Promoters associated with expression of specific enzymes in the glycolytic pathway are used for expression of alien DNA, particularly yeast promoters known to provide high enzyme levels of enzymes in the glycolytic pathway are employed for expressing a mammalian protein, such as .alpha..sub.1 -antitrypsin. The promoters include promoters involved in expression of pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, phosphoglycerate mutase, hexokinase 1, hexokinase 2, glucokinase, phosphofructokinase, and aldolase, as well as the glycolytic regulation gene. Particularly, the glycolytic regulation gene can be used in conjuction with promoters in the glycolytic pathway for regulated production of desired proteins.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The ability to obtain expression of foreign, i.e., exogenous, DNA in 
unicellular microorganisms provided the opportunity to conveniently 
prepare long polypeptide chains of interest. Almost immediately, varied 
polypeptides, such as the small hormone somatostatin and more 
sophisticated polypeptides, such as insulin, interferons, thymosin and a 
variety of vaccines having capsid proteins, were prepared and reported in 
the literature. For the most part, the initial work was performed in the 
bacterium E. coli which had been the subject of intensive study because 
scientists were familiar with many aspects of its genetic structure and 
properties. Initial attention was therefore directed to producing foreign 
proteins in E. coli. Once the ability to employ E. coli as a host was 
established, the limitations and disadvantages of employing E. coli 
encouraged the use of other hosts. 
One host which appeared to be particularly attractive because it lacked 
many of the shortcomings of E. coli was yeast. However, yeast is a 
eukaryote and, therefore, has a more sophisticated genetic system. 
Furthermore, less is known about the yeast genome than is known about E. 
coli. In order to use yeast as a host for the production of proteins 
foreign to yeast, a number of discoveries are required, and new materials 
must be made available. 
Initially, a replication system was required which provided stability in 
yeast, either as an extrachromosomal element or by integration into the 
yeast chromosome. In addition, the regulatory functions concerned with 
transcription and expression had to be developed in order to allow for 
expression of the desired protein. There was also the uncertainty whether 
foreign DNA sequences would be transcribed and translated and, if 
expressed, whether the resulting polypeptides would survive in the yeast 
cell. Also remaining to be determined was the effect of the foreign 
proteins on the viability of the yeast cell, such as the effect of 
recombinant DNA (RDNA) on mitosis, sporulation and vegetative growth. 
There have, therefore, been substantial efforts to develop novel RDNA 
systems in yeast, which will allow for regulated expression of a protein 
of interest, as well as highly efficient production of such proteins. 
2. Description of the Prior Art 
Hitzeman et al., J. Biol. Chem. (1980) 255:12073-12080 describe a plasmid 
having a yeast 3-phosphoglycerate kinase (PGK) gene and accompanying 
regulatory signals capable of expression in yeast. Other references of 
interest include Clifton, et al., Genetics (1978) 88:1-11; Clark and 
Carbon, Cell (1976) 9:91-99; Thomson, Gene (1977) 1:347-356; Holland and 
Holland, J. Biol. Chem. (1979) 254:5466-5474; Holland and Holland, ibid. 
(1979) 254:9830-9845; Nasmyth and Reed, Proc. Nat. Acad. Sci. (1980) 
77:2119-2123; Broach, et al., Gene (1979) 8:121-133; and Williamson, et 
al., Nature (1980) 283:214-216. 
SUMMARY OF THE INVENTION 
Novel yeast promoters are provided which control the transcription of genes 
in the glycolytic pathway and which find use in the regulated production 
of proteins foreign to the yeast. Promoters of particular interest include 
the promoters for triose phosphate isomerase, pyruvate kinase, 
phosphoglucose isomerase, phosphoglycerate mutase, hexokinase 1, 
hexokinase 2, glucokinase, phosphofructokinase, and aldolase, as well as 
the glycolytic regulatory gene. The protease inhibitor, mammalian 
.alpha..sub.1 -antitrypsin, is expressed using the promoter for triose 
phosphate isomerase. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Methods and compositions are provided for regulated efficient expression of 
alien or foreign DNA in a yeast host. (Alien or foreign DNA is DNA not 
naturally occurring in the wild type particularly from a different species 
and which does not normally exchange genetic information with the host.) 
Novel promoters are employed which are involved in the glycolytic pathway 
and provide for high levels of protein production, so that a substantial 
proportion of the total protein produced by the yeast cells can be 
dedicated to the protein of interest. In addition, regulatory mechanisms 
associated with regulation of production of the glycolytic enzymes are 
achieved, so that production of the desired products may be modulated. 
Furthermore, viable cells can be maintained to enhance the efficiency and 
amount of expression. 
The promoters of interest are particularly those promoters involved with 
expression of triose phosphate isomerase, pyruvate kinase, phosphoglucose 
isomerase, phosphoglycerate mutase, hexokinase 1, hexokinase 2, 
glucokinase, phosphofructokinase, and aldolase, which are controlled by 
the glycolytic regulation gene GCR1. The genes of the glycolytic pathway 
include hexokinase 1 and 2 (HXK1,2); phosphoglucose isomerase (PGI), 
triose phosphate isomerase (TPI); phosphoglycerate kinase (PGK), 
phosphoglycerate mutase (GPM), pyruvate kinase (PYK); phosphofructokinase 
(PFK), enolase (ENO); fructose 1,6-diphosphate aldolase (FDA); 
glyceraldehyde 3-phosphate dehydrogenase (PGK); and glycolysis regulation 
protein (GCR). 
The promoters may be obtained by employing a gene bank having large 
fragments of yeast DNA. By introducing the fragments into appropriate 
vectors, particularly shuttle vectors having replicons for prokaryotes and 
yeast, one can readily amplify and clone the yeast DNA in a bacterium and 
then introduce the yeast DNA into mutant yeast cells for complementation. 
In this manner, yeast fragments can be identified which complement 
auxotrophic lesions or mutations in a yeast host. 
Of particular interest, is where the host is auxotrophic in both the 
glycolytic pathway step of interest and a separate biochemical pathway, 
which is complemented by a marker in the vector. Once having established a 
DNA segment having the desired gene, one may reclone by various techniques 
to shorten the DNA segment and provide for a segment which is primarily 
the gene of interest in conjunction with its regulatory signals for 
transcription and expression. 
In order to retain the promoter, it is essential that the initiator 
methionine be determined and this codon be used for developing the 
strategy for introducing the alien DNA downstream from the promoter. 
Various techniques can be employed for providing a site for introduction 
of the alien DNA so as to be under the regulatory control of the promoter 
in the glycolytic pathway. 
Where a restriction site is conveniently adjacent to the initiator 
methionine codon, the glycolytic gene may be cleaved at that site and the 
DNA chewed back with Ba131 for varying periods of time, so as to chew into 
or past the initiator methionine codon or retain the initiator methionine 
codon. 
Where there is no convenient restriction site, other splicing techniques 
such as primer repair may be employed. Also, by employing in vitro 
mutagenesis, one can introduce a restriction site adjacent the initiator 
methionine, which encodes for the initial amino acids of the desired 
protein. In each instance, a linearized DNA segment is obtained having the 
intact promoter for the glycolytic product and normally includes other DNA 
sequences, such as an intact replicon, one or more markers, and the like. 
Exemplary of the above procedure is the development of a vector having the 
promoter for the TPI1 gene. An exemplary vector CV13 having the replicons 
or replication systems from pBR322 and 2.mu.-plasmid of yeast, as well as 
the LEU2 gene was employed for insertion of a yeast fragment which was 
shown to have the TPI1 gene. This was achieved by employing double 
selection with a mutant yeast which was leu.sup.-, tpi.sup.-. The TPI1 
gene was found to have a unique KpnI site. The vector was cleaved at the 
KpnI site and then treated with the double stranded exonuclease Ba131 for 
varying times to chew back the DNA to about the f-met codon. Linkers were 
then inserted providing desired restriction sites. Alien DNA could then be 
inserted providing a sequence having a f-met codon in the appropriate 
position for initiation. Alternatively, the foreign DNA can be expressed 
using the f-met codon of the TPI1 gene. 
Similar procedures can be performed with the other subject glycolytic genes 
in order to provide the promoters associated with those genes. The PYK 
sequence has a convenient XbaI site for restriction, where the few 
additional bases may be removed, if required, using Ba131 for a short 
period of time to chew to or through the methionine codon. Of particular 
interest is the use of the GCR promoter to control the expression of the 
other genes involved in the glycolytic pathway. By employing the GCR gene, 
in conjunction with other glycolytic promoters regulating expression of 
alien DNA, one can turn on and off the other promoters, so as to regulate 
the expression of the alien DNA. Thus, one can allow vegetative growth to 
proceed until a desired cell density is achieved, before permitting 
production of the desired polypeptide. 
By employing appropriate auxotrophs, one can further regulate the 
expression of the polypeptides of interest in choosing the appropriate 
nutrient medium. Where the chosen promoter is repressed by the particular 
nutrient because of a metabolic block, a change in the nature of the 
nutrient can induce expression. Furthermore, the activity of a number of 
promoters in the glycolytic pathway can be affected by the repression or 
activation of expression by the GCR gene or other regulatory controls. 
Also, the GCR regulatory signals can be used to titrate the polypeptide 
functioning as the regulator for expression of GCR. By having vectors 
whose copy number can be controlled, one can vary the activity of the wild 
type GCR gene. 
In order to obtain expression, an extrachromosomal element construct will 
be prepared having a number of sequences defining different functions. One 
function is the replication system, which forms part of a vector. Another 
function is a promoter by itself or in conjunction with the alien DNA. 
Other functions include initiators and terminators of expression. Also, 
there will be selectable markers. 
In developing an appropriate vector, while not necessary, it will be common 
to have both a replication system for yeast and a replication system for a 
prokaryote (a shuttle vector). The replication system for yeast may be one 
which provides for stable maintenance of an extrachromosomal element or 
one which provides a sufficient lifetime for the DNA in the host, that 
there is an acceptable probability of integration of the DNA into the 
host. Integration can be greatly aided by providing for a sequence 
homologous to the host DNA, so as to provide for recombination. Generally, 
the homologous sequence will be at least about 800bp usually not more than 
about 2000bp. Therefore, either integration or an autonomous replication 
system, such as the use of the ARS1 gene, may be employed to provide for 
the maintenance of the alien DNA in the yeast host. The replication system 
which is chosen should provide for a reasonable copy number usually 
greater than 1, preferably greater than 5. A wide variety of replication 
systems are available on a wide variety of prokaryotic vectors, such as 
pBR322, pACYC184, pSC101, pMB9, etc. Alternatively, one or more copies of 
the DNA construct can be integrated into the host chromosome. The 
replication systems may also be conditionally regulated, usually being 
temperature sensitive so that replication can be turned on and off by 
varying the temperature. 
In addition to the replication system, there will also be one or more 
selectable markers, there usually being at least one marker in addition to 
the alien DNA, which may serve as a marker. Conventional markers include 
biocidal markers providing antibiotic resistance and those providing 
resistance to toxins and heavy metal. Also useful is employing an 
auxotrophic host and providing prototrophy by complementation. In addition 
to the conventional selection systems just described, the glycolytic genes 
of the present invention are particularly desirable markers since they can 
provide for selection, using sugars as selective substrates, in 
appropriate mutant host strains. 
Other genes may also be inserted into the extrachromosomal element for a 
variety of purposes. Where integration is desirable in the genome of the 
host, a homologous sequence for a particular region of the host genome may 
be included in the extrachromosomal element. Where amplification of one or 
more sequences is desired, genes known to provide such amplification, such 
as dihydrofolate reductase genes, which respond to methotrexate stress or 
metallothionein genes, which respond to heavy metal stress, may be 
included in the extrachromosomal element, flanked by the DNA regions to be 
reiterated. Other regulatory signals may also be included, such as 
centromeres, autonomously replicating segments, etc. 
In order to isolate the promoters of interest, clones can be made of yeast 
chromosomal DNA by random digestion or mechanical shearing of the yeast 
genome. The presence of the desired gene is then determined by introducing 
a homogeneous clone of a yeast fragment into an auxotrophic host for 
complementation. Desirably, the cloning vehicle may have another gene 
which allows for an additional basis for selection, so that double 
selection techniques can be used. The mutants are substantially incapable 
of growing on limited nutrient medium, so that one can select for the 
presence of the desired glycolytic gene by the choice of medium. After 
isolating the yeast fragment having the desired gene, the fragment may be 
subcloned so as to remove superfluous DNA flanking regions and provide for 
a fragment which is more easily manipulated. The smaller fragment 
containing the desired gene, of a size less than about 500 base pairs may 
then be further cloned, restriction mapped and sequenced, so as to provide 
a useful source for the desired promoters and insertion of the alien DNA. 
Also, as indicated, the promoters in themselves may be useful, in acting 
as a titrater for repressor or activator, where it is desirable to 
modulate the production of a particular enzyme in the yeast host. 
The alien DNA may be from any source, either naturally occurring or 
synthetic, either prokaryotic or eukaryotic. Of particular interest are 
mammalian genes which express a poly(amino acid), that is, polypeptide or 
protein which has physiological activity. To varying degrees, poly(amino 
acids) prepared in yeast may be modified by glycosylation, where the 
glycosylation may not occur or may occur at different sites from the 
naturally occurring mammalian polypeptide and/or in different degrees with 
different saccharides. It is therefore of great interest to be able to 
prepare polypeptides which are different from the naturally occurring 
polypeptide by the degree and manner of glycosylation and in many 
instances may differ in one or more ways as to the amino acid sequences, 
where there may be deletions of one or more amino acids or substitutions 
of one or more amino acids. Mammalian genes may come from a wide variety 
of mammalian sources, such as domestic animals (e.g. bovine, porcine, 
ovine and equine) and primates e.g. humans and monkeys. 
As exemplary of the use of the subject promoters in preparing an active 
polypeptide composition, as well as being of particular interest for a 
variety of purposes, a protease inhibitor is described and made. The 
protease inhibitor has the same or substantially the same amino acid 
sequence of human .alpha..sub.1 -antitrypsin and is capable of inhibiting 
a number of proteolytic enzymes. The human .alpha..sub.1 -antitrypsin gene 
appears to reside within a 9.6 kb EcoRI DNA fragment in the human genome. 
The mature mRNA appears to have about 1400 nucleotides. One human 
.alpha..sub.1 -antitrypsin cDNA has the following sequence, although other 
naturally-occurring forms (polymorphisms) are known. 
3 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
##STR15## 
##STR16## 
##STR17## 
##STR18## 
##STR19## 
The human .alpha..sub.1 -AT has a BamHI restriction site which allows the 
cutting of the gene with the removal of information for a single glutamic 
acid from the mature protein. Various schemes can be employed for 
introducing the human .alpha..sub.1 -AT gene adjacent the glycolytic 
promoter to be under the regulation of the promoter. Where the promoter 
does not have a convenient restriction site near the f-met codon, the 
glycolytic gene may be cleaved and chewed back to the promoter with Ba131. 
A linker may then be introduced downstream from the promoter to provide a 
convenient cohesive end or flush end for joining to the human 
.alpha..sub.1 -AT gene. The linker can also provide one or more codons for 
amino acids at the N-terminus of the .alpha..sub.1 -AT gene, which may be 
the same or different from the naturally occurring amino acids. 
The gene for human .alpha..sub.1 -AT may then be inserted into the 
extrachromosomal element downstream from the glycolytic promoter, where an 
f-met codon is provided for initiation of expression of the human 
.alpha..sub.1 -AT. 
The resulting extrachromosomal element containing the human .alpha..sub.1 
-AT gene may then be introduced into a yeast host, particularly an 
auxotrophic host and the yeast host grown for expression of a polypeptide 
having .alpha..sub.1 -antitrypsin activity. The resulting polypeptide 
differs from the naturally occurring human .alpha..sub.1 -AT in its degree 
of glycosylation. 
The .alpha..sub.1 -antitrypsin can be used as an antigen for production of 
polyclonal and monoclonal antibodies to human .alpha..sub.1 -AT, for 
introduction into a host having a deficiency of .alpha..sub.1 -AT, or for 
modulating proteolytic activity in a mammalian host. In particular, the 
.alpha..sub.1 -antitrypsin can be administered to humans to replace 
.alpha..sub.1 -antitrypsin which has been inactivated (oxydized) by 
tobacco and other smoke.

The following examples are offered by way of illustration and not by way of 
limitation. 
EXPERIMENTAL 
Materials and Methods 
Strains. 
Isogenic strains carrying mutations in PGI1, PGK1, GPM1, PYK1, and GCR1 
where obtained by ethyl methane sulfonate (EMS) mutagenesis of S. 
cerevisiae (S. c.) X2180-1A (MATa SUC2 CUP1 ga12, from the Berkeley Yeast 
Stock Center). 35,000 independent colonies were grown on YEP-3% 
glycerol-2% ethanol and were screened by replica plating for the inability 
to grow on YEP-4% dextrose (Table 1). 
Identification of specific lesions was made by complementation tests with 
known glycolysis mutants (Ciriacy and Breitenbach (1979) J.Bacteriol. 
139:152-60), while at least 15 additional complementation groups were 
found by intercrossing mutant strains. Enzyme assays (Clifton et al. 
(1980) Genetics 88:1-11) confirmed the glycolytic defects in pgi1, pgk1, 
gpm1, pyk1, and gcr1 mutants. 
A LEU2 mutant was also derived from S. cerevisiae X2180-1A by EMS treatment 
and was crossed to X2180-1B (an isogenic MAT.alpha. strain) to produce 
N501-1B (MAT.alpha. leu2 SUC2 CUP1 ga12). Cycloheximide (cyh2) and 
canavanine (can1) resistances were then selected as spontaneous mutations 
in N501-1B. The glycolysis mutants were crossed to N501-1B to produce a 
series of isogenic leu2 strains each defective in a single glycolytic 
function or in GCR1. 
A tpil mutant, S. cerevisiae GLU77 was crossed to N551-1A (MATa leu2 SUC2 
CUP1 ga12); strains derived from this mating were crossed twice to N501-1B 
to produce a tpil leu2 strain, N587-2D, which was similar in genetic 
background to the other glycolysis mutants. 
Mutations in three glucose phosphorylating enzymes produce a strain which 
is unable to grow on dextrose as the sole carbon source and which is 
resistant to catabolite repression by 2-deoxyglucose and glucosamine. 
N517-6C (hxk1 hxk2 glk1 leu2 can1-100 cyh2 ade2-1) was derived from a hxk1 
hxk2 glk1 strains, D308.3, by screening for glucosamine-resistant spore 
colonies. Defects in glucose kinasing activities were confirmed by assay. 
TABLE 1 
______________________________________ 
Complementation Groups of glu.sup.- Derivatives of X2180-1A 
Gene No. of Mutants 
______________________________________ 
PYK1 14 
PDC1 9 
GCR1 4 
PGI1 3 
GPM1 3 
PGK1 1 
TPI1 0 
FDP 0 
(LEU2) (1) 
I 11 60 other mutations not in 
II 10 the complementation groups 
III 3 
IV 5 
V 1 
VI 1 
VII 2 
VIII 3 
IX 2 
X 3 
XI 2 
XII 1 
XIII 1 
XIV 5 
XV 1 
______________________________________ 
27 sterile glu.sup.- strains 
35,000 colonies screened (EMS mutagenized for 50% kill) 
The homothallic diploid strain, S. c. AB320 was the source of the yeast DNA 
pool (Nasmyth and Reed, Proc. Nat. Acad. Sci. (1980) 77:2119-2123 and was 
used as a control in some experiments. 
The triose phosphate isomerase gene (including the upstream sequence having 
the regulatory signals) is as follows: 
3 
##STR20## 
##STR21## 
##STR22## 
##STR23## 
##STR24## 
##STR25## 
##STR26## 
##STR27## 
##STR28## 
##STR29## 
##STR30## 
##STR31## 
##STR32## 
##STR33## 
##STR34## 
##STR35## 
##STR36## 
##STR37## 
##STR38## 
##STR39## 
##STR40## 
##STR41## 
##STR42## 
##STR43## 
##STR44## 
##STR45## 
##STR46## 
##STR47## 
The pyruvate kinase gene upstream sequence having the regulatory signals is 
as follows: 
__________________________________________________________________________ 
##STR48## 
##STR49## 
##STR50## 
##STR51## 
##STR52## 
##STR53## 
CTTAAGTCGT 
ACTATCGATG 
CATTTACACA AGGCGTGGCA 
GTGTTTCACA AAACATGACA 
##STR54## 
##STR55## 
##STR56## 
##STR57## 
##STR58## 
##STR59## 
AGAAAGAAGA 
AAGCAAGTAA 
GTCAACTCAA CTCACTCACG 
AAACAAGTTA CCTAGAATCG 
##STR60## 
##STR61## 
##STR62## 
##STR63## 
##STR64## 
##STR65## 
ATTTTACGTA 
TAAAAAAGAG 
AACCATTTAC TTACGAACAC 
TACAGAAGGT TCACTAAAGG 
##STR66## 
##STR67## 
##STR68## 
##STR69## 
##STR70## 
##STR71## 
AAAGGAAGGG 
TATACTACGA 
TCCATGGAAA TCACAGAAGG 
ATTTTTTTTT TTTTCCGAGC 
##STR72## 
##STR73## 
##STR74## 
##STR75## 
##STR76## 
##STR77## 
GGTAGTTTTG 
CTATAAGCAA 
CCGAAAAAAA AGACTTAATA 
TTTATGAGAA ACCATTGAAA 
##STR78## 
##STR79## 
##STR80## 
##STR81## 
##STR82## 
##STR83## 
AGTAAAGGTT 
CTTGGAGAAA 
AAAGGTCAAT ATAGTACCAG 
GGGAAAGTTT CAATAAGAGA 
##STR84## 
##STR85## 
##STR86## 
##STR87## 
##STR88## 
##STR89## 
TGAGAAAAAG 
TATAAGTAAG 
AAAAAGTAGG AAACCAAAAA 
ATAAGAATTG AACAAATAAT 
##STR90## 
##STR91## 
##STR92## 
##STR93## 
##STR94## 
##STR95## 
AAGAGAGAAC 
AAAGATAAAT 
GTTCTGTGGT TAGTTTTGTT 
TATTTTGTAG TAGTGTTACA 
##STR96## 
##STR97## 
##STR98## 
##STR99## 
##STR100## 
##STR101## 
GATCTAATCT 
TTCTAACTGG 
AGTAATTTGC AACAACGACC 
AAGACTGAAC TCTTCTTGGA 
##STR102## 
##STR103## 
##STR104## 
##STR105## 
##STR106## 
##STR107## 
GGTAGTAACC 
ATGGTAGCCA 
AGTTTCTGGT TGTTGGGTCT 
TTGGAACCAA CGAAACTCTT 
##STR108## 
##STR109## 
##STR110## 
##STR111## 
##STR112## 
##STR113## 
TCCGACCAAA 
CTTGTAACAA 
GCATACTTGA AGAGAGTGCC 
AACAATGCTT ATGGTGTTCA 
##STR114## 
##STR115## 
##STR116## 
##STR117## 
GACAGCAACT 
GTTGCGGTCT 
TTCAGGCTTC TTAACATGGG 
__________________________________________________________________________ 
Screening of clone bank. 
The leu2 glycolysis mutants were transformed with a yeast DNA pool inserted 
into pYE13, a high copy plasmid carrying a selectable LEU2 wild-type gene 
(Broach et al., Gene (1979) 8:121-133). The glycolytic genes were obtained 
by complementation, involving the simultaneous selection for growth on 
glucose and leucine prototrophy. A synthetic medium containing yeast 
nitrogen base, 4% glucose, and the following supplements was used: per 
liter, 40 mg adenine, 20 mg arginine, 50 mg aspartate, 10 mg histidine, 60 
mg isoleucine, 40 mg lysine, 10 mg methionine, 60 mg phenylalanine, 50 mg 
threonine, 40 mg tryptophan, 50 mg tyrosine, 20 mg uracil, and 60 mg 
valine. 
The transformants were purified on leucineless media and were then grown on 
a non-selective medium (YEPGE) to allow mitotic segregation of the 
plasmids. Strains which cosegregated the leu2 and glycolysis mutant 
phenotypes, as determined by replica plating on selective media, were 
assayed for glycolytic enzyme activities. Yeast DNA preps were made, and 
the E. coli strain, RR1, was transformed, selecting for ampicillin 
resistance, to verify the presence of plasmid DNAs in these yeast 
glycolytic transformants. 
Enzyme Assays. 
The transformed yeast strains were selectively grown on minimal medium with 
8% glucose (adenine was added to a final concentration of 50 mg/1). The 
wild-type control, N501-1B, was grown on the same medium plus leucine (100 
mg/1). The glycolysis mutant strains were grown on YEP-5% glycerol-1% 
lactate. Overnight cultures were fed fresh media and were aerobically 
grown at 30.degree. for four hours before harvesting. The cells were 
washed two times with water and resuspended in 50mM K.sub.2 HPO.sub.4 2mM 
EDTA 3mM 2-mercaptoethanol (adjusted to pH 7.4 with HCl). Extracts were 
obtained by vortexing the cells with an equal volume of glass beads (0.45 
mm diam.) at high speed for two minutes. The cell debris was removed by 
centrifugation in a microfuge for 15 min. at 4.degree.. Enzymes were 
assayed as described by Clifton and Breitenbach, supra. Protein 
concentrations were determined by the Biuret-TCA method. 
In order to determine the activity of the various glycolytic genes in the 
transformants, the various enzymes were assayed and the results for the 
transformants were compared to mutant and wild-type strains. The gcr1 
mutant had 5-10% of the wild-type levels of most glycolytic activities 
(exemplified by PGI, aldolase and enolase) and grows very poorly on 
glucose media. In contrast, the GCR1 transformants had nearly wild-type 
levels of enzymes and were virtually identical to wild-type for growth on 
glucose media. The other glycolysis mutants had less than 5% of the normal 
levels of their respective enzyme activities. However, when transformed 
with a complementing high copy plasmid, the specific enzyme activities 
were substantially elevated above wild-type levels (typically 5-10 fold 
higher). The following Table 2 indicates the results. 
TABLE 2 
______________________________________ 
Comparison of Glycolytic Activities in Wild-type, 
Mutant, and Transformed Strains 
Ratio: 
Enzyme Activities Transf/Wt 
______________________________________ 
Wild-type.sup.a 
Mutant.sup.b 
Transformant.sup.c 
PGI 2.85 .0065 31.49 (10) 
11.1 
TPI 18.3 .0000 167.8 (10) 
9.2 
PGK 1.99 .0046 17.67 (3) 8.9 
GPM 0.74 .0000 4.80 (10) 
6.5 
PYK 4.02 .0057 14.77 (10) 
3.7 
______________________________________ 
gcr1 
Wild-type.sup.a 
Mutant.sup.d 
GCR1 Transf.sup.c 
PGI 2.85 .2436 2.42 (10) .85 
Aldolase 
4.33 .4415 2.96 (10) .68 
Enolase 
0.43 .0274 .316 (10) .74 
______________________________________ 
.sup.a Wild-type is N5011B. 
.sup.b The respective mutant strains are N5439D (pgi1 leu2), N5872D (tpi1 
leu2), N5488A (pkg1 leu2), N5832C (gpm1 leu2), and N5493A (pyk1 leu2). 
.sup.c The activities of the transformants are averages for many differen 
isolates. The numbers in parentheses represent the numbers pf independent 
transformants assayed. 
.sup.d The gcr1 leu2 mutant strain is N5252C. 
In order to demonstrate that the hyperproduction of glycolytic enzymes was 
specific to the mutational defect complemented by the particular plasmid, 
assays for ten different glycolytic proteins were conducted on the various 
transformants. The following Table 3 reports the results for one 
transformant for each of the six different glycolysis genes which were 
examined in detail. 
TABLE 3 
__________________________________________________________________________ 
RELATIVE ENZYME ACTIVITIES OF WILD-TYPE AND TRANSFORMED STRAINS 
GLYCOLYTIC ENZYMES 
Strains GLK PGI 
PFK 
FBA 
TPI GLD PGK 
GPM ENO PYK 
__________________________________________________________________________ 
N501-1B 1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
Transformant GCR-8 
1.05 
0.63 
1.44 
0.79 
0.62 
0.63 
0.75 
0.56 
0.51 
1.36 
Transformant PGI-19 
0.64 
5.63 
1.26 
0.57 
0.58 
0.75 
0.51 
0.32 
0.54 
0.82 
Transformant TPI-10 
0.99 
0.77 
1.35 
0.99 
13.85 
0.87 
0.64 
1.01 
0.64 
1.14 
Transformant PGK-2 
0.54 
0.45 
1.05 
0.54 
0.46 
0.63 
2.99 
0.24 
0.43 
0.83 
Transformant GPM-2 
0.97 
0.82 
1.69 
1.02 
1.02 
0.85 
0.97 
12.75 
0.72 
2.00 
Transformant PYK-1 
1.02 
0.83 
1.09 
0.89 
1.22 
0.84 
1.23 
0.49 
0.85 
6.53 
__________________________________________________________________________ 
The GCR-8 transformant gave nearly wild-type levels of all ten enzymes, 
while PGI-19, TPI-10, PGK-2, GPM-2 and PYK-1 transformants overproduced 
their respective glycolytic proteins, but not other enzymes. 
It was noted that the plasmids readily segregated (typically 5-50% 
segregation in fully grown cultures even under selective pressure of 
leucine prototrophy, so the assayed cultures probably contain cells with a 
range of number of plasmids. By complementation in E. coli and/or 
sequencing, TPI1 and PYK1 have both been shown to be the structural gene. 
Exploitation of the promoter for TPI1 for the production of human 
.alpha..sub.1 -antitrypsin was demonstrated as follows. The plasmid CV13 
was employed. CV13 can be maintained by selection of yeast with an average 
of about ten copies per cell. CV13 is comprised of pBR322, the replicon 
for the 2.mu.-plasmid and the yeast LEU2 gene. TPI1 promoter fragment was 
obtained by cutting the TPI1 gene at the unique KpnI site (bases 511 to 
518); and the resulting linearized DNA was then treated with Ba131 for 
four to five minutes in order to remove the TPI1 structural sequences. 
Linkers, either EcoRI, Hind III or BamHI, were then inserted. The linkers 
will then cleave with the appropriate restriction enzyme to provide 
cohesive ends for insertion of human .alpha..sub.1 -antitrypsin genes. The 
human .alpha..sub.1 -antitrypsin gene was digested with BamHI, which 
cleaves at the 5'-terminus of the coding strand to remove the information 
for a single glutamic acid codon from the mature protein. Four different 
constructions were prepared, as set forth in the following Table 4. From 
this table it is noted that the glutamic acid codon is substituted by the 
codons for alanine and proline in three of the constructions having the 
initiator methionine. 
After ligation of the human .alpha..sub.1 -AT construction into the CV13 
plasmid, the resulting plasmid was transformed into S. c. N501-1B. The 
resulting yeast cells were then grown on a minimal synthetic medium. 
TABLE 4 
______________________________________ 
Plasmid N--terminal amino acid 
Orientation in CV13 
______________________________________ 
CAT1 met glu + hAT* clockwise 
C-T.alpha.2 
met ala pro + hAT 
counterclockwise 
C-T.alpha.1 
met ala pro + hAT 
clockwise 
C-TS.alpha.2 
met ala pro + hAT, but 
counterclockwise 
missing part of TPI 
promoter 
______________________________________ 
*remainder of approximately 400 amino acids of human .alpha..sub.1 
-antitrypsin 
Yeast cells containing the human .alpha..sub.1 -AT genes were broken open 
by vortexing with glass beads (0.45mm) at high speed for 2-3 minutes. The 
extraction buffer contained 50mM K.sub.2 HPO.sub.4, 2mM EDTA, 2mM 
2-mercaptoethanol and 1 mM PMSF (pH7.4) cell debris was removed by 
centrifugation and the extracts contain 3-4mg/ml protein as determined by 
Lowry assays. 
The presence of human .alpha..sub.1 -antitrypsin was determined using a 
RIA, employing tritium-labeled human .alpha..sub.1 -AT and antibody 
directed against the protein. The following Table 5 indicates the results. 
TABLE 5 
______________________________________ 
Competition assay for alpha-1 antitrypsin 
______________________________________ 
Tritium Average .alpha..sub.1 -AT 
Total % Total 
Plasmid 
Counts Count [.mu.g] 
Protein (.mu.g) 
Protein 
______________________________________ 
CAT1 46010 49133.5 
0.75 420 .18 
52257 
C-T.alpha.2 
12268 12799 3.35 380 .88 
13330 
C+ T.alpha.1 
41635 40353 0.95 360 .26 
39071 
C-TS.alpha.2 
66490 68264 0 345 0 
70038 
______________________________________ 
Controls** 
Counts 
______________________________________ 
0 .mu.g .alpha.-1 
68440 
0.25 .mu.g .alpha.-1 
65333 
0.5 .mu.g .alpha.-1 
58928 
1.0 .mu.g .alpha.-1 
38468 
2.0 .mu.g .alpha.-1 
19559 
3.0 .mu.g .alpha.-1 
14432 
4.0 .mu.g .alpha.-1 
11155 
5.0 .mu.g .alpha.-1 
9615 
______________________________________ 
*Plasmids were grown in yeast strain, N5011B. 100 .mu.l of extracts were 
assayed. 
**Non-radioactive alpha1 antitypsin mixed with 100 .mu.l of yeast extract 
(330 .mu.g protein) 
It is evident from the above results that an immunologically active product 
is obtained, which is capable of competing with naturally occurring human 
.alpha..sub.1 -AT for antibodies to the native protein. Furthermore, the 
expression of the .alpha..sub.1 -AT gene is regulated by the TPI promoter, 
for as is seen, where a portion of the TPI promoter is removed, no 
.alpha..sub.1 -AT is produced. In addition, the production of the 
mammalian protein human .alpha..sub.1 -AT has not been optimized in the 
above study, so that the results indicate a minimum production of product 
which can be further enhanced. Thus, the TPI promoter is found to be an 
effective promoter for efficiently producing high yields of expression 
products of alien DNA. 
It is evident from the above results that yeast promoters can be 
efficiently used for the production of foreign proteins by regulating the 
expression of alien DNA in yeast. The promoters are found to be strong 
promoters, so as to provide for a high degree of expression. Furthermore, 
it would appear that the messengers are sufficiently stable as to allow 
for a significant degree of translation into the desired expression 
product. Furthermore, by employing the glycolytic promoters and 
appropriate nutrient media, the expression of the alien DNA can be 
modulated. In this way, production of the alien DNA can be turned on and 
off. Thus, the subject invention provides a method for using yeast as 
efficient host in the production of foreign proteins, where the production 
may be modulated. In addition, by using the glycolytic regulation gene, 
one can turn on and off a plurality of glycolytic promoters. 
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 modification may be practiced 
within the scope of the appended claims.