WO2011024065A1 - Procédé de développement de plantes tolérantes au stress présentant des propriétés autoglucogènes pour une utilisation dans la production de bioéthanol à partir de biomasse lignocellulosique - Google Patents

Procédé de développement de plantes tolérantes au stress présentant des propriétés autoglucogènes pour une utilisation dans la production de bioéthanol à partir de biomasse lignocellulosique Download PDF

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WO2011024065A1
WO2011024065A1 PCT/IB2010/002107 IB2010002107W WO2011024065A1 WO 2011024065 A1 WO2011024065 A1 WO 2011024065A1 IB 2010002107 W IB2010002107 W IB 2010002107W WO 2011024065 A1 WO2011024065 A1 WO 2011024065A1
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plant
cellulase
enzymes
promoter
xylanase
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WO2011024065A8 (fr
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Villoo Morawala Patell
Mahesh Venkataramaih
Suhas Nimbalkar
Naveen Sharma
Suresh Sadasivam
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Avesthagent Limited
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    • 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)
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    • C12N9/2405—Glucanases
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    • 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
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    • 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
    • C12N9/2485—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1,3-beta-xylanase
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    • 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
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01015—Glutamate decarboxylase (4.1.1.15)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the method of development of stress tolerant plants, which also exhibit self-glucogenic properties and thus can be utilized in the bio ethanol production from the lignocellulosic biomass.
  • Ethanol is gradually becoming one of the chosen fuels for the automotive industry both as a fuel and as a fuel additive in blends. Its easy availability combined with its suitability in spark-ignited internal combustion systems as a fuel and as a fuel additive in petrol blends makes it one of the renewable carbon source of choice.
  • This proposal is geared towards providing farmers with a non food-competitive crop adapted to the biofuel industry for marginal and rainfed areas.
  • This proposal draws on the expensive nature of agricultural crop waste digestion by externally applied cellulose degrading enzymes rendering the process non-feasible at remote locations and thus the expensive transportation cost of an otherwise low priced post-harvest plant waste. Pearl millet has been selected as the target of this technology as it is a marginal crop with about 9.3 million Ha cultivation and a good adaptation to arid drought-prone cultivation.
  • This present application will entail the development of Agrobacterium mediated plant transformation vectors encoding a peroxisomal or vacuolar-targeted cellulase, cellobiohydrolase and xylanase.
  • pearl millet Pierennisetum glaucum (L.)
  • a peroxisomally or vacuolar- targeted cellulase, cellobiohydrolase ⁇ -glucosidase and xylanase towards storing the hydrolytic enzymes for post-harvest glucogenesis and with a rice glutamate decarboxylase for enhancing biomass under stress conditions.
  • pearl millet cultivation yields around 8.3 million tons of grain annually and the stovers are used as fodder for farm animals. It is a preferred crop in areas where irrigation is not available and scanty rainfall around planting time is sufficient to assure the farmer of some yield. While a number of technologies are centered around the use of grain for glucogenesis, the competition of this process with food production makes it unattractive. This technology will thus facilitate the decentralized conversion of bulk farm agricultural residue into high glucose broths that can further be fermented to ethanol at local centers. The fuel thus produced may be directly used locally or used in the national transportation grid. Pearl millet is a commonly cultivated marginal land crop providing both feed and fodder to farmers.
  • This invention entails the enhancement of leaf biomass under marginal conditions to enhance the recovery of sugars after degradation of the cell wall components by glycolases expressed and sequestered in the peroxisome/vacuole.
  • Glutamate decarboxylase is also a known alleviator of salinity stress. Taken together, glutamate decarboxylase is a valid target to enhance biomass under stress conditions. Glycolysis by enzymes in transgenic plants has been reported in maize. The combination of these enzymes is targeted for crops under environmental stress.
  • Maize is the target crop in most cases as the American acreage of Maize covers 33 million hectares.
  • the expression of Cellulase El from Acidothermus cellulyticus has been carried out in a number of plants including tobacco, Arabidopsis, rice, maize and duckweed.
  • the localization of this enzyme has been evaluated in various organelles including the chloroplast, the apoplast, the vacuole and the cytosol.
  • Xylanase has also been evaluated in various crops including rice, barley, Arabidopsis, potato, and tobacco. Its localization has been evaluated in the cytosol, chloroplast, peroxisome, and apoplast.
  • a method to express ligninases and cellulase in maize has been described.
  • glutamate decarboxylase to enhance biomass has never been reported before and is novel. This enzyme has been addressed as a modulator of stress, as a modulator of nitrogen metabolism. Its application towards enhancing biomass under stress conditions has not been reported and is novel. To further tie this application to biofuel production using endogenously expressed cellulases and xylanase for autoglucone ⁇ esis has not been reported and is novel.
  • India is moving towards blending petrol fuel with ethanol at levels of 5% or 10%. Such a step calls for a projected demand of 2.3 billion liters of ethanol by 2009-2010 at 5% blending levels. The corresponding level for 10% would then be approximately double. It has been estimated that if the entire molasses production of the country is diverted to ethanol production then the requirement in 2009-2010 will touch 10.4 million tons at 5% blending and 13.8 million tons at 10% blending. Considering the peak molasses production of 2002-2003 at 8.9 million tons, a significant shortfall in production will be encountered. This shortfall may be partially met by pearl millet production in marginal areas, which cannot be used for crop production, let alone sugarcane production. The combined increased biomass under marginal cultivation and the auto-glucolytic nature of the crop waste will enable both increased feed stock production and reduced processing costs towards ethanol production.
  • cellulase in maize has resulted in a 2.1% yield of cellulase. Similar levels have been achieved in numerous other plants indicating that the expression of cellulase in plants results in active protein, which is capable of degrading cellulose to smaller saccharides.
  • the targeting of this hydrolase to organelles has resulted in significant protection of the plant cell from inhibitory responses during the growth of the plant and no untoward results have been reported indicating that plants expressing in an organelle, limited cellulases are not affected in their growth patterns.
  • xylanase likewise has resulted in 2-2.5% expression levels in a number of crop plants, though the list is not as extensive as that of cellulase, it is impressive in its own right.
  • the role of this enzyme is to degrade hemicellulose, a major component of the cell wall with a significant pentose composition. Again the success of this strategy lies in few indications of intolerance by the plant in spite of the relative high level of expression.
  • the primary object of the invention is to develop stress tolerant plants exhibiting self-glucogenic properties for use in bioethanol production from lignocellulosic biomass.
  • Another object of the invention is to make the lignocellulosic biomass self-glucogenic by employing a combination of genes namely cellulase (1-3 endoglucanase), cellobiohydrolase, ⁇ -glucosidase and xylanase.
  • the object of the invention is to develop a method wherein the transformed plant expresses glutamate decarboxylase (GAD), cellulase, xylanase, cellobio hydrolase(CBH), beta glucosidase enzymes, at significantly higher level than the level of the enzymes expressed by a non-transformed plant of the same species under the same conditions.
  • Yet another object is to select the target plants from the group consisting of monocots, dicots, cereals, forage crops, legumes, pulses, vegetables, fruits, oil seeds, fiber crops, flowers, horticultural, medicinal and aromatic plants and transform it with the DNA construct mediated by Agrobacterium-me ⁇ iated transformation.
  • Yet another object of the instant invention is to obtain a transformed plant with a DNA construct comprising a promoter operably linked to a nucleotide sequence that encodes a functional glutamate decarboxylase (GAD), cellulase, xylanase, cellobio hydrolase(CBH), beta glucosidase enzymes, wherein the plant exhibits significantly improved growth characteristics, yield, reproductive function and other morphological or agronomic characteristic compared to a non-transformed plant in addition to self glucogenic properties.
  • GAD glutamate decarboxylase
  • CBH cellobio hydrolase
  • the present invention relates to a method of increasing stress tolerance in plants (monocotyledons and dicotyledons) with a glutamate decarboxylase gene and making the lignocellulosic biomass self- glucogenic.
  • This strategy in the instant invention will sequester the hydrolytic enzymes from the cellulose fibril polymerizing machinery and protect the plant from auto-degradation during the growth and maturation of the crop. After harvest and threshing of the grain, the crop residue will be homogenized to release the enzymes from the peroxisome or vacuole. It is expected of this technology to digest the crop residue without further addition of cellulose digesting enzymes using minimal resources that may be implemented at the level of the farm.
  • Betaglucosidase amino acid sequence from A. naeslundii SEQ ED No. 14 Betaglucosidase amino acid sequence from A. naeslundii
  • FIGURE 1 Shows Assembly of each gene cassette is as follows: Schematic representation of each cassette (i-v). i) a rice glutamate decarboxylase (GAD) gene driven by 35S CaMv promoter; ii) cellulase (Endoglucanase) gene driven by a RUBISCO promoter and a C-terminal vacuolar targeting sequence terminated by a proteinase inhibitor terminator; iii) Cellobiohydrolase (CBH-I) gene driven by UBIQUITIN promoter and a C-terminal vacuolar targeting sequence terminated by a proteinase inhibitor terminator; iv) ⁇ -glucosidase (BGL) gene driven by the ZMVBO promoter and a C-terminal vacuolar targeting sequence terminated by a proteinase inhibitor terminator; v) Xylanase (XynZ) gene driven by COMT promoter .
  • GAD rice glutamate decarboxylase
  • CBH-I
  • FIGURE 2 Shows vector map (pAGTSG) of binary plant transformation vector harboring the vacuolar targeted - cellulase gene driven by a RUBISCO promoter, xylanase gene driven by COMT promoter, cellobiohydrolase driven by UBIQUITIN promoter, ⁇ -glucosidase gene driven by the ZMVBOMT promoter and a GAD gene driven by 35S CaMv promoter within a single T-DNA region.
  • FIGURE 3 Shows growth performance of three cultivars of pearl millet ABI13B, ABI56B and 843B (L- R); seed germination in three cultivars (A); Plant growth (B); Leaf size (C)
  • FIGURE 4 Shows callogenic potential of different explants in pearl millet; callus growth from immature embryo, arrows denote embryogenic calli (A); callus growth from seeds (B); inset shows the explant. Multiple shoot regeneration from embryogenic calli in pearl millet; Shoot regeneration on SRM with activated charcoal (C); root proliferation on charcoal free medium (D); different stages of shoot regeneration (E & F)
  • FIGURE 5 Shows xylose sensitivity Assay in pearl millet; 2% xylose concentration was effective selection for seed germination (A); 0.5% xylose concentration was effective selection for callus growth (B)
  • FIGURE 6 Shows successful transformation of pearl millet was achieved and confirmed with transient GUS assay in transformed immature embryo (A-D) using Agrobacterium strain GV3101.
  • the invention is directed to generating stress tolerant plants, which express one or more exogenous cellulose-degrading (cellulase) enzymes.
  • the invention is further drawn to a method of producing cellulases in plants.
  • the invention is further drawn to a method of sorting the produced cellulases in plants to organelle compartments like vacuole or peroxisomes.
  • the invention is further directed to generating stress tolerant plants whose biomass is amenable for cellulose digestion without addition of external cellulose-degrading (cellulase) enzymes.
  • the invention allows the production of cellulases using the means and methods of large-scale agriculture rather than the conventional route of large-scale fermentation of the bacteria or fungi, which are native producers of the cellulases.
  • the recombinant plants are produced by incorporating into a plant host genome one or more expression constructs comprising a DNA sequence which encodes a protein having cellulose-degrading activity.
  • Introduction of the exogenous gene or genes into the plant is accomplished by any means known to the art.
  • the expression constructs described herein below enable the stable transformation of plants with one or more genes, which encode cellulose-degrading enzymes.
  • the constructs include a DNA coding sequence which encodes a cellulase (as that term is described herein) which is operatively linked to regulatory sequences which direct constitutive, stage-specific, or tissue-specific expression of the cellulase DNA.
  • Cellulose-Degrading Enzymes Cellulases
  • Genes Cellulose-Degrading Enzymes (Cellulases) and Genes: As noted above, the term “cellulase” shall be used herein to refer to any and all enzymes, which catalyze the cleavage of cellulosic or lignocellulosic materials. As used herein, “cellulase”is synonymous with “cellulose-degrading enzymes.” Explicitly, but not exclusively, included within the term cellulases are those enzymes, which fall under the Enzyme
  • EC 3.2.1.6 enzymes hydrolyze internal 1,3 glycosidic bonds of the polysaccharide chain, which also results in the formation of new chain ends at the surface of cellulose crystals.
  • EC 3.2.1.21 enzymes hydrolyze cellobiose into glucose, a readily fermentable substrate.
  • EC 3.2.1.91 enzymes (1, 4-exocellulases) cleave cellobiosyl residues (cellobiose is a glucose dimer) from the chain ends of cellulose.
  • Particularly preferred enzymes for use in the present invention are glutamate decarboxylase gene from rice, cellulase (1-3 endoglucanase) gene from ⁇ . cellulyticus, cellobiohydrolase gene from T. reesei, ⁇ -glucosidase gene from A. naeslundii and xylanase gene from T. reesei.
  • Stress refers to a factor, which externally causes a change in the growth of plants.
  • Environmental stress refers to a stress provided by a change in an external environment, including salts, high osmotic pressure, drying, high temperature, low temperature, intense light, air pollution, and the like.
  • oligonucleotide sequences using restriction endonucleases to cleave DNA molecules into fragments and DNA ligase enzymes to unite compatible fragments into a single DNA molecule with subsequent incorporation into a suitable plasmid, cosmid, or other transformation vector are well-known to the art.
  • a transcription regulatory sequence must be included in the expression construct in order to direct the transformed plant cells to transcribe the inserted coding sequences.
  • Transcriptional regulators may be inducible or constitutive. Inducible transcription regulators direct transcription of the downstream coding sequences in a tissue-specific or growth-stage specific manner. Constitutive regulators provide for sustained transcription in all cell tissues. For purposes of the present invention, constructs, which provide constituitive expression of the coding sequence, are preferred.
  • the expression construct contain a transcription initiation sequence from the tumor-inducing plasmid (Ti) of Agrobacterium.
  • T-DNA transcription initiation sequences are well known and include, without limitation, the RUBISCO, actin, 35S Cauliflower mosaic virus (CaMv), Cassava vein mosaic virus (CvMv), octopine synthase, nopaline synthase, and mannopine synthase initiators.
  • the expression construct may be manipulated to contain a signal sequence which directs the resulting polypeptide to a particular organelle or targets the expressed product for secretion (or to signal post-transcriptional or post- translational modification of the gene product).
  • the expression construct should also include a termination sequence to signal transcription termination.
  • the expression construct should also include one or more selectable markers.
  • the neomycin phosphotransferase gene (NPT EI) is a well-characterized and widely employed antibiotic resistance selection marker. This marker provides resistance to kanamycin. A large number of other markers are known and can be used with equal success (e. g., other antibiotic resistance markers, dihydrofolate reductase, hygromycin phosphotransferase and the like). Many non-anti biotic selection markers like luciferase, ⁇ -glucuronidase and the like are also widely employed. A previously described positive selection marker - xylose isomerase, which employs the xylose sugar as a selection means is preferred in this invention.
  • Fig. 2 depicts schematic representations of suitable expression construct for transformation of plants. These construct is intended for use with Agrobacterium- mediated transformation using the binary vector approach. However, similar constructs can be coated onto micro-projectiles for transformation by particle bombardment.
  • Fig. 2 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain glutamate decarboxylase gene from rice, cellulase (1-3 endoglucanase) gene from A. cellulyticus, cellobiohydrolase gene from T. reesei, ⁇ -glucosidase gene from A. naeslundii and xylanase gene from T. reesei.
  • Transformation of Plants can be accomplished by any means known to the art, including Agrobacterium-mediated transformation, particle bombardment, electroporation, and virus- mediated transformation. The method of transformation is not critical to the functionality of the present invention insofar as the method chosen successfully incorporates the oligonucleotide construct containing the desired protein-encoding region and any accompanying regulatory sequences into the plant host.
  • Agrobacterium-mediated transformation utilizing protoplasts or leaf disks is most preferred.
  • any crop plant including monocots, can be utilized. Transformation of monocots is typically achieved by particle bombardment of embryogenic cell lines or cultured embryos. See, for instance, Vasil et al. (1993) and Castillo et al. (1994). Recent developments in"super- binary"vectors, however, also allow for the use of Agrobacterium-mediated gene transfer in most of the major cereal crops. See, for instance, Ishida et al. (1996). In this case, the explant source is typically immature embryos.
  • Agrobacterium-mediated transformation of the plant host using explants is preferred for its relative ease, efficiency, and speed as compared to other methods of plant transformation.
  • disks are punched from the leaves of the plant host and cultured in a suitable medium where they are then exposed to Agrobacterium containing the expression construct and (preferably) a disarmed tumor-inducing (Ti) plasmid.
  • Agrobacterium tumefaciens LBA 4404 is the preferred strain for transformation.
  • the preferred binary vector is the pCGN1578 binary vector (McBride and Summerfelt (1990)).
  • the binary vector transformation method is well known and needs only be briefly described herein.
  • the T-DNA portion of the Ti plasmid is flanked by two border regions (the right and left borders), which act as recognition sites for the excision of the T-DNA from the plasmid prior to its transfer to the plant host. Excision of the T-DNA is mediated by the vir genes of the Ti plasmid and involves nicking of the right and left borders of the T-DNA, which frees a single-stranded oligonucleotide fragment. This fragment is then mobilized out of the Agrobacterium and into the plant host target.
  • the T-DNA with its right and left border regions is cloned into E. coli in known fashion, and the wild-type genes normally found between the two border regions is excised.
  • the expression construct encoding the genes of interest are inserted between the right and left border regions. This construct is designated the "binary plasmid.” Construction of the binary plasmid is accomplished utilizing the well-characterized recombinant genetic methods applicable to E. coli. Successful transformants are selected utilizing a co-transformed marker appropriate for E. coli.
  • the binary plasmid is then mobilized back into Agrobacterium. This is accomplished by direct transformation procedures well known to those skilled in the art.
  • the Agrobacterium itself such as the preferred GV3101 strain, is genetically manipulated to contain a Ti plasmid (called the helper plasmid) which lacks the T-DNA and the tumor-inducing regions (i. e., the Ti plasmid is"disarmed") but which still encodes the virulence proteins necessary for DNA transfer.
  • the helper plasmid By cooperation between the helper plasmid and the binary plasmid, the length of DNA between the two border regions of the binary plasmid is excised and mobilized into the plant host, where it is incorporated into the plant host genome.
  • the binary method derives its name from the fact that the plasmid containing the expression construct to be transferred is maintained within Agrobacterium as a distinct and independently replicating vector from the Ti plasmid itself.
  • Selection of successful transformants is accomplished using the co-transformed selection marker discussed above. If the marker is Xi, selection is accomplished by growing the transformants on a media supplemented with xylose sugar.
  • the most preferred plants for transformation are pearl millet and tobacco.
  • cauliflowers artichokes
  • apples bananas
  • cherries cucumbers
  • grapes lemons
  • melons nuts
  • oranges peaches
  • plums strawberries
  • tomatoes cabbages
  • endive leeks
  • lettuce spinach
  • arrowroot beets
  • radishes yams
  • sweet potatoes beans, peas, soya, wheat, barley, corn, rice, rapeseed, millet, sunflower, oats, tubers, kohlrabi, potatoes, and the like.
  • the plants to be transformed are preferably common green field plants, such as the preferred alfalfa and tobacco, as well as soya, corn, and the like. Equally preferred are plant hosts, which are grown specifically for "biomass energy," such as switchgrass, poplar, and the like. In this instance, the enzymes would not be recovered from the plants. The plants are then transformed and regenerated into whole plants, which express fully functional, cellulose-degrading enzymes in economically significant quantities.
  • Pearl millet is one of the most preferred plant species for use in the present invention because pearl millet is a hardy plant, which grows well with minimal fertilization and irrigation. In the tropics pearl millet is a commonly cultivated marginal land crop providing both feed and fodder to farmers. It has been largely ignored by the biofuel community, which concentrates on maize, which is an input intensive crop, for bioethanol production. This invention entails the enhancement of leaf biomass under marginal conditions to enhance the recovery of sugars after degradation of the cell wall components by glycolases expressed and sequestered in the vacuole or peroxisome.
  • the cellulase enzymes described in the instant invention are most preferred because they are native to thermo-tolerant bacteria and are relatively heat stable. This allows to maintain the enzyme activity intact of the cellulases expressed in the plant material during the bio-ethanol production, where the process includes relatively rigorous heat treatments which other wise would adversely effect the activity of the cellulase incase the cellulase were not thermo stable.
  • Stage-Specific and Tissue-Specific Expression of Cellulases Because the enzymes to be expressed by the transformed plant hosts hydrolyze components of the plant cell wall, high levels of expression might have a deleterious effect on the plant host. Therefore, targeting of the expressed enzyme to particular subcellular compartments may be preferred. Targeting of the expressed enzyme may also be preferred to avoid expression of the enzyme in sub-cellular compartments where proteolytic activity is high. Targeting of the expressed enzyme may also be preferred if the exogenous cellulase activity interferes with the normal cellular metabolism of certain compartments.
  • Plant lignocellulosic biomass is a complex matrix of polymers composing of polysachharides cellulose and hemicellulose and a polyphenolic complex lignin as the major structural components.
  • Cellulose the most abundant biopolymer on the earth, is a simple, linear polymer of glucose.
  • Nature has developed effective cellulose hydrolytic machinery for recycling of carbon from plant biomass in the environment, without it the global carbon cycle would not function.
  • cellulase genes have been cloned and sequenced from a wide variety of fungi, bacteria, protozoans and plants.
  • Cellulase refers to a class of enzymes that catalyze the hydrolysis of cellulose (cellulolysis).
  • cellulases which differ structurally and mechanistically.
  • Cellulose is degraded through the synergistic action of two genearal types of cellulase enzymes.
  • Enzymes that cleave the cellulose chain are referred to as endo-1,4 ⁇ -D-glucanases (endoglucanase, EG; EC 3.2.1.4) and serve to provide new reducing and non- reducing chain termini on which exo-1,4- ⁇ -D glucanases (cellobiohydrolase, CBH; EC 3.2.1.91) can operate.
  • a third activity, ⁇ -D Glucosidase (EC 3.2.1.21) is required to cleave cellobiose and other oligomers to glucose.
  • the glucosidase activity is required at 100-1000 times lower concerntration than the cellulases.
  • Clostridium thermocellum exhibits a highly active and thermostable (optima 7O 0 C) xylanase activity when cells are grown on cellobiose.
  • Xylanase Endo ⁇ -1, 4- xylanase, XynZ; EC 3.2.1.8
  • enzymes secreted by during growth on cellobiose may make cellulose accessible to cellulolytic enzymes. Based on this criterion T. reesei xylanase SEQ ID No 4 was chosen.
  • T. reesei produces at least five endoglucanases (EI, EII, EIU, EIV, and EV), two exoglucanases (CBHI and CBHII), and two ⁇ -glucosidases (BGLI and BGLII). The need for five endoglucanase species in the T.
  • T. reesei produces ⁇ -glucosidases at low levels compared to other fungi such as Aspergillus species. Furthermore, the ⁇ -glucosidases of T. reesei are subject to product (glucose) inhibition whereas those of Aspergillus niger are more glucose tolerant. The levels of T. reesei ⁇ -glucosidase are presumably sufficient for growth on cellulose, but not sufficient for extensive in vitro saccharification of cellulose. T.
  • reesei cellulase preparations are considered most often for cellulose saccharification on an industrial scale.
  • A. naeslundii ⁇ -glucosidase SEQ ID No. 8 was chosen.
  • Enzyme should be thermostable to at least 45-5O 0 C, and (iii) Enzyme should have compatible pH optima.
  • CBHI and CBHII are the principal components of the T.
  • glycolases for lignocellulosic degradation are Endoglucanase (El) SEQ ID No. 2, Cellobiohydrolase (CBHl) SEQ ID No. 6, Beta Glucosidase (BGL) SEQ ID No. 8, Xylanase (XynZ) SEQ ID No. 4; and GAD gene SEQ ID No. for stress tolerance:
  • glycolases genes for lignocellulosic degradation used for plant transformation after codon optimization for efficient expression in the pearl millet system are Endoglucanase (El) SEQ ID No. 3, Cellobiohydrolase (CBHl) SEQ ID No. 7, Beta Glucosidase (BGL) SEQ ID No. 9, Xylanase (XynZ) SEQ ID No. 5; and GAD gene SEQ ID No. 1 for stress tolerance, which code for the cellulose degrading enzymes Endoglucanase (El) SEQ ID No.
  • ABI- 13B, ABI-56B and 843B Three parental inbred lines of pearl millet (P. glaucum L.) viz. ABI- 13B, ABI-56B and 843B were procured from Atash Seeds Private Ltd. India. They were initially screened for their agronomic performance in green house at Avesthagen. Selection of elite cultivar was based on the criteria like seed germination percentage and higher biomass yield. Seed germination percentage was highest (97%) in ABI- 13B followed by 843B (68 %) and ABI-56B (58 %) (Fig-3A). However, biomass yield was highest in ABI 56 B (82.7 g) followed by ABI13 B (76 g) and 843 B (41 g) respectively (Fig-3 B & C).
  • DW/FW ratio was 0.43 in ABI13B and 0.4 in ABI 56B, however in 843B this was 0.32 only. Seed germination and dry biomass yield is highest in ABI 13B. On this basis of over all productivity ABI 13B genotype was selected for further tissue culture studies (Fig 3).
  • the fascicles were manually separated from the rachis of the pearl millet spike, then young green seeds were cleaned and as surface sterilized as mature seeds. Then immature embryos (2 mm in size) were aseptically excised and inoculated on CIM. Immature embryos were excised from young seeds at around 90% efficiency. A rapid callus induction and growth thereafter was recorded on CIM. About 40% of the callus obtained from these embryos was embryogenic as seen in Fig-4A.
  • Embryogenic callus distinguishable by its compact and globular texture was transferred to Shoot Regeneration Medium (SRM) containing 1, 2, 3 mg/L of Benzyl amino purine (BAP), Kinetin (Kn) zeatin (1, 2, 3 mg/L) alone or in combination and with (0.2%) or without activated charcoal.
  • SRM Shoot Regeneration Medium
  • BAP and Kn were also supplemented with low concentrations (0.1 mg/L) of IAA, IBA and NAA.
  • media was supplemented with 10 mg/L AgNO 3 as ethylene inhibitor. All the cultures were incubated under 16h photoperiod in culture room.
  • selection and reporter gene has been vital for developing strategies for the production of transgenic plants and to increase their frequency.
  • selectable marker genes confer resistance to antibiotics or herbicides, enabling the transformed cells to be selected by growth on a media containing the corresponding compound.
  • GMO genetically modified organisms
  • antibiotic resistance markers which encompass: i) Inherent toxicity of the marker gene product; ii) Transfer and expression of the marker gene in gut microorganism and subsequent transfer to pathogen; iii) Toxicity or allergenicity of the gene product; iv) Intake of genetically modified plant products might compromise orally administrated antibiotics.
  • XyIA xylose isomerase
  • This selection favors the regeneration and growth of transgenic cells, while non-transgenic cells are starved but not killed.
  • XyIA catalyzes the reversible isomerization of D-xylose to D-xylulose as a part of the xylose metabolic pathway in microorganisms.
  • D-xylose is the major component of hydrolysis of hemicellulose from biomass.
  • Plant cells are not capable of metabolizing D- xylose as such completely.
  • 2% xylose concentration was regarded as the threshold for seed germination, beyond that xylose was found to inhibit the seed germination (Fig- 5A). However, in case of callus induction from seed and proliferation, all the concentrations 0.5 to 4.0 % were found to inhibit the callus proliferation (Fig- 5B). Callus growth was recorded only in control 3% sucrose. Thus 2% xylose concentration is the preferred concentration to be used for selection of transgenic pearl millet during seed germination. And 0.5% concentration is the preferred concentration to be used for selection of transgenic pearl millet during callus induction and proliferation.
  • Agrobacterium mediated transformation has become an extensively established mode of producing transgenic plants. There are only a very few reports on genetic transformation in pearl millet but those have been executed only via biolistic method. There is no published report available on AMT in pearl millet so far. Therefore, the current invention has focused towards developing an efficient AMT protocol in pearl millet.
  • A. tumefaciens strains EHA105 and GV 3101 were transformed with pl305.1 vector by electroporation.
  • Immature embryos were excised and infected with GV-3101 (OD ⁇ oo 0.5-0.6) and EHA-105 (OD 600 0.25-

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Abstract

La présente invention porte sur un procédé de développement de plantes tolérantes au stress qui présentent également des propriétés autoglucogènes et peuvent donc être utilisées dans la production de bioéthanol à partir de biomasse lignocellulosique.
PCT/IB2010/002107 2009-08-28 2010-08-28 Procédé de développement de plantes tolérantes au stress présentant des propriétés autoglucogènes pour une utilisation dans la production de bioéthanol à partir de biomasse lignocellulosique WO2011024065A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017053164A1 (fr) * 2015-09-22 2017-03-30 Dow Agrosciences Llc Promoteur et 3'utr de plante pour l'expression d'un transgène
WO2017180180A3 (fr) * 2015-09-22 2018-01-04 Dow Agrosciences Llc Promoteur de plante et 3'utr pour l'expression de transgènes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040067555A1 (en) * 1999-05-26 2004-04-08 University Of Florida Research Foundation, Incorporated Recombinant hosts suitable for simultaneous saccharification and fermentation
US20070192900A1 (en) * 2006-02-14 2007-08-16 Board Of Trustees Of Michigan State University Production of beta-glucosidase, hemicellulase and ligninase in E1 and FLC-cellulase-transgenic plants
WO2008095033A2 (fr) * 2007-01-30 2008-08-07 Verenium Corporation Enzymes pour le traitement de matières lignocellulosiques, des acides nucléiques les codant et procédés pour leur fabrication et leur utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040067555A1 (en) * 1999-05-26 2004-04-08 University Of Florida Research Foundation, Incorporated Recombinant hosts suitable for simultaneous saccharification and fermentation
US20070192900A1 (en) * 2006-02-14 2007-08-16 Board Of Trustees Of Michigan State University Production of beta-glucosidase, hemicellulase and ligninase in E1 and FLC-cellulase-transgenic plants
WO2008095033A2 (fr) * 2007-01-30 2008-08-07 Verenium Corporation Enzymes pour le traitement de matières lignocellulosiques, des acides nucléiques les codant et procédés pour leur fabrication et leur utilisation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017053164A1 (fr) * 2015-09-22 2017-03-30 Dow Agrosciences Llc Promoteur et 3'utr de plante pour l'expression d'un transgène
WO2017180180A3 (fr) * 2015-09-22 2018-01-04 Dow Agrosciences Llc Promoteur de plante et 3'utr pour l'expression de transgènes

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