp Phe Asn Met

385 390 395 400

Thr Phe Thr Val Asn Gly Thr Thr Asp Val Thr Gln Lys Asp Ser Val 405 410 415

Thr Phe Glu Leu Arg He Lys Be Asn Gln Be Asp Glu Ala He Ala 420 425 430

Leu Gly Tyr Asp Tyr Asn Asn Glu GIn Phe Tyr He Asn Arg Ala Thr

435 440 445

Glu Ser Tyr Phe Gln Arg Thr Asn Gln Phe Phe Gln Glu Arg Trp Ser

450 455 460

Thr Tyr Val Gln Pro Leu Thr He Thr Glu Ser Giy Asp Lys Gln Tyr 465 470 475 480

Gln Leu Tyr Gly Leu Val Asp Asn Asn He Leu Giu Leu Tyr Phe Asn 485 490 495

Asp Gly Ala Phe Thr Ser Thr Asn Thr Phe Phe Leu Glu Lys Gly Lys 500 505 510 Pro Be Asn Val Asp He Val Ala Be Be Be Lys Glu Ala Tyr His 515 520 525

Arg Gly Pro Wing Asp 530

Claims

1. A Candida utilis yeast transformation system characterized in that it employs a host yeast cell capable of being transformed with a recombinant DNA material where said host is defective in at least one biosynthetic pathway. 2. A Candida utilis yeast transformation system according to claim 1 characterized in that it employs a host yeast cell that is defective in at least the biosynthetic pathway of an amino acid. 3. A transformation system for Candida or til yeast according to claim 2, characterized in that it employs a host yeast cell that is defective in the biosynthetic pathway of uracil. 4. A transformation system according to claim 3 characterized in that said host yeast is defective in the activity of the enzyme orotidin-5-phosphate decarboxylase. 5. A transformation system according to claim 4 characterized in that said host yeast cell is Candida or useful is NRRL Y-1084 CUT35 (Pending Deposit Number). 6. A Candida utilis yeast transformation system according to claim 2 characterized in that it employs a host yeast cell that is defective in at least the biosynthetic pathway of histidine. 7. A transformation system according to claim 6 characterized in that said host yeast is defective in the activity of the enzyme imidazole glycerol phosphate dehydratase. 8. A transformation system according to claim 7 characterized in that said host yeast cell is Candida utilis NRRL Y-1084 TMN3. 9. A transformation system according to claim 1 characterized in that said recombinant DNA material contains a functional gene that complements the defect in the biosm pathway in which the host is defective. 10. A transformation system according to claim 9 characterized in that said functional gene is the Candida utilis URA3 gene. 11. A transformation system according to claim 10 characterized in that said recombinant DNA material is the pURA5 plasmid and the UOC3 pOC plasmid. 12. A transformation system according to claim 9 characterized in that said functional gene is the HIS3 gene of Candida utilis. 13. A transformation system according to claim 12 characterized in that said recombinant DNA material is the plasma pHCU37 and pHCU40. 14. A Candida utilis yeast host cell capable of being transformed with a recombinant DNA where said host is defective in at least one biosmotic pathway. 15. A yeast host cell of claim 14 characterized in that said host cell is defective in at least the biosm pathway of an amino acid. 16. A yeast host cell according to claim 15 characterized in that said host cell is defective in the biosm pathway of the uracil. 17. A yeast host cell according to claim 16 characterized in that said host cell is defective in the activity of the enzyme orotιdιn-5-phosphate decarboxylase. 18. The yeast host cell according to claim 17 characterized in that it is the strain Candida utilis NRRL Y-1084 CUT35 (Pending Deposit Number). 19. A yeast host cell according to claim 15 characterized in that said host cell is defective in the biosmtic pathway of histidma. 20. A yeast host cell according to claim 19 characterized in that said host cell is defective in the activity of the enzyme imidazole glycerol phosphate dehydratase. 21. A yeast host cell according to claim 20 characterized in that it is the strain Candida utilis NRRL Y-1084 TMN3. 22. A recombinant DNA material capable of transforming a host cell of Candida yeast or tilis characterized in that said recombinant DNA material contains a functional gene that complements the defect in the biosynthetic pathway in which the host is defective. 23. A recombinant DNA material according to claim 22 characterized in that said functional gene is the Candida utilis URA3 gene. 24. A recombinant DNA material according to claim 23 characterized in that said recombinant DNA material are plasmids pURA5 and pUREC3. 25. A recombinant DNA material according to claim 22 characterized in that said functional gene is the HIS3 gene of Candida or tilis. 26. A recombinant DNA material according to claim 25 characterized in that said recombinant DNA material is the plasma pHCU37 and pHCU40. 27. Procedure for the transformation of the yeast Candida u til is characterized in that it comprises: (a) Treatment of a host strain of Candida utilis yeast with alkali metal salts; (b) Contact the cellular product of step (a) with the recombinant DNA material under conditions suitable for transformation; (c) Proceed under appropriate conditions for electroporation of the host strain. (d) Plating of the cellular product from step (c) under selective conditions of the culture medium. 28. Method according to claim 27 characterized in that the alkali metal salts used in the step (a) they are from Lithium Acetate at a concentration of 50 mM. 29. The method according to claim 27, characterized in that said conditions suitable for the transformation of step (b) comprise: - Equal volume of 70% polyethylene glycol (PEG) solution per volume of mixture of the cell suspension treated with salts of Lithium Acetate and recombinant DNA; - Incubation at 30 ° C for 60 minutes; - Thermal shock caused by treatment of the mixture at 42 ^{* "} C for 5 minutes; and - Cooling on ice for 5 minutes. 30. The method according to claim 27, characterized in that the appropriate conditions for electroporation of the host strain of step (c) are: - electric field of 3.5 kV / cm; - 800 ^ resistance, and - 25 ^ F capacitance. 31. Method for the transformation of the yeast Candida or tilis according to claim 27 characterized in that it employs a host yeast cell capable of being transformed with a recombinant DNA material where said host is defective in at least one biosm pathway. 32. A method according to claim 31, characterized in that it employs a host yeast cell that is defective in at least the biosm pathway of an amino acid. 33. Method according to claim 32 characterized in that it employs a host yeast cell that is defective in the biosynthetic pathway of uracil. 34. The method according to claim 33, characterized in that said host yeast is defective in the activity of the enzyme orotmdm-5-phosphate decarboxylase. 35. The method according to claim 34, characterized in that said host yeast cell is Candida utilis NRRL Y-1084 CUT35 (Pending Deposit Number). 36. Method according to claim 32 characterized in that it employs a host yeast cell that is defective in at least the biosmtic pathway of histidma. 37. Method according to claim 36 characterized in that said host yeast is defective in the activity of the enzyme imidazole glycerol phosphate dehydratase. 38. Method according to claim 37 characterized in that said host yeast cell is Candida or tilis NRRL Y-1084 TMN3. 39. Method according to claim 31 characterized in that said recombinant DNA material contains a gene functional that complements the defect in the biosynthetic pathway in which the host is defective. 40. The method according to claim 39, characterized in that said functional gene is the Candida or useful is URA3 gene. 41. A method according to claim 40, characterized in that said recombinant DNA material is plasmids pURA5 and piasmid pUC URA3. 42. Method according to claim 39, characterized in that said functional gene is the HIS3 gene of Candida or til is. 43. Method according to claim 42 characterized in that said recombinant DNA material is plasmids pHCU37 and pHCU40. 44. A DNA sequence encoding the URA3 gene of Candida or til s (No. Id. Sec. 1). 45. A DNA sequence encoding the HIS3 gene of Candida or tilis (No. Id. Sec. 3). 46. A DNA sequence encoding the INVl gene of Candida useful is (No. Id. Sec. 5).