CA2015046C - Recombinant dna and expression vector - Google Patents
Recombinant dna and expression vector Download PDFInfo
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- CA2015046C CA2015046C CA002015046A CA2015046A CA2015046C CA 2015046 C CA2015046 C CA 2015046C CA 002015046 A CA002015046 A CA 002015046A CA 2015046 A CA2015046 A CA 2015046A CA 2015046 C CA2015046 C CA 2015046C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The recombinant DNA according to the present invention contains a regulator region which is at least 50 %
homologous to the sections -969 to -991 base pairs and/or -1308 to -1330 base pairs of the sequence in Fig.
and in the 3' direction from the regulation sequence contains at least one promoter region which in turn contains a -35/-10 promoter sequence. An expression vector according to the present invention contains a recombinant DNA according to the present invention ligated into a suitable vector molecule. To prepare the recombinant DNA according to the present invention, the pfl gene with its upstream regions is isolated from the gene bank of a microorganism, the desired parts are obtained according to well-known methods and they are combined with the other desired sequences or promoters.
When using a recombinant DNA according to the present invention or an expression vector according to the present invention for the inducible and the repressible expression of a foreign gene, the induction is effected under anaerobic conditions and by pyruvate and the repression is effected by oxygen.
homologous to the sections -969 to -991 base pairs and/or -1308 to -1330 base pairs of the sequence in Fig.
and in the 3' direction from the regulation sequence contains at least one promoter region which in turn contains a -35/-10 promoter sequence. An expression vector according to the present invention contains a recombinant DNA according to the present invention ligated into a suitable vector molecule. To prepare the recombinant DNA according to the present invention, the pfl gene with its upstream regions is isolated from the gene bank of a microorganism, the desired parts are obtained according to well-known methods and they are combined with the other desired sequences or promoters.
When using a recombinant DNA according to the present invention or an expression vector according to the present invention for the inducible and the repressible expression of a foreign gene, the induction is effected under anaerobic conditions and by pyruvate and the repression is effected by oxygen.
Description
--..
~' 2 0 1 5 0_!~ 6 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 ,~., 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 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.
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 .
'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.
- 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, _ 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 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.
,...
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 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.
~' 2 0 1 5 0_!~ 6 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 ,~., 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 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.
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 .
'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.
- 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, _ 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 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.
,...
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 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.
(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 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 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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U v M Lf7 U Ca r~ u~
U
U U C
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cd E' ~ E1 ~y U
H ~ H
U
U
H U
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Ei H ~ H
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2015x46 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 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 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 .._ 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 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 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 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 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 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 Z~ ~5Q4 6 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 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 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.
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 .
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 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 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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2015x46 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 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 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 .._ 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 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 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 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 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 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 Z~ ~5Q4 6 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 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 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.
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 .
Claims (33)
1. A recombinant DNA comprising:
a) a regulator region, said regulator region containing at least one of a sequence selected from 5'-GAGATATGATCTATATCAATTTC-3' (I), 5'-CTGGGCAAAATAAAATCAAATAG-3' (II) , a functional variant of sequence (I) and a functional variant of sequence (II), wherein the sequence of said functional variant of sequence (I) is at least 50% homologous to that of sequence (I) and wherein said sequence of said functional variant of sequence I,II) is at least 50% homologous to that of sequence (II); and b) in the 3' direction from said regulator region, at least one promoter region, wherein said promoter region contains a -35/-10 pro-moter sequence.
a) a regulator region, said regulator region containing at least one of a sequence selected from 5'-GAGATATGATCTATATCAATTTC-3' (I), 5'-CTGGGCAAAATAAAATCAAATAG-3' (II) , a functional variant of sequence (I) and a functional variant of sequence (II), wherein the sequence of said functional variant of sequence (I) is at least 50% homologous to that of sequence (I) and wherein said sequence of said functional variant of sequence I,II) is at least 50% homologous to that of sequence (II); and b) in the 3' direction from said regulator region, at least one promoter region, wherein said promoter region contains a -35/-10 pro-moter sequence.
2. Recombinant DNA as claimed in claim 1, wherein the sequence of saict functional variant of (I) and said functional variant of (II) is at least 65% homologous to that of sequence (I) and sequence (II), respectively.
3. Recombinant DNA as claimed in claim 1 or 2, wherein said -35/-10) promoter sequence is at least 80%
homologous to the following consensus sequence:
homologous to the following consensus sequence:
4. Recombinant DNA as claimed in claim 1, 2 or 3, wherein the promoter sequence is selected from lac, lambda, PL, trp or mgl promoter sequence.
5. Recombinant DNA as claimed in claim 1, 2 or 3, wherein the promoter sequence is selected from AATGTAGGCTTAATGATTAGTCTGAGTTATATTACGGGGCG;
TATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCG;
TGGTATCCTGGCAAACCTGATGGTATGTCTGGCAGTATGGA;
GTTCATTATGGTGCTGCCGGTCGCGATGTTTGTTGCCAGCG.
CGGAATTTTGGACCGCAGTCGGTTCTGCACCGGAAAATTTT
CATTATCGGTGGTGGTTTGTTGGTTGGGTTGACATACTGGG ;and ACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAA
or a functional variant thereof, wherein the functional variant of the promoter sequence is at least 50% homolo-gous to said promoter sequence.
TATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCG;
TGGTATCCTGGCAAACCTGATGGTATGTCTGGCAGTATGGA;
GTTCATTATGGTGCTGCCGGTCGCGATGTTTGTTGCCAGCG.
CGGAATTTTGGACCGCAGTCGGTTCTGCACCGGAAAATTTT
CATTATCGGTGGTGGTTTGTTGGTTGGGTTGACATACTGGG ;and ACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAA
or a functional variant thereof, wherein the functional variant of the promoter sequence is at least 50% homolo-gous to said promoter sequence.
6. Recombinant DNA as claimed in claim 5, wherein the functional variant of the promoter sequence is at least 65% homologous to said promoter sequence.
7. Recombinant DNA as claimed in claim 5 or 6, wherein it contains at least one of the promoter sequence TATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCG and AATGTAGGCTTAATGATTAGTCTGAGTTATATTACGGGGCG.
8. Recombinant :DNA as claimed in claim 3, wherein the regulator region, promoter sequence and sequence (III) are located upstream of a transcription start site of a foreign gene too be expressed.
9. Expression vector, comprising a recombinant DNA
according to claim 1, 2, 3, 4, 5, 6, 7 or 8, which is ligated into a suitable vector molecule.
according to claim 1, 2, 3, 4, 5, 6, 7 or 8, which is ligated into a suitable vector molecule.
10. Expression vector as claimed in claim 9, compris-ing a polylinker or a single restriction cleavage site for the insertion of a foreign gene downstream of the recombinant DNA.
11. Expression erector as claimed in claim 10, comprising a Shine-Dalgarno sequence between the recombinant DNA and the polylinker or between the recombinant DNA and the foreign gene.
12. Expression vector as claimed in claim 10 or 11, comprising at least:: a part: of the sequences upstream of sequence (I) and :sequence (II) of a pyruvate formate-lyase (pfl) gene .
13. Expression vector as claimed in claim 9, compris-ing a Shine-Dalgarno sequence.
14. Expression v,rector as claimed in claim 10, 11 or 12, comprising a Shine-Dalgarno sequence.
15. Expression vector as claimed in claim 13 or 14, further comprising a start codon of a pyruvate formate-lyase (pfl) gene.
16. Expression vector as claimed in claim 14, further comprising a pyruvate formate-lyase (pfl) gene, or part thereof, between the recombinant DNA and the polylinker or the restriction cleavage site.
17. Expression vector as claimed. in claim 9, 10, 11, 12, 13, 14, 15 or 16, comprising the sequence:
ACGCGTTTGCTGCACATCAGTCGTTGTTGAAGGCCTACGAAAAGCTGCAGCGCGCCAAAG
CAGCATTCTGGGCAAAATAAAATCAAATAGCCTACGCAATGTAGGCTTAATGATTAGTCT
GAGTTATATTACGGGGCGTTTTTTTAATGCCCCGCTTTACATATATTTGCATTAATAAAA
TAATTGTAATTATAAGGTTAAATATCGGTAATTTGTATTTAATAAATACGATCGATATTG
TTACTTTATTCGCCTGATGCTCCCTTTTAATTAACTGTTTTAGCGGAGGATGCGGAAAAA
ATTGAACTGATTTGTTAATTTTTAAAATTTATTTTTATTTGGATAATCAAATATTTACTC
CGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAGCCAGGCGAGATATGATCTAT
ATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCGCCGACTTAATAAAGAGAGAG
TTAGTGTGAAAGCTGACAACCCTTTTGATCTTTTACTTCCTGCTGCAATGGCCAAAGTGG
CCGAAGAGGCGGGTGTCTATAAAGCAACGAAACATGCGCTTAAGACTTTCTATCTGGCGA
TTACCGCCGGTGTTTTGATCTCAATCGCATTCGTCTTCTATATCACAGCAACCACTGGCA
CAGGCACAATGCCCTTCGGCATGGCAAAACTGGTTGGCGGCATTTGCTTCTCTCTGGGGC
TGATTCTTTGTGTTGTCTGCGGAGCCGATCTCTTTAGTTCCACCGTGTTGATTGTTGTTG
CTAAGGCGAGTGGGCGCATCACCTGGGGTCAGTTGGCGAAAAACTGGCTAAATGTCTATT
TTGGCAACCTGGTCGGCGCACTGCTGTTTGTACTTTTAATGTGGCTTTCCGGCGAGTATA
TGACCGCAAATGGTCAATGGGGACTAAACGTCCTACAAACCGCCGACCACAAAGTGCACC
ATACTTTTATTGAGGCCGTCTGTCTTGGTATCCTGGCAAACCTGATGGTATGTCTGGCAG
TATGGATGAGTTATTCTGGCCGCAGCCTGATGGACAAAGCGTTCATTATGGTGCTGCCGG
TCGCGATGTTTGTTGCCAGCGGTTTTGAGCACAGTATCGCAAACATGTTTATGATCCCGA
TGGGTATTGTAATCCGCGACTTCGCATCCCCGGAATTTTGGACCGCAGTCGGTTCTGCAC
CGGAAAATTTTTCTCACCTGACCGTGATGAATTTCATCACTGATAACCTGATTCCGGTTA
CGATCGGCAACATTATCGGTGGTGGTTTGTTGGTTGGGTTGACATACTGGGTCATTTACC
TGGGTGAAAACGACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAATCCAGTT
AAGAAGGTAGGTGTTACATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTT
TTACCAAAGGTGACTGGCAGAATGAAGTAAACGTCCGTGACTTCATTCAGAAAAACTACA
CTCCGTACGAGGGTGACGAGTCCTTCCTGGCTGGCGCTACTGAAGCGACCACCACCCTGT
GGGACAAAGTAATGGAAGGCGTTAAACTGGAAAACCGCACTCACGCGCCAGTTGACTTTG
AGAAAATCGTTGGTCTGCAGACTGAAGCTCCGCTGAAACGTGCTCTTRTCCCGTTCGGTG
GTATCAAAATGATCGAAGGTTCCTGCAAAGCGTACAACCGCGAACTGGATCC.
ACGCGTTTGCTGCACATCAGTCGTTGTTGAAGGCCTACGAAAAGCTGCAGCGCGCCAAAG
CAGCATTCTGGGCAAAATAAAATCAAATAGCCTACGCAATGTAGGCTTAATGATTAGTCT
GAGTTATATTACGGGGCGTTTTTTTAATGCCCCGCTTTACATATATTTGCATTAATAAAA
TAATTGTAATTATAAGGTTAAATATCGGTAATTTGTATTTAATAAATACGATCGATATTG
TTACTTTATTCGCCTGATGCTCCCTTTTAATTAACTGTTTTAGCGGAGGATGCGGAAAAA
ATTGAACTGATTTGTTAATTTTTAAAATTTATTTTTATTTGGATAATCAAATATTTACTC
CGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAGCCAGGCGAGATATGATCTAT
ATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCGCCGACTTAATAAAGAGAGAG
TTAGTGTGAAAGCTGACAACCCTTTTGATCTTTTACTTCCTGCTGCAATGGCCAAAGTGG
CCGAAGAGGCGGGTGTCTATAAAGCAACGAAACATGCGCTTAAGACTTTCTATCTGGCGA
TTACCGCCGGTGTTTTGATCTCAATCGCATTCGTCTTCTATATCACAGCAACCACTGGCA
CAGGCACAATGCCCTTCGGCATGGCAAAACTGGTTGGCGGCATTTGCTTCTCTCTGGGGC
TGATTCTTTGTGTTGTCTGCGGAGCCGATCTCTTTAGTTCCACCGTGTTGATTGTTGTTG
CTAAGGCGAGTGGGCGCATCACCTGGGGTCAGTTGGCGAAAAACTGGCTAAATGTCTATT
TTGGCAACCTGGTCGGCGCACTGCTGTTTGTACTTTTAATGTGGCTTTCCGGCGAGTATA
TGACCGCAAATGGTCAATGGGGACTAAACGTCCTACAAACCGCCGACCACAAAGTGCACC
ATACTTTTATTGAGGCCGTCTGTCTTGGTATCCTGGCAAACCTGATGGTATGTCTGGCAG
TATGGATGAGTTATTCTGGCCGCAGCCTGATGGACAAAGCGTTCATTATGGTGCTGCCGG
TCGCGATGTTTGTTGCCAGCGGTTTTGAGCACAGTATCGCAAACATGTTTATGATCCCGA
TGGGTATTGTAATCCGCGACTTCGCATCCCCGGAATTTTGGACCGCAGTCGGTTCTGCAC
CGGAAAATTTTTCTCACCTGACCGTGATGAATTTCATCACTGATAACCTGATTCCGGTTA
CGATCGGCAACATTATCGGTGGTGGTTTGTTGGTTGGGTTGACATACTGGGTCATTTACC
TGGGTGAAAACGACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAATCCAGTT
AAGAAGGTAGGTGTTACATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTT
TTACCAAAGGTGACTGGCAGAATGAAGTAAACGTCCGTGACTTCATTCAGAAAAACTACA
CTCCGTACGAGGGTGACGAGTCCTTCCTGGCTGGCGCTACTGAAGCGACCACCACCCTGT
GGGACAAAGTAATGGAAGGCGTTAAACTGGAAAACCGCACTCACGCGCCAGTTGACTTTG
AGAAAATCGTTGGTCTGCAGACTGAAGCTCCGCTGAAACGTGCTCTTRTCCCGTTCGGTG
GTATCAAAATGATCGAAGGTTCCTGCAAAGCGTACAACCGCGAACTGGATCC.
18. Process for producing a recombinant DNA as claimed in claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein a pyruvate formate-lyase (pfl) gene is isolated with an upstream region of said pfl gene from a gene bank of a microorganism containing the pfl gene, said upstream region containing the regulator region, and said pfl gene with its upstream region ligated to a promoter sequence and a gene to be expressed, for producing said recombinant DNA.
19. Process as claimed in claim 18, wherein the gene bank is a gene bank of a microorganism of Enterobac-teriaceae family.
20. Process as claimed in claim 19, wherein the gene bank of a microorganism of the Enterobacteriaceae family is a gene bank of E. coli.
21. Process for the production of an expression vector as defined in claim 9, 10, 11, 12, 13, 14, 15, 16 or 17, wherein the recombinant DNA along with a polylinker, a Shine-Dalgarno sequence, a start codon and a gene to be expressed, is inserted into a suitable vector.
22. Use of a recombinant DNA as defined in claim 1, 2, 3, 4, 5, 6, 7 on 8, or of an expression vector as defined in claim 9, 10, 11, 12, 13, 14, 15, 16 or 17, for the inducible and repressible expression of a foreign gene, wherein induction is effected under anaerobic conditions and by pyruvate and repression is effected by oxygen.
23. Use as claimed in claim 22, wherein the expression is carried out in microorganisms of the genus Enterobacteriaceae or in other gram-negative or gram-positive bacteria.
24. Use as claimed in claim 23, wherein the expression is carried out in E. coli, Salmonella or Pseudomonas.
25. Use as claimed in claim 23 or 24, wherein the expression is carried out in a host strain which is FNR-positive.
26. Use as claimed in claim 25, wherein the expression is carried out in E. coli FM420, deposited at the Deutsche Sammlung f~r Mikroorganismen (DSM).
27. Use as claimed in claim 23 or 24, wherein when a FRN-negative host strain is used, a FNR gene is intro-duced into a host cell, in addition, of an expression vector.
28. Use as claimed in claim 27, wherein the FNR gene is introduced on a different expression vector than the recombinant DNA.
29. A recombinant DNA, comprising:
a gene to be expressed, wherein said gene is dif-ferent from a pfl gene;
a promoter region 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 said regulator region contains sequence (I) 5 ' -GAGATATGATCTATATCAATTTC-3' or a 23 base pair sequence which is identical at posi-tions 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.
a gene to be expressed, wherein said gene is dif-ferent from a pfl gene;
a promoter region 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 said regulator region contains sequence (I) 5 ' -GAGATATGATCTATATCAATTTC-3' or a 23 base pair sequence which is identical at posi-tions 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.
30. The recombinant DNA according to claim 29, wherein said regulator region further comprises a sequence which is identical to a sequence (II) 5'-CTGGGCAAAATAAAATCAAATAG-3' operably linked to sequence (I) or a 23 base pair sequence which is identical 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.
31. A recombinant DNA comprising:
a gene to be expressed wherein said gene is dif-ferent from a pfl gene;
the sequence:
ACGCGTTTGCTGCACATCAGTCGTTGTTGAAGGCCTACGAAAAGCTGCAGCGCGCCAAAG
CAGCATTCTGGGCAAAATAAAATCAAATAGCCTACGCAATGTAGGCTTAATGATTAGTCT
GAGTTATATTACGGGGCGTTTTTTTAATGCCCCGCTTTACATATATTTGCATTAATAAAA
TAATTGTAATTATAAGGTTAAATATCGGTAATTTGTATTTAATAAATACGATCGATATTG
TTACTTTATTCGCCTGATGCTCCCTTTTAATTAACTGTTTTAGCGGAGGATGCGGAAAAA
ATTCAACTCATTTGTTAATTTTTAAAATTTATTTTTATTTGGATAATCAAATATTTACTC
CGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAGCCAGGCGAGATATGATCTAT
ATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCGCCGACTTAATAAAGAGAGAG
TTAGTGTGAAAGCTGACAACCCTTTTGATCTTTTACTTCCTGCTGCAATGGCCAAAGTGG
CCGAAGAGGCGGGTGTCTATAAAGCAACGAAACATCCGCTTAAGACTTTCTATCTGGCGA
TTACCGCCGGTGTTTTCATCTCAATCGCATTCGTCTTCTATATCACAGCAACCACTGGCA
CAGGCACAATGCCCTTCGGCATGGCAAAACTGGTTGGCGGCATTTGCTTCTCTCTGGGGC
TGATTCTTTGTGTTGTCTGCGGAGCCGATCTCTTTACTTCCACCGTGTTGATTGTTGTTG
CTAAGGCGAGTGGGCGCATCACTGGGGTCAGTTGGCGAAAAACTGGCTAAATGTGCTATT
TTGGCAACCTGGTCGGCGCACTGCTGTTTGTACTTTTAATGTGGCTTTCCGGCGAGTATA
TGACCGCAAATGGTCAATGGGGACTAAACGTCCTACAAACCGCCGACCACAAAGTGCACC
ATACTTTTATTGAGGCCGTCTGTCTTGGTATCCTGGCAAACCTGATGGTATGTCTGGCAG
TATGGATGAGTTATTCTGGCCGCAGCCTGATGGACAAAGCGTTCATTATGGTGCTGCCGG
TCGCGATGTTTGTTGCCAGCGGTTTTGAGCACAGTATCGCAAACATGTTTATGATCCCGA
TGGGTATTGTAATCCGCGACTCCGCATCCCCGGAATTTTGGACCGCAGTCGGTTCTGCAC
CGGAAAATTTTTCTCACCTGACCGTGATGAATTTCATCACTGATAACCTGATTCCGGTTA
CGATCGGCAACATTATCGGTGGTGGTTTGTTGGTTGGGTTGACATACTGGGTCATTTACC
TGCGTGAAAACGACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAATCCACTT
AAGAAGGTAGGTGTTACATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTT
TTACCAAAGGTGACTGGCAGAATGAAGTAAACGTCCGTGACTTCATTCAGAAAAACTACA
CTCCGTACGAGGGTGACGAGTCCTTCCTGGCTGGCGCTACTGAAGCGACCACCACCCTGT
GGGACAAAGTAATGGAAGGCGTTAAACTGGAAAACCGCACTCACGCGCCAGTTGACTTTG
ACACCGCTGTTGCTTCCACCATCACCTCTCACGACGCTGGCTACATCAACAAGCAGCTTG
AGAAAATCGTTGGTCTGCAGACTGAAGCTCCGCTGAAACGTGCTCTTATCCCGTTCGGTG
GTATCAAAATGATCGAAGGTTCCTGCAAAGCGTACAACCGCGAACTGGATCC
upstream from said gene to be expressed; and a promoter sequence upstream from said sequence, wherein said promoter sequence is selected from the group consisting of lac, lambda, Pl, trp, and mgl, or is selected from the group consisting of the following sequences:
41.
AATGTAGGCTTAATGATTAGTCTGAGTTATATTACGGGGCG, TATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCG, TGGTATCCTGGCAAACCTGATGGTATGTCTGGCAGTATGGA, GTTCATTATGGTGCTGCCGGTCGCGACGTTTGTTGCCAGCG, CGGAATTTTGGACCGCAGTCGGTTCTGCACCGGAAAATTTT, CATTATCGGTGCTGGTTTGTTGGTTGGGTTGACATACTGGG, and ACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAA.
a gene to be expressed wherein said gene is dif-ferent from a pfl gene;
the sequence:
ACGCGTTTGCTGCACATCAGTCGTTGTTGAAGGCCTACGAAAAGCTGCAGCGCGCCAAAG
CAGCATTCTGGGCAAAATAAAATCAAATAGCCTACGCAATGTAGGCTTAATGATTAGTCT
GAGTTATATTACGGGGCGTTTTTTTAATGCCCCGCTTTACATATATTTGCATTAATAAAA
TAATTGTAATTATAAGGTTAAATATCGGTAATTTGTATTTAATAAATACGATCGATATTG
TTACTTTATTCGCCTGATGCTCCCTTTTAATTAACTGTTTTAGCGGAGGATGCGGAAAAA
ATTCAACTCATTTGTTAATTTTTAAAATTTATTTTTATTTGGATAATCAAATATTTACTC
CGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAGCCAGGCGAGATATGATCTAT
ATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCGCCGACTTAATAAAGAGAGAG
TTAGTGTGAAAGCTGACAACCCTTTTGATCTTTTACTTCCTGCTGCAATGGCCAAAGTGG
CCGAAGAGGCGGGTGTCTATAAAGCAACGAAACATCCGCTTAAGACTTTCTATCTGGCGA
TTACCGCCGGTGTTTTCATCTCAATCGCATTCGTCTTCTATATCACAGCAACCACTGGCA
CAGGCACAATGCCCTTCGGCATGGCAAAACTGGTTGGCGGCATTTGCTTCTCTCTGGGGC
TGATTCTTTGTGTTGTCTGCGGAGCCGATCTCTTTACTTCCACCGTGTTGATTGTTGTTG
CTAAGGCGAGTGGGCGCATCACTGGGGTCAGTTGGCGAAAAACTGGCTAAATGTGCTATT
TTGGCAACCTGGTCGGCGCACTGCTGTTTGTACTTTTAATGTGGCTTTCCGGCGAGTATA
TGACCGCAAATGGTCAATGGGGACTAAACGTCCTACAAACCGCCGACCACAAAGTGCACC
ATACTTTTATTGAGGCCGTCTGTCTTGGTATCCTGGCAAACCTGATGGTATGTCTGGCAG
TATGGATGAGTTATTCTGGCCGCAGCCTGATGGACAAAGCGTTCATTATGGTGCTGCCGG
TCGCGATGTTTGTTGCCAGCGGTTTTGAGCACAGTATCGCAAACATGTTTATGATCCCGA
TGGGTATTGTAATCCGCGACTCCGCATCCCCGGAATTTTGGACCGCAGTCGGTTCTGCAC
CGGAAAATTTTTCTCACCTGACCGTGATGAATTTCATCACTGATAACCTGATTCCGGTTA
CGATCGGCAACATTATCGGTGGTGGTTTGTTGGTTGGGTTGACATACTGGGTCATTTACC
TGCGTGAAAACGACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAATCCACTT
AAGAAGGTAGGTGTTACATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTT
TTACCAAAGGTGACTGGCAGAATGAAGTAAACGTCCGTGACTTCATTCAGAAAAACTACA
CTCCGTACGAGGGTGACGAGTCCTTCCTGGCTGGCGCTACTGAAGCGACCACCACCCTGT
GGGACAAAGTAATGGAAGGCGTTAAACTGGAAAACCGCACTCACGCGCCAGTTGACTTTG
ACACCGCTGTTGCTTCCACCATCACCTCTCACGACGCTGGCTACATCAACAAGCAGCTTG
AGAAAATCGTTGGTCTGCAGACTGAAGCTCCGCTGAAACGTGCTCTTATCCCGTTCGGTG
GTATCAAAATGATCGAAGGTTCCTGCAAAGCGTACAACCGCGAACTGGATCC
upstream from said gene to be expressed; and a promoter sequence upstream from said sequence, wherein said promoter sequence is selected from the group consisting of lac, lambda, Pl, trp, and mgl, or is selected from the group consisting of the following sequences:
41.
AATGTAGGCTTAATGATTAGTCTGAGTTATATTACGGGGCG, TATCAATTTCTCATCTATAATGCTTTGTTAGTATCTCGTCG, TGGTATCCTGGCAAACCTGATGGTATGTCTGGCAGTATGGA, GTTCATTATGGTGCTGCCGGTCGCGACGTTTGTTGCCAGCG, CGGAATTTTGGACCGCAGTCGGTTCTGCACCGGAAAATTTT, CATTATCGGTGCTGGTTTGTTGGTTGGGTTGACATACTGGG, and ACCACCATTAATGGTTGTCGAAGTACGCAGTAAATAAAAAA.
32. An expression vector, comprising a recombinant DNA according to claim 29, 30 or 31.
33. A process for inducibly and repressibly expressing a gene which is different from pfl gene, comprising the steps of:
a) inserting said gene to be expressed into the expression vector of claim 32, wherein the gene to be expressed is inserted downstream of:
(i) said promoter region which contains a -35/-10 promoter sequence; and (ii) said regulator region, which regulates the expression of said gene upstream from said promoter region;
b) transforming a microbial host which is FNR
positive and in which said promoter sequence is operable with said expression vector; and c) culturing said host under conditions suitable to induce and repress expression of said gene to be expressed.
a) inserting said gene to be expressed into the expression vector of claim 32, wherein the gene to be expressed is inserted downstream of:
(i) said promoter region which contains a -35/-10 promoter sequence; and (ii) said regulator region, which regulates the expression of said gene upstream from said promoter region;
b) transforming a microbial host which is FNR
positive and in which said promoter sequence is operable with said expression vector; and c) culturing said host under conditions suitable to induce and repress expression of said gene to be expressed.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3913201.3 | 1989-04-21 | ||
| DE3913201 | 1989-04-21 | ||
| DE3926076A DE3926076A1 (en) | 1989-04-21 | 1989-08-07 | RECOMBINANT DNA AND EXPRESSION VECTOR |
| DEP3926076.3 | 1989-08-07 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2015046A1 CA2015046A1 (en) | 1990-10-21 |
| CA2015046C true CA2015046C (en) | 2003-09-16 |
Family
ID=25880155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002015046A Expired - Lifetime CA2015046C (en) | 1989-04-21 | 1990-04-20 | Recombinant dna and expression vector |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0393684B1 (en) |
| JP (1) | JP2774989B2 (en) |
| AT (1) | ATE154639T1 (en) |
| CA (1) | CA2015046C (en) |
| DE (2) | DE3926076A1 (en) |
| DK (1) | DK0393684T3 (en) |
| ES (1) | ES2104568T3 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992015689A1 (en) * | 1991-03-05 | 1992-09-17 | The Wellcome Foundation Limited | Expression of recombinant proteins in attenuated bacteria |
| GB9104596D0 (en) * | 1991-03-05 | 1991-04-17 | Wellcome Found | Production of recombinant proteins |
| DE4136389A1 (en) * | 1991-03-18 | 1992-10-08 | Hoechst Ag | PROCESS FOR THE PRODUCTION OF GLUTARYLACYLASE IN LARGE QUANTITIES |
| US5830692A (en) * | 1995-03-24 | 1998-11-03 | Consortium Fur Elektrochemische Industrie Gmbh | Expression system which can be regulated |
| KR101643195B1 (en) * | 2014-10-06 | 2016-07-27 | 경일대학교산학협력단 | Helmet apparatus for detecting object under water and method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3735381A1 (en) * | 1987-03-31 | 1989-05-03 | Boehringer Mannheim Gmbh | RECOMBINANT DNA FOR REPRESSIBLE AND INDUCIBLE EXPRESSION OF FOREIGN GENES |
-
1989
- 1989-08-07 DE DE3926076A patent/DE3926076A1/en not_active Withdrawn
-
1990
- 1990-04-19 DK DK90107465.8T patent/DK0393684T3/en active
- 1990-04-19 EP EP90107465A patent/EP0393684B1/en not_active Expired - Lifetime
- 1990-04-19 DE DE59010726T patent/DE59010726D1/en not_active Expired - Lifetime
- 1990-04-19 ES ES90107465T patent/ES2104568T3/en not_active Expired - Lifetime
- 1990-04-19 AT AT90107465T patent/ATE154639T1/en not_active IP Right Cessation
- 1990-04-20 CA CA002015046A patent/CA2015046C/en not_active Expired - Lifetime
- 1990-04-20 JP JP2103227A patent/JP2774989B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0380088A (en) | 1991-04-04 |
| DK0393684T3 (en) | 1998-01-26 |
| DE3926076A1 (en) | 1990-10-25 |
| EP0393684B1 (en) | 1997-06-18 |
| DE59010726D1 (en) | 1997-07-24 |
| CA2015046A1 (en) | 1990-10-21 |
| EP0393684A1 (en) | 1990-10-24 |
| ATE154639T1 (en) | 1997-07-15 |
| ES2104568T3 (en) | 1997-10-16 |
| JP2774989B2 (en) | 1998-07-09 |
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| EEER | Examination request | ||
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