WO2010018251A1 - Méthode pour améliorer l'expression des protéines dans des chloroplastes - Google Patents

Méthode pour améliorer l'expression des protéines dans des chloroplastes Download PDF

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WO2010018251A1
WO2010018251A1 PCT/ES2009/000385 ES2009000385W WO2010018251A1 WO 2010018251 A1 WO2010018251 A1 WO 2010018251A1 ES 2009000385 W ES2009000385 W ES 2009000385W WO 2010018251 A1 WO2010018251 A1 WO 2010018251A1
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protein
seq
chimeric
sequence
plants
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PCT/ES2009/000385
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Spanish (es)
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María Julieta DEL PRETE
Jon Mirena Veramendi Charola
Ignacio María MORENO ECHANOVE
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Fundación Para El Desarrollo De La Investigación En Genómica Y Proteómica
Plant Bioproducts, S.L.
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Publication of WO2010018251A1 publication Critical patent/WO2010018251A1/fr

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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention belongs to the field of technology of transgenic plants that express proteins. Specifically, it refers to a new method for increasing the expression of proteins in plants, preferably cellulolytic enzymes and more specifically focusing on the production of the CeIY and CeIIZ endoglucanases of
  • Dickeya dadantii in tobacco chloroplasts by means of the integration of genes in the genome of the plant chloroplast.
  • the present invention is useful in any industrial process that needs to use a large amount of enzymes, for example in the conversion of cellulose biomass into ethanol.
  • Plants have great potential for use as biofactories to obtain various products of commercial interest.
  • the potential of the plants lies in the possible reduction in production costs and in the scalability of production.
  • the present invention relates to the production of enzymes in genetically modified plants (PGMs) for industrial use and has focused on the production of a series of cellulases, enzymes involved in the hydrolysis of cellulosic biomass, which have various applications in the industries of fuels, food, animal production, textile and paper.
  • PGMs genetically modified plants
  • a method for the expression of enzymes, and in general of any protein, in plants is based on the integration of genes in the chloroplast (Svav et al., 1991, Svav and Maliga, 1993).
  • Numerous examples of stable gene integration in tobacco chloroplasts with very high gene expression levels have been described (For example: McBride et al., 1995, Khan and Maliga, 1999, Kota et al. ,, 1999, Staub et al. ., 2000, Kuroda and Maliga, 2001a and 2001b, Lutz et al., 2001, Ye GN et al., 2001).
  • Cellulases are a group of enzymes that catalyze the hydrolysis of cellulose, transforming it into glucose. Fundamentally cellulases are produced in fungi, bacteria and protozoa although they are also found in plants and animals.
  • denominating cellulases are: endoglucanases, endo-1,4-beta-glucanase, CMCase, carboxymethyl cellulase, endo-1,4-beta-D-glucanase, beta-1, 4- glucanase, beta-1, 4-endoglucan hydrolase, celudextrinase, and avicelase.
  • WO 00/05381 A2 describes a construct that can be produced in the cells of a plant and encodes an enzyme capable of degrading cellulose.
  • a method for altering the cellulose content in a plant's tissue comprises growing a plant whose cells have been transformed with a construction that has the following components that operate together in the 5'a to 3 ' direction of transcription: i) a functional promoter in a plant cell; i ⁇ ) a DNA sequence encoding Acidthermus cellulolyticus E1 cellulase; and iii) a region of termination of the transcription, under the conditions in which said enzyme is expressed in the cells of the plant, resulting that the cellulose of said plant partially degrades and, therefore, decreases its content.
  • the method describes the expression of cellulase E1 in plastids of plants for the production of enzymes that hydrolyze the polysaccharides, specifically in chloroplasts.
  • plants have been obtained that accumulate the catalytic subunit of E1 in the tobacco apoplast so that it constitutes up to 1.6% of the total soluble protein (Ziegelhoffer and col., 2001). It is an acceptable level of accumulation but insufficient for some industrial applications and has the disadvantage that, in order to reach this level of expression, the cellulase binding domain of the E1 gene was eliminated, which may reduce its usefulness in certain applications.
  • Ziegglehoffer et al. 2001 Jin et al.
  • PGMs were obtained in which the product of the E1 gene is produced in the cytoplasm and accumulates in the chloroplast, but no significantly elevated levels of expression were obtained.
  • Ziegler et al. (2000) obtained Arabidopsis thaliana plants that accumulate, on average, 7.5% of endoglucanase E1 but this plant species is not an appropriate host for large-scale enzyme production, due to the low capacity of biomass production
  • Corn plants have also been obtained that accumulate up to 2.1% of endoglucanase E1 in corn (Biswas et al., 2006) that have an interest in being one of the most widespread crops and therefore, an important source of biomass for The production of enzymes or their transformation into fuels.
  • the works carried out with other genes that encode hydrolytic enzymes integrated in the nuclear genome of the plant have not provided encouraging results (Ziegelhoffer 1999, Dai, 1999).
  • WO 01/16338 A2 refers to the production of cellulases using gene constructs whose expression gives rise to a fusion protein comprising endoglucanase E1 and a transient peptide. Said plants produce cellulase by means of the expression of the gene construct.
  • the document mentions that the cellulase produced can be collected and purified but does not mention the amount or the enzymatic activity thereof.
  • Patent document WO 03/012094 A1 describes a process for the production of thermostable xylanase in plants (XynA of Thermospora fusca) that is based on the expression of the gene in tobacco chloroplast.
  • XynA of Thermospora fusca thermostable xylanase in plants
  • XynA of Thermospora fusca thermostable xylanase in plants
  • xylanase accumulates in tobacco plants and constitutes approximately 11% of the soluble protein, mentioning that xylanase is recovered from an extract that has undergone heat treatment, denaturing at least part of the tissue proteins but maintaining Stable xylanase, recovering significant enzymatic activity after drying the plant material.
  • the chloroplast integration vectors contain segments of DNA derived from the chloroplast that determine the place of integration of the gene of interest in the chloroplast genome (cpDNA) by homologous recombination. These sequences flank the genes of interest so that they are integrated into the cpDNA after recombination.
  • the transformation vectors contain an additional gene necessary for the positive selection of cells containing recombinant cpDNA. The most commonly used is the aac / A gene, which provides resistance to spectinomycin and streptomycin (Svav and Maliga 1993). The expression of exogenous genes requires that these be operatively linked to a series of DNA sequences that function as expression control elements in the chloroplast.
  • Such elements include, at least, a transcription promoter that contains elements recognized by the chloroplast RNA polymerases (whether of nuclear or plastidial origin) that determine the beginning of the transcription, a messenger mRNA leader sequence immediately prior to the coding sequence of the gene of interest, a triplet of initiation of the translation in reading phase with the coding sequence of the gene of interest, a triplet of termination of the translation, and a non-coding region containing transcription termination signals recognized in the chloroplast.
  • the most commonly used gene expression control elements derive from gene sequences of the chloroplast itself (Svav and Maliga, 2003 and others).
  • the accumulation of an enzyme of interest in the chloroplast depends on a number of factors, including the following: i) efficient transcription of the gene and stability of the messenger RNA (mRNA), ii) correct onset and translation into the triplet corresponding iii) speed of elongation during translation, iv) adequate folding of the protein in the stroma of the chloroplast and v) stability of the product in the stroma.
  • mRNA messenger RNA
  • ii) correct onset and translation into the triplet corresponding iii) speed of elongation during translation iv) adequate folding of the protein in the stroma of the chloroplast and v) stability of the product in the stroma.
  • the leader sequence of the tobacco psbA gene promotes the efficient translation of exogenous mRNAs in vitro (Hirose Si Sugiura, 1996) and in vivo, and results in the efficient expression of a series of genes integrated in the tobacco cpDNA (Staub and Maliga 1994, Eibl et al., 1999, Daniell et al., 1998, Kota et al., 1999, Daniell et al., 2001, De Cosa et al. 2001).
  • the expression levels achieved are variable depending on the gene of interest and efficient expression of the introduced gene is not always observed (Whitney et al., 2001).
  • sequences derived from genes of the chloroplast itself (Kuroda and Maliga, 2001b), of the green fluorescent protein (GFP) of the jellyfish (Ye, Staub et al., 2001) mentioned below, or simply a synthetic sequence encoding a pentapeptide (Herz et al. 2005).
  • the international patent application WO 01/04331 A2 (hereinafter D1) and the corresponding non-patent literature article (Ye, Staub et al., 2001) describe nucleic acid sequences useful for promoting the expression of a wide variety of genes , both eukaryotic and prokaryotic.
  • the invention focuses on the translational fusion of 14 amino acids derived from the green fluorescent protein (GFP) (Seq Id No. 26) of jellyfish to the N-terminal domain of a protein.
  • GFP green fluorescent protein
  • the document presents the invention as useful for increasing the level of expression of any enzyme, in particular, it performs the translational fusion of said sequence of the GFP to the EPSPS enzyme, resulting in high levels of expression of said protein; It presents a construction comprising the DNA sequence corresponding to the 14 amino acids of the GFP (Seq Id No. 27) fused to the N-terminal domain of the C4 protein gene. Said construction is expressed in plastids.
  • the high levels of expression obtained through the invention refer to levels of more than 10% of the total soluble protein.
  • the patent application claims the cell that comprises at least, approximately 12% of protein of interest, of the total soluble protein of the cell.
  • the publication corresponding to the patent application in addition to the above, refers to the adaptation of the codons of the CP4 gene to the codons of the chloroplast. To do this, they carry out an inspection of the native sequence of CP4, observing that it only has 45% of codons preferred by plastids. In order to verify the effect of the use of preferred codons for expression in plastids, they construct a synthetic CP4 gene that predominantly has (77%) codons preferred by plastids. The results obtained demonstrate that the optimization through the use of Plastid codons results in an increase in protein accumulation of approximately 10% more (that is, about 50 times more than without codon optimization).
  • the elongation rate may depend on the triple bias of the gene that is intended to be expressed, on the GC pair content of the DNA and on the secondary structure of the mRNA, which in turn is a determinant of messenger stability (Stern et al., 1991).
  • the efficient expression of exogenous genes in a heterologous organism may require the adaptation of the primary sequence of the gene to the characteristic triple bias of the host organism and the alteration of structural elements of the mRNA.
  • the bias in the use of triplets of an organism is an indicator of the relative frequency of the different triplets that encode an amino acid or a termination signal.
  • triplet bias there have been significant differences in the bias in the use of triplets between different organisms, which are in turn related to the frequency of nucleotides in the DNA (frequency of GC or AT pairs in the DNA). In some cases it has been observed that the relative frequency of "rare" triplets is limiting in the efficiency of the translation, but in other cases it is not. This effect of the triplet bias used may be related to the frequency of complementary transfer RNAs (tRNAs) in the cytoplasm (reviewed by Gustafsson et al., 2004).
  • tRNAs complementary transfer RNAs
  • genes of different origins human, bacterial, etc.
  • triplet use bias is not an important determinant for gene expression in this system, but the absence of detectable expression from others.
  • genes may be related to factors such as the content in GC pairs and / or the secondary structure of the mRNA, which can influence the elongation of the nascent protein during translation.
  • fusion products such as those mentioned above (Ye, Staub et al., 2001) may also have combined effects on the expression and stability of the product.
  • the best example that demonstrates the importance of product stability is that of the accumulation of an insecticide in tobacco chloroplasts (De Cosa et al., 2001).
  • the high accumulation of the product in this specific case is due to the stabilization of the insecticide thanks to the co-expression of a chaperone that facilitates the folding of the insecticide.
  • the solution provided by the present invention focuses on the translational fusion of a modified version (with low GC pair content) of the first 15 amino acids of the green fluorescent protein (GFP) (Seq Id No. 28, 29), as well as The fusion of the tetramerization domain of the p53 protein (Seq Id No. 35, 36) (fusion that can be performed between functional domains or at the C-terminal end), to the sequence of the protein in question whose production is to be increased.
  • chloroplasts are transformed that will express the protein of interest in an increased way and with improved stability so that in mature tissues the product of interest can be the most abundant extractable protein, said protein being recovered in a range between 25-30 % of the total extractable protein of the plant.
  • any protein of interest preferably an enzyme, of animal or plant origin, is understood as the enzymes involved in the hydrolysis of cellulose and other components of the plant cell wall - endoglucanses, cellobiohydrolases, betaglucosidases, endoxylanases, arabinofuranosidases etc-, and other enzymes for industrial use such as proteases, phytase, lipoxygenase, laccase, phytase and others.
  • the present invention relates to a method for increasing the production of functional proteins, and more specifically functional enzymes in chloroplasts.
  • D1 describes the strategy of carrying out the translational fusion of the first 14 amino acids of the sequence of the GFP (Seq Id No. 26) to the N-terminal domain of a protein, achieving expression levels of said protein in plants (specifically tobacco plants) of the order of 12% of the total soluble protein of the cell, and that in document D2, also mentioned above, the fusion of a fragment of 41 amino acids (TD) is presented ( Seq Id No. 35) which contains the tetramerization domain of the human transcription factor p53 to a 21 amino acid sequence of the 2L21 protein derived from VP2 of the canine parvovirus, thus being achieved (in A.
  • document D2 does not indicate possible effects of the tetramerization domain of p53 (Seq Id No. 35) on any functionality of the protein of interest other than its use as an antigen, or other possible effects of said domain on the expression of genes in the chloroplast.
  • the results of document D2 indicate the stabilization of peptides of small size, and therefore, highly susceptible to proteolytic degradation, but their results are not directly extrapolated to the production of larger proteins and with stable structural domains such as CeIYo endoglucanases.
  • the proteins / enzymes obtained in the present invention have improved product stability properties during the reaction, indicating that they are new products, with differentiated properties with respect to native enzymes.
  • the chimeric DNA sequence of interest (sequence of the gene of interest simultaneously fused to the modified version of the sequence coding for the first 15 amino acids of the GFP (Seq Id No. 28) and the tetramerization domain of p53, Seq Id No. 35) is operatively linked to the transcription promoter (Seq Id No. 2), leader sequence (Seq Id No. 3) and transcription termination sequence derived from the psbA gene.
  • the method that constitutes the object of the invention involves the transformation of the plant cells with the aforementioned gene constructs, the subsequent growth of the cells and / or plants and the recovery of the chimeric protein expressed in the chloroplasts.
  • Said methodology promotes the expression (efficiency of the translation) as well as the stabilization of the products in the plant by means of the expression of genes introduced in a genetically manipulated organism, specifically the manipulation by introduction of genes in the genome of the plant chloroplast.
  • the plants of the invention have an increase in the accumulation in chloroplasts of the protein in question of up to 30% of the TSP, which is greater than the sum of the increments obtained through the individual fusion strategies of said sequences.
  • An object of the present invention relates to the nucleotide construction characterized by comprising a.
  • a chimeric DNA sequence comprising a DNA or protein sequence simultaneously fused to two synthetic fragments of coding DNA that consist of i) a modified version of the sequence encoding the first 15 amino acids of the GFP (Seq Id No. 28) and ii) the sequence coding for the tetramerization domain of p53 (Seq Id No. 35).
  • the protein is an enzyme, preferably a cellulase, more preferably an endoglucanase and in particular CeIY and / or CeHZ, or said proteins lacking the secretion peptide.
  • the synthetic DNA fragments encoding stage a) have a low GC pair content, preferably the GC content is 40%.
  • the sequence coding for the first 15 amino acids of the GFP consists of Seq Id No. 28, being carried out at the N-terminal end of the protein or DNA sequence whose expression is to be increased, and the The tetramerization domain sequence of p53 consists of Seq Id No. 35, the fusion being performed within the separation arm, replacing the separation arm or in C-terminal, specifically at the C-terminal end when the enzyme to be expressed does not it presents separate functional domains and in the separation arm or in replacement thereof when the enzyme to be expressed has separate functional domains.
  • the promoter (Seq Id No. 2) and leader sequence (Seq Id No. 3) are those of the psbA gene.
  • Another object of the invention relates to the synthetic fragment of coding DNA, characterized in that it consists of a modified version of the sequence coding for the first 15 amino acids of the GFP, specifically (Seq Id No. 28)
  • Another object of the invention refers to the synthetic fragment of coding DNA, characterized in that it consists of the tetramerization domain of p53, specifically Seq Id No.35
  • Another object of the invention relates to the method for increasing protein expression in plants, by integrating genes into the genome of the plant chloroplast characterized in that it comprises the steps of:
  • the chimeric proteins whose expression has been increased remain functionally active and the increase in gene expression is greater than that obtained through the sum of the increments obtained by fusing the synthetic fragments of coding DNA (GFP and p53).
  • the accumulation of the protein of interest expressed is 25-30% of the TPS.
  • said increase is at least two orders of magnitude greater than when expressing the native gene product, twice greater than when expressing the gene product fused to GFP, and 10 times greater than when expressing the gene product fused to p53.
  • Another object of the invention relates to the cell, specifically a plant cell characterized by comprising the nucleotide construction defined above.
  • the cell has been obtained through the method of the invention.
  • the chimeric protein expressed from the chimeric DNA sequence of the nucieotidic construction corresponds to a percentage between 25% and 30% of the total soluble protein comprised in said cell.
  • Another object of the invention relates to the transgenic plant comprising the nucieotidic construction of the invention, specifically that obtained by the method of the invention.
  • the transgenic plant presents as a removable majority product the protein of interest encoded by the chimeric DNA sequence of the described nucieotidic construction, preferably between 25% and 30% of the total soluble protein, said accumulation occurring in all the leaves of the plant regardless of their maturation state, the accumulation in mature leaves being especially greater, between 5 and 10% higher in terms relative to the total protein content than in the young leaves.
  • Another object of the invention relates to the seed produced by the described plants.
  • Another object of the invention relates to the chimeric protein obtained by the described method.
  • said chimeric protein is an enzyme, preferably chimeric CeIY (Seq Id No. 30) or Chimeric Ce1 1 Z (Seq Id No. 43).
  • Another object of the invention relates to the use of the plants described as biofactories, for the mass production of enzymes of industrial interest, in the biodiesel industry.
  • Another object of the invention relates to the use of the synthetic fragment that encodes p53 for obtaining chimeric fusion products in the chloroplast whose expression is more stable with respect to variants that lack this fragment.
  • Another object of the invention relates to the use of the synthetic fragment derived from GFP to increase gene expression and accumulation of the product in the chloroplast.
  • Another object of the invention relates to the described method, for the production of chimeric proteins in genetically modified plants (PGMs).
  • Said proteins are characterized by having improved stability properties, and activity on an insoluble substrate with respect to the native enzyme.
  • said chimeric protein is Chimeric CeIY (Seq Id No. 42) or Chimeric CeMZ (Seq Id No. 56 ).
  • said chimeric enzymes do not need to be purified.
  • Another object of the invention relates to the use of the method in the fuel, food, animal production, textile and paper industries.
  • Another object of the invention relates to the use of the method for generating second generation bioethanol.
  • Another object of the invention relates to the use of the CeIy and / or CeI 1 Z chimeric proteins for the conversion of cellulose into ethanol.
  • CeIY secretion peptide sequence (simple underlined bold)
  • CeI Y non-coding region (simple) Ce / Z secretion peptide sequence (bold, italic, simple underline)
  • Fig 1 Shows sequences of the intermediate vectors used, the complete sequence of the vector pPLAg (A1), and the partial sequences of pPLAg (A2), pPLAf (B) and pPLA (C), indicating in each of them the part of The sequence that represents the control elements of the expression.
  • psbA promoter silica
  • psbA leader sequence silica
  • Restriction sites silic
  • Kanamycin resistance gene underline interspersing single dots and dashes
  • starting site of Ia translation gray shading
  • the modifications correspond to GFP derived sequence (double underlined bold), ferredoxin derived sequence (interrupted underlined bold).
  • Fig 2 Oligonucleotides used in the different stages of development.
  • A Shows the different oligonucleotides used for the construction of pPLA, pPLAg and pPLAf, distinguishing between those used for A.1 amplification of the promoter and leader sequence of the psbA gene, A.2 construction of the GFP synthetic linker and A.3 of Ia ferredoxin
  • B Shows the oligonucleotides used to perform the gene integration analysis by PCR.
  • C and D show the different oligoucleotides used for the constructions with CeIY and CeMZ respectively.
  • Fig 3 Homoplasmic analysis by RFLP (restriction fragment polymorphism) in 1% agarose.
  • Fig 4 Differences in the sequence of the GFP used in Ye et al. (2001) and in this work. Positions that differ are represented in lowercase. ATG (gray shading) Represents the start codon of the translation.
  • Fig 5 PCR product sequence of the CeIY gene (999 nucleotides) cloned in pGEM-Easy. In this sequence, different areas stand out: Start (ATG) and end of translation (ATT) codons, transciption start (A) (gray shading), CeIY secretion peptide sequence (bold underlined simple) (nucleotides 1-69 ), Sequence of the mature enzyme CeI Y (nucleotides 70-996) (bold). The rest, the first 7 nucleotides and the last 100 represent non-coding regions that flank the CeIY open reading frame.
  • Fig 6 Represents the different CeIY constructs in intermediate plasmids.
  • Promoter psbA (simple underlined italic), Leader sequence psbA (simple italic), Restriction sites (Simple underline), Start codons (ATG) and end of translation (ATT), start of transciption (A) (gray shading), CeI Y catalytic domain sequence (first 27 nucleotides and last 23) (bold), GFP coding sequence (amino acids 2-15) (double underlined bold), ferredoxin coding sequence (amino acids29-48) (bold underlined interrupted), a sequence coding for the tetramerization domain of p53 (wavy underlined bold) separated from the last 23 amino acids of the CeI Y catalytic domain by
  • Fig 7 Visulization of Yf, Yg and Yt in 10% acrylamide.
  • Fig 8 Comparison of the accumulation of Yg and Ygt in leaves of different ages in A) gels with coomassie blue staining and B) zymogram.
  • the streets of the zymogram (B) correspond to the streets that have been used in the gel (A).
  • the most intensely stained protein is the chloroplast ribulose carboxylase (RUBISCO) with an apparent molecular weight of 54kDa.
  • Fig 9 Sequence (A) and mature protein (B) of CeHZ and its different functional domains respectively.
  • Start ATG
  • end of translation ATT
  • transciption start A
  • CeI Z __ secretion peptide sequence nucleotides 4-129
  • CeI Z catalytic domain nucleotides 130-996
  • CeI Z cellulose binding arm nucleotides 1100-1282
  • Fig 10 DNA sequence (A) and (B) sequence deduced from chimeric Endoglucanase Z amino acids and their different functional domains respectively.
  • the following domains stand out: Start (ATG) and end of translation (ATT) codons, transciption start (A) (gray shadow), CeI Z secretion peptide sequence (bold, italic, simple underline), Catalytic domain CeI Z (bold, italic), tetramerization domain (bold wavy underline), CeI Z cellulose binding arm, cellulose binding domain (bold, italic, dotted underline), Restriction sites (Simple underline).
  • Fig 11 Zimogram of plant extract expressing Zg and Zgt separated by 8% acrylamide-SDS. It is observed, both in Zg and Zgt extracts several bands with catalytic activity, some with an electrophoretic mobility lower than the expected molecular weight of the endoglucanase Z. In the case of Zgt, and not in Zg, discrete bands of high molecular weight endoglucanase activity (indicated with white arrows) are detected that may correspond to multimeric complexes of Zgt stable during the extraction and electrophoresis.
  • Fig 12 Endoglucanase activity on cellulose. This graph compares the reaction kinetics of the Zg and Zgt chimeric proteins at 37 0 C. The reaction is quantified by measuring reducing sugars released by the Nelson-Somogyi method. The values represented by each correspond to the average of 3 independent reactions.
  • Fig 13 Nucleotide sequence of the chimeric Ygt Endoglucanas. This sequence highlights: GFP coding sequence (nucleotides 1-48) (double underlined bold), Start (ATG) and end of translation (ATT) codons, transciption start (A) (gray shadow), Catalytic domain sequence CeI Y (nucleotides 52-980) (bold), Restriction site (Simple underline), Sequence coding sequence of the tetramerization domain of p53 (nucleotides 988-1107) (bold underlined wavy).
  • Fig 14 Nucleotide sequence of the chimeric Endoglucanase Zgt. This sequence highlights: Start (ATG) and end of translation (ATT) codons, transciption start (A) (gray shading), GFP coding sequence (nucleotides 1-48) (double underline bold), Peptide sequence of CeI Z secretion (bold, italic, simple underline), CeI Z catalytic domain (nucleotides 52-918) (bold, italic), CeI Z separation arm (nucleotides 919-969) (bold, italic underlined by collating dots and dashes ), tetramerization domain (nucleotides 976-1095) (wavy underlined bold), CeI Z separation arm (nucleotides 1096, 1146) (bold, italic underlined by inserting dots and dashes), CeI Z cellulose binding arm (bold, italic, dotted underline), Restriction sequence (S
  • the technology that is intended to be patented refers to the genetic manipulation of plants, specifically the manipulation by introducing genes into the genome of the plant chloroplast.
  • the development has been carried out using tobacco (Nicotiana tabacum var. Petit Havanna) as a model plant.
  • the present invention describes the combination of two methods to increase the production of proteins in the chloroplast.
  • the invention preferably relates to enzymes, more preferably endoglucanases by carrying out practical embodiments specifically with CeIY and CeHZ.
  • One of the methods is based on strategies to increase the expression of genes in the chloroplast and the second is based on strategies that increase the stability of the proteins produced in plants.
  • the methods described apply to the production of proteins, preferably enzymes, and particularly endoglucanases, the method is applicable for the production of any protein that remains functionally active.
  • pAF an integration vector in the chloroplast called pAF was used, which directs the integration in the tobacco chloroplast between the trnl and trnA genes (Fernandez-San Millán et al, 2008).
  • the genes of interest are first cloned into an intermediate plasmid, called pPLA (Seq Id No. 1), so that they are operatively linked to the promoter (Seq Id No. 2) and the leader sequence (Seq Id No. 3) of the gene tobacco psbA (FIGURE 1a).
  • the sequence corresponding to the promoter and leader of psbA represents nucleotides 1594-1780 of the genome of tobacco chloroplast (accession Z00044).
  • the pPLA vector (Seq Id No. 1) was constructed by amplification by the reaction of the chain polymerase (PCR) of fragment 1594-1780 of the chloroplast using as a template a preparation of tobacco genomic DNA and the oligonucleotides Ppsba-D (Seq Id No. 6) and Lpsba-R (Seq Id No. 7) (FIGURE 2).
  • the PCR product was cloned into plasmid pGEM-T Easy (Promega) following the manufacturer's instructions and its sequence was determined.
  • the sequence corresponding to the psbA promoter is shown and the transcription start site and the translation start site that is included in a sequence representing the restriction target ⁇ / col are marked.
  • the expression control elements derived from psbA promote the efficient expression of exogenous genes in vivo (for example, De Cosa et al. 2001, Fernandez-San Millán et al. 2008) and in vito (Hirose & Sugiura, 1996).
  • the cassette consisting of the gene of interest, the promoter and the leader sequence of subclone psbA in the pAF vector, between the aadA gene and the psbA transcription terminator, forming an operational transcriptional unit.
  • Chloroplast gene integration experiments are performed as described in Svab and Maliga, 1993. It is based on a DNA preparation of the vector with the gene of interest and covered with a suspension of gold particles of 0.6 you love The particles are fired on leaves of tobacco plants grown in sterile conditions. The leaves subjected to firing with gold particles are grown in RMOP medium, in the presence of spectinomycin (500 mg / Iiter), and green shoots, resistant to spectinomycin, are selected that grow between 3 and 8 weeks after gene firing.
  • spectinomycin 500 mg / Iiter
  • the outbreaks that are obtained in this first round of selection are usually heteroplastic (they contain cells with recombinant chloroplastic DNA next to cells with unmodified chloroplastic DNA, Svab and Maliga, 1990 and 1993), and are subjected to a second round of selection between RMOP with spectinomycin (500 mg / liter).
  • the shoots obtained in the second round of selection are grown in sterilized rooting medium and in the presence of spectinomycin at 500 mg / liter.
  • the rooting medium is Murashigue-Skooge pH5.7 with 30% sucrose)
  • the seedlings grown in vitro are passed to the ground and the following analyzes are carried out: i) the integration of the vector into the chloroplast through the reaction of the chain polymerase (PCR) on DNA extracts of the plant, using an oligonucleotide that annuls in the cDNA but not in the DNA of the pAF vector next to a second oligonucleotide that annuls in pAF but not in the cpDNA (Respectively oligonucleotides Int-R (Seq Id No. 13) and Int-D (Seq Id No. 12), FIGURE 2).
  • PCR chain polymerase
  • the CMCase activity is quantified by measuring reducing sugars released by the Nelson Somogyi method.
  • the expression of endoglucanases is also analyzed by zymograms.
  • the zymograms consist of visualization of the endoglucanase activity of previously electrophoretically separated proteins. After separation, the gels are incubated in the presence of 1% CMC and stained with Congo red, so that the protein bands corresponding to endoglucanases are visualized as clear areas in a gel stained red.
  • CeIY and CeHZ genes of Dickeya dadantii have been cloned in the transformation vector (pAF) operatively linked to the promoter (Seq Id No. 2), leader sequence (Seq Id No. 3) and termination sequence of The transcription of the tobacco psbA gene.
  • pAF transformation vector
  • Other vectors and promoters known from the state of the art can be used.
  • Modified versions of the native genes have been cloned, lacking the region encoding the secretion peptide (nucleotides +4 to +69 of the open reading frame of CeIY and nucleotides +4 to +130 of CeHZ).
  • the function of the secretion peptide in Dickeya dadantii is to promote the secretion of the product to the culture medium in a process that includes the proteolytic elimination of said peptide and which gives rise to the mature peptide. Since the accumulation in the stroma of the chloroplast does not require any secretion process, only the sequences corresponding to the mature peptides were cloned following the translation triplet. As a control, a construct containing the complete native CeIY gene was obtained. This construction resulted in plants selected by In vito cultivation but the shoots proved unfeasible in the absence of an added carbon source.
  • CeHZ Ceq Id No. 43 native, without secretion peptide, integrated into the chloroplast.
  • the recoverable endoglucanase activity of these plants is very low (CeIY) or undetectable (CeIIZ), indicating that the accumulation of functional product is very reduced in both cases.
  • the inefficient accumulation of CeIY and CeHZ may be related to an inefficient expression (translation), with the instability of the product expressed in the chloroplast or with both factors.
  • the efficient accumulation of genes in the chloroplast may therefore require the development of the methods proposed to stimulate the genetic expression and increase the stability of the product.
  • the first fragment used represents the first 15 amino acids of the green fluorescent protein of the jellyfish (GFP) (Seq Id No. 28), and resembles the fragment used in Ye et al (2001) (Seq Id No. 26) with Ia difference that the fragment used in the present invention has a composition of CG pairs (40%) similar to that of tobacco chloroplast (FIGURE 4).
  • This fragment differs from the fragment used by Ye et al. 2001 in 15 positions on 45 total nucleotides, in the GC content (40% vs. 62%), and in the first amino acid it encodes, which has been altered (glycine by serine) to be able to introduce a restriction target ⁇ / col suitable for cloning CeIY and CeHZ (FIGURE 4).
  • the second synthetic fragment that is proposed encodes a peptide derived from tobacco ferredoxin, also adapted to the use of triplets and the GC pair content of tobacco chloroplast (FIGURE 1).
  • the two fragments described in this invention are synthetic. They were constructed using two pairs of phosphorylated and complementary oligonucleotides.
  • Figure 2 were incubated at a concentration of 50 pmol / microliter in 2OmM TrispH ⁇ .O, 1mM EDTA for 2 minutes at 100 0 C and 30 minutes at 37 0 C to facilitate banding.
  • the banding of each pair causes a double stranded DNA fragment with cohesive ends compatible with those left by the ⁇ / col restriction target and phosphorylated.
  • These fragments were ligated to plasmid pPLA (Seq Id No. 1) digested with Ncol using Rapid DNA Ligation Kit (Roche Cat No. 11635379001) obtaining the intermediate vectors pPLAg (Seq Id No. 4 and 57) and pPLAf (Seq Id No.
  • the present invention describes the use of an element, the tetramerization domain of the p53 protein, hereinafter referred to as DTp53 (Seq Id No. 35), to favor the stability of enzymes produced in plants, similar to the use of the same domain to favor the accumulation of an antigen in plants as described in patent D2.
  • the tetramerization domain favors the formation of multimeric structures (dimers and tetramers) by the interaction established between the DTp53 domains of different subunits. This interaction gives rise to very stable complexes whose structure has been analyzed in detail in the case of p53, and which expose the different effector domains of the protein in spatially defined positions (Tidow et al. 2007).
  • CeHZ The mature CeHZ peptide, and that of many other cellulases, is constituted by structural and functional domains separated by short peptide arms (spacer arm) that separate these domains and, probably, provide a flexible structure that reduces steric interference between functional domains (Brun et al. 1997).
  • CeHZ like many cellulases described, consists of a large catalytic domain (Seq Id No. 50), involved in the enzymatic activity of interest, and a second domain of smaller size, with affinity for the substrate, which is called the domain of cellulose binding (Seq Id No. 47), and favors the access of the catalytic domain to the substrate.
  • Some cellulases have additional structural domains.
  • the tetramerization domain was fused in the separation arm (Seq Id No. 46) so that the hypothetical resulting multimeric structures have an arrangement spatial analogous to the different structural and functional domains of p53: a large aminoterminal domain, which in the case of CeHZ corresponds to the catalytic domain (Seq Id No. 50), separated from the cellulose binding domain (Seq Id No. 52) by DTp53 (Seq Id No. 36) and the native separation peptide (Seq Id No. 51).
  • CeIIZ the tetramerization domain was fused in the separation arm (Seq Id No. 46) so that the hypothetical resulting multimeric structures have an arrangement spatial analogous to the different structural and functional domains of p53: a large aminoterminal domain, which in the case of CeHZ corresponds to the catalytic domain (Seq Id No. 50), separated from the cellulose binding domain (Seq Id No. 52) by DTp53 (Seq
  • the fusion of the DTp53 domain affects the stability of the products in plants.
  • the carboxyterminal fusion of the DTp53 domain significantly affects the accumulation of the product in young and old expanded leaves, but the most significant differences are observed when comparing old leaves.
  • CeIY fused to the fragment derived from the GFP no significant differences are observed in the expression of CeI Y in expanded sheets, however, the accumulation of the product and the extractable activity in old sheets is approximately double in the case of the variant fused to DTp53.
  • the fusion of the DTp53 domain increases the extraction of endoglucanases.
  • the increase is 300% in the case of the native CeHZ and 50% in the case of CeHZ fused to the fragment derived from the GFP.
  • the zymograms show that CeIIZ suffers partial proteolysis in the chloroplast of tobacco and it is observed that the fusion of DTp53 alters the proteolysis pattern of the protein. Only in the case of CeIIZ fused to DTp53 are high molecular weight bands with endoglucanase activity observed that may correspond to complexes CeHZ multimerics.
  • the results suggest that the separation arm of CeHZ is a domain susceptible to proteolysis in the chloroplast and that the tetramerization domain partially protects the proteolysis and increases the recoverable activity.
  • Affinity for the substrate in the case of cellulosic enzymes, which have separate functional domains, the organization in tetramers alters the spatial configuration of the cellulose binding domains by increasing the affinity for the substrate (cellulose in this case).
  • Activity on an insoluble substrate may also be affected by the aggregation of catalytic subunits in the same complex. This is the case of some cellulolytic microorganisms - for example, ruminant bacteria - that produce enzymes that are organized in aggregate form.
  • the method proposed by the invention allows the obtaining of chimeric proteins / enzymes that, thanks to the simultaneous fusion strategies of the modified version of the first 15 amino acids of the GFP, together with the fusion of the tetramerization domain of p53, they have improved properties in terms of product stability, and activity on an insoluble substrate.
  • Example 1 Expression in chloroplasts of the endoglucanase encoded by the CeIY gene of Dickeya dadantii
  • the CeIY gene (Accession n ° M74044) of Dickeya dadantii encodes a small endoglucanase (EC3.2.1.4) that hydrolyzes glucose ⁇ (1-4) cellulose bonds favoring the depolymerization of the long glucose chains that constitute this polymer of the cell wall of the plants. This activity is usable in the industrial process of converting cellulosic biomass into bioethanol and in other processes of the textile or paper industry that use endoglucanases.
  • an amplified DNA product was used by means of the polymerase chain reaction in the laboratory of Dr. Pablo Rodr ⁇ guez Palenzuela (E.T.S.I. Agronomists, Polytechnic University of Madrid) who kindly provided the mentioned product.
  • the PCR product was cloned into a bacterial plasmid and sequenced (FIGURE 5). The sequence turned out to be identical to the one published (accession M74044).
  • CeIY constructs were obtained, operatively linked to the promoter (Seq Id No. 2) and leader sequence (Seq Id No. 3) of psbA.
  • the six constructs were obtained by linking PCR products representing the complete CeIY gene, or a version lacking the secretion peptide, at the ⁇ / col and Xba ⁇ positions of the intermediate plasmid pPLA or the pPLAg and pPLAf variants described in The previous section.
  • pPLA-Yc a 1010 base pair fragment (PCR Yc) was amplified by PCR (VENT R DNA polymerase, New England Biolabs, cat #: M0254S) using the cellYID oligonucleotides (Seq Id No. 14) and celYR (Seq Id No. 15) (FIGURE 2) representing the complete CeIY gene and flanked by the targets
  • the PCR product was purified by preparative agarose electrophoresis and extracted from the agarose fragment with QIAquik Extraction Kit (QIAGEN, Cat: 28704).
  • the purified fragment was digested with ⁇ / col and Xba ⁇ (New England Biolabs) and ligated to the similarly digested plasmid pPLA, using a commercial "rapid linkage kit" (Rapid Ligation Kit, Roche Cat.).
  • Chemically competent DH5 ⁇ cells were transformed and selected colonies that contain the plasmid linked to the fragment of interest and whose sequence coincides with the original.
  • pPLA-Yi (FIGURE 6): a 956 bp fragment (PCR-Yi) representing nucleotides +70 to +999 of the CeIY reading frame preceded by a translation start triplet was amplified by PCR flanked by the targets ⁇ / col and Xba ⁇ . This fragment encodes a protein equivalent to mature CeIY cellulase, without secretion peptide.
  • the oligonucleotides celY2D (Seq Id No. 16) and celYR (Seq Id No. 17) were used and the PCR fragment was cloned into pPLA similarly to pPLA-Yc.
  • PCR-Yi mentioned in the previous section, in plasmid pPLAg (Seq Id No. 4) using the same restriction targets.
  • the 5 'end of the open reading frame of the CeIY gene equivalent to mature cellulase, is preceded by a 45 base pair DNA fragment that encodes the first 15 amino acids of the GFP (Seq Id No. 28). This fragment determines the translation start context (FIGURE_6).
  • the resulting open reading frame encodes a fusion protein that represents the first 15 amino acids of the GFP (Seq Id No. 29) followed by a methionine residue and the mature CeIY cellulase.
  • the 5 'end of the open reading frame of the CeIY gene representing the mature peptide (Seq Id No. 31), is preceded by a synthetic DNA fragment, 54 base pairs, which encodes amino acids 28-47 of the transit peptide of tobacco ferredoxin (Seq Id No. 33) and which determines the context of initiation of translation (FIGURE 1).
  • the resulting open reading frame encodes a fusion protein that represents the amino acids 28-47 of the ferredoxin (Seq Id No. 34) followed by a methionine residue and the mature CeIY cellulase.
  • a CeIY fragment representing the mature CeIY protein was amplified by PCR without the original stop triplet by elimination of residue 999 (thymidine) from the CeIY open reading frame.
  • the PCR product was cloned into pPLA (Seq Id No. 1) digested with Ncol and Xbal similar to the previous constructs.
  • pPLA Seq Id No. 1
  • a sequence containing the tetramerization domain of p53 was amplified by PCR using two oligonucleotides
  • TetraXX-D (Seq Id No. 19) and TetraXX-R (Seq Id No. 20), which amplify a 140 bp fragment flanked by Xba targets at both ends containing 120 bp representing the tetramerization domain of p53 human (accession P04637).
  • the PCR fragment was cloned into the Xba site located at the 3 'end of the clone obtained in the first stage and the constructions that inserted the fragment in the appropriate direction were selected.
  • the resulting construction contains, under the promoter (Seq Id No. 2) and leader (Seq Id No.
  • pPLA-Ygt (Seq Id No. 41): it was obtained similarly to pPLA-Yt (Seq Id No. 40) with the difference that cloning was performed in pPLAg (Seq Id No. 4).
  • the resulting construction contains, under the promoter (Seq Id No. 2) and leader (Seq Id No. 3) of psbA, a reading frame that represents the first 15 amino acids of the GFP (Seq Id No. 28), the protein CeIY matures (Seq Id No. 31), a tyr-leu-asp spacer triplet and the tetramerization domain of p53 (DTp53) (Seq Id No. 35).
  • FIGURE 6 shows the sequences of the variants obtained.
  • CeIY chloroplast integration DNA corresponding to the promoter and leader of psbA operatively linked to the open reading frame that includes the CeIY gene (Seq Id No. 31) by digestion of the intermediate plasmids with Kpn ⁇ and Noti. These fragments were cloned, using the methods described above, in similarly digested pAF, obtaining the following clones: pAF-Yc, pAF-Yi, pAF-Yf, pAF-Yg, pAF-Yt and pAF-Ygt. The sequence of the integrated fragment was verified by partial sequencing of the resulting plasmids. CeIY chloroplast integration:
  • plasmid DNA preparations were obtained using QIAprep Spin Miniprep Kit from QIAGEN (Cat: 27106).
  • the plasmid DNA of the preparations was quantified by measuring the optical density at 260 nm. 1 ⁇ g of each DNA preparation was mixed with 3 mg of gold particles (stored at 60 mg / ml in 50% glycerol at -2O 0 C) in a volume of 60 ⁇ l.
  • Each outbreak is considered a clone.
  • 0.5 cm fragments of these shoots were cut and subjected to a second round of selection in RMOP-spectinomycin similar to the first.
  • RMOP-spectinomycin similar to the first.
  • numerous green shoots were obtained that were passed to rooting medium (M&S pH5.7, 30% sucrose, 0.6% agar) with spectinomycin at 500 mg / liter. After rooting, about 5-10 shoots per clone were grown on land.
  • DNA extracts were made from 0.1 g leaf fragments of first generation plants grown on land.
  • the plant material was transferred to an eppendorf and homogenized fresh with a plastic buril in the presence of 0.2 ml of extraction buffer (1% CTAB, 5OmM Tris-HCI pH8.0, 0.7M NaCI, 1OmM EDTA, 0.5% PVP and 0.1% 2-Mercaptoethanol) and the homogenate was incubated 1 hour at 6O 0 C.
  • the extract was extracted on 0.5 ml of chloroform: isoamyl (24: 1) mixing with a vortexer and the phases were separated by centrifugation (5 min at 10,000 rpm).
  • the aqueous phase was transferred to new tubes and the DNA was precipitated by adding a volume of isopropanol and incubating 20 minutes at -2O 0 C.
  • the DNA of the plant was recovered by centrifugation (10 min at 14,000 rpm), washed with 1 ml. 70% ethanol and resuspended in 20 ⁇ l of sterile water.
  • the integration of the vector into the plant was analyzed by PCR using the oligonucleotides Int-D (Seq Id No. 12) and Int-R (Seq Id No. 13) that respectively ring with pAF and cpDNA sequences. PCR products of expected size were obtained in plants that have integrated the vector. Integration and homoplasmia was also analyzed by restriction fragment polymorphism analysis based on the digestion of plant DNA with an appropriate restriction enzyme, whose restriction pattern is altered by the integration of the vector and the detection of the fragments by molecular hybridization techniques (FIGURE 3). The presence of the CeIY gene in plant DNA preparations was also analyzed by PCR with the oligonucleotides celY2D (Seq Id No. 16) and celYR (Seq Id No. 15).
  • the expression analyzes were made by assays of recoverable enzyme activity of raw plant extracts and, in some cases, by quantitative analysis of proteins separated by denaturing electrophoresis in polyacrylamide gels (SDS-PAGE).
  • SDS-PAGE polyacrylamide gels
  • the endoglucanase activity of crude protein extracts obtained from the first generation of homoplasmic plants was compared. Be They used plants that have developed at least 6 leaves during cultivation on land. Samples of leaf were taken consisting of leaf disks taken with punch of 8 mm in diameter. In this particular example, each plant was analyzed separately by taking samples of 8 discs (about 80 mg on average) from the various expanded leaves of each plant. The samples were homogenized in fresh, in extraction buffer (0.1M TrisCIH pH6.8, 0.5M NaCI, 1mM EDTA, 0.1% 2-mercaptoethanol), using plastic bricks that adapt to the shape of the eppendorf. The weight: volume ratio of the extracts is 1: 5 (80 mg in 0.4 ml buffer).
  • the proteins were solubilized by mixing 3 times with a vortexer, for 30 seconds, at maximum speed.
  • the extracts were centrifuged for 3 minutes at 12,000 rpms to separate the plant residue.
  • Serial dilutions of the supernatant are used to measure endoglucanase activity on carboxymethyl cellulose (CMC).
  • CMC carboxymethyl cellulose
  • the reactions were mounted in a volume of 0.5 ml of 1% CMC, 5OmM NaOAc pH5.2 and 2mM MgCl2 for 1 hour at 5O 0 C.
  • the activity was measured by quantifying the reducing sugars released by the Somogyi Nelson method (DNS dye ).
  • the CeIY gene is expressed more efficiently since an endoglucanase activity is recovered 80 times and 12 times higher, respectively, with respect to to the plants that contain the native version of CeIY and iv) the carboxyterminal fusion of the tetramerization domain of p53 promotes a recovery of an activity 2 times higher, both in the context of native CeIY and in the context of CeIY fused to the derived fragment of the GFP.
  • CeIY expression was also analyzed by electrophoretic separation of proteins from the extracts and staining with Coomassie blue.
  • each sample contains 8 leaf discs from the first two or the last two leaves of 4 different plants (4 discs per sheet).
  • the extraction and measurement of endoglucanase activity was performed as in the previous example. Different dilutions of each sample were tested in triplicate and the most appropriate one was selected for the quantification of released reducing sugars.
  • the measured activity averages are summarized in Table 2.
  • Control 1 20 - - pAF-Yi 1: 20 0.38 + 0.08 0 pAF-Yt 1: 40 3.41 ⁇ 0.30 3.60 ⁇ 0.50 pAF-Yg 1: 2000 50.61 ⁇ 2.86
  • the stabilization of CeIY mediated by the fusion with the DTp53 domain was analyzed in greater detail, comparing the expression of Yg and Ygt, since the high expression of CeIY in these plants allows a quantitative analysis of the expression.
  • "young" leaf samples (the first expanded leaf of about 15 cm) were taken from 5 normal plants, 5 "Yg” plants and 5 "Ygt” plants and compared with "mature” leaf samples from the same plants. Each sample consists of 10 leaf discs (2 discs per sheet). The samples were homogenized in 5 volumes (0.5 ml) of extraction buffer (0.1 M Tris pH6.8, 0.5M NaCI, 1mM EDTA, 0.1% 2 mercapto ethanol).
  • the protein content of the extracts was quantified by the Bradford method (BIO-RAD PROTEIN ASSAY Cat: 500-0006) by comparing against a standard of BSA (bovine serum albumin).
  • BSA bovine serum albumin
  • the protein content of the extracts was analyzed by electrophoresis in a denaturing gel and Coomassie blue staining.
  • the proteins of the extracts with endoglucanase activity were identified by a zymogram of the electrophoretically separated proteins and the endoglucanase activity of the extracts was analyzed by testing appropriate dilutions on CMC as described in the previous examples.
  • the second gel was processed for the zymogram in the following manner: 200 ml of 1% Triton X-100 was washed 3 times to remove the SDS, equilibrated in 5OmM NaOAc pH 5.2 for 1 hour at 4 0 C to favor Ia renaturation of the proteins, it was incubated for 1 hour in 1% CMO 5OmM NaOAc at 37 0 C, stained with 1% Congo Red (which joins CMC) for 20 min at room temperature, and stained with several washes in 0.5M NaCI. The result is a red gel with faded areas corresponding to proteins with endoglucanase activity that was photographed with a digital camera on a fluorescent light transilluminator (Figure 8b).
  • Protein quantification indicates that "young" leaf samples have a higher protein content (about 1 mg / ml) than mature leaves (0.6 mg / ml). This difference is manifested in the Coomassie blue stained gel in which equivalent volumes of extract, not protein, were loaded (Fig. 8a). In young leaves of normal plant (lane 1) the most intensely stained protein is the chloroplast ribulose carboxylase (RUBISCO) with an apparent molecular weight of 54kDa.
  • RUBISCO chloroplast ribulose carboxylase
  • Example 2 Expression in chloroplasts of endoglucanase encoded by the CeHZ gene of Dickeya dadantii.
  • the CeHZ gene (Seq Id No. 43) was cloned and isolated in a similar way as described in the case of CeIY from a PCR product kindly provided by Professor Pablo Rodr ⁇ guez Palenzuela. The product was cloned into the pGEM-Teasy vector (Promega, Cat n °: A3610) and the complete sequence of the fragment was obtained ( Figure 9).
  • the sequence (Seq Id No. 43) turned out to be almost identical to the published sequence of the endoglucanase Z of Erwinia chrysanthemi (Now Dickeya dadantii, Accession Y00540). 2 differences were found.
  • the sequence obtained has a deletion of an adenine residue (A) in the +1096 position and an addition of an adenine residue in the +1162 position of the CeHZ open reading frame.
  • a fragment representing nucleotides 130-1282 corresponding to the mature endoglucanase Z preceded by the translation initiation ATG triplet was amplified by means of the chain polymerase reaction (PCR).
  • PCR chain polymerase reaction
  • the oligonucleotides Cel1Z-D2 (Seq Id No. 21) and Cel1Z-R2 (Seq Id No. 22) (FIGURE 2) were added which add BspH ⁇ and Xba ⁇ targets flanking, respectively, the 5 'and 3 ends 'of the open reading frame of endoglucanase Z.
  • the PCR product was cloned into plasmids pPLA (Seq Id No.
  • the sequence context immediately after the translation start triplet corresponds to a synthetic fragment that encodes the first 15 amino acids of the GFP described (Seq Id No. 29) in the previous example (FIGURE 1 ) and iii) in the particular case of pPLAZf, the sequence context immediately after the translation start triplet corresponds to a synthetic fragment encoding amino acids 28-47 of the tobacco ferredoxin (Seq Id No. 33) (FIGURE 1 ).
  • the purpose of these constructions is to analyze the effects of the sequences immediately following the ATG triplet in the expression of genes under the control of the promoter and leader sequence of psbA.
  • Endoglucanase Z unlike Ce / Yes a protein of modular structure with differentiated structural-functional domains (FIGURE 9).
  • the mature protein consists of an aminoterminal domain with catalytic function (amino acids 1-289, Seq Id No. 50) followed by a separation arm (amino acids 290-323 Seq Id No. 51) and a cellulose affinity domain (amino acids 324 -383, Seq Id No. 52) (Brun et al, 1997).
  • the DTp53 domain can be fused at the aminoterminal end, at the carboxy terminal end, or at the separation arm.
  • the fusion to the aminoterminal end was ruled out because it can interfere with the expression of the gene.
  • the fusion of DTp53 in the separation arm of the endoglucanase Z mimics the structure of the p53, which consists of a large aminoterminal domain and a smaller aminoterminal domain separated by the tetramerization domain (Tidow et al. 2007).
  • the resulting complex would have a structure that exposes 2 cellulose binding domains spatially close to each other, and may affect the affinity of the complex for cellulose.
  • DTp53 (Seq Id No. 35) to the cellulose binding domain (Seq Id No. 47) by combined PCR.
  • This method consists in the amplification, separately, of two partially overlapping DNA fragments.
  • One of the PCR fragments (199 bp) represents the DTp53 domain amplified with the oligonucleotides TXXL-D (Seq Id No. 23) and TXXL-R (Seq Id No. 24) (FIGURE 2) flanked by an Xba restriction target ⁇ at its end 5 'and by a sequence of 12 nucleotides at its 3' end complementary to nt 1048-1059 of the CeHZ gene (FIGURE 9).
  • the second PCR fragment represents CeHZ 1048-1285 nucleotides encoding part of the separation arm (Seq Id No. 46) and the cellulose binding domain (Seq Id No. 47), amplified with the TCBD oligonucleotides (Seq Id No. 25) and Cel1Z-R2 (Seq Id No. 21) (FIGURE 2).
  • This fragment contains, at its 5 'end, a segment of 12 nucleotides representing the last 4 triplets of DTp53.
  • PCR products which overlap in 24 bp, were purified from agarose gels and combined as a template in a short 5-cycle amplification reaction, which generates a product that represents the fusion of both fragments by the overlapping of their overlapping regions. .
  • This product flanked by Xba? Targets, was PCR reactivated with VENT polymerase (New England Biolabs) using the oligonucleotides TXXL-D (Seq Id No. 23) and Cel1Z-R2 (Seq Id No. 21) which ring at both ends and was cloned in pGEM-T Easy following the manufacturer's instructions (Promega Cat n ° A3610).
  • the sequence of the resulting clone was analyzed by checking the correct fusion of the fragments. From a DNA preparation of this plasmid, the Xba I fragment representing DTp53 fused to the cellulose binding domain was purified and ligated to similarly digested plasmids pPLA-Zcat and pPLA-Zgcat to obtain pPLA-Zt and pPLA-Zgt which they contain chimeric genes that represent the mature endoglucanase Z of Dickeya dadantii fused to the DTp53 domain in the separation arm, whose sequence was analyzed (FIGURE 10).
  • the chimeric Zi, Zf, Zg, Zt and Zgt genes operatively linked to the promoter (Seq Id No. 2) and leader sequence (Seq Id No. 3) of psbA were cloned into the pAF integration vector (Fernández-San Millán y Col 2008) analogously to example 1, obtaining plasmids pAF-Zi, pAF-Zf, pAF-Zg, pAF-Zt and pAF-Zgt (Seq Id No. 55).
  • Tobacco plants that integrate Zi, Zf, Zg, Zt and Zgt were obtained with the methodology described in example 1.
  • the analysis of integration and homoplasmia of the plants was performed as described in said example.
  • endoglucanase Z variants The expression and accumulation of endoglucanase Z variants was analyzed by measurements of endoglucanase activity from raw plant extracts homoplasmic that integrate the different variants.
  • the extracts were obtained as described in example 1, from 80 mg samples of fresh tissue and endoglucanase activity tests on CMC were also performed as described in said example.
  • the test of serial dilutions of plant extracts found that the linear range of endoglucanase Z activity is much broader than in the case of CeIY. This allowed a comparative analysis of the endoglucanase activity using the same amount of extract in all cases.
  • samples of first-generation homoplasmic plants were tested in a state of 8-10 leaves. The results obtained are summarized in table 4:
  • CeIY Unlike CeIY, no significant activity was observed in plants that integrate the native CeHZ gene (Seq Id No. 48) (Zi). Similar to what was observed with CeI Y, the aminoterminal fusions of two synthetic fragments with low proportion of CG pairs favored the expression of CeIIZ cloned under the promoter (Seq Id No. 2) and leader sequence (Seq Id No. 3) of psbA. As in example 1, the sequence derived from the GFP (Zg) stimulated the expression of Ia. endoglucanase in greater amount than the sequence derived from ferredoxin (Zf).
  • Endoglucanase Z expression protein stability.
  • Control 1 1000 0 - pAF-Zg 1, 97 ⁇ 0.40 1: 1000 7.7 + 0.8 0.78 + 0.08 pAF-Zgt 1, 52 + 0.20 1: 1000 9 , 6 + 0.8 1, 27 + 0.10
  • Z fused to DTp53 was 25% higher in terms relative to the fresh weight extracted and 60% in terms relative to the protein extracted.
  • the gel was processed similar to the zymogram detailed in example 1 and the proteins of the extracts with electrophoretic activity were visualized (FIGURE 10). In this case the pattern of endoglucanases is much more complex than in the case of example 1.
  • Zg and Zgt extracts Seq Id No.
  • the activity data and the zymograms indicate that the fusion of DTp53 does not prevent the proteolysis of CeIZ in the chloroplast but probably increases the stability, due to the formation of complex structures. It has been described that the arms that separate domains of cellulases are susceptible to the action of proteases in the absence of glycosylation (Langsford et al. 1987). These results show that in the chloroplast, lacking glycosylation machinery, the cellulase separation arms can be a target of proteases, so it is important to develop strategies that reduce the accessibility of these separator segments to proteases.
  • the natural and industrial substrate of cellulases is insoluble and semi-crystalline in nature.
  • the construction of chimeric enzymes that form complex structures can affect the activity on this class of substrates.
  • the activity of endoglucanase Z on microcrystalline cellulose (avicel) was analyzed.
  • the formation of multimeric structures can reduce the specific activity of the protein, but this hypothetical reduction can be compensated by a greater stability of the product in the reaction and by a greater affinity for the substrate since the multimeric complexes also have several copies of the cellulose binding domain.
  • the extracts were obtained from 2 grams of plant material (expanded leaves) homogenized fresh in a ceramic mortar in 10 ml of 10OmM Tris pH6.8, 0.5M NaCI, 1mM EDTA, 0.1% 2-mercaptoethanol.
  • the reactions were incubated for 8 h in 10 ml of 2% avicel, 5OmM pH 5.2 and 1 mM MgCl2, in an orbital shaker at 37 0 C with stirring at 200 rpm
  • Each reaction contains 1 ml of extract and 3 replicates were made for each treatment. Samples of 1 ml were taken at 1, 2, 3, 6 and 8 hours from the start of the reaction to compare the reaction kinetics of the chimeric proteins.
  • the reducing sugars released by the Nelson Somogyi method were determined and the micromoles released per milliliter were quantified:
  • the activity of the same extracts on CMC was determined as described in example 1.
  • the activity of Zg and Zgt on CMC was 8.2 and 13.6 lU / gram of fresh tissue respectively.
  • the data shows that in one hour of reaction, the difference in activity of Zg or Zgt extracts on poultry is much smaller than on CMC, which indicates that the specific activity of Zgt on solid substrates is less than that of Zg.
  • the difference in activity observed is 60-70%, while on poultry, in one hour, the difference in favor of Zgt is reduced to 20%.
  • a greater increase in reducing sugars with Zgt extracts is observed so that at 8 hours the difference in activity of the extracts increases up to 40%.

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Abstract

La présente invention concerne le domaine de la technologie des plantes transgéniques qui expriment des protéines. L'invention concerne concrètement une nouvelle méthode pour augmenter l'expression de protéines dans des plantes, de préférence des enzymes cellulolytiques et plus particulièrement la production des endoglucanases CeIY et CeIIZ de Dickeya dadantii dans les chloroplastes du tabac par intégration de gènes dans le génome du chloroplaste végétal. L'invention est destinée à être utilisée dans tout procédé industriel nécessitant une grande quantité d'enzymes comme par exemple dans la conversion de biomasse de cellulose en éthanol.
PCT/ES2009/000385 2008-07-31 2009-07-21 Méthode pour améliorer l'expression des protéines dans des chloroplastes WO2010018251A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004331A2 (fr) * 1999-07-09 2001-01-18 Calgene Llc Amelioration de l'expression de proteines
WO2002006497A2 (fr) * 2000-07-14 2002-01-24 International Centre For Genetic Engineering And Biotechnology Plantes transplastomiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004331A2 (fr) * 1999-07-09 2001-01-18 Calgene Llc Amelioration de l'expression de proteines
WO2002006497A2 (fr) * 2000-07-14 2002-01-24 International Centre For Genetic Engineering And Biotechnology Plantes transplastomiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GIL F ET AL.: "Multimerization of peptide antigens for production of stable immunogens in transgenic plants.", JOURNAL OF BIOTECHNOLOGY., vol. 128, 2007, pages 512 - 518 *
YE G-N ET AL.: "Plastid-expressed 5- enolpyruvylshikimate-3- phosphate synthase genes provide high levthe glyphosate tolerance in tobacco.", THE PLANT JOURNAL., vol. 25, no. 3, 2001, pages 261 - 270 *

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