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US00516976OA
United States Patent [19]
[11] Patent Number:
Wilcox
[45]
[54] METHOD, VECTORS, AND HOST CELLS
FOR THE CONTROL OF EXPRESSION OF
HETEROLOGOUS GENES FROM LAC
OPERATED PROMOTERS
[75] Inventor:
Edward R. Wilcox, Escondido, Calif.
[73] Assignee:
Mycogen Corporation, San Diego,
[21] Appl. No.: 386,821
[51]
[52]
Dec. 8, 1992
Chief, American Society for Microbiology, Washing
ton, D.C.
Freifelder, D., (1987), “Molecular Biology”, Jones and
Bartlett Publishers, Inc., Portola Valley, Calif.
Jobe, A., Bourgeois, S., (1972), “The Natural Inducer of
the lac Operon”, J. Mol. Biol., 69:397-408.
Muller-Hill, H. V. Rickenberger and K. Wallenfels,
(1964), “Speci?ecity of the Induction of the Enzymes of
the lad Operon in Escherichia coll", J. Mol. Biol.,
10:303-318.
Calif.
[22] Filed:
Date of Patent:
5,169,760
Jul. 27, 1989
Primary Examiner-David L. Lacey
Int. Cl.5 ..................... .. C12N 15/11; C12N 15/67
US. Cl. .............................. .. 435/691; 435/252.3;
435/320.1; 536/27
[58]
Field of Search ................... .. 435/691, 172.3, 39,
[56]
435/2523, 320.1; 536/27
References Cited
PUBLICATIONS
Gene, 51:255-267, 1987, Stark, Multicopy Expression
Vectors Carrying the lac Repressor Gene for Regulated
High-Level Expression of Genes in Escherichia coli.
Beckwith, J ., (1987), “The Lactose Operon", in Escher
ichia coli and Salmonella typhimurium Cellular and Mo
lecular Biology, vol. 2, Neidhardt, F. C., Editor in
Assistant Examiner—John D. Ulm
Attorney, Agent, or Firm—-S_aliwanchik 8t Saliwanchik
[57]
ABSTRACT
The invention is a process for controlling the expression
of heterologous genes from lac-operated promoters by
removing the CAP binding site and lac promoter from
the lac operon. Illustrated is the fusion of the lacZ, Y,
and A genes of the lac operon to the 3' end of the lacl
structural gene. This elimination of the natural regula
tory elements of the lac operon results, advantageously,
in the production of lac operon gene products in a con
stitutive mode from the lac] promoter.
16 Claims, 9 Drawing Sheets
U.S. Patent
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Stoneham, MA) is to have enough lac repressor present
METHOD, VECTORS, AND HOST CELLS FOR
THE CONTROL OF EXPRESSION OF
'
HETEROLOGOUS GENES FROM LAC
OPERATED PROMOTERS
BACKGROUND OF THE INVENTION
The lactose (lac) operon consists of three protein
products under the control of a lac promoter-operator.
These gene products are B-galactosidase (Z), permease
(Y), and thiogalactoside transacetylase (A). The protein
product of the lad transcript (repressor), an indepen
dent gene product, interacts with the operator of the lac
operon and keeps synthesis off until allolactose (1,6-0-[3
in the cell, so that transcription from the tac promotor
is off until IPTG or another proper inducing B-galacto
side is added to the cell. Although IPTG is the current
inducer of choice, it is expensive and has been labeled a
potential carcinogen. Thus, there is a need to replace
IPTG in commercial systems where control of the ex
pression of heterologous genes from lac operated pro
moters is used.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns an improved method
for controlling the expression of heterologous genes
from lac operated promoters. Speci?cally, the subject
D-galactopyranosyl-D-glucose), a product of the B
invention concerns a method for controlling the expres
galatosidase reaction, accumulates in the cell and binds
sion of heterologous genes from lac operated promoters
to the repressor. The allolactose repressor complex has
which comprises removing the CAP binding site and
a changed conformation, allowing the repressor to be
lac promoter/operator from the lac operon. The subject
displaced from the operator. RNA transcription then
invention is exempli?ed herein by use of a novel recom
begins from the lac promoter (Beckwith, J. [1987] In 20 binant DNA construct comprising the lacZ, Y, and A
Escherichia coli and Salmonella typhimurium Cellular
genes of the lac operon, to control the expression of
and Molecular Biology. Vol. 2, Neidhardt, F. C., Editor
heterologous genes from lac operated promoters. The
in Chief, American Society for Microbiology, Washing
ton, DC).
lacZ, Y, and A genes of the lac operon were fused to the
3’ end of the lacI structural gene, thereby eliminating all
A few molecules of the lac operon transcript are 25 of the natural regulatory elements of the lac operon. By
present in E. coli, even in the absence of lactose. Hence,
doing this, lac operon gene products are produced in a
permease and B-galactosidase are always present, at
constitutive mode from the lacI promoter and are not
least at a low level. Allolactose, the natural inducer of
responsive to catabolite repression or to allolactose
the operon, is made in the cell when lactose (1,4-O-B-D
galactopyanosyl-D-glucose) enters the cell by the per 30 induction. When lactose is added, the constitutively
synthesized B-galactosidase converts as much as 20% to
mease reaction and is converted through transgalactosi
allolactose, which in turn derepresses the tac promoter
dation by B-galactosidase into allolactose (Freifelder,
and
any associated heterologous gene. As long as the
D., [1987] Molecular Biology. Jones and Bartlett Publish
allolactose concentration in the cell is above 104M,
ers, Inc, Portola Valley, CA; Beckwith, supra). It was
calculated that greater than 20% of the lactose acted 35 the tac promoter provides high expression .of the pro
tein of interest. The complete DNA sequence of the
upon by B-galactosidase is converted to allolactose
transcriptional fusion of the lad gene to the lacZ, Y,
(Jobe, A., Bourgeois, S., [1972] J. Mol. Biol.
and A genes of the subject invention is as shown in
69:397-408). The majority of the remaining lactose is
converted to glucose and galactose. Allolactose is a
Table 1.
better substrate for B-galactosidase than lactose (Jobe 40 The novel lacIZYA operon (Table 1) can be inserted
in any plasmid, or it can be inserted into any microbial
and Bourgeois, supra), and is itself rapidly converted to
chromosome to improve control of heterologous gene
glucose and galactose.
expression from a lac operated promoter (as long as a
Another control element of the lac operon is catabo
promoter is present to drive lacIZYA transcription). It
lite repression. In the presence of glucose the cell is able
to repress many operons. For example the lac operon is 45 will be apparent to a person skilled in the art that other
constructs can be used to fuse the genes.
only transcribed at 2% of its maximum level in the
The lac operator, which has very high affinity for the
lac] repressor (KDNA=2—5>< 1013 M-l), is a sequence
21 hp long as follows; AATTGTGAGC
found to act as a very poor effector molecule. The 50 GGATAACAATT (Barkley, M. D. and S. Bourgeois
presence of glucose (Beckwith, supra).
When several B-galactosides were compared for their
ability to induce the lac operon in vivo, lactose was
synthetic non-metabolized B-galactoside, isopropyl-B
D-thiogalactoside (IPTG), was found to induce the
lactose operon 5 times better than lactose (Monod, J.,
G. Cohen-Bazire and M. Cohn [1951] Biochirn. Bio
phys. Acta. 7:585-599). Yet, allolactose itself is as good 55
an inducer of the operon as IPTG (Muller-Hill, B., H.
V. Rickenberg and K. Wallenfels [1964] J. Mol. Biol.‘
10:303-318; Jobe and Bourgeois, supra).
Given the efficiency of lactose conversion to allolac
[1978] In The Operon Miller, J. H. and W. S. Reznikoff,
eds. Cold Spring Harbor). Many mutations within the
lac operator exist which have higher or lower affinities
to the lad repressor (Barkley and Bourgeois, supra;
Sadler, J. R. et al. [1983] Proc. Natl. Acad. Sci. USA
80:6785-6789; Simons, A. et al. [1984] Proc. Natl. Acad.
Sci. USA 81:1624-1628). Many mutations exist within
the lad repressor that allow for greater affinity to the
lac operator with no significant effect on derepression
tose, one might expect lactose to work very well as an
(Barkley and Bourgeois, supra). Using any of the many
inducer of the lac operon. However, prior to the present
disclosure, this has never occurred in practice (Monod
et al., supra).
The current way to control the expression of heterol
different combinations of lac operator and lacI repres
sor, the control of heterologous gene expression is ap
parent to a person skilled in the art. Many different
promoters (eukaryotic as well as prokaryotic) have
ogous genes from the lac promoter or lac concensus 65 been placed under lac operator regulation (Yansura, D.
promoters such as tac (Rezinkoff, W. S. and W. R.
G. and D. J. Henner [1984] Proc. Natl. Acad. Sci. USA
McClure [1986] Maximizing Gene Expression, W. Rez
81:439-443; Herrin Jr., G. L. and G. N. Bennett [1984]
nikoff and L. Gold, eds., Butterworth Publishers,
Gene 321349-356; Deuschle, U., et al. [1986] Proc. Natl.
3
5,169,760
4
HindIII fragment (at the 3' end of the coleopteran
active sequence). The 5' end of the tac promoter had
been previously inserted by blunt-end ligation into the
EcoRI site of pBR322.
Acad. Sci. USA 83:4134-4137; Hu, M. C-T. and N.
Davidson [1988] Gene 62:301-314; Figge, J., et al.
[1988] Cell 52:713-722). The subject invention is de
signed to improve lactose induction from such lac oper
ated promoters.
This invention enables cells to make enough lacZ and
DETAILED DISCLOSURE OF THE INVENTION
Upon removal of the CAP binding site and lac
glucose and galactose, as well as to the true lac inducer,
promoter/operator from the lac operon, there is real
allolactose. In addition, the invention enables the cell to
ized an improved control of expression of a heterolo
synthesize enough repressor (lacl protein) to bind to the 10 gous gene. Exempli?ed herein is the insertion of a lacl
lac operator upstream from a heterologous gene, and
ZYA fusion construct into a plasmid containing a tac
Y protein to ef?ciently take up lactose and convert it to
keep heterologous gene expression off in the absence of
promoted gene encoding a lepidopteran insect toxin.
inducer. With excess lactose present in the medium, the
cell can accumulate suf?cient concentrations of allolac
tose for ef?cient derepression of the lac operator.
The plasmid (pMYC47l) was then used to transform
Pseudomonas?uorescens, which was designated Pseudo
monas?uorescens MR47l (pMYC471).
One element of plasmid pMYC47l provides its host
BRIEF DESCRIPTION OF THE DRAWINGS
with constitutive levels of lac operon gene products.
FIG. l-pMYC 2005, LacI and Z operon fusion
For this purpose, the operon could be fused to any of a
A synthetic fragment of DNA was cloned into
variety of available constitutive promoters. In this in
pUCl8 to replace the normal sequence found at Hind 20 stance, it was fused to the lacl promoter. In addition, a
111-403 to HaeII-524. This synthetic sequence removes
new construct containing a lacIQ promoter, instead of
the lac promoter and operator sequences and replaces
the lacl promoter, was made (pMYC‘ASS). This new
them with a Shine-Dalgarno site.
construct provides higher levels of the repressor as well
FIGS. 2a to d-pMYC 2101, construction of the lacI
as
of lac operon gene products. The plasmid,
ZYA operon
25
pMYC485, functions like pMYC471.
Three fragments of DNA were ligated and then in
As a demonstration that lactose inducibility is not
troduced by transformation into MC1061 (Casadaban,
dependent on the speci?c gene under tac promoter
M. and S. Cohen [1980] J. Mol. Biol. 138:179-207) to
control, another gene encoding a coleopteran-active
construct the plasmid pMYC2lOl. The ?rst fragment
came from pMYCZOOS as a HindIII-403 to Eael-485 30 toxin was cloned into the pMYC47l vector replacing
the lepidopteran-active toxin gene, resulting in
piece, the second fragment came from pMC9 as an
pMYCl 161. Pseudomonas?uorescens strain MBlOl car
EcoRI-l to EaeI-ll03 piece and the ?nal fragment
ryin g this new plasmid also permits lactose induction of
comes from pSKS107 as a 9889 bp HindlIl-3l to
toxin expression. This will hold true for any gene under
EcoRl-l piece. Once constructed, the EcoRI and SalI
ends were converted to BamHI and BglII ends, respec 35 control of a lac operated promoter.
Another aspect of this fusion is that the lac operon is
tively, using oligonucleotide linkers, creating
no longer regulated by the normal control elements
pMYCZlOl-B.
associated with lactose. This means that synthesis of lac
FIG. 3 -pMYC467, a tac promoted toxin-containing
operon ZYA gene products in these constructs
plasmid
The tac promoted toxin gene found in pMYC436 40 (pMYC47l, pMYC485, and pMYC1l6l) is constitutive
and occurs independently of the concentrations of lac
(NRRL deposit no. B-l8292) was cloned as a 4.5 Kbp
tose or glucose present in the cell. For example, in the
BamHI to PstI fragment in the vector pTJ S260. Not all
presence or absence of IPTG or lactose, a new protein
the sequence of pTJS260 or of the 3' ?anking sequences
band corresponding to the mass of B-galactosidase is
of the toxin genes are available, hence some restriction
sites are approximated in FIGS. 3, 4, 5 and 6.
45 apparent on SDS-PAGE and the clones cleave X-gal,
forming blue colonies under the same conditions.
FIG. 4 -pMYC47l, a lactose inducible plasmid
For ease in construction, the lacA gene has been
The lacIZYA operon was cloned into the BamHI site
included on all the plasmids contining lacIZYA men
of pMYC467. The resulting plasmid, pMYC471, was
tioned in this disclosure. The lacA gene product has no
then induced by transformation into MBlOl, a P. ?uo
rescens.
50
FIG. 5-pMYC485, an alternate lactose inducible plas
mid
A lacIQ ZYA operon was constructed by replacing
lacl with lacIQ as a 638 bp BamHI to ApaI fragment.
role (Freifelder, supra) in production of allolactose and
can easily be removed from the lacIZYA operon. For
example, there is a nonunique AflII (CTTAAG) restric
tion enzyme site a the stop codon of the lacY gene. By
using this AflII restriction site, it is possible to remove
The lacIQZYA operon was then cloned into the BamHI 55 the lacA gene. The lacIZY operon has the same lactose
induction properties as those found in the lacIZYA
operon.
was tested in M13101 for lactose inducibility. The lacIQ
The data of Table 2 demonstrate toxin production is
ZYA operon is shown in Table 1 wherein base 16 is a T
instead of a C as shown in the table.
regulated by lactose or IPTG. Although the amount of
lactose used for induction is 10 to 20 fold higher than
FIG. 6-pMYCll6l, a coleopteran toxin-lactose in- V
site of pMYC467. The resulting plasmid, pMYC485,
ducible plasmid
IPTG induction levels, the signi?cantly reduced cost of
The plasmid pMYC471 was cut with BamHI (at posi
tion 12103) and KpnI (at position 7933) to remove the
tac-promoted lepidopteran-active toxin and then both
ends were ?lled in with T4 DNA polymerase, resulting 65
lactose ($7.00 per kilo, Sigma Chemical Co.) compared
to IPTG ($20,000 per kilo, Sigma Chemical Co.) make
the former inducer highly economically advantageous.
in blunt termini. The tac promoted toxin gene disclosed
in US. Pat. No. 4,771,131, was inserted into the above
DNA as a blunt-ended Seal (of pBR322) to ?lled-in
strate, lactose, must be replenished during cell growth
For maximum production levels the metabolized sub
or the culture will decrease the synthesis of the tac-pro
moted gene.
5,169,760
5
6
380A oligonucleotide synthesizer. This sequence starts
Materials and Methods
at BP 1073 of the lacl sequence (Farabaugh, P. J ., supra)
Cloning and DNA manipulation techniques are de
as seen in Table l.
Hae 11
Bee 1
CC
CGCGGG
AAT
TTA
ACG
TGC
CAA
GT1‘
ACC
TGG
GCC
CGG
TCT
AGA
CCC
GGG
CGC
GCG
GCG
CGC
T'I'G
AAC
GCC
CGG
GAT
CTA
AGA
TCT
CTG
GAC
GAA
CTT
AGC
TCG
..Xh_o1
TCA
AGT
TTA
AAT
ATG
TAC
CAA
GTT
CT C
GAG
GCA
CGT
CGA
GCT
CAG
GTC
la_cl
CAG
GTC
TGA
ACT
TCG
AGC
lacZ
Stop‘
666
CCC
GTC
CAG
S/D
GCGCTAGGAGGTAACTI'
CGCGATCCTCCATTGAA
scribed in Maniatis, T., E. F. Fritsch and J. Sambrook 20
(1982) Molecular Cloning, a Laboratory Manual. Cold
Spring Harbor Laboratory Publishers, Cold Spring
Harbor, NY.
A plasmid containing the lac operon, pSKSl07, was
received from Dr. M. Casadaban at the University of
Chicago (Shapira, S. K., J. Chou, P. V. Richaud and M.
J. Casadaban [1983] Gene 25:71-82). The sequence of
the lac operon is published (Kalnins, A., K. Otto, U.
Ruther and B. Muller-Hill [1983] EMBO .1. 2:593-597;
Hediger, M. A., D. F. Johnson, D. P. Nierlich and I.
Zabin [1985] Pro. Nat. Acad. Sci. 82:6414-6418). The
lad gene, on a 1.7 KB EcoRl restriction fragment
cloned into pBR322, is available from the ATCC, as
v‘pMC9, catalogue no. 37195, 37196 and 37197. The se
quence of the lac] gene is also published (Farabaugh, P.
J. [1978] Nature 274:765-769). The broad host range
vector, pMMB22, was received from Dr. M. Bag
dasarian (Bagdasarian, M. M., E. Amann, R. Lurz, B.
Ruckert and M. Bagdasarian [1983] Gene 26:273-282).
A Hindlll fragment of pMMB22,v containing the lacIQ
gene, was relinkered to BamHl. The sequence of the
lacIQ gene has also been published (Calos, M. P. [1978]
Nature 274:762-765). Analysis here is largely based on
the published DNA sequences. Some of the critical
portions of the constructs were con?rmed by DNA
sequencing, e.g., the lacl and laclQ promoters and the
fusion region between the lacl transcriptional unit and
the lac operon. The vector, pUCl8, is available from a
variety of sources, such as catalogue no. 27-4949-01 of
Pharmacia Corporation. The broad host range vector,
pTJS260, is available from Dr. D. R. Helinski at the
University of California, San Diego, La Jolla CA._92093
(Schmidhauser, T. J. [1986] Ph.D Thesis, UCSD).
Following are examples which illustrate procedures,
including the best mode, for practicing the invention.
These examples should not be construed as limiting. All
percentages are by weight and all solvent mixture pro
portions are by volume unless otherwise noted.
EXAMPLE 1
Fusions of the LacI Gene and Lac Operon.
Fusions of the lacI gene and lac operon were ?rst
carried out in pUCl8. To do so, pUCl8 was linearized
with Hindlll and then partially cut with HaeII. The
l;1i_nd III
Start
ATG
TAC
GAA
C l l ICGA
The above synthetic sequence removes the PvuII site
normally found at BP 1123 of lacI. A new XhoI site was
inserted near the 3' end of the lacl gene (BP 1137).
These changes were introduced for ease in identi?ca
tion of the new construct. No amino acid changes
would occur as a result of the mutations introduced in
making the base pair changes for the above two restric
tion sites. In this construct, the distance between the
stop codon of lacl and the start codon of lacZ is 17 base
pairs. This region contains a ribosome binding site
(marked as S/D) such that a ribosome translating the
lacI transcript will be able to continue synthesis of the
lacZ gene product from the same transcript. A plasmid
diagram of pMYC2005 is seen in FIG. 1.
EXAMPLE 2
Con?rmation of Sequence.
The synthetic portion of pMYC2005 was sequenced
to validate its structure and was used in a three~piece
ligation to tie the full lacl gene of pMC9 to the lac
operon found in pSKSl07 (FIGS. 20 to d). After trans
formation, the lacI/Z fusion region was sequenced from
a blue colony on an LB+X-gal (5-bromo-4-chloro-3
indolyl~B-D-galactosidase, a colorimetric substrate for
the presence of B-galactosidase) plate. Those restriction
sites that were examined yielded fragments of the pre
dicted sized (FIGS. 20 to d). The lacl promoter region
was also con?rmed by sequence analysis.
EXAMPLE 3
Cloning
The EcoRI and Sall ends of the lacIZYA construct
(FIGS. 2a to d) were respectively relinkered to yield
BamHI and BglII restriction sites. The laclZYA operon
was cloned into the unique BamHI site of pMYOi67
(FIG. 3) yielding pMYC471 (FIG. 4). The plasmid,
pMYC467, contains the tac promoted lepidopteran
toxin gene of pMYC436 (a cryIA(c)-like toxin gene
[NRRL deposit no. B48292] Adang, M. J. et. al. [1985]
Gene 36:289-300) cloned into the broad host range
vector, pTJS260. pMYC471 was introduced by trans
formation into P. ?ourescens MBlOl and the resulting
clone was designated MR471. MR471 was tested for the
key elements of the resident pMYC47l plasmid. A func
DNA was resolved by electrophoresis on a 1.4% aga
rose gel. The 2402 bp band was eluted and ligated to a
tional repressor is synthesized because in the absence of
double-stranded synthetic DNA insert produced by
B-cyanoester chemistry on an Applied Biosystems
duced (Table l). The cells are blue when plated on
IPTG or lactose no signi?cant amount of toxin is pro
X-gal becausea functional B-galactosidase is present. A
5,169,760
11
12
TABLE l-continued
6801
6851
6901
6951
7001
7051
GGCACCAATC
AACCGCCG'IT
GTGAATACCA
CTCGCGGGCG
GCGCCACCAG
AGGGCGTTAT
TCAATATAGA
AATCAGACCG
CGCGAACCGG
CTITGCCACC
GCGAGTGTTT
GGCGGCACCA
' 710]
CACAGCCCCA
TCACCAGTGG
CGTGCGCTGC
TGAAACCGCC
GT'ITAATCAC
CCGCCTGAAT
AGCAGCGCAC
CAGCAACAGA
GCCAGCTTCC
Sall site in pSKSlO7
GTCGAC
GATGCCGATC
'I'TTGCGGGTA
CT GATGGCGA
GGCCAGCGCC
CCCACCCCAC
AAGCTCACGC
CACTGCGACG
AGCCCGCCCA
CAAGCAGTGC
ATCAATGCAC
'ITCGCTGACA
TGGTAACCAC
7151 CGAAGCATCA
7201 GCTGCTAAGC
7251 CGACGGCAAT
7301 TGCTGATGAA
7351
CGGCAGAGCG
AAGCGGGTAA
AAGTAAACAC
AGTGGTTGTT
ACCAGGCAAA
GATACCAGGT
AGCCCACCGC
CCAGGCAATC
CCAGCGTCCA
GTCTTGTGGG
GAGCGCAACA
'ITCGCTATGT
CGCCCATCAG
AGGCTGGCGT
CGCGCGGGGA
AAGAGGCGAC
ACGGCAGGCA
TGAACTAACC
AGCCGCGGAC
(l) sequence in bold. between the two ' marks. is synthetic DNA used to fuse the lac operon to the lac l or lac I9 gene operon.
(2) pSKSl07 contains an unsequenced mutation at the EcoRl site normally found in the lac Z gene.
TABLE 2
Lactose lnducibility of Various Constructs
Hours after“)
Lactose
Toxinm
Induction
(mM)
Cells/ml
(ug/ml)
40
40 no lactose
15
24
15
24
23(3)
390)
O
2 mM IPTG
20
20
40
40
8.314)
8.314)
24
24
4O
0
24
24
4o
0
MR471
1.6 X 10‘0
2.3 x l0l0
9.1 X to9
1.2 >< 1010
1.8 X 1010
1.1 X 1010
ZYA or laclQZYA operon wherein the CAP binding
site and lac promoter/operator is not present in the lac
operon.
20
none detected
1003
1011
737
887
1025
1.9 x 1010
2.0 X 1010
6. A transfer vector comprising a lacIZYA or lacIQ'
ZYA operon wherein the CAP binding site and lac
promoter/operator is not present in the lac operon.
7. A microbial host transformed by the transfer vec
tor of claim 6.
25
938
1555
8. The transfer vector, according to claim 6, being a
plasmid.
MBlOl containing pMYC485
9. A process for preparing a protein product which
comprises culturing a microbe comprising a heterolo
—
-
from a lac promoter, and a lacIZYA or lacIQZYA op
gous gene encoding said product which is expressed
775
none detected
MBlOl containing pMYCl 161
—
eron wherein the CAP binding site and lac promoter
/operator is not present in the lac operon, until a suffi
300
none detected
cient amount of desired product is produced.
10. A process for controlling the expression of a het
erologous gene from a lac operated promoter which
comprises culturing a microbe comprising a heterolo
gous gene expressed from a lac operated promoter, and
(“Cultures were induced upon reaching stattonary phase.
(“Toxin concentration was determined by laser densitometr) of Coomassie-stained
protein bands after electrophoresis of disrupted cells on SDS-PAGE (LKB lnstruc
ttonal Manual 222-010).
(“This experiment used MR471 grown in a 10L fennentor. All other experimental
data were generated using same medium in 250 ml ba?led shake ?asks.
min the ferrnentor. 8.3 mM lactose/hour was fed into the culture. It was found that
MR4?! did not metabolize this lactose level in a fermentor. resulting in increased
concentrations during the experiment. The experiments done in shake flasks were
site and lac promoter/operator is not present in the lac
gt\ en a single dose of lactose or lPTG at the indicated times.
operon.
a lacIZY or lacIQZY operon wherein the CAP binding
11. The process, according to claim 10, wherein said
microbe has been transformed with a plasmid compris
ing a heterologous gene.
12. The process according to claim 10, wherein said
ability to control expression of heterologous genes from 45 microbe comprises a chromosome comprising said
lac operated promoters as does the parent operon.
lacIZY or lacIQZY operon wherein the CAP binding
2. A novel laclQ ZYA operon having the sequence
site and lac promoter/operator is not present in the lac
I claim:
1. A novel lacl ZYA operon having the sequence
shown in Table l, or mutations thereof which retain the
shown in Table 1, wherein base 16 is a T instead of a C
as shown in the table, or mutations thereof which retain
operon.
13. A transfer vector comprising a lacIZY or la
the ability to control expression of heterologous genes
cIQZY operon wherein the CAP binding site and lac
promoter/operator is not present in the lac operon.
14. A microbial host transformed by the transfer vec
from lac operated promoters as does the parentoperon.
3. A process for controlling the expression of a heter
ologous gene from a lac operated promoter which com
tor of claim 13.
prises culturing a microbe comprising a heterologous
15. The transfer vector, according to claim 13, being
gene expressed from a lac operated promoter, and a 55 a plasmid.
lacIZYA or lacIQZYA operon wherein the CAP bind
16. A process for preparing a protein product which
ing site and lac promoter/operator is not present in the
comprises culturing a microbe comprising a heterolo
lac operon.
gous gene encoding said product which is expressed
4. The process, according to claim 3, wherein said
from a lac promoter, and a laclZY or lacIQZY operon
microbe has been transformed with a plasmid compris 60 wherein the CAP binding site and lac promoter/opera
ing the heterologous gene.
‘tor is not present in the lac operon, until a sufficient
5. The process according'to claim 3, wherein said
amount of desired product is produced.
microbe comprises a chromosome comprising a lacl
it
65
t
it
t
it