Note: Descriptions are shown in the official language in which they were submitted.
--..
~' 2 0 1 5 0_!~ 6
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D a s c r i p t i o n
Recombinant DNA and Expression Vector Derived from
the pfl Gene
The invention concerns recombinant DNA and expression
vectors, processes for the production of such
recombinant DNA and expression vectors, as well as their
use for the inducible and repressible expression of a
foreign gene.
An important aim of applied genetic engineering is the
production of proteins from recombinant DNA. A special
class of vectors, the so-called expression vectors, are
necessary for this. These have not only the structural
requirements for the cloning, the transfer and the
multiplication of the recombinant DNA, but also for the
expression of the protein. These recombinant DNA
molecules contain special regulation sequences for this,
the promoters, which effect the transcription of the DNA
sequence into RNA, the translation of which by the
ribosomes leads to the finished protein.
DNA regions, to which bacterial RNA polymerase binds,
for the transcription of one or more genes are
designated promoters. Many of these promoters have
structures in common which are presumed to be important,
inter alia, for interactions with particular proteins.
Such interactions with cellular proteins or other
molecules can cause a repression, or also an induction,
of the activity of a promoter. An example of this is the
interaction of the lambda promoter P1 with the lambda
repressor cI.
2015p46
,~.,
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In the production of proteins by genetic engineering, it
is particularly advantageous if a promoter present in an
expression vector can be regulated by the presence or
addition of a repressor or inducer.
This regulation can be effected by suppressing the
activity of the promoter at the beginning of the
fermentation so that a large biomass can be produced
with only minimal impairment of the vitality of the
cells. Subsequently, the promoter is stimulated by
suitable means and the synthesis of the product can then
take place. Thus the fermentation process may be
basically divided into a growth phase and a production
phase.
The PL promoter of the bacteriophage lambda, the lac
promoter, the trp promoter, the tac promoter, the trc
promoter and the rac promoter may, for example, be used
for the controllable gene expression.
These expression systems are, however, only of limited
suitability for a large-scale technical application.
Above all, a temperature increase to 42°C, which is
necessary for the induction of the lambda PL promoter,
is very difficult in a technical application with
volumes of more than 50 1. In addition, it has been
shown that the induction of the lambda PL promoter has
to take place at an early stage of the growth phase and
it is therefore possible that not enough biomass is
obtained for a large-scale production.
When using the lac promoter for expression vectors it is
not possible, in contrast to the PL promoter, to
completely repress the system using a copy of the
~..~
20 150'6
- 3 -
repressor gene because the repressor is titrated by the
copy number of the lac operator. Although the repression
can be re-established by the use of repressor over-
producers, such strains are only partially inducible. If
the expression vector does not contain the corresponding
repressor gene then one is limited in the selection of
host strains for lambda PL, for lac and the other
derivatives of the lac promoter. Moreover, the addition
of inducers during the fermentation, which is necessary
in these systems, is not only expensive but also causes
fundamental difficulties, above all if inducers which
can be metabolized, e.g. lactose, are concerned.
The trp promoter is also a system which cannot be
completely repressed. The addition of tryptophan for the
repression also considerably increases the cost of the
fermentation as is the case when using inducers.
The inducers known up to now are therefore only of
limited suitability for an industrial application since
these inducers are in general very expensive and the
methods used for induction or repression are complicated
and are only of limited suitability for the regulation.
of the repression of a protein.
A promoter which can be repressed by oxygen is known
from German Patent Application DE-A 37 10 633, published
on November 10, 1988, which originates from the fdhF
gene and can be induced by formate. With this a simple
repression and induction is indeed possible; however, it
is a relatively weak promoter.
20 1 5p4 6
f~'',. _ 4 _
The Figures show:
Fig. 1: A) Section from the plasmid p29:
Complete~regulatory region and pfl
structural gene with the adjoining
terminators.
fnr: binding site for the fnr gene
product: tr: transcription start;
t: terminator.
B) Section from the plasmid p29:
The most important cleavage sites for
restriction~enzymes are marked.
Fig. 2: Construction of the M13 derivative
Ml3pec23. (Explanation in the text).
Fig. 3: Diagrams of the deletion mutagenesis for
coupling the creatinase gene to the pfl.
promoter; shown for Ml3pec23S as an
example.
Creatinase sequences are shaded; the EcoK
cassette is represented by the black bar.
(Explanation in the text).
Fig. 4: Summary of the pfl-creatinase fusions: The
diagrams each show a section from the
plasmids pPFL23S-C, pPFL23A-C and
pPFL39-C.
SD: Shine-Dalgarno region.
20 ~5a~s
Fig. 5: Recloning of the the pfl-creatinase
fusions from M13 in the plasmid pGH-C,
shown for M13pc23S as an example.
Fig. G: A) Plasmid map of pBTac1
B) Plasmid map of pBT2a-1
C) Plasmid map of pBTdtac
D) Plasmid map of pGH-C
Fig. 7: A) Plasmid map of pRS552
B) Plasmid map'of pRS551
Fig. 8: Construction of plasmid pRM23:
translational coupling of the complete pfl
promoter fragment to lacZ.
B: BamHI; E: EcoRI; S: SaII: H: HindIII:
M: MluI; .
P: PstI; Pv: PvuI.
Fig. 9: Kinetics of a fermentation run: FM420
(pPFL23S-C). Fermenter: Bioflo Ifs (New
Brunswick);
Filled volume: 4.0 1; stirring rate:
constant at 300 rpm: aeration: constant at
4.0 1/min;
the curves show:
- time-course of growth (cell density as
log OD600);
- expression of the creatinase gene at an
increasing deficiency of oxygen (volume
activity: log units/ml medium);
* Trademark
20150;4_8
,~ _
- Decrease in the content of dissolved
oxygen (%) during the course of the
growth.
Fig. 10: Sequence of the pfl promoter region.
The start codon of the pfl structural gene
is underlined; the position of the first
nucleotide (A of the ATG) is numbered +1.
Fig. 11: Promoter regions 1 - 7
It is therefore the object of this invention to
provide recombinant DNA and expression vectors which
enable a regulation of the expression and synthesis
of a desired gene product in a simple manner as well
as a particularly high expression rate of the gene.
CA 02015046 2002-02-20
This object is achi~~~~ed by a recombinant DNA which is
characterized in that: it contains:
a) a regulator rE~gion which is at least 50%
homologous t.o the sections -969 to -991 base
pairs and/or --1308 to --1330 base pairs of the
sequence of Figure 10 and
b) a promoter region i:n the 3' direction from the
regulation secxuence which has a -35/-10 promoter-
consensus sequence (Rosenberg, M. and Court, I7.
(1979) Ann. Rf=_v. Genet. 13:319-353).
Still in accordance with the present invention, there is
provided a recombinant. DNA comprising:
a) a regulator region, said regulator region
containing at least one of a sequence
selected l:rom
' -GAGATA7.'GATCTATATCAATTTC- 3 ' ( I ) ,
5 ' - CTGGGCAAAATAAAATCAA.ATAG- 3 ' ( I I ) ,
a functional variant of_ sequence ( I ) and a
functiona:~. variant of sequence (II), wherein
the sequence of said functional variant of
sequence (I) is at least 50% homologous to
that of sF~quence ( I ) and wherein said
sequence of= said functional variant of
sequence (II) is at least 50% homologous t.o
that of sequence (II); and
b) in the 3' direction from said regulator
region, at least. one promoter region, wherein
said prornoter region contains a -35/-10 pro-
moter seqoience .
CA 02015046 2002-02-20
'7 a -
Further in accordance with the present invention, there
is provided a recombinant L>NA, ~~omprising:
a gene to be expressed, wherein said gene is dif-
ferent from a pfl gene;
a promoter reunion upstream from said gene to be
expressed, wherein said promoter region contains a -35/-
promoter sequence; and
a regulator region, which regulates the
expression of said gene, upstream from said promoter
region, wherein sai~~ regulator region contains sequence
(I)
5'-GAGATATGAT~~TATATCAATTTC--3'
or a 23 base pair sec[uence which is identical at posi-
tions 6-10 and 15-19 to sequence (I) or a 23 base pair
sequence which diffez~s from sequence (I) at positions 6-
10 and 15-19 by two nucleotides or less.
In one embodiment, the regulator region may further
comprise a sequence which is identical to a sequence
(II)
5 ' -CT~3C-~GCAAAATAAAATCAAATAG- 3 '
operably linked to sequence (I) or a 23 base pair
sequence which is idEentical at positions 6-10 and 15-19
to sequence (I) or a 23 base pair sequence which differs
from sequence (I) at positions 6-10 and 15-19 by two
nucleotides or less.
CA 02015046 2002-02-20
- 7b -
In a preferred embodz.ment of the invention the regulator
region is at least 5~» homologous to the above-mentioned
sequences in Figure :1.d.
The numbering of the bases in this connection relates to
the ATG start codon c>f the pfl gene as +1 (for adenine)
which is underlined in Figure 10. Nucleotides which are
on the 5' side of this have negative numbers.
In a further preferred embodiment of the invention the
recombinant DNA contains in addition at least a third
2015p~6
sequence which is at least 80 % homologous to the
following consensus sequence:
c t
TATTTG AT AA
c -
This third sequence can thereby be included once or
several fold in the recombinant DNA.
Suitable promoters of the promoter region of the
recombinant DNA according to the present invention are
all promoters which contain a consensus sequence as
defined above in the -35/-10 region. These are, e.g. the
lac promoter, lambda PL promoter, trp promoter, mgl
promoter (European Patent Application EP-A 0 285 152,
published on October 5, 1988) or the promoters from Figure
11 or 50 $, and preferably 65 % homologues thereof.
A sequence is preferably used as the promoter region
which is 50 % and particularly preferably 65
homologous to one of the sequences shown in Figure 11.
In an especially preferred embodiment at least one of
the promoters from transcript 6 and transcript 7 is used
which have the sequences shown in Figure 11.
A>further embodiment of the invention is an expression
vector which contains a recombinant DNA according to the
present invention ligated into a suitable vector.
These DNA sequences of the recombinant DNA sequence
according to the present invention are located in the
expression vector, according to the present invention,
2015048
_ g _
upstream (i.e. on the 5' side) of the transcription
start of the gene to ~be expressed which is controlled by
this promoter whereby an ATG codon, and preferably also
a Shine-Dalgarno sequence, is located between promoter
and the gene which is to be expressed.
In a preferred embodiment of the invention the
expression vector according to the present invention
contains a polylinker or a single restriction cleavage
site, i.e. a restriction cleavage site which is present
only once in the expression vector, at the site at which
the foreign gene to be expressed is to be inserted.
In a further preferred embodiment the PFL gene or parts
of it and, if desired, untranslated upstream regions of
the PFL gene which contain e.g. the ATG codon and the
Shine-Dalgarno sequence, are present between the
promoter region and the foreign gene to be expressed. It
is particularly preferable to use the sequence shown in
Figure 10 for this, especially the entire sequence. In
another preferred embodiment the expression vector
contains the recombinant DNA according to the present
invention, untranslated sequences of the upstream region
of the PFL gene of Figure 10 as well as the Shine-
Dalgarno sequence and ATG of the foreign gene and, if
desired, already the foreign gene itself. It is,
however, also possible, according to the present
invention, to couple the foreign gene including its
start codon directly to the recombinant DNA according to
the present invention.
A further embodiment of the invention is a process for
the production of a recombinant DNA according to the
present invention in which the PFL gene together with
its upstream regions is isolated from the gene bank of a
20 ~5a~~s
-~o-
microorganism containing this gene, if desired, the
parts which are not required are removed by well-known
methods, and the desired sequences are combined with a
promoter region or third sequences.
The ligation, restriction and deletion of DNA sequences
is carried out according to the usual methods for this
purpose.
In a preferred embodiment of the invention the DNA
sequence is isolated from the gene bank of a
microorganism of the Enterobacteriaceae family and
preferably from E.coli.
Another further embodiment of the invention is a process
for the production of an expression vector according to
the present invention. For this, the recombinant DNA
according to the present invention and, if desired, also
a polylinker, a Shine-Dalgarno sequence, a start codon
and/or further desired sequences are inserted into a
suitable vector. Suitable vector molecules for this are
known to the expert e.g. pBR322 or derivatives thereof.
The use, according to the present invention, of a
recombinant DNA or expression vector as described above
for the inducible and repressible expression of a
foreign gene is characterized in that the induction is
effected under anaerobic conditions and by pyruvate and
the repression is effected by oxygen.
In this process the expression can be carried out in
suitable microorganisms of the Enterobacteriaceae genus
such as preferably E.coli and Salmonella, or other gram-
2 0 ~ 5 0- ~~ s
negative bacteria such as preferably Pseudomonas, or in
gram-positive bacteria.
The expression is preferably carried out in a host
strain which is FNR-positive and which thus forms a
functional FNR gene product. This is preferably E.coli
FM 420 which is deposited at the German Collection for
Microorganisms, DSM 5312.
The FNR protein (Stewart, V. Microbiol. Rev. 52 (1988)
190-232), which is produced by FNR-positive
microorganisms, is a dimeric protein which can interact
with the operator of the promoter according to the
present invention and thus activates the expression.
It is of course possible to use a host strain which is
FNR-negative; however, in this case the cell has to be
supplied with the FNR protein in order to achieve an
activation. For this purpose the FNR gene can be ligated
into the expression vector according to the present
invention which also carries or will carry the desired
foreign gene and by this means expression of the FNR
protein is achieved simultaneously with the expression
of the foreign gene. The FNR gene can also be present in
the host cells on an additional vector. In this case the
production of the FNR protein is independent of the
production of the foreign gene whereby this is however
induced by the FNR protein. This embodiment i.e. the
incorporation of at least one FNR gene on a separate
vector is therefore preferred according to, the present
invention.
It is possible to regulate the expression of a foreign
gene in a simple manner by the recombinant DNA according
20 15048
- 12 -
to the present invention and the expression vectors
according to the present invention. Thus, for example,
an interfering expression of the foreign gene is
suppressed in the aerobic early growth phase of the
microorganism used. In the transition to the late
logarithmic anaerobic growth phase, the expression is
then induced by the pyruvate formed by the microorganism
and an enhancement of the expression is achieved by the
addition of pyruvate in the growth medium. A further
simplification is that in the late anaerobic growth
phase the cells are already fully grown and have reached
an optimal density. In this case a limitation of oxygen
automatically occurs which can be amplified or regulated
by the fermentation technique. An optimal expression can
be achieved by the high cell density of the
microorganism.
The use of the recombinant DNA and the expression vector
according to the present invention for the production of
proteins thus represents an inexpensive and simple
alternative for the regulation since expensive and
complicated inductions (addition of inducer, temperature
shift etc.) are no longer necessary. The invention is
further elucidated by the following Examples in
conjunction with the Figures.
201500
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General comments on the construction of the ex ression
plasmids v~.
1.) All anaerobic cultures were carried out in serum
flasks according to Balch W.E. and Wolfe R.S.
(1976) Appl. Environ. Microbiol. 32: 781-791.
Aerobic cultures were carried out in Erlenmeyer
flasks which were shaken vigorously (the flasks
were filled to a maximum of 1/10 of their nominal
volume).
The cultures were incubated at 37°C.
2.) Medium: TGYEP (pH 6.5; 0.4 % glucose) (Begg Y.A.,
Whyte J.N., Haddock B.A. (1977) FEMS Microbiol.
Lett. 2: 47-50).
3.) Transformation of the strains used with plasmid DNA
was carried out according to standard procedures
(Maniatis T., Fritsch E.F., Sambrook (1982)
Molecular cloning: a laboratory manual. Cold Spring
Harbor Laboratory, Cold Spring Harbor N.Y.).
4.) Determination of the pfl enzyme activity was
carried out according to Conradt et al. (1984)
Arch. Biochem. and Biophysics 228: 133.
2p ~5Q46
- 14 -
5.) Determination of the creatinase enzyme activity was
carried out according to Schmitt J. (1984)
Diplomarbeit, Univ. Wiirzburg. The specific activity
(U/mg protein) is~,~~;quoted.
6.) Determination of the p-galactosidase enzyme
activity was carried out according to Miller J.H.
(1972) Experiments in molecular genetics. Cold
Spring Harbor Laboratory, Cold Spring Harbor N.Y.
The activities are quoted as Miller units.
7.)~ The integration of the pfl-lacZ fusion into the
chromosome was carried out according to the method
of Simons R.W., Houman F., Kleckner N. (1987) Gene
53: 85-96. Starting with strain RM102 (fnr-, DSM
5311), the following~transductants were obtained:
RM135, RM136, RM409, RM415, RM412. Starting with
strain FM420 (fnr+, DSM 5312), the following
transductants were obtained: RM123, RM124, RM401,
RM404, RM407.
Starting vectors: 1.) M13mp18 (Yanisch-Perron, Vieira,
Messing (1985) Gene 33:
103-119)
2.) M13111RX (Waye M.M.Y. et al.
(1985) Nucleic Acids Res.
13: 8561-8571)
3.) pBT2a-1 (DSM 3148 P)
4.) p29 (Christiansen L.,
Pedersen S. (1981) Mol.
Gen. Genet. 181: 548-551:
DSM 5380)
The fusion of the promoter to the creatinase structural
gene was carried out by directed deletion mutagenesis on
the single-stranded DNA of a M13 construction. This
contains a pfl fragment (regulatory sequence and the
2015~r~6
,,,-.
f
- 15 -
beginning of the structural gene), a selection marker
for the mutagenesis (EcoK cassette: contains 4 times in
sequence the recognition sequence for the restriction
system K from E.coli) ~xtd a fragment of the creatinase
gene (beginning of thelstructural gene with a part of
the 5' untranslated sequence).
The plasmids p29 (Christiansen L., Pedersen S. (1981)
Mol. Gen. Genet. 181: 548-551; DSM 5380) pRS551 (DSM
5382), pRS552 (DSM 5381) (Simons R.W., Houman F.,
Kleckner N. (1987) Gene 53: 85-96) were used for the
pfl-lacZ fusions (Examples 8 - 12).
E x a m p 1 a 1
Cloning of the EcoK cassette and the creatinase fragment
in M13mp18
In preparation for the deletion mutagenesis, the
individual components were cloned in M13mp18. In the
first step, the EcoK cassette and the creatinase
fragment were inserted into the XbaI/SphI cleaved
vector. The EcoK cassette was isolated as a 90 by
XbaI/BamHI fragment from M13k11RX (Waye M.M.Y. et al.
(1985) Nucleic Acids Res. 13: 8561-8571). The creatinase
fragment was isolated as a 580 by XhoII/SphI fragment
from pBT2a-1. It contains the first 460 nucleotides of
the structural gene and 120 nucleotides of the 5'
untranslated sequence. The 5' protruding end of the
XhoII cleavage site is compatible with the protruding
end of the BamHI cleavage site (EcoK cassette). EcoK
cassette, creatinase fragment and vector were added
together and ligated via the corresponding cleavage
sites (Fig. 2). E. coli RR1dM15 (rk-, mk-; ATCC 35102)
2015a~ fi
f
- 16 -
was transfected.
The construction which was obtained was denoted Ml3ec.
E x a m p 1 a 2 "w
Cloning of the pfl promoter fragment in Ml3ec
The promoter region of the pfl gene was isolated as a
178~6bp MluI/BamHI fragment from the plasmid p29 (pfl
sequence: +390 to -1396, in relation to the first
nucleotide of the pfl structural gene, A of the ATG of
Fig. 10). p29 was cut with MluI, the 5' protruding end
was filled in with T7 polymerase in the presence of all
4 dNTP's and then cut again with BamHI. The vector Ml3ec
was first cut with AvaII, the protruding end was also
filled in and then cut again with BamHI. The isolated
pfl fragment was inserted in Ml3ec in a directed manner
and the construction obtained was denoted Ml3pec23 (Fig.
2). E. coli RRldMlS (rk-, mk-; ATCC 35102) was
transfected. The orientation of the creatinase fragment
in Ml3pec23 corresponds to the direction of
transcription of the pfl promoter.
E x a m p 1 a 3
Deletion mutagenesis
Two deletion mutageneses were carried out using
oligonucleotides (analogous to the method of Waye M.M.Y.
et al. (1985) Nucleic Acids Res. 13: 8561-8571) in order
to fuse the creatinase structural gene to the promoter.
One translational fusion (replacement of the pfl
structural gene by the creatinase structural gene from
the start codon onwards) and one transcriptional fusion
2015(~4fi
-m-
(fusion of the creatinase gene with its own Shine-
Dalgarno sequence (SD-sequence) to the promoter) were
prepared.
These were denoted M13p~23A and M13pc23S (see Table 1).
T a b 1 a 1
Construction Fusion Fusion point 1)
Promoter Creatinase
M13pc23A translational -1 +1
M13pc23S transcriptional -12 -14
1) the numbering is relative to the
respective structural gene whose first
nucleotide (A) was numbered +1.
2015p,~8
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4I
-. ~"'~ M
U v
M Lf7
U Ca
r~ u~
U
U U C
7
~
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r-
V
U C~ H
U H
H CJ U
cd
E' ~ E1
~y
C9 U C7 U
U
H ~ H
U
U
H U
E~
~
U
H E1
tc3 ~ C~ U H
Ei
H ~ H
b E~
E1
U
H
N U H
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U
~ M ~ M
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!4.a.,..~
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U
2015x46
- 19 -
Procedure for the mutagenesis: (see Fig. 3)
a) Hybridization of the mutagenic oligonucleotide to the
M13 plus-strand. The DNA sequence to be deleted between
the fusion points remains unpaired.
b) The remainder of the M13 single-stranded DNA is
filled in to a double-strand with Klenow and dNTP's.
c) The selection against the non-mutated parent strand
is carried out in vivo after transformation in E.coli
JM83 (rk+, mk+; ATCC 35607).
E x a m p 1 a 4
Directed mutagenesis in the orf gene
In order to prevent an over-expression of the orf gene
product by the gene dose effect in the later expression
vectors (high copy plasmids), two sequential stop codons
were introduced into the reading frame of the orf gene
by directed mutagenesis according to Kunkel (Kunkel T.
(1985) Proc. Natl. Acad. Sci. 82: 488-492; Kunkel T.,
Roberts J., Zakour R. (1987) Methods in Enzymol. 154:
367-382).
The base substitutions were carried out with a mutagenic
oligonucleotide on the plus-strand DNA of the
construction M13pc23S. The substituted bases and their
positions are shown in the following diagram:
5'.. ...=AAC-TAG-TAA-...-... ... ...-3'
5'CG-AAA-AAC-TGG-CTA-AAT-GTC-TAT-TTT-3'
3'-TTT-TTG-ATC-ATT-TTA-CAG-A5'
2015~r6
- 20 -
The middle strand corresponds to the sequence in the
plus-strand of M13pc23S (5'-->3'), the lower strand (3'-
->5') corresponds to that of the mutagenic
oligonucleotide. The upper strand corresponds to the
sequence after mutagenesis; the substituted bases are
marked by colons (:). The asterisk marks the position
-570 relative to the pfl structural gene.
No mutagenesis was carried out on the construction
M13pc23A. In this case the pfl promoter region,
including the intact orf gene, was removed via the
cleavage sites AvaI and HindII and replaced by the
corresponding segment from the orf mutant of M13pc23S.
E x a m p 1 a 5
Construction of the creatinase plasmid pGH-C without
promoter
Starting vectors: 1.) pBTacl (Boehringer Mannheim GmbH,
Order No. 1081 365)
2.) pBT2a-1
pGH-C serves as the initial vector for the preparation
of the expression vectors. This plasmid contains the
complete creatinase structural gene and termination
sequences: the expression cassette may be completed by
recloning the fusions from the M13 derivatives into
pGH-C.
a) Elimination of the tac promoter from pBTacl
In order to eliminate the tac promoter from pBTacl, the
plasmid was cut with EcoRI and the 5' protruding end was
filled in with T7 polymerase. Subsequently it was cut
20~~~o~s
- 21 -
with PvuII and the vector part was isolated. The EcoRI
cleavage site was regenerated by ligation of the blunt
ends. The construction obtained was denoted pBTdtac.
b) Insertion of the creatinase gene in pBTdtac
The creatinase gene was isolated from pBT2a-1. The
plasmid was cut with Aval and the fragment with the
creatinase gene (ca. 1600bp) was isolated. The
protruding ends were filled in and provided with BamHI
linkers (BM). The fragment was inserted into the BamHI
cleavage site of pBTdtac.
The construction obtained was denoted pGH-C.
E x a m p 1 a 6
Recloning of the fusions from Ml3 into pGH-C
The fusion fragments from M13pc23S and M13pc23A were
isolated via the cleavage sites StuI and MluI.
The plasmid pGH-C was cut with SmaI and Mlul and the
vector part was isolated. The fusion fragments were
inserted into the vector in such an orientation that the
direction of transcription of the pfl promoter
corresponds to the orientation of the creatinase gene
(Fig. 5).
The expression plasmids were denoted pPFL23S-C and
pPFL23A-C corresponding to the M13 construction.
These vectors contain the following parts of the pfl
promoter region:
pPFL23A Pos. -1 to -1364 inclusive
pPFL23S Pos. -12 to -1364 inclusive
20 ~ ~o~ s
.._
- 22 -
E x a m v 1 a 7
Construction of the expression vector pPFL39-C
This plasmid contains only a shortened promoter element
at the 5' end (promoters 6 and 7, fnr boxes 1 and 2;
Fig. 1). This element corresponds to the 577 by
MluI/AflII fragment from p29 and contains the pfl
sequence from position -819 to -1396 inclusive relative
to the pfl start codon (A of the ATG = +1). The fragment
was isolated via the EcoRI cleavage sites from the
plasmid pRM39 (Example 10) and inserted into the EcoRI
site of the plasmid pGH-C. The construction obtained was
denoted pPFL-39C.
E x a m p 1 a 8
Construction of pRM23
Translational coupling of the complete promoter fragment
with the lacZ gene.
For the isolation of the promoter fragment, p29 was cut
with Mlul and the protruding end was filled in with
Klenow in the presence of all 4 dNTP's. Subsequently a
BamHI linker (8-mer) was ligated on and cut with BamHI.
The 1791bp fragment (including BamHI linker) was
isolated and inserted into the BamHI cleavage site of
pRS552 (Fig. 8) .
The pfl fragment consists of the bases -1396 to + 390
inclusive (relative to the first nucleotide of the pfl
structural gene) and contains the promoters 1 to 7 and
the fnx boxes 1 and 2.
2015t~46
- 23 -
E x a m p 1 a 9
Construction of pRM24
Translational coupling of a shortened promoter fragment
with the lacZ gene.
The promoter element was isolated via the cleavage sites
SspI and BamHI from p29 and inserted into pRS552 via the
cleavage sites SmaI and BamHI. The pfl fragment consists
of the bases -1045 to +390 inclusive (relative to the
pfl start codon) and contains the promoters 1 to 7 and
the fnr box 2.
E x a m p 1 a 10
Construction of pRM39
Transcriptional coupling of a shortened promoter
fragment at the 5' end with the lacZ gene.
The promoter element was isolated from p29 via the
cleavage sites Mlul and AflII, provided with EcoRI
linkers (10-mer) and inserted into the EcoRI cleavage
site of pRS551.
The pfl fragment consists of the bases -819 to -1396
inclusive (relative to the first nucleotide of the pfl
structural gene) and contains the promoters 6 and 7 and
the fnr boxes 1 and 2.
20 1 5 0:4 6
~.,""' - 2 4 -
E x a m p 1 a 11
Construction of pRM43
Transcriptional coupling of a shortened promoter
fragment at the 5' end with the lacZ gene.
The promoter element was isolated from p29 via the
cleavage sites MluI and DraI, provided with EcoRI
linkers (10-mer) and inserted into the EcoRI cleavage
site of pRS551.
The pfl fragment consists of the bases -1075 to -1396
inclusive (relative to the first nucleotide of the pfl
structural gene) and contains the promoter 7 and the fnr
box 2.
E x a m p 1 a 12
Construction of pRM46
Transcriptional coupling of a shortened pfl fragment
with the lacZ gene.
The promoter element was isolated from p29 via the
cleavage sites NIaIII and Bgll, provided with EcoRI
linkers and inserted into the EcoRI cleavage site of
pRS551.
The pfl fragment consists of the bases -861 to -1016
inclusive (relative to the first nucleotide of the pfl
structural gene) and contains the promoter 6 and the fnr
box 1.
2p ~:5~4 6
E x a m p 1 a 13
Expression of pyruvate-formate lyase by the complete
promoter fragment (promoter 1 - 7, fnr boxes 1 and 2):
homologous expression.
The proportion of pyruvate-formate lyase is quoted in
relation to the total cell protein under anaerobic
conditions.
The determination was carried out by measuring the
specific acitivity of the pyruvate-formate lyase formed.
Strain: Strain:
HB101 (free of plasmid) HB101 (p29)
_02 1) 3 ~ ca. 30 %
1) -02: under anaerobic conditions.
20~5p~6
- 26 -
E x a m p 1 a 14
Expression of pyruvate-formate-lyase-(3-galactosidase
fusion protein by the complete promoter fragment (MluI-
BamHI, promoters 1 - 7, fnr boxes 1 and 2):
translational coupling.
Strain: Strain:
FM420 (pRM23). , RM123 (fnr+) RM135 (fnr-)
+02 2) 10937 302 255
-p2 >45000 4418 845
3) >45000 6642 869
-02, +Pyr.
2) +02: under aerobic conditions
3) +Pyr.: addition of 0.8 % (w/v) pyruvate
20 1 5 Q 4 6
- 27 -
E x a m p 1 a 15
Expression of pyruvate-formate-p-galactosidase fusion
protein by the shortened promoter fragment (Sspl-BamHI,
promoters 1 - 6, fnr box 1): translational coupling.
Strain: Strain:
FM420 (pRM24) RM124 (fnr+) RM136 (fnr-)
+02 8993 217 389
-02 >45000 2669 610
-02, +pyr. >45000
~.., 2 0 1 5 Q: '4 6
- 28 -
E x a m p 1 a 16
Expression of ~i-galactosidase protein by the shortened
promoter fragment (MluI - AflII, promoters 6 - 7, fnr
boxes 1 and 2): transcriptional coupling.
' Strain: Strain:
FM420 (pRM39) RM401 (fnr+) RM409 (fnr-)
+02 3441 203 439
-02 >38000 3238 1813
-02, +pyr. >45000
20150;8
- 29 -
E x a m p 1 a 17
Expression of ~i-galactosidase protein by the shortened
promoter fragment (MluI - DraI, promoter 7, fnr box 2):
transcriptional coupling.
Strain: Strain:
FM420 (pRM43) RM404 (fnr+) RM412 (fnr-)
+02 220 7 6
-02 1050 71 17
Z~ ~5Q4 6
- 30 -
E x a m p 1 a 18
Expression of ,Q-galactosidase protein by the shortened
promoter fragment (NIaIII - BglI, promoter 6, fnr
box 1): transcriptional coupling.
Strain: Strain:
FM420 (pRM46) RM407 (fnr+) RM415 (fnr-)
+02 11000 542 400
-p2 >40000 3126 815
-02, +pyr. >40000
20 15p~ 6
- 31 -
E x a m p 1 a 19
Expression of creatinase protein by the complete
promoter fragment (promoters 1 - 7, fnr boxes 1 and 2):
transcriptional coupling (region of translation
initiation of the creatinase gene).
Strain: Strain:
JM83 (pPFL23S-C) 1) FM420 (pPFL23S-C) 2)
+02 0.008 ~ 0.181 3)
-OZ 0.050 1.476
1) Cells harvested at OD600 = 0.5 (start to
middle of the log phase).
2) Cells harvested at OD600 = 4.0 (stationary
growth phase).
3) Creatinase enzyme activity: units/mg
soluble protein.
20 ~ 5~~ s
- 32 -
Fermentation of FM420 (pPFL23S-C):
Fig. 9 shows the course of the creatinase expression
(volume activity: units/ml) during the course of a
fermentation process.
Fermenter . Bioflo II (New Brunswick, 5 litre
fermenter)
Filled volume. 4 litres
Medium . K2HP04 (3 H20): 8 g/litre;
KH2PO4 2 g/litre
Peptone: 10 g/litre:
yeast extract (Ohly Kav):
32.4 g/litre;
MgS04 (1M): 4 ml/litre;
glucose: 0.4 %.
Temperature . 32C
pH . 7.0 (constant)
The stirring rate (300 rpm) and the rate of aeration
(4 1/min)
were kept
constant
during the
entire run.
Shown are: - growth curve (cell density expressed
as OD600).
- creatinase expression (units/ml).
- content of dissolved oxygen
(DO = dissolved oxygen: %).
It can be seen from the graph that:
- The DO value is lowered as the cell density increases.
- Growth is not influenced negatively when conditions of
oxygen limitation occur (DO value = 0).
- Creatinase expression is induced at a low DO value.
20 1 504 6
- 33 -
E x a m p 1 a 20
Expression of creatinase protein by the complete
promoter fragment (promoters 1 - 7, fnr boxes 1 and 2)
with the region of translation initiation of the pfl
gene (translational coupling).
Strain:
JM83 (pPFL23A-C)
+02 0.024
-02 0.195 -
Cells were harvested in the stationary growth phase.
E x a m p 1 a 21
Expression of creatinase protein by the shortened
promoter fragment (promoters 6 and 7, fnr boxes 1 and 2)
with the region of translation initiation of the
creatinase gene (transcriptional coupling).
Strain:
JM83 (pPFL39-C)
+02 0.006 4)
-p2 0.045
4) Cells were harvested at the beginning of the log
.phase .
