EP1104476A1 - Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequence - Google Patents
Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequenceInfo
- Publication number
- EP1104476A1 EP1104476A1 EP99939866A EP99939866A EP1104476A1 EP 1104476 A1 EP1104476 A1 EP 1104476A1 EP 99939866 A EP99939866 A EP 99939866A EP 99939866 A EP99939866 A EP 99939866A EP 1104476 A1 EP1104476 A1 EP 1104476A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- nucleotide sequence
- sequence
- operator
- isolated
- nucleotide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 239000002773 nucleotide Substances 0.000 title claims abstract description 93
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 91
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 47
- 125000003275 alpha amino acid group Chemical group 0.000 title claims description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 11
- 239000013598 vector Substances 0.000 claims description 23
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- 230000004913 activation Effects 0.000 claims description 6
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- 241000187180 Streptomyces sp. Species 0.000 description 8
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- 241000187747 Streptomyces Species 0.000 description 6
- 229920001221 xylan Polymers 0.000 description 6
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- 108010042407 Endonucleases Proteins 0.000 description 2
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
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- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
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- 241000192125 Firmicutes Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
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- 229920002472 Starch Polymers 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
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- 230000000692 anti-sense effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229920000617 arabinoxylan Polymers 0.000 description 1
- 150000004783 arabinoxylans Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
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- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
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- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
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- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
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- 230000009466 transformation Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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- 101150034760 xlnR gene Proteins 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
Classifications
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
-
- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
Definitions
- the present invention is related to a new nucleotide sequence controlling in trans the expression of a xylanase promoter-operator nucleotide sequence, the amino-acid sequence encoded by said new nucleotide sequence, the vector comprising said new nucleotide sequence and the cell, preferably a Streptomyces strain, transformed by said vector.
- the aim of the present invention is to provide a method and system which improve the control upon the expression of nucleotide sequence encoding enzymes such as xylanase, as well as homologous or heterologous sequences of said enzymes whose transcription is also activated by a xylanase promoter-operator regulatory sequence .
- a specific aim of the present invention is to provide such a method and system for improving enzymatic processes, especially for improving production of antibiotics, malting processes of cereals such as barley, sorghum and wheat, production of beers, of baked or extruded cereals products, animal feed stuff, the production of starch derived from syrups, sorbitol, xylose and xylitol, and for the improvement of paper and pulp technologies.
- the present invention is related to a new nucleotide sequence 1 which controls the expression of any xylanase promoter-operator nucleotide sequence 2.
- Said control upon the activation of a xylanase promoter-operator nucleotide sequence 2 is advantageously obtained by trans- activation (said new nucleotide sequence 1 encoding a trans-activated factor which controls the activation of said xylanase promoter-operator nucleotide sequence 2) .
- the present invention is also related to said factor, preferably a peptidic factor 3 which is an activator and/or repressor encoded by said nucleotide sequence 1 and which controls positively and/or negatively the expression of a xylanase promoter-operator nucleotide sequence 2.
- said factor could be present in a composition with other cofactors 4 that induce positively and/or negatively said mechanism.
- said cofactors 4 present in said composition are selected from the group consisting of glucose, xylan or a mixture thereof.
- the Inventors have discovered unexpectedly that the presence of glucose induces a repressive mechanism upon the activation of a xylanase promoter-operator nucleotide sequence, while the presence of xylan induces a positive mechanism of said expression.
- the simultaneous presence of said two cofactors in a medium induces also positively the expression of a xylanase promoter-operator nucleotide sequence.
- a xylanase promoter-operator nucleotide sequence any nucleotide sequence 2 which cis- activates any nucleotide sequence 5 encoding a xylanase enzyme .
- a classification of the xylanase enzymes in the categories F/10 and G/ll is described by Henrissart et al. (Bioche . J. 293, pp. 781-788).
- Said xylanase promoter-operator nucleotide sequences comprise at least one 5 base pairs pattern : 5 ' - CGAAA-3 ' .
- said xylanase promoter-operator nucleotide sequence is the Strepto yces sp . strain EC3 xlnC xylanase promoter-operator nucleotide sequence SEQ ID NO 2 also described by Giannotta F. et al . (FEMS Microbiol . Letters 142, pp. 91-97 (1996)).
- the isolated and purified nucleotide sequence according to the invention is a (DNA) sequence which presents more than 60%, advantageously more than 80%, preferably more than 90%, and more preferably more than 95%, homology (i.e. sequence identity) with the nucleotide sequence SEQ ID NO 1 or its complementary strand described hereafter.
- said isolated and purified nucleotide sequence corresponds to the nucleotide sequence SEQ ID NO 1 or its complementary strand or a portion thereof; preferably a sequence having more than 100 nucleotides and encoding a peptide which still controls positively and/or negatively the expression of a xylanase promoter-operator nucleotide sequence.
- said sequence portion comprises at least the nucleotides of SEQ ID NO 3 or any nucleotide sequence encoding for its corresponding peptidic sequence.
- the terms "a portion of the nucleotide sequence SEQ ID NO 1 or its complementary strand” mean any kind of nucleic acid molecule (DNA, RNA, antisense nucleotide sequence, etc.) which is specific of SEQ ID NO 1, comprises more than 15 nucleotides (such as a probe or one or several primers) , and which may be used to identify, reconstitute or block the transcription of said specific isolated and purified nucleotide sequence SEQ ID NO 1 or its complementary strand.
- sequence identity as above-described with SEQ ID NO 1, SEQ ID NO 3 or their complementary strands, or any nucleotide sequence which preferably allows an hybridisation with SEQ ID NO 1, SEQ ID NO 3 or their complementary strands under standard stringent hybridisation conditions, and which may encode the same or a similar amino-acid sequence due to the redundancy of the genetic code.
- Exemplary standard stringent hybridisation conditions are as follows : hybridisation at 40 °C in 50% formamide, 5x SSC, 20 m ol sodium phosphate, pH 6.8, washing in 0.2x SSC at 50 °C . Variations in these conditions may occur based on the length and the GC nucleotide content of the sequence to be hybridised.
- Another aspect of the present invention is related to the amino-acid sequence encoded by said nucleotide sequence, and which present more than 60%, advantageously more than 80%, preferably more than 90%, more preferably more than 95% homology (i.e. sequence identity), with SEQ ID NO 2.
- amino-acid sequence according to the invention corresponds to the amino-acid sequence of SEQ ID NO: 1
- said portion is an amino-acid sequence that comprises at least the amino-acid sequence encoded by the nucleotide sequence SEQ ID NO 3 above- described. It is meant by “controlling (positively and/or negatively) in trans the expression of a xylanase promoter-operator nucleotide sequence", the possibility for any nucleotide sequence 1 or any amino-acid sequence 3 encoded by said nucleotide sequence 1 to induce or reduce (preferably in the presence of the other cofactors 4 such as glucose and/or xylan) the expression of a xylanase promoter-operator nucleotide sequence 2 and obtain thereafter a control upon the cis-activation of a downstream nucleotide sequence 5 (for instance a gene encoding a xylanase enzyme) which is controlled in cis by said xylanase promoter-operator nucleotide sequence.
- a downstream nucleotide sequence 5 for instance a gene
- the inducing or reduction of said expression is observed preferably by a positive or a negative modification of said cis-activation (for instance by an increasing or a decreasing of the synthesis of said xylanase enzyme by a cell) .
- Said mechanism is also illustrated in the enclosed Fig. 3.
- the present invention is also related to a nucleotide construct 6 comprising the isolated and purified nucleotide sequence 1 according to the invention, linked to a xylanase promoter-operator nucleotide sequence 2 and possibly any homologous or heterologous nucleotide sequence 5 of a gene encoding a xylanase enzyme, which is cis- activated by said xylanase promoter-operator nucleotide sequence 2.
- Another aspect of the present invention is related to the vector 7 comprising said isolated and purified nucleotide sequence 1 or the nucleotide construct 6 according to the invention.
- said vector 7 is a plasmid comprising the necessary elements (origin of replication ORI) for the transfection of said nucleotide sequence 1 or said nucleotide construct 6 into a cell, preferably into a Streptomyces sp . strain.
- the vector according to the invention can be also a shuttle vector comprising the necessary elements for the expression of said shuttle vector in E. coli and Streptomyces sp .
- Another aspect of the present invention is related to the cell such as a gram-positive bacteria, preferably a Streptomyces strain, transformed by said vector 7 or by said shuttle vector, which allows the expression of the isolated and purified nucleotide sequence 1 according to the invention controlling the activation of the xylanase promoter-operator nucleotide sequence 2 present in said cell and therefore the transcription of any nucleotide sequence 5 which could be cis-activated by said xylanase promoter-operator nucleotide sequence 2.
- nucleotide construct 6, the vector 7 and/or the cell transformed by said vector as well as specific portions of the isolated and purified nucleotide sequence 1 according to the invention can be advantageously used in several industrial biochemical processes such as production of antibiotics, malting processes of cereals, preparation of beers, baked or extruded cereals products, for the improving of animal feed stuff and for the improvement of paper and pulp technologies.
- the products of the invention are advantageously present in a bioreactor, and will allow the controlled synthesis of proteins or peptides of interest or possibly avoid or reduce the synthesis of said proteins or peptides by specific cells in the above-identified biochemical industrial processes .
- FIGS 1 to 3 represent the steps for the construction of the vector according to the invention.
- box 1 at -200 BP
- box 2 at -210 BP
- box 3 at -350 BP from the ATG codon.
- the box B3 is extremely conserved between the Streptomyces strain. (83% of identity of sequence upon 12 bases) .
- consensus nucleotide sequence is not present in other known xylanase nucleotide sequence of other bacteria such as Bacillus strains.
- the gene coding for xylanase C of Streptomyces sp has been cloned into a multicopy vector which confers positive xylanase phenotype when the host strain is under repression conditions.
- Repressed clones which may be a genomic fragment encoding the repressor according to the invention, will be characterised by a wild type phenotype .
- Repressors from a genomic bank in the vector pDML614 were isolated.
- Step 2 30 cycles 94 °C 30 sec
- Step 3 54 °C 1 min
- Step 4 72 °C 3 min 30 sec
- Step 5 72 °C 10 min
- Step 6 4 °C Table 2 Size of the insert
- a sequence of 1022 nucleotides obtained from the clone S6 allows the identification of an open reading frame with several bacterial regulator systems.
- a first polypeptide of 164 amino acids was identified and the corresponding nucleotide sequence was used as a probe for the isolation of the complete nucleotide sequence SEQ ID NO 1.
- the cloning of the carboxy terminal portion was obtained by Southern blotting. 2,5 genomic DNA of Streptomyces sp . EC3 are cleaved by several restriction enzymes and have been transferred upon a nylon membrane.
- a fragment of 720 BP has been amplified and labelled with biotine by PCR, and is used as a probe for the specific hybridisation of the genomic DNA.
- a portion of the genomic DNA of Streptomyces sp . was cleaved by restriction enzymes and the generated fragments by PCR were introduced in a plasmid pUC for sequencing.
- the sequenced nucleotide sequence comprises four open reading frames .
- the longest open reading frame hereafter called xlnR was implicated in the regulation of the xylanase enzyme, and the corresponding amino-acid sequence was identified by the BLAST software.
- the complete isolated and purified nucleotide sequence 1 according to the invention was introduced in a vector 7 having incorporated also a xylanase promoter- operator nucleotide sequence 2 linked to a gene encoding a xylanase enzyme 5.
- said xylanase promoter- operator nucleotide sequence 2 comprises a poly-linker sequence (nucleotide sequence with several cleaving sites) which improves the insertion of homologous or heterologous sequences.
- the characteristics of the vector according to the invention were improved by incorporating a specific marker (such as the thiostreptone) which is used for the specific selection of transformed cells.
- said shuttle vector was prepared according to the method comprising the following steps.
- the pUC18 polylinker was replaced by a dsDNA fragment containing endonuclease restriction sites and the following dsDNA fragment was entered in a Hi ⁇ dlll-EcoRI-digested pUC18 (L08752, Norrander et al . , Gene 26, pp. 101-106
- dsDNA fragment SEQ ID NO 5 : 5'- AGC TAG GCC TAT CGA TGG
- streptomycine/spectinomycine resistance gene (Str/Spm) from an omega interposon (Prentki, P. & Krisch, H.M. Gene 29 pp. 303-313 (1984)) was introduced at the HindiII restriction site.
- the pUC18 sequence was deleted from the construction and replaced by the C2aI-.Kp.nl Streptomyces replication origin from the pIJ702 vector (Katz et al . , J. Gen. Microbio. 129 pp. 2703-2714) .
- the construction was achieved to be a shuttle vector: a 1242 bp Asel -Ndel D ⁇ A fragment, containing the E. coli D ⁇ A replication origin from the pBR322 vector (J01749, Sutcliffe, J.G., Proc . ⁇ atl . Acad. Sci . U.S.A. 75(8), pp. 3737-3741 (1978)) was treated by klenow and introduced in EcoRV.
- the regulatory sequence xlriR was introduced in a Pad - Seal -digested vector and the xlnC structural gene with its promoter in the Ascl-Pstl restriction sites in order to obtain the shuttle vector "Vpro" according to the invention (see enclosed Fig. 3).
- the vector according to the invention may also comprise one or more mutations in the xylanase promoter-operator nucleotide sequence 2 in order to improve (increase) a cis-activating by said xylanase promoter-operator nucleotide sequence.
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- General Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
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- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention is related to an isolated and purified genetic sequence (1) controlling in trans the expression of a xylanase promoter-operator nucleotide sequence (2).
Description
NUCLEOTIDE AND/OR AMINO-ACID SEQUENCE CONTROLLING THE EXPRESSION OF A XYLANASE PROMOTE -OPERATOR NUCLEOTIDE
SEQUENCE
Field of the invention
The present invention is related to a new nucleotide sequence controlling in trans the expression of a xylanase promoter-operator nucleotide sequence, the amino-acid sequence encoded by said new nucleotide sequence, the vector comprising said new nucleotide sequence and the cell, preferably a Streptomyces strain, transformed by said vector.
Background of the invention
In beer production, efficient hydrolysis of xylans and other saccharides is important because said compounds can be involved in production problems such as wort viscosity (Ducroo, P. & Frelon, P.G., Proceedings of the European Brewery Convention Congress , Zurich, 1989, 445; Viέtor, R.J. & Voragen, A.G.J., Journal of the Insti tute of Brewing, 1993, 99, 243) and filterability and haze formation (Coote N. & Kirsop, B.H., Journal of the Insti tute of Brewing, 1976, 82, 34; Izawa, M. , Kano, Y. & Kanimura, M. , Proceedings Aviemore Conference on Mal ting, Brewing and Distilling, 1990, 427) .
In other areas, efficient hydrolysis of xylans and/or arabinoxylans is highly desirable as well . Examples include rye and wheat breadmaking processes, paper and pulp technologies (see US patent 5,116,746). It follows that a lot of research efforts have been devoted to the xylan hydrolysis enzymes due to their applications as described above.
Aims of the present invention The aim of the present invention is to provide a method and system which improve the control upon the expression of nucleotide sequence encoding enzymes such as xylanase, as well as homologous or heterologous sequences of said enzymes whose transcription is also activated by a xylanase promoter-operator regulatory sequence .
A specific aim of the present invention is to provide such a method and system for improving enzymatic processes, especially for improving production of antibiotics, malting processes of cereals such as barley, sorghum and wheat, production of beers, of baked or extruded cereals products, animal feed stuff, the production of starch derived from syrups, sorbitol, xylose and xylitol, and for the improvement of paper and pulp technologies.
Summary of the invention
The present invention is related to a new nucleotide sequence 1 which controls the expression of any xylanase promoter-operator nucleotide sequence 2. Said control upon the activation of a xylanase promoter-operator nucleotide sequence 2 is advantageously obtained by trans- activation (said new nucleotide sequence 1 encoding a
trans-activated factor which controls the activation of said xylanase promoter-operator nucleotide sequence 2) .
Therefore, the present invention is also related to said factor, preferably a peptidic factor 3 which is an activator and/or repressor encoded by said nucleotide sequence 1 and which controls positively and/or negatively the expression of a xylanase promoter-operator nucleotide sequence 2.
Advantageously, said factor could be present in a composition with other cofactors 4 that induce positively and/or negatively said mechanism.
Preferably, said cofactors 4 present in said composition are selected from the group consisting of glucose, xylan or a mixture thereof. The Inventors have discovered unexpectedly that the presence of glucose induces a repressive mechanism upon the activation of a xylanase promoter-operator nucleotide sequence, while the presence of xylan induces a positive mechanism of said expression. The simultaneous presence of said two cofactors in a medium induces also positively the expression of a xylanase promoter-operator nucleotide sequence.
It is meant by "a xylanase promoter-operator nucleotide sequence", any nucleotide sequence 2 which cis- activates any nucleotide sequence 5 encoding a xylanase enzyme .
A classification of the xylanase enzymes in the categories F/10 and G/ll is described by Henrissart et al. (Bioche . J. 293, pp. 781-788). Said xylanase promoter-operator nucleotide sequences comprise at least one 5 base pairs pattern : 5 ' - CGAAA-3 ' .
Preferably, said xylanase promoter-operator nucleotide sequence is the Strepto yces sp . strain EC3 xlnC
xylanase promoter-operator nucleotide sequence SEQ ID NO 2 also described by Giannotta F. et al . (FEMS Microbiol . Letters 142, pp. 91-97 (1996)).
According to a preferred embodiment of the present invention, the isolated and purified nucleotide sequence according to the invention is a (DNA) sequence which presents more than 60%, advantageously more than 80%, preferably more than 90%, and more preferably more than 95%, homology (i.e. sequence identity) with the nucleotide sequence SEQ ID NO 1 or its complementary strand described hereafter.
According to another preferred embodiment of the present invention, said isolated and purified nucleotide sequence corresponds to the nucleotide sequence SEQ ID NO 1 or its complementary strand or a portion thereof; preferably a sequence having more than 100 nucleotides and encoding a peptide which still controls positively and/or negatively the expression of a xylanase promoter-operator nucleotide sequence. Preferably, said sequence portion comprises at least the nucleotides of SEQ ID NO 3 or any nucleotide sequence encoding for its corresponding peptidic sequence.
According to a further preferred embodiment of the present invention, the terms "a portion of the nucleotide sequence SEQ ID NO 1 or its complementary strand" mean any kind of nucleic acid molecule (DNA, RNA, antisense nucleotide sequence, etc.) which is specific of SEQ ID NO 1, comprises more than 15 nucleotides (such as a probe or one or several primers) , and which may be used to identify, reconstitute or block the transcription of said specific isolated and purified nucleotide sequence SEQ ID NO 1 or its complementary strand. Said identification, reconstitution or blocking is obtained with known techniques by the person skilled in the art, such as the
use of antisense RNA, specific labelled probe hybridisation or genetic amplification, preferably by PCR (as described in the US patent 4,965,188) or by LCR (as described by Landgren et al . ( Sciences 241, pp. 1077-1080 (1988)). Therefore, the present invention is also related to any nucleotide sequence which presents an homology (i.e. sequence identity) as above-described with SEQ ID NO 1, SEQ ID NO 3 or their complementary strands, or any nucleotide sequence which preferably allows an hybridisation with SEQ ID NO 1, SEQ ID NO 3 or their complementary strands under standard stringent hybridisation conditions, and which may encode the same or a similar amino-acid sequence due to the redundancy of the genetic code. Exemplary standard stringent hybridisation conditions are as follows : hybridisation at 40 °C in 50% formamide, 5x SSC, 20 m ol sodium phosphate, pH 6.8, washing in 0.2x SSC at 50 °C . Variations in these conditions may occur based on the length and the GC nucleotide content of the sequence to be hybridised. Formula standard in the art are approved for determining exact hybridisation conditions such as the one described by Sambrook et al . (Molecular Cloning : A Laboratory Manual , Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, New York, §9.47-9.51 (1989)).
Another aspect of the present invention is related to the amino-acid sequence encoded by said nucleotide sequence, and which present more than 60%, advantageously more than 80%, preferably more than 90%, more preferably more than 95% homology (i.e. sequence identity), with SEQ ID NO 2.
According to another embodiment of the present invention, the amino-acid sequence according to the
invention corresponds to the amino-acid sequence of SEQ ID
NO 2 or any portion thereof having preferably more than
50 amino-acids and which is still capable of controlling
(positively or negatively) in trans the expression of a xylanase promoter-operator nucleotide sequence.
Preferably, said portion is an amino-acid sequence that comprises at least the amino-acid sequence encoded by the nucleotide sequence SEQ ID NO 3 above- described. It is meant by "controlling (positively and/or negatively) in trans the expression of a xylanase promoter-operator nucleotide sequence", the possibility for any nucleotide sequence 1 or any amino-acid sequence 3 encoded by said nucleotide sequence 1 to induce or reduce (preferably in the presence of the other cofactors 4 such as glucose and/or xylan) the expression of a xylanase promoter-operator nucleotide sequence 2 and obtain thereafter a control upon the cis-activation of a downstream nucleotide sequence 5 (for instance a gene encoding a xylanase enzyme) which is controlled in cis by said xylanase promoter-operator nucleotide sequence. The inducing or reduction of said expression is observed preferably by a positive or a negative modification of said cis-activation (for instance by an increasing or a decreasing of the synthesis of said xylanase enzyme by a cell) . Said mechanism is also illustrated in the enclosed Fig. 3.
The present invention is also related to a nucleotide construct 6 comprising the isolated and purified nucleotide sequence 1 according to the invention, linked to a xylanase promoter-operator nucleotide sequence 2 and possibly any homologous or heterologous nucleotide sequence 5 of a gene encoding a xylanase enzyme, which is cis-
activated by said xylanase promoter-operator nucleotide sequence 2.
Another aspect of the present invention is related to the vector 7 comprising said isolated and purified nucleotide sequence 1 or the nucleotide construct 6 according to the invention. Advantageously, said vector 7 is a plasmid comprising the necessary elements (origin of replication ORI) for the transfection of said nucleotide sequence 1 or said nucleotide construct 6 into a cell, preferably into a Streptomyces sp . strain.
The vector according to the invention may comprise also other elements, such as a marker (thiostreptone = tsr) for the identification of a possible transformation by the vector according to the invention in said specific cell. The vector according to the invention can be also a shuttle vector comprising the necessary elements for the expression of said shuttle vector in E. coli and Streptomyces sp .
Another aspect of the present invention is related to the cell such as a gram-positive bacteria, preferably a Streptomyces strain, transformed by said vector 7 or by said shuttle vector, which allows the expression of the isolated and purified nucleotide sequence 1 according to the invention controlling the activation of the xylanase promoter-operator nucleotide sequence 2 present in said cell and therefore the transcription of any nucleotide sequence 5 which could be cis-activated by said xylanase promoter-operator nucleotide sequence 2.
The nucleotide construct 6, the vector 7 and/or the cell transformed by said vector as well as specific portions of the isolated and purified nucleotide sequence 1 according to the invention can be advantageously used in several industrial biochemical processes such as production of antibiotics, malting processes of cereals,
preparation of beers, baked or extruded cereals products, for the improving of animal feed stuff and for the improvement of paper and pulp technologies.
The products of the invention, possibly combined with the above-described cofactors, are advantageously present in a bioreactor, and will allow the controlled synthesis of proteins or peptides of interest or possibly avoid or reduce the synthesis of said proteins or peptides by specific cells in the above-identified biochemical industrial processes .
The various aspects of the present invention will be described in details in the enclosed non-limiting examples in reference to the following figures.
Brief description of the drawings
Figures 1 to 3 represent the steps for the construction of the vector according to the invention.
Detailed description of the invention The alignment of various nucleotide sequences upstream xylanase gene in the strain Streptomyces sp . EC3 , shows the presence of three repetitive units of five BP : 5 ' -CGAAA-3 ' observed among all xylanase sequences (except in the strain Actino adura sp . which comprises only one repetitive unit) .
In the specific strain Streptomyces sp . EC3 , three boxes in the promoter-operator regions of 390 BP are defined : box 1 (Bl) at -200 BP, box 2 (B2) at -210 BP and box 3 (B3) at -350 BP from the ATG codon. The box B3 is extremely conserved between the Streptomyces strain. (83% of identity of sequence upon 12 bases) .
The identification of the repetitive consensus sequence is presented in the following table 1.
Table 1
Cons. Bl C G A A A C T G T T G A
Cons. B2 T T T C C G A A A G T T T G C C
Cons. B3 T C G A A A C T T T C G Consensus t C G A A A g c c
However, it seems that said consensus nucleotide sequence is not present in other known xylanase nucleotide sequence of other bacteria such as Bacillus strains.
The Inventors have discovered that the proteinic trans-activation factor according to the invention affects the regulation of said specific portions
(B3 > B2 > Bl) of the xylanase promoter-operator nucleotide sequence of the Streptomyces sp. EC3.
The Inventors have also discovered a modification of the trans factor affinity for the B2 box in repression and induction. Repression : B3 > B2 > Bl Induction : B3 >> B2 = Bl
Additional competitive experiments have identified as a preferred fixation site of the trans- activation factor according to the invention, the above- identified specific regions (boxes 3, 2 and 1) . It should be noted also that the above- described boxes present inverse repeated sequence and a palindrome of 4 BP that seems to be specifically recognised by the proteinic trans -activation factor according to the invention. Therefore, it seems that the main fixation site of said proteinic trans-activation factor is the box B3 , which allows thereafter a fixation upon the box B2 even when a mutation is present in said box B2.
According to said preliminary results, it seems that the control upon the activation of a xylanase gene is based upon operative sites which are specifically recognised by a trans-activation factor which is working as a repressor and which allows the formation of a repressive loop (connection between the B2 and B3 boxes by the trans- activation factor) and avoids the fixation of the RNA- polymerase and thereafter the transcription of a downstream coding nucleotide sequence. Genetic identification of the proteinic trans -activation factor and i ts encoding nucleotide sequence
The gene coding for xylanase C of Streptomyces sp . has been cloned into a multicopy vector which confers positive xylanase phenotype when the host strain is under repression conditions. Repressed clones, which may be a genomic fragment encoding the repressor according to the invention, will be characterised by a wild type phenotype .
Repressors from a genomic bank in the vector pDML614 were isolated.
After plasmid purification, an amplification by PCR allows a raw estimation of the insert size, which is presented in Table 2.
PCR conditions : Step 1 : 96 °C 4 min
Step 2 : 30 cycles 94 °C 30 sec
Step 3 : 54 °C 1 min
Step 4 : 72 °C 3 min 30 sec
Step 5 : 72 °C 10 min Step 6 : 4 °C
Table 2 Size of the insert
A sequence of 1022 nucleotides obtained from the clone S6 allows the identification of an open reading frame with several bacterial regulator systems. A first polypeptide of 164 amino acids was identified and the corresponding nucleotide sequence was used as a probe for the isolation of the complete nucleotide sequence SEQ ID NO 1. The cloning of the carboxy terminal portion was obtained by Southern blotting. 2,5 genomic DNA of Streptomyces sp . EC3 are cleaved by several restriction enzymes and have been transferred upon a nylon membrane. A fragment of 720 BP has been amplified and labelled with biotine by PCR, and is used as a probe for the specific hybridisation of the genomic DNA. A portion of the genomic DNA of Streptomyces sp . was cleaved by restriction enzymes and the generated fragments by PCR were introduced in a plasmid pUC for sequencing. The sequenced nucleotide sequence comprises four open reading frames . The longest open reading frame hereafter called xlnR was implicated in the regulation of the xylanase enzyme, and the corresponding amino-acid sequence was identified by the BLAST software.
The complete isolated and purified nucleotide sequence 1 according to the invention was introduced in a vector 7 having incorporated also a xylanase promoter- operator nucleotide sequence 2 linked to a gene encoding a xylanase enzyme 5. Advantageously, said xylanase promoter- operator nucleotide sequence 2 comprises a poly-linker sequence (nucleotide sequence with several cleaving sites) which improves the insertion of homologous or heterologous sequences. The characteristics of the vector according to the invention were improved by incorporating a specific marker (such as the thiostreptone) which is used for the specific selection of transformed cells.
The vector according to the invention was advantageously a shuttle vector comprising the necessary elements for the transfection of said vector in a
Streptomyces strain and in E. coli (see also U.S. patent
4,992,371 incorporated hereafter by reference).
Preferably, said shuttle vector was prepared according to the method comprising the following steps. The pUC18 polylinker was replaced by a dsDNA fragment containing endonuclease restriction sites and the following dsDNA fragment was entered in a Hiπdlll-EcoRI-digested pUC18 (L08752, Norrander et al . , Gene 26, pp. 101-106
(1983) ) . dsDNA fragment: SEQ ID NO 5 : 5'- AGC TAG GCC TAT CGA TGG
CGC GCC AAG CTA GCA ACT TAA GTA GAT CTA ACT AGT CTG CAG CAG
AAG CTT AAT ATT TAA TTA AGC GGC CGC AGT ACT CTC GAG CCG CCA
TGG GCC CGA TAT CGG TAC CAG GCC T- 3' (or SEQ ID NO 4)
(Endonuclease restriction sites: 5 ' - Clal -Ascl -Nhel -Aflll - Bgl11 - Spel - Pstl -Hindi11 -Sspl - Pad -Notl - Seal -Xhol -Ncol -Apal -
EcoRV-KpnI -3 ' ) .
Thereafter, the streptomycine/spectinomycine resistance gene (Str/Spm) from an omega interposon
(Prentki, P. & Krisch, H.M. Gene 29 pp. 303-313 (1984)) was introduced at the HindiII restriction site.
The pUC18 sequence was deleted from the construction and replaced by the C2aI-.Kp.nl Streptomyces replication origin from the pIJ702 vector (Katz et al . , J. Gen. Microbio. 129 pp. 2703-2714) .
The construction was achieved to be a shuttle vector: a 1242 bp Asel -Ndel DΝA fragment, containing the E. coli DΝA replication origin from the pBR322 vector (J01749, Sutcliffe, J.G., Proc . Νatl . Acad. Sci . U.S.A. 75(8), pp. 3737-3741 (1978)) was treated by klenow and introduced in EcoRV.
The regulatory sequence xlriR was introduced in a Pad - Seal -digested vector and the xlnC structural gene with its promoter in the Ascl-Pstl restriction sites in order to obtain the shuttle vector "Vpro" according to the invention (see enclosed Fig. 3).
For the analysis of an heterologous expression of foreigner genes in Streptomyces , the person skilled in the art may refer to the US patent 5,641,663 and the US patent 5,435,730.
Furthermore, the vector according to the invention may also comprise one or more mutations in the xylanase promoter-operator nucleotide sequence 2 in order to improve (increase) a cis-activating by said xylanase promoter-operator nucleotide sequence.
Claims
1. Isolated and purified genetic sequence (1) controlling in trans the expression of a xylanase promoter- operator nucleotide sequence (2) .
2. Isolated and purified genetic sequence according to claim 1, being a nucleotide sequence which presents more than 60% homology with the nucleotide sequence SEQ ID NO 1 or its complementary strand.
3. Isolated and purified genetic sequence according to claim 2, which presents more than 80%, preferably more than 90%, more specifically more than 95%, homology with the nucleotide sequence SEQ ID NO 1 or its complementary strand.
4. Isolated and purified genetic sequence according to any one of the preceding claims, being the nucleotide sequence SEQ ID NO 1, its complementary strain or a portion thereof having more than 100 nucleotides and encoding a peptide controlling positively and/or negatively the activation of a xylanase promoter-operator nucleotide sequence.
5. Isolated and purified genetic sequence according to claim 1, being an amino-acid sequence which presents more than 60% homology with SEQ ID NO 2.
6. Isolated and purified genetic sequence according to claim 5, being an amino-acid sequence which presents more than 80%, preferably more than 90%, more specifically more than 95%, homology with SEQ ID NO 2.
7. Isolated and purified genetic sequence according to claim 1, being the amino-acid sequence SEQ ID NO 2 or a portion thereof having more than 50 amino-acids which is capable of controlling positively and/or negatively in trans the expression of a xylanase promoter- operator nucleotide sequence.
8. Nucleotide construct (6) comprising the isolated and purified nucleotide sequence according to any one of the claims 1 to 4, linked to a xylanase promoter- operator nucleotide sequence (2) and possibly a nucleotide sequence (5) which is cis-activated by said xylanase promoter-operator nucleotide sequence (2) .
9. Vector (7), preferably a plasmid, comprising the isolated and purified nucleotide sequence (2) according to any one of the claims 1 to 7 or the nucleotide construct (6) according to claim 8.
10. Cell transformed by the vector according to claim 9 and which allows the expression of the isolated and purified genetic sequence according to any one of the claims 1 to 7.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9655698P | 1998-08-14 | 1998-08-14 | |
| US96556P | 1998-08-14 | ||
| PCT/BE1999/000105 WO2000009717A1 (en) | 1998-08-14 | 1999-08-12 | Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequence |
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| Publication Number | Publication Date |
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| EP1104476A1 true EP1104476A1 (en) | 2001-06-06 |
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| EP99939866A Withdrawn EP1104476A1 (en) | 1998-08-14 | 1999-08-12 | Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequence |
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| EP (1) | EP1104476A1 (en) |
| JP (1) | JP2002522086A (en) |
| AU (1) | AU5402799A (en) |
| BR (1) | BR9914311A (en) |
| CA (1) | CA2339414A1 (en) |
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| WO (1) | WO2000009717A1 (en) |
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| US6534286B1 (en) | 2000-06-15 | 2003-03-18 | University Of Georgia Research Foundation, Inc. | Protein production in Aureobasidium pullulans |
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1999
- 1999-08-12 EP EP99939866A patent/EP1104476A1/en not_active Withdrawn
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- 1999-08-12 AU AU54027/99A patent/AU5402799A/en not_active Abandoned
- 1999-08-12 WO PCT/BE1999/000105 patent/WO2000009717A1/en not_active Application Discontinuation
- 1999-08-12 CA CA002339414A patent/CA2339414A1/en not_active Abandoned
- 1999-08-12 BR BR9914311-9A patent/BR9914311A/en not_active Application Discontinuation
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| CA2339414A1 (en) | 2000-02-24 |
| WO2000009717A1 (en) | 2000-02-24 |
| BR9914311A (en) | 2001-10-16 |
| AU5402799A (en) | 2000-03-06 |
| IL141288A0 (en) | 2002-03-10 |
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