Method for producing recombinant proteins and host cells used therein

Novel genetically engineered yeast strains of genus Kluyveromyces lactis, their preparation, and the use thereof for producing recombinant proteins, are described.

The present invention relates to new yeast strains modified by genetic 
engineering, their preparation and their use for the production of 
recombinant proteins. More particularly, the invention relates to new 
yeast strains of the genus Kluyveromyces. 
There is a large selection of host organisms, such as mammalian cells or 
microorganisms, which may potentially be used with a view to the 
production of recombinant proteins. 
The use of mammilian cells modified by recombinant DNA techniques has the 
advantage of leading to products very close to those of natural origin. 
However, the culture of these cells is delicate, expensive and can be 
carried out only in limited volumes. 
The use of microorganisms, such as bacteria, allows production on a larger 
scale but has the disadvantage of leading to products which, in some 
cases, differ substantially from products of natural origin. Thus, the 
proteins which are normally glycosylated in man are not, generally, 
glycosylated by bacteria [P. Berman and L. A. Laskey, Trends Biochem. 
Sci., (1985) 10, p. 51 et seq.]. Moreover, human proteins expressed at a 
high level in bacteria such as E. coli often acquire an unnatural 
structure which is accompanied by intracellular precipitation [R. G. 
Schoner et al., Bio. Technol. (1985), 3, p. 151 et seq.; J. M. Schoemaker 
et al., EMBO J. (1985), 4, p. 775 et seq.]. Finally, for a gene to be able 
to be expressed in a bacterium, such as E. coli, it is essential to 
position an initiator codon ATG, generating a methionine before the coding 
sequence of the mature protein. Frequently, this residue is not excised by 
the methionyl aminopeptidase of E. coli [P. H. Seeburg et al., 1985, 2, p. 
37 et seq.; J. M. Schoner et al., Proc. Natl. Acad. Sci. USA (1981), 81, 
p. 5403]. The protein obtained then has an abnormal amino acid as first 
residue, which may give rise to the steric inhibition of a biological 
activity if the start of the protein is involved in this activity. The 
residue may also have an immunogenic character adverse to the subsequent 
administration of the protein. 
The use of eukaryotic microbial systems such as yeasts or mushrooms 
represents an interesting alternative to the use of bacterial hosts for 
the preparation of recombinant proteins. In fact, these organisms have all 
the structural and cellular organisation characteristics of more complex 
eukaryotic organisms, such as mammalian cells. In particular, yeasts are 
capable of effecting post-transcriptional and post-translational 
modifications important for the activity of numerous proteins. Moreover, 
yeasts are well known on the industrial scale: they may be cultured in 
high cell density, they are not pathogenic, they do not produce endotoxins 
and they have been used in the foodstuffs industry for a very long time. 
Yeasts are already used as host organisms for the production of recombinant 
proteins (cf. "Yeast Genetic Engineering" Barr et al. (Eds), Butterworths, 
Stoneham, 1989). 
In particular, yeasts of the genus Saccharomyces cerevisiae have been the 
subject of numerous studies (see, in particular, European Patent 60057), 
and the systems employing this yeast allow heterologous genes to be 
expressed at fairly high levels. However, the secretory capacity of S. 
cerevisiae constitutes a limiting factor in the exploitation of this 
yeast. 
Other production systems have been developed with the yeasts Pichia 
pastoris (European Patent 402 847) or Schizosaccharomyces pombe (European 
Patent 385 391), and studies have also been carried out on the yeast 
Schwanniomyces (European Patent 394 538). 
Recently, yeasts of the genus Kluyveromyces have been used as host 
organisms for the production of recombinant proteins. The proteins 
produced by this yeast are, in particular, chymosin (European Patent 96 
430), thaumatin (European Patent 96 910), albumin, interleukin-1.beta., 
tPA and TIMP (European Patent 361 991) and albumin derivatives having a 
therapeutic function (European Patent 413 622). 
However, although relatively powerful expression vectors have been 
developed for the use of this yeast, no studies have been carried out with 
the aim of improving the intrinsic performance of the cell used. In 
particular, there are numerous species of yeasts of the genus 
Kluyveromyces and, within these species, numerous different strains. The 
Applicant has now shown that these different strains behave in a very 
heterogeneous manner for the production of recombinant proteins, some of 
them being completely unusable. 
The present invention results from the identification of a yeast strain 
taxonomically allied to the species Kluyveromyces lactis possessing 
particularly advantageous properties for the production of recombinant 
proteins. 
The present invention thus describes the production of yeast strains 
modified by genetic engineering, which can be cultured in bulk and are 
capable of efficiently producing, and optionally of secreting into the 
culture medium, biologically active recombinant proteins. 
One aspect of the invention therefore relates to new yeast strains for the 
production of recombinant proteins. More precisely, one subject of the 
invention relates to a host cell for the production of recombinant 
proteins, characterised in that the said host cell is the yeast K. lactis 
CBS 293.91 or a derivative or mutant thereof, containing a heterologous 
DNA fragment comprising a structural gene and signals permitting its 
expression. 
In the meaning of the present invention, derivative or mutant is understood 
to be any strain obtained from K. lactis CBS 293.91 capable of being used 
for the production of recombinant proteins. In particular, such 
derivatives or mutants may be obtained by genetic modifications 
(alterations at the DNA level) or by biochemical modifications. To this 
end, various mutagenesis tools can be used, such as, for example, 
non-specific tools: 
physical agents (X-rays, ultraviolet rays etc) or, 
chemical agents (alkylating or dialkylating agents, intercalating agents 
etc), 
or specific tools, such as the mutational insertion systems aimed at the 
DNA (transposons, retrotransposons, integrative plasmids, etc.). 
One example of such derivatives is the strain K. lactis Y616, obtained from 
the strain CBS 293.91 by deletion at the level of the URA3 gene. Other 
mutants may be obtained by mutation at the level of genes coding for 
proteases, and in particular proteases conveyed by the endoplasmic 
reticulum, such as A and B proteases, carboxypeptidase Y, or convertases 
(Kex 1 in particular). 
The heterologous DNA fragment may be introduced into the cell by various 
techniques. In general, transformation or electroporation are suitable, 
but it is understood that the invention is not restricted to a particular 
technique. 
More preferentially, the heterologous DNA fragment also comprises signals 
permitting the secretion of the recombinant protein. These signals may 
correspond to the natural secretion signals of the protein under 
consideration, but they may also be of different origin. In particular, 
secretion signals derived from yeast genes may be used, such as those of 
the genes of the killer toxin (Stark and Boyd, EMBO J. 5 (1986) 1995) or 
of the alpha pheromone (Kurjan and Herskowitz, Cell 30 (1982) 933; Brake 
et al., Yeast 4 (1988) S436). 
Still in a particular embodiment of the invention, the heterologous DNA 
fragment also comprises a selection marker. A marker of this type in fact 
enables the cells of the invention to be easily identified. The markers 
concerned may be, in particular, markers imparting resistance to 
antibiotics (such as, for example, the aph gene (Jimenez and Davies, 
Nature 287 (1980) 869)) or to other compounds toxic for the cell (copper 
ions in particular), or markers complementing the auxotrophies of the host 
cell (such as, for example, the URA3 gene (De Louvencourt et al., J. 
Bacteriol. 154 (1983) 737)). 
In general, the signals permitting the expression of the structural gene 
are chosen from transcription promoters and terminators. It is understood 
that these signals are chosen depending on the structure gene and the 
desired result. In particular, it may be preferable in some cases to use a 
controllable promoter so as to be able to uncouple the growth phases of 
the host and that of expression of the gene. Similarly, on strength and 
compatibility grounds it may be preferable to use natural promoters of the 
structure genes in some cases and promoters of different origin in other 
cases. 
Preferentially, the promoters used are derived from yeast genes and still 
more preferentially from yeast glycolytic genes. Promoters which are very 
particularly valuable are the promoters derived from glycolytic genes of 
yeasts of the genus Saccharomyces or Kluyveromyces. In particular, the 
promoters of genes coding for phosphoglycerate kinase (PGK), 
glyceraldehyde 3-phosphate dehydrogenase (GPD), enolases (ENO) or alcohol 
dehydrogenases (ADH) may be mentioned. Promoters derived from strongly 
expressed genes, such as the lactase gene (LAC4), the acid phosphatase 
gene (PHO5) or translation elongation factors (TEF) may also he mentioned. 
Moreover, these promoter regions may be modified by mutagenesis, for 
example by adding supplementary transcription control elements, such as, 
in particular, UAS (Upstream Activating Sequence) regions. By way of 
example, a hybrid promoter between the promoters of the PGK and GAL1/GAL10 
genes of S. cerevisiae gives good results. 
In a preferred embodiment of the invention, the heterologous DNA fragment 
forms part of an expression plasmid, which may be autonomously replicating 
or integrative. 
With regard to autonomously replicating vectors, these may be obtained by 
using autonomously replication sequences of K. lactis CBS 293.91 or its 
derivatives or mutants. In particular, the sequences concerned may be 
chromosomal sequences (ARS) originating, for example, from S. cerevisiae 
(Stinchcomb et al., Nature 282 (1979) 39) or from K. lactis (Das and 
Hollenberg, Curr. Genet. 6 (1982) 123). The vectors may also be origins of 
replication derived from plasmids and, for example, from the plasmid pKD1 
of K. drosophilarum (European Patent 361991) or the 2 .mu. plasmid of S. 
cerevisiae (for review see Futcher, Yeast 4 (1988) 27). 
With regard to integrative vectors, the latter are generally obtained using 
sequences homologous to certain regions of the genome of the host cell, 
permitting integration of the plasmid by homologous recombination. 
Advantageously, according to the present invention, the structural gene 
codes for a protein which is pharmaceutically valuable or of value in the 
agri-foodstuffs industry. By way of example, the following may be 
mentioned: enzymes (such as, in particular, superoxide dismutase, 
catalase, amylases, lipases, amidases, chymosin, etc.), blood derivatives 
(such as serum albumin or molecular variants of the latter, alpha- or 
beta-globin, factor VIII, factor IX, yon Willebrand's factor or fragments 
thereof fibronectin, 1-alpha-antitrypsin, etc.), insulin and its variants, 
lymphokines [such as the interleukins, interferons, colony stimulation 
factors (G-CSF, GM-CSF, M-CSF . . . ), TNF, TRF, MIPs, etc.], growth 
factors (such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, 
etc.), apolipoproteins, antigenic polypeptides for the production of 
vaccines (hepatitis, cytomegalovirus, Epstein-Barr, herpes, etc.), vital 
receptors, or alternatively fusions of polypeptides, such as, in 
particular, fusions comprising an active part fused to a stablising part 
(for example fusions between albumin or albumin fragments and the receptor 
or part of a virus receptor (CD4, etc.)). 
Preferentially, the structural gene codes for human serum albumin, a 
precursor of the latter or one of its molecular variants. Molecular 
variants of albumin are understood to be the natural variants resulting 
from the polymorphism of albumin, structural derivatives possessing an 
activity of the albumin type, truncated forms or any hybrid protein based 
on albumin. 
Another subject of the invention relates to a process for the production of 
recombinant proteins, according to which process a recombinant cell as 
defined above is cultured and the protein produced is recovered. 
As shown in the examples, this process surprisingly enables very high 
production levels of recombinant proteins to be obtained. 
Advantageously, the process of the invention also allows the secretion of 
the recombinant protein into the culture medium. 
The process of the invention allows the production of large amounts of 
recombinant proteins which are pharmaceutically valuable or of value in 
the agri-foodstuffs industry. It is particularly suitable for, although 
not restricted to, the production of human serum albumin or its molecular 
variants. 
Further advantages of the present invention will become apparent on reading 
the examples which follow, which must be regarded as illustrative and 
non-limiting.

EXAMPLES 
Example 1 
Construction of expression cassettes and/or vectors for recombinant 
proteins. 
1.1. Construction of expression vectors for human albumin. 
1.1.1. Construction of an albumin expression vector under the control of a 
PGK/GAL hybrid promoter. 
An expression vector for human serum albumin was prepared from the plasmid 
pYG19 (European Patent 361 991). The latter comprises the following 
elements: 
the plasmid pKD1 sequence, which, from pYG19, makes a plasmid having 
multiple copies which is stable and capable of replicating in yeasts of 
the genus Kluyveromyces (European Patent 361 991), 
an expression cassette for human serum albumin comprising the structural 
gene coding for the prepro form under the control of the promoter of the 
PGK gene of S. cerevisiae; 
a bacterial replicon and a bacterial selection marker (bla gene conferring 
resistance to ampicillin); and 
the aph gene conferring resistance to G418 to yeast. 
The vector pYG401 was constructed from the plasmid pYG19 by modification at 
the level of the expression cassette for human serum albumin. In pYG401 
the albumin gene is no longer under the control of the promoter of the PGK 
gene of S. cerevisiae but under the control of a hybrid promoter between 
the promoters of the PGK and GAL1/GAL10 genes of S. cerevisiae. This 
hybrid promoter was obtained by replacing the UAS ("Upstream Activating 
Sequence") region of the PGK promoter (Stanway et al., Nucl. Acid. Res. 15 
(1987) 6855) by the UAS region of the GAL1/GAL10 promoter (Johnston and 
Davies, Mol. Cell. Biol. 4 (1984) 1440; West et al., Mol. Cell. Biol. 4 
(1984) 2467). 
This hybrid promoter was constructed in the following way (cf. FIG. 1): 
The production of the plasmid pYG29 has been described in detail in 
European Patent Application 361 991. This plasmid contains the promoter of 
the PGK gene of S. cerevisiae isolated from the plasmid pYG19 in the form 
of a SalI-HindIII fragment and cloned into the bacteriophage M13mp18. It 
was then modified by directed mutagenesis to introduce the following 
restriction sites: 
1 supplementary HindIII site in position -25 with respect to the ATG. The 
introduction of this site makes it possible to restore, after the various 
cloning steps, a nucleotide sequence close to ATG identical to the natural 
sequence of the PGK promoter. The environment of the ATG codon is, in 
fact, known to have a substantial influence on the efficiency of the 
initiation of the translation of eukaryotic genes (Kozak, M., Microbiol. 
Rev. 47 (1983) 1-45; Hamilton, R., Nucl. Acid. Res 15 (1987) 3581-3593). 
2 NotI sites on either side of the UAS. 
The UAS of the GAL1/GAL10 promoter has been isolated from the plasmid pG1 
described by Miyajima et al. (Nucl. Acid. Res 12 (1984) 6397-6414; Cloning 
Vectors, Pouwels et al., Elsevier (1985) VI-B-ii-2). This plasmid has been 
deposited with ATCC under the number 37305. 
The plasmid pG1 contains a 0.8-kb fragment containing the GAL1/GAL10 
promoter of S. cerevisia, inserted in the HindII site of the plasmid pUC8, 
from which it may be excised in the form of a BamHI-PstI fragment (FIG. 
1). 
This fragment was excised from pG1, purified and then digested with the 
enzymes RsaI and AluI, the respective cutting sites of which are located 
on either side of the UAS region. A 143-bp fragment was then isolated by 
electroelution and then brought into the form of a NotI fragment by adding 
a 5'-GCGGCCGC-3' linker. This fragment was then cloned in the plasmid 
pYG29, previously digested with NotI. 
The resulting plasmid is named pYG32 (FIG. 1). 
In order to obtain the expression vector carrying this hybrid promoter, the 
SalI-HindIII fragment carrying the hybrid promoter was isolated from pYG32 
and ligated with a synthetic HindIII-BstEII adaptor composed of the 
following 2 complementary strands: 5'-AGC TTT ACA ACA AAT ATA AAA ACA ATG 
AAG TGG-3' (SEQ ID NO: 1) and 5'-GT TAC CCA CTT CAT TGT TTT TAT ATT TGT 
TGT AA-3' (SEQ ID NO: 2) (the codon initiating transcription is 
represented in bold letters). This adaptor reconstitutes the 22-bp 
situated immediately upstream of the PGK structural gene of S. cerevisia 
and contains the first codons of the gene coding for the preproHSA, up to 
a BstEII site present in the natural gene (FIG. 1). 
The expression cassette for human albumin was then reconstituted by 
ligating the SalI-BstEII fragment thus obtained, carrying the hybrid 
promoter and the 5' end of the albumin structural gene, with the 
BstEII-SacI fragment isolated from the plasmid pYG19, carrying the 
remainder of the albumin gene and the terminator of the PGK gene of S. 
cerevisia (FIG. 2). 
The cassette thus obtained was used to replace the SalI-SacI expression 
cassette carried by the plasmid pYG19. 
The resulting vector is named pYG401 (FIG. 2). 
1.1.2. Construction of an albumin expression vector under the control of 
the LAC4 promoter of K. lactis. 
To test the efficiency of the inducible promoter LAC4 in the production 
system of the present invention, the expression vector pYG107 was 
constructed. This vector is identical to the pYG401 vector, in which: 
(i) The EcoRI site located at the junction between the pKD1 part and the 
bacterial replicon is destroyed. This destruction has been obtained by 
cutting the pYG401 plasmid by EcoRI, filling in the cohesive ends by means 
of the Klenow fragment of DNA polymerase of E. coli and religating. 
(ii) The HindIII site present in the aph gene conferring resistance to G418 
is destroyed: this modification has been obtained, after sub-cloning of 
the aph gene into the bacteriophage M13mp7, by directed mutagenesis using 
the method described by Taylor et al. (Nucleic. Acid. Res. 13 (1985) 
8749), using the following oligodeoxynucleotide: 5'-GAA ATG CAT AAG CTC 
TTG CCA TTC TCA CCG-3'SEQ ID NO: 3 . This oligodeoxynucleotide converts 
the CTT codon coding for leucine 185 to CTC. This change does not modify 
the resulting protein sequence. 
Modifications (i) and (ii) result in minor changes, intended solely to 
facilitate the subsequent cloning steps, but which do not interfere in the 
expression efficiency of the vectors. 
(iii) The albumin expression cassette (SalI-SacI fragment) has been 
replaced by the SalI-SacI cassette originating from the plasmid pYG404 
(European Patent 361 991), in which the gene coding for human albumin 
(prepro form) is under the control of the LAC4 promoter of K. lactis. 
The structure of the vector pYG107 is shown in FIG. 3. 
Example 2 
Transformation of Kluyveromyces by expression vectors for recombinant 
proteins. 
Various techniques permitting the introduction of a DNA fragment into yeast 
may be used. 
Advantageously, the various Kluyveromyces strains used have been 
transformed by treating whole cells in the presence of lithium acetate and 
polyethylene glycol, using the technique described by Ito et al. (J. 
Bacteriol. 153 (1983) 163-168). 
An alternative method has also been described in detail in European Patent 
Application 361 991. 
Example 3 
Expression and secretion of recombinant proteins in various yeasts of the 
genus Kluyveromyces. 
This example demonstrates that the use of the new genetically modified 
yeasts of the invention makes it possible to obtain particularly high 
levels of production and secretion of recombinant proteins. 
3.1. Human serum albumin 
3.1.1. The following Kluyveromyces lactis strains have been transformed by 
the pYG401 vector, using the method described in Example 2: 
K. lactis CBS293.91 
K. lactis CBS739 
K. lactis CBS762 
K. lactis CBS1067 
K. lactis CBS1797 
K. lactis CBS2619 
K. lactis CBS2621 
K. lactis CBS6315 
K. lactis CBS8043 
K. lactis CBS683 
K. lactis CBS579.88 
The recombinant albumin production has been determined by the method 
described in European Patent Application 361 991 after a culture time of 
120 hours at 28.degree. C. with constant stirring, in YPD medium (yeast 
extract 10 g/l; peptone 20 g/l; glucose 20 g/l) in the presence of 2 % 
geneticin. The culture supernatants were obtained after centrifuging twice 
in succession (5 minutes at 4000 rpm and then 10 minutes at 12,000 rpm), 
enabling all cellular contamination to be removed. A 0.5 ml sample was 
then heated at 95.degree. C. for 15 minutes in the presence of an equal 
volume of the following buffer: 0.125M Tris-HCl, 20% glycerol, 10% 
2-mercaptoethanol, 4.6% sodium dodecyl sulphate (SDS) and 0.4% of 
bromophenol blue (Laemli 2.times.buffer, Laemli, Nature 227 (1970) 680)); 
and 50 .mu.l of the solution obtained were deposited on 8.5% 
SDS-polyacrylamide gel. After migration, the gel was visualised using 
Coomassie blue. 
FIG. 4 shows an increase of about 200% in the amount of albumin produced in 
the system of the invention, compared with the best systems previously 
described (European Patent 361 991). 
3.1.2. The following Kluyveromyces lactis strains were transformed by the 
pYG107 vector, using the method described in Example 2: 
K. lactis CBS4574 
K. lactis CBS683 
K. lactis CBS2359 
K. lactis CBS293.91 
K. lactis CBS579.88 
The recombinant albumin production was determined using the method 
described in European Patent Application 361 991 and in Example 3.1.1. 
after a culture time of 120 hours at 28.degree. C. with constant stirring, 
in M9EL10 medium in the presence of 20 g/l glucose or 20 g/l lactose. The 
M9EL10 medium consists of M9 medium (Maniatis et al., mentioned above) 
supplemented by 10 g/l of yeast extract. 
FIG. 5 shows that the amount of albumin produced is always higher in the 
system of the invention. It also shows that the production is 2 to 3 times 
better in a medium containing lactose (LAC4 promoter inducer) than in a 
glucose medium. Moreover, it finally shows that, surprisingly, the strain 
CBS293.91 is semi-constitutive for the production of recombinant proteins 
under the control of the LAC4 promoter. 
3.2. Interleukin-1 .beta. 
The following Kluyveromyces lactis strains have been transformed by the 
pSPHO-IL35 vector (cf. European Patent 361 991) using the method described 
in Example 2: 
K. lactis CBS683 
K. lactis CBS293.91 
K. lactis CBS579.88 
Example 4 
Construction of a ura3 mutant of K. lactis CBS293.91. 
A ura3 derivative has been prepared from K. lactis CBS293.91. Such a 
derivative retains the properties of the initial strain, with, in 
addition, an auxotrophy for uracil, which may be used as selection marker. 
In order to prevent the occurrence of any reversion, this mutant was 
prepared by deletion of part of the chromosomal URA3 allele of CBS293.91. 
This mutagenesis technique also makes it possible to avoid the use of 
non-specific mutagenic agents capable of modifying other genomic regions 
of the cell. 
4.1. Cloning and modification of the URA3 gene of K. lactis CBS2359 (FIG. 
6). 
The URA3 gene of K. lactis coding for orotidine-5-phosphate decarboxylase 
(Shuster et al., Nucl. Acid. Res. 15 (1987) 8573) has been cloned in the 
form of a 1.2-kb BamHI-PstI fragment using the PCR technique (cf. general 
cloning techniques), from a genomic DNA extract (Rose et al., "Methods in 
Yeast Genetics" Cold Spring Harbor Laboratory Press, N.Y., 1990) from K. 
lactis CBS2359 using the following oligodeoxynucleotides: 
5'-GGAAGCTTGGCTGCAGGAATTGTCGTTCATGGTGACAC-3' and (SEQ ID NO.: 4) 
5'-CCGAATTCCCGGATCCCATAATGAAAGAGAGAGAGAGAAGCAAAC-3' (SEQ ID NO. 5). 
The fragment obtained was then sub-cloned in the BamHI and PstI sites of 
the pIC-20H plasmid (Marsh et al., Gene 32 (1984) 481) to give the plasmid 
pYG1007 (FIG. 6). This fragment was then modified by deletion of an 
internal fragment of the URA3 gene comprising 286 bp, located between the 
StyI sites, followed by religation in the presence of ligase. This new 
plasmid is named pYG1010 (FIG. 6). 
4.2 Transformation of K. lactis CBS293.91 by the deleted URA3 9 gene. 
The CBS293.91 strain was transformed by 10 .mu.g of the PstI-BamHI fragment 
isolated from the plasmid pYG1010 by electroelution and containing the 
deleted URA3 gene. After a sudden rise in temperature to 42.degree. C. 
("heat shock") and 2 successive washings with water, 600 .mu.l of YPD 
medium were added and the cells were incubated overnight. The cells were 
then plated out on synthetic minimal SD medium ("Bacto-yeast nitrogen 
base" without amino acids (Difco) 6.7 g; glucose 20 g; Bacto-agar 20 g, 
distilled water 1,000 ml) in the presence of uracil (100 .mu.g/ml), 
uridine (100 .mu.g/ml) 15 mM 5-fluoroorotate (5FO). 
Clones appeared at the end of 4 to 5 days. They were subcultured on YPD 
medium so as to obtain isolated colonies. 
From the first subculture, 3 clones resulting from the colony which 
initially appeared on SD+5FO medium were reisolated on YPD medium 
(secondary subculture). 
The clones resulting from the secondary subculture were then tested for the 
Ura3.sup.- phenotype using the drop test on SD and SD+uracil medium (Jund 
and Lacroute, J. of Bact. 102 (1970) 607-615; Bach and Lacroute, Mol. Gen. 
Genet. 115 (1972) 126-130). The ura3 genotype of the clones thus obtained 
was checked by: 
PCR reaction using the oligodeoxynucleotides described for cloning under 
4.1., which allows identification of the clones carrying only the deleted 
URA3 gene; 
complementation using the pKan707 plasmid (European Patent 361 991) 
carrying the intact URA3 gene of S. cerevisia, known for its ability to 
complement the ura3 auxotroph in K. lactis (De Louvencourt, mentioned 
above); and 
Southern blotting on genomic DNA of the identified clones, using, as probe, 
the URA3 gene of K. lactis isolated in Example 4.1., labelled with .sup.32 
p using the technique described by Feinberg and Vogelstein (Anal. Biochem. 
132 (1983) 6). 
The selected ura3 mutant is named K. lactis Y616. 
A sample of the strain K. lactis Y616 was deposited on 11 Jun. 1991 with 
the Centraalbureau voor Schimmelkulturen (CBS) in Baarn in the Netherlands 
under the conditions of the Budapest Treaty, under number CBS294.91. The 
strain K. lactis CBS293.91 corresponds to the strain CBS1065 refiled on 11 
Jun. 1991 under the conditions of the Budapest Treaty. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGCTTTACAACAAATATAAAAACAATGAAGTGG33 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GTTACCCACTTCATTGTTTTTATATTTGTTGTAA34 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAAATGCATAAGCTCTTGCCATTCTCACCG30 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GGAAGCTTGGCTGCAGGAATTGTCGTTCATGGTGACAC38 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCGAATTCCCGGATCCCATAATGAAAGAGAGAGAGAGAAGCAAAC45 
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