AU744698B2 - Candida utilis transformation system - Google Patents

Description

METHOD AND MATERIALS FOR THE TRANSFORMATION OF THE CANDIDA UTILIS YEAST.

Technical Sector.

The present invention is related to the field of the genetic engineering and biotechnology, and in particular with the development of a system host-vector for the genetic transformation of the yeast Candida utilis which permits the expression and secretion of heterologous proteins in this yeast, which can be further used with several purposes.

Prior Art.

The genetic engineering and biotechnology have opened an unprecedented goal in the production of many interest proteins with medical, nutritious or industrial purpose, which report excellent benefits.

The bacteria Escherichia coli has been the most employed microorganism with these purposes by diverse biotechnology companies, due to the knowledge of their genetics, its easy manipulation and their systems of cultivation at high densities.

However, the hopes of the production of proteins of interest in this microorganism are affected for diverse factors. First of all the pirogenic and toxic compounds in the cell wall of Escherichia coli have provoked regulations limiting its use when the products obtained are intended to be used as medicaments or food in humans. In addition, the proteins that are over-expressed in Escherichia coli generally appear in an insoluble form that could not be secreted. On the other hand, the mechanisms of transcription, translation and postranslation modifications differ from the eukaryotic systems, resulting in recombinant proteins that in a certain way differ from that of the natural sources.

The possibility of producing heterologous proteins in eukaryotic systems, such as the yeast, has some advantages in relation to the prokaryotic systems. Among these, they could mention the capacity to grow to high cellular densities and the possibility of adapting their cultivation to continuous systems. Also the yeast are able to secrete proteins to the culture medium in considerable bigger amounts, in comparison with Escherichia coli, also the growth medium used for the growth of yeast are more economic than those used in bacteria (Lemoine, 1988. Heterologous expression in yeast. 8th International Biotechnology Symposium, Paris, July 17-22).

Also, these systems can carry out other postraductional modifications as it is the case of the glycosylation, which is absent in the bacterial systems (Fiers, 1988.

Engineering Maximal Expression of Heterologous Gene in Microorganism. 8th International Biotechnology Symposium, Paris, July 17-22). In addition, these systems generally have certain preference for the same codon use that the higher eukaryotic systems (Kigsman, S.M. et al., 1990. Heterologous Gene Expression in Saccharomyces cerevisiae, Biotechnology Genetic Engineering Reviews, 3, Ed. G.E. Russell).

All this has led to the development and dissemination of new eukaryotic transformation systems and with particular interest in yeast, firstly described for species of the Saccharomyces genus, with special emphasis in Saccharomyces cerevisiae. However, the expression of proteins in Saccharomyces has confronted problems with the expression levels obtained using their homologous promoters as well as the hyperglycosylation observed in the proteins secreted to the medium. That is the main reasons that in the last years it has been intensified the search of non-conventional yeast for their use in the expression of heterologous proteins.

With the development of transformation system in other non- Saccharomyces yeast, like Hansenula polymorpha, Pichia pastoris, and yeast of the Kluyveromyces genus (Sudbery, P., 1994. Yeast 10: 1707-1726) has permitted a quick advance in the knowledge and development of these systems, as well as increased the number of foreign proteins expressed with vaccination, diagnoses and industrial purposes in these system.

Also inside the genus Candida several transformation and expression systems have been reported, including Candida tropicalis, Candida boidiini, Candida glabrata, Candida parapsilosis, Candida maltosa and Candida albicans all with a marked medical interest, because many of these species are the causing of opportunists illnesses in humans.

Candida utilis, in the Candida genus, has special interest due to its particular characteristics. First of all, Candida utilis uses a great variety of inexpensive carbon sources such as xylose, sucrose and maltose among other. Another interesting feature is that it is possible produce efficiently a great amount of cells in continuous cultures.

Also Candida utilis, as well as Saccharomyces cerevisiae and Kluyveromyces lactis, have been authorized for the FDA (Food and Drug Administration) like safe sources in foodstuff.

Besides, Candida utilis has been used in the industry for the production of L-glutamina, etil acetate and invertase among other products.

A preliminary system for a transformation system in Candida utilis has been described by Ho, I. et. al., 1984, (Biotechnology and Bioengineering Symp. 14: 295-301). This report is incomplete because the presence of the drug resistance marker and direct evidence of the transformation process are not disclosed. Recently, a novel strategy concerning a transformation system for Candida utilis has been reported by Kondo, K. et al., 1995, Bacteriol. 177: 7171-7177). They obtained cycloheximide (CYH) resistant transformants by using a marker gene containing a mutated -4form of the ribosomal protein L41, which conferred resistance, and also used ribosomal DNA (rDNA) fragment as a multicopy target for plasmid integration because the marker need to be present in multiple copies for selection of CHY-resistant transformants.

Many attempts has been done to use Candida utilis as host for heterologous gene expression, nevertheless, a transformation procedure in Candida utilis using auxotrophyc mutants has not been developed up to now.

It if is taken into account the knowledge obtained in the industrial exploitation of Candida utilis and the novelty of its genetics, it could be considered an attractive mircoorganism for its commercial utilization as expression system of heterologous S. 10 proteins.

Summary of the Present Invention.

In a first aspect of the present invention there is provided a Candida utilis transformation system comprising a host Candida utilis yeast cell capable of being transformed with recombinant DNA material wherein the host cell has at least one 15 defective biosynthetic pathway obtained by chemical mutagenesis.

In a second of the present invention there is provided a method of transforming a Candida utilis host cell with at least one defective biosynthetic pathway obtained by chemical mutagenesis, comprising: transforming the host cell with recombinant DNA material; wherein the recombinant DNA material comprises a functional gene that complements the defect in the biosynthetic pathway.

In a third aspect of the present invention there is provided a yeast host cell comprising a Candida utilis cell capable of being transformed with recombinant DNA wherein said host has at least one defective biosynthetic pathway obtained by chemical SRAm f/ 'pmutagenesis.

21987-GO.DOC 4a- In a fourth aspect of the present invention there is provided recombinant DNA material when used in a method of the present invention wherein the recombinant DNA material is capable of integration into the genome of the Candida utilis yeast host cell, and contains a functional gene that complements the defect in said biosynthetic pathway in which the host cell is defective.

In addition, disclosed herein is an isolated DNA sequence encoding the HIS3 gene of Candida utilis.

There is also provided an isolated DNA sequence comprising SEQ ID NO:3.

Any discussion of the prior art throughout the specification should in no way be 10 considered as an admission that such prior art is widely known or forms part of common S general knowledge in the field.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Brief Description of the Accompanying Figures.

Figure 1. Plasmid pURA5, obtained in the Candida utilis genomic library by complementation of the Escherichia coli MC 1066 pyrF Saccharomyces cerevisiae SEY 2202 ura3 mutations.

Figure 2. Restriction enzyme map, sequence strategy and complementation analysis of the URA3 gene from Candida utilis.

Figure 3. Plasmid pUREC3 obtained by cloning of the 1.9 kb EcoRI fragment of the plasmid pURA5 in the pBLUESCRIPT 21987-OO.DOC 4b Figure 4. Amino acid sequence deduced from the DNA sequence of the URA3 gene, and the DNA sequence of the DNA encoding it.

Figure 5. Plasmid pUT64 obtained for the complementation experiment in the Sacharomyces cerevisiae ura3 mutant strain.

Figure 6. Plasmid pUCURA3 used in the Candida utilis transformation experiments.

Figure 7. Predicted arrangements of the vector DNA integrated at the URA3 locus by homologous recombination.

DNA-blot hybridisation of genomic DNA from some transformants.

Figure 8. DNA sequence of the primers used in the isolation of the HIS3 gene, and the 10 deduced amino acid sequence encoding it.

Figure 9. Plasmid pHCU37, obtained in the Candida utilis genomic library by complementation of the Escherichia coli KC8 hisb463 mutation.

Figure 10. Amino acid sequence deduced from the DNA sequence of the HIS3 gene, and the DNA sequence of the DNA encoding it.

Detailed Description of Preferred Embodiments of the Present Invention The present invention if various embodiments provides a method and materials for the transformation of Candida utilis yeast, use for expressing heterologous proteins in this yeast and based on the provision of auxotrophic mutants in this species, as well as the isolation of different genes from a genomic library which complement said auxotrophs.

The transformation process described herein provides means to introduce DNA fragments or sequences into Candida utilis host cells and allows Candida utilis to be used as a host system for gene expression and protein production.

21987-00.DOC 4c- Furthermore transformed yeast cells can be identified and selected by the methods described in the present invention. Novel strains of Candida utilis, vectors and subclones are provided. Novel yeast strains are used as hosts for introduction of recombinant DNA fragments.

The invention further relates to stable transformation and maintenance of DNA in host cells, where the marker is homologously integrated in the genome of the yeast.

e* *o 21987-OO.DOC Concretely the present invention consists in a transformation system in the yeast Candida utilis, which uses as hosts new auxotrophyc mutants isolated from the strain NRRL Y-1084 of said yeast. These mutants are defective in the enzyme orotidin-5' phosphate decarboxylase of the biosynthetic pathway of the uracil or in the biosynthetic pathway of the histidine, and were obtained by classical mutagenesis using both UV and NTG as mutagenic agents known from the prior art (Sherman, F. et al., 1986. Laboratory course: Manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, NY). These mutants present a high stability (frequency of reversion approximately of 10-8) and can be efficiently transformed with the procedure described in this invention.

In addition, it was isolated as selection markers for the mutants of Candida utilis the gene URA3, encoding for the orotidin 5'-phosphate decarboxylase enzyme and HIS3 encoding for the Imidazol-glycerol-phosphate dehydratase enzyme which were isolated from a gene library of Candida utilis in PUC19 and identified by complementation of the pyrF and hisb463 mutations respectively in the strain Escherichia coli MC1066.

Similarly the mutation ura3 of Saccharomyces cerevisiae strain SEY 2202 was used to identify this gene came from C.

utilis. The complete sequence of these genes was determined and the predicted amino-acid sequences show high similarities with that of the same gene from other yeast and fungi.

Some of the vectors used in the transformation system were the plasmids pURA5 and pUREC3 which comprise the URA3 gene, capable of being integrated into the homologous locus of the Candida utilis mutant host by homologous recombination.

The present invention also provides a set of plasmids based on those described formerly, which are used for the transformation of the mutants isolated from C. utilis in order to obtain heterologous proteins.

The transformation system of the present invention uses as hosts new auxotrophyc mutants obtained from the strain NRRL Y-1084 of Candida utilis, which are defective mainly in the uracil and histidine pathways, and among them were selected for their characteristics the mutant CUT-35 (ura-) and the mutant TMN-3 (his-).

EXAMPLES:

Example 1: Mutagenesis of Candida utilis To develop a transformation system in a microorganism they are generally required three elements: a marker for the selection of the transformants, that could be an auxotrophyc or a dominant marker, a mutant or appropriate host for this selection and a method to reproducible introduce the extranuclear DNA in the host in an efficient form.

In order to achieving the second objective, it was carried out a classical mutagenesis in the yeast Candida utilis.

Cultures of the of the selected yeast strain (NRRL Y-1084) were inoculated in 100 ml of YPG medium (Yeast extract 1%, peptone glucose and they were incubated in a shaker at 30 0 C for 10-20 hours. 50 ml of the culture was centrifuged to 3000 rpm for 5 minutes. Later the cells were washed 2 times with citrate buffer 0.1M (pH 5.5) sterile and resuspended in 50 ml of the same buffer. After, 10 ml of this suspension was incubated with a solution of NTG to a final concentration of 50 mg/ml. The suspension was incubated for minutes at 30 0 C in repose.

The NTG was removed of the suspension washing 2 times with distilled water. The cells were resuspended in 50 ml of YPG and then they were transferred to an erlenmeyer with 100 ml of YPG. This culture of mutant cells was incubated at 30 0

C

for 48 hours.

Enrichment with nystatin Approximately 5 ml of the 48 hours YPG expressed culture was used to inoculate 100 ml of minimum medium. The minimum medium (YNB, Yeast Nitrogen Base) used for the enrichment with the antibiotic was not supplement with the metabolic produced by the biosynthetic via in which the defect is looked for. For example, for the isolation of auxotrophics mutants for uracil, it is not added to the medium.

The incubation was continued until the optical density (OD) of the culture reached 20 to 30% of the initial OD. When the culture reached the desire OD, the cellular suspension was treated with 25 units/ml a solution of nystatin. The solution with the antibiotic was incubated at 30 0 C for 30 minutes without agitation. The nystatin was eliminated of the medium washing the cellular suspension with distilled water 2 times and later the cells were resuspended in an appropriate volume in order to obtain 150 to 200 colonies per plate.

Screening and selection.

The plates containing the mutagenized colonies according to the example 1, were plated in YNB mediums with and without between uracil. The colonies that did not grow in absence of uracil were taken for further analysis.

Specifically in order to identify the presence ura3 or mutants, the cells were grown in presence of 5-fluorotic acid (5FOA). The resistant colonies were selected as ura3 or like mutants.

Example 2: Isolation of ura3 mutants.

After the nystatin enrichment the culture was washed twice with distilled water and plate directly on YNB plates containing 0.75 tg/ml of 5-FOA (5-fluoro-orotic acid, Fluka) and 40 gg/ml of uracil. The plates were incubated four days and the colonies that grew were analyzed in order to check the ura- phenotype. From the 4x10 4 viable cells, after the nystatin enrichment, 79 colonies showed resistance to the FOA. These colonies could be ura3, ura5, or simply resistant to the 5-FOA. To confirm the uracil auxotrophy the supposed mutants were plated in YPG medium incubated 48 hours to and replicated in YNB plates with and without uracil. A total of 67 colonies were unable to grow in YNB without uracil, shown a ura- phenotype.

The frequency of reversion of all these mutants were determined standing out a group of 23 mutants by presenting a frequency of reversion in the order of 10-8, what confers them certain stability to be used as a host for transformation system.

The orotidin 5'-monophosphate decarboxylase (ODCase) activity of all the uracil auxotrophyc mutants was determined by the method of Yoshimoto et. al., 1978 (Methods Enzymol. 51: 74- 79), as well as it was determined their growth conditions.

These results are shown in Table 1.

Table 1. Summary of the characteristics of more significant ura3 mutants.

Name Reversion OMPDCase Growth Frequency Activity 5x10 7 CUT43 1x10 7 CUT61 x10 8 1x10 8 1x10 8 CUT88 7x107 CUT93 1x10 8 CUT166 6x10 8 Example 3: Isolation of other mutants different of the phenotype ura-.

With the objective of having a variety of auxotrophyc mutants different than uracil, the cellular suspension obtained according to the nystatin enrichment were plated in YPG and incubated at 30 0 C for 50 hours. Later the colonies contained in the YPG plates were replicated on plates containing YNB medium and incubated at 30 0 C for 48 hours. The colonies unable to grow in the YNB plates were taken for further analysis.

Around 2411 colonies were screened and consequently was obtained a 2% of appearance of auxotrophyc mutants. These mutants were checked using the Holliday and the Finchan tests. It was obtained 90% of his- mutants, 2% responded to the phenotype lys-, 1% to the phenotype leu-, 1% to the phenotype met-, 1% to the phenotype ade- and the 5% did not show a simple auxotrophyc phenotype (Naa).

The mutants having frequency of reversion between 10 and 10' were selected for further analysis (Table 2).

.Table 2: Name Phenotype Reversion Frequency TMN3 his- 1x10 8 TMN31 his- 1x10 8 TMN64 his- 1xl10 8 TMN9 his- 4x10-7 TMN12 his- 5x10-7 TMN13 his- 2.5x10 7 TMN62 his- 8x10-7 TMN74 his- 2x10 7 TMN78 his- 2x10 7 lys- 8x10' TMN71 his- 2x10-6 TMN82 Naa 2x10 6 Example 4: Construction of a Candida utilis genomic library.

The chromosomal DNA extracted from Candida utilis NRRL Y-1084 was partially digested with the enzyme Sau3A and fragments with sizes between 6 and 9 kb were isolated by electrophoresis in low point gel temperature agarose (LGT).

These fragments were ligated in the pUC19 vector previously digested with BamHI and treated with alkaline phosphatase.

This ligation were transformed in Escherichia coli MC 1066 D Lac x74, hsr, hsm, rpsl, galU, galK, trip C 9030F, leuB, pyrF::tn5) strain. Aproximately 95% of recombinants were obtained in the genomic library.

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

As a marker for transformation of the Candida utilis ura3 host, the URA3 gene from Candida utilis was isolated and characterized. DNA fragments which contained the Candida utilis URA3 gene were isolated from a Candida utilis pUC19 genomic library by the ability to complement Escherichia coli pyrF mutation, taking into account that URA3 gene from Saccharomyces cerevisiae complements the pyrF mutation of E.

coli, using fortuitous promoter activity in Escherichia coli.

When this library was spread on uracil-deficient medium, 12 independent pyrF+ colonies were isolated. Two of these clones (pURA-2 and pURA-5) had the same 2.6-kb genomic Candida utilis insert DNA on pUC19 using HindIII and EcoRI restriction digestions. DNA from both plasmids transformed Escherichia coli MC1066 to Ura' at a high frequency. The map of one of the Candida utilis URA3 gene-pUC19 recombinant plasmid (pURA-5) is shown in Figure 1. This plasmid was used for further complementation and sequence analysis.

Example 6: Demarcation and sequence analysis of Candida utilis URA3 gene.

The plasmid pURA5 was digested with several restriction enzymes. The fragments corresponding to the EcoRI digestions (1,9 kb), HincII (1,3 kb), SacI (1,1 kb) were subcloned in pBluescript SK giving rise the plasmids pUREc-3, pURHinc- 1 pURSac-4, respectively. Fragment corresponding to the plasmid pURSac-4 was not able to complement the pyrF mutation of Escherichia coli (Figure 2).

The 1,9 kb EcoRI fragment of (pUREc-3, Figure 3) containing the URA3 gene of Candida utilis was completely double strand sequenced by the method of Sanger et. al. (1977, Proc. Natl.

Acad. Sci USA 74: 5463-5467).

With this end, the universal oligonucleotides of the series M13mp/pUC, as well as internal oligonucleotides derived of the sequence were used. The complete sequence of 1179 bp of the EcoRI fragment is shown in Figure 4 (Seq. Id. No.: 1, 2).

This fragment contains an open reading frame of of 800 bp (266 codons) The Candida utilis URA3 gene codes for a protein with theoretical molecular mass of 29 436 Da. The nucleotide sequence flanking to the ATG initiation codon (GAAAATG) corresponds well with the consent reported in yeast (A/YAA/YAATG), by Cigan y Donehue, 1987 (Gene 59: 1-18).

The 3'-non translated region contains a putative polyadenilation site (TATAAAA, consensus AATAAAA) present in the 3 terminal region in most of the eukaryotic genes (Guo, Z y Sherman, 1995. Mol. Cell. Biol. 15: 5983-5990).

Example 7: Complementation analysis in Saccharomyces cerevisiae.

With the objective to verify that the fragment cloned, correspond to the Candida utilis URA3 gene and not a DNA fragment with suppressor activity, the 2.8 kb KpnI/XbaI fragment of the pURA5 plasmid was cloned in a pBR322 derivative vector (pBSARTR-3). The pBSARTR-3 vector posses an autonomous replicating sequence (ARS1) and the TRP1 gene selection marker both from Saccharomyces cerevisiae.

Consequently, the plasmid pUT64 (Figure 5) was obtained and used to transform the Saccharomyces cerevisiae strain SEY2202 (ura3-52-, leu2-112, his3) using the lithium acetate method, previously reported by Ito. et. al., 1983 Bacteriol. 153: 163-168).

The transformants were obtained 48 hours after the transformation. The presence of the replicative plasmid was checked using both colony hybridization and southern-blot experiments.

The frequency of transformation obtained (2-5 x 102 transf/mg) is in agreement with that of reported in the literature for other auxotrophic markers from other yeast.

Consequently, it was demonstrated that the gene URA3 from Candida utilis is able to complement the ura3 mutation of Saccharomyces cerevisiae Example 8: Transformation of Candida utilis NRRL Y-1084 with the plasmids pURA5 and pUCURA3 using the LiAc method.

The ura3 mutant strain of Candida utilis CUT35, phenotype ura- and deposited under accession number CBS 100085 at Centraalbureau voor Schimmelcultures, The Netherlands, on October 1, 1997, was transformed using the method of lithium acetate reported by Ito. et. al. (1983, J. Bacteriol. 153: 163-168), and using the previously isolated URA3 gene from Candida utilis as a selection marker. The vectors (pURA5 and pUCURA3), used in the transformation system were designed to directly integrate into the homologous locus of the Candida utilis mutant strain by homologous recombination. The plasmid pUCURA3 was obtained by cloning the 1,8 kb EcoRI fragment of the Candida utilis URA3 gene in the corresponding site of the vector pUC19 (Figure Previously to the transformation procedure, both plasmids were digested in XhoI site, which is located in the 5' prime of the structural gene. The linearization of the plasmids favors the homologous integration in the genomic locus.

The transformation procedure was performed as previously described by Ito. et al., for lithium acetate procedure, except that the concentration used for LiAc was 50 mM in the present case. In order to carry out this method, after the colony growth on YPD plate, the selected strain is cultured with shaking in 5 ml of YPD liquid medium at 30 0 C for about 8 hours, it is inoculated in 100 ml of YPD liquid medium at a concentration of OD 600 =0.003 and cultured with shaking at 30 0

C

for about 16 hours. After the cells have grown to logarithmic phase (DO 600 they are collected by centrifugation at 3000 rpm for 5 minutes. The cells were washed once with 3 ml of sterilized water. After, the cells are suspended into 1 ml of 50 mM LiAc. Afterward they are incubated with agitation at 0 C for 1 hour. Consequently 100 il of cells were aliquoted in microfuge tube and 5 4g of DNA was added. Then the mix cells/DNA was incubated at 30°C for 30 minutes. Consequently the cells/DNA treated with 0.7 ml of 40% of PEG 4000 and mM of LiAc were incubated at 30 0 C for 1 hour. Afterward a heat shock at 42 0 C for 5 min was applied in water bath.

Immediately the mix was spin for 30 seconds and the mix cells/DNA was washed twice in Tris 10mM, EDTA 1mM pH 8.

Finally the supernatant is removed, then the cells are suspended in 200 il of Tris 10 mM pH 8, EDTA 1 mM pH 8 so as the final volume.

The selection of the transformants was carried in YNB minimum medium lacking uracil. The mitotic stability of these transformants was high, due to the mechanism of homologous integration. The transformation frequency coincides with that of reported for Saccharomyces cerevisiae and other nonconventional yeast using integrative vectors (table 2).

Example 9: Transformation of Candida utilis CUT35 with the plasmids pURA5 and pUCURA3 using electroporation method.

The ura3 mutant strain of Candida utilis CUT35 was transformed using the electroporation method reported by Kondo, K. et al., 1995, Bacteriol. 177: 7171-7177), using the previously isolated URA3 gene from Candida utilis as a selection marker. The vectors (pURA5 and pUCURA3), used in the transformation system were designed to directly integrate into the homologous locus of the Candida utilis mutant strain by homologous recombination.

The procedure used is based on the treatment of the intact cell yeast with an electric field. The following conditions were used: 0.7 kV (3,5 kV/cm) as a pulse, a resistance of 800 9 and a capacitance of 25 iF.

Previously to the transformation procedure, both plasmids were digested with XhoI which is located in the 5' prime of the structural gene facilitating the homologous integration in the genomic locus.

The selection of the transformants was done in YNB minimal medium without uracil.

The frequency of transformation using both pURAS and pUCURA3 depended of the plasmid concentration. A comparison of both methods (LiAc and electroporation) is shown in Table 3.

Table 3 Transformation frequency Vector DNA Concentration transf./g) (pg) LiAc Electroporation pUCURA-3 0.1 -70-90 640 22 0.1 -40-50 -670 21 The mitotic stability of these transformants was high, due to the mechanism of integration.

The frequencies of transformation coincide with that of reported for Saccharomyces cerevisiae and other nonconventional yeast using integrative vectors.

In figure 7 is shown the outline of the possible integration events in the genome of Candida utilis as well as the Southern-blot of some transformants.

Example 10: Isolation of the HIS3 gene of Candida utilis.

The HIS3 gene from Candida utilis was isolated and characterized from the library previously described in the Example 4. DNA fragments, which contained the Candida utilis HIS3 gene, were isolated from a Candida utilis genomic library by the ability to complement the hisb463 mutation in the Escherichia coli KC8 (hsd, hisB463, leuB6, pyrF::Tn5 Kmr, trp (9830 (lact YA), stm, galU, gal), taking into account that HIS3 gene from Saccharomyces cerevisiae complements the hisb463 mutation of Escherichia coli, using fortuitous promoter activity in Escherichia coli.

In order to isolate the HIS3 gene, 10 5 cells were spread on minimum medium (M9) supplemented with uracil, tryptophan and leucine. Plasmid DNA was extracted from colonies able to growth in this medium and consequently capable to complement the hisb463 mutation in the Escherichia coli KC8 mutant strain. The plasmid DNAs isolated were used to retransform the Escherichia coli KC8 mutant strain. All the plasmid able to supplement the histidine requirement of the mutant strain were denominated pHCU. In order to confirm that the his' colonies contained the HIS3 gene of Candida utilis and not a fragment of ADN with suppressor activity, two of the plasmids obtained from the his' transformants (pHCU37 pHCU40) were subjected to a PCR reaction. Two degenerate oligonucleotides from two regions highly conserved in five IGPDasas sequences from yeast and fungi were used. The oligonucleotide as well as amino-acid sequences of the degenerate oligonucleotides are shown in the figure 8.

Approximately a 500-bp PCR band corresponding to the coding sequence of the HIS3 gene from Candida utilis was amplified.

The approximately 500 bp PCR fragment, which was shown by Southern blot to hybridize the Candida utilis genomic DNA, was cloned in T-Vector (pMOSBLUE, Amershan) and the predicted amino-acid translation of its sequence was shown to be highly identical to His3p from other yeast and fungi. The plasmid pHCU3-7 (Figure 9) was used for the determination of the entire sequence of the HIS3 gene from Candida, utilis.

Example 11: Sequencing of the EIB3 gene of Candida utilia.

The HIS3 gene from Candida utilis was completely double strand sequenced using the method of Sanger et. al. (1977).

Oligonucleotides of the universal series M13mp/pUC were used.

Primers taken from the PCR fragment were used to initiate sequencing of the entire gene. A total 1190 bp of the pHCU37 was sequenced. The entire sequence HIS3 from Candida utilis 15 is shown in Figure 10 (Seq. Id. No.: 5, 6).

This fragment contains an open reading frame of 210 codons.

The Candida utilis HIS3 gene code for a protein with theoretical molecular mass of 24 518 Da.

NEXT PAGE IS PAGE 19 *~oo *o ooa SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA STREET: AVE. 31 ENTRE 158 Y 190, CUBANACAN, PLAYA.

CITY: CIUDAD DE LA HABANA STATE: CIUDAD DE LA HABANA COUNTRY: CUBA POSTAL CODE (ZIP) 12100 TELEPHONE: 53 7 216013 TELEFAX: 53 7 336008 (ii) TITLE OF INVENTION: TRANSFORMATION SYSTEM IN CANDIDA UTILIS.

(iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: 82/96 FILING DATE: 03-OCT-1996 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1179'base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Candida utilis STRAIN: NRRL Y-1084 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..1179 OTHER INFORMATION:/product= "Enzyme decarboxylase /gene= "URA3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CAAATAGCTC TCTACTTGCT TCTGCTCAAC AAGCTGCTGG AACTGCTGCT GCTCTTTTGG GTTCAATTGG TCCATCCTTG CTACTTTTCC AGCCCAGCTA TGAATGGAAG AAATTTTTCA TGCTTCGGAC AAAAAAATAG TGGAGGCACT AATTTCAAGC TCATCTCATC GTCCAAGTGG AGGAAAATGG TCACCACGTT ATCGTACACA AAGCGTCTGT TTTCGCTTAT GGAGTCCAAG CGTACCACAG AGGAGTTGCT CAAGCTCGTT

GCCTAGTTTC

CTTTTGTATG

CGGTGGAGGG

GACAGCAAGC

GAGAGGGCAT

AAGACGAACC

GATTCCGATT

TCCTTTTTTT

AAGCTATCCT

TGAGGCTTCT

CGCACCCTTC

TGTGTGCCAG

AAGACGCATA

GAGCTTTCAA

AACACCGTCA

ACCAACGCCC

GAAACCACGG

TTCGCTCACG

TGTATTGGAT

ATCATGACAC

ACTGTCGATG

GGAAAGGGAA

3 5 TATCTCAAGA

ATAAAATGTT

TTGATATCAT TGATGACTTC AAGAGCACAA TTTCCTCATC AGGCACAGTA CGCCGGTGGT ACGGTGTCAC CGGTCGAGGT ATGAGCCAAG AGGGCTGTTG GGACATATAC CGAGGAGACC TCATCGCACA GAGAGACATG CAGGCGTGGG ACTCGACGAT AGGTTGTCAG TGGTGGCTGT GAGATCCAAC AGTGGAAGGT GATACTCAGC TCAATAAACG CATTACTGTT TTCGGAAGTT GATACGCTTG GTCCTTATAT TCTATGGAGT CTACTGTGGC TTTGAGGACC GTAAGTTTGC GCGTTCAAGA TTGCACAATG ATCGTCAAGG GGTTGAAGGA ATGCTTGCTG AGCTAAGCTC GTGGAGATTG CCAAAACTGA GGTGGCAGAG AAGATGGGTT AAGGGCGACT CCCTGGGCCA GACATCATCA TCGTTGGTAG GAGCGTTATA GAAAAGCAGG TTGAGCTCTG GCTTGTATAG

GTAGATTGC

CTGATAGAGA

CACGCTTCGT

CGAGATGAAA

GAAGAGGTTG

GCCACTTGCT

TGTCGATGTT

CTGTCTGTTG

TCCAC!TGTTG

TGATATCGGC

GGCAGACATC

GG3CTGCACAG

CAAGGGCTCC

TAAGGACTTT

CGACTGGATC

ACAGTACAGA

AGGCTTGTTT

CTGGGATGCT

GTTCACTTGT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1179 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 266 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Candida utilis STRAIN: NRRL Y-1084 (ix) FEATURE: NAME/KEY: Protein 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 Leu Ala Lys Arg Leu Phe Ser Leu Met Glu Ser Lys Lys Thr Asn Leu 25 Cys Ala Ser Val Asp Val Arg Thr Thr Glu Glu Leu Leu Lys Leu Val 35 40 Asp Thr Leu Gly Pro Tyr Ile Cys Leu Leu Lys Thr His Ile Asp Ile 55 Ile Asp Asp Phe Ser Met Glu Ser Thr Val Ala Pro Leu Leu Glu Leu 70 75 Ser Lys Glu His Asn Phe Leu Ile Phe Glu Asp Arg Lys Phe Ala Asp 90 Ile Gly Asn Thr Val Lys Ala Gin Tyr Ala Gly Gly Ala Phe Lys Ile 100 105 110 Ala Gin Trp Ala Asp Ile Thr Asn Ala His Gly Val Thr Gly Arg Gly 115 120 125 Ile Val Lys Gly Leu Lys Glu Ala Ala Gin 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 Ile Ala Lys Thr Asp Lys 165 170 175 Asp Phe Cys Ile Gly Phe Ile Ala Gin Arg Asp Met Gly Gly Arg Glu 180 185 190 Asp Gly Phe Asp Trp Ile Ile Met Thr Pro Gly Val Gly Leu Asp Asp 195 200 205 Lys Gly Asp Ser Leu Gly Gin Gin Tyr Arg Thr Val Asp Glu Val Val 210 215 220 Ser Gly Gly Cys Asp Ile Ile Ile 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 Ala Gly Trp 245 250 255 Asp Ala Tyr Leu Lys Arg Tyr Ser Ala Gin 260 265 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1190 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Candida utilis STRAIN: NRRL Y-1084 (ix) FEATURE: NAME/KEY: matpeptide LOCATION:1..1190 OTHER INFORMATION: /product= "Enzyme imidazol-glycerol phosphate dehydratase /gene= "HIS3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ACCTCCCAAT CGCACAGGCA ACGATACAAA TTCAACGAGT ATTAACCATC TTGTGTGCTA

-'U

AAAAGAGTCG

GCTCTCGGAA

GAGTCATCAC

AGAGAAATGG

AACGAAACGA

TCAATCTTCA

TCAATCAACA

GGGTGGAGTT

GAGGACGTTG

AAGAGATTTG

CTGAGTAACC

TTGTCATGTG

ATGCATGTTG

GCCCTGGCAG

ACAAAGGGTG

AAGAACAACA GTGCGCCAAA TATCCCTCGG AATGCGCCAC CATCGTACTT TAACGACTTA CTGAACGAAC GGTGAAACCC AGATCCAGAT TTCCTTGAGT AGGATAAGAA GTACGACGAT CGGGCGTTGG ATTCCTGGAC TGATTGTGGA GTGTATTGGT GTATTGCGCT GGGAGACGCC GTAGCGGGTT TGCTCCATTG GTCCGTTTGC CGTTGTTGAG AGATGATTCC TCACTTCTTG ACTGTTTGAG AGGCTTCAAC TCGCCATTAA GGAATCCATC TTTTGTTCTA GATAGCAGTC

AAAAAAACTC

TTCCGGGTGC

CTATTCTCAT

CAGAGAAGAG

TTGGATGGTG

GCTACTCAAG

CACATGATCC

GATTTGCACA

GTCAAGGAGG

GACGAGGCTC

CTGGGACTCA

GAGAGTTTTG

GACCATCACA

TCCAGTAACG

TTTCTGTCTC

CGGACCGCAC

GTGGCCATCG

TGAGTATTGA

CTCTTGTGAA

GATACGTAAC

TCACCTCTTC

ATGCTCTTGC

TTGACGACCA

CCTTGGCATA

TGAGCAGAGC

AGAGGGAAAA

CCCAAGCAGC

GAGCTGAATC

GCACCAATGA

TCTATTTATT

ACGACTCATC

GAAGAGCGAA

GAAGAAGGAT

TCGTACAACA

GGTTCCGGAG

TCAGGTGATT

GAAGCATGGT

CCACACCACC

TAGAGGTGTC

CGTTGTTGAT

GATCGGTGAC

TCATATCACG

CGCATTCAAG

TGTTCCCTCA

CGATAAATAA

GAACTATGTA TATCTTTCTC TTTTAATTGT ATATGTACAT GCACAGCTGA CTTCATCAAC GGAAGATGTT ATTGAGTGCA GCCATTGTCT GACTGTCGTT ATCCTTCTTT GCGGATTTAC CAAGGACTCT ACGACCACTG GTGGCTTTGA TATGATTTCC TGCCAGTACT TGTAAGAGGT GCAACGTCAA TGGAAACGGC ACCGTTAGCC TTGATGGTTG, CACGGGTAGG INFORMATION FOR SEQ ID NO: 4: 1020 1080 1140 1190 Wi SEQUENCE CHARACTERISTICS: LENGTH: 210 amino acids TYPE: amino acid STRA1NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Candida utiiis STRAIN: NRRL Y-1084 (ix) FEATURE: NAME/KEY: Protein LOCATION:l. .210 (xi) Met 1 Thr SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ala Glu Arg Thr Val Lys Pro Gln Axg Arg Ala Leu Vai Asn Arg Thr Asn Glu Thr Lys Ile Pro Glu Ser Gin Ile 25 Ile Phe Ser Leu Ser Leu Tyr Val Thr Ala Thr Gin Gly Phe Leu Val Lys Asp Lys Lys Asn Asp Gly Gly Tyr Asp Asp Thr Gly Vai Val Thr Ser Asp His Met 70 Gln Val Ile Ser His Ala Leu Ser Leu Ile Val Thr Thr Glu Leu Ala Tyr 115 Glu Cys Ile Gly Asp Val Gly Ile Ala Leu 105 Gly Val Lys Arg Phe 120 His Ile Asp Ala His Gly Gly Trp, Asp Asp His His Val Lys Glu Ala 110 Ala Pro Leu Gly Ser Giy Asp Glu Ala Leu Ser Arg Ala Vai Val Asp Leu Ser Asn Arg Pro Phe 130 135 '140 24 Ala Val Val Glu Leu Gly Leu Lys Arg Glu Lys Ile Gly Asp Leu Ser 145 150 155 160 Cys Glu Met Ile Pro His Phe Leu Glu Ser Phe Ala Gin Ala Ala His 165 170 175 Ile Thr Met His Val Asp Cys Leu Arg Gly Phe Asn Asp His His Arg 180 185 190 Ala Glu Ser Ala Phe Lys Ala Leu Ala Val Ala Ile Lys Glu Ser Ile 195 200 205 Ser Ser 210

Claims (10)

1. A Candida utilis transformation system comprising a host Candida utilis yeast cell capable of being transformed with recombinant DNA material wherein the host cell has at least one defective biosynthetic pathway obtained by chemical mutagenesis.

2. A Candida utilis transformation system according to claim 1 wherein the host yeast cell is defective in at least a biosynthetic pathway for an amino acid.

3. A Candida utilis transformation system according to claim 2 wherein the host yeast cell is defective in a biosynthetic pathway for uracil.

4. A Candida utilis transformation system according to claim 3 wherein said host 10 yeast cell is defective in the activity of the enzyme orotidin-5-phosphate decarboxylase. *oo

5. A Candida utilis tranformation system according to claim 4 wherein said host yeast cell is Candida utilis NRRL T-1084 phenotype ura-, deposited with accession number CBS 100085 at Centraalbureau voor Schimmelcutures on October 1, 1997.

6. Candida utilis transformation system according to claim 2 wherein the host yeast S59-*S cell is defective in a biosynthetic pathway for histidine.

7. Candida utilis transformation system according to claim 6 wherein the host yeast cell is defective in the activity of the enzyme imidazol-glycerol phosphate dehydratase.

8. A Candida utilis transformation system according to claim 1 wherein the recombinant DNA material comprises a functional gene that complements the defect in the biosynthetic pathway in which the host is defective.

9. A Candida utilis transformation system according to claim 8 wherein the functional gene is the URA3 gene of Candida utilis. A Candida utilis transformation system according to claim 8 wherein the functional gene is the HIS3 gene of Candida utilis.

21987-DO.DOC -26- 11. A Candida utilis transformation system according to claim 1 or 2 further comprising the recombinant DNA material for transformation of the host cell. 12. A Candida utilis transformation system according to claim 8 wherein said recombinant DNA material comprises a pUC19 plasmid with a 2.6 kb fragment containing the URA3 gene from Candida utilis inserted into a BamHI site, and a pUC19 plasmid with a 1.8 kb fragment containing the URA3 gene from Candida utilis inserted into an EcoRI site. 13. A Candida utilis transformation system according to claim 10 wherein the recombinant DNA material comprises a pUC19 plasmid with a 4.3 kb fragment 10 containing the HIS3 gene from Candida utilis inserted into a BamHI site and a pUC19 plasmid with a 4.5 kb fragment containing the HIS3 gene from Candida utilis inserted into BamHI site. 14. A method of transforming a Candida utilis host cell with at least one defective biosynthetic pathway obtained by chemical mutagenesis, comprising: transforming the host cell with recombinant DNA material; wherein the recombinant DNA material comprises a functional gene that Scomplements the defect in the biosynthetic pathway. A method according to claim 14 wherein the host cell is defective in at least a biosynthetic pathway for an amino acid. 16. A method according to claim 15 wherein the host cell is defective in a biosynthetic pathway for uracil. 17. A method according to claim 16 wherein the host cell is defective in the activity of the enzyme orotidin-5-phosphate decarboxylase. 21987-OO.DOC -27- 18. A method according to claim 17 wherein the host cell is Candida utilis NRRL T- 1084 phenotype ura-, deposited with accession number CBS 100085 at Centraalbureau voor Schimmelcutures on October 1, 1997. 19. A method according to claim 15 wherein the host cell is defective in a biosynthetic pathway for histidine. A method according to claim 19 wherein the host yeast cell is defective in the activity of the enzyme imidazol-glycerol phosphate dehydratase. 21. A method according to claim 14 wherein the recombinant DNA material comprises a functional gene that complements the defect in the biosynthetic pathway in which the 10 host is defective. *i 22. A method according to claim 14 wherein the functional gene is the URA3 gene of Candida utilis. 23. A method according to claim 21 wherein the recombinant material comprises a *oo pUC19 plasmid with a 2.6 kb fragment containing the URA3 gene from Candida utilis inserted into a BamHI site, and a pUC19 plasmid with a 1.8 kb fragment containing the URA3 gene from Candida utilis into an EcoRI site. 24. A method according to claim 14 wherein the functional gene is the HIS3 gene of Candida utilis. A method according to claim 21 wherein the recombinant material comprises a pUC19 plasmid with a 4.3 kb fragment containing the HIS3 gene from Candida utilis inserted into a BamHI site and a pUC19 plasmid with a 4.5 kb fragment containing the HIS3 gene from Candida utilis inserted into BamHI site. 21987-00.DOC -28- 26. A yeast host cell comprising a Candida utilis cell capable of being transformed with recombinant DNA wherein said host has at least one defective biosynthetic pathway obtained by chemical mutagenesis. 27. A yeast host cell according to claim 26 wherein said host cell is defective in at least a biosynthetic pathway for an amino acid. 28. A yeast host cell according to claim 27 wherein said host cell is defective in at least a biosynthetic pathway for uracil. 29. A yeast host cell according to claim 28 wherein said host cell is defective in the activity of the enzyme orotidin-5-phosphate decarboxylase.

10 30. A yeast host cell according to claim 29 wherein said host cell is Candida utilis NRRL Y-1084 phenotype ura-, deposited with accession number CBS 100085 at Centraalbureau voor Schimmelcutures on October 1, 1997. 31. A yeast host cell according to claim 27 wherein this host cell is defective in a .biosynthetic pathway for histidine. 15 32. A yeast host cell according to claim 31 wherein said host cell is defective in the activity of the enzyme imidazol-glycerol phosphate dehydratase. 33. Recombinant DNA material when used in a method as defined in claim 14 or wherein the recombinant DNA material is capable of integration into the genome of the Candida utilis yeast host cell, and contains a functional gene that complements the defect in said biosynthetic pathway in which the host cell is defective. 34. Recombinant DNA material according to claim 33 wherein the functional gene is the URA3 gene of Candida utilis. Recombinant DNA material according to claim 34 comprising pUC19 plasmid Swith a 2.6 kb fragment containing the URA3 gene from Candida utilis inserted into a 21987-00DOC iY/0a/0i 12:ii BbW bYUNEY ubu^aa u NO.079 P004/007 -29- BamHI site, and a pUC19 plasmid with a 1.8 kb fragment containing the URA3 gene from Candida utilis inserted into an EcoRI site. 36. Recombinant DNA material according to claim 33 wherein the functional gene is the HIS3 gene of Candida utilis. 37. Recombinant DNA material according to claim 36 wherein the recombinant DNA material comprises a pUC19 plasmid with a 4.3 kb fragment containing the HIS3 gene from Candida utilis inserted into a BamHI site and a pUC19 plasmid with a 4.5 kb fragment containing the HIS3 gene from Candida utilis inserted into BamHI site. 38. A Candida utilis transformation system comprising a host Candida utilis yeast cell capable of being transformed with recombinant DNA material wherein the host cell has at least one defective biosynthetic pathway obtained by chemical mutagenesis, substantially as hereinbefore described with reference to one or more of the Examples and accompanying figures. 39. A method of transforming a Candida utilis host cell with at least one defective biosynthetic pathway obtained by chemical mutagenesis, comprising transforming the host cell with recombinant DNA material comprising a functional gene that complements the defect in the biosynthetic pathway, substantially as hereinbefore described with reference to one or more of the Examples and accompanying figures. A yeast host cell comprising a Candida utilis cell capable of being transformed with recombinant DNA wherein said host cell has at least one defective biosynthetic pathway obtained by chemical mutagenesis, substantially as hereinbefore described with reference to one or more of the Examples and accompanying figures. 41. Recombinant DNA material when used in a method of transforming a Candida utilis host cell with at least one defective biosynthetic pathway obtained by chemical 2197.00.DOC 17/01/02 12:31 BSW SYDNEY 0262837999#789 NO.0?9 P005/00? mutagenesis, wherein the recombinant DNA material is capable of integration into the genome of the host cell, and contains a functional gene that complements the defect in said biosynthetic pathway in which the host cell is defective, substantially as hereinbefore described with reference to one or more of the Examples and accompanying figures. 42. An isolated DNA sequence comprising SEQ ID NO:3, substantially as hereinbefore described with reference to one or more of the Examples and accompanying figures. DATED the 17th day of January 2002 CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA Attorney: DAVID A. ADAMTHWAITE Fellow Institute of Patent and Trade Mark Attorneys of Australia of BALDWIN SHELSTON WATERS 2 1987-00.DOC