Cloning and expression vector, yeast transformed by such vector and applications thereof

Cloning and expression vector of a heterologous gene in a yeast, characterized in that it comprises at least; all or part of DNA of the plasmide k.sub.1 of Kluyveromyces lactis, a DNA segment incorporating the heterologous gene as well as the sequences providing the expression of said gene in said yeast.

The present invention relates to novel cloning and expression vectors in 
yeasts, and to yeasts transformed with the aid of these vectors and their 
application in the synthesis of proteins. 
Certain strains of yeasts of the species Kluyveromyces lactis contains a 
linear plasmid couple, the presence of which imparts "killer" character to 
the cell, this cell producing a toxin which prevents the growth of other 
so-called sensitive cells. 
Sensitive cells which should be mentioned are the K. lactis cells deficient 
in plasmids, and also the cells of different species, such as 
Saccharomyces cerevisiae. 
PRIOR ART 
These plasmids were first described by Gunge et al. (J. Bacteriol. 145, 
382-390 (1981), 147, 155-166 (1981), and independently by H. Fukuhara et 
al. (Current Genetics, in the press). 
These are the two double-stranded linear DNA plasmids called k.sub.1 (8.8 
kb) and k.sub.2 (13.4 kb). 
The genetic studies carried out at the Laboratoire de M. Fukuhara reveal 
great analogy with the "killer" system of S. cerevisiae, except for the 
fact that the latter contains two RNA plasmids. 
The plasmid k.sub.1 is essential for expression of the "killer" character 
and of the immunity to the toxin, since the lack of this plasmid causes 
the disappearance of these two characteristics. It seems that the plasmid 
k.sub.2 is necessary to maintain k.sub.1 in the cell. 
Replication of these DNAs involves several chromosomal genes of the yeast; 
the expression of the toxin also depends on nuclear genes. There are 
therefore interactions between the two plasmids and between the plasmids 
and the nucleus. 
It has now been discovered that it is possible to use the plasmid k.sub.1 
as a cloning and expression vector. 
DESCRIPTION OF THE INVENTION 
The present invention thus proposes a cloning and expression vector of a 
heterologous gene in a yeast, which contains at least all or some of the 
DNA of the plasmid k.sub.1 of K. lactis, a DNA segment incorporating the 
heterologous gene and sequences which ensure expression of the said gene 
in the said yeast. 
The use of such a cloning and expression vector is of particular interest, 
firstly because few vectors which can be used in yeasts exist. In 
addition, the plasmid k.sub.1 has several unique restriction sites, in 
particular EcoRI, BamHI and ClaI, which is of particular interest for the 
construction of recombinant hybrids. 
Finally, it is known that the plasmids k.sub.1 are present in a high copy 
number per cell, it being possible for this number of copies to reach 100 
to 150. Under these conditions, it may be hoped that the gene which will 
be inserted in the vector will be amplified. 
The heterologous genes are more particularly provided in the context of the 
present invention are genes which encode the synthesis of peptides or 
proteins of industrial interest. 
In certain cases, the vector will of course contain various heterologous 
genes, some of which will not be expressed but of which certain sequences 
will ensure the expression of another gene. 
Preferably, the gene to be cloned and expressed is inserted in one of the 
unique restriction sites, in particular in Cla1 site. 
It is of course possible, if this is advantageous, to insert in an 
appropriate site of the vector (that is to say in general downstream of 
the principal elements ensuring expression) a sequence containing multiple 
unique restriction sites as is known in this field, in order to be able 
conveniently to insert the heterologous genes. 
Vectors according to the present invention which should be mentioned are 
those which incorporate, as the heterologist gene, the URA.sub.3 gene of 
yeast, in particular in the form of a DNA segment of about 1.1 kb, limited 
by two HindIII sites. 
Experiments have shown that integrality of the plasmid k.sub.1 was not 
essential for cloning and expression. Thus, it was possible to use as the 
vector the plasmid k.sub.1 .delta., which originates from a "non-killer" 
mutant NK2 of K. lactis and is resistant to the toxin secreted. This 
plasmid k.sub.1 .delta. has a deletion of 2.9 kb, with respect to the 
plasmid k.sub.1, between the two HindIII sites. 
The vectors according to the invention can also contain bacterial DNA 
fragments, in particular bacterial DNA fragments containing an origin of 
replication and/or a gene having resistance to an antibiotic, the latter 
in particular to enable selection of the clones in the bacterial strains. 
It is thus possible to insert a pBR322 restriction fragment containing the 
origin of replication and the ampicillin resistance gene. 
The particular vectors according to the invention which are of special 
interest are circular plasmids such as pL3 which contains, as the DNA of 
plasmid k.sub.1, a Cla1 restriction fragment of the plasmid k.sub.1 
.delta. and, in addition, preferably a Cla1 restriction fragment of the 
clone 6 plasmid. 
The various plasmids mentioned above can of course be prepared using known 
techniques. 
The present invention also relates to the transformed yeasts incorporating 
a vector according to the present invention, and in particular, although 
not uniquely, strains of the genus Kluyveromyces, and especially K. 
lactis. 
The invention also relates to the application of the transformed yeasts to 
the expression of the protein encoded by the heterologous gene carried by 
the vector. 
Finally, the invention relates to a process for the preparation of a 
protein or a peptide, wherein a yeast transformed by a vector according to 
the invention containing, as the heterologous gene, the gene which encodes 
the said protein or the said peptide is grown on a nutrient medium. 
DESCRIPTION OF THE DRAWINGS

I--CONSTRUCTION OF THE HYBRID PLASMIDS k.sub.1 -URA.sub.3 
The plasmids k.sub.1 and k.sub.1 .delta. have already been described in the 
abovementioned articles. 
The restriction plan of plasmids k.sub.1 and k.sub.1 .delta. has been shown 
in attached FIG. 1. 
As can be seen, the plasmid k.sub.1 carries three unique restriction sites, 
EcoRI, BamHI and ClaI, and carries a double restriction site, HindIII. 
In contrast, plasmid k.sub.1 .delta., which carries a deletion of 2.9 kb, 
contains only a single unique restriction site, ClaI, but has preserved 
the two HindIII restriction sites. 
This explains why, if cloning of a gene on the ClaI site is desired, it 
would be possible to effect total restriction, whilst if restriction 
effected by HindIII is desired, it would be appropriate to effect only 
partial restriction. 
In order to avoid inactivation of a region necessary for replication, the 
marker URA.sub.3 has been introduced in various sites of k.sub.1 and 
k.sub.1 .delta.. 
First method of cloning: by HindIII 
Partial restriction of the plasmids k.sub.1 and k.sub.1 .delta. is effected 
by HindIII. 
The URA.sub.3 gene is obtained by total restriction of the clone 6 plasmid 
by HindIII (described by Bach et al., 1979, PNAS, 76, 386-390). 
The whole URA.sub.3 gene, carried by a DNA fragment of 1.1 kb, is thus 
obtained. This fragment is treated with alkaline phosphatase in order to 
prevent recircularization of the latter plasmid. 
The following ligations are effected: 
(1) k.sub.1 cut by HindIII+clone 6 cut by HindIII, 
(2) k.sub.1 .delta. cut by HindIII+clone 6 cut by HindIII. 
Second method of cloning: by ClaI 
Since the ClaI site is unique on the two plasmids used, that is to say 
k.sub.1 .delta. and clone 6, total restriction of the two plasmids may be 
effected. 
The following ligation is effected: 
(1) k.sub.1 .delta. cut by ClaI+clone 6 cut by ClaI. 
The combination of these ligation mixtures contains a certain proportion of 
hybrid plasmids. 
In order to be able to demonstrate cloning and expression of the URA.sub.3 
gene, it is appropriate to use a mutant strain of K. lactis, uraA.sup.-. 
In addition, in order to ensure that the vector according to the invention 
is maintained, it is useful for this receptor strain also to possess the 
plasmid k.sub.2. 
II--CONSTRUCTION OF A MUTANT STRAIN OF K. lactis urA.sup.-, WITHOUT k.sub.1 
The K. lactis CBS 2360-6 uraA.sup.- strain which has been depleted in OMP 
decase activity and has been obtained from the strain CBS 2360 by 
mutagenesis with UV, is used as the starting material. 
The degree of reversion of the mutation is less than 1.10.sup.-8 cells. 
This strain is "killer" and resistant to the toxin. It possesses the two 
plasmids k.sub.1 and k.sub.2 and is called (k.sub.1 +, k.sub.2 +). 
The plasmid k.sub.1 is eliminated from this strain by crossing with the 
"non-killer" strain VM2, which has been isolated by mutagenesis and has 
K.sub.1 O, k.sub.2 + character. 
The following crossing: 
##EQU1## 
leads to about 1% of diploid cells which have lost k.sub.1 by mitotic 
segregation and have become "non-killer". 
After sporulation of these diploid strains, a "non-killer" haploid strain 
ura.sup.- is obtained, for which it can be verified by extraction of the 
DNA that it has lost k.sub.1 and still possesses k.sub.2. 
A "non-killer" strain uraA.sup.- (K.sub.1.sup.O, K.sub.2.sup.+) has thus 
been obtained and will be used to demonstrate the expression of the gene 
cloned on the vector according to the present invention. 
III--TRANSFORMATION OF K. lactis 
The receptor strain constructed above is grown on a minimum medium (YNB 
0.67%, glucose 2%) supplemented with uracil, up to a concentration of 1 to 
2.10.sup.8 cells/ml. 
After washing twice, the cells are suspended in a concentration of 10.sup.9 
cells/ml in a protoplast formation buffer (0.6M KCl, pH 5). 
Zymolyase 5000 (0.5 mg/ml) is added and protoplasts are obtained within 5 
to 10 minutes at 34.degree. C. 
After washing twice, 2.10.sup.8 cells and 1 to 10 .mu.g of DNA of the 
bonding mixtures are brought together. 
2 ml of PEG 40% and 10 mM CaCl.sub.2 are added and the mixture is left at 
the ambient temperature for 20 minutes. 
After removal of the PEG, the mixture is incubated for 1 hour in a complete 
osmotically buffered medium; the protoplasts are included in the gelose by 
surfusion and are spread out on a minimum KCl medium. 
The colonies appear after incubation at 30.degree. C. for one week, and are 
small in size and, after subculture on minimum medium, many do not 
increase in size. 
The efficiency of the transformation is one transformant per .mu.g of DNA, 
and the degree of regeneration of the protoplasts is about 10%. 
The results observed are as follows: 
TABLE I 
__________________________________________________________________________ 
Number of colonies Stability in 
per .mu.g of DNA which selective 
increase in size 
Name of the medium 
Transformant DNA 
after subculture 
transformants 
"Killer" resistant 
(%) 
__________________________________________________________________________ 
k.sub.1 .differential. and clone 6 
3 L1 "non-killer" sensitive 
0.13 
by Cla L2 " 35 
L3 " 28 
k.sub.1 and clone 6 
4 L4 " 35 
by Hind III L5 " 61 
L6 " 1.5 
L7 " 0.5 
k.sub.1 .differential. and clone 6 
1 L8 " 50 
by Hind III 
__________________________________________________________________________ 
All the transformants given in Table 1 are UPA.sup.+, since they increase 
in size on a minimum medium without uracil. This shows that the URA.sub.3 
gene of S. cerevisiae complements the uraA.sup.- mutation deficient in 
OMP decarboxylase of K. lactis. 
The transformants thus obtained have different stabilities: when grown in 
minimum medium without uracil, between 40 and 99% of the ura.sup.- they 
segregate within 15 generations; when grown for the same number of 
generations on minimum medium+uracil, between 94 and 99% of the ura.sup.- 
they segregate. 
It is, however, possible to stabilize the URA.sup.+ character by 
successive subcultures on minimum medium. 
The presence of plasmids carrying k.sub.1 and a foreign DNA, whether pBR322 
or URA.sub.3, has been confirmed by the results of hybridization on gel or 
in situ. 
By using three radioactive probes, that is to say k.sub.1, pBR325 and 
URA.sub.3, it has been possible to show that: L4 carries a free plasmid of 
4.4 kb, visible on gel, containing part of the k.sub.1 and the URA.sub.3 
gene; L3 carries a free plasmid of 9 kb containing part of the k.sub.1 
.delta. and part of the DNA of pBR322. 
The total DNA of the L.sub.2 and L.sub.3 transformants is extracted and 
this DNA is used to transform a pyr F strain of E. coli (mutation 
complemented by the URA.sub.3 gene of S. cerevisiae). The URA.sup.+ and 
Amp.sup.r transformants are then selected on suitable media, and 
transformants which have incorporated the plasmids according to the 
invention are obtained. 
The extraction of the plasmids issued from the E. coli strains transformed 
by the DNA of L.sub.3 shows that these are plasmids conforming to the 
representation in the attached figure, these plasmids being called p.sup.L 
3. These are circular plasmids which have a size of the order of 7.3 kb 
and possess a k.sub.1 end cut by ClaI. 
The attached figure shows the restriction plan of the plasmid p.sup.L 3, 
such as has been possible to determine by analysis. 
This plasmid includes a bacterial fragment originating from the plasmid pBR 
322 and a HindIII restriction fragment corresponding to the URA3.sup.+ 
gene of the yeast S. cerevisiae (these two DNA fragments originating from 
the restriction of the clone 6 plasmid by ClaI), and, finally, a ClaI 
restriction fragment of the plasmid k.sub.1 .delta. of K. lactis. 
It is appropriate to note that in p.sup.L 3, as shown in the figure, one of 
the ClaI sites has disappeared in the vicinity of the DNA of the URA3 
gene. 
Under these conditions, the plasmid p.sup.L 3 comprises a large number of 
unique restriction sites: ClaI, EcoRI and BamHI, which makes it 
particularly suitable as a cloning vector for the gene in K. lactis. 
In addition, this plasmid has the enormous advantage of being able to be 
amplified in E. coli and easily extracted, which makes it a particularly 
useful plasmid. 
Finally, the plasmid p.sup.L 3 extracted from E. coli transforms the K. 
lactis ura.sup.- yeasts for ura.sup.+ character with an efficiency ten 
times greater than that obtained with the bonding mixtures such as is 
described in the main patent. 
The procedures below are intended to illustrate the detailed preparation of 
vectors according to the invention, but without limiting the invention. 
The strains 
The K. lactis yeast strains used are: the wild strain CBS 2360 a 
(k.sub.1.sup.+ k.sub.2.sup.+), and the mutant CBS 2360 a uraA.sup.- 
obtained by induction with UV and deficient in OMP decase activity; and 
the strain VM2 .alpha.lys.sup.- (k.sub.1.sup.0, K.sub.2.sup.+) derived 
from the strain CBS 2359. 
The Escherichia coli strain HB 101 Ap.sup.R Tet.sub.5.sup.R containing the 
clone 6 plasmid carrying the URA.sub.3 fragment of S. cerevisiae of 1.1 
kb. 
The media 
The yeasts are grown on minimum medium (2% of glucose, 0.67% of "yeast 
nitrogen base" Difco depleted in aminoacids, 2% of agar Difco). This 
medium can be supplemented by uracil (50 mg/liter) or lysine (40 
mg/liter). The YPG medium contains 2% of glucose, 1% of yeast extract and 
1% of bactopeptone. The crossing and sporulation medium is ME medium 
composed of 5% of malt extract and 2% of agar Difco. The "killer" test is 
carried out on GAL medium: 2% of galactose, 1% of bactopeptone, 1% of 
yeast extract, 0.05M KH.sub.2 PO.sub.4 and 2% of agar. 
Extraction of the plasmids 
The extraction of the bacterial clone 6 plasmid is carried out in 
accordance with the method of Guerry et al. 1973 (J. Bact. 116, 
1064-1066). 
The extraction and purification of the plasmids of K. lactis were developed 
by M. Wesolowski, P. Dumazert and H. Fukuhara, 1982, Current Genetics (in 
the press). 
The cells of a culture of 200 ml of YPG at the end of the exponential phase 
are washed once with water, weighed and suspended in 1M sorbitol (2 ml/g 
of cells) with zymolyase 60,000 (0.5 mg/g of cells). 
After incubation at 30.degree. C. for 45 minutes, the cells are centrifuged 
and suspended in a solution of 0.15M NaCl and 0.10M EDTA (2 ml/g of 
cells). 0.5 mg/g of cells of pronase and a final 1% of SDS are added. 
After 1 hour of incubation at 37.degree. C. and 1 hour at 50.degree. C., 
the entire mixture is cooled and a final amount of 0.5M potassium acetate 
is added. The mixture is left in the cold for at least half an hour. 
After centrifugation, the supernatant liquor is treated with RNase at 
37.degree. C. for half an hour. Sevag extraction (chloroform:isoamyl 
alcohol, 24:1, volume/volume) is followed by precipitation with ethanol 
(1.2 volumes). 
The DNA strands collected with a Pasteur pipette are dissolved in TE (10 mM 
tris, 1 mM EDTA, pH 8). The DNA strands are reprecipitated with 0.56 
volume of redistilled isopropanol and are redissolved in TE. This 
solution, to which a colorant (bromophenol blue) is added, is deposited on 
0.6% agarose gel. 
After migration at 60 V for 18 hours, the bands shown up by ethidium 
bromide which correspond to each plasmid are cut out. They are 
electro-eluted overnight at 100 mA. 
The DNA is then introduced onto a DAEA-cellulose column and the column is 
washed with 0.3 to NaCl and eluted with 2M NaCl. The DNA is then subjected 
to CsCl gradient centrifugation, dialyzed and precipitated with ethanol. 
Restrictions, ligations 
The DNAs are restricted by restriction enzymes at 37.degree. C. for 1 hour 
in the buffer corresponding to each enzyme. Partial restrictions are 
effected with a limiting amount of enzymes at 37.degree. C. for 5 to 10 
minutes. 
Dephosphorylation is carried out at 60.degree. C. for half an hour, with 
bacterial alkaline phosphatase. 
Ligation is carried out after purification of the DNA with phenol and 
precipitation with alcohol, in the presence of the T4 DNA ligase at 
10.degree. C. overnight. 
The transformation of K. lactis 
We were inspired by the works of Hinnen et al. (1978) PNAS, 75, 1929-1933 
and Gerbaud, Fournier, Blanc, Aigle, Heslot, Guerineau (1979) Gene 5, 
233-253. The cells grown in minimum medium+uracil up to the exponential 
phase (1-2.10.sup.8 /ml) are washed once with distilled water and once 
with the protoplasticization buffer (0.6M KCl, pH 5). 
The cells are resuspended at 10.sup.9 cells/ml in this buffer with 0.5 
mg/ml of zymolyase 5000, and the suspension is incubated at 35.degree. C. 
for a short time (5 to 10 minutes). 
More than 90% of protoplasts are obtained. 
The protoplasts are washed twice with a buffer of tris HCl, pH 7.5, 0.6M 
KCl and 10 mM CaCl.sub.2 by centrifuging at 1,800 g for 5 minutes and 
delicately resuspending the cells; they are concentrated to 10.sup.9 
cells/ml in the latter buffer. 1 to 10 .mu.g of ligation mixture are added 
to 0.2 ml of cells (2.10.sup.8 cells) and the components are mixed well. 
After incubation at the ambient temperature for 15 minutes, 2 ml of 30% 
strength (weight/volume) of PEG 4000 are added and the mixture is left at 
the ambient temperature for 15 minutes. 
After centrifugation at 1,800 g for 6 minutes, the product is resuspended 
in 2 ml of 0.6M KCl, 6 g/liter of glucose, 6 g/liter of bactopeptone and 4 
g/liter of yeast extract and the suspension is stirred gently at 
30.degree. C. for 1 hour. 
The cells are then centrifuged and resuspended in 0.2 ml of the 
protoplasticization buffer. 5 ml of gelose are added, with surfusion at 
46.degree. C. (0.6M KCl, 2% of glucose, 0.67% of YNB "without aminoacids" 
Difco, 30 g/liter of agar Difco "purified" grade). The entire mixture is 
poured into a container which has been preheated at 55.degree. C. and 
contains a mixture of the same composition as the above gelose but with a 
normal agar Difco. 
Hybridization 
(1) Preparation of the hybridization filters on the colony (in situ) 
(Grunstein M. and Hogness D.S. (1975) PNAS 72, 3961-3965). 
The colonies growing on W are replicated on a Schleicher and Schull BA 85 
filter placed on a container of W, and are incubated at 30.degree. C. for 
2 days. 
Protoplasticization is effected at 30.degree. C. in the course of half an 
hour with zymolyase 60,000 (1 mg/ml), in 0.6M KCl. After drying on Whatman 
paper, the filters are transferred successively to Whatman paper 
impregnated with: 0.5N NaOH (5 minutes), 1M tris HCl, pH 7.5 (2.times.2 
minutes) and 1.5M NaCl and 0.5M tris HCl, pH 7.5 (2.times.5 minutes) . 
The filters are dried in vacuo at 80.degree. C. for 3 hours. 
(2) Hybridization on gel is carried out in accordance with the method of 
Southern E. M. (1975) J. mol. Biol. 98, 503-517. 
(3) The filters are treated at 65.degree. C. in various baths: 
The prehybridization is effected in 3.times.SSC (20.times.SSC=3M NaCl, 0.3M 
Na citrate) for 30 minutes, then in the buffer of 3.times.SSC and 
10.times.Denhardt (Denhardt=0.2% of Ficoll 400, 0.2% of bovine serum 
albumin and 0.2% of polyvinylpyrrolidone) for 3 hours and finally in the 
same buffer to which 50 .mu.g/ml of the competitor DNA, 0.1% of SDS, 0.1M 
NaH.sub.2 PO.sub.4, pH 5.6 and 10% of dextran sulfate have been added, for 
2 hours. 
For hybridization of the filters with the radioactive probe labelled at 
.sup.32 P by nick translation, the denatured probe is added to the last 
bath of the prehybridization and is incubated at 65.degree. C. overnight. 
Nick translation is described by Cameron et al. 1979, Cell, 16, 739-751, 
and in information from the Amersham Centre of Radiochemistry. 
The first 6 washings of the filters are effected with 3.times.SSC, 
10.times.Denhardt and 0.1% of SDS (with 50 .mu.g/ml of the competitor DNA 
during just the first washing), and the last two washings are effected in 
1.times.SSC. The filters are dried and recorded by Kodak films.