WO2000009717A1 - 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 sequence Download PDFInfo
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- WO2000009717A1 WO2000009717A1 PCT/BE1999/000105 BE9900105W WO0009717A1 WO 2000009717 A1 WO2000009717 A1 WO 2000009717A1 BE 9900105 W BE9900105 W BE 9900105W WO 0009717 A1 WO0009717 A1 WO 0009717A1
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- nucleotide sequence
- sequence
- operator
- isolated
- nucleotide
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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
- C12N15/74—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
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- 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)
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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)
- C12N9/24—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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Abstract
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000565151A JP2002522086A (en) | 1998-08-14 | 1999-08-12 | Nucleotide and / or amino acid sequence controlling expression of xylanase promoter operator nucleotide sequence |
BR9914311-9A BR9914311A (en) | 1998-08-14 | 1999-08-12 | Nucleotide or amino acid sequence that controls the expression of a xylanase promoter-operating nucleotide sequence |
CA002339414A CA2339414A1 (en) | 1998-08-14 | 1999-08-12 | Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequence |
AU54027/99A AU5402799A (en) | 1998-08-14 | 1999-08-12 | Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequence |
IL14128899A IL141288A0 (en) | 1998-08-14 | 1999-08-12 | Nucleotide and/or amino-acid sequence controlling the expression of a promoter-operator nucleotide sequence |
EP99939866A EP1104476A1 (en) | 1998-08-14 | 1999-08-12 | Nucleotide and/or amino-acid sequence controlling the expression of a xylanase promoter-operator nucleotide sequence |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US9655698P | 1998-08-14 | 1998-08-14 | |
US60/096,556 | 1998-08-14 |
Publications (1)
Publication Number | Publication Date |
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WO2000009717A1 true WO2000009717A1 (en) | 2000-02-24 |
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Application Number | Title | Priority Date | Filing Date |
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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 |
Country Status (7)
Country | Link |
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EP (1) | EP1104476A1 (en) |
JP (1) | JP2002522086A (en) |
AU (1) | AU5402799A (en) |
BR (1) | BR9914311A (en) |
CA (1) | CA2339414A1 (en) |
IL (1) | IL141288A0 (en) |
WO (1) | WO2000009717A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001096578A2 (en) * | 2000-06-15 | 2001-12-20 | University Of Georgia Research Foundation, Inc. | Protein production in aureobasidium pullulans |
-
1999
- 1999-08-12 EP EP99939866A patent/EP1104476A1/en not_active Withdrawn
- 1999-08-12 IL IL14128899A patent/IL141288A0/en unknown
- 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
- 1999-08-12 JP JP2000565151A patent/JP2002522086A/en not_active Withdrawn
Non-Patent Citations (3)
Title |
---|
DATABASE EMBL 7 April 1998 (1998-04-07), HAGEGE J M: "S.ambofaciens plasmid pSAM2 gene encoding KorSA", XP002122813 * |
GIANNOTTA F ET AL: "A sequence-specific DNA-binding protein interacts with the xlnC upstream region of Streptomyces sp. strain EC3", FEMS MICROBIOLOGY LETTERS, vol. 142, 1996, AMSTERDAM, NL, pages 91 - 97, XP000853721, ISSN: 0378-1097 * |
VAN PEIJ N N M E ET AL: "Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger", MOLECULAR MICROBIOLOGY., vol. 27, no. 1, January 1998 (1998-01-01), OXFORD., GB, pages 131 - 142, XP000853720 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001096578A2 (en) * | 2000-06-15 | 2001-12-20 | University Of Georgia Research Foundation, Inc. | Protein production in aureobasidium pullulans |
WO2001096578A3 (en) * | 2000-06-15 | 2002-05-10 | Univ Georgia Res Found | Protein production in aureobasidium pullulans |
US6534286B1 (en) | 2000-06-15 | 2003-03-18 | University Of Georgia Research Foundation, Inc. | Protein production in Aureobasidium pullulans |
Also Published As
Publication number | Publication date |
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JP2002522086A (en) | 2002-07-23 |
CA2339414A1 (en) | 2000-02-24 |
BR9914311A (en) | 2001-10-16 |
AU5402799A (en) | 2000-03-06 |
IL141288A0 (en) | 2002-03-10 |
EP1104476A1 (en) | 2001-06-06 |
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