Kluyveromyces as a host strain

Kluyveromyces hosts and DNA expression cassettes for use in Kluyveromyces are provided for transcription of endogenous and/or exogenous DNA, and production of peptides, for enhancing production of an endogenous product, or producing an exogenous product. The Kluyveromyces hosts find particular use for secretion of a desired peptide product, where signal sequences may be native to the peptide or provided from endogenous or exogenous signal sequences, including synthetic sequences, functional in Kluyveromyces. A transformation procedure is provided for efficiently transforming Kluyveromyces.

INTRODUCTION 
1. Technical Field 
This invention relates to methods for preparing and using Kluyveromyces for 
the production of polypeptides of interest which preferentially are 
secreted into the growth medium. The invention is exemplified by sequences 
useful in the production of chymosin and precursors thereof in 
Kluyveromyces. 
1. Background 
The bright promise of production of peptides in microorganisms has been 
tarnished by a number of factors. In many instances, where the peptide has 
been produced and retained in the cytoplasm, inclusion bodies have 
resulted requiring denaturation and renaturation of the protein, 
frequently with only partial or little success. In other instances, the 
peptide has been subjected to substantial degradation, so that not only 
are yields low, but also complicated mixtures are obtained which are 
difficult to separate. As a potential solution to these difficulties, the 
possibility of secretion of the desired peptide into the nutrient medium 
has been investigated. Secretion has met with limited success, since not 
all proteins have been found to be capable of secretion in the hosts which 
have been employed. Even when secreted, the processing of the peptide may 
result in a product which differs from the composition and/or conformation 
of the desired peptide. There is therefore, substantial interest in being 
able to develop systems for the efficient and economic production of 
active peptides under conditions which allow for the use of the peptides 
in a wide variety of environments, both in vitro and in vivo. 
RELEVANT LITERATURE 
European Patent Application No. 0,096,430, the European analog of the 
subject parent application and the references cited therein. The leader 
sequence of amyloglucosidase for Aspergillus is described in Boyle et al., 
EMBO J. (1984) 3:1581-1585 and Innis et al., Science 1985) 228:21-26. 
Lactase promoters are described in Bruenig et al., Nucleic Acids Res. 
(1984) 12:2327-2341. See also European Patent Application 0,123,544. The 
use of signal peptides associated with mating-type alpha factor and of the 
enzymes invertase and acid phosphatase to direct the secretion of 
heterologous proteins in Saccharomyces has been described by Singh 
(European Patent Application 84 302723.6) and by Smith et al. (Science 
(1985) 229:1219). 
Production of preprochymosin, prochymosin and chymosin in Saccharomyces has 
been studied by Mellor et al., Gene (1983) 24:1-14. When prochymosin is 
made intracellularly in Saccharomyces, only a low percentage of the 
prochymosin obtained is activatable. See Moir et al. in Developments in 
Industrial Biology (1985) 26:75-85; Mellor et al., Gene (1983) 24:1-14; 
Kingsman et al. in Biotechnology and Genetic Engineering Reviews Vol. 3 
(1985) pp. 376-418. The aggregated prochymosin produced by Saccharomyces 
required complicated methods of denaturation and renaturation to 
solubilize the prochymosin. See WO 83/04418 and EP-A-114506. 
SUMMARY OF THE INVENTION 
Peptide production systems are provided comprising Kluyveromyces host 
strains, expression cassettes which include efficient transcriptional 
initiation and termination regions for use in Kluyveromyces and a gene, 
optionally containing a signal sequence for secretion, under the 
transcriptional and translational regulation of the regulatory regions. 
The cassettes are introduced into the Kluyveromyces host strain under 
conditions whereby the resulting transformants stably maintain the 
expression cassettes. Naturally occurring DNA and synthetic genes may be 
employed for the production of peptides of interest.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
In accordance with the subject invention, expression cassettes are provided 
which allow for the efficient and economic production of polypeptides by 
Kluyveromyces yeast cells. The expression cassettes have transcriptional 
and translational regulatory sequences functional in a Kluyveromyces host 
cell and an open reading frame coding for a peptide of interest under the 
transcriptional and translational control of the regulatory regions. The 
open reading frame also may include a leader sequence recognized by the 
Kluyveromyces host which provides for secretion of the polypeptide into 
the growth medium. The Kluyveromyces cells used may be either laboratory 
or industrial strains. 
The expression cassette will include in the 5'-3' direction of 
transcription, a transcriptional and translational initiation regulatory 
region, an open reading frame encoding a peptide of interest, desirably 
having a signal sequence for secretion recognized by Kluyveromyces, and a 
translational termination region. The expression cassette will further 
comprise a transcriptional termination regulatory region. The initiation 
and termination regulatory regions are functional in Kluyveromyces and 
provide for efficient expression of the peptide of interest without 
undesirable effects on the viability and proliferation of the 
Kluyveromyces host. 
The transcriptional and translational initiation regulatory region may be 
homologous or heterologous to Kluyveromyces. Of particular interest are 
transcriptional initiation regions from genes which are present in 
Kluyveromyces or other yeast species, such as Saccharomyces, for example, 
cerevisiae, Schizosaccharomyces, Candida, etc., or other fungi, for 
example, filamentous fungi such as Aspergillus, Neurospora, Penicillium, 
etc. The transcriptional initiation regulatory regions may be obtained for 
example from genes in the glycolytic pathway, such as alcohol 
dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 
phosphoglucoisomerase, phosphoglycerate kinase, etc., or regulatable genes 
such as acid phosphatase, lactase, glucoamylase, etc. 
Any one of a number of regulatory sequences may be preferred in a 
particular situation, depending upon whether constitutive or induced 
transcription is desired, the particular efficiency of the promoter in 
conjuction with the open reading frame of interest, the ability to join a 
strong promoter with a control region from a different promoter which 
allows for inducible transcription, ease of construction, and the like. 
These regulatory regions find ample precedent in the literature. See, for 
example, EPA 164,556, incorporated herein by reference. 
Secretion of heterologous proteins in genetically modified microorganisms 
is generally accomplished by one of two methods. In the first, the leader 
sequence is homologous to the protein; in the second, the leader sequence 
is homologous to the host organism. Other alternatives of particular 
interest in the present invention for secretion of heterologous protein in 
Kluyveromyces include the use of a synthetic leader sequence or a leader 
sequence heterologous to both Kluyveromyces and the peptide of interest. 
Thus, the open reading frame usually will include a wild-type or mutated 
gene, where the signal sequence is normally associated with the remainder 
of the coding sequence, a hybrid or chimeric open reading frame, where the 
signal sequence is normally not associated with the remaining portion of 
the open reading frame, or a synthetic sequence, where the signal sequence 
and the remainder of the open reading frame are synthesized to provide for 
preferred codons, convenient restriction sites, novel amino acid 
sequences, and the like, or combinations thereof. Signal sequences which 
may be employed may be obtained from genes, such as alpha-factor, 
invertase, amyloglucosidase, native or wild type signal sequences present 
in structural genes and recognized by Kluyveromyces. Saccharomyces, other 
fungi, e.g. Neurospora, Aspergillus, and other eukaryotes. 
Of particular interest is the use of a signal sequence which provides for 
secretion of the peptide of interest into the nutrient medium, rather than 
into the periplasmic space. For the most part, the signal sequence will be 
the 5'-terminus of the open reading frame. However, in some situations, it 
may be desirable to have the signal sequence internal to the open reading 
frame. For use of internal signal sequences for secretion see U.S. Pat. 
No. 4,338,397 and Perara and Lingappa, J. Cell Biology (1985) 
101:2292-2301. Genes into which the open reading frame of interest may be 
inserted include highly expressed constitutive genes, for example, genes 
encoding enzymes of the glycolytic pathway, or highly expressed 
regulatable genes such as lactase, amyloglucosidase, or the like. 
For optimal gene expression, the nucleotide sequences surrounding the 
translational initiation codon ATG have been found to be important in 
yeast cells and in animal cells. For example, M. Kozak (Microbiol. Revs. 
(1983) 47:1-45) has studied extensively the effect of these regions on the 
expression of insulin in COS cells. Similarly, specific nucleotides are 
found more frequently in highly expressed yeast proteins than others 
indicating the important effect of these nucleotides on the level of 
expression of these genes. 
For optimal gene expression of exogenous genes it will be important to 
modify the nucleotide sequences surrounding the initiation codon ATG. This 
can be done by site-directed mutagenesis or by fusing the exogenous gene 
in frame to an endogenous Kluyveromyces gene, preferably a highly 
expressed gene, such as the lactase gene. 
Normally, it will be desirable to provide that the signal leader is cleaved 
from the peptide of interest during the secretory process, rather than 
subsequent to the secretory process, although either procedure may find 
use. Usually, the processing signal employed will be the processing signal 
naturally occurring with the signal sequence or a processing signal which 
has been modified from the naturally occurring one, which is still 
effective for providing for a peptide signal resulting in hydrolytic 
cleavage of the signal peptide and processing signal peptide from the 
peptide of interest. Various processing signals have been sequenced and 
defined, such as .alpha.-factor (see for example U.S. Patent No. 
4,546,082, which is incorporated herein by reference), amyloglucosidase, 
.alpha.-amylase, etc. In some instances, other peptidase-recognized 
sequences may be employed which may require subsequent cleavage for 
isolation of the desired peptide. These sequences include dibasic 
peptides, e.g. KR, (D).sub.4 K, and EA, which are cleaved by KEX2, bovine 
enterokinase, and a yeast membrane peptidase, respectively. 
The peptide of interest may be native to the host or heterologous, being 
derived from prokaryotic or eukaryotic sources, which eukaryotic sources 
may involve fungi, protists, vertebrates, non-vertebrates, and the like. 
The peptide products may include enzymes, such as lactase, 
.alpha.-amylase, .beta.-amylase, amyloglucosidase, chymosin, etc., 
mammalian peptides, such as hormones, interleukins, cytokines, cachexin, 
growth factors, e.g. platelet derived, epidermal, skeletal, etc., growth 
hormone, follicle stimulating hormone, interferons (.alpha.-.beta.-, and 
.gamma.-), blood factors such as factor V, VI, VII, VIII (vW or c) IX, X, 
XI or XII, plasminogen activator, (tissue or urinary), serum albumin, 
colony growth factor (e.g. GM), erythropoietin, thaumatin, insulin, etc. 
These structural genes may be obtained in a variety of ways. Where the 
amino acid sequence is known, the structural gene may be synthesized in 
whole or in part, particularly where it is desirable to provide 
yeast-preferred codons. Thus, all or a portion of the open reading frame 
may be synthesized using codons preferred by Kluyveromyces. Preferred 
codons may be determined by those codons which are found in the proteins 
produced in greatest amount by the Kluyveromyces host e.g. glycolytic 
enzymes. Methods for synthesizing sequences and bringing the sequences 
together are well established in the literature. Where a portion of the 
open reading frame is synthesized, and a portion is derived from natural 
sources, the synthesized portion may serve as a bridge between two 
naturally occurring portions, or may provide a 3'- terminus or a 
5'-terminus. Particularly where the signal sequence and the open reading 
frame encoding the peptide are derived from different genes, synthetic 
adaptors commonly will be employed. In other instances, linkers may be 
employed, where the various fragments may be inserted at different 
restriction sites or substituted for a sequence in the linker. 
For the most part, some or all of the open reading frame will be from a 
natural source. Methods for identifying sequences of interest have found 
extensive exemplification in the literature, although in individual 
situations, different degrees of difficulty may be encountered. Various 
techniques involve the use of probes, where at least a portion of the 
naturally occurring amino acid sequence is known, where genomic or cDNA 
libraries may be searched for complementary sequences. Alternatively, 
differential transcription can be detected when the gene of interest can 
be induced or when cells are from the same host but of different 
differentiation, by comparing the messenger RNA's produced. Other 
techniques have also been exemplified. 
The termination region may be derived from the 3'- region of the gene from 
which the initiation region was obtained or from a different gene. A large 
number of termination regions are known and have been found to be 
satisfactory in a variety of hosts from the same and different genera and 
species. The termination region is usually selected more as a matter of 
convenience rather than because of any particular property. Preferably, 
the termination region will be derived from a yeast gene, particularly 
Saccharomyces or Kluyveromyces. 
In developing the expression cassette, the various fragments comprising the 
regulatory regions and open reading frame may be subjected to different 
processing conditions, such as ligation, restriction, resection, in vitro 
mutagenesis, primer repair, use of linkers and adaptors, and the like. 
Thus, nucleotide transitions, transversions, insertions, deletions, or the 
like, may be performed on the DNA which is employed in the regulatory 
regions and/or open reading frame. 
During the construction of the expression cassette, the various fragments 
of the DNA will usually be cloned in an appropriate cloning vector, which 
allows for expansion of the DNA, modification of the DNA or manipulation 
by joining or removing of the sequences, linkers, or the like. Normally, 
the vectors will be capable of replication in at least a relatively high 
copy number in E. coli. A number of vectors are readily available for 
cloning, including such vectors as pBR322, pACYC184, pUC7-19, M13, Charon 
4A, and the like. 
The cloning vectors are characterized by having an efficient replication 
system functional in E. coli. Also, the cloning vector will have at least 
one unique restriction site, usually a plurality of unique restriction 
sites and may also include multiple restriction sites, particularly two of 
the same restriction sites for substitution. In addition, the cloning 
vector will have one or more markers which provide for selection for 
transformants. The markers will normally provide for resistance to 
cytotoxic agents such as antibiotics, heavy metals, toxins or the like, 
complementation of an auxotrophic host, or immunity to a phage. By 
appropriate restriction of the vector and cassette, and, as appropriate, 
modification of the ends, by chewing back or filling in overhangs, to 
provide for blunt ends, by addition of linkers, by tailing, complementary 
ends can be provided for ligation and joining of the vector to the 
expression cassette or component thereof. 
After each manipulation of the DNA in the development of the cassette, the 
plasmid will be cloned and isolated and, as required, the particular 
cassette component analyzed as to its sequence to ensure that the proper 
sequence has been obtained. Depending upon the nature of the manipulation, 
the desired sequence may be excised from the plasmid and introduced into a 
different vector or the plasmid may be restricted and the expression 
cassette component manipulated, as appropriate. 
In some instances a shuttle vector will be employed where the vector is 
capable of replication in different hosts requiring different replication 
systems. This may or may not require additional markers which are 
functional in the two hosts. Where such markers are required, these can be 
included in the vector, where the plasmid containing the cassette, the two 
replication systems, and the marker(s) may be transferred from one host to 
another, as required. In the present situation, the second replication 
system would be a replication system functional in Kluyveromyces. The 
replication systems which may be used may be derived from plasmids, 
viruses, or the chromosome of Kluyveromyces or other species, particularly 
one associated with Kluyveromyces, such as Saccharomyces. Thus, 
replication systems include the replication system of the 2 micron plasmid 
found in Saccharomyces and an autonomously replicating sequence (ARS) 
gene, for example when used in conjunction with a centromere sequence, or 
the like. If desired, regions of homology may be provided to encourage 
integration of the expression cassette into the genome of the 
Kluyveromyces host. 
Of particular interest in the constructs of the subject invention is a 
sequence derived from Kluyveromyces DNA chromosomes referred to as KARS, 
which provide for high transformation frequency. The KARS gene may be 
obtained by screening a library of Kluyveromyces DNA fragments for 
enhanced transformation efficiency. In this manner, fragments can be 
obtained which contain KARS sequences, which fragments can be further 
modified by restriction, resection, or primer repair, to provide a 
fragment of approximately 200 bp and not more than about 5000 bp, more 
usually from about 200 bp to 2000 bp which provides for enhanced 
transformation efficiency. The presence of the KARS gene can provide 
transformation of K. lactis auxotrophic species to prototophy at a 
frequency of at least about 10.sup.3 per microgram of DNA, usually at a 
frequency of 10.sup.4 per microgram of DNA or higher. 
The manner of transformation of E. coli with the various DNA constructs 
(plasmids and viruses) for cloning is not critical to this invention. 
Conjugation, transduction, transfection or transformation, e.g. calcium 
phosphate mediated transformation, may be employed. By contrast, for 
yeast, for the most part the prior art has relied on transformation of 
protoplasts employing combinations of calcium and polyethylene glycol of 
from about 2000 to 8000, usually 4000 to 7000 daltons. 
An alternative method of transformation involves growing Kluyveromyces in a 
standard yeast nutrient medium to a density of 1 to 25, desirably 4 to 10 
OD.sub.610. The Kluyveromyces cells are then harvested, washed and 
pretreated with chaotropic ions particularly the alkali metal ions, 
lithium, cesium, or rubidium particularly as the chloride or sulfate, more 
particularly the lithium salts, at concentrations of about 2 mM to 1 M, 
preferably about 0.1 M. After incubating the cells in the presence of 
polyethylene glycol of 2000 to 6000 daltons, preferably 4000, for from 
about 5 to 120 min. preferably about 60 min. with the chaotropic ion(s), 
the cells are then incubated with DNA for a short period of time at a 
moderate temperature, generally from about 20.degree. C. to 35.degree. C., 
for about 5 min. to 60 min. Desirably, polyethylene glycol is added at a 
concentration to about 25 to 50%, where the entire medium may be diluted 
by adding an equal volume of a polyethylene glycol concentrate to result 
in the desired final concentration. The polyethylene glycol will be of 
from about 2000 to 8000 daltons, preferably about 4000 to 7000 daltons. 
Incubation will generally be for a relatively short time, generally from 
about 5 to 60 min. after the second addition of the polyethylene glycol. 
Desirably, the incubation medium is subjected to a heat treatment of from 
about 1 to 10 min. at about 35.degree. C. to 45.degree. C., preferably 
about 42.degree. C. 
For selection, any useful marker may be used, although the number of 
markers useful with Kluyveromyces is narrower than the markers used for 
Saccharomyces. Desirably, resistance to kanamycin and the aminoglycoside 
G418 are of interest, as well as complementation of a gene in the 
tryptophan metabolic pathway, particulary TRP1 or the lactase gene, 
particularly LAC4. 
Although a marker for selection is highly desirable for convenience, other 
procedurees for screening transformed cells have been described. See for 
example G. Reipen et al., Current Genetics (1982) 189-193. Besides the use 
of an indicator enzyme such as beta-lactamase, transformed cells may be 
screened by the specific products they make. In the case of chymosin, for 
example, synthesis of the product may be determined by an immunological or 
an enzymatic method. 
The vector used may be capable of extra chromosomal maintenance in 
Kluyveromyces or result in integration into the Kluyveromyces gene. It has 
been found that the 2 micron plasmid replication system from Saccharomyces 
provides for extrachromosonal maintenance in Kluyveromyces. In addition, 
one may use a combination of a centromere, such as the Saccharomyces CEN3 
and a high transformation frequency sequence, such as ARS or KARS. If 
selective maintenance is provided, such as complementation or resistance 
to an antibiotic to which Kluyveromyces is susceptible, the ARS-like 
sequences will usually suffice for extrachromosomal maintenance. 
For large scale fermentation even a small loss of plasmid stability will 
greatly effect the final yield of the desired protein. To increase the 
stability of recombinant molecules in host cells, for example 
Kluyveromyces, integration of the recombinant molecules into the host 
chromosome may be used. 
Where integration is desired, it will usually be desirable to have a 
sequence homologous to a sequence of the chromosome of the host, so that 
homologous recombination may occur. It is understood, that random 
integration also occurs, so that the homologous sequence is optional. 
Where an homologous sequence is employed, the homologous sequence will 
usually be at least about 200 bp and may be 1000 bp or more. In addition, 
where integration is involved, one may wish to have amplification of the 
structural gene. Amplification has been achieved by providing for a gene 
in tandem with the desired structural gene, which provides for selective 
advantage for the host in a selective medium. Thus, the genes expressing 
dihydrofolate reductase, metallothioneins, thymidine kinase, etc., have 
proven useful in a variety of hosts to provide for amplification, where 
the gene provides protection from a toxin, such as methotrexate, heavy 
metals, such as copper and mercury, and the like. 
Vectors of interest providing for stable replication include KARS vectors 
originating from K. lactis, e.g. pKARS12 and pKARS2, which plasmids 
comprise a K. lactis DNA fragment containing the KARS12 or KARS2 sequence 
in the S. cerevisiae plasmid YRp7. A vector employed for integration is 
illustrated by pL4, a hybrid plasmid of the ARS1 carrying plasmid YRp7 and 
K. lactis XhoI DNA fragment carrying the LAC4 gene. 
Plasmids of particular interest include plasmids having the 2 micron 
plasmid replication system, the LAC4 gene, the Tn601 and Tn5 kanamycin 
resistance gene, which also provides resistance to the antibiotic G418 in 
Kluyveromyces (Jimenez and Davis, Nature (1980) 287:869,871). This plasmid 
provides for autonomous replication in Kluyveromyces and can be selected 
for by resistance to G418 on regeneration plates containing glucose, 
sorbitol, and 0.2 .mu.g/ml G418, while avoiding elevated concentrations of 
KCl, which interferes with the sensitivity of Kluyveromyces to G418. 
Preferred plasmids include the TRP1 gene, particularly from S. cerevisiae, 
the LAC4 gene, particularly K. lactis, Kan.sup.R gene providing for 
resistance against antibiotic G418 from Tn5, or the like. 
The subject vectors and constructs are introduced into an appropriate host 
for cloning and expression of the desired structural genes. After 
transformation, colonies will normally appear on regeneration medium 
within about 5 to 6 days. Where an antibiotic is employed for selection, 
the colonies should be screened to ensure the absence of spontaneous 
mutation to a resistant strain. Employing the plasmids and the methods of 
the subject invention, about 5% of resistant colonies were found to 
contain the plasmid construct providing for at least about 4 transformants 
per .mu.g of plasmid DNA. Where selection was based on the presence of the 
LAC4 gene, using plates containing lactose as the sole carbon source and 
0.6M KCl as an osmotic stabilizer, all of the surviving colonies were 
found to be transformants and not spontaneous revertants. About 20 
transformants were obtained after about 4 to 5 days of incubation at 
moderate temperature, e.g. 30.degree. C. 
As a host organism, Kluyveromyces is especially suitable for the production 
of heterologous proteins, in particular for the production and extraction 
of the enzyme chymosin and its precursors preprochymosin, pseudochymosin 
and prochymosin. Although other organisms such as Saccharomyces produce 
prochymosin in reasonable amounts, the produced prochymosin cannot be 
extracted in an active or activatable form. We have surprisingly found 
that more than 90% of the total amount of the prochymosin produced by 
Kluyveromyces can be extracted in an active form with very simple standard 
techniques. 
Any of the many Kluyveromyces species may be employed. Either laboratory or 
industrial, preferably industrial, strains may be used. By industrial 
species is intended, Kluyveromyces strains from organisms which may be 
isolated from natural sources or may be available from depositories or 
other sources or obtained by modification, e.g. mutation, of such strains. 
The industrial strains are characterized by being resistant to genetic 
exchange, being protrotophic or made protrotophic by a single gene being 
introduced into the host strain, and are usually selected for improved 
production of peptides. Among the Kluyveromyces species which may find use 
are K. lactis, K. fragilis, K. bulgaricus, K. thermotolerans, K. 
marxianus, etc. It should be further noted that the Kluyveromyces 
organisms are on the GRAS (Generally Recognized As Safe) list. Their use 
for production of products to be used in vivo or to be ingested normally 
will not require special governmental review and approval. 
Both wild type and mutant Kluyveromyces, particularly Kluyveromyces lactis 
or Kluyveromyces fragilis may be employed as hosts. Hosts of particular 
interest include K. lactis SD11 lac4 trp1 and K. lactis SD69 lac4. These 
strains, derived from the wild-type CBS2360, were deposited under rule 28, 
resp. 28A of the European Patent Convention with Centraal Bureau Voor 
Schimmelcultures, Oosterstraat 1, 3742 SK Baarn, Netherlands, under 
numbers CBS8092 and CBS8093, respectively, on May 19, 1982. 
For maintaining selective pressure on the transformants for maintenance of 
the plasmids, selective media may be used such as a yeast nitrogen-based 
medium, 2% lactose instead of glucose for K. lactis SD69 lac4 (PTY75-LAC4) 
and for K. lactis SD69 lac4 (pL4) and a yeast nitrogen-based medium 
(Difco) plus 2% glucose for K. lactis SD11 lac4 trp1 (pKARS12). Similarly, 
strains containing plasmids conferring antibiotic resistance, for example 
against gentamycin 418, may be cultivated in a medium containing said 
antibiotic. 
Where the hybrid plasmids are employed for large scale production of the 
desired protein, it would generally be useful to remove at least 
substantially all of the bacterial DNA sequences from the hybrid plasmids. 
Depending upon the nature of the structural gene of interest, the 
expression product may remain in the cytoplasm of the host cell or be 
secreted. It has been found that not only the proteins that remain in the 
cell but also those that are secreted are soluble. Where the expression 
product is to remain in the host cell, it may generally be desirable to 
have an inducible transcription initiation region, so that until the 
transformant has reached a high density, there is little or no expression 
of the desired product. After sufficient time for the expression product 
to form, the cells may be isolated by conventional means, e.g. 
centrifugation, lysed and the product of interest isolated. Depending upon 
the nature and use of the product, the lysate may be subjected to various 
purification methods, such as chromatography, electrophoresis, solvent 
extraction, crystallization, or the like. The degree of purity may vary 
from about 50%, to 90% or higher, up to essential purity. 
Alternatively, the expression product may be secreted into the culture 
medium, and produced on a continuous basis, where the medium is partially 
withdrawn, the desired product extracted, e.g., by affinity 
chromatography, or the like, and the spent medium discarded or 
recirculated by restoring essential components. When the product is to be 
secreted, normally a constitutive transcriptional initiation region will 
be employed. 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill of those skilled in the art to which 
this invention pertains. All publications and patent applications are 
herein incorporated by reference to the same extent as if each individual 
publication or patent application was specifically and individually 
indicated to be incorporated by reference. 
The following examples are offered by way of illustration and not by 
limitation. 
EXPERIMENTAL 
Example 1 
Recombinant plasmid PTY75-LAC4 
0.5 .mu.g of the plasmid pK16 described by R. Dickson, (Gene (1980) 
10:347-356) and 0.5 .mu.g of the plasmid PTY75 described by C.P. 
Hollenberg et al. (Gene (1976) 1:33-47) were digested with the restriction 
enzyme SalI. The two digests were mixed and after inactivation of the 
restriction enzyme the solution was incubated with T4-ligase, yielding a 
solution with recombinant DNA. 
This ligated mixture was used to transform the E. coli strain DG75 
(hsdS1leu-6 ara-14 galK2 xyl-5 15 mt-1 rpsL20 thi-1 
supE44-.lambda.-lac.DELTA.z 39) according to R. C. Dickson et al., Cell 
(1978) 15:123-130, resulting in kanamycin resistance (Kan.sup.R). 
Kan.sup.R colonies were further selected on supplemented minimal plates, 
containing lactose as the sole carbon source, for the formation of 
lac.sup.+ colonies. The plasmid PTY75-LAC4 was isolated from one of the 
selected Kan.sup.R lac.sup.+ transformants, using the method according to 
L. Katz et al., J. Bacteriol. (1973) 114:577-591. 
Example 2 
Recombinant pKARS plasmids 
5 .mu.g of plasmid YRp7 (Struhl et al., Proc. Natl. Acad. Sci., USA (1979) 
76:1035-39) was digested with restriction enzyme SalI. 14 .mu.g of DNA 
from the wild strain K. lactis CBS2360 was digested with enzyme XhoI. The 
fragments of the plasmid and the K. lactis DNA were mixed in a molar ratio 
of 1:3. After inactivation of the restriction enzymes the solution was 
brought to a DNA concentration of 25 .mu.g/ml and incubated with T4-ligase 
(Boehringer) under standard conditions. 
Transformation of E. coli DG75 with the ligated mixture under the usual 
conditions yielded a mixture of 4.5.times.10.sup.5 Amp.sup.R 
transformants, 9.times.10.sup.3 of which contained K. lactis inserts, as 
can be deduced from their sensitivity to tetracycline. The proportion of 
tetracycline-sensitive cells can be increased to 85% by cycloserine 
treatment. See F. Bolivar and K. Bachman, Methods in Enzymology (1979) 
68:245-267. Fourteen different plasmids were isolated according to the 
method of Katz et al. (see Example 1). These plasmids are referred to as 
pKARS 1-14. All were capable of transforming K. lactis SD11 lac4 trp1 
strain to Trp.sup.+ phenotype with a frequency of 3.times.10.sup.4 per 
microgram of DNA, but plasmid pKARS2 appeared to be more convenient in 
further processing. 
The recombinant plasmids obtained could also be transferred to E. coli 
JA221 (.DELTA.trp E5, leu B6, lac y, rec A, hsdM.sup.+, hsdR.sup.-). 
Example 3 
Recombinant plasmid pL4 
A mixture of YRp7 and K. lactis DNA fragments was prepared as described in 
Example 2. E. coli DG75 strain was transformed with the ligated mixture 
and subsequently plated on M9 minimal agar, which medium contained lactose 
as the sole carbon source, to which leucine was also added. Lac.sup.+ 
colonies appeared after 8 days at 30.degree. C. Plasmid pL4 was isolated 
from one of these Lac.sup.+ colonies using the method of Katz et al. (see 
Example 1). 
Example 4 
Kluyveromyces lactis SD69 lac4 transformed to G418.sup.R Lac.sup.+ with 
plasmid PTY75-LAC4 
Cells of the Kluyveromyces lactis mutant SD69 lac4 were suspended in a 
growth medium (pH 6.8) containing 1% yeast extract, 2% peptone and 2% 
glucose. Growth was continued until the exponential phase 
(3-5.times.10.sup.7 cells per ml) had been reached. 
The yeast cells were collected by centrifugation, washed with water and 
resuspended in a solution (pH 8.0) containing 1.2 M sorbitol, 25 mM EDTA 
and 0.2 M fresh mercaptoethanol. After incubation for 10 min. at 
30.degree. C. the cells were centrifuged, washed two times with a 1.2 M 
sorbitol solution and resuspended in 20 ml of a solution (pH 5.8) 
containing 1.2 M sorbitol, 10 mM EDTA, 0.1 M sodium citrate and 10 mg 
helicase. 
Protoplasts were formed and after 15 to 20 minutes they were centrifuged, 
washed three times with 1.2 M sorbitol and resuspended to a concentration 
of about 5.times.10.sup.10 cells per ml in 0.1 ml of a solution containing 
10 mM CaC1.sub.2 and 1.2 M sorbitol. 
10 .mu.g of PTY75-LAC4 was added and the mixture was incubated for 15 min. 
at 25.degree. C. Thereafter 0.5 ml of a solution (pH 7.5) containing 10 mM 
Tris, 10 mM CaC1.sub.2 and 20% (w/v) polyethylene glycol 4000 was added, 
followed by a 20 min. incubation. 
Protoplasts were precipitated by centrifugation and then resuspended to a 
concentration of about 5.times.10.sup.10 protoplasts per ml in a solution 
(pH 6.8) containing 7 mM CaC1.sub.2, 1.2 M sorbitol, 0.5 mg/ml yeast 
extract, 1 mg/ml peptone and 2 mg/ml glucose. 
After incubation for 60 min. at 30.degree. C., the protoplasts were 
centrifuged, washed with 0.6 M KCl solution and resuspended in 0.6 M KCl 
solution. 
To select G418 resistant transformants, 1x10.sup.9 protoplasts were plated 
in a 3% agar overlay on 2% minimal agar plates containing 2% glucose, 1.2 
M sorbitol and 0.2 mg/ml G418. In order to simultaneously select for 
Lac.sup.+ transformants, 5.times.10.sup.8 protoplasts were plated in 3% 
agar overlay on 2% minimal agar plates, Difco yeast nitrogen base medium, 
containing 2% lactose as the sole carbon source and 0.6 M KCl instead of 
1.2 M sorbitol. 
Colonies appeared within 4 to 5 days. On sorbitol plates without G418, 
protoplast regeneration was usually 0.2-0.5%, whereas on the 0.6 M KCl 
plates with glucose as carbon source this increased to 0.5-1.5%. 
When G418 was used for selection, one transformant was obtained per 
10.sup.7 regenerated protoplasts. Simultaneous selection on lactose plates 
yielded 10 transformants per 10.sup.7 regenerated protoplasts or 20 
transformants per microgram of plasmid DNA (see Table, p. 21). 
The presence of PTY75-LAC4 in the yeast cells could be proved by means of 
Southern hybridization with 32P-labeled pCR1. 
DNA preparations were made according to Struhl et al. (Proc. Natl Acad. 
Sci. USA (1979) 76:1035-1039). 
Example 5 
Kluyveromyces lactis SD11 lac4 trp1 transformed to Trp.sup.+ with plasmid 
pKARS12 
Cells of the strain K. lactis SD11 lac4 trp1 were transformed as described 
in Example 4 with 10 .mu.g of pKARS12 DNA. Transformants were selected on 
2% agar minimal plates containing 2% glucose and 0.6 M KCl. Per microgram 
of pKARS12 DNA, 3.4.times.10.sup.4 Trp.sup.+ transformants were obtained. 
Example 6 
Kluyveromyces lactis SD69 lac4 transformed to Lac.sup.+ with plasmid pL4 
K. lactis strain SD69 lac4 was transformed with plasmid pL4 using the same 
method as described for PTY75-LAC4 in Example 4. The transformants were 
selected on yeast nitrogen base plates (Difco) containing 2% lactose. The 
transformation frequency was 20 transformants per microgram of plasmid 
DNA. 
Examples 7-13 
Kluyveromyces lactis strains transformed to Trp.sup.+ with KARS-type 
plasmids 
Analogous to the method described in Example 5, transformation experiments 
were carried out with other KARS-type plasmids. The results of the 
experiments are summarized in the following Table. 
TABLE 1 
______________________________________ 
Transfor- 
mants per 
Size of 
microgram 
KARS frag- 
Ex. Strain Genotype Plasmid 
DNA ments (kb) 
______________________________________ 
4. SD69 lac4 PTY75- 20 -- 
LAC4 
7. SD11 lac4 trp1 pKARS1 1.5 .times. 10.sup.3 
2.24 
8. SD11 lac4 trp1 pKARS2 5 .times. 10.sup.3 
1.24 
9. SD11 lac4 trp1 pKARS7 10.sup.3 
2.3 
10. SD11 lac4 trp1 pKARS8 5 .times. 10.sup.3 
1.85 
11. SD11 lac4 trp1 pKARS10 
2.4 .times. 10.sup.4 
3.15 
12. SD11 lac4 trp1 pKARS12 
3.4 .times. 10.sup.4 
5.0 
13. SD11 lac4 trp1 pKARS13 
1.5 .times. 10.sup.4 
2.0 
______________________________________ 
The molecular weights of pKARS plasmids were determined after digestion 
with endonucleases EcoRI and HindIII, using an 0.8% agarose gel and 
standard molecular weight markers. 
Example 14 
Kluyveromyces lactis SD11 lac4 trp1 transformed to Trp.sup.+ with plasmids 
containing the KARS-2 sequence using a transformation procedure with whole 
cells 
Plasmid pEK2-7 (see FIG. 2) was used to transform K. lactis SD11. This 
plasmid consists of plasmid YRp7 into which a 1.2 kb fragment containing 
the autonomously replicating sequence derived from KARS-2 has been cloned. 
K. lactis SD11 was grown overnight at 30.degree. C. in 1% yeast extract, 
2% peptone and 
2% glucose (pH 5.3). The cells were harvested at OD.sub.610 nm of 4-8 by 
centrifugation at 1000xg for 5 min. The cells were washed with TE-buffer 
(10 mM Tris-HC1, 1 mM EDTA, pH 8.0) and the pellet was resuspended in 
TE-buffer at a concentration of 2.times.10.sup.8 cells per ml. This 
suspension was diluted with one volume of 0.2 M LiCl and shaken at 
30.degree. C. for 60 min. 
Plasmid pEK2-7 DNA (10 .mu.g) was added to 0.1 ml of Li-treated cells and 
the incubation was continued at 30.degree. C. for 30 min. One volume of 
70% polyethylene glycol 7000 was added and the mixture was incubated for 
another 60 min. at 30.degree. C. The mixture was exposed to heat treatment 
at 42.degree. C. for 5 min. and the cells were plated onto minimal agar 
containing 2% glucose and 0.67% yeast nitrogen base (YNB). Transformants 
were observed after incubation at 30.degree. C. for 36-48 hrs. 
Example 15 
Kluyveromyces fragilis transformed with plasmids containing the KARS-2 
sequence 
Two types of plasmids were used to transform K. fragilis. The first 
plasmid, pGB 180, was constructed by cloning the 3.5 kb BglII fragment 
from plasmid pEK2-7 (FIG. 2) containing the KARS-2 autonomously 
replicating sequence from K. lactis and the TRP1 gene from S. cerevisiae 
into the BamHI site of pJDB 207 (J.D. Beggs, Alfred Benzon Symposium 
(1981) 16:383). About 36 K. fragilis leu mutants obtained after 
UV-treatment of K. fragilis were transformed with pGB 180 by the Li.sup.+ 
method as described in Example 14. One mutant, K. fragilis leu 24, was 
transformed to Leu.sup.+ with a frequency of about 10.sup.3 transformants 
per .mu.g of plasmid DNA. 
The second plasmid, pGL2, was constructed by cloning the 3.5 kb BglII 
fragment from pEK2-7 as described above into the BamHI site of plasmid 
pACYC177 (Chang et al., J. Bacteriol. (1972) 134:1141-1156) which contains 
the transposon Tn601 conferring resistance to kanamycin and the gentamycin 
derivative G418. K. fragilis strain 21 was transformed with plasmid pGL2 
by the Li.sup.+ method as described in Example 14. The transformed cells 
were plated onto YNPD-agar (YNB medium to which 2% dextrose and 2% agar 
was added) containing 50 .mu.g of G418 per ml. Transformants were detected 
after incubation at 30.degree. C. for 48 hours, whereas spontaneously 
resistant mutants were detected only after 6 days. DNA was extracted from 
K. fragilis transformants and transformed into suitable E. coli DG 75 
cells. DNA extracted from kanamycin-resistant E. coli cells showed the 
presence of plasmid pGL2. These experiments show that K. fragilis strains 
can be transformed by plasmids containing KARS-sequences and that these 
plasmids are autonomously replicating in K. fragilis. 
Example 16 
Kluyveromyces lactis SD11 lac4 trp1 expressing preprochymosin and its 
various maturation forms after being transformed with plasmids containing 
the KARS-2 sequence, the structural genes encoding preprochymosin and its 
various maturation forms, and various promoters directing the expression 
of the structural genes 
This Example comprises a number of steps the most essential of which are: 
1. Addition of SalI linkers in front of the cloned structural genes 
encoding preprochymosin, prochymosin, pseudochymosin and chymosin. 
2. Introduction of a DNA fragment in plasmids obtained above containing the 
KARS-2 autonomously replicating sequence from K. lactis and the TRP1 gene 
from S. cerevisiae. 
3. Introduction of various promoters, which direct the synthesis of the 
various maturation forms of preprochymosin, into the plasmids obtained 
above. 
Starting materials for the expression of bovine preprochymosin and its 
various maturation forms in K. lactis were the following cloned structural 
genes: 
pUR1531: methionyl-pseudochymosin 
pUR1522: methionyl-prochymosin 
pUR1523: methionyl-preprochymosin 
pUR1524: methionyl-preprochymosin 
The construction and structure of these plasmids has been described in 
detail in European Patent Application No. 82 201272.0, published on Apr. 
20, 1983, EPA Serial No. 0 077 109, which disclosure is herein 
incorporated by reference. The genes were isolated and the plasmids 
constructed as described in the above reference. 
A. Introduction of SalI linkers in plasmids pUR1531, pUR1522, and pUR1523 
and pUR1524 (FIG. 1) 
The plasmids pUR1531, pUR1522, pUR1523, pUR1524 contain an EcoRI 
restriction site just in front of the ATG initiation codon. Because an 
additional EcoRI site was present within the chymosin gene, a SalI linker 
molecule was introduced just in front of the first EcoRI site to 
facilitate the introduction of various promoter sequences directing the 
expression of the distal structural genes. 
About 50 .mu.g of DNA was incubated with 50 units of endonuclease EcoRI in 
the presence of 125 .mu.g/ml ethidium bromide in 10 mM Tris-HCl, 50 mM 
NaCl, 6 mM betamercaptoethanol, 10 mM MgCl.sub.2 and 100 .mu.g/ml bovine 
serum albumin, pH 7.5, at 37.degree. C. for 60 min. Plasmid DNA was 
predominantly converted to linear and circularized linear molecules under 
these conditions. The DNA was extracted with one volume of phenol and one 
volume of chloroform and precipitated with one volume of 2-propanol. 
The DNA was dissolved in TE-buffer and completely digested with 
endonuclease SalI. A DNA fragment of about 1800 bp was isolated from an 
agarose gel by electroelution. The fragments were extracted with phenol 
and chloroform and precipitated with 2-propanol. The precipitates were 
dissolved in TE-buffer. The cohesive ends were filled-in with DNA 
polymerase as follows: To 15 .mu.l containing the 1800 bp DNA fragment 
(about 1-2 .mu.g) was added 1 .mu.l of a 2 mM solution of dATP, dGTP, dCTP 
and dTTP, 6.5 .mu.l of 4x nick-buffer containing 0.2 M Tris-HC1 (pH 7.2), 
40 mM MgSO.sub.4, 4 mM dithiothreitol, 200 mg/ml bovine serum albumin, and 
2.5 .mu.l of water. Two units of DNA polymerase (Klenow fragment) were 
added and the mixture was incubated at 20.degree. C. for 30 min. DNA 
polymerase was then inactivated by heating at 70.degree. C. for 5 min. A 
phosphorylated SalI-linker (prepared as described in Maniatis et al., 
Molecular Cloning, (1982) Cold Spring Harbor Laboratory was added to this 
mixture together with T4 DNA ligase (10.sup.3 Units) and ATP. After 
incubation at 22.degree. C. for 4 hours the mixture was incubated at 
4.degree. C. for an additional 16 hours. The mixture was then incubated 
with endonucleases SalI and HindIII and a DNA fragment of about 1500 bp 
was recovered from an agarose gel by electroelution. The fragments (A, B, 
C, D) were purified by phenol and chloroform extraction followed by 
precipitation with 2-propanol. These fragments were ligated to a 3.3 kb 
HindIII-SalI fragment (about 0.5 .mu.g) derived from plasmid p53-209 
(Andreoli, Mol. Gen. Genetics (1985) 199:372) containing a 
temperaturesensitive replicon and an ampicillin resistant gene (encoding 
beta-lactamase), and purified from an agarose gel by electroelution. The 
ligated molecules were transformed into E. coli HB 101. 
Ampicillin-resistant, tetracycline-sensitive clones were cultured and the 
plasmid DNA extracted. Digestion of plasmid DNA with endonucleases SalI, 
EcoRI and HindIII confirmed that the plasmids pGB131, pGB122, pGB123 and 
pGB124 (FIG. 1) were obtained. 
B. Introduction of a KARS-2 and TRP1 gene in the plasmids pGB131, pGB122, 
pGB123, pGB124, respectively 
Autonomously replicating sequences derived from and replicating in 
Kluyveromyces were obtained as described in Examples 2 and 7-15. The 
autonomously replicating sequence in plasmid pKARS-2 is located on a 1.24 
kb fragment and this fragment was cloned into the well-known plasmid YRp7 
and a new plasmid pEK2-7 was obtained (FIG. 2). Digestion of pEK2-7 with 
endonuclease ClaI resulted in fragments of 3.5 and 5.5 kb, respectively. 
The 3.5 kb fragment containing both the TRP1 gene derived from S. 
cerevisiae and the KARS-2 sequence derived from K. lactis (FIG. 2) was 
isolated from an agarose gel by electroelution and ligated to 
ClaI-digested plasmids pGB131, pGB122, pGB123 and pGB124. The resulting 
mixture was transformed into E. coli JA300 (trpC). Characterization of 
plasmid DNA extracted from Trp.sup.+ transformants confirmed the 
construction of plasmids pGB151, pGB152, pGB153 and pGB154 (FIG. 2). 
C. Introduction of various promoter sequences in the plasmids directing the 
synthesis of the various maturation forms of preprochymosin 
The SalI-digested plasmids containing the KARS-2 sequence, the TRP1 gene 
and the structural gene of preprochymosin or its various maturation forms 
are well suited to accept SalI-linked promoter sequences to direct the 
synthesis of the distal structural gene in K. lactis transformants. In 
most cases the promoter sequences have to be provided with SalI linkers. 
Any promoter sequence can be provided with such a SalI linker and in the 
following Examples this is illustrated with: 
1 the isocytochrome cI promoter from S. cerevisiae 
2. the lactase promoter from K. lactis. 
C1. Addition of Sall linkers to the isocytochrome cI promoter from S. 
cerevisiae and introduction into plasmids 
Plasmid pYeCYC1 consisting of the isocytochrome c1 gene cloned into plasmid 
pBR322 was used as the starting material (G. Faye et al., Proc. Natl. 
Acad. Sci. USA (1981) 78:2258). From nucleotide sequence data it is known 
that an EcoRI site is present in the isocytochrome cI gene at nucleotide 
+8 (Ibid.). Plasmid pYeCYCI was cleaved with endonuclease EcoRI, ligated 
with T4 DNA ligase and transformed into E. coli HB101, yielding a plasmid 
pC15 containing the 1930 bp fragment carrying the promoter and 8 
nucleotides of the isocytochrome cI gene. 
Plasmid pC15 was cleaved with endonuclease EcoRI and incubated briefly with 
nuclease Bal31 to remove just a few nucleotides. The Bal31 digested ends 
were converted to blunt-ends with DNA polymerase (Klenow fragment) and a 
phosphorylated EcoRI linker was ligated to this DNA. After incubation with 
endonuclease EcoRI, ligation and transformation into E. coli, a 
transformant, pC15-R12, was identified in which 12 nucleotides from the 
cytochrome cI gene had been removed. A SalI linker was introduced by 
cleaving plasmid pC15-R12 with endonuclease EcoRI, filling in the cohesive 
ends with DNA polymerase, ligation of a phosphorylated SalI linker, 
incubation with endonuclease SalI and recloning the resulting 1070 bp 
fragment in the SalI digested plasmids pGB151, pGB152, pGB153 and pGB154, 
respectively. This yielded the isocytochrome cI promoter-containing 
plasmids pGB161, pGB162, pGB163 and pGB164 (FIG. 3), respectively, as 
identified by colony hybridization with the .sup.32 P-labeled 1070 bp 
fragment as probe. Plasmid DNA was prepared from the positive clones and 
the correct orientation of the isocytochrome cI promoter was confirmed by 
the presence of a 850 bp fragment after digestion with endonuclease SmaI. 
C2. Addition of SalI linkers to the lactase promoter from Kluyveromyces 
lactis and introduction into plasmids 
The starting material was plasmid pK16 containing the lactase gene from K. 
lactis cloned into the EcoRI site of plasmid pBR322 (R. C. Dickson and J. 
S. Markin, Cell (1978) 15:123). Sequencing of large parts of the lactase 
structural gene and its promoter established the presence of a ClaI site 
at about 450 bp in the lactase structural gene. Plasmid pK16 was digested 
with endonuclease ClaI and the fragment containing the promoter and about 
450 bp of the structural gene were recloned into the plasmid pBR322 
digested with endonucleases ClaI and AccI (partially). In one plasmid, 
pGB182, the retained Clal site at about 450 bp in the lactase structural 
gene was opened by incubation with endonuclease ClaI and trimmed by 
incubation with nuclease Bal31. The Bal31 ends were rendered blunt-ends by 
incubation with DNA polymerase. A phosphorylated EcoRI linker was ligated 
to this trimmed fragment. Digestion with endonuclease EcoRI and recloning 
of the trimmed fragment resulted in the plasmid pGB183, which had retained 
the lactase promoter but was devoid of the structural gene. SalI linkers 
were added to this fragment as described above (see Example 16.C1). The 
SalI-linked lactase promoter was ligated to SalI-cleaved plasmids pGB151, 
pGB152, pGB153 and pGB154, respectively, yielding plasmids pGB171, pGB172, 
pGB173 and pGB174, respectively. 
Plasmids obtained as described in this Example 16 were introducted into 
Kluyveromyces lactis SD11 lac4 trp1 by the Li.sup.+ method as described in 
Example 14, and Trp.sup.+ transformants selected for. The presence of 
preprochymosin or its maturation forms in Kluyveromyces extracts was 
demonstrated by immunological ELISA techniques e.g., by spotting aliquots 
of the extracts on nitrocellulose filters and assaying the filters as 
described by D. J. Kemp and A. F. Cowman (Proc. Natl. Acad. Sci. USA 
(1981) 78:4520-4524). 
Cell-extracts were prepared as follows: K. lactis transformants were grown 
at 30.degree. C. for about 16-24 hours in YNB-medium containing 2% 
dextrose. Cells were harvested at OD.sub.610 nm between 2.2-6.0 by 
centrifugation at 6000 rpm for 10 min. in a Sorvall G-S3 rotor. The pellet 
was resuspended in sterile distilled water to OD.sub.610 nm of 600 and 
chilled on ice. 0.5 ml of this cell suspension was diluted with 0.5 ml of 
ice-cold water and mixed with 2 g of Ballotini Beads (diameter 0.25-0.35 
mm; Braun-Melsungen GMBH, GFR). The cells were disrupted by shaking for 4 
min on a Vortex shaker at maximum speed. More than 95% of the cells were 
disrupted by this method as verified by phase contrast microscopy. Cell 
debris was removed by centrifugation for 1 min in an Eppendorf centrifuge. 
Aliquots of the extracts were frozen in liquid nitrogen and stored at 
-80.degree. C. 
1-5 .mu.l aliquots of the cell extracts were spotted on nitrocellulose 
membrane filters. The filters were dried, wetted with 192 mM glycine, 25 
mM Tris, 20% ethanol (pH 8.3) and incubated for 60 min. at 22.degree. C. 
The filters were subsequently incubated with preincubation buffer (0.35M 
NaC1, 10 mM Tris-HCl (pH 7.6), 2% bovine serum albumin) for 30 min. The 
filters were washed 3 times for 5 min with RIA-buffer (0.125M NaC1, 10 mM 
Tris-HCl, pH 7.6, 0.1 mM PMSF, 1% Triton X-100, 0.5% sodium desoxycholate, 
0.1% sodium dodecylsulfate (SDS) and 0.3% gelatin). The filters were 
incubated overnight at 4.degree. C. in 1 ml RIA buffer containing 10 .mu.l 
of chymosin antiserum. Antiserum was removed by washing with RIA buffer 
(three times) and incubated with 1 .mu.Ci .sup.125 I-protein A in 1 ml of 
RIA-buffer for 60 min at 22.degree. C. .sup.125 I-protein A was removed by 
washing with RIA buffer (5 times). The filters were dried and 
autoradiographed overnight. The presence of preprochymosin or its 
maturation forms in K. lactis transformants was clearly observed. 
The presence of chymosin activity in cell extracts from K. lactis 
transformants was determined by high performance liquid chromatography 
(HPLC) as described by A. C. M. Hooydonk and C. Olieman, Netherl. Milk 
Dairy (1982) 36:153. 50 .mu.l of enzyme solution or extract was added to 1 
ml of a 10% solution of milkpowder (Difco) in 10 mM CaC1.sub.2. The 
solution was incubated for 15 min. at 31.degree. C. The reaction was 
stopped by adding 2 ml of 12% trichloroacetic acid (TCA). Almost all 
proteins are precipitated by TCA except glycomacropeptide (GMP) that has 
been cleaved from K casein by chymosin action. Denatured proteins were 
pelleted by centrifugation and 1 ml of the clear supernatant was 
neutralized with 0.4 ml of 1N NaOH. The solution was centrifuged again and 
the amount of GMP produced was detected with HPLC, monitoring the 
extinction coefficient at 214 nm. Extracts from K. lactis transformants 
containing prochymosin were first incubated at pH 2 for 2 hours and 
subsequently neutralized before performing the chymosin activity test. 
Chymosin was found only after the pH 2 treatment. 
Example 17 
Construction of chymosin expression plasmids containing a long lactase 
promoter sequence 
A. Construction of pUCla56 
Chromosomal DNA was isolated from Kluyveromyces lactis strain CBS 2360 (Das 
and Hollenberg, Current Genetics (1982) 5:123-128), cleaved with XhoI, and 
separated according to size on a sucrose gradient. Fractions containing 
the lactase gene were detected with a LAC4 probe from plasmid pK16 (see 
Example 16.C2) after spotting the DNA on a nitrocellulose filter. DNA 
containing the LAC4 gene was cloned into the XhoI site of plasmid 
p53-215 (Andreoli, Mol. Gen. Gen (1985) 199:372-380) giving rise to 
plasmid p1. An XbaI fragment of p1 containing the lactase gene was 
subcloned in the XbaI site of pUC19 (Yanisch-Perron et al., Gene (1985) 
33:103-119) which yields plasmid pUCla56. 
B. Introduction of the G418 resistance gene in the terminator of the 
lactase gene 
The terminator fragment containing the G418 resistance marker was obtained 
from plasmid pGBTeG418. E. coli containing pGBTeG418 were deposited at 
Centraal Bureau voor Schimmelcultures under number CBS184.87 on Feb. 26, 
1987. Plasmid pGBTeG418 (see FIG. 4) consists of the plasmid p53-215 as 
described (Andreoli, Mol. Gen. Gen. (1985) 199:372-380), and a 5.6 kb 
fragment consisting of the 3.7 kb BamHI K. lactis lactase terminator 
fragment (Breunig et al., Nucl. Acid Res. (1984) 12:2327-2341) and the Tn5 
gene (Reiss et al., EMBO J. (1984) 3:3317) conferring resistance to 
gentamycin G418 under the direction of the promoter alcohol dehydrogenase 
I (ADH) from yeast, similar to that described by Bennetzen and Hall, J. 
Biol. Chem. (1982) 257:3018-3025. 
C. Construction of plasmids containing the G418 resistance gene and 
chymosin encoding DNAs 
The 3.6 kb HindIII-XbaI fragment from plasmid pGBTeG418 containing the G418 
resistance gene (see Example 17B) and the SalI-HindIII fragment containing 
the prochymosin gene from pGB123 were ligated in pUC19 cleaved with SalI 
and XbaI. This yielded plasmid pGB900. 
D. Construction of plasmid pGB901 
Plasmid pGB901 was constructed by ligating the following four fragments: 
(1) A 3.6 kb XbaI-HaeII fragment containing the lactase promoter to about 
position -90 from the lactase ATG start codon isolated from pUCla56, 
(2) a HaeII-SalI fragment of about 70 bp which extends from the above HaeII 
site to a SalI site which was ligated to position -26 in a similar Bal31 
experiment as described in Example 16.C2. However, only a SalI linker was 
used in the present case. 
(3) the 5.1 kb SalI-XbaI fragment containing prochymosin and G418 from 
pGB900 (see Example 17C), 
(4) pUC 19 cleaved with XbaI. 
During the construction of the plasmid the CG sequence from the HaeII site 
was inadvertently removed, thereby creating a HindIII site at this 
position. 
Prochymosin-encoding DNA is present in plasmid pGB901. This may readily be 
converted to plasmids with preprochymosin, pseudochymosin or chymosin DNA 
by using the SalI-BglII fragments from pGB 131, 122 or 124, respectively. 
Example 18 
Secretion of prochymosin from Kluveromyces lactis transformants 
To direct the synthesis of prochymosin in Kluyveromyces, plasmid pGB901 was 
used to transform K. lactis strains SD11 and CBS2360 with similar results. 
The transformation was carried out essentially as described in Examples 4 
and 14 by using intact plasmid DNA or plasmid DNA cut with restriction 
endonucleases. In the latter case restriction endonucleases were used 
which cut in the promoter region, e.g., Sacll, NdeI, SnaBI or SpeI, or in 
the terminator region, e.g., EcoRV, or both the promoter and terminator 
regions. 
K. lactis strains CBS2360 and SD11 were grown in 100 ml of YEPD medium 
containing 2.5 ml 10xYNB to OD.sub.610 of 0.75. After collecting and 
washing the cells and incubating with 0.1 M lithium acetate, 15 .mu.g of 
plasmid DNA, cut at the unique SacII site in the lactase promoter, was 
added to the cells. After a heat shock of 5 min at 42.degree. C., the 
transformed cells were spread on agar plates containing 15 ml of YEPD agar 
with 50 .mu.g/ml of G418 and were overlayered 1 hr before use with 15 ml 
YEPD without G418. Colonies were grown for 3 days at 30.degree. C. 
In one of the experiments K. lactis strain CBS2360 was transformed with 
pGB901, linearized by cutting with SacII. Transformants were selected on 
G418 containing agar plates and grown at 30.degree. C. in YEPmedium 
containing 2% galactose. After 60 hours, cells and medium were separated 
by centrifugation. Cells were disrupted by treatment with glass beads. 
Culture medium and cell extract were treated at pH 2 before assaying for 
chymosin activity. Foltman, Methods in Enzymology (1970) 19:421-426. 
Cells were removed from cultures by centrifugation and the resulting 
supernatants were acidified to pH 2 by the addition of 1 M H.sub.2 
SO.sub.4 and incubated for 2 hours at room temperature. The solutions were 
then neutralized to pH 6 by the addition of 2 M Tris base. A 50 .mu.l 
volume of an appropriate dilution was added to a suspension of 12% non-fat 
dry milk in 10 mM CaC1.sub.2 and incubated at 37.degree. C. until a clot 
formed. A unit of chymosin activity is defined as the amount of active 
chymosin required to produce a clot in 10 min. under these conditions. The 
supernatant contained milk-clotting activity due to the production and 
secretion of prochymosin by K. lactis transformants although no signal 
sequence for protein secretion was added to prochymosin. About 30-60% of 
the total prochymosin produced was found in the medium as determined by 
the above-described milk-clotting assay. Similar results were obtained 
when K. lactis strain SD11 was used. 
Example 19 
Construction of lactase-chymosin fusion proteins giving enhanced chymosin 
expression 
By taking various SnaBI - SalI fragments (from a Bal31 experiment similar 
to the one described in Example 16.C2 but using a single SalI linker only) 
variants of pGB901 containing a fusion between the lactase and chymosin 
proteins were obtained (Table 2). The extra amino acids provided by 
lactase DNA and linker sequences can be removed, along with the pro 
sequence of prochymosin, by treatment with acid. It was observed that a 
fusion containing 4 amino acids from the lactase coding sequence (pGB902) 
resulted in enhanced chymosin production. 
TABLE 2 
__________________________________________________________________________ 
Nucleotide Sequence at the Junction Between 
the Lactase Promoter and Prochymosin 
in pGB901 and pGB902 
__________________________________________________________________________ 
##STR1## 
##STR2## 
__________________________________________________________________________ 
Protein synthesis starts at the boxed ATG codon. 
Example 20 
Secretion of preprochymosin by Kluyveromyces transformants 
The SalI site from the polylinker of pGB902 (see Example 19) was removed 
for convenience. pGB902 was partially digested with SalI, followed by a 
short incubation with Bal31 (Boehringer). Linear fragments were isolated 
from an agarose gel, ligated and transformed into E. coli. A correct 
plasmid, pGB903, was obtained. Restriction analysis showed that this 
plasmid also has the XbaI and HindIII sites removed from the polylinker. 
To construct a plasmid containing and expressing preprochymosin, plasmid 
pGB903 was digested with the restriction endonucleases SalI and BglII. The 
11 kb DNA fragment was isolated from an agarose gel by electroelution. 
Similarly, plasmid pGB124 (see Example 16) containing the preprochymosin 
gene was digested and the 0.3 kb SalI-BglII fragment containing the 
N-terminal part of the preprochymosin gene was isolated. The 11 kb and the 
0.3 kb DNA fragments were mixed, ligated with DNA ligase and transformed 
into E. coli. Plasmid pGB904 was isolated which contained the 
preprochymosin gene fused to a small part of the lactase gene (Table 3). 
TABLE 3 
__________________________________________________________________________ 
Nucleotide Sequence at the Junction Between the 
Lactase Promoter and Preprochymosin 
in pGB904 
__________________________________________________________________________ 
##STR3## 
__________________________________________________________________________ 
Protein synthesis starts at the boxed ATG codon. 
K. lactis CBS2360 cells were transformed with pGB904, which had been 
linearized with SacII. Transformants were selected, grown and assayed for 
chymosin activity as described in Example 18. In the following Table a 
comparison is made between K. lactis CBS2360 cells transformed with pGB902 
(see Example 19) and with pGB904. Chymosin production is expressed in 
arbitrary units per ml of cells at OD.sub.610 of 200. 
TABLE 4 
______________________________________ 
Secretion of Prochymosin by K. Lactis Cells 
Transformed With pGB902 and pGB904 
pGB902 pGB904 
Transformant 
Supernatant 
Pellet Supernatant 
Pellet 
______________________________________ 
1 3.2 &lt;0.4 22.4 1.7 
2 1.3 &lt;0.4 33.3 3.0 
3 7.1 1.4 28.0 2.3 
4 4.4 0.66 53.8 5.8 
______________________________________ 
Example 21 
Secretion of prochymosin by Kluyveromyces using heterologous leader 
sequences 
A. Chemical synthesis of an amyloglucosidase leader sequence and 
construction of a plasmid containing said leader sequence 
The leader sequence of amyloglucosidase (AG) from Aspergillus awamori was 
published by Innis et al., Science (1985) 228:21-26. Based on the protein 
sequence, oligonucleotides were derived to permit insertion of the leader 
sequence in front of the prochymosin gene (see FIG. 5). 
The oligonucleotides were synthesized with an Applied Biosystems DNA 
synthesizer. The oligonucleotides were purified by electrophoresis on a 
denaturing polyacrylamide gel, then electroeluted from the gel. 
Plasmid pGB903 (see Example 20) was cut at the unique SalI site. The 
oligonucleotides were hybridized at 65.degree. C., 50.degree. C. and 
37.degree. C. for one hour each in 2.times.SSC. The oligonucleotides had 
no phosphate at the 5' end to prevent formation of multimers. The DNA was 
ligated into the SalI site using T4 polynucleotide ligase. The ligation 
mixture was transformed into E. coli HB101. Twenty-four of the colonies 
were cultured and plasmid DNA isolated. One of the plasmids, pGB905, was 
shown to have the correct orientation of the oligonucleotides by 
restriction enzyme analysis. Plasmid pGB905 was transformed to K. lactis 
CBS2360. Chymosin production was analyzed according to the procedure 
described in Example 18. The results are shown in the Table below. 
Chymosin production, in arbitrary units/ml of cells of OD.sub.610 at 200, 
is shown in the following Table. 
TABLE 5 
______________________________________ 
Secretion of Prochymosin by K. Lactis Cells 
Transformed with pGB902 and pGB905 
902 905 
Transformant 
Sup. Pellet Sup. Pellet 
______________________________________ 
1. 3.2 &lt;0.4 60.6 &lt;0.4 
2. 1.3 &lt;0.4 56.4 &lt;0.4 
3. 7.1 1.4 56.7 &lt;0.4 
4. 4.4 0.66 57.6 &lt;0.4 
______________________________________ 
B. Chemical Synthesis of a Novel Synthetic Leader Sequence into Contruction 
of a Plasmid Containing the Novel Synthetic Leader Sequence 
A synthetic leader sequence was prepared which has a sequence different 
from any known known leader sequence. Using this leader sequence, all 
prochymosin synthesized was secreted by Kluyveromyces as shown below. 
The synthetic leader sequence was devised using frequently occuring amino 
acids from position -6 to +2 of the signal sequence cleavage site (Von 
Heyne, Eur. J. Biochem. (1983) 133:17-21). Frequently occuring yeast 
codons were also employed and extra nucleotides were incorporated in front 
of the ATG sequence to make up for the deletion of 26 nucleotides in 
pGB902. The oligonucleotides used and the resulting leader sequence are 
shown in FIG. 6. 
The synthetic leader sequence DNA was synthesized within an Applied 
Biosystems DNA synthesizer. The resulting oligonucloetides were run on a 
40 cm long, 1 mm thick polyacrylamide gel, containing TBE buffer (50 mM 
Tris, 50 mM borate, 1 mM EDTA, pH 8.3) and 7 M urea until the bromphenol 
had traveled 2/3 of the gel length. The DNA was visualized, eluted from 
the gel and precipitated with ethanol. 
Also from pGB901 a derivative was made with a deletion around the SalI site 
resulting from the polylinker of pUC19. This was done by replacing the 0.5 
kb SnaBI-BglII fragment from pGB903 by the corresponding fragment from 
pGB901. The resulting plasmid was cut at the unique SalI site. The 
oligonucleotides were hybridized at 65.degree. C., 50.degree. C. and 
37.degree. C. for one hour each in 2.times.SSC. The DNA was ligated into 
the SalI site using T4 polynucleotide ligase. The plasmid was then 
transformed into E. coli HB101. Of the colonies obtained, 24 were cultured 
and plasmid DNA isolated. One of the plasmids, pGB906, was shown to have 
the nucleotides in the correct orientation by restriction enzyme 
digestion. It was found that K. lactis transformed with pGB906 secreted 
more than 95% of the prochymosin produced. 
C. Analysis of Chymosin Protein Produced by K. lactis Transformed with 
pGB905 
Transformants were grown for 3 days at 30.degree. C. and samples were 
collected from the supernatant of the cultures. Protein samples were 
electrophoresed on a polyacrylamide gel according to Laemmli (Nature 
(1970) 227:680-685). Proteins were blotted onto a nitrocellulose filter 
according to the method of Towbin et al. (Proc. Natl. Acad. Sci. USA 
(1979) 76:4350-4354). Chymosin protein was detected by incubating the 
filter with a polyclonal antiserum against chymosin (Chr. Hansen), 
followed by donkey anti-rabbit antibodies coupled to a peroxidase 
(Amersham) and finally with 0.6 mg/ml 4-chloronaphthol and 0.015% hydrogen 
peroxide in a buffer solution (50 mM Tris-HCl pH 7.5, 0.9% NaCl) 
containing 40% methanol Prochymosin excreted by the AG signal sequence is 
correctly cleaved after pH 2 treatment as demonstrated by this assay (FIG. 
7). 
Example 22 
Construction of plasmids containing the Saccharomyces cerevisiae 
.alpha.-factor sequence for efficient secretion 
A. Saccharomyces .alpha.-factor Expression Plasmids 
1. Construction of Plasmids 
pDM100-PC: The starting material was plasmid pGB163 (see Example 16.C1, 
above). Plasmid pGB163 was digested with BamHI and ligated to an 
XbaI-BamHI, .alpha.-factor leader-prochymosin adaptor. The resulting 
mixture was then treated with PstI and a 96 bp fragment encoding the 
pro-.alpha.-factor processing site and the N-terminal region of 
prochymosin was isolated. A 1900 bp fragment encoding bovine prochymosin 
was isolated from plasmid pJS111 following digestion with PstI and SalI. 
Plasmid pJS111 is a pBR322 derivative containing the prochymosin gene from 
pGB163 under the regulatory control of the ADH-2 promoter and the 
glyceraldehyde 3-phosphate (GAP) terminator. The 1900 bp PstI to SalI 
fragment that was removed contains the prochymosin gene and the GAP 
terminator. The yeast GAP 49 gene promoter and transcription terminator 
are essentially as described by Travis, J. Biol. Chem. (1985) 
260:4384-4389. 
Plasmid pDM100, containing a fusion of the GAP promoter the S. cerevisaie 
.alpha.-factor leader, and a synthetic gene for human .gamma.-interferon 
flanked by XbaI and SalI sites and the .alpha.-factor terminator, was 
digested with XbaI and SalI, treated with alkaline phosphatase, then 
ligated to the 96 bp and 1900 bp fragments described above. The 
.alpha.-factor leader and terminator are essentially as described by 
Brake, Proc. Natl. Acad. Sci. USA (1984) 81:4642-4646. The resulting 
plasmid pDM100-PC was isolated and contained a fusion of the GAP promoter, 
the .alpha.-factor leader and prochymosin gene. The complete sequence of 
the BamHI insert is shown in FIG. 8. 
To allow selection of yeast transformants, two plasmids, pKS100 and pAB300, 
were constructed. 
pKS100: pKS100 was constructed by insertion into pDM100-PC of an 1170 bp 
HindIII fragment from YEp24 containing the S. cerevisiae URA3 gene. 
pAB300: pAB300 was produced by insertion into pDM100-PC of a 3500 
HindIII-SalI fragment from pGB901 containing the 3' region of the K. 
lactis LAC4 gene and the G418 resistance marker. The 
GAP/.alpha.-factor/Prochymosin BamI insert in pDM100PC is illustrated in 
FIG. 8. 
2. Transformation of K. lactis and S. cerevisiae 
Plasmid pKS100 was digested at the BglII site in the prochymosin coding 
region and used to transform K. Lactis strain KRN201-6. This strain is a 
derivative of strain 2UV21 (a lac4 trp1 ura3 [kil.sup.0 ]) in which the 
lac4 gene has been replaced by the LAC4 promoter-prochymosin gene fusion 
from pGB901. Integration of pKS100 is thus targeted to the integrated 
prochymosin coding region. Plasmid pKS100 was also used to transform S. 
cerevisiae strain AB110 (.alpha. ura3 leu2 his4 pep4-3 [cir.sup.0 ]), in 
this case targeting to the SacII site in the 3' region of the GAPDH gene. 
The resulting transformants were grown to saturation in liquid YEPD medium, 
and the culture supernatants and cell lysates assayed for chymosin 
activity after activation at pH 2. As shown by the results summarized in 
Table 6 below, the K. lactis transformants efficiently secreted 
prochymosin into the medium, whereas the S. cerevisiae transformants 
secreted only a small fraction of the prochymosin produced. 
TABLE 6 
______________________________________ 
Prochymosin Production in K. lactis 
and S. cerevisiae Transformants 
Chymosin Activity 
(relative units/ml culture) 
Cell Culture 
Strain Extract Supernatant 
______________________________________ 
AB110 &lt;0.25 &lt;1.0 
AB110::pKS100 15.5 2.3 
KRN201-6 &lt;0.25 &lt;1.0 
KRN201-6::pKS100 
12.0 333.0 
______________________________________ 
Plasmid pAB300 was used to transform K. lactis strain 2UV21 to G418 
resistance, targeting integration to the EcoRV site in the 3' region of 
the LAC4 gene. These transformants were also found to efficiently secrete 
prochymosin into the culture medium as shown in Table 7 below. 
TABLE 7 
______________________________________ 
Prochymosin Secretion From 
.alpha.-Factor/Prochymosin Fusions 
Host Transforming 
Secreted Chymosin Activity 
Strain Plasmid (relative units/ml culture) 
______________________________________ 
2UV21 -- &lt;2 
KRN201-6 -- &lt;2 
KRN201-6 pKS100 385 
2UV21 pAB300 294 
______________________________________ 
B. Construction of LAC4 Promoter-.alpha.-Factor Leader-Prochymosin Fusions 
In order to produce this fusion, two intermediate plasmids were 
constructed. Plasmid pDM100-PC was partially digested with PstI, ligated 
to a SalI-PstI adaptor encoding a portion of the .alpha.-factor leader and 
26 bp of the region 5' to the LAC4 gene, and then digested with HindIII. A 
1500 bp fragment was isolated from this mixture and then cloned into pUC18 
digested with HindIII and SalI to produce pKS102. 
A synthetic E. coli lac operator was ligated into the SalI site just 5' to 
the .alpha.-factor leader coding sequence in pKS102 to produce the plasmid 
pKS103. This was done because the LAC4 promoter-.alpha.-factor 
leader-prochymosin fusion may be toxic to E. coli. 
A 490 bp SalI-BglII fragment from pKS103 was isolated and ligated to 
SalI-BglII-digested pJD15R. pJD15R is derived from pGB901 by deletion of 
the SalI site in the pUC19 polylinker by filling-in to produce pJD15, and 
then recloning the 8800 bp XbaI fragment in the opposite orientation. From 
this reaction the plasmid pKS105 was isolated. These plasmids are 
illustrated in FIG. 9. 
Plasmid pKS105 was then used to transform K. lactis strain CBS2360 to G418 
resistance, using the SacII site in the LAC4 5' region as a targeting site 
for the integrative transformation. Chymosin production is expressed in 
units per ml of cells at OD.sub.610 of 200 (Table 8). 
TABLE 8 
______________________________________ 
Secretion of Prochymosin by K. Lactis Cells 
Transformed with pKS105* 
Transformant Supernatant 
Pellet 
______________________________________ 
1 111 3.3 
2 147 4.5 
3 124 3.7 
4 125 3.0 
______________________________________ 
*Chymosin activity in relative units/ml culture. 
Example 23 
Isolation and use of K. lactis .alpha.-factor signal sequence 
Biological assays of culture supernatants were carried out as described 
(Julius, et al, Cell (1983) 32:839) using as a tester strain the S. 
cerevisiae Mata sst2-3 strain RC687. K. lactis strain CBS141 (.alpha.) was 
grown in medium consisting of 0.5% glucose, 0.17% yeast nitrogen base 
without ammonium sulfate (Difco), and 0.002% ammonium sulfate. After 
removal of cells by centrifugation, acetic acid was added to the culture 
supernatant to a concentration of 0.1 M, and the supernatant was passed 
over a column of BioRex 70 (Biorad). The column was washed with 0.1 M 
acetic acid and then the .alpha.-factor was eluted with 80% ethanol/10 mM 
HCl. The eluate was evaporated to dryness and then dissolved in 0.1% 
trifluoroacetic acid (TFA)/20% acetonitrile and applied to a reverse=phase 
HPLC guard column. The column was washed stepwise with solutions 
containing 0.1% TFA and 20%, 40%, 60% and 80% acetonitrile. The 60% 
fraction, containing the .alpha.-factor activity, was then applied to an 
analytical C-18 HPLC column and eluted with a gradient of 20% to 80% 
acetonitrile in 0.1% TFA. Fractions were collected and assayed for 
.alpha.-factor activity. The fractions containing .alpha.-factor activity 
were dried and subjected to amino acid sequence analysis. 
Hybridization screening of plasmid libraries 
Pools of oligonucleotides were labeled using .alpha.-[.sup.32 P]-ATP and T4 
polynucleotide kinase. These oligonucleotide probes were used to probe 
Southern blots or bacterial colonies at 42.degree. C. in the following 
hybridization solution: 4.times.SSC, 50 mM KH.sub.2 PO.sub.4 pH 7, 1% 
sarkosyl, 10% dextran sulfate, 200 .mu.g/ml sonicated, denatured salmon 
sperm DNA. Filters were washed in 2.times.SSC, 0.1% SDS at 42.degree. C. 
A plasmid library in the vector pJS109, containing inserts resulting from a 
limited Sau3AI digest of genomic DNA from K. lactis strain SD11 (a trpl 
lac4), size-fractionated to purify fragments &gt;5000 bp was screened with 
these probes by plating transformants of E. coli strain HB101 at a density 
of 500-2000 colonies per 80 mm plate of L-agar containing 100 .mu.g/ml 
ampicillin. DNA was transferred from the colonies to nitrocellulose 
filters and these filters hybridized as described above. Areas on the 
original plates corresponding to regions of hybridization signals on the 
filters were picked, then replated and retested by hybridization to 
isolate single colonies with plasmids containing hybridizing sequences. 
Positive colonies were further tested by Southern blot analysis of DNA 
purified from small cultures. 
Plasmids purified from hybridization-positive colonies were digested with a 
variety of restriction enzymes and the resulting fragments analyzed by 
Southern blot analysis using the same hybridization probes in order to 
identify restriction fragments of size suitable for DNA sequence analysis. 
Fragments thus identified were purified by agarose gel electrophoresis and 
cloned into appropriate MP18 and MP19 vectors. DNA sequence analysis was 
then performed. 
Isolation of Kluyveromyces .alpha.-factor 
The first 10 amino acids of the K. lactis .alpha.-factor showed a definite 
homology to that from S. cerevisiae, with 6 identical residues. This 
sequence is shown below: 
Trp-Ser-Trp-Ile-Thr-Leu-Arg-Pro-Gly-Gln 
This protein sequence was used to design a set of oligonucleotides deduced 
to be complementary to the structural gene for the corresponding 
structural gene as shown in FIG. 10. Oligonucleotides including all of the 
possible codons for a segment of the .alpha.-factor peptide were 
sythesized as 2 pools of 96 and 48 different molecules. 
These 2 pools were radioactively labelled and were each used to probe a 
Southern blot of restriction digests of K. lactis DNA Pool #2 gave strong 
hybridization to a single fragment and much weaker hybridization to a 
second fragment in several different digests. Thus, pool 2 was chosen to 
screen plasmid libraries of K. lactis genomic DNA. 
Use of these probes to screen plasmid libraries resulted in the isolation 
of a number of hybridizing clones. DNA sequence analysis of one of these 
clones, .alpha.fk18b, showed it encodes an .alpha.-factor related peptide 
which bears a strong similarity to the precursor of the S. cerevisiae 
.alpha.-factor peptide. The hybridizing segment was located on a 
PstI-EcoRI fragment of about 1000 bp. The sequence of this fragment is 
shown in FIG. 11. The K. lactis precursor contains only 2 sites for the 
addition of N-linked carbohydrate chains. In addition, the spacers of the 
K. lactis repeats are longer than those of the S. cerevisiae repeats and 
show a more diverse sequence with the pattern X-Ala/Pro rather than the 
Glu/Ala-Pro sequences found in S. cerevisiae. A comparison of the DNA 
sequences showed a strong degree of homology throughout the coding region. 
Construction of Plasmids 
A series of plasmids (shown in FIG. 12) were constructed in order to 
provide a fusion of the K. lactis .alpha.-factor leader to prochymosin 
expressed under the transcriptional control of a strong promoter. 
pAB307: A 673 bp SspI-EcoRI fragment from .alpha.fk18b (FIG. 11) was 
modified by filling the EcoRI overhang by Klenow enzyme and addition of 
BglII linkers to the blunt ends. This fragment was then inserted into a 
BglII site joining the promoter and terminator regions of the S. 
cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This 
cassette was cloned as a BamHI fragment in pUC18, resulting in pAB307. 
pAB309: Fusion of sequences encoding the .alpha.-leader and bovine 
prochymosin was then performed. First pAB307 was digested with NcoI and 
the cohesive ends made blunt by treatment with mung bean nuclease. The 
resulting product was then digested with SalI. To this was ligated a 2000 
bp EcoRV-SalI fragment containing sequences encoding prochymosin and the 
S. cerevisiae transcriptional termination region. This fragment was 
derived from plasmid pJS111 in which a XbaI-BamHI adaptor had been added 
to the 5' end of a fragment containing prochymosin cDNA fused to the S. 
cerevisiae GAPDH transcriptional termination region. This ligation mixture 
was used to transform E. coli strain HB101 and a transformant carrying the 
plasmid pAB309 was isolated. The sequences around the junction of this 
fusion are shown in FIG. 13 and the sequence of the entire BamHI insert of 
pAB309 is shown in FIG. 14. 
pAB312a: In order to obtain transformation of K. lactis strains, a 3600 bp 
HindIII fragment derived from pGB901 was inserted into pAB309 producing 
plasmid pAB312a. The HindIII fragment contains the 3' region of the K. 
lactis LAC4 gene and a fusion of the S. cerevisiae ADH1 promoter to the 
bacterial G418-resistance structural gene. 
pAB313 and pAB314: A 1900 bp SacI-HindIII was isolated from pAB309 and 
cloned into MP19 (YanischPerron et al., Gene (1985) 33:103). 
Single-stranded phage DNA was prepared and used as a template for invitro 
mutagenesis with one of the two oligonucleotide primers shown in FIG. 15. 
The M13 phage MP19/.alpha.k11.5 and MP19/.alpha.k12.2 were prepared using 
Primer #1 and Primer #2, respectively. 
Double-stranded RF DNA was prepared from these phage, and 1100 bp SacI-StuI 
fragments isolated from each. These fragments were ligated to a 7100 bp 
SacI-StuI fragment from pAB312. The resulting plasmids pAB313 and pAB314 
were isolated with the sequence alterations illustrated in FIG. 13. 
Transformation of Kluyveromyces 
Plasmid pAB312a was digested with EcoRV (to target integration to the LAC4 
region of the K. lactis genome) and was then used to transform K. lactis 
strain 2UV21 (a ura3 trp1 lac4 [kil.sup.0 ]) to G418 resistance. 
The plasmids pAB313 and pAB314 were used to transform strain 2UV21 to G418 
resistance. Cultures of transformants 2UV21::pAB312, 2UV21::pAB313 and 
2UV21: :pAB314 were grown and culture supernatants assayed for chymosin 
activity as above. 
A number of these transformants, as well as an untransformed control 
strain, were grown for 36 hours in 1 ml of medium composed of 1% yeast 
extract, 2% peptone, 2% glucose, 0.17% yeast nitrogen base, 50 .mu.g/ml 
tryptophan and 50 .mu.g/ml uracil. Culture supernatants were then assayed 
for chymosin activity after acid activation. All of the transformants were 
found to secrete activatable chymosin. The results are shown below. 
TABLE 9 
______________________________________ 
Secretion of Prochymosin 
in Kluyveromyces 
Chymosin Activity 
Strain Host Plasmid (relative units/ml culture) 
______________________________________ 
2UV21 2UV21 -- &lt;2 
KRN303-1 2UV21 pAB312 256 
KRN304-4 2UV21 pAB313 175 
KRN305-2 2UV21 pAB314 206 
______________________________________ 
Each of the transformants was found to secrete a single prochymosin-related 
species as judged by SDS polyacrylamide gel electrophoresis of 
trichloroacetic acid-precipitated culture supernatants. The 
prochymosin-related protein secreted by pAB312 transformants appeared to 
be of slightly higher molecular weight than those secreted by pAB313 and 
pAB314 transformants as determined by electrophoretic mobility. 
The major species secreted by KRN303-1 and KRN304-4 were purified by 
preparative SDS polyacrylamide gel electrophoresis and subjected to gas 
phase amino acid sequence analysis. The N-terminal sequences of these 
species are given below. 
##STR4## 
These results indicate that the prochymosin-related species secreted by 
KRN303-1 has undergone no processing of the amino-terminal spacer 
sequence, while the species secreted from KRN304-4 has the authentic 
mature prochymosin amino terminus. 
Example 24 
Kluyveromyces SD11 lac4 trp1 expressing preprothaumatin and its various 
maturation forms after being transformed with plasmid pURK 528-01 
containing the structural gene encoding preprothaumatin, the KARS-2 
sequence from K. lactis, the glyceraldehyde-3-phosphate dehydrogenase 
promoter from S. cerevisiae and the TRP1 gene from S. cerevisiae 
This Example comprises a number of steps, the most essential of which are: 
1. Isolation of clones containing the glyceraldehyde-3-phosphate 
dehydrogenase (GAPDH) operon of S. cerevisiae 
A DNA pool of the yeast S. cerevisiae was prepared in the hybrid E. 
coli-yeast plasmid pFL (Chevallier et al, Gene (1980) 11:11-19) by a 
method similar to the one described by Carlson and Botstein, Cell (1982) 
28:145-154. Purified yeast DNA was partially digested with restriction 
endonuclease Sau3A and the resulting DNA fragments (with an average length 
of 5 kb) were ligated by T4 DNA ligase in the dephosphorylated BamHI site 
of pFL 1. After transformation of CaC1.sub.2 -treated E. coli cells with 
the ligated material, a pool of about 30,000 ampicillin resistant clones 
was obtained. 
The clones were screened by a colony hybridization procedure (Thayer, Anal. 
Biochem. (1979) 98:60-63) with a chemically synthesized, .sup.32 P-labeled 
oligomer with the sequence 5'TACCAGGAGACCAACTT3'. According to data 
published by J. P. Holland and M. J. Holland (J. Biol. Chem. (1980) 
225:2596-2605) this oligomer is complementary with the DNA sequence 
encoding amino acids 306-310 (the wobble base of the last amino acid was 
omitted from the oligomer) of the GAPDH gene. Using hybridization 
conditions described by Wallace et al, Nucleic Acid Res. (1981) 9:879-894, 
six positive transformants could be identified. One of these harbored 
plasmid pFL 1-33. The latter plasmid contained the GAPDH gene including 
its promoter/ regulation region and its transcription termination/ 
polyadenylation region. The approximately 9 kb long insert of pF2 1-33 has 
been characterized by restriction enzyme analyses (FIG. 16) and partial 
nucleotide sequence analysis (FIGS. 17 and 18). 
2. Isolation of the GAPDH promoter/regulation region and its introduction 
into a preprothaumatin encoding plasmid 
On the basis of the restriction enzyme analysis and the nucleotide sequence 
data of the insert of plasmid pFL 1-33, the DNA initiation/regulation 
region of the GAPDH gene was isolated as an 800 nucleotide long DdeI 
fragment. To identify this promoter fragment, plasmid pFL 1-33 was 
digested with SalI and the three resulting DNA fragments were subjected to 
a Southern blot hybridization test with chemically synthesized oligomer 
(Southern, J. Mol. Biol. (1975) 98:503-517). A positively hybridizing 4.3 
kb long restriction fragment was isolated on a preparative scale by 
electroelution from a 0.7% agarose gel and was then cleaved with DdeI. Of 
the resulting DdeI fragments, only the largest one has a recognition site 
for PvuII, a cleavage site located within the GADPH promoter region (FIG. 
16). The largest DdeI fragment was isolated and incubated with Klenow DNA 
polymerase and 4 dNTP's (Davis et al., Gene (1980) 10:205-218) to generate 
a blunt-ended DNA molecule. After extraction of the reaction mixture with 
phenol/chloroform (50/50 v/v), passage of the aqueous layer through a 
Sephadex G50 column and ethanol precipitation of the material present in 
the void volume, the DNA fragment was joined to the .sup.32 P-labeled 
EcoRI linker 5'GGAATTCC3' by incubation with T4 DNA ligase. Using the 
Klenow polymerase reaction and subsequent ligation of the EcoRI linker, 
the original DdeI sites were reconstructed at the end of the 
promoter-containing fragment. 
To inactivate the ligase, the reaction mixture was heated to 65.degree. C. 
for 10 min, then sodium chloride was added (final concentration 50 mM) and 
the whole mix was incubated with EcoRI. Incubation was terminated by 
extraction with phenol/chloroform. The DNA was precipitated twice with 
ethanol, resuspended and then ligated into a suitable vector molecule. 
Since the DdeI promoter-containing fragment has EcoRI sites, it can easily 
be introduced into the EcoRI site of pUR528 (Edens et al., Gene (1982) 
18:1-12) to create a plasmid in which the yeast GAPDH promoter is adjacent 
to the structural gene encoding preprothaumatin. The latter plasmid was 
obtained by cleavage of pUR528 with EcoRI, treatment of the linearized 
plasmid molecule with calf intestinal phosphatase to prevent 
self-ligation, and incubation of each of these vector molecules as well as 
the purified DdeI promoter fragment with T4 DNA ligase Transformation of 
the various ligation mixes in CaC1.sub.2 -treated E. coli HB101 cells 
yielded several ampicillin resistant colonies. From some of these colonies 
plasmid DNA was isolated (Birnboim and Doly, Nucleic Acids Res. (1979) 
7:1513-1523) and incubated with PvuII to test the orientation of the 
insert. 
In the naming of the plasmids, plasmids containing the EcoRI (DdeI) GAPDH 
promoter fragment in the correct orientation (i.e. transcription from the 
GAPDH promoter occurs in the direction of a downstream located structural 
gene) are indicated by the addendum-01 to the original code of the plasmid 
(for example pUR528 is changed into pUR528-01; see FIG. 19). 
To facilitate manipulation of plasmids containing the EcoRI promoter 
fragment, one of the two EcoRI sites was destroyed. Two .mu.g of plasmid 
DNA (e.g. pUR 528-01) was partially digested with EcoRI and then incubated 
with 5 units mung bean nuclease (obtained from P. L. Biochemicals Inc.) in 
a total volume of 200 .mu.l in the presence of 50 mM sodium acetate (pH 
5.0), 50 mM sodium chloride and 1 mM zinc chloride for 30 min. at room 
temperature to remove sticky ends. The nuclease was inactivated by 
addition of SDS to a final concentration of 0.1% (Kowalski et al, 
Biochemistry (1976) 15:4457-4463) and the DNA was precipitated by the 
addition of 2 volumes of ethanol (in this case the addition of 0.1 volume 
of 3 M sodium acetate was omitted). Linearized DNA molecules were then 
religated by incubation with T4 DNA ligase and used to transform 
CaC1.sub.2 -treated E. coli cells. Plasmid DNA isolated from 
ampicillinresistant colonies was tested by cleavage with EcoRI and MluI 
for the presence of a single EcoRI site adjacent to the thaumatin gene 
(see FIG. 19). 
Plasmids containing the GAPDH promoter fragment but having only a single 
EcoRI recognition site adjacent to the ATG initiation codon of a 
downstream located structural gene are referred to as -02 type plasmids 
(for example: pUR 528-01 is changed into pUR 528-02; see FIG. 19). 
3. Reconstitution of the original GAPDH promoter/regulation region in 
plasmids encoding preprothaumatin by introduction of a synthetic DNA 
fragment 
As shown by the nucleotide sequence depicted in FIG. 20, the EcoRI (DdeI) 
GAPDH promoter fragment contains the nucleotides -850 to -39 of the 
original GAPDH promoter/regulation region. Not contained in this promoter 
fragment are the 38 nucleotides preceding the ATG initiation codon of the 
GAPDH encoding gene. The latter 38-nucleotide long fragment contains the 
PuCACACA sequence, which is found in several yeast genes. Said PuCACACA 
sequence situated about 20 bp upstream of the translation start site 
(Dobson et al, Nucleic Acids Res. (1982) 10:2625-2637) provides the 
nucleotide sequence upstream of the ATG codon which is optimal for protein 
initiation (Kozak, Nucleic Acids Res. (1981) 9:5233-5252). Moreover, these 
nucleotides allow the formation of a small loop structure which might be 
involved in the regulation of expression of the GAPDH gene. On this basis, 
introduction of the 38 nucleotides in between the DdeI promoter-fragment 
and the ATG codon of a downstream located structural gene was considered 
necessary to improve promoter activity as well as translation initiation. 
As outlined in FIG. 21, the missing DNA fragment was obtained by the 
chemical synthesis of two partially overlapping oligomers. The SacI site 
present in the overlapping part of the 2 oligonucleotides was introduced 
for two reasons: (i) to enable manipulation of the nucleotide sequence 
immediately upstream of the ATG codon including the construction of poly 
A-tailed yeast expression vectors: (ii) to give a cleavage site for an 
enzyme generating 3'-protruding ends that can easily and reproducibly be 
removed by incubation with T4 DNA polymerase in the presence of the 4 
dNTP's. Equimolar amounts of the two purified oligomers were 
phosphorylated at their 5'-termini, hybridized (Rossi et al, J. Biol. 
Chem. (1982) 257:9226-9229) and converted into a double-stranded DNA 
molecule by incubation with Klenow DNA polymerase and the 4 dNTP's under 
conditions which have been described for double-stranded DNA synthesis 
(Davis et al., Gene (1980) 10:205-218). Analysis of the reaction products 
by electrophoresis through a 13% acrylamide gel followed by 
autoradiography showed that more than 80 % of the starting single-stranded 
oligonucleotides were converted into double-stranded material. The DNA was 
isolated by passage of the reaction mix over a Sephadex G50 column and 
ethanol precipitation of the material present in the void volume. The DNA 
was then phosphorylated by incubation with polynucleotides cleaved off in 
the latter reaction and the reaction mix subjected to two precipitations 
with ethanol. 
As shown in FIG. 20, cloning of the resulting synthetic DNA fragment was 
carried out by the simultaneous ligation of this fragment and a BglII-DdeI 
GAPDH promoter regulation fragment in a vector molecule from which the 
EcoRI site preceding the ATG initiation codon was removed by mung bean 
nuclease digestion (see Step 2). The BglII-DdeI promoter/regulation 
fragment was obtained by digestion of plasmid pUR528-02 with DdeI and 
BglII. Separation of the resulting restriction fragments by 
electrophoresis through a 2% agarose gel and subsequent isolation of the 
fragment from the gel yielded the purified 793 nucleotides long promoter/ 
regulation fragment. In the plasmid pUR528-02 the nucleotide sequence 
preceding the ATG codon is 5'-GAATTC(T)ATG-3' (EPA 54330 and EPA 54331), 
which is different from the nucleotide sequence given by Kozak (Nucleic 
Acids Res. (1981) 9:5233-5252). Since our aim was to reconstitute the 
original GAPDH promoter/regulation/protein initiation region as accurately 
as possible, the EcoRI site was removed in order to ligate the synthetic 
DNA fragment to the resulting bluntend. Removal of the EcoRI site was 
accomplished by mung bean nuclease digestion of EcoRI-cleaved pUR528-02 
DNA. 
Subsequently, the plasmid DNA was digested with BglII and incubated with 
phosphatase. After separation of the two DNA fragments by electrophoresis 
through a 0.7% agarose gel, the largest fragment was isolated and used as 
the vector into which the BglII-DdeI promoter fragment as well as the 
DdeI-treated-synthetic DNA fragment were ligated. Plasmids in which the 
DdeI promoter/regulation fragment together with the SacI recognition site 
containing synthetic DNA fragment are introduced are indicated by the 
addendum-03 (for example: pUR528-02 is changed into pUR528-03). 
4. Introduction of the KARS-2 replicon from K. lactis and the TRP1 gene 
from S. cerevisiae in preprothaumatin encoding plasmids 
The KARS-2 replicon and the TRP1 gene were excised from pEK2-7 by digestion 
with BglII, followed by isolation from an 0.7% agarose gel of the 3.5 kb 
fragment. This purified fragment was inserted in the dephosphorylated 
BglII cleavage site of pUR528-03 by incubation with T4 DNA ligase. 
Transformation of the ligation mix in E. coli yielded plasmid pURK528-03 
(FIG. 22). Transformants generated by the introduction of plasmid pURK 
528-03 into K. lactis SD11 cells by the Li.sup.+ method were shown to 
synthesize thaumatin-like proteins assayed as described by L. Edens et 
al., Gene (1982) 18:1-12 (see FIG. 23). 
The above results demonstrate that one can obtain efficient, convenient 
expression of exogenous genes in Kluyveromyces strains. Furthermore, the 
Kluyveromyces strains appear to be particularly useful for providing 
highly efficient secretion and processing of a wide variety of proteins, 
as illustrated by the results with prochymosin. Constructs and vectors are 
provided which allow for the introduction of an exogenous gene under the 
regulatory control of efficient promoters in Kluyveromyces and, as 
desired, joining to signal sequences which provide for translocation of 
the exogenous gene, particularly secretion. Thus, a fermentation system is 
provided for commercial production of a wide variety of exogenous proteins 
in an active or activatable form. 
The following organisms have been deposited with the American Type Culture 
Collection on June 30, 1987: 2UV21, ATCC Accession No. 20855; KRN201-6, 
ATCC Accession No. 20854; HB101 pAB307, ATCC Accession No. 67454; HB101 
pAB312, ATCC Accession No. 67455. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.