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WO2009090379A2 - Polypeptides de phenol oxydase - Google Patents

Polypeptides de phenol oxydase Download PDF

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Publication number
WO2009090379A2
WO2009090379A2 PCT/GB2009/000093 GB2009000093W WO2009090379A2 WO 2009090379 A2 WO2009090379 A2 WO 2009090379A2 GB 2009000093 W GB2009000093 W GB 2009000093W WO 2009090379 A2 WO2009090379 A2 WO 2009090379A2
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WIPO (PCT)
Prior art keywords
nucleic acid
cell
polypeptide
acid molecule
acid sequence
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PCT/GB2009/000093
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WO2009090379A3 (fr
Inventor
Andrew King
Ian Alexander Graham
Simon Mqueen Mason
Neil Bruce
Dianna Joy Bowles
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University of York
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University of York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to nucleic acid molecules that encode haemocyanin polypeptides that have phenol oxidase activity; polypeptides encoded by said nucleic acids, transgenic cells transfected with said nucleic acids and uses thereof in the manufacture of biofuels and the modification of lignocellulose materials.
  • Invertebrates lack adaptive immunity and rely on innate immune responses, for example the secretion of pro-inflammatory cytokines, complement activation, pattern recognition receptors e.g. Toll like receptors.
  • An innate immune response found in invertebrates is the prophenol oxidase activating system which is triggered by microbial infection and is therefore an important part of invertebrate defence wherein it functions to detect and destroy invading pathogens as well as an involvement in melanin biosynthesis which is utilized in wound healing and pathogen encapsulation.
  • the cascade involves a series of enzymes which exists in the body fluid of insects.
  • lignocellulose enter coastal marine ecosystems particularly from large river systems and mangrove forests.
  • Wood in particular, has provided an important diet for a range of organisms.
  • Particularly prominent in this context are two families of specialist wood borers. These are the teredinid bivalves (known commonly as shipworms) and isopod crustaceans of the Limnoriidae. These borers ingest wood, but have highly contrasted approaches to lignocellulose degradation.
  • teredinid wood borers have a close mutualistic association with microbes that appear to play a critical role in lignocellulose degradation. In contrast the limnorid digestive tract appears to be effectively sterile with few or no resident microbes.
  • This disclosure relates to the identification and isolation of nucleic acid sequences that encode polypeptides that function in the modification and degradation of lignocellulose.
  • Our sequencing has identified a large number of DNA sequences that encode polypeptides that facilitate the breakdown of lignocellulosic material.
  • a surprising finding is the abundant representation of hemocyanins in the sequence data. Hemocyanins are part of a small group of copper complexed proteins involved in oxygen transport in many invertebrate species and also phenol oxidation. Not only do hemocyanins share key structural features with tyrosinases and phenol oxidases, but it has been shown that limited proteolysis of hemocyanins can render them into active phenol oxidases.
  • Plant biomass is one of the greatest untapped reserves on the planet and is largely composed of cell walls.
  • Saccharification is a process by which plant lignocellulosic materials (e.g., lignin, cellulose, hemicellulose) are hydrolysed to glucose through chemical and enzymatic means. Typically this involves the pre-treatment of plant material with alkali to remove lignin followed by enzyme digestion of cellulose.
  • the enzymes currently available for industrial lignocellulose saccharification involve a cocktail of endoglucanases and cellobiohydrolases, the activity of which is severely limited by access to their substrates due to the lignification of the cell wall and cellulose crystallinity.
  • endoglucanases and cellobiohydrolases the activity of which is severely limited by access to their substrates due to the lignification of the cell wall and cellulose crystallinity.
  • ammonia, or dilute acid hydrolysis at high temperature are required.
  • commercial cellulases have to be used at loadings of several grams per kg of lignocellulose, and as a consequence, the cost and energy inputs of producing liquid biofuels from plant biomass are currently too high to be economically competitive.
  • the identification of large numbers of hemocyanins comprising phenol oxidase activities provides the means by which lignocellulosic materials can be treated prior to enzymatic digestion using glycosyl hydrolases and the like.
  • an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a nucleic acid molecule consisting of the nucleic acid sequence as represented in i) Figures 19a, 19c, 20a, 20c, 21a, 21c, 22a, 22c, 23a 24a or 25a; ii) a nucleic acid molecule comprising nucleic acid sequences that hybridise to the sequence identified in (i) above under stringent hybridisation and encode a hemocyanin polypeptide with phenol oxidase activity; iii) a nucleic acid molecule comprising nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) and (ii).
  • Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993).
  • the T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
  • Hybridization 5x SSC at 65°C for 16 hours
  • Hybridization 5x-6x SSC at 65°C-70°C for 16-20 hours
  • Hybridization 6x SSC at RT to 55°C for 16-20 hours
  • nucleic acid is cDNA.
  • said nucleic acid is genomic DNA.
  • said polypeptide comprises or consists of an amino acid sequence presented in Figures 19b, 1 ⁇ d, 20b, 2Od, 21b, 21 d, 22b, 22d, 23b, 24b or 25b.
  • said polypeptide is a variant polypeptide and comprises the amino acid sequence represented in Figures 19b, 19d, 20b, 2Od, 21b, 21 d, 22b, 22d, 23b, 24b or 25b, which sequence has been modified by deletion, addition or substitution of at least one amino acid residue wherein said modification retains or enhances the enzyme activity of said polypeptide.
  • polypeptide is a fragment comprising wherein said fragment has phenol oxidase activity.
  • a variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination.
  • substitutions are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics.
  • amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies. Variant polypeptides include natural polymorphic variants that exist, for example as illustrated in the figures and examples.
  • the invention features polypeptide sequences having at least 60-75% identity with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof.
  • the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated herein.
  • a vector comprising a nucleic acid molecule according to the invention.
  • said vector is adapted for the recombinant expression of said nucleic acid molecule.
  • nucleic acid constructs which operate as plant vectors.
  • Suitable vectors may include plant viral-derived vectors (see e.g. EP194809).
  • vectors that are adapted for bacterial and fungal expression.
  • said adaptation is the provision of a regulatable promoter; preferably an inducible promoter.
  • promoter is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription.
  • Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design.
  • Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-Ia promoter, which is activated by salicylic acid.
  • promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) MoI. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.
  • tissue-specific promoters can be utilised.
  • Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792- 803; Hansen et al. (1997) MoI. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol.
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • the promoter is an inducible promoter or a developmental ⁇ regulated promoter.
  • said promoter sequence is a senescence inducible promoter sequence.
  • Senescence specific promoters are known in the art. For example, WO0070061 ; US2004025205; WO2006102559; US6, 359, 197; WO2006025664 the contents of which are incorporated by reference in their entirety, describe various plant promoters that become activated when senescence is induced.
  • the present disclosure also describes two promoters that control the expression of genes involved in triacylglycerol metabolism. The genes that encode ACX 1 and KAT 2 are both induced during the induction of senescence and are therefore considered a least in part, senescence inducible.
  • vectors are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Cray RRD ed) Oxford, BIOS Scientific Publishers, pp 121- 148. Suitable vectors may include plant viral-derived vectors (see e.g. EP-A-194809).
  • Vectors may also include a selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).
  • a selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).
  • an isolated cell comprising a nucleic acid molecule according to the invention.
  • said cell is a eukaryotic cell.
  • said cell is a fungal cell; preferably a yeast cell.
  • said fungal cell is selected from the group consisting of: Aspergillus spp, Neurospora spp or Saccaromyces spp.
  • said cell is a plant cell.
  • a plant comprising a plant cell according to the invention.
  • said plant cell is selected from the group consisting of:
  • said plant is selected from the group consisting of: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton ⁇ Gossypium hirsutum), sweet potato ⁇ lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut
  • plants of the present invention are biomass crops (switchgrass, alfalfa, willow, poplar, eucalyptus, miscanthus, wheat, maize or barley.), other crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea), and other root, tuber or seed crops.
  • Important seed crops are oilseed rape, sugar beet, maize, sunflower, soybean, sorghum, and flax (linseed).
  • Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassica including cabbage, broccoli, and cauliflower.
  • the present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper.
  • said cell is transfected with a further nucleic acid molecule wherein said further nucleic acid molecule encodes a polypeptide that has glycosyl hydrolase activity.
  • said nucleic acid molecule is selected from the group consisting of: i) a nucleic acid molecule consisting of the nucleic acid sequence as represented in Figures 1a, 1c, 2a, 3a, 3c, 4a, 5a, 5c, 6a, 7a, 7c, 8a, 8c, 9a, 9c, 10a, 10c, 11a, 11c, 12a, 13a, 13c, 14a, 15a, 15c, 16a, 17a, 17c, 18a or 18c; ii) a nucleic acid molecule comprising nucleic acid sequences that hybridise to the sequence identified in (i) above under stringent hybridisation and encodes a polypeptide with glycosyl hydrolase activity; iii) a nucleic acid molecule comprising nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) and (ii).
  • said polypeptide comprises or consists of an amino acid sequence presented in Figures 1b, 1d, 2b, 3b, 3d, 4b, 5b, 5d, 6b, 7b,7d, 8b, 8d, 9b, 9d, 10b, 10d, 11b, 11d, 12b, 13b, 13d, 14b, 15b, 15d, 16b, 17b, 17d, 18b or 18d.
  • said polypeptide is a variant polypeptide and comprises the amino acid sequence represented in Figures 1b, 1d, 2b, 3b, 3d, 4b, 5b, 5d, 6b, 7b,7d, 8b, 8d, 9b, 9d, 10b, 10d, 11b, 11 d, 12b, 13b, 13d, 14b, 15b, 15d, 16b, 17b, 17d, 18b or 18d, which sequence has been modified by deletion, addition or substitution of at least one amino acid residue wherein said modification retains or enhances the glycosyl hydrolase enzyme activity of said polypeptide.
  • said further nucleic acid molecule encodes a protease that digests said hemocyanin polypeptide to provide a phenol oxidase polypeptide.
  • a seed comprising a plant cell according to the invention.
  • said cell is a prokaryotic cell; preferably a bacterial cell.
  • a method for the recombinant production of a polypeptide according to the invention comprising: i) providing a prokaryotic cell according to the invention; ii) culturing said cell under cell culture conditions conducive to the production of said polypeptide; and optionally iii) isolating said polypeptide from the cell or cell culture.
  • said cell is a bacterial cell, for example an E.coli cell.
  • a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of the DNA sequence as represented in Figure 19a, 19c, 20a, 20c, 21a, 21c, 22a, 22c, 23a 24a or 25a ; ii) a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above under stringent hybridisation and encodes a hemocyanin polypeptide with phenol oxidase; iii) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in (i) and (ii), for use in the modification of lignocellulosic material.
  • a method to prepare plant biomass for saccharification comprising: i) providing a transgenic plant according to the invention; ii) inducing expression of at least one of said nucleic acid molecules; and/or harvesting the plant biomass; and optionally iii) contacting the plant biomass with a protease that digests said hemocyanin polypeptide to provide a phenol oxidase polypeptide.
  • Plant biomass refers to plant tissue and lignocellulosic materials that comprise the plant and includes plant organs (e.g. stems, leaves, flowers, roots, seeds) which may increase in size, number or quality to increase yield and also plant material that has been pre-treated prior to further processing.
  • plant organs e.g. stems, leaves, flowers, roots, seeds
  • Genes that encode proteins that enhance the growth characteristics of a plant are well known in the art.
  • WO92/09685 the content of which is incorporated by reference, describes the plant homologue of the yeast cell-cycle control gene cdc2 referred to as p34Cd 2 and is an important regulator of cell proliferation, particularly in leaf tissue.
  • WO2005/085452 the content of which is incorporated by reference, describes the shoot specific expression of cyclin D3, a cell growth regulator and the enhancement of plant yield.
  • WO2004/087929 the content of which is incorporated by reference, describes the expression of the CCS52 gene, a gene that encodes a cell-cycle regulatory protein, and the enhancement of plant size and increased organ size and number.
  • WO2005/059147 the content of which is incorporated by reference, describes a growth regulatory protein, GRUBX and the effect of over-expression on plant morphology.
  • WO2005/083094 describes a D-type cyclin dependent kinase which when over-expressed results in increased seed yield, also see WO2005/085452, WO2005/061702 and WO2006/100112 each of which is incorporated by reference in their entirety.
  • the transgenic plant material is further processed by saccharification to sugar.
  • a method to prepare plant biomass for saccharification comprising: i) forming a preparation comprising a plant biomass material and at least one polypeptide according to the invention; ii) incubating said preparation; and optionally iii) processing said plant biomass material by saccharification to sugar.
  • said sugar is used as a feedstock in the production of ethanol by microbial fermentation.
  • Microorganisms used in the process according to the invention are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism.
  • microorganisms are grown in a liquid medium comprising a carbon source (e.g. sugar as formed during the saccharification process), a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0 0 C and 100 0 C, preferably between 10 0 C and 60 0 C, while gassing in oxygen.
  • a carbon source e.g. sugar as formed during the saccharification process
  • a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0 0 C and 100 0 C, preferably between 10 0 C
  • the pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not.
  • the cultures can be grown batchwise, semi-batchwise or continuously.
  • Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously.
  • the products produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction or distillation.
  • the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.
  • the culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
  • these media which can be employed in accordance with the invention usually comprise one or more, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds.
  • nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur- containing fine chemicals, in particular of methionine.
  • Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.
  • Chelating agents may be added to the medium in order to keep the metal ions in solution.
  • Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.
  • the fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
  • growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium.
  • the exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical
  • Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
  • All media components are sterilized, either by heat (20 min at 1.5 bar and 121 0 C) or by filter sterilization.
  • the components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
  • the culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40 0 C, and may be kept constant or may be altered during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid.
  • Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters.
  • the fermentation broth can then be processed further.
  • the biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth.
  • a method for disrupting lignin in woody plant materials comprising: i) forming a preparation comprising at least one polypeptide according to the invention and woody material; ii) incubating said preparation under conditions that disrupt lignin in said woody material to facilitate pulping.
  • a method to modify surface properties of cotton materials comprising: i) forming a preparation comprising a polypeptide according to the invention and a cotton based material; ii) incubating said preparation under conditions that modify the surface properties of said cotton based material.
  • polypeptides according to the invention have utility as brightenmers or texturising agents for improving textile properties during manufacture or in laundry applications.
  • Figure 1a is the nucleic acid sequence of a GH5a glycosyl hydrolase;
  • Figure 1b is the corresponding amino acid sequence;
  • Figure 1c is the nucleic acid sequence of a GH5a glycosyl hydrolase;
  • Figure 1d is the corresponding amino acid sequence;
  • Figure 2a is the nucleic acid sequence of GH5b glycosyl hydrolase;
  • Figure 2b is the corresponding amino acid sequence;
  • Figure 3a is the nucleic acid sequence of GH5c glycosyl hydrolase;
  • Figure 3b is the corresponding amino acid sequence including natural polymorphic variant;
  • Figure 3c is the nucleic acid sequence of a GH5c glycosyl hydrolase;
  • Figure 3d is the corresponding amino acid sequence
  • Figure 4a is the nucleic acid sequence of GH5d glycosyl hydrolase
  • Figure 5b is the corresponding amino acid sequence including natural polymorphic variants
  • Figure 5a is the nucleic acid sequence of GH5e glycosyl hydrolase;
  • Figure 5b is the corresponding amino acid sequence including natural polymorphic variant;
  • Figure 5c is the nucleic acid sequence of a GH5e glycosyl hydrolase;
  • Figure 5d is the corresponding amino acid sequence
  • Figure 6a is the nucleic acid sequence of GH5f glycosyl hydrolase;
  • Figure 6b is the corresponding amino acid sequence including natural polymorphic variants;
  • Figure 7a is the nucleic acid sequence of GH7a glycosyl hydrolase;
  • Figure 7b is the corresponding amino acid sequence including natural polymorphic variants;
  • Figure 7c is the nucleic acid sequence of a GH7a glycosyl hydrolase;
  • Figure 7d is the corresponding amino acid sequence;
  • Figure 8a is the nucleic acid sequence of GH7b glycosyl hydrolase;
  • Figure 8b is the corresponding amino acid sequence including natural polymorphic variants;
  • Figure 8c is the nucleic acid sequence of a GH7b glycosyl hydrolase;
  • Figure 8d is the corresponding amino acid sequence;
  • Figure 9a is the nucleic acid sequence of GH7c glycosyl hydrolase; Figure 9b is the corresponding amino acid sequence; Figure 9c is the nucleic acid sequence of a GH7c glycosyl hydrolase; Figure 9d is the corresponding amino acid sequence
  • Figure 10a is the nucleic acid sequence of GH9a glycosyl hydrolase;
  • Figure 10b is the corresponding amino acid sequence;
  • Figure 10c is the nucleic acid sequence of a GH9a glycosyl hydrolase;
  • Figure 10d is the corresponding amino acid sequence
  • Figure 11a is the nucleic acid sequence of GH9b glycosyl hydrolase; Figure 11b is the corresponding amino acid sequence; Figure 11c is the nucleic acid sequence of a GH9b glycosyl hydrolase; Figure 11d is the corresponding amino acid sequence
  • Figure 12a is the nucleic acid sequence of GH9c glycosyl hydrolase
  • Figure 12b is the corresponding amino acid sequence and natural polymorphic variant
  • Figure 13a is the nucleic acid sequence of GH9d glycosyl hydrolase;
  • Figure 13b is the corresponding amino acid sequence and natural polymorphic variants;
  • Figure 13c is the nucleic acid sequence of a GH9d glycosyl hydrolase;
  • Figure 13d is the corresponding amino acid sequence
  • Figure 14a is the nucleic acid sequence of GH9e glycosyl hydrolase;
  • Figure 14b is the corresponding amino acid sequence and natural polymorphic variants;
  • Figure 15a is the nucleic acid sequence of GH9f glycosyl hydrolase;
  • Figure 15b is the corresponding amino acid sequence and natural polymorphic variant;
  • Figure 15c is the nucleic acid sequence of a GH9f glycosyl hydrolase;
  • Figure 15d is the corresponding amino acid sequence
  • Figure 16a is the nucleic acid sequence of GH9g glycosyl hydrolase;
  • Figure 16b is the corresponding amino acid sequence;
  • Figure 17a is the nucleic acid sequence of GH9h glycosyl hydrolase;
  • Figure 17b is the corresponding amino acid sequence and natural polymorphic variants;
  • Figure 17c is the nucleic acid sequence of a GH9h glycosyl hydrolase;
  • Figure 17d is the corresponding amino acid sequence
  • Figure 18a is the nucleic acid sequence of GH9i glycosyl hydrolase;
  • Figure 18b is the corresponding amino acid sequence;
  • Figure 18c is the nucleic acid sequence of a GH9i glycosyl hydrolase;
  • Figure 18d is the corresponding amino acid sequence;
  • Figure 19a is nucleic acid sequence of hemocyanin HC1 ;
  • Figure 19b is the corresponding amino acid sequence and natural polymorphic variants;
  • 19c is the nucleic acid sequence of a hemocyanin HC1 ;
  • Figure 19d is the corresponding amino acid sequence;
  • Figure 20a js nucleic acid sequence of hemocyanin HC2;
  • Figure 20b is the corresponding amino acid sequence;
  • 20c is the nucleic acid sequence of a hemocyanin HC2;
  • Figure 2Od is the corresponding amino acid sequence;
  • Figure 21a is nucleic acid sequence of hemocyanin HC3;
  • Figure 21b is the corresponding amino acid sequence and natural polymorphic variants;
  • 21c is the nucleic acid sequence of a hemocyanin HC3;
  • Figure 21 d is the corresponding amino acid sequence
  • Figure 22a is nucleic acid sequence of hemocyanin HC4;
  • Figure 22b is the corresponding amino acid sequence and natural polymorphic variants;
  • 22c is the nucleic acid sequence of a hemocyanin HC4;
  • Figure 22d is the corresponding amino acid sequence
  • Figure 23a is nucleic acid sequence of hemocyanin HC5; Figure 23b is the corresponding amino acid sequence and natural polymorphic variants; Figure 24a is nucleic acid sequence of hemocyanin HC6; Figure 24b is the corresponding amino acid sequence and natural polymorphic variants; and
  • Figure 25a is nucleic acid sequence of hemocyanin HC7;
  • Figure 25b is the corresponding amino acid sequence.
  • the midgut from 30 Limnoria were dissected and placed into a microfuge tube containing PBS (on ice). The microfuge tube was then spun at 500 r.p.m. for 1 minute. The excess PBS was removed, and 200 ⁇ l of Trizol (Invitrogen, Carlsbad, California, US) was added. The tube was vortexed for 10 seconds to homogenise the contents, and a further 200 ⁇ l of Trizol were added. The tubes were then frozen at -70 0 C.
  • the tube was thawed, and 80 ⁇ l of chloroform were added. The tube was then vortexed for 15 secconds. The samples were centrifuged at 14,000 g for 5 minutes at room temperature. The upper aqueous phase containing the RNA was transferred to an RNase free microfuge tube, lsopropanol (200 ⁇ l) was added and the tube mixed by inversion. The tube was then incubated at room temperature for 15 minutes.
  • the tube was then centrifuged (14,000 g, 10 minutes) to pellet the RNA. The supernatant was removed, and 500 ⁇ l of 70 % ethanol was added. The tube was vortexed, and centrifuged again (14,000 g, 10 minutes). The tube washed twice more with 500 ⁇ l of 70 % ethanol. After the final wash, the RNA pellet was allowed to dry. The RNA was then dissolved in 30 ⁇ l of RNase free water.
  • RNA quality and concentration was determined using a Nanodrop spectrophotometer. The RNA was stored at -70 0 C until required. cDNA synthesis and 454 sequencing.
  • cDNA was prepared using the Dualsystems Biotech (Schlieren, Switzerland) EasyClone cDNA library construction kit.
  • the initial cDNA synthesis reaction was performed in a 20 ⁇ l reaction using 700 ng of Limnoria total RNA, CDS-1 adapter and PlugOligo-1 adapter according to the manufacter's instructions.
  • the final PCR reactions were carried out in 5 x 50 ⁇ l reactions using 2 ⁇ l of double-stranded DNA and 17 cycles of PCR.
  • the PCR products were pooled and concentrated by ethanol precipitation. 25 ⁇ l of 3M sodium acetate (pH 5.2), 625 ⁇ l of ethanol and 30 ⁇ g of glycogen was added.
  • the tubes were placed at -70 0 C for 1 hour.
  • the DNA was pelleted by centrifugation at 13,000 g for 15 minutes.
  • the DNA pellet was washed twice with 500 ⁇ l of 70 % ethanol.
  • the DNA was then allowed to dry.
  • the cDNA was then dissolved in 31.2 ⁇ l of low TE (10 mM Tris-HCI, pH 8.0, 0.1 mM EDTA). The cDNA concentration was determined using a 1.2 ⁇ l aliquot. The remaining cDNA (7.5 ⁇ g in 30 ⁇ l) was sent to Cogenics, France for 454 sequencing. A single run was conducted using the Roche 454 GS-FLX platform, yielding 431,836 sequences with a median read length of 250 bases. Sequence assembly was performed using CAP3.

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Abstract

Cette invention concerne des molécules d'acides nucléiques codant pour des polypeptides d'hàmocyanine ayant une activité phénol oxydase, des polypeptides codés par ces acides nucléiques, des cellules transgéniques transfectées avec ces acides nucléiques et leurs utilisations pour la fabrication de biocarburants et la modification de matériaux lignocellulosiques.
PCT/GB2009/000093 2008-01-17 2009-01-15 Polypeptides de phenol oxydase Ceased WO2009090379A2 (fr)

Applications Claiming Priority (4)

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GB0800832.8 2008-01-17
GBGB0800832.8A GB0800832D0 (en) 2008-01-17 2008-01-17 Enzymes 2
US8946908P 2008-08-15 2008-08-15
US61/089,469 2008-08-15

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WO2009090379A3 WO2009090379A3 (fr) 2010-01-28

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* Cited by examiner, † Cited by third party
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WO2002014484A1 (fr) * 2000-08-15 2002-02-21 Valtion Teknillinen Tutkimuskeskus Enzyme de tyrosinase
GB0800831D0 (en) * 2008-01-17 2008-02-27 Univ York Enzymes 1

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