WO2000071728A1 - Recombinant bacterial phytases and uses thereof - Google Patents

Recombinant bacterial phytases and uses thereof Download PDF

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Publication number
WO2000071728A1
WO2000071728A1 PCT/US2000/014846 US0014846W WO0071728A1 WO 2000071728 A1 WO2000071728 A1 WO 2000071728A1 US 0014846 W US0014846 W US 0014846W WO 0071728 A1 WO0071728 A1 WO 0071728A1
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Prior art keywords
sequence
phytase
enzyme
phytate
sequences
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PCT/US2000/014846
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French (fr)
Inventor
Jay M. Short
Keith A. Kretz
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Diversa Corporation
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Priority to AU53044/00A priority Critical patent/AU783041B2/en
Priority to JP2000620105A priority patent/JP2003500057A/en
Priority to CA002374532A priority patent/CA2374532A1/en
Priority to KR1020017015109A priority patent/KR20020018672A/en
Priority to EP00937932A priority patent/EP1180152A1/en
Priority to BR0010946-0A priority patent/BR0010946A/en
Publication of WO2000071728A1 publication Critical patent/WO2000071728A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
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    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30—Removing undesirable substances, e.g. bitter substances
    • A23L11/33—Removing undesirable substances, e.g. bitter substances using enzymes; Enzymatic transformation of pulses or legumes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A23L11/65—Soy drinks
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/101—Addition of antibiotics, vitamins, amino-acids, or minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12G2200/00—Special features
    • C12G2200/15—Use of particular enzymes in the preparation of wine

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention have been identified as phytases and in particular, microbial enzymes having phytase activity
  • Minerals are essential elements for the growth of all organisms. Dietary minerals can be derived from many source materials, including plants E.g , plant seeds are a rich source of minerals since they contain ions that are complexed with the phosphate groups of phytic acid molecules. These phytate-associated minerals satisfy the dietary needs of some species of farmed organisms, such as multi-stomached ruminants. Accordingly, ruminants do not require dietary supplementation with inorganic phosphate and minerals because microorganisms in the rumen produce enzymes that catalyze conversion of phytate (myo-inositol-hexaphosphate) to inositol and inorganic phosphate.
  • the instantly disclosed phytase molecules - either alone or in combination with other reagents (including but not limited to enzymes, including proteases) - are serviceable not only in this application (e g , for prevention of the unwanted slugde) but also in other applications where phytate hydrolysis is desirable
  • the instantly disclosed phytase molecules - either alone or in combination with other reagents (including but not limited to enzymes, including proteases) - are serviceable not only in this application (e g , for increasing the growth rate of farmed animals) but also in other applications where phytate hydrolysis is desirable
  • Phytate occurs as a source of stored phosphorous in virtually all plant feeds (Graf (Ed ), 1986)
  • Phytic acid forms a normal part of the seed in cereals and legumes It functions to bind dietary minerals that are essential to the new plant as it emerges from the seed
  • the phosphate groups of phytic acid are removed by the seed enzyme phytase, the ability to bind metal ions is lost and the minerals become available to the plant
  • the trace minerals bound by phytic acid are largely unavailable for abso ⁇ tion by monogast ⁇ c animals, which lack phytase activity
  • phytate-associated nutrients are comprised of not only phosphate that is covalently linked to phytate, but also other minerals that are chelated by phytate as well Moreover, upon injestion, unhydrolyzed phytate may further encounter and become associated with additional minerals. The chelation of minerals may inhibit the activity of enzymes for which these minerals serve as co-factors.
  • Phytate Conversion of phytate to inositol and inorganic phosphorous can be catalyzed by microbial enzymes refe ⁇ ed to broadly as phytases.
  • Phytases such as phytase #EC 3.1.3.8 are capable of catalyzing the hydrolysis of myo-mositol hexaphosphate to D-myo-mositol 1, 2,4,5, 6-pentaphosphate and orthophosphate.
  • Certain fungal phytases reportedly hydrolyze inositol pentaphosphate to tetra-, t ⁇ -, and lower phosphates. E.g., A.
  • ficuum phytases reportedly produce mixtures of myoinositol di- and mono-phosphates (Ullah, 1988).
  • Phytase-producing microorganisms are comprised of bacteria such as Bacillus subtilis (Powar and Jagannathan, 1982) and Pseudomonas (Cosgrove, 1970); yeasts such as Sacchoromyces cerevisiae (Nayini and Markakis, 1984); and fungi such as Aspergillus terreus (Yamada et al, 1968).
  • Acid phosphatases are enzymes that catalytically hydrolyze a wide variety of phosphate esters and usually exhibit pH optima below 6.0 (Igarashi & Hollander, 1968). E.g., #EC 3.1.3.2 enzymes catalyze the hydrolysis of orthophospho ⁇ c monoesters to orthophosphate products.
  • An acid phosphatase has reportedly been purified from A. ficuum. The deglycosylated form of the acid phosphatase has an apparent molecular weight of 32.6 kDa (Ullah et al . 1987)
  • Phytase and less specific acid phosphatases are produced by the fungus Aspergillus ficuum as extracellular enzymes (Shieh et al, 1969). Ullah reportedly purified a phytase from wild-type A. ficuum that had an apparent molecular weight of 61.7 kDA (on SDS- PAGE: as co ⁇ ected for glycosylation); pH optima at pH 2.5 and pH 5.5; a Km of about 40 ⁇ m; and, a specific activity of about 50 U/mg (Ullah, 1988).
  • PCT patent application WO 91/05053 also reportedly discloses isolation and molecular cloning of a phytase from Aspergillus ficuum with pH optima at pH 2.5 and pH 5.5, a Km of about 250 ⁇ m, and specific activity of about 100 U/mg protein.
  • the specific activity cited for these previously repo ⁇ ed microbial enzymes has been approximately in the range of 50-100 U/mg protein.
  • the phytase activity disclosed in the instant invention has been measured to be approximately 4400 U/mg. This co ⁇ esponds to about a 40-fold or better improvement in activity
  • Microbial phytases may also reportedly be useful for producing animal feed from certain industrial processes, e g., wheat and corn waste products
  • the wet milling process of corn produces glutens sold as animal feeds.
  • the addition of phytase may reportedly improve the nutritional value of the feed product.
  • a transgenic plant can be formed that is comprised of an expression system for expressing a phytase molecule
  • factors that are not directly related to the activity of the expressed molecule proper such as the expression level
  • only a finite - and potentially insufficient - level of optimization may be maximally achieved Accordingly, there is also a need for obtaining molecules with improved characteristics
  • DirectEvolution® comprises a) the subjection of one or more molecular template to mutagenesis to generate novel molecules, and b) the selection among these progeny species of novel molecules with more desirable characteristics
  • the power of directed evolution depends on the starting choice of starting templates, as well as on the mutagenesis process(es) chosen and the screening process(es) used
  • the approach of generating and evaluating a full range of mutagenic permutations on randomly chosen molecular templates &/or on initial molecular templates having overly suboptimal properties is often a forbiddingly large task
  • the use of such templates offers, at best, a circuitously suboptimal path and potentially provides very poor prospects of yielding sufficiently improved progeny molecules
  • our cu ⁇ ent body of knowledge is very limited with respect to the ability to rigorously predict beneficial modifications
  • the present invention provides a polynucleotide and a polypeptide encoded thereby which has been identified as a phytase enzyme having phytase activity
  • a novel recombinant enzyme as well as active fragments, analogs and derivatives thereof
  • this invention relates to the use of recombinant phytase molecules of bacterial origin that are serviceable for improving the nutntional value of phytate-containing foodsmffs
  • Previous publications have disclosed the use of fungal phytases. but the use of bacterial phyatases for this pu ⁇ ose is novel
  • this invention relates to the use of newly identified recombinant phytase molecules of E coli origin that are serviceable for improving the nutritional value of phytate-containing foodstuffs
  • This use is comprised of employing the newly identified molecules to hydrolyze phytate m foodstuffs Hydrolysis may occur before injestion or after injestion or both before and after injestion of the phytate
  • This application is particularly relevant, but not limited, to non-ruminant organisms and includes the expression of the disclosed novel phytase molecules in transformed hosts, the contacting of the disclosed novel phytase molecules with phytate in foodstuffs and other materials, and the treatment of animal digestive systems with the disclosed novel phytase molecules
  • hydrolysis may occur mdependently of consumption, e g in an in vitro application, such as in a reaction vessel
  • the treatment of phytate-containing materials includes the treatment of a wide range of mate ⁇ als, including ones that are not intended to be foodsmffs, e g the treatment of excrementary (or fecal) material
  • Prefe ⁇ ed molecules of the present invention include a recombinant phytase isolated from Eschenchia coli B that improves the efficiency of release of phosphorous from phytate and the salts of phytic acid when compared to previosuly identified fungal phytases
  • a phytase enzyme that is serviceable for inco ⁇ ortion into foodstuffs More specifically, there is provided a phytase enzyme that is serviceable for improving the nutritional value of phytate-containing foodstuffs More specifically still, there is provided a phytase enzyme that, when applied to phytate-containing foodstuffs, measurably improves the growth performance of an organism that consumes it It is theorized that the beneficial mechanism of action of the phytase activity is compnsed appreciably if not substantially of the hydrolysis of phytate It is provided that the beneficial action may occur before injestion or alternatively after injestion or alternatively both before and after injestion of the phytate-containing foodstuff In the case where the beneficial action occurs after injestion, it is an object of the present invention to provide a phytase enzyme that has activity that is retained upon consumption by non-ruminant organisms
  • nucleic acid molecules encoding the enzyme of the present invention - including mRNA, DNA, cDNA, genomic DNA - as well as active derivatives, analogs and fragments of such enzyme
  • a process for producing such polypeptides by recombinant techniques comprising cultu ⁇ ng recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding an enzyme of the present invention, under conditions promoting expression of said enzyme and subsequent recovery of said enzyme
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention
  • hybridization-based uses of these probes include, but are by no means limited to, PCR, Northern and Southern types of hybridizations, RNA protection assays, and in situ types of hybridizations
  • the uses of the instantly disclosed molecules further include, in a non-limiting manner, diagnostic applications
  • these processes compnse the generation of antibodies to the disclosed molecules, and uses of such antibodies, including, for example, for the identification and isolation of similar sequences in enzymes from other organisms
  • these processes include the use of the present enzymes as templates for directed evolution, comprising the generation of novel molecules by followed by screening-based approaches for discoverying of progeny molecules with improved properties
  • transgenic non-human organism whose genome compnses a heterologous nucleic acid sequence encoding a polypeptide having phytase activity, wherein said transgene results in expression of a phytase polypeptide.
  • Figure 1A and B show the nucleotide and deduced amino acid sequences the enzyme of the present invention. Sequencing was performed using a 378 automated DNA sequencer (Applied Biosystems, Inc.).
  • Figure 2 shows the pH and temperature profile and stability data for the phytase enzyme of the present invention.
  • the assay used for these analysis is the following for the detection of phytase activity Phytase activity is measured by incubating 150 ⁇ l of the enzyme preparation with 600 ⁇ l of 2 mM sodium phytate in 100 mM Tris HCl buffer pH 7.5, supplemented with ImM CaCl 2 for 30 minutes at 37°C. After incubation the reaction is stopped by adding 750 ⁇ l of 5% t ⁇ chloroacetic acid.
  • antibody refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab') 2 , Fv, and SCA fragments, that are capable of binding to an epitope of a phytase polypeptide.
  • Fab fragments of immunoglobulin molecules
  • Fab' fragments of immunoglobulin molecules
  • Fv fragments of a phytase polypeptide.
  • SCA fragments fragments, which retain some ability to selectively bind to the antigen (e g., an phytase antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g , Harlow and Lane, supra), and are descnbed further, as follows.
  • An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain
  • An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
  • An (Fab') 2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a (Fab') 2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds (4)
  • An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains
  • SCA single chain antibody
  • degradation effective amount refers to the amount of enzyme which is required to degrade at least 50% of the phytate, as compared to phytate not contacted with the enzyme. Preferably, at least 80% of the phytate is degraded
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical pu ⁇ oses typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the pu ⁇ ose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a gel to isolate the desired fragment.
  • epitope refers to an antigemc determinant on an antigen, such as a phytase polypeptide, to which the paratope of an antibody, such as an phytase-specific antibody, binds
  • Antigemc determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics
  • fragments when refernng to the enzyme of Figure 1 comprise a enzyme which retains at least one biological function or activity that is at least essentially same as that of the reference enzyme
  • fragment fragment, “derivative” or “analog” are exemplified by a "pro-form” molecule, such as a low activity proprotem that can be modified by cleavage to produce a mamre enzyme with significantly higher activity
  • gene means the segment of DNA involved in producing a polypeptide chain, it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons)
  • isolated means that the material is removed from its original environment (e g , the natural environment if it is naturally occumng)
  • a naturally-occu ⁇ ing polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated
  • Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment
  • isolated nucleic acid is meant a nucleic acid, e g , a DNA or RNA molecule, that is not immediately contiguous with the 5' and 3' flanking sequences with which it normally is immediately contiguous when present in the naturally occumng genome of the organism from which it is derived
  • a nucleic acid that is inco ⁇ orated into a vector such as a plasmid or viral vector
  • a nucleic acid that is inco ⁇ orated into the genome of a heterologous cell or the genome of a homologous cell, but at a site different from that at which it naturally occurs
  • a nucleic acid that exists as a separate molecule e g , a DNA fragment produced by PCR amplification or restnction enzyme digestion, or an RNA molecule produced by in vitro transcription
  • a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used
  • nucleic acid molecule is comprised of at least one nucleotide base or one nucleotide base pair, depending on whether it is single-stranded or double- stranded, respectively.
  • a nucleic acid molecule may belong exclusively or chimencally to any group of nucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally occumng and not naturally occumng nucleic acids, and synthetic nucleic acids.
  • nucleic acids associated with any organelle such as the mitochondria, nbosomal RNA, and nucleic acid molecules comprised chimencally of one or more components that are not naturally occurring along with naturally occumng components.
  • nucleic acid molecule may contain in part one or more non- nucleotide-based components as exemplified by, but not limited to, amino acids and sugars
  • a ribozyme that is in part nucleotide- based and in part protein-based is considered a "nucleic acid molecule”.
  • nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive or alternatively a non-radioactive label, is likewise considered a "nucleic acid molecule”.
  • nucleic acid sequence coding for or a "DNA coding sequence of or a “nucleotide sequence encoding” a particular enzyme - as well as other synonymous terms - refer to a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences
  • a "promotor sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcnption of a downstream (3' direction) coding sequence The promoter is part of the DNA sequence This sequence region has a start codon at its 3 ' terminus The promoter sequence does include the minimum number of bases where elements necessary to initiate transcnption at levels detectable above background bind However, after the RNA polymerase binds the sequence and transcription is initiated at the start codon (3' terminus with a promoter), transcription proceeds downstream in the 3' direction Within the promotor sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S I) as well as protein binding domains (cons
  • nucleic acid encoding an enzyme (protein) or “DNA encoding an enzyme (protein)” or “polynucleotide encoding an enzyme (protein)” and other synonymous terms encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non- coding sequence
  • a "specific nucleic acid molecule species” is defined by its chemical structure, as exemplified by, but not limited to, its primary sequence
  • a specific "nucleic acid molecule species” is defined by a function of the nucleic acid species or by a function of a product derived from the nucleic acid species
  • a “specific nucleic acid molecule species” may be defined by one or more activities or properties attributable to it, including activities or properties attributable its expressed product
  • the instant definition of "assembling a working nucleic acid sample into a nucleic acid library” includes the process of inco ⁇ orating a nucleic acid sample into a vector- based collection, such as by ligation into a vector and transformation of a host
  • a description of relevant vectors, hosts, and other reagents as well as specific non-limiting examples thereof are provided hereinafter
  • the instant definition of "assembling a working nucleic acid sample into a nucleic acid library” also includes the process of inco ⁇ orating a nucleic acid sample into a non-vector-based collection, such as by ligation to adaptors.
  • the adaptors can anneal to PCR pnmers to facilitate amplification by PCR.
  • a "nucleic acid library” is compnsed of a vector-based collection of one or more nucleic acid molecules
  • a "nucleic acid library” is comprised of a non-vector-based collection of nucleic acid molecules.
  • a "nucleic acid library” is compnsed of a combined collection of nucleic acid molecules that is in part vector-based and in part non-vector-based.
  • the collection of molecules comprising a library is searchable and separable according to individual nucleic acid molecule species
  • the present invention provides a "nucleic acid construct” or alternatively a “nucleotide construct” or alternatively a "DNA construct”.
  • construct is used herein to describe a molecule, such as a polynucleotide (e g., a phytase polynucleotide) may optionally be chemically bonded to one or more additional molecular moieties, such as a vector, or parts of a vector.
  • a nucleotide construct is exemplified by a DNA expression DNA expression constructs suitable for the transformation of a host cell.
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those that do not will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • a coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences
  • the coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein
  • phytate refers to an anionic form of phytic acid
  • phytic acid refers to inositol hexaphosphate, a compound that occurs naturally in plants, including particularly plant leaves, and that may serve as a substrate for the enzyme phytase
  • phytin refers to a salt of phytic acid, such as a calcium-magnesium salt of phytic acid It is understood, accordingly, that “phytate”, “phytic acid”, and “phytin” are chemically related and interconvertible forms having a shared chemical structure As used herein, therefore, “phytate”, “phytic acid”, and “phytin” are interchangeable terms in as much as they are highly related, similar, chemically interconvertible, and may all (either with or without said chemical interconversion) be subject to degredation by the novel phytase enzyme disclosed instantly Accordingly, where only one of the terms “phytate”, “phytic acid”, or “phytin” is used in the descnptions of the methods disclosed herein, it is understood to function as a representative term that further refers to any substrate of the enzyme phytase including "phytase", “phytic acid”, and "phytin”
  • a "polynucleotide” is a molecule composed of 2 or more nucleotide bases or nucleotide base pairs
  • a molecule having a "pre-form” or a “pro-form” refers to a molecule that undergoes any combination of one or more covalent and noncovalent chemical modifications (e g glycosylation, proteolytic cleavage, dime ⁇ zation or ohgomenzation, temperature-induced or pH-induced conformational change, association with a co-factor, etc ) en route to attain a more mamre molecular form having a property difference (e g an increase in activity) in comparison with the reference pro-form molecule
  • a precursor molecule in "pre-form” or in "pro-form” is able to undergo two or more chemical modification (e g two proteolytic cleavages, or a proteolytic cleavage and a change m glycosylation) en route to the production of a mature molecule
  • the term "pre- pro-form” may also be used in reference to the precursor molecule Accordingly, a pre- pro-enzyme is an enzyme in "pre
  • the term "reagent” includes phytase molecules of the instant invention.
  • phytase molecules catalyze the hydrolysis of phytate to inositol and free phosphate with release of minerals from the phytic acid complex
  • An exemplary phytase molecule is a phytase derived from Escherichia coli B This exemplary enzyme is shown in Figure 1. SEQ ID NO 2.
  • the term "reagent” includes substrate reagents molecules of the instant invention, such as phytate molecules.
  • such phytate molecules are found in foodsmffs, potential foodstuffs, byproducts of foodsmffs (both in vitro byproducts and in vivo byproducts, e g ex vivo reaction products and animal excremental products), precursors of foodstuffs, and any other source of phytate.
  • Recombinant enzymes refer to enzymes produced by recombinant DNA techniques, i e , produced from cells transformed by an exogenous DNA construct encoding the desired enzyme
  • Synthetic enzymes are those prepared by chemical synthesis
  • similarity between two enzymes is determined by companng the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information)
  • the members of a pair of molecules are said to "specifically bind" to each other if they bind to each other with greater affinity than to other, non-specific molecules.
  • an antibody raised against an antigen to which it binds more efficiently than to a non-specific protein can be described as specifically binding to the antigen.
  • a nucleic acid probe can be described as specifically binding to a nucleic acid target if it forms a specific duplex with the target by base pairing interactions (see above).)
  • “Stringent hybridization conditions” means hybridization will occur only if there is at least 90%) identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences. See Sambrook et al, 1989, which is hereby inco ⁇ orated by reference in its entirety.
  • polypeptides having sequences that are "substantially identical" to the sequence of a phytase polypeptide, such as one of SEQ ID NO: l are also included in the invention.
  • a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence or sequences only by conservative amino acid substimtions, for example, substimtions of one amino acid for another of the same class (e.g., substimtion of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substimtion of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).
  • substantially identical amino acid sequence is a sequence that differs from a reference sequence or sequences or by one or more non-conservative substimtions, deletions, or insertions, particularly when such a substimtion occurs at a site that is not the active site the molecule, and provided that the polypeptide essentially retains its behavioural properties.
  • one or more amino acids can be deleted from a phytase polypeptide, resulting in modification of the strucmre of the polypeptide, without significantly altering its biological activity.
  • amino- or carboxyl-terminal amino acids that are not required for phytase biological activity can be removed. Such modifications can result in the development of smaller active phytase polypeptides.
  • the present invention provides a "substantially pure enzyme".
  • substantially pure enzyme is used herein to describe a molecule, such as a polypeptide (e g., a phytase polypeptide, or a fragment thereof) that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated.
  • a substantially pure molecule such as a polypeptide. can be at least 60%, by dry weight, the molecule of interest.
  • the purity of the polypeptides can be determined using standard methods including, e g , polyacrylamide gel electrophoresis (e g , SDS-PAGE), column chromatography (e g., high performance liquid chromatography (HPLC)), and amino-terminal ammo acid sequence analysis.
  • the present invention provides purified a recombinant phytase enzyme, shown in Figure 1. Additionally, the present invention provides isolated nucleic acid molecules (polynucleotides) which encode for the mature enzyme having the deduced amino acid sequence of Figure 1.
  • the phytase molecules of the instant invention are patentably novel with respect to their structures and with respect to their origin Additionally, the instant phytase molecules are patentably novel with respect to activity
  • an assay as desc ⁇ bed in Food Chemicals Codex, 4 th Ed.
  • the activity of the instant phytase enzyme was demonstrated to be far superior in comparison to a fungal (Aspergillus) phytase control.
  • a plurality of experiments showed the E.
  • the present invention provides purified a recombinant enzyme that catalyzes the hydrolysis of phytate to inositol and free phosphate with release of minerals from the phytic acid complex.
  • An exemplary purified enzyme is a phytase derived from Escherichia coli B. This exemplary enzyme is shown in Figure 1, SEQ ID NO-2
  • the enzymes of the present invention include, in addition to an enzyme of Figure 1 (in particular the mamre enzyme), polypeptides having sequences that are "substantially identical" to the sequence of a phytase polypeptide, such as one of SEQ ID 1
  • the phytase enzyme of SEQ ID NO.2 of the present invention has a molecular weight of about 47,056 kilodaltons as measured by SDS-PAGE and infe ⁇ ed from the nucleotide sequence of the gene.
  • the pi is 6.70
  • the pH and temperature profile and stability data for this enzyme is presented in Figure 2
  • This purified enzyme may be used to catalyze the hydrolysis of phytate to inositol and free phosphate where desired.
  • the phytase enzyme of the present invention has a high thermostabihty, thus it is particularly serviceable for raised temperature and/or pressure applications including, but not limited to, the preparation of fish foodstuff pellets that will not dissolve prematurely in water.
  • the phytase polypeptide included in the invention can have the amino acid sequences of the phytase shown in Figure 1 (SEQ ID NO:2).
  • Phytase polypeptides. such as those isolated from E cob B can be characterized by catalyzing the hydrolysis of phytate to inositol and free phosphate with the release of minerals from the phytic acid complex.
  • phytase polypeptides included in the invention are polypeptides having amino acid sequences that are at least about 50% identical to the amino acid sequence of a phytase polypeptide, such as any of the phytase in SEQ ID NO:2.
  • the length of comparison in determining amino acid sequence homology can be, for example, at least 15 amino acids, and for example, at least 20, 25, or 35 amino acids
  • sequence analysis software e g ,
  • sequence companson typically one sequence acts as a reference sequence, to which test sequences are compared, however a database of reference sequences can be used.
  • sequence companson algonthm test and reference sequences are entered mto a computer, subsequence coordinates are designated, if necessary, and sequence algonthm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence companson algonthm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “companson window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequence for companson are well-known in the art. Optimal alignment of sequences for companson can be conducted, eg , by the local homology algonthm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algonthm of Needleman & Wunsch, J. Mol.
  • algonthms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS.
  • FNAT Forward Nucleotide Alignment Tool
  • Frameahgn Framesearch
  • DYNAMIC FILTER
  • FNAT Forward Nucleotide Alignment Tool
  • Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences.
  • a number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (J. Roach, http://weber.u.Wash ⁇ ngton.edu/ ⁇ roach/human_ genome_ progress 2.html) (Gibbs, 1995). At least twenty-one other genomes have already been sequenced, including, for example, M genitahum (Fraser et al, 1995), M. jannaschu (Bult et al, 1996), H. influenzae (Fleischmann et al, 1995), E. co (Blattner et al, 1997).
  • BLAST and BLAST 2.0 algonthms are descnbed in Altschul et al. Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al, J. Mol. Biol. 215:403-410, 1990, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm nih gov/)
  • This algonthm involves first identifying high sconng sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length m a database sequence.
  • HSPs high sconng sequence pairs
  • T is refe ⁇ ed to as the neighborhood word score threshold (Altschul et al , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) For amino acid sequences, a sconng matnx is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when- the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-sconng residue alignments; or the end of either sequence is reached.
  • the BLAST algonthm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algonthm also performs a statistical analysis of the simila ⁇ ty between two sequences (see, eg , Karhn & Altschul, Proc. Natl. Acad. Sci. USA 90:5873, 1993).
  • One measure of similanty provided by BLAST algonthm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a references sequence if the smallest sum probability in a companson of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001
  • protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") In particular, five specific BLAST programs are used to perform the following task
  • BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands)
  • TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database
  • the BLAST programs identify homologous sequences by identifying similar segments, which are refe ⁇ ed to herein as "high-sconng segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database
  • High-scoring segment pairs are preferably identified (i e , aligned) by means of a scoring matrix, many of which are known in the art
  • the scoring matrix used is the BLOSUM62 matrix (Gonnet et al Science 256 1443-1445, 1992, Henikoff and Henikoff, Proteins J 49-61, 1993)
  • the PAM or PAM250 matrices may also be used (see, e g , Schwartz and Dayhoff, eds , 1978, Matrices for Detecting Distance Relationships Atlas of Protein Sequence and Structure, Washington National Biomedical Research Foundation)
  • BLAST programs are accessible through the U S National Library of Medicine, e g , at
  • the parameters used with the above algonthms may be adapted depending on the sequence length and degree of homology smdied In some embodiments, the parameters may be the default parameters used by the algonthms in the absence of instructions from the user.
  • the present invention further relates to an enzyme which has the deduced amino acid sequence of Figure 1 , as well as analogs, de ⁇ vatives, and fragments of such enzyme.
  • An analog, derivative, or fragment of the enzyme of Figure 1 may be (a) one in which one or more of the amino acid residues are substituted with an amino acid residue which is not encoded by the genetic code, or (b) one in which one or more of the amino acid residues includes a substiment group, or (c) one in which the mature enzyme is fused with another compound, such as a compound to increase the half-life of the enzyme (for example, polyethylene glycol), or (d) to provide a label or a tag, such as a 6xH ⁇ s tag or a green fluorescent protein tag, (e) one in which the additional amino acids are fused to the mature enzyme, such as a leader or secretory sequence or a sequence which is employed for purification of the mature enzyme or a proprotem sequence.
  • Such analogs, denvatives, and fragments are deemed to be within the scope of those skilled in the art from the teachings herein.
  • a vanant e.g., a "fragment”, “analog” or “derivative” enzyme, and reference enzyme may differ in amino acid sequence by one or more substimtions, additions, deletions, fusions and truncations, which may be present in any combination.
  • a substimtion can be comprised of a substimtion of one amino acid by another amino acid with a like property
  • a substimtion can be comprised of a substimtion of an amino acid by an unlike amino acid, where the change is non-inhibitory or silent or improved with respect to
  • an addition can be comprised of an addition either at the amino or the carboxy terminal of the protein or alternatively between the terminal sites, where the change is change is non-inhibitory or silent or improved with respect to at least one enzyme property
  • a change can be comprised of a plurality of modifications, including substimtions, additions, deletions, fusions and/or truncations, in the enzyme encoded by the reference polynucleotide (SEQ ID NO 1, such that, l ⁇ espective of the effects of the individual modifications, when taken together as a set, the effect of the modifications is non-inhibitory or silent or improved with respect to at least one enzyme property
  • variable refers to polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of a phytase of the invention
  • Va ⁇ ants can be produced by any number of means including methods such as, for example, e ⁇ or- prone PCR, shuffling, ohgonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, ligation reassembly, GSSM and any combination thereof as discussed more fully below 6.
  • isolated nucleic acid molecules polynucleotides which encode for the mamre enzyme having the deduce
  • the polynucleotide encoding SEQ ID NO 2 was originally isolated from genomic DNA
  • DNA recovered from Escherichia cob B as described below It contains an open reading frame encoding a protein of 432 amino acid residues
  • an isolated polynucleotide encoding an exemplary enzyme of the present invention comprising the DNA of Figure 1
  • the present invention also relates to polynucleotides which differ from the reference polynucleotide such that the changes are silent changes, for example the changes do not alter the amino acid sequence encoded by the polynucleotide
  • the present invention also relates to nucleotide changes which result in amino acid substitutions, additions, deletions, fusions and truncations in the enzyme encoded by the reference polynucleotide (SEQ ID NO 1) In a prefe ⁇ ed aspect of the invention these enzymes retain about the same biological action as the enzyme encoded by the reference polynucleotide
  • nucleic acids that encode SEQ ID NO 2 are included in the invention. These nucleic acids can contain naturally occurring nucleotide sequences, or sequences that differ from those of the namrally occumng nucleic acids that encode phytases, but encode the same amino acids, due to the degeneracy of the genetic code
  • the nucleic acids of the invention can contain DNA or RNA nucleotides, or combinations or modifications thereof Exemplary nucleic acids of the invention are shown in SEQ ID NO 1
  • the polynucleotide of the present invention may be in the form of DNA which DNA includes cDNA, genomic DNA, and synthetic DNA The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand
  • the coding sequence which encodes the mamre enzyme may be identical to the coding sequences shown in Figure 1 and/or that of
  • SEQ ID NO 1 may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mamre enzyme as the DNA of Figure 1 (e g , SEQ ID NO 1)
  • ID NO 2 may include, but is not limited to only the coding sequence for the mamre enzyme, the coding sequence for the mamre enzyme and additional coding sequence such as a leader sequence or a proprotem sequence, the coding sequence for the mature enzyme (and optionally additional coding sequence) and non-cod g sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mamre enzyme
  • the present invention further relates to vanants of the hereinabove descnbed polynucleotides which encode for fragments, analogs and derivatives of the enzyme having the deduced amino acid sequence of Figure 1 (e g , SEQ ID NO 2)
  • the variant of the polynucleotide may be a naturally occumng allelic v ariant of the polynucleotide or a non-naturally occumng variant of the polynucleotide
  • the present invention includes polynucleotides encoding the same mamre enzyme as shown in Figure 1 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the enzyme of Figure 1
  • nucleotide variants include deletion variants, substimtion variants and addition or insertion variants
  • the polynucleotide may have a coding sequence which is a namrally occumng allelic variant of the coding sequence shown in Figure 1
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substimtion, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded enzyme.
  • variants can be produced by any number of means including methods such as, for example, e ⁇ or-prone PCR, shuffling, ohgonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis. cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site- specific mutagenesis, ligation reassembly, GSSM and any combination thereof.
  • methods such as, for example, e ⁇ or-prone PCR, shuffling, ohgonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis.
  • cassette mutagenesis recursive ensemble mutagenesis, exponential ensemble mutagenesis, site- specific mutagenesis, ligation reassembly, GSSM and any combination thereof.
  • a non-stochastic method termed synthetic ligation reassembly (SLR), that is somewhat related to stochastic shuffling, save that the nucleic acid building blocks are not shuffled or concatenated or chime ⁇ zed randomly, but rather are assembled non- stochastically can be used to create variants.
  • SLR synthetic ligation reassembly
  • the SLR method does not depend on the presence of a high level of homology between polynucleotides to be shuffled.
  • the invention can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10 100 different chimeras. Conceivably, SLR can even be used to generate libraries compnsed of over , Q IOOO different progeny chimeras.
  • the invention provides a non-stochastic method of producing a set of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design, which method is comprised of the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
  • the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
  • the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends and. if more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
  • the annealed building pieces are treated with an enzyme, such as a ligase (e g , T4 DNA ligase) to achieve covalent bonding of the building pieces.
  • a ligase e g , T4 DNA ligase
  • the design of nucleic acid building blocks is obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules
  • progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, i e chimenzed or shuffled
  • the invention provides for the chime ⁇ zation of a family of related genes and their encoded family of related products
  • the encoded products are enzymes. Enzymes and polypeptides of the invention can be mutagenized in accordance with the methods desc ⁇ bed herein.
  • the sequences of a plurality of progenitor nucleic acid templates are aligned in order to select one or more demarcation points, which demarcation points can be located at an area of homology
  • the demarcation points can be used to delineate the boundaries of nucleic acid building blocks to be generated
  • the demarcation points identified and selected in the progenitor molecules serve as potential chime ⁇ zation points in the assembly of the progeny molecules
  • a serviceable demarcation point is an area of homology (comprised of at least one homologous nucleotide base) shared by at least two progenitor templates, but the demarcation point can be an area of homology that is shared by at least half of the progenitor templates, at least two thirds of the progenitor templates, at least three fourths of the progenitor templates, and preferably at almost all of the progenitor templates Even more preferably still a serviceable demarcation point is an area of homology that is shared by all of the progenitor templates
  • the ligation reassembly process is performed exhaustively in order to generate an exhaustive library
  • all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules
  • the assembly order i e the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
  • the assembly order is by design (or non-stochastic) Because of the non-stochastic nature of the method, the possibility of unwanted side products is greatly reduced
  • the method provides that, the ligation reassembly process is performed systematically, for example in order to generate a systematically compartmentalized library, with compartments that can be screened systematically, e g , one by one
  • the invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, an expenmental design can be achieved where specific sets of progeny products are made in each of several reaction vessels This allows a systematic examination and screening procedure to be performed Thus, it allows a potentially very large number of progeny molecules to be examined systematically in smaller groups
  • the instant invention provides for the generation of a library (or set) comprised of a large number of progeny molecules
  • the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design
  • such a generated library is comprised of greater than 10 3 to greater than 10 1000 different progeny molecular species
  • a set of finalized chimeric nucleic acid molecules, produced as described is comprised of a polynucleotide encoding a polypeptide.
  • this polynucleotide is a gene, which may be a man-made gene.
  • this polynucleotide is a gene pathway, which may be a man-made gene pathway.
  • the invention provides that one or more man-made genes generated by the invention may be inco ⁇ orated into a man-made gene pathway, such as pathway operable in a eukaryotic organism (including a plant).
  • the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g , one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e g., by mutageneis) or in an in vivo process (e.g , by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.
  • nucleotides e.g , one or more nucleotides, which may be, for example, codons or introns or regulatory sequences
  • the invention provides that a nucleic acid building block can be used to introduce an intron.
  • the invention provides that functional introns may be introduced into a man-made gene of the invention.
  • the invention also provides that functional introns may be introduced into a man-made gene pathway of the invention.
  • the invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced ⁇ ntron(s).
  • the invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced ⁇ ntron(s).
  • the artificially introduced ⁇ ntron(s) are functional in one or more host cells for gene splicing much in the way that namrally-occumng introns serve functionally in gene splicing.
  • the invention provides a process of producing man-made intron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing
  • a man-made genes produced using the invention can also serve as a substrate for recombination with another nucleic acid.
  • a man-made gene pathway produced using the invention can also serve as a substrate for recombination with another nucleic acid.
  • the recombination is facilitated by, or occurs at, areas of homology between the man-made intron-containing gene and a nucleic acid with serves as a recombination partner.
  • the recombination partner may also be a nucleic acid generated by the invention, including a man-made gene or a man-made gene pathway. Recombination may be facilitated by or may occur at areas of homology that exist at the one (or more) artificially introduced ⁇ ntron(s) in the man-made gene.
  • the synthetic ligation reassembly method of the invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends.
  • the two ligatable ends on each nucleic acid building block may be two blunt ends (i.e. each having an overhang of zero nucleotides), or preferably one blunt end and one overhang, or more preferably still two overhangs.
  • a serviceable overhang for this pu ⁇ ose may be a 3 ' overhang or a 5' overhang.
  • a nucleic acid building block may have a 3' overhang or alternatively a 5' overhang or alternatively two 3' overhangs or alternatively two 5' overhangs
  • the overall order in which the nucleic acid building blocks are assembled to form a finalized chimeric nucleic acid molecule is determined by pu ⁇ oseful experimental design and is not random.
  • a nucleic acid building block is generated by chemical synthesis of two single-stranded nucleic acids (also referred to as single- stranded ohgos) and contacting them so as to allow them to anneal to form a double- stranded nucleic acid building block
  • a double-stranded nucleic acid building block can be of variable size
  • the sizes of these building blocks can be small or large Prefe ⁇ ed sizes for building block range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs)
  • Other prefe ⁇ ed size ranges are also provided, which have lower limits of from 1 bp to 10,000 bp (including every integer value in between), and upper limits of from 2 bp to 100, 000 bp (including every integer value in between)
  • a double-stranded nucleic acid building block is generated by first generating two single stranded nucleic acids and allowing them to anneal to form a double-stranded nucleic acid building block
  • the two strands of a double-stranded nucleic acid building block may be complementary at every nucleotide apart from any that form an overhang, thus containing no mismatches, apart from any overhang(s)
  • the two strands of a double-stranded nucleic acid building block are complementary at fewer than every nucleotide apart from any that form an overhang
  • a double-stranded nucleic acid building block can be used to introduce codon degeneracy
  • the codon degeneracy is introduced using the site-saturation mutagenesis described herein, using one or more N,N,G/T cassettes or alternatively using one or more N,N,N cassettes
  • the in vivo recombination method of the invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide
  • the approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA and growth hormone
  • This approach may be used to generate proteins having altered specificity or activity
  • the approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes
  • This approach may be used to generate genes having increased rates of expression.
  • This approach may also be useful in the study of repetitive DNA sequences.
  • this approach may be useful to mutate ⁇ bozymes or ap tamers.
  • variants of the polynucleotides and polypeptides described herein are obtained by the use of repeated cycles of reductive reassortment, recombination and selection which allow for the directed molecular evolution of highly complex linear sequences, such as DNA, RNA or proteins thorough recombination.
  • In vivo shuffling of molecules is useful in providing variants and can be performed utilizing the natural property of cells to recombine multimers While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.
  • the invention includes a method for producing a hybnd polynucleotide from at least a first polynucleotide and a second polynucleotide.
  • the invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell.
  • the regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide.
  • hybrid polynucleotide is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence withm a DNA molecule
  • the invention provides a means for generating hybrid polynucleotides which may encode biologically active hybrid polypeptides (e g , a hybrid phytase).
  • biologically active hybrid polypeptides e g , a hybrid phytase
  • the onginal polynucleotides encode biologically active polypeptides.
  • the method of the invention produces new hybrid polypeptides by utilizing cellular processes which integrate the sequence of the original polynucleotides such that the resulting hybnd polynucleotide encodes a polypeptide demonstrating activities derived from the original biologically active polypeptides
  • the original polynucleotides may encode a particular enzyme from different microorganisms.
  • An enzyme encoded by a first polynucleotide from one organism or variant may, for example, function effectively under a particular environmental condition, e g , high salinity
  • An enzyme encoded by a second polynucleotide from a different organism or variant may function effectively under a different environmental condition, such as extremely high temperatures.
  • a hybnd polynucleotide containing sequences from the first and second original polynucleotides may encode an enzyme which exhibits charactenstics of both enzymes encoded by the onginal polynucleotides.
  • the enzyme encoded by the hybrid polynucleotide may function effectively under environmental conditions shared by each of the enzymes encoded by the first and second polynucleotides, e g , high salinity and extreme temperatures
  • Enzymes encoded by original polynucleotides include, but are not limited to, phytases
  • a hybrid polypeptide resulting from the method of the invention may exhibit specialized enzyme activity not displayed in the original enzymes.
  • the resulting hybnd polypeptide encoded by a hybrid polynucleotide can be screened for specialized hydrolase activities obtained from each of the onginal enzymes, i e , the type of bond on which the hydrolase acts and the temperature at which the hydrolase functions
  • the hydrolase may be screened to ascertain those chemical functionalities which distinguish the hybrid hydrolase from the original hydrolyases, such as: (a) amide (peptide bonds), i.e., proteases; (b) ester bonds, i.e., esterases and lipases; (c) acetals, i.e., glycosida
  • Sources of the original polynucleotides may be isolated from individual organisms
  • isolated collections of organisms that have been grown in defined media (“enrichment cultures”), or, uncultivated organisms (“environmental samples”).
  • enrichment cultures collections of organisms that have been grown in defined media
  • uncultivated organisms uncultivated organisms
  • Environmental libraries are generated from environmental samples and represent the collective genomes of namrally occu ⁇ ing organisms archived in cloning vectors that can be propagated in suitable prokaryotic hosts. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture. Additionally, a normalization of the environmental DNA present in these samples could allow more equal representation of the DNA from all of the species present in the original sample. This can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample which may be under-represented by several orders of magnimde compared to the dominant species.
  • gene libraries generated from one or more uncultivated microorganisms are screened for an activity of interest.
  • Potential pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries.
  • Polynucleotides encoding activities of interest are isolated from such libraries and introduced into a host cell. The host cell is grown under conditions which promote recombination and/or reductive reassortment creating potentially active biomolecules with novel or enhanced activities.
  • the microorganisms from which the polynucleotide may be prepared include prokaryotic microorganisms, such as Xanthobacter, Eubacteria and Archaebactena, and lower eukaryotic microorganisms such as fungi, some algae and protozoa
  • Polynucleotides may be isolated from environmental samples in which case the nucleic acid may be recovered without culturmg of an organism or recovered from one or more cultured organisms
  • such microorganisms may be extremophiles, such as hyperthermophiles, psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles
  • Polynucleotides encoding enzymes isolated from extremophihc microorganisms are particularly prefe ⁇ ed.
  • Such enzymes may function at temperamres above 100°C in te ⁇ estnal hot springs and deep sea thermal vents, at temperamres below 0°C in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 1 1 in sewage sludge.
  • esterases and lipases cloned and expressed from extremophihc organisms show high activity throughout a wide range of temperatures and pHs.
  • polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell
  • a suitable host cell is any cell which is capable of promoting recombination and or reductive reassortment
  • the selected polynucleotides are preferably already in a vector which includes appropriate control sequences
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bactenal cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al, 1986)
  • bacterial cells such as E cob, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma, adenoviruses
  • plant cells such as E cob, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma, adenoviruses
  • plant cells such as E cob, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma, adenoviruses
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, descnbed in "SV40-transformed simian cells support the replication of early SV40 mutants" (Gluzman, 1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscnbed sequences DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscnbed genetic elements
  • Host cells containing the polynucleotides of interest can be cultured in conventional nut ⁇ ent media modified as approp ⁇ ate for activating promoters, selecting transformants or amplifying genes
  • the culmre conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinanly skilled artisan
  • the clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the enhanced activity
  • methods can be used to generate novel polynucleotides encoding biochemical pathways from one or more operons or gene clusters or portions thereof
  • bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes
  • the genes are clustered, in strucmres refe ⁇ ed to as "gene clusters," on a single chromosome or immediately adjacent to one another and are transcnbed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster
  • a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function
  • An example of a biochemical pathway encoded by gene clusters are polyketides
  • Polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (such as tetracychnes and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and vetennary products (monen
  • Polyketides produced by polyketide synthases are valuable as therapeutic agents.
  • Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of an enormous variety of carbon chains differing in length and patterns of functionality and cyclization
  • Polyketide synthase genes fall into gene clusters and at least one type (designated type I) of polyketide synthases have large size genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins
  • Gene cluster DNA can be isolated from different organisms and ligated into vectors, particularly vectors containing expression regulatory sequences which can control and regulate the production of a detectable protein or protein-related a ⁇ ay activity from the ligated gene clusters.
  • vectors which have an exceptionally large capacity for exogenous DNA introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E cob.
  • This f-factor of E. cob is a plasmid which affects high-frequency transfer of itself dunng conjugation and is ideal to achieve and stably propagate large DNA fragments, such as gene clusters from mixed microbial samples.
  • two or more vectors containing different phytase gene clusters can be introduced into a suitable host cell Regions of partial sequence homology shared by the gene clusters will promote processes which result in sequence reorganization resulting in a hybrid gene cluster.
  • the novel hybrid gene cluster can then be screened for enhanced activities not found in the original gene clusters.
  • the invention relates to a method for producing a biologically active hybrid polypeptide and screening such a polypeptide for enhanced activity by.
  • expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e g , vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus and yeast).
  • the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences.
  • suitable vectors are known to those of skill in the art, and are commercially available.
  • the following vectors are provided by way of example; Bacterial: pQE vectors (Qiagen), pBluesc ⁇ pt plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).
  • any other plasmid or other vector may be used so long as they are replicable and viable in the host.
  • Low copy number or high copy number vectors may be employed with the present invention.
  • a prefe ⁇ ed type of vector for use in the present invention contains an f-factor ongin replication
  • the f-factor (or fertility factor) in E cob is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself.
  • a particularly prefe ⁇ ed embodiment is to use cloning vectors, refe ⁇ ed to as "fosmids" or bacterial artificial chromosome (BAC) vectors These are derived from E cob f-factor which is able to stably integrate large segments of genomic DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable "environmental DNA library "
  • Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA. Cloning into cosmid vectors is described in detail in "Molecular Cloning' A laboratory Manual” (Sambrook et al , 1989)
  • the DNA sequence in the expression vector is operatively linked to an approp ⁇ ate expression control sequence(s) (promoter) to direct RNA synthesis.
  • a bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcnption terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracychne or ampicillin resistance in E. cob.
  • the method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector, and their subsequent introduction into an appropnate host cell
  • the reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasi-repeated units.
  • the reassortment process recombines and/or reduces the complexity and extent of the repeated sequences, and results in the production of novel molecular species.
  • Various treatments may be applied to enhance the rate of reassortment.
  • the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their own evolution.
  • Quadsi-repeats are repeats that are not restncted to their ongmal unit strucmre. Quasi-repeated units can be presented as an a ⁇ ay of sequences in a construct; consecutive units of similar sequences Once ligated, the junctions between the consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level. The deletion process the cell performs to reduce the complexity of the resulting construct operates between the quasi- repeated sequences.
  • the quasi-repeated units provide a practically limitless repertoire of templates upon which slippage events can occur. The constructs containing the quasi- repeats thus effectively provide sufficient molecular elasticity that deletion (and potentially insertion) events can occur virtually anywhere within the quasi-repetitive units.
  • Sequences can be assembled in a head to tail orientation using any of a variety of methods, including the following a) Primers that include a poly-A head and poly-T tail which when made single- stranded would provide onentation can be utilized This is accomplished by having the first few bases of the primers made from RNA and hence easily removed RNAseH b) Pnmers that include unique restriction cleavage sites can be utilized Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required c) The inner few bases of the primer could be thiolated and an exonuclease used to produce properly tailed molecules
  • Encoding sequences for example, genes
  • Encoding sequences may demonstrate a high degree of homology and encode quite diverse protein products. These types of sequences are particularly useful in the present invention as quasi-repeats. However, while the examples illustrated below demonstrate the reassortment of nearly identical original encoding sequences (quasi-repeats). this process is not limited to such nearly identical repeats.
  • the following example demonstrates a method of the invention.
  • Encoding nucleic acid sequences (quasi-repeats) derived from three (3) unique species are depicted. Each sequence encodes a protein with a distinct set of properties. Each of the sequences differs by a single or a few base pairs at a unique position in the sequence which are designated "A", "B” and "C”.
  • the quasi-repeated sequences are separately or collectively amplified and ligated into random assemblies such that all possible permutations and combinations are available in the population of ligated molecules.
  • the number of quasi-repeat units can be controlled by the assembly conditions.
  • the average number of quasi-repeated units in a construct is defined as the repetitive index (RJ)
  • the constructs may, or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell.
  • the cells are then propagated and "reductive reassortment" is effected.
  • the rate of the reductive reassortment process may be stimulated by the introduction of DNA damage if desired Whether the reduction in RJ is mediated by deletion formation between repeated sequences by an "intra-molecular” mechanism, or mediated by recombination-like events through “mter-molecular” mechanisms is immaterial.
  • the end result is a reassortment of the molecules into all possible combinations.
  • the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact, or catalyze a particular reaction (e g , such as catalyzing the hydrolysis of a haloalkane)
  • a particular reaction e g , such as catalyzing the hydrolysis of a haloalkane
  • polypeptides that are identified from such libraries can be used for therapeutic, diagnostic, research and related pu ⁇ oses (e g , catalysts, solutes for increasing osmola ⁇ ty of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or selection
  • polynucleotides of the invention or polynucleotides generated by the method descnbed herein can be subjected to agents or processes which promote the introduction of mutations into the o ⁇ ginal polynucleotides
  • agents or processes which promote mutagenesis can include, but are not limited to (+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenme, see Sun and Hurley, 1992), an N-acelylated or deacetylated 4'-fluro-4-am ⁇ nob ⁇ phenyl adduct capable of inhibiting DNA synthesis (see.
  • the invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions according to the invention which provide for the production of hybrid or re-assorted polynucleotides.
  • the invention also provides for the use of proprietary codon primers (containing a degenerate N,N,N sequence) to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substimtions is represented at each amino acid position (gene site saturated mutagenesis (GSSM)).
  • GSSM gene site saturated mutagenesis
  • the ohgos used are comprised contiguously of a first homologous sequence, a degenerate N,N,N sequence, and preferably but not necessarily a second homologous sequence.
  • the downstream progeny translational products from the use of such ohgos include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,N sequence includes codons for all 20 amino acids.
  • one such degenerate oligo (comprised of one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substimtions.
  • at least two degenerate N,N,G/T cassettes are used - either in the same oligo or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substimtions.
  • more than one N,N,G/T sequence can be contained in one oligo to introduce amino acid mutations at more than one site.
  • This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
  • ohgos serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and or substimtions.
  • the present invention provides for the use of degenerate cassettes having less degeneracy than the N,N,G/T sequence
  • degenerate cassettes having less degeneracy than the N,N,G/T sequence
  • Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet
  • this invention provides a means to systematically and fairly easily generate the substitution of the full range of possible amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide
  • the invention provides a way to systematically and fairly easily generate 2000 distinct species (/ e , 20 possible amino acids per position times 100 amino acid positions)
  • an oligo containing a degenerate N,N,G/T or an N.N, G/C tnplet sequence 32 individual sequences that code for 20 possible amino acids
  • a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using one such oligo, there are generated 32 distinct progeny polynucle
  • This invention also provides for the use of nondegenerate ohgos, which can optionally be used in combination with degenerate primers disclosed It is appreciated that in some situations, it is advantageous to use nondegenerate ohgos to generate specific point mutations in a working polynucleotide This provides a means to generate specific silent point mutations, point mutations leading to co ⁇ esponding amino acid changes, and point mutations that cause the generation of stop codons and the co ⁇ esponding expression of polypeptide fragments
  • each samration mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 amino acids are represented at the one specific amino acid position co ⁇ esponding to the codon position mutagenized in the parental polynucleotide.
  • the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g., cloned into a suitable E cob host using an expression vector) and subjected to expression screening.
  • clonal amplification e.g., cloned into a suitable E cob host using an expression vector
  • an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide), it can be sequenced to identify the co ⁇ espondingly favorable amino acid substimtion contained therein.
  • site-saturation mutagenesis can be used together with shuffling, chime ⁇ zation, recombination and other mutagenizing processes, along with screening.
  • This invention provides for the use of any mutagenizing process(es), including samration mutagenesis, in an iterative manner In one exemplification, the iterative use of any mutagenizing process(es) is used in combination with screening.
  • polynucleotides and polypeptides of the invention can be derived by samration mutagenesis in combination with additional mutagenization processes, such as process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment
  • mutagenesis can be used to replace each of any number of bases in a polynucleotide sequence, wherein the number of bases to be mutagenized is preferably every integer from 15 to 100,000
  • the number of bases to be mutagenized is preferably every integer from 15 to 100,000
  • a separate nucleotide is used for mutagenizing each position or group of positions along a polynucleotide sequence
  • a group of 3 positions to be mutagenized may be a codon
  • the mutations are preferably introduced using a mutagenic primer, containing a heterologous cassette, also refe ⁇ ed to as a mutagenic cassette
  • Prefe ⁇ ed cassettes can have from 1 to 500 bases
  • Each nucleotide position in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G
  • samration mutagenesis is comprised of mutagenizing a complete set of mutagenic cassettes (wherein each cassette is preferably about 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is preferably from about 15 to 100,000 bases in length)
  • a group of mutations (ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized
  • a grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of samration mutagenesis
  • Such groupings are exemplified by deletions, additions, groupings of particular codons and groupings of particular nucleotide cassettes
  • sequences to be mutagenized include a whole gene, pathway, cDNA, an entire open reading frame (ORF), and entire promoter, enhancer, repressor/transactivator, origin of replication, intron, operator
  • this invention specifically provides for degenerate codon substitutions (using degenerate ohgos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each position, and a library of polypeptides encoded thereby
  • the present invention also includes polynucleotides, wherein the coding sequence for the mamre enzyme may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of an enzyme from a host cell, for example, a leader sequence which functions to control transport of an enzyme from the cell
  • a leader sequence which functions to control transport of an enzyme from the cell
  • An enzyme having a leader sequence is an example of a preprotein and may have the leader sequence cleaved by the host cell to form the mamre form of the enzyme
  • the polynucleotides may also encode for a proprotem which is exemplified by a mamre protein plus additional 5' amino acid residues
  • An otherwise mamre protein having a prosequence is exemplified by a proprotem that is an inactive form of the protein Once the prosequence is cleaved an active mamre protein remains
  • the polynucleotide of the present invention may encode for a mamre enzyme, or for an enzyme having a prosequence or for an enzyme having both a prosequence and a presequence (e g leader sequence) 6.1.4 - Methods of isolation:
  • the coding sequences for the phytase enzymes of the present invention were identified by preparing E cob B genomic DNA, for example, and recovenng (via, for example, PCR amplification) from the genomic DNA, DNA encoding phytase activity Such methods for recovery are well-known in the art.
  • One means for example, comprises designing amplification primers to recover the coding sequence, amplifying the gene from the genomic DNA, subcloning the DNA into a vector, transforming the resulting construct into a host strain, and expressing the phytase enzyme for evaluation
  • amplification primers to recover the coding sequence
  • the enzyme of the present invention was isolated from an E.coli B genomic DNA by the following technique-
  • E.coli B genomic DNA was obtained commercially (Sigma Catalog # D-2001, St. Louis, New Jersey). The following pnmers were used to amplify the gene directly from the genomic DNA:
  • PCR product and pQE60 vector were both digested with EcoRI and Bglll restnction endonucleases (New England Biolabs) according to manufacturers protocols. Ligation and transformation into, and expression in Ml 5 pREP4 host cells (Qiagen) yields c-term 6X-H ⁇ s tagged protein.
  • the isolated nucleic acid sequences and other enzymes may then be measured for retention of biological activity characteristic to the enzyme of the present invention, for example, in an assay for detecting enzymatic phytase activity (Food Chemicals Codex, 4 lh Ed.)
  • Such enzymes include truncated forms of phytase, and variants such as deletion and insertion variants.
  • an in vitro example of such an assay is the following assay for the detection of phytase activity- Phytase activity can be measured by incubating 150 ⁇ l of the enzyme preparation with 600 ⁇ l of 2 mM sodium phytate in 100 mM Tris HCl buffer pH 7.5, supplemented with ImM CaCl 2 for 30 minutes at 37°C. After incubation the reaction is stopped by adding 750 ⁇ l of 5% t ⁇ chloroacetic acid.
  • Phosphate released was measured against phosphate standard spectrophotometncally at 700nm after adding 1500 ⁇ l of the color reagent (4 volumes of 1.5% ammonium molybdate in 5.5% sulfu ⁇ c acid and 1 volume of 2.7% fe ⁇ ous sulfate; Shimizu, 1992).
  • One unit of enzyme activity is defined as the amount of enzyme required to liberate one ⁇ mol Pi per min under assay conditions. Specific activity can be expressed in units of enzyme activity per mg of protein.
  • the enzyme of the present invention has enzymatic activity with respect to the hydrolysis of phytate to inositol and free phosphate.
  • the enzymes and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • the phytase polypeptide of the invention can be obtained using any of several standard methods.
  • phytase polypeptides can be produced in a standard recombinant expression system (see below), chemically synthesized (this approach may be limited to small phytase peptide fragments), or punfied from organisms in which they are namrally expressed.
  • Serviceable recombinant expression methods include the use of mammalian hosts, microbial hosts, and plant hosts.
  • the recombinant expression of the instant phytase molecules may be achieved in combination with one or more additional molecules such as, for example, other enzymes.
  • This approach is serviceable for producing combination products, such as a plant or plant part that contains the instant phytase molecules as well as one or more additional molecules - preferably said phytase molecules and said additional molecules are serviceable in a combination treatment
  • the resulting recombinantly expresssed molecules may be used in homogenized and/or purified form or alternatively in relatively unpurified form (e g as consumable plant parts that are serviceable when admixed with other foodstuffs for catalyzing the degredation of phytate).
  • an enzyme of the present invention may be a recombinant enzyme, a natural enzyme, or a synthetic enzyme, preferably a recombinant enzyme.
  • the present invention also relates to vectors which include polynucleotides of the present mvention, host cells which are genetically engineered with vectors of the invention, and the production of enzymes of the invention by recombinant techniques.
  • Host cells are genetically engineered (e g transduced or transformed or transfected) with the vectors containing the polynucleotides of this invention.
  • Such vectors may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, a p ⁇ on, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, &/or selecting transformants or amplifying the genes of the present invention.
  • the culmre conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan
  • the polynucleotides of the present invention may be employed for producing enzymes by recombinant techniques
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing an enzyme
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e g , denvatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors denved from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovims, fowl pox virus, and pseudorabies
  • any other vector may be used as long as it is replicable and viable in the host
  • the approp ⁇ ate DNA sequence may be inserted into the vector by a vanety of procedures
  • the DNA sequence is inserted into an approp ⁇ ate restnction endonuclease s ⁇ te(s) by procedures known in the art Inclusive in this meaning is the use of blunt-ended molecules which could be generated by the use of restriction digestion as well as restriction digestion-independent means
  • the insert may be inco ⁇ orated into a vector by so called "hgase-independent" means
  • a "hgase-independent" means is exemplified by the use of topoisomerase-mediated ligation at room temperature, for example according to the commercially available kit termed TOPO-TA Cloning® (Invitrogen Co ⁇ oration, Carlsbad, CA)
  • Alteranative enzymes including isomers of topoisomerase as well as more distantly related recombination enzymes (e g recombinases), may also be serviceable for mediating this type of "hgas
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis
  • promoter an LTR or SV40 promoter, an E cob lac or trp, a phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator
  • the vector may also include appropriate sequences for amplifying expression
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracychne or ampicilhn resistance in E cob.
  • the vector containing the appropnate DNA sequence as hereinabove desc ⁇ bed, as well as an appropriate promoter or control sequence, may be employed to transform an appropnate host to permit the host to express the protein
  • bactenal cells such as E cob, Streptomyces, Bacillus subtilis
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera 5/9
  • animal cells such as CHO, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs compnsmg one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further compnses regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • One or more additional inserts may also be inco ⁇ orated that lead to expression of one or more aditional molecules, such as another phytase or a protease enzyme, preferably said one or more additional molecules are serviceable in combination with the instant phytase in a combination treatment.
  • Plasmids are designated by a lower case p preceded and or followed by capital letters and or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • the following vectors are provided by way of example, Bactenal pQE70, pQE60, pQE-9 (Qiagen), pBluesc ⁇ pt II (Stratagene), pTRC99a, pKK223-3, pDR540, pRIT2T (Pharmacia), Eukaryotic pXTl, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia)
  • any other plasmids or other vectors may be used as long as they are replicable and viable in the host
  • Promoter regions can be selected from any desired gene using CAT
  • bactenal promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and t ⁇
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionem-I Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art
  • the present invention relates to host cells containing the above-desc ⁇ bed constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, 1986).
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the enzymes of the invention can be synthetically produced by conventional peptide synthesizers
  • Mamre proteins can be expressed in mammalian cells, yeast, bactena, or other cells under the control of appropriate promoters Cell-iree translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Approp ⁇ ate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described (e.g Sambrook et al , 1989, the disclosure of which is hereby inco ⁇ orated by reference).
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication ongin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovims enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g. , the ampicilhn resistance gene of E. cob and 5. cerevisiae TRP1 gene, and a promoter derived from a highly- expressed gene to direct transcnption of a downstream structural sequence.
  • promoters can be denved from operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), A-factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in approp ⁇ ate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated enzyme.
  • the heterologous sequence can encode a fusion enzyme including an N-terminal identification peptide imparting desired charactenstics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bactenal use are constructed by inserting a structural
  • DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will compnse one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host
  • Suitable prokaryotic hosts for transformation include E cob, Bacillus subtilis, Salmonella typhunurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids compnsmg genetic elements of the well known cloning vector pBR322 (ATCC 37017)
  • plasmids compnsmg genetic elements of the well known cloning vector pBR322 (ATCC 37017)
  • Such commercial vectors include, for example, pKK223- 3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e g , temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
  • mammalian cell culmre systems can also be employed to express recombinant protein
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, as described (Gluzman, 1981), and other cell lines capable of expressing a compatible vector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscnbed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscnbed genetic elements
  • the enzyme can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Protein refolding steps can be used, as necessary, in completing configuration of the mamre protein Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps
  • the enzymes of the present invention may be a namrally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) Depending upon the host employed in a recombinant production procedure, the enzymes of the present invention may be glycosylated or may be non-glycosylated Enzymes of the invention may or may not also include an initial methiomne amino acid residue
  • the enzyme of the present invention is a phytase enzyme which is stable to heat and is heat resistant and catalyzes the enzymatic hydrolysis of phytate, / e , the enzyme is able to renature and regain activity after a brief (. e , 5 to 30 seconds), or longer period, for example, minutes or hours, exposure to temperamres of up to about 50 °C or slightly above 50 °C
  • a method for producing an phytase enzyme such as those shown in Figures 1.
  • the method includes growing a host cell which contains a polynucleotide encoding the enzyme (e g , SEQ ID NO 1), under conditions which allow the expression of the nucleic acid, and optionally isolating the enzyme encoded by the nucleic acid
  • a host cell which contains a polynucleotide encoding the enzyme (e g , SEQ ID NO 1)
  • Methods of culturmg the host cell are described in the Examples and are known by those of skill in the art
  • transgenic plants and plant organs In a particular embodiment, the present invention provides for the expression of phytase in transgenic plants or plant organs and methods for the production thereof DNA expression constructs are provided for the transformation of plants with a gene encoding phytase under the control of regulatory sequences which are capable of directing the expression of phytase These regulatory sequences include sequences capable of directing transcription in plants, either constitutively. or in stage and/or tissue specific manners
  • the manner of expression depends, in part, on the use of the plant or parts thereof
  • the transgenic plants and plant organs provided by the present invention may be applied to a vanety of industrial processes either directly, e g in animal feeds or alternatively, the expressed phytase may be extracted and if desired, punfied before application Alternatively, the recombinant host plant or plant part may be used directly
  • the present invention provides methods of catalyzing phytate-hydrolyzing reactions using seeds containing enhanced amounts of phytase The method involves contacting transgenic, non-wild type seeds, preferably in a ground or chewed form, with phytate- containing substrate and allowing the enzymes in the seeds to increase the rate of reaction
  • the invention provides a solution to the expensive and problematic process of extracting and purifying the enzyme
  • the present invention also provides methods of treatment whereby an organism lacking a sufficient supply of
  • Petunia, Brassica Additionally, for example, strategies for the expression of foreign genes in plants are available Additionally still, regulatory sequences from plant genes have been identified that are serviceable for the construction of chimeric genes that can be functionally expressed in plants and in plant cells (e g Klee et al , 1987, Clark et al , 1990, Smith et al , 1990)
  • Non-limiting examples of plant tissues that can be transformed thusly include protoplasts, microspores or pollen, and explants such as leaves, stems, roots, hypocotyls, and cotyls
  • DNA can be introduced directly into protoplasts and plant cells or tissues by microinjection, electnporation, particle bombardment, and direct DNA uptake
  • Proteins may be produced in plants by a variety of expression systems
  • a constitutive promoter such as the 35S promoter of Cauliflower Mosaic ims (Guilley et al , 1982) is serviceable for the accumulation of the expressed protein in virtually all organs of the transgenic plant
  • promoters that are highly tissue-specific and/or stage-specific are serviceable for this invention (Higgins, 1984, Shotwell, 1989) in order to bias expression towards desired tissues and/or towards a desired stage of development
  • Further details relevant to the expression in plants of the phytase molecules of the instant invention are disclosed, for example, in USPN 5,770,413 (Van Ooijen et al ) and USPN 5,593,963 (Van Ooijen et al ), although these reference do not teach the inventive molecules of the instant application and instead teach the use of fungal phytases
  • a variety of means can be used to achieve the recombinant expression of phytase in a transgenic plant or plant part
  • a transgenic plant and plant parts are serviceable as sources of recombinantly expressed phytase, which can be added directly to phytate-containing sources
  • the recombinant plant-expressed phytase can be extracted away from the plant source and, if desired, purified prior to contacting the phytase substrate
  • plants to be selected include, but are not limited to crops producing edible flowers such as cauliflower (Brassica oleracea), artichoke (Cynara scolvmus), fruits such as apple (Malus, e g domesticus), banana (Musa, e g acuminata), bernes (such as the cu ⁇ ant, Ribes, e g rubrum), che ⁇ ies (such as the sweet cherry, Prunus, e g avium), cucumber (Cucumis, e g sativus), grape (Vitis, e g vimfera), lemon (Citrus limon), melon (Cucumis melo), nuts (such as the walnut, Juglans, e g t egia, peanut, Arachis hypogeae), orange (Citrus, e g maxima), peach (Prunus, e g persica), pear (Pyra
  • phytase construct involves such details as transcription of the gene by plant polymerases, translation of mRNA, etc that are known to persons skilled in the art of recombinant DNA techniques Only details relevant for the proper understanding of this invention are discussed below Regulatory sequences which are known or are found to cause expression of phytase may be used in the present invention
  • the choice of the regulatory sequences used depends on the target crop and/or target organ of interest
  • Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized
  • Such regulatory sequences are promoters active in directing transcription in plants, either constitutively or stage and or tissue specific, depending on the use of the plant or parts thereof
  • promoters include, but are not limited to promoters showing constitutive expression, such as the 35S promoter of Cauliflower Mosaic Virus (CaMV) (Guilley et al , 1982), those for leaf- specific expression, such as the promoter of the ⁇ bulose bisphosphate carboxylase
  • those for seed-specific expression such as the crucife ⁇ n A promoter from Brassica napus (Ryan et al , 1989), those for tuber-specific expression, such as the class-I patatin promoter from potato (Koster-Topfer et al , 1989; Wenzler et al , 1989) or those for fruit-specific expression, such as the polygalacturonase (PG) promoter from tomato (Bird et al , 1988).
  • seed-specific expression such as the crucife ⁇ n A promoter from Brassica napus (Ryan et al , 1989)
  • tuber-specific expression such as the class-I patatin promoter from potato (Koster-Topfer et al , 1989; Wenzler et al , 1989) or those for fruit-specific expression, such as the polygalacturonase (PG) promoter from tomato (Bird et al , 1988).
  • PG polygalacturonas
  • regulatory sequences such as terminator sequences and polyadenylation signals include any such sequence functioning as such in plants, the choice of which is within the level of the skilled artisan
  • An example of such sequences is the 3' flanking region of the nopahne synthase (nos) gene of Agrobacterium tumefaciens (Bevan, supra).
  • the regulatory sequences may also include enhancer sequences, such as found in the 35S promoter of CaMV, and mRNA stabilizing sequences such as the leader sequence of Alfalfa Mosaic Cirus (A1MV) RNA4 (Brederode et al , 1980) or any other sequences functioning in a like manner
  • the phytase should be expressed in an environment that allows for stability of the expressed protein.
  • the choice of cellular compartments, such as cytosol, endoplasmic reticulum, vacuole, protein body or periplasmic space can be used in the present invention to create such a stable environment, depending on the biophysical parameters of the phytase.
  • Such parameters include, but are not limited to pH-optimum, sensitivity to proteases or sensitivity to the mola ⁇ ty of the prefe ⁇ ed compartment.
  • the expressed enzyme should not contain a secretory signal peptide or any other target sequence.
  • the expressed enzyme should contain specific so-called transit peptide for import into these organelles.
  • Targeting sequences that can be attached to the enzyme of interest in order to achieve this are known (Smeekens et al , 1990; van den Broeck et al , 1985, Wolter et al , 1988). If the activity of the enzyme is desired in the vacuoles a secretory signal peptide has to be present, as well as a specific targeting sequence that directs the enzyme to these vacuoles (Tague et al, 1990). The same is true for the protein bodies in seeds.
  • the DNA sequence encoding the enzyme of interest should be modified in such a way that the enzyme can exert its action at the desired location in the cell
  • the expression construct of the present invention utilizes a secretory signal sequence
  • signal sequences which are homologous (native) to the plant host species are prefe ⁇ ed
  • heterologous signal sequences i e those originating from other plant species or of microbial origin, may be used as well.
  • signal sequences are known to those skilled in the art Appropriate signal sequences which may be used within the context of the present invention are disclosed in Blobel et al , 1979; Von Heijne, 1986, Garcia et al , 1987; Sijmons et al , 1990. Ng et al , 1994, and Powers et al , 1996).
  • the instant invention provides a method (and products thereof) of achieving a highly efficient overexpression system for phytase and other molecules.
  • the instant invention provides a method (and products thereof) of achieving a highly efficient overexpression system for phytase and pH 2.5 acid phosphatase in T ⁇ choderma.
  • the instant invention provides a method (and products thereof) of producing stabilized aqueous liquid formulations having phytase activity that exhibit increased resistance to heat inactivation of the enzyme activity and which retain their phytase activity during prolonged periods of storage.
  • the liquid formulations are stabilized by means of the addition of urea and or a polyol such as sorbitol and glycerol as stabilizing agent.
  • feed preparations for monogast ⁇ c animals and methods for the production thereof that result from the use of such stabilized aqueous liquid formulations. Additional details regarding this approach are in the public literature and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes EP 0626010 (W0 9316175 Al) (Barendse et al ), although references in the publicly available literature do not teach the inventive molecules of the instant application.
  • the instant invention provides a method of hydrolyzing phytate comprised of contacting the phytate with one or more of the novel phytase molecules disclosed herein Accordingly, the invention provides a method for catalyzing the hydrolysis of phytate to inositol and free phosphate with release of minerals from the phytic acid complex.
  • the method includes contacting a phytate substrate with a degrading effective amount of an enzyme of the invention, such as the enzyme shown in SEQ ID NO:2
  • a degrading effective amount refers to the amount of enzyme which is required to degrade at least 50% of the phytate, as compared to phytate not contacted with the enzyme. Preferably, at least 80%o of the phytate is degraded.
  • the invention provides a method for hydrolyzing phospho- mono-ester bonds in phytate
  • the method includes administering an effective amount of phytase molecules of the invention (e g , SEQ ID NO 2), to yield inositol and free phosphate
  • an "effective" amount refers to the amount of enzyme which is required to hydrolyze at least 50% of the phospho-mono-ester bonds, as compared to phytate not contacted with the enzyme
  • at least 80% of the bonds are hydrolyzed
  • the phytase molecules may be used in combination with other reagents, such as other catalysts, in order to effect chemical changes (e g hydrolysis) in the phytate molecules and/or in other molecules of the substrate source(s)
  • the phytase molecules and the additional reagent(s) will not inhibit each other, more preferably the phytase molecules and the additional reagent(s) will have an overall additive effect, and more preferably still the phytase molecules and the additional reagent(s) will have an overall synergistic effect
  • Relevant sources of the substrate phytate molecules include foodsmffs, potential foodsmffs, byproducts of foodsmffs (both in vitro byproducts and in vivo byproducts, e g ex vivo reaction products and animal excremental products), precursors of foodsmffs, and any other matenal source of phytate
  • the recombinant phytase can be consumed by organisms and retains activity upon consumption
  • transgenic uzes can be used to achieve expression of the recombinant phytase - preferably in a controlled fashion (methods are available for controlling expression of transgenic molecules in time-specific and tissue specific manners)
  • the phytase activity in the source material may be increased upon consumption, this increase in activity may occur, for example, upon conversion of a precursor phytase molecule in pro-form to a significantly more active enzyme in a more mamre form, where said conversion may result, for example, from the injestion and digestion of the phytase source
  • Hydrolysis of the phytate substrate may occur at any time upon the contacting of the phytase with the phytate; for example, this may occur before injestion or after injestion or both before and after injestion of either the substrate or the enzyme or both
  • the phytate substrate may be contacted with - in addition to the phytase - one or more additional reagents, such as another enzyme, which may be also be applied either directly or after purification from its source matenal.
  • the phytase source mate ⁇ al(s) can be contacted directly with the phytate source mate ⁇ al(s); e g upon in vitro or in vivo grinding or chewing of either or both the phytase source(s) and the phytate source(s).
  • the phytase enzyme may be purified away from source matenal(s), or the phytate substrate may be purified away from source mate ⁇ al(s), or both the phytase enzyme and the phytate substrate may be purified away from source mate ⁇ al(s) pnor to the contacing of the phytase enzyme with the phytate substrate. It is appreciated that a combination of purified and unpunfied reagents - including enzyme(s) or substrates(s) or both - may be used.
  • more than one source material may be used as a source of phytase activity This is serviceable as one way to achieve a timed release of reagent(s) from source mate ⁇ al(s), where release from different reagents from their source mate ⁇ als occur differentially, for example as injested source materials are digested in vivo or as source matenals are processed in in vitro applications.
  • the use of more than one source material of phytase activity is also serviceable to obtain phytase activities under a range of conditions and fluctuations thereof, that may be encountered - such as a range of pH values, temperamres, salinities, and time intervals - for example during different processing steps of an application.
  • a single source material such as a trangemc plant species (or plant parts thereof), may be a source material of both phytase and phytate, and that enzymes and substrates may be differentially compartmentalized within said single source - e g secreted vs. non-secreted, differentially expressed &/or having differential abundances in different plant parts or organs or tissues or in subcellular compartments within the same plant part or organ or tissue.
  • Purification of the phytase molecules contained therein may comprise isolating and/or further processing of one or more desirable plant parts or organs or tissues or subcellular compartments.
  • this invention provides a method of catalyzing in vivo and/or in vitro reactions using seeds containing enhanced amounts of enzymes.
  • the method comprises adding transgenic, non-wild type seeds, preferably in a ground form, to a reaction mixmre and allowing the enzymes in the seeds to increase the rate of reaction.
  • the method provides a solution to the more expensive and cumbersome process of extracting and purifying the enzyme.
  • Methods of treatment are also provided whereby an organism lacking a sufficient supply of an enzyme is administered the enzyme in the form of seeds from one or more plant species, preferably transgenic plant species, containing enhanced amounts of the enzyme. Additional details regarding this approach are in the public literature and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes USPN 5,543,576 (Van Ooijen et al) and USPN 5,714,474 (Van Ooijen et al ), although these reference do not teach the inventive molecules of the instant application and instead teach the use of fungal phytases.
  • the instant phytase molecules are serviceable for generating recombinant digestive system life forms (or microbes or flora) and for the administration of said recombinant digestive system life forms to animals.
  • Administration may be optionally performed alone or in combination with other enzymes &/or with other life forms that can provide enzymatic activity in a digestive system, where said other enzymes and said life forms may be may recombinant or otherwise.
  • the present invention provides a method for steeping corn or sorghum kernels in warm water containing sulfur dioxide in the presence of an enzyme preparation comprising one or more phytin- degrading enzymes, preferably in such an amount that the phytin present in the com or sorghum is substantially degraded
  • the enzyme preparation may compnse phytase and/or acid phosphatase and optionally other plant material degrading enzymes
  • the steeping time may be 12 to 18 hours
  • the steeping may be interrupted by an intermediate milling step, reducing the steeping time
  • com or sorghum kernels are steeped in warm water containing sulfur dioxide in the presence of an enzyme preparation including one or more phytin-degradmg enzymes, such as phytase and acid phosphatases, to eliminate or greatly reduce phytic acid
  • the present invention provides a method to obtain a bread dough having desirable physical properties such as non-tackiness and elasticity and a bread product of superior quality such as a specific volume comprising adding phytase molecules to the bread dough
  • phytase molecules of the instant invention are added to a working bread dough preparation that is subsequently formed and baked Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan
  • such publicly available literamre includes JP 03076529 (Hara et al ), although this reference does not teach the inventive phytase molecules of the instant application
  • the present invention provides a method to produce improved soybean foodsmffs Soybeans are combined with phytase molecules of the instant invention to remove phytic acid from the soybeans, thus producing soybean foodsmffs that are improved in their supply of trace nutrients essential for consuming organisms and in its digestibility of proteins
  • phytase molecules of the instant invention are added to or brought into contact with soybeans in order to reduce the phytic acid content
  • the application process can be accelerated by agitating the soybean milk together with the enzyme under heating or by a conducting a mixing-type reaction in an agitation container using an immobilized enzyme Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-limiting exemplification, such publicly available literamre includes JP 59166049 (Kamikubo e
  • the instant invention provides a method of producing an admixture product for d ⁇ nking water or animal feed in fluid form, and which comprises using mineral mixtures and vitamin mixtures, and also novel phytase molecules of the instant invention
  • a prefe ⁇ ed embodiment there is achieved a co ⁇ ectly dosed and composed mixmre of necessary nutrients for the consuming organism without any risk of precipitation and destruction of important minerals/vitamins, while at the same time optimum utilization is made of the phytin-bound phosphate in the feed
  • Additional details regarding this approach are in the public literature and/or are known to the skilled artisan
  • such publicly available literamre includes EP 0772978 (Bendixen et al ), although this reference does not teach the inventive molecules of the instant application
  • the phytase molecules of the instant invention may also be used to produce other alcoholic and non-alcoholic drinkable foodsmffs (or dnnks) based on the use of molds &/or on grains &/or on other plants
  • drinkable foodsmffs mclude liquors, wines, mixed alcoholic drinks (e g wine coolers, other alcoholic coffees such as Irish coffees, etc ), beers, near-beers, juices, extracts, homogenates, and purees
  • the instantly disclosed phytase molecules are used to generate transgenic versions of molds &/or grains &/or other plants serviceable for the production of such drinkable foodsmffs
  • the instantly disclosed phytase molecules are used as additional ingredients in the manufacturing process &/or in the final content of such drinkable foodsmffs. Additional details regarding this approach are in the public literamre and/or are known
  • the present invention provides a means to obtain refined sake having a reduced amount of phytin and an increased content of inositol Such a sake may have - through direct &/or psychoge c effects - a preventive action on hepatic disease, arteriosclerosis, and other diseases.
  • a sake is produced from rice Koji by multiplying a rice Koji mold having high phytase activity as a raw material. It is appreciated that the phytase molecules of the instant invention may be used to produce a serviceable mold with enhanced activity (preferably a transgenrc mold) &/or added exogenously to augment the effects of a Koji mold.
  • the strain is added to boiled rice and Koji is produced by a conventional procedure
  • the prepared Koji is used, the whole nee is prepared at two stages and Sake is produced at constant Sake temperamre of 15°C to give the objective refined Sake having a reduced amount of phytin and an increased amount of inositol. Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literamre includes JP 06153896 (Soga et al ) and JP 06070749 (Soga et al ), although these references do not teach the inventive molecules of the instant application
  • the present invention provides a method to obtain an absorbefacient capable of promoting the abso ⁇ tion of minerals including ingested calcium without being digested by gastric juices or intestinal juices at a low cost
  • said mineral absorbefacient contains a partial hydrolysate of phytic acid as an active ingredient
  • a partial hydrolyzate of the phytic acid is produced by hydrolyzing the phytic acid or its salts using novel phytase molecules of the instant invention
  • the treatment with said phytase molecules may occur either alone &/or in a combination treatment (to inhibit or to augment the final effect), and is followed by inhibiting the hydrolysis within a range so as not to liberate all the phosphate radicals Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes JP
  • the present invention provides a method (and products therefrom) to produce an enzyme composition having an additive or preferably a synergrstic phytate hydrolyzing activity; said composition comprises novel phytase molecules of the instant invention and one or more additional reagents to achieve a composition that is serviceable for a combination treatment
  • the combination treatment of the present invention is achieved with the use of at least two phytases of different position specificity, i.e any combinations of 1-, 2-, 3-, 4-, 5-, and 6-phytases.
  • compositions such as food and feed or food and feed additives comprising such phytases in combination are also included in this invention as are processes for their preparation. Additional details regarding this approach are in the public literature and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes W09 830681 (Ohmann et al ), although references in the publicly available literature do not teach the use of the inventive molecules of the instant application.
  • the combination treatment of the present invention is achieved with the use of an acid phosphatase having phytate hydrolyzing activity at a pH of 2 5, in a low ratio co ⁇ esponding to a pH 2.5 '5.0 activity profile of from about 0.1 1 0 to 10T , preferably of from about 0 5.1 0 to 5 1 , more preferably still of from about 0 8 1 0 to 3 1 , and more preferably still of from about 0 8 1 0 to 2 1
  • Said enzyme composition preferably displays a higher synergetic phytate hydrolyzing efficiency through thermal treatment
  • Said enzyme composition is serviceable in the treatment of foodsmffs (drinkable and solid food, feed and fodder products) to improve phytate hydrolysis Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literamre includes USPN 5,554.399 (Vanderbeke et al ) and USPN
  • the present invention provides a method (and products therefrom) to produce composition comprised of the instant novel phytate-acting enzyme in combination with one or more additional enzymes that act on polysaccha ⁇ des
  • polysaccharides can be selected from the group consisting of arabinans, fmctans, fucans, galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan, ca ⁇ ageenan, galactocarolose, pectin, pectic acid, amylose, pullulan, glycogen, amylopectin, cellulose, carboxylmethylcellulose, hydroxypropylmethylcellulose, dextran, pustulan, chitin, agarose, keratan, chondroitin, dermatan, hyaluronic acid, alginic acid, and poly
  • the present invention provides a method (and products therefrom) to produce composition having a synergistic phytate hydrolyzing activity comprising one or more novel phytase molecules of the instant invention, a cellulase (including preferably but not exclusively a xylanase), optionally a protease, and optionally one or more additonal reagents
  • a synergistic phytate hydrolyzing activity comprising one or more novel phytase molecules of the instant invention, a cellulase (including preferably but not exclusively a xylanase), optionally a protease, and optionally one or more additonal reagents
  • such combination treatments are serviceable in the treatment of foodsmffs, wood products, such as paper products, and as cleansing solutions and solids
  • the instant phytase molecules are serviceable in combination with cellulosome components. It is known that cellulases of many cellulolytic bacteria are organized into discrete multienzyme complexes, called cellulosomes. The multiple subunits of cellulosomes are composed of numerous functional domains, which interact with each other and with the cellulosic substrate One of these subunits comprises a distinctive new class of noncatalytic scaffolding polypeptide, which selectively integrates the various cellulase and xylanase subunits into the cohesive complex. Intelligent application of cellulosome hybrids and chimeric constructs of cellulosomal domains should enable better use of cellulosic biomass and may offer a wide range of novel applications in research, medicine and industry.
  • the instant phytase molecules are serviceable - either alone or in combination treatments - in areas of biopulpmg and biobleaching where a reduction in the use of environmentally harmful chemicals traditionally used in the pulp and paper industry is desired Waste water treatment represents another vast application area where biological enzymes have been shown to be effective not only in colour removal but also in the bioconversion of potentially noxious substances into useful bioproducts.
  • the instant phytase molecules are serviceable for generating life forms that can provide at least one enzymatic activity - either alone or in combination treatments - in the treatment of digestive systems of organisms.
  • Particularly relevant organisms to be treated include non-ruminant organisms.
  • this approach may be performed alone or in combination with other biological molecules (for example, xylanases) to generate a recombinant host that expresses a plurality of biological molecules.
  • the administration of the instant phytase molecules &/or recombinant hosts expressing the instant phytase molecules may be performed either alone or in combination with other biological molecules, &/or life forms that can provide enzymatic activities in a digestive system - where said other enzymes and said life forms may be may recombinant or otherwise.
  • administration may be performed in combination with xylanolytic bacteria
  • Hemicelluloses or xylans are major components (35%) of plant materials
  • Hemicelluloses or xylans are major components (35%) of plant materials
  • the major xylanolytic species are Butvrivibno fibrisolvens and Bacteroides rumimcola
  • Bacteroides ovatus and Bacteroides fragilis subspecies "a" are major xylanolytic bacteria Xylans are chemically complex, and their degradation requires multiple enzymes.
  • the present phytase molecules are serviceable for 1) transfemng into a suitable host (such as Bact. fragilis or Bact. umformis), 2) achieving adequate expression in a resultant recombinant host; and 3) administering said recombinant host to organisms to improve the ability of the treated organisms to degrade phytate.
  • a suitable host such as Bact. fragilis or Bact. umformis
  • administering said recombinant host to organisms to improve the ability of the treated organisms to degrade phytate.
  • the present invention provides a method (and products therefrom) for enhancement of phytate phosphorus utilization and treatment and prevention of tibial dyschondroplasia in animals, particularly poultry, by administering to animals a feed composition containing a hydroxylated vitamin D 3 derivative
  • the vitamin D 3 derivative is preferably administered to animals in feed containing reduced levels of calcium and phosphorus for enhancement of phytate phosphorus utilization
  • the vitamin D 3 derivative is preferably administered in combination with novel phytase molecules of the instant invention for further enhancement of phytate phosphorus utilization Additional details regarding this approach are in the public literamre and/or are known to the skilled artrsan In a particular non-limiting exemplification, such publicly available literature includes USPN 5,516,525 (Edwards et al ) and USPN 5,366.736 (Edwards et al ), USPN 5,316,770 (Edwards et al ) although these reference do not
  • Said foodstuff is comprised of an admixture of a phytin-contaimng grain, a lactic acid-producing microorganism, and a novel phytase molecule of the instant invention.
  • said foodstuff is produced by compounding a phytin- containing grain (preferably, e.g rice bran) with an effective microbial group having an acidophilic property, producing lactic acid, without producing butyric acid, free from pathogenicity, and a phytase.
  • an effective microbial group include e g Streptomyces sp.
  • a preferable amount of addition of an effective microbial group is 0.2 wt. % in terms of bactenal body weight based on a grain material.
  • the amount of the addition of the phytase is preferably 1-2 wt. % based on the phytin in the grain material. Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-hmiting exemplification, such publicly available literamre includes JP 08205785 (Akaho ⁇ et al ), although references in the publicly available literamre do not teach the inventive molecules of the instant application.
  • the present invention provides a method for improving the solubility of vegetable proteins. More specifically, the invention relates to methods for the solubilization of proteins in vegetable protein sources, which methods comprise treating the vegetable protein source with an efficient amount of one or more phytase enzymes - including phytase molecules of the instant invention - and treating the vegetable protein source with an efficient amount of one or more proteolytic enzymes.
  • the invention provides animal feed additives compnsmg a phytase and one or more proteolytic enzymes Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan.
  • such publicly available literamre includes EP 0756457 (WO 9528850 Al) (Nielsen and Knap), although references in the publicly available literamre do not teach the inventive molecules of the instant application
  • the present invention provides a method of producing a plant protein preparation comprising dispersing vegetable protein source matenals in water at a pH in the range of 2 to 6 and admixing phytase molecules of the instant invention therein
  • the acidic extract containing soluble protein is separated and dried to yield a solid protein of desirable character
  • proteases can also be used to improve the characteristics of the protein Additional details regarding this approach are in the public literamre and or are known to the skilled artisan In a particular non-limiting exemplification, such publicly available literamre includes USPN 3.966,971 (Morehouse et al ), although references in the publicly available literamre do not teach the inventive molecules of the instant application
  • the present invention provides a method (and products thereof) to activate inert phosphorus in soil and/or compost, to improve the utilization rate of a nitrogen compound, and to suppress propagation of pathogenic molds by adding three reagents, phytase, saponin and chitosan, to the compost
  • the method can comprise treating the compost by 1) adding phytase-contaimng microorganisms in media - preferably recombinant hosts that overexpress the novel phytase molecules of the instant invention - e g at 100 ml media/ 100 kg wet compost, 2) alternatively also adding a phytase-contaimng plant source - such as wheat bran - e g at 0 2 to 1 kg/100 kg wet compost, 3) adding a saponin-containing source
  • Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA or a genomic library to isolate the full length DNA and to isolate other DNAs which have a high sequence similarity to the gene or similar biological activity.
  • Probes of this type have at least 10, preferably at least 15, and even more preferably at least 30 bases and may contain, for example, at least 50 or more bases.
  • the probe may also be used to identify a DNA clone co ⁇ esponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns.
  • the present invention provides methods for identifying nucleic acid molecules that encode members of the phytase polypeptide family in addition to SEQ ID NO: l .
  • a sample e.g., a nucleic acid library, such as a cDNA library, that contains a nucleic acid encoding a phytase polypeptide is screened with a phytase-specific probe, e g., a phytase-specific nucleic acid probe
  • Phytase-specific nucleic acid probes are nucleic acid molecules (e.g , molecules containing DNA or RNA nucleotides, or combinations or modifications thereof) that specifically hybridize to nucleic acids encoding phytase polypeptides, or to complementary sequences thereof
  • the invention facilitates production of phytase-specific nucleic acid probes.
  • Methods for obtaining such probes can be designed based on the amino acid sequences shown in Figure 1
  • the probes which can contain at least 12, e g ,at least 15, 25, 35, 50, 100, or 150 nucleotides, can be produced using any of several standard methods (see, e g., Ausubel et al , supra)
  • the probes are generated using PCR amplification methods
  • primers are designed that co ⁇ espond to phytase- conserved sequences (see Figure 1), which can include phytase-specific amino acids, and the resulting PCR product is used as a probe to screen a nucleic acid library, such as a cDNA library
  • This invention can be used to isolate nucleic acid sequences substantially similar to the isolated nucleic acid molecule encoding an phytase enzyme disclosed in Figure 1 (SEQ ID NO 1)
  • Isolated nucleic acid sequences are substantially similar if (I) they are capable of hybridizing under stnngent conditions, hereinafter described, to SEQ ID NO 1 , or (n) they encode a phytase polypeptide as set forth in SEQ ID NO 2 due to the degeneracy of the genetic code (e g Regenerate to SEQ ID NO 1 )
  • nucleotide sequences encode the amino acid sequence of SEQ ID NO 2, but have vanations in the nucleotide coding sequences
  • substantially similar refers to the sequences having similar identity to the sequences of the instant invention
  • the nucleotide sequences that are substantially similar can be identified by hybridization or by sequence comparison
  • Enzyme sequences that are substantially similar can be identified by one or more of the following proteolytic digestion, gel electrophoresis and/or microsequencing
  • One means for isolating a nucleic acid molecule encoding a phytase enzyme is to probe a genomic gene library with a natural or artificially designed probe using art recognized procedures (see, e g , Ausubel et al , supra) It is appreciated to one skilled in the art that SEQ ID NO 1 , or fragments thereof (comprising at least 15 contiguous nucleotides), is a particularly useful probe.
  • Other particular useful probes for this pu ⁇ ose are hybndizable fragments to the sequences of SEQ ID NO 1 (/ e , comprising at least 15 contiguous nucleotides)
  • probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe
  • Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other animal sources or to screen such sources for related sequences.
  • hybndization may be earned out under conditions of reduced stringency, medium stringency or even stringent conditions
  • a polymer membrane containing immobilized denatured nucleic acid is first prehyb ⁇ dized for 30 minutes at 45°C in a solution consisting of 0 9 M NaCI, 50 mM NaH : P0 4 , pH 7 0, 5 0 mM Na 2 EDTA, 0.5% SDS, 10X Denhardt's.
  • nucleic acid molecules of the invention can be used as templates in standard methods for production of phytase gene products (e g , phytase RNAs and phytase polypeptides ).
  • nucleic acid molecules that encode phytase polypeptides (and fragments thereof) and related nucleic acids - such as (1) nucleic acids containing sequences that are complementary to, or that hybridize to, nucleic acids encoding phytase polypeptides, or fragments thereof (e g , fragments containing at least 12, 15, 20, or 25 nucleotides), and (2) nucleic acids containing sequences that hybridize to sequences that are complementary to nucleic acids encoding phytase polypeptides, or fragments thereof (e g , fragments containing at least 12, 15, 20, or 25 nucleotides) - can be used in methods focused on their hybridization properties For example, as is desc ⁇ bed in further detail herein, such nucleic acids
  • Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention. Accordingly, fragments or portions of the enzymes of the present invention may be employed for producing the co ⁇ esponding full-length enzyme by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length enzymes. Size separation of the cleaved fragments is generally performed using 8 percent polyacrylamide gel as descnbed in the literamre (e g by Goeddel et al , 1980)
  • This invention provides enzymes, as well as fragments, other derivatives, and analogs thereof, and the co ⁇ esponding nucleotides for use in directed evolution.
  • the discovery and use of a plurality of templates as disclosed herein may significantly increase the potential yield of directed evolution in companson to the directed evolution of a single template protein.
  • the need for discovery is based on the premise that nature provides a wealth of potentially unattainable or unpredictable feamres in distinct but related members of molecular groupings, and that the exploitation of these feamres may greatly facilitate directed evolution.
  • related but distinct molecules may serve as unique starting templates for the directed evolution of a desired charactenstic
  • they may serve as repositories of structure- function information including, but not limited to, a variety of consensus motifs. Both utilities help to obviate the logistically impractical task of at-once exploring an overly wide range of mutational permutations on any given molecule. For example, the full range of mutational permutations on a 100 amino acid protein includes over 10' 30 possibilities (assuming there are 20 amino acid possibilities at each position), a number too large for practical consideration
  • the enzyme activities of the novel molecules generated can then be screened by a vanety of methods including, by way of non-hmiting exemplification: a) molecular biopanning; b) recombinant clone screening, and c) extract screening.
  • This invention provides enzymes, as well as fragments, other derivatives, and analogs thereof, and cells expressing them that can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimenc, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Vanous procedures known in the art may be used for the production of such antibodies and fragments
  • Antibodies generated against the enzymes co ⁇ esponding to a sequence of the present invention can be obtained by direct injection of the enzymes into an animal or by administering the enzymes to an animal, preferably a nonhuman The antibody so obtained will then bind the enzymes itself. In this manner, even a sequence encoding only a fragment of the enzymes can be used to generate antibodies binding the whole native enzymes. Such antibodies can then be used to isolate the enzyme from cells expressing that enzyme.
  • any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybndoma technique (Kohler and Milstein, 1975), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al , 1983), and the EBV-hyb ⁇ doma technique to produce human monoclonal antibodies (Cole et al . 1985, pp 77-96).
  • Techniques desc ⁇ bed for the production of single chain antibodies (USPN 4,946 778 Ladner et al ) can be adapted to produce single chain antibodies to lmmunogenic enzyme products of this invention
  • transgenic mice may be used to express humanized antibodies to lmmunogenic enzyme products of this invention
  • Antibodies generated against the enzyme of the present invention may be used in screening for similar enzymes from other organisms and samples Such screening techniques are known in the art Antibodies may also be employed as a probe to screen gene libraries generated from this or other organisms to identify this or cross reactive activities
  • antigens that can be used in producing phytase-specific antibodies include phytase polypeptides, e g , any of the phytase shown in Figures 1 polypeptide fragments
  • the polypeptide or peptide used to immunize an animal can be obtained by standard recombinant, chemical synthetic, or purification methods
  • an antigen in order to increase immunogenicity, an antigen can be conjugated to a earner protein
  • Commonly used earners include keyhole limpet hemocyamn (KLH), thyroglobuhn, bovine serum albumin (BSA), and tetanus toxoid
  • KLH keyhole limpet hemocyamn
  • BSA bovine serum albumin
  • tetanus toxoid The coupled peptide is then used to immunize the animal (e g , a mouse, a rat, or a rabbit)
  • well known adjuvants can be administered with the anti
  • Phytase-specific polvclonal and monoclonal antibodies can be purified, for example, by binding to and elution from, a matrix containing a phytase polypeptide, e g , the phytase polypeptide (or fragment thereof) to which the antibodies were raised
  • Anti-idiotype antibodies co ⁇ esponding to phytase-specific antigens are also included in the invention, and can be produced using standard methods These antibodies are raised to phytase-specific antibodies, and thus mimic phytase-specific epitopes.
  • This invention also includes additonal uses of fragments of the phytase polypeptides that retain at least one phytase-specific activity or epitope. Phytase activity can be assayed by examining the catalysis of phytate to inositol and free phosphate. Such fragments can easily be identified by companng the sequences of phytases found in Figure 1
  • a phytase polypeptide fragment containing, e g , at least 8-10 amino acids can be used as an immunogen in the production of phytase- specific antibodies.
  • the fragment can contain, for example, an amino acid sequence that is conserved in phytases, and this amino acid sequence can contain amino acids that are conserved in phytases.
  • the above-described phytase fragments can be used in immunoassays, such as ELISAs, to detect the presence of phytase-specific antibodies in samples.
  • transgenic animals of the subject invention can be employed. Generally speaking, three such methods may be employed. In one such method, an embryo at the pronuclear stage (a "one cell embryo") is harvested from a female and the transgene is microinjected into the embryo, in which case the transgene will be chromosomally integrated into both the germ cells and somatic cells of the resulting mature animal. In another such method, embryonic stem cells are isolated and the transgene inco ⁇ orated therein by electroporation, plasmid transfection or microinjection, followed by reintroduction of the stem cells into the embryo where they colonize and contribute to the germ line. Methods for microinjection of mammalian species is described in U.S. Pat. No.
  • embryonic cells are infected with a retrovirus containing the transgene whereby the germ cells of the embryo have the transgene chromosomally integrated therein
  • the animals to be made transgenic are avian, because avian fertilized ova generally go through cell division for the first twenty hours in the oviduct, microinjection into the pronucleus of the fertilized egg is problematic due to the inaccessibility of the pronucleus. Therefore, of the methods to make transgenic animals described generally above, retrovims infection is prefe ⁇ ed for avian species, for example as described in U.S Pat No 5,162,215.
  • micro-injection is to be used with avian species, however, a published procedure by Love et al , (Biotechnology, 12, Jan 1994) can be utilized whereby the embryo is obtained from a sacrificed hen approximately two and one-half hours after the laying of the previous laid egg, the transgene is microinjected into the cytoplasm of the germinal disc and the embryo is cultured in a host shell until matunty
  • the animals to be made transgenic are bovine or porcine
  • microinjection can be hampered by the opacity of the ova thereby making the nuclei difficult to identify by traditional differential interference-contrast microscopy.
  • the ova can first be centrifuged to segregate the pronuclei for better visualization.
  • the "non-human animals” of the invention bovine, porcine, ovine and avian animals (e.g., cow, pig, sheep, chicken).
  • the "transgenic non-human animals” of the invention are produced by introducing "transgenes" into the germhne of the non-human animal. Embryonal target cells at vanous developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. The use of zygotes as is target for gene transfer has a major advantage in that in most cases the injected DNA will be inco ⁇ orated into the host gene before the first cleavage (Bnnster et al, Proc. Natl.
  • transgenic non- human animal will carry the inco ⁇ orated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
  • transgenic is used to describe an animal which includes exogenous genetic material within all of its cells
  • a “transgenic” animal can be produced by crossbreeding two chimenc animals which include exogenous genetic matenal within cells used in reproduction Twenty-five percent of the resulting offspring will be transgenic i e., animals which include the exogenous genetic material within all of their cells in both alleles, 50% of the resulting animals will include the exogenous genetic material withm one allele and 25% will include no exogenous genetic material
  • the transgene is digested and purified free from any vector DNA, e g , by gel electrophoresis It is prefe ⁇ ed that the transgene include an operatively associated promoter which interacts with cellular proteins involved in transcription, ultimately resulting in constitutive expression
  • Promoters useful in this regard include those from cytomegalovirus (CMV) , Moloney leukemia virus (MLV), and he ⁇ es virus, as well as those from the genes encoding metallothionin, skeletal actin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine kinase.
  • Promoters for viral long terminal repeats such as Rous Sarcoma Virus can also be employed.
  • prefe ⁇ ed promoters include those for the chicken ⁇ -globin gene, chicken lysozyme gene, and avian leukosis virus.
  • Constructs useful in plasmid transfection of embryonic stem cells will employ additional regulatory elements well known in the art such as enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, and ribosome binding sites to permit translation
  • Retroviral infection can also be used to introduce transgene into a non-human animal, as described above
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R , Proc Natl Acad. Sci USA 73 1260- 1264, 1976).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida
  • the viral vector system used to introduce the transgene is typically a replication-defective retro virus carrying the transgene (Jahner, et al , Proc Natl Acad. Sci USA 82 6927-6931, 1985, Van der Putten, et al , Proc. Natl. Acad Sci USA 82' 6148-6152, 1985) Transfection is easily and efficiently obtained by culmnng the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al, EMBO J. 6: 383-388, 1987).
  • infection can be performed at a later stage.
  • Virus or virus-producing cells can be injected into the blastocoele (D. Jahner et al , Nature 298: 623-628, 1982).
  • Most of the founders will be mosaic for the transgene since inco ⁇ oration occurs only in a subset of the cells which formed the transgenic nonhuman animal. Further, the founder may contain various retro viral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
  • ES embryonal stem cell
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al, Namre 292: 154-156, 1981 ; M. O. Bradley et al, Namre 309:255-258, 1984; Gossler, et al, Proc. Natl. Acad. Sci USA 83:9065-9069, 1986; and Robertson et al, Namre 322:445-448, 1986).
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction.
  • Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimenc animal.
  • Transformed means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid molecule.
  • Heterologous refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form p ⁇ manly expressed in the cell.
  • Transgene means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i.e., either stably integrated or as a stable extrachromosomal element) which develops from that cell.
  • a transgene may include a gene which is partly or entirely heterologous (i e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
  • transgene created by the providing of an RNA sequence which is transcnbed into DNA and then inco ⁇ orated into the genome
  • the transgenes of the invention include DNA sequences which encode phytases or polypeptides having phytase activity, and include polynucleotides, which may be expressed in a transgenic non-human animal
  • transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout.
  • transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination
  • transgenic includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or "knocked out.”
  • the transgene to be used the practice of the subject invention is a DNA sequence compnsmg a sequence coding for a phytase or a polypeptide having phytase activity.
  • a polynucleotide having a sequence as set forth in SEQ ID NO 1 or a sequence encoding a polypeptide having a sequence as set forth in SEQ ID NO 2 is the transgene as the term is defined herein.
  • DNA sequences that encode proteins having phytase activity but differ in nucleic acid sequence due to the degeneracy of the genetic code may also be used herein, as may truncated forms, allelic vanants and interspecies homologues.
  • the present invention includes methods for increasing the phosphorous uptake in the transgenic animal and/or decreasing the amount of polltant in the manure of the transgenic organism by about 15%, typically about 20%o, and more typically about 20% to about 50%
  • the animals contemplated for use in the practice of the subject invention are those animals generally regarded as domesticated animals including pets (e g., canines, felines, avian species etc.) and those useful for the processing of food stuffs, i e , avian such as meat bred and egg laying chicken and turkey, ovme such as lamb, bovine such as beef cattle and milk cows, piscine and porcine
  • these animals are refe ⁇ ed to as "transgenic” when such animal has had a heterologous DNA sequence, or one or more additional DNA sequences normally endogenous to the animal (collectively refe ⁇ ed to herein as "transgenes") chromosomally integrated into the germ cells of the animal.
  • the transgenic animal (including its progeny) will also have the transgene fortuitously integrated into the chromosomes of somatic cells.
  • a phytase sequence of the invention may be advantageous to deliver and express locally (e g , within a particular tissue or cell type)
  • a phytase sequence of the invention may be directly delivered to the salivary glands, tissue and cells and/or to the epithelial cells lining the gut, for example
  • Such delivery methods are known in the art and include electroporation, viral vectors and direct DNA uptake.
  • nucleic acid constructs of the present invention will comprise nucleic acid molecules in a form suitable for uptake into target cells within a host tissue
  • the nucleic acids may be in the form of bare DNA or RNA molecules, where the molecules may comprise one or more structural genes, one or more regulatory genes, antisense strands, strands capable of tnplex formation, or the like
  • the nucleic acid construct will include at least one structural gene under the transcriptional and translational control of a suitable regulatory region
  • nucleic acid constructs of the present invention will comprise nucleic acids inco ⁇ orated in a delivery vehicle to improve transfection efficiency, wherein the delivery vehicle will be dispersed within larger particles comprising a dried hydrophilic excipient material
  • One such delivery vehicles comprises viral vectors, such as retroviruses, adenoviruses, and adeno-associated viruses, which have been inactivated to prevent self- replication but which maintain the native viral ability to bind a target host cell, deliver genetic matenal into the cytoplasm of the target host cell, and promote expression of structural or other genes which have been inco ⁇ orated in the particle.
  • viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses
  • adenovims gene delivery is described in Rosenfeld et al (1991 ) SCIENCE 252 431-434, the disclosure of which is inco ⁇ orated herein by reference
  • Both retroviral and adenovims delivery systems are described in Friedman (1989) SCIENCE 244 1275-1281 , the disclosure of which is also inco ⁇ orated herein by reference.
  • a second type of nucleic acid delivery vehicle comprises hposomal transfection vesicles, including both anionic and cationic hposomal constructs.
  • anionic hposomes requires that the nucleic acids be entrapped within the hposome.
  • Cationic hposomes do not require nucleic acid entrapment and instead may be formed by simple mixing of the nucleic acids and hposomes.
  • hposomes avidly bind to the negatively charged nucleic acid molecules, including both DNA and RNA, to yield complexes which give reasonable transfection efficiency in many cell types
  • hpofectin which is composed of an equimolar mixmre of dioleylphosphatidyl ethanolamine (DOPE) and dioleyloxypropyl-t ⁇ ethylammomum (DOTMA), as described in Feigner and Ringold (1989) NATURE 337 387-388, the disclosure of which is inco ⁇ orated herein by reference.
  • DOPE dioleylphosphatidyl ethanolamine
  • DOTMA dioleyloxypropyl-t ⁇ ethylammomum
  • Kahn et al (1992), supra . teaches that a retrovims vector may be combined in a cationic DEAE-dextran vesicle to further enhance transformation efficiency. It is also possible to inco ⁇ orate nuclear proteins into viral and or hposomal delivery vesicles to even further improve transfection efficiencies. See. Kaneda et al ( 1989) SCIENCE 243-375-378, the disclosure of which is inco ⁇ orated herein by reference
  • a digestive aid containing an enzyme either as the sole active ingredient or in combination with one or more other agents and/or enzymes is provided (as desc ⁇ bed in co-pending application U.S. Serial No , entitled
  • feed for livestock e g , certain poultry feed
  • minerals e g , inorganic phosphorous
  • enzymes e.g , enzymes, growth factors, drugs, and other agents for delivery to the livestock.
  • These supplements replace many of the calories and natural nutrients present in grain, for example
  • the feed By reducing or eliminating the inorganic phosphorous supplement and other supplements (e g , trace mineral salts, growth factors, enzymes, antibiotics) from the feed itself, the feed would be able to carry more nutrient and energy Accordingly, the remaining diet would contain more usable energy
  • gram-oilseed meal diets generally contain about 3,200 kcal metabohzable energy per kilogram of diet, and mineral salts supply no metabohzable energy Removal of the unneeded minerals and substitution with grain would therefore increase the usable energy in the diet
  • the invention can be differentiated over commonly used phytase containing feed
  • a biocompatible material is used that is resistant to digestion by the gastrointestinal tract of an organism.
  • the digestive tract includes a gizzard which stores and uses hard biocompatible objects (e g , rocks and shells from shell fish) to help in the digestion of seeds or other feed consumed by a bird.
  • a gizzard which stores and uses hard biocompatible objects (e g , rocks and shells from shell fish) to help in the digestion of seeds or other feed consumed by a bird.
  • a typical digestive tract of this general family of organisms includes the esophagus which contains a pouch, called a crop, where food is stored for a brief penod of time. From the crop, food moves down into the true stomach, or proventriculus, where hydrochloric acid and pepsin starts the process of digestion.
  • the small intestine of birds is similar to mammals There are two blind pouches or ceca, about 4-6 inches in length at the junction of the small and large intestine.
  • the large intestine is short, consisting mostly of the recmm about 3-4 inches in length.
  • the rectum empties mto the cloaca and feces are excreted through the vent.
  • Hard, biocompatible objects consumed (or otherwise introduced) and presented in the gizzard provide a useful vector for delivery of various enzymatic, chemical, therapeutic and antibiotic agents. These hard substances have a life span of a few hours to a few days and are passed after a period of time
  • the invention provides coated, impregnated (e g , impregnated matrix and membranes) modified dietary aids for deliver ' of useful digestive or therapeutic agents to an organism
  • Such dietary aids include objects which are typically ingested by an organism to assist in digestion within the gizzard (e g , rocks or grit)
  • the invention provides biocompatible objects that have coated thereon or impregnated therein agents useful as a digestive aid for an organism or for the delivery of a therapeutic or medicinal agent or chemical
  • the invention provides a dietary aid, having a biocompatible composition designed for release of an agent that assists in digestion, wherein the biocompatible composition is designed for oral consumption and release in the digestive tract (e g , the gizzard) of an organism
  • Biocompatible means that the substance, upon contact with a host organism (e g , a bird), does not elicit a detnmental response sufficient to result in the rejection of the substance or to render the substance inoperable Such inoperabihty may occur, for example, by formation of a fibrotic structure around the substance limiting diffusion of impregnated agents to the host organism therein or a substance which results in an increase in mortality or morbidity in the organism due to toxicity or infection
  • a biocompatible substance may be non-biodegradable or biodegradable
  • the biocompatible composition is resistant to degradation or digestion by the gastrointestinal tract
  • the biocompatible composition has the consistency of a rock or stone
  • a non-biodegradable material useful in the invention is one that allows attachment or impregnation of a dietary agent
  • non-biodegradable materials include, for example, thermoplastics, such as acrylic, modacryhc, polyamide, polycarbonate, polyester, polyethylene, polypropylene, polystyrene, polysulfone, polyethersulfone, and polyvinyhdene fluonde Elastomers are also useful matenals and include, for example, polyamide, polyester, polyethylene, polypropylene, polystyrene, polyurethane, polyvinyl alcohol and sihcone (e g , sihcone based or containing silica)
  • the biocompatible composition can contain a plurality of such materials, which can be, e g , admixed or layered to form blends, copolymers or combinations thereof
  • biodegradable material means that the composition will erode or degrade in vivo to form smaller chemical species Degradation may occur, for example, by enzymatic, chemical or physical processes
  • Suitable biodegradable materials contemplated for use in the invention include poly(lact ⁇ de)s, poly(glycohde)s, poly(lact ⁇ c ac ⁇ d)s, poly(glycohc ac ⁇ d)s, polyanhyd ⁇ des, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides. polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate. Such materials can be admixed or layered to form blends, copolymers or combinations thereof
  • biocompatible substances may be ingested or otherwise provided to the same organism simultaneously, or in various combinations (e.g , one material before the other).
  • the biocompatible substance may be designed for slow passage through the digestive tract.
  • large or fatty substances tend to move more slowly through the digestive tract, accordingly, a biocompatible material having a large size to prevent rapid passing in the digestive tract can be used
  • Such large substances can be a combination of non- biodegradable and biodegradable substances.
  • a small non-biodegradable substance can be encompassed by a biodegradable substance such that over a penod of time the biodegradable portion will be degraded allowing the non-biodegradable portion to pass through the digestive trace.
  • any number of flavonngs can be provided to the biocompatible substance to assist in consumption
  • any number of agents alone or in combination with other agents can be coated on the biocompatible substance including polypeptides (e g , enzymes, antibodies, cytokines or therapeutic small molecules), and antibiotics, for example. Examples of particular useful agents are listed in Table 1 and 2, below.
  • cells can be encapsulated into the biocompatible material of the invention and used to deliver the enzymes or therapeutics.
  • porous substances can be designed that have pores large enough for cells to grow in and through and that these porous materials can then be taken into the digestive tract.
  • the biocompatible substance can be compnsed of a plurality of microfloral environments (e g , different porosity, pH etc.) that provide support for a plurality of cell types.
  • the cells can be genetically engineered to deliver a particular drug, enzyme or chemical to the organism.
  • the cells can be eukaryotic or prokaryotic.
  • agents can be designed to become active or activated under certain conditions (e g , at certain pH's, in the presence of an activating agent etc.)
  • pro-enzymes can be activated by a protease (e g., a salivary protease that is present in the digestive tract or is artificially introduced into the digestive tract of an organism).
  • a protease e g., a salivary protease that is present in the digestive tract or is artificially introduced into the digestive tract of an organism.
  • the agents delivered by the biocompatible compositions of the invention can be activated or inactivated by the addition of an activating agent which may be ingested by, or otherwise delivered to, the organism.
  • Another mechanism for control of the agent in the digestive tract is an environment sensitive agent that is activated in the proper digestive compartment.
  • an agent may be inactive at low pH but active at neutral pH. Accordingly, the agent would be inactive in the gut but active in the intestinal tract. Alternatively, the agent can become active in response to the presence of a microorganism specific factor (e g , microorganisms present in the intestine)
  • a microorganism specific factor e g , microorganisms present in the intestine
  • the potential benefits of the present invention include, for example, ( 1) reduction in or possible elimination of the need for mineral supplements (e g , inorganic phosphorous supplements), enzymes, or therapeutic drugs for animal (including fish) from the daily feed or grain thereby increasing the amount of calories and nutrients present in the feed, and (2) increased health and growth of domestic and non-domestic animals including, for example, poultry, porcine, bovine, equine, canine, and feline animals.
  • enzymes can be used in the methods and compositions of the present invention. These enzymes include enzymes necessary for proper digestion of consumed foods, or for proper metabolism, activation or derivation of chemicals, prodrugs or other agents or compounds delivered to the animal via the digestive tract.
  • enzymes that can be delivered or incorporated into the compositions of the invention, include, for example, feed enhancing enzymes selected from the group consisting of ⁇ - galactosidases, ⁇ -galactosidases, in particular lactases, phytases, ⁇ -glucanases, in particular endo- ⁇ -l,4-glucanases and endo- ⁇ -l,3(4)-glucanases, cellulases, xylosidases, galactanases, in particular arabmogalactan endo-l,4- ⁇ -galactos ⁇ dases and arabinogalactan endo-l ,3- ⁇ -galactos ⁇ dases.
  • feed enhancing enzymes selected from the group consisting of ⁇ - galactosidases, ⁇ -galactosidases, in particular lactases, phytases, ⁇ -glucanases, in particular endo- ⁇ -l,4-glucanases
  • endoglucanases in particular endo-l ,2- ⁇ -glucanase, endo-1,3- u-glucanase, and endo-l ,3- ⁇ -glucanase
  • pectin degrading enzymes in particular pectinases, pectinesterases, pectin lyases, polygalacturonases, arabinanases, rhamnogalacturonases, rhamnogalacturonan acetyl esterases, rhamnogalacturonan- ⁇ - rhamnosidase.
  • pectate lyases and ⁇ -galacturomsidases, mannanases, ⁇ -mannosidases, mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases, arabinoxylanases and lipolytic enzymes such as lipases, phospholipases and cutinases.
  • SEQ ID NO' 1 and 2 and in Table 3 below are preferred.
  • the sequences desc ⁇ bed in Table 3 are SEQ ID NO 1 and 2 having the amino acid substimtions and nucleotide substimtions as described therein. TABLE 3
  • the enzymes used in the invention can be modified to enhance their activity, delivery, activation and degradation Such modifications can be performed in vivo or in vitro and use methods and processes generally known in the art as desc ⁇ bed more fully below
  • Such methodology generally uses polynucleotide or polypeptide sequences that are either synthesized by automated machines or are cloned, expressed, or manipulated by recombinant DNA techniques
  • the enzyme used in the compositions (e g., a dietary aid) of the present invention is a phytase enzyme which is stable to heat and is heat resistant and catalyzes the enzymatic hydrolysis of phytate, 1 e , the enzyme is able to renature and regain activity after a brief (1 e , 5 to 30 seconds), or longer period, for example, minutes or hours, exposure to temperamres of above 50 C C
  • a “feed” and a “food,” respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by an animal and a human being, respectively "Dietary Aid,” as used herein, denotes, for example, a composition containing agents that provide a therapeutic or digestive agent to an animal or organism
  • Dietary Aid typically is not a source of calo ⁇ c intake for an organism, in other words, a dietary aid typically is not a source of energy for the organism, but rather is a composition which is taken in addition to typical "feed” or "food”
  • An agent or enzyme e g , a phytase may exert its effect in viti o or in vivo, i e before intake or in the stomach or gizzard of the organism, respectively Also a combined action is possible
  • a dietary aid of the invention includes an enzyme (e g , a phytase)
  • a dietary aid containing a phytase composition is liquid or dry
  • Liquid compositions need not contain anything more than the enzyme (e g a phytase), preferably in a highly purified form Usually, however, a stabilizer such as glycerol. sorbitol or mono propylen glycol is also added
  • the liquid composition may also compnse other additives, such as salts, sugars, preservatives, pH-adjusting agents, proteins, phytate (a phytase substrate)
  • Typical liquid compositions are aqueous or oil- based slurries
  • the liquid compositions can be added to a biocompatible composition for slow release
  • the enzyme is added to a dietary aid composition that is a biocompatible matenal (e g , biodegradable or non-biodegradable) and includes the addition of recombinant cells into, for example, porous microbeads
  • Dry compositions may be spray dried compositions, in which case the composition need not contain anything more than the enzyme in a dry form
  • dry compositions are so-called granulates which may readily be mixed with a food or feed components, or more preferably, form a component of a pre-mix
  • the particle size of the enzyme granulates preferably is compatible with that of the other components of the mixmre This provides a safe and convenient means of incorporating enzymes into animal feed
  • the granulates are biocompatible and more preferably they biocompatible granulates are non-biodegradable
  • Agglomeration granulates coated by an enzyme can be prepared using agglomeration technique in a high shear mixer Abso ⁇ tion granulates are prepared by having cores of a carrier material to absorp/be coated by the enzyme
  • the carrier material is a biocompatible non-biodegradable material that simulates the role of stones or grit in the gizzard of an animal
  • Typical filler materials used in agglomeration techniques include salts, such as disodmm sulphate Other fillers are kaolin, talc, magnesium aluminium silicate and cellulose fibres
  • binders such as dextnns are also included in agglomeration granulates
  • the carrier materials can be any biocompatible matenal including biodegradable and non-biodegradable materials (e g , rocks, stones, ceramics, various polymers)
  • the granulates are coated with a coating mixmre Such mixmre compnses coating agents, preferably hydrophobic coating agents, such as hydrogenated
  • the dietary aid compositions may contain other substituents such as colouring agents, aroma compounds, stabilizers, vitamins, minerals, other feed or food enhancing enzymes etc
  • substituents such as colouring agents, aroma compounds, stabilizers, vitamins, minerals, other feed or food enhancing enzymes etc
  • a typical additive usually compnses one or more compounds such as vitamins, minerals or feed enhancing enzymes and suitable earners and/or excipients
  • the dietary aid compositions of the invention additionally compnses an effective amount of one or more feed enhancing enzymes, in particular feed enhancing enzymes selected from the group consisting of ⁇ -galactosidases, ⁇ - galactosidases, in particular lactases, other phytases, ⁇ -glucanases, in particular endo- ⁇ - 1,4-glucanases and endo- ⁇ -l,3(4)-glucanases.
  • feed enhancing enzymes selected from the group consisting of ⁇ -galactosidases, ⁇ - galactosidases, in particular lactases, other phytases, ⁇ -glucanases, in particular endo- ⁇ - 1,4-glucanases and endo- ⁇ -l,3(4)-glucanases.
  • cellulases xylosidases, galactanases, in particular arabinogalactan endo-l ,4- ⁇ -galactos ⁇ dases and arabinogalactan endo-l,3- ⁇ - galactosidases, endoglucanases, in particular endo-l,2- ⁇ -glucanase, endo-l,3- ⁇ -glucanase, and endo-l,3- ⁇ -glucanase, pectin degrading enzymes, in particular pectinases, pectinesterases, pectin lyases, polygalacturonases, arabinanases, rhamnogalacturonases, rhamnogalacmronan acetyl esterases, rhamnogalacturonan- ⁇ -rhamnosidase, pectate lyases, and ⁇ -galacmronisidases.
  • the animal dietary aid of the invention is supplemented to the mono-gast ⁇ c animal before or simultaneously with the diet.
  • the dietary aid of the invention is supplemented to the mono-gastric animal simultaneously with the diet.
  • the dietary aid is added to the diet in the form of a granulate or a stabilized liquid
  • An effective amount of an enzyme in a dietary aid of the invention is from about 10-20,000; preferably from about 10 to 15,000, more preferably from about 10 to 10,000, in particular from about 100 to 5,000, especially from about 100 to about 2,000 FYT/kg dietary aid
  • Examples of other specific uses of the phytase of the invention is in soy processing and in the manufacture of inositol or derivatives thereof.
  • the invention also relates to a method for reducing phytate levels in animal manure, wherein the animal is fed a dietary aid containing an effective amount of the phytase of the invention.
  • a dietary aid containing an effective amount of the phytase of the invention.
  • the dietary aid is a magnetic carrier.
  • a magnetic carrier containing an enzyme e.g , a phytase
  • a magnetic carrier e g., a porous magnetic bead
  • an enzyme e.g , a phytase
  • a porous magnetic bead e.g., a porous magnetic bead
  • Such distribution and recollection of beads reduces additional pollution and allows for reuse of the beads.
  • use of such magnetic beads in vivo allows for the localization of the dietary aid to a point in the digestive tract where, for example, phytase activity can be carried out.
  • a dietary aid of the invention containing digestive enzymes can be localized to the gizzard of the animal by juxtapositionmg a magnet next to the gizzard of the animal after the animal consumes a dietary aid of magnetic earners.
  • the magnet can be removed after a period of time allowing the dietary aid to pass through the digestive tract.
  • the magnetic carriers are suitable for removal from the organism after sacrificing or to aid in collection
  • the dietary aid is a porous particle
  • such particles are typically impregnated by a substance with which it is desired to release slowly to form a slow release particle
  • Such slow release particles may be prepared not only by impregnating the porous particles with the substance it is desired to release, but also by first dissolving the desired substance in the first dispersion phase.
  • slow release particles prepared by the method in which the substance to be released is first dissolved in the first dispersion phase are also within the scope and spirit of the invention
  • the porous hollow particles may, for example, be impregnated by a slow release substance such as a medicine, ag ⁇ cultural chemical or enzyme.
  • porous hollow particles impregnated by an enzyme are made of a biodegradable polymers
  • the particles themselves may be used as an ag ⁇ cultural chemical or fertilizer, and they have no adverse effect on the environment.
  • the porous particles are magnetic in namre.
  • the porous hollow particles may be used as a bioreactor support, in particular an enzyme support Therefore, it is advantageous to prepare the dietary aid utilizing a method of a slow release, for instance by encapsulating the enzyme of agent in a microvesicle, such as a hposome, from which the dose is released over the course of several days, preferably between about 3 to 20 days.
  • the agent e g , an enzyme
  • the agent can be formulated for slow release, such as incorporation into a slow release polymer from which the dosage of agent (e g , enzyme) is slowly released over the course of several days, for example from 2 to 30 days and can range up to the life of the animal
  • hposomes are generally derived from phosphohpids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium Any non-toxic, physiologically acceptable and metabohzable lipid capable of forming liposomes can be used.
  • the present compositions in hposome form can contain stabilizers, preservatives, excipients, and the like in addition to the agent
  • the preferred lipids are the phosphohpids and the phosphatidyl chohnes (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. ( 1976), p. 33 et seq.
  • a phytase of the invention during the preparation of food or feed preparations or additives, i e , the phytase excerts its phytase activity during the manufacture only and is not active in the final food or feed product.
  • This aspect is relevant for instance in dough making and baking. Accordingly, phytase or recombinant yeast expressing phytase can be impregnated in, on or through a magnetic earners, distributed in the dough or food medium, and ret ⁇ eved by magnets.
  • the dietary aid of the invention may be administered alone to animals in an biocompatible (e.g , a biodegradable or non-biodegradable) earner or in combination with other digestion additive agents.
  • the dietary aid of the invention thereof can be readily administered as a top dressing or by mixing them directly into animal feed or provided separate from the feed, by separate oral dosage, by injection or by transdermal means or in combination with other growth related edible compounds, the proportions of each of the compounds in the combination being dependent upon the particular organism or problem being addressed and the degree of response desired.
  • forms of the dietary aid can be prepared by combining them with non-toxic pharmaceutically acceptable edible earners to make either immediate release or slow release formulations, as is well known in the art
  • edible carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol
  • a solid earner is used the dosage form of the compounds may be tablets, capsules, powders, troches or lozenges or top dressing as micro-dispersable forms.
  • a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form.
  • the dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeuticaUy valuable substances.
  • a significant advantages of the invention include for example, 1) ease of manufacture of the active ingredient loaded biocompatible compositions; 2) versatility as it relates to the class of polymers and/or active ingredients which may be utilized; 3) higher yields and loading efficiencies; and 4) the provision of sustained release formulations that release active, intact active agents in vivo, thus providing for controlled release of an active agent over an extended penod of time.
  • another advantage is due to the local delivery of the agent with in the digestive tract (e g., the gizzard) of the organism.
  • the phrase "contained within” denotes a method for formulating an agent into a composition useful for controlled release, over an extended period of time of the agent.
  • sustained release or slow release refers to the gradual release of an agent from a biocompatible material, over an extended period of time.
  • the sustained release can be continuous or discontinuous, linear or non-linear, and this can be accomplished using one or more biodegradable or non-biodegradable compositions, drug loadings, selection of excipients, or other modifications.
  • the slow release encompasses slow activation or continual activation of an agent present on the biocompatible composition
  • a phytase need not be released from the biocompatible composition to be effective.
  • the phytase is immobilized on the biocompatible composition.
  • the animal feed may be any protein-containing organic meal normally employed to meet the dietary requirements of animals.
  • Many of such protein-containing meals are typically pnma ⁇ ly composed of com, soybean meal or a corn soybean meal mix.
  • typical commercially available products fed to fowl include Egg Maker Complete, a poultry feed product of Land O'Lakes AG Services, as well as Country Game & Turkey Grower a product of Agwa, Inc.
  • the present invention is applicable to the diet of numerous animals, which herein is defined as including mammals (including humans), fowl and fish.
  • the diet may be employed with commercially significant mammals such as pigs, cattle, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), fur-bearing animals such as mink and fox, and zoo animals such as monkeys and apes, as well as domestic mammals such as cats and dogs
  • Typical commercially significant avian species include chickens, turkeys, ducks, geese, pheasants, emu, ostrich, loons, kiwi, doves, parrots, cockatiel, cockatoo, canaries, penguins, flamingoes, and quail.
  • fish such as trout would also benefit from the dietary aids disclosed herein.
  • Other fish that can benefit include, for example, fish (especially in an aquarium or acquaculture environment, e.g , tropical fish), goldfish and other ornamental ca ⁇ , catfish, trout, salmon, shark, ray, flounder, sole, tilapia, medaka, guppy, molly, platyfish, swordtail, zebrafish, and loach.
  • E cob B genomic DNA was obtained from Sigma (Catalog # D-2001), St Louis, New Jersey
  • PCR product was purified and punfied product and pQE60 vector (Qiagen) were both digested with EcoRI and Bglll restriction endonucleases (New England Biolabs) according to manufacmrers protocols Overnight ligations were performed using standard protocols to yield pQE60
  • the amplified sequences were inserted in frame with the sequence encoding for the RBS
  • the ligation mixmre was then used to transform the E cob strain M15/pREP4 (Qiagen, Ine ) by electroporation M15/pREP4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan 1 )
  • Plasmid DNA was isolated and confirmed by rest ⁇ ction analysis
  • Clones containing the desired constructs were grown overnight (O/N) in liquid culmre in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml)
  • the O/N culmre was used to inoculate a large culmre at a ratio of 1 100 to 1 250
  • the cells were grown to an optical density 600 (O D 600 ) of between 0 4 and 0 6 IPTG ("Isopropyl-B-D-thi
  • primer sequences set out above may also be employed to isolate the target gene from the deposited material by hybndization techniques described above
  • This invention also provides for the isolation and use of phytase molecules (nucleic acids and phytase enzymes encoded thereby) from all other strains of E coli (whether vimlent or non-virulent, including K12, W, C), as well as all bacteria These include all known species and strains belonging to
  • Pu ⁇ le bacteria including the following subdivisions Delta & Epsilon, including
  • Desulfuromonas acetoxidans Desulfosarcina va ⁇ abihs
  • Hehcobacter pylori Alpha including
  • Rhodospi ⁇ llum rubrum Gamma including
  • Beta including Eikenella corrodens
  • Such phytase molecules can be isolated from these bacteria by know methods, including library screening methods, e.g. expression screening, hybridization methods, PCR (e.g see Sammbrook, 1989).
  • Cosgrove DJ Inositol phosphate phosphatases of microbiological origin. Inositol phosphate intermediates in the dephosphorylation of the hexaphosphates of myo- inositol, scyllo-inositol, and D-chiro-inositol by a bacterial (Pseudomonas sp.) phytase Aust JBwl Sci 23(6): 1207-1220, 1970.
  • Gluzman Y S V40-transformed simian cells support the replication of early SV40 mutants Cell 23(1) 175-182, 1981
  • Prade RA Xylanases: from biology to biotechnology. Biotechnol Genet Eng Rev; 13: 101- 31, 1996.
  • Tague BW, Dickinson CD, Chrispeels MJ A short domain of the plant vacuolar protein phytohemagglutimn targets invertase to the yeast vacuole.
  • Vasil IK Vasil V: Totipotency and embryogenesis in plant cell and tissue culmres. In Vitro 8(3) 1 17-27, (Nov-Dec) 1972.
  • Vasil V Vasil IK- Regeneration of tobacco and pemnia plants from protoplasts and culmre of corn protoplasts. In Vitro 10:83-96, (Jul-Aug) 1974.
  • Yamada K, et al. Agricultural and Biological Chemistry 32.1275-1282, 1968.
  • Hooykaas RJJ A process of the inco ⁇ oration of foreign DNA into the genome of dicotyledonous plants; Agrobacte ⁇ um tumefaciens bacteria and a process for the production thereof, plants and plant cells with modified genetic properties; a process for the preparation

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Abstract

A purified recombinant phytase enzyme derived from Escherichia coli B. The enzyme has a molecular weight of about 47.1 kilodaltons and has phytase activity. The enzyme can be produced from native or recombinant host cells and can be used to aid in the digestion of phytate where desired. In particular, the phytase of the present invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients.

Description

RECOMBINANT BACTERIAL PHYTASES AND USES THEREOF
1. FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention have been identified as phytases and in particular, microbial enzymes having phytase activity
2. BACKGROUND
2.1.1 - Brief Summary: Minerals are essential elements for the growth of all organisms. Dietary minerals can be derived from many source materials, including plants E.g , plant seeds are a rich source of minerals since they contain ions that are complexed with the phosphate groups of phytic acid molecules. These phytate-associated minerals satisfy the dietary needs of some species of farmed organisms, such as multi-stomached ruminants. Accordingly, ruminants do not require dietary supplementation with inorganic phosphate and minerals because microorganisms in the rumen produce enzymes that catalyze conversion of phytate (myo-inositol-hexaphosphate) to inositol and inorganic phosphate. In the process, minerals that have been complexed with phytate are released. The majority of species of farmed organisms, however, are unable to efficiently utilize phytate-associated minerals. Thus, for example, in the livestock production of monogastπc animals (e g , pigs, birds, and fish), feed is commonly supplemented with minerals &/or with antibiotic substances that alter the digestive flora environment of the consuming organism to enhance growth rates.
As such, there are many problematic burdens - related to nutrition, ex vivo processing steps, health and medicine, environmental conservation, and resource management - that are associated with an insufficient hydrolysis of phytate in many applications. The following are non-limiting examples of these problems
1) The supplementation of diets with inorganic minerals is a costly expense 2) The presence of unhydrolyzed phytate is undesirable and problematic in many ex vivo applications (e g by causing the presence of unwanted sludge)
3) The supplementation of diets with antibiotics poses a medical threat to humans and animals alike by increasing the abundance of antibiotic-tolerant pathogens
4) The discharge of unabsorbed fecal minerals into the environment disrupts and damages the ecosystems of surrounding soils, fish farm waters, and surface waters at large
5) The valuable nutritional offerings of many potential foodstuffs remain significantly untapped and squandered
2.1.2 - Nutritional Concerns: Many potentially nutritious plants, including particularly their seeds, contain appreciable amounts of nutrients, e g phosphate, that are associated with phytate in a manner such that these nutrients are not freely available upon consumption The unavailability of these nutrients is overcome by some organisms, including cows and other ruminants, that have a sufficient digestive ability - largely denved from the presence of symbiotic life forms in their digestive tracts - to hydrolyze phytate and liberate the associated nutπents However, the majority of species of farmed animals, including pigs, fish, chickens, turkeys, as well as other non-ruminant organisms including man, are unable to efficiently liberate these nutrients after ingestion
Consequently, phytate-contaimng foodstuffs require supplementation with exogenous nutrients and/or with a source of phytase activity in order to ammend their deficient nutritional offerings upon consumption by a very large number of species of organisms
2.1.3 - Ex vivo Processing Concerns: In yet another aspect, the presence of unhydrohzed phytate leads to problematic consequences in ex vivo processes including but not limited to - the processing of foodstuffs In but merely one exemplification, as described in EP0321004-B 1 (Vaara et al ), there is a step in the processing of corn and sorghum kernels whereby the hard kernels are steeped in water to soften them Water- soluble subtances that leach out during this process become part of a corn steep liquor, which is concentrated by evaporation Unhydrohzed phytic acid in the corn steep liquor, largely in the form of calcium and magnesium salts, is associated with phosphorus and deposits an undesirable sludge with proteins and metal ions This sludge is problematic in the evaporation, transportation and storage of the corn steep liquor. Accordingly, the instantly disclosed phytase molecules - either alone or in combination with other reagents (including but not limited to enzymes, including proteases) - are serviceable not only in this application (e g , for prevention of the unwanted slugde) but also in other applications where phytate hydrolysis is desirable
2.1.4 - Medical Concerns: The supplementation of diets with antibiotic substances has many beneficial results in livestock production. For example, in addition to its role as a prophylactic means to ward off disease, the administration of exogenous antibiotics has been shown to increase growth rates by upwards of 3-5%. The mechanism of this action may also involve - in part - an alteration in the digestive flora environment of farmed animals, resulting in a microfloral balance that is more optimal for nutπent absoφtion.
However, a significant negative effect associated with the overuse of antibiotics is the danger of creating a repository of pathogenic antibiotic-resistant microbial strains This danger is imminent, and the rise of drug-resistant pathogens in humans has already been linked to the use of antibiotics in livestock For example, Avoparcin, the antibiotic used in animal feeds, was banned in many places in 1997, and animals are now being given another antibiotic, virginiamycin, which is very similar to the new drug, Synercid, used to replace vancomycin in human beings. However, studies have already shown that some enterococci in farm animals are resistant to Synercid. Consequently, undesired tolerance consequences, such as those already seen with Avoparcin and vancomycin, are likely to reoccur no matter what new antibiotics are used as blanket prophylactics for farmed animals. Accordingly, researchers are calling for tighter controls on drug use in the industry The increases in growth rates achieved in animals raised on foodstuffs supplemented with the instantly disclosed phytase molecules matches - if not exceeds - those achieved using antibiotics such as, for example. Avoparcin Accordingly, the instantly disclosed phytase molecules - either alone or in combination with other reagents (including but not limited to enzymes, including proteases) - are serviceable not only in this application (e g , for increasing the growth rate of farmed animals) but also in other applications where phytate hydrolysis is desirable
2.1.5 - Environmental Concerns: An environmental consequence is that the consumption of phytate-containing foodstuffs by any organism species that is phytase- deficient - regardless of whether the foodstuffs are supplemented with minerals - leads to fecal pollution resulting from the excretion of unabsorbed minerals This pollution has a negative impact not only on the immediate habitat but consequently also on the surrounding waters The environmental alterations occur primarily at the bottom of the food chain, and therefore have the potential to permeate upwards and throughout an ecosystem to effect permanent and catastrophic damage - particularly after years of continual pollution This problem has the potential to manifest itself in any area where concentrated phytate processing occurs - including in vivo (e g by animals in areas of livestock production, zoological grounds, wildlife refuges, etc ) and in vitro (e g in commercial corn wet milling, ceral steeping processes, etc ) processing steps
2.1.6 - Financial Concerns: The decision to use exogenously added phytase molecules - whether to fully replace or to augment the use of exogenously administered minerals &/or antibiotics - ultimately needs to pass a test of financial feasibility & cost effectiveness by the user whose livelihood depends on the relevant application, such as livestock production
Consequently, there is a need for means to achieve efficient and cost effective hydrolysis of phytate in various applications Particularly, there is a need for means to optimize the hyrolysis of phytate m commercial applications In a particular aspect, there is a need to optimize commercial treatment methods that improve the nutritional offerings of phytate-containmg foodstuffs for consumption by humans and farmed animals
Previous reports of recomoinant phytases are available, but their infeπor activities are eclipsed by the newly discovered phytase molecules of instant invention Accordingly, the instantly disclosed phytase molecules are counted upon to provide substantially superior commercial performance than previously identified phytase molecules, e g phytase molecules of fungal origin
2.2 - General Overview of Phytate & Phytate Hydrolysis
2.2.1 - Phytate Hydrolysis Leads to Release of Nutrients: Phytate occurs as a source of stored phosphorous in virtually all plant feeds (Graf (Ed ), 1986) Phytic acid forms a normal part of the seed in cereals and legumes It functions to bind dietary minerals that are essential to the new plant as it emerges from the seed When the phosphate groups of phytic acid are removed by the seed enzyme phytase, the ability to bind metal ions is lost and the minerals become available to the plant In livestock feed grains, the trace minerals bound by phytic acid are largely unavailable for absoφtion by monogastπc animals, which lack phytase activity
Although some hydrolysis of phytate occurs in the colon, most phytate passes through the gastrointestinal tract of monogastπc animals and is excreted in the manure contributing to fecal phosphate pollution problems in areas of intense livestock production Inorganic phosphorous released in the colon has an appreciably diminished nutπtional value to livestock because inorganic phosphorous is absorbed mostly - if not virtually exclusively - in the small intestine Thus, an appreciable amount of the nutπtionally important dietary minerals in phytate is unavailable to monogastπc animals
In sum, phytate-associated nutrients are comprised of not only phosphate that is covalently linked to phytate, but also other minerals that are chelated by phytate as well Moreover, upon injestion, unhydrolyzed phytate may further encounter and become associated with additional minerals. The chelation of minerals may inhibit the activity of enzymes for which these minerals serve as co-factors.
2.2.2 - Microbial Enzymes Can Hydrolyze Phytate: Conversion of phytate to inositol and inorganic phosphorous can be catalyzed by microbial enzymes refeπed to broadly as phytases. Phytases such as phytase #EC 3.1.3.8 are capable of catalyzing the hydrolysis of myo-mositol hexaphosphate to D-myo-mositol 1, 2,4,5, 6-pentaphosphate and orthophosphate. Certain fungal phytases reportedly hydrolyze inositol pentaphosphate to tetra-, tπ-, and lower phosphates. E.g., A. ficuum phytases reportedly produce mixtures of myoinositol di- and mono-phosphates (Ullah, 1988). Phytase-producing microorganisms are comprised of bacteria such as Bacillus subtilis (Powar and Jagannathan, 1982) and Pseudomonas (Cosgrove, 1970); yeasts such as Sacchoromyces cerevisiae (Nayini and Markakis, 1984); and fungi such as Aspergillus terreus (Yamada et al, 1968).
Acid phosphatases are enzymes that catalytically hydrolyze a wide variety of phosphate esters and usually exhibit pH optima below 6.0 (Igarashi & Hollander, 1968). E.g., #EC 3.1.3.2 enzymes catalyze the hydrolysis of orthophosphoπc monoesters to orthophosphate products. An acid phosphatase has reportedly been purified from A. ficuum. The deglycosylated form of the acid phosphatase has an apparent molecular weight of 32.6 kDa (Ullah et al . 1987)
Phytase and less specific acid phosphatases are produced by the fungus Aspergillus ficuum as extracellular enzymes (Shieh et al, 1969). Ullah reportedly purified a phytase from wild-type A. ficuum that had an apparent molecular weight of 61.7 kDA (on SDS- PAGE: as coπected for glycosylation); pH optima at pH 2.5 and pH 5.5; a Km of about 40μm; and, a specific activity of about 50 U/mg (Ullah, 1988). PCT patent application WO 91/05053 also reportedly discloses isolation and molecular cloning of a phytase from Aspergillus ficuum with pH optima at pH 2.5 and pH 5.5, a Km of about 250 μm, and specific activity of about 100 U/mg protein. Summarily, the specific activity cited for these previously repoπed microbial enzymes has been approximately in the range of 50-100 U/mg protein. In contrast, the phytase activity disclosed in the instant invention has been measured to be approximately 4400 U/mg. This coπesponds to about a 40-fold or better improvement in activity
2.3 - Solving the Problem of Insufficient Phytate Hydrolysis
2.3.1 - Enzyme Additives in Commercial Applications: The possibility of using microbes capable of producing phytase as a feed additive for monogastnc animals has been reported previously (USPN 3,297,548 Shieh and Ware, Nelson et al , 1971) The cost-effectiveness of this approach has been a major limitation for this and other commercial applications. Therefore improved phytase molecules are highly desirable
Microbial phytases may also reportedly be useful for producing animal feed from certain industrial processes, e g., wheat and corn waste products In one aspect, the wet milling process of corn produces glutens sold as animal feeds. The addition of phytase may reportedly improve the nutritional value of the feed product. For example, the use of fungal phytase enzymes and process conditions (t~50°C and pH -5.5) have been reported previously in (e g EP 0 321 004) Bπefly, in processing soybean meal using traditional steeping methods, i e , methods without the addition of exogenous phytase enzyme, the presence of unhydrolyzed phytate repoπedly renders the meal and wastes unsuitable for feeds used in rearing fish, poultry and other non-ruminants as well as calves fed on milk. Phytase is reportedly useful for improving the nutnent and commercial value of this high protein soy material (see Finase Enzymes by Alko, Rajamaki, Finland). A combination of fungal phytase and a pH 2.5 optimum acid phosphatase form A mger has been used by Alko, Ltd as an animal feed supplement in their phytic acid degradative product Finas F and Finase S However, the cost-effectiveness of this approach has remained a major limitation to more widespread use. Thus a cost-effective source of phytase would greatly enhance the value of soybean meals as an animal feed (Shieh et al , 1969) 2.3.2 - Optimization ot Enzyme Additives Is Needed: To solve the problems disclosed, the treatment of foodstuffs with exogenous phytase enzymes has been proposed, but this approach was not been fully optimized, particularly with respect to feasibility and cost efficiency This optimization requires the consideration that a wide range of applications exists, particularly for large scale production For example, there is a wide range of foodstuffs, preparation methods thereof, and species of recipient organisms
In a particular exemplification, it is appreciated that the manufacture of fish feed pellets requires exposure of ingedients to high temperatures &/or pressure in order to produce pellets that do not dissolve &/or degrade prematurely (e g e g pnor to consumption) upon subjection to water It would thus be desirable for this manufacturing process to obtain additive enzymes that are stable under high temperature and/or pressure conditions Accordingly it is appreciated that distinct phytases may be differentially preferable or optimal for distinct applications
It is furthermore recognized that an important way to optimize an enzymatic process is through the modification and improvement of the pivotal catalytic enzyme For example, a transgenic plant can be formed that is comprised of an expression system for expressing a phytase molecule It is appreciated that by attempting to improve factors that are not directly related to the activity of the expressed molecule proper, such as the expression level, only a finite - and potentially insufficient - level of optimization may be maximally achieved Accordingly, there is also a need for obtaining molecules with improved characteristics
A particular way to achieve improvements in the characteristics of a molecule is through a technological approach termed directed evolution, including Diversa Coφoratιon"s proprietary approaches for which the term DirectEvolution® has been coined and registered These approaches are further elaborated in Diversa's co-owned patent (US 5,830,696) as well as in several co-pending patent applications In bnef, DirectEvolution® comprises a) the subjection of one or more molecular template to mutagenesis to generate novel molecules, and b) the selection among these progeny species of novel molecules with more desirable characteristics
However, the power of directed evolution depends on the starting choice of starting templates, as well as on the mutagenesis process(es) chosen and the screening process(es) used For example, the approach of generating and evaluating a full range of mutagenic permutations on randomly chosen molecular templates &/or on initial molecular templates having overly suboptimal properties is often a forbiddingly large task The use of such templates offers, at best, a circuitously suboptimal path and potentially provides very poor prospects of yielding sufficiently improved progeny molecules Additionally, it is appreciated that our cuπent body of knowledge is very limited with respect to the ability to rigorously predict beneficial modifications
Consequently, it is a desirable approach to discover and to make use of molecules that have pre-evolved properties - preferably pre-evolved enzymatic advantages - in nature It is thus appreciated in the instant disclosure that nature provides (through what has sometimes been termed "natural evolution") molecules that can be used immediately in commercial applications, or that alternatively, can be subjected to directed evolution to achieve even greater improvements
In sum, there is a need for novel, highly active, physiologically effective, and economical sources of phytase activity Specifically, there is a need to identify novel phytases that a) have superior activities under one or more specific applications, and are thus serviceable for optimizing these specific applications, b) are serviceable as templates for directed evolution to achieve even further improved novel molecules, and c) are serviceable as tools for the identification of additional related molecules by means such as hybndization-based approaches This invention meets these needs in a novel way 3. SUMMARY OF THE INVENTION
The present invention provides a polynucleotide and a polypeptide encoded thereby which has been identified as a phytase enzyme having phytase activity In accordance with one aspect of the present invention, there is provided a novel recombinant enzyme, as well as active fragments, analogs and derivatives thereof
More particularly, this invention relates to the use of recombinant phytase molecules of bacterial origin that are serviceable for improving the nutntional value of phytate-containing foodsmffs Previous publications have disclosed the use of fungal phytases. but the use of bacterial phyatases for this puφose is novel
More particularly still, this invention relates to the use of newly identified recombinant phytase molecules of E coli origin that are serviceable for improving the nutritional value of phytate-containing foodstuffs
This use is comprised of employing the newly identified molecules to hydrolyze phytate m foodstuffs Hydrolysis may occur before injestion or after injestion or both before and after injestion of the phytate This application is particularly relevant, but not limited, to non-ruminant organisms and includes the expression of the disclosed novel phytase molecules in transformed hosts, the contacting of the disclosed novel phytase molecules with phytate in foodstuffs and other materials, and the treatment of animal digestive systems with the disclosed novel phytase molecules
Additionally, hydrolysis may occur mdependently of consumption, e g in an in vitro application, such as in a reaction vessel Thus, the treatment of phytate-containing materials includes the treatment of a wide range of mateπals, including ones that are not intended to be foodsmffs, e g the treatment of excrementary (or fecal) material
Prefeπed molecules of the present invention include a recombinant phytase isolated from Eschenchia coli B that improves the efficiency of release of phosphorous from phytate and the salts of phytic acid when compared to previosuly identified fungal phytases
In accordance with one aspect of the present invention, there is provided a phytase enzyme that is serviceable for incoφortion into foodstuffs More specifically, there is provided a phytase enzyme that is serviceable for improving the nutritional value of phytate-containing foodstuffs More specifically still, there is provided a phytase enzyme that, when applied to phytate-containing foodstuffs, measurably improves the growth performance of an organism that consumes it It is theorized that the beneficial mechanism of action of the phytase activity is compnsed appreciably if not substantially of the hydrolysis of phytate It is provided that the beneficial action may occur before injestion or alternatively after injestion or alternatively both before and after injestion of the phytate-containing foodstuff In the case where the beneficial action occurs after injestion, it is an object of the present invention to provide a phytase enzyme that has activity that is retained upon consumption by non-ruminant organisms
In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding the enzyme of the present invention - including mRNA, DNA, cDNA, genomic DNA - as well as active derivatives, analogs and fragments of such enzyme
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising cultuπng recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding an enzyme of the present invention, under conditions promoting expression of said enzyme and subsequent recovery of said enzyme
In accordance with yet a further aspect of the present invention, there is provided a process for expressing such enzymes, or polynucleotides encoding such enzymes in transgenic plants or plant organs and methods for the production of such plants This is achievable by introducing into a plant an expression construct comprised of a nucleic acid sequence encoding such phytase enzymes
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes for use in commercial processes, such as, for example, processes that liberate minerals from phytates in plant materials either in vitro, . e , m feed treatment processes, or in vivo, i e , by administering the enzymes to animals
In accordance with yet a further aspect of the present invention, there are provided foodstuffs made by the disclosed feed treatment processes
In accordance with yet a further aspect of the present invention, there are provided a processes for utilizing such enzymes, or polynucleotides encoding such enzymes, for in \ιtro puφoses related to research, discovery, and development In a non-limiting exemplification, such processes compnse the generation of probes for identifying and isolating similar sequences which might encode similar enzymes from other organisms
In a particular non-limiting examplification. there are also provided processes for generating nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention By way of prefeπed exemplification, hybridization-based uses of these probes include, but are by no means limited to, PCR, Northern and Southern types of hybridizations, RNA protection assays, and in situ types of hybridizations The uses of the instantly disclosed molecules further include, in a non-limiting manner, diagnostic applications
In accordance with a non-limiting exemplification, these processes compnse the generation of antibodies to the disclosed molecules, and uses of such antibodies, including, for example, for the identification and isolation of similar sequences in enzymes from other organisms In another non-hmitmg examplification, these processes include the use of the present enzymes as templates for directed evolution, comprising the generation of novel molecules by followed by screening-based approaches for discoverying of progeny molecules with improved properties
Also provided is a transgenic non-human organism whose genome compnses a heterologous nucleic acid sequence encoding a polypeptide having phytase activity, wherein said transgene results in expression of a phytase polypeptide.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
4. BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1A and B show the nucleotide and deduced amino acid sequences the enzyme of the present invention. Sequencing was performed using a 378 automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 shows the pH and temperature profile and stability data for the phytase enzyme of the present invention. The assay used for these analysis is the following for the detection of phytase activity Phytase activity is measured by incubating 150μl of the enzyme preparation with 600μl of 2 mM sodium phytate in 100 mM Tris HCl buffer pH 7.5, supplemented with ImM CaCl2 for 30 minutes at 37°C. After incubation the reaction is stopped by adding 750μl of 5% tπchloroacetic acid. Phosphate released was measured against phosphate standard spectrophotometπcally at 700nm after adding 1500μl of the color reagent (4 volumes of 1.5% ammonium molybdate in 5.5% sulfuπc acid and 1 volume of 2.7%o ferrous sulfate, Shimizu, 1992) OD at 700nm is indicated on the Y-axis of the graphs in Figure 2. Temperature or pH is indicated on the X-axis of the graphs 5. DEFINITIONS OF TERMS
In order to facilitate understanding of the examples provided herein, certain frequently occumng methods and/or terms will be described. In addition, the headings and sub-headings used herein are provided for the convenience of the reader and are not to be construed to limit the invention in any manner whatsoever.
The term "antibody," as used herein, refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that are capable of binding to an epitope of a phytase polypeptide. These antibody fragments, which retain some ability to selectively bind to the antigen (e g., an phytase antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g , Harlow and Lane, supra), and are descnbed further, as follows.
(1) An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain
(2) An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
(3) An (Fab')2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds (4) An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains
(5) An single chain antibody ("SCA") is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker
The term "degrading effective" amount refers to the amount of enzyme which is required to degrade at least 50% of the phytate, as compared to phytate not contacted with the enzyme. Preferably, at least 80% of the phytate is degraded
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical puφoses, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the puφose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a gel to isolate the desired fragment.
As used in this invention, the term "epitope" refers to an antigemc determinant on an antigen, such as a phytase polypeptide, to which the paratope of an antibody, such as an phytase-specific antibody, binds Antigemc determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics The terms "fragment", "derivative" and "analog" when refernng to the enzyme of Figure 1 comprise a enzyme which retains at least one biological function or activity that is at least essentially same as that of the reference enzyme Furthermore, the terms "fragment", "derivative" or "analog" are exemplified by a "pro-form" molecule, such as a low activity proprotem that can be modified by cleavage to produce a mamre enzyme with significantly higher activity
The term "gene" means the segment of DNA involved in producing a polypeptide chain, it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons)
The term "isolated" means that the material is removed from its original environment (e g , the natural environment if it is naturally occumng) For example, a naturally-occuπing polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment
By "isolated nucleic acid" is meant a nucleic acid, e g , a DNA or RNA molecule, that is not immediately contiguous with the 5' and 3' flanking sequences with which it normally is immediately contiguous when present in the naturally occumng genome of the organism from which it is derived The term thus describes, for example, a nucleic acid that is incoφorated into a vector, such as a plasmid or viral vector, a nucleic acid that is incoφorated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that at which it naturally occurs), and a nucleic acid that exists as a separate molecule, e g , a DNA fragment produced by PCR amplification or restnction enzyme digestion, or an RNA molecule produced by in vitro transcription The term also describes a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion protein "Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Sambrook et al, 1989). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.
As used herein, a "nucleic acid molecule" is comprised of at least one nucleotide base or one nucleotide base pair, depending on whether it is single-stranded or double- stranded, respectively. Furthermore, a nucleic acid molecule may belong exclusively or chimencally to any group of nucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally occumng and not naturally occumng nucleic acids, and synthetic nucleic acids. This includes, by way of non-limitmg example, nucleic acids associated with any organelle, such as the mitochondria, nbosomal RNA, and nucleic acid molecules comprised chimencally of one or more components that are not naturally occurring along with naturally occumng components.
Additionally, a "nucleic acid molecule" may contain in part one or more non- nucleotide-based components as exemplified by, but not limited to, amino acids and sugars Thus, by way of example, but not limitation, a ribozyme that is in part nucleotide- based and in part protein-based is considered a "nucleic acid molecule".
In addition, by way of example, but not limitation, a nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive or alternatively a non-radioactive label, is likewise considered a "nucleic acid molecule".
The terms "nucleic acid sequence coding for" or a "DNA coding sequence of or a "nucleotide sequence encoding" a particular enzyme - as well as other synonymous terms - refer to a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences A "promotor sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcnption of a downstream (3' direction) coding sequence The promoter is part of the DNA sequence This sequence region has a start codon at its 3 ' terminus The promoter sequence does include the minimum number of bases where elements necessary to initiate transcnption at levels detectable above background bind However, after the RNA polymerase binds the sequence and transcription is initiated at the start codon (3' terminus with a promoter), transcription proceeds downstream in the 3' direction Within the promotor sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S I) as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
The terms "nucleic acid encoding an enzyme (protein)" or "DNA encoding an enzyme (protein)" or "polynucleotide encoding an enzyme (protein)" and other synonymous terms encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non- coding sequence
In one prefeπed embodiment, a "specific nucleic acid molecule species" is defined by its chemical structure, as exemplified by, but not limited to, its primary sequence In another prefeπed embodiment, a specific "nucleic acid molecule species" is defined by a function of the nucleic acid species or by a function of a product derived from the nucleic acid species Thus, by way of non-limiting example, a "specific nucleic acid molecule species" may be defined by one or more activities or properties attributable to it, including activities or properties attributable its expressed product
The instant definition of "assembling a working nucleic acid sample into a nucleic acid library" includes the process of incoφorating a nucleic acid sample into a vector- based collection, such as by ligation into a vector and transformation of a host A description of relevant vectors, hosts, and other reagents as well as specific non-limiting examples thereof are provided hereinafter The instant definition of "assembling a working nucleic acid sample into a nucleic acid library" also includes the process of incoφorating a nucleic acid sample into a non-vector-based collection, such as by ligation to adaptors. Preferably the adaptors can anneal to PCR pnmers to facilitate amplification by PCR.
Accordingly, in a non-limiting embodiment, a "nucleic acid library" is compnsed of a vector-based collection of one or more nucleic acid molecules In another prefeπed embodiment a "nucleic acid library" is comprised of a non-vector-based collection of nucleic acid molecules. In yet another prefeπed embodiment a "nucleic acid library" is compnsed of a combined collection of nucleic acid molecules that is in part vector-based and in part non-vector-based. Preferably, the collection of molecules comprising a library is searchable and separable according to individual nucleic acid molecule species
The present invention provides a "nucleic acid construct" or alternatively a "nucleotide construct" or alternatively a "DNA construct". The term "construct" is used herein to describe a molecule, such as a polynucleotide (e g., a phytase polynucleotide) may optionally be chemically bonded to one or more additional molecular moieties, such as a vector, or parts of a vector. In a specific - but by no means limiting - aspect, a nucleotide construct is exemplified by a DNA expression DNA expression constructs suitable for the transformation of a host cell.
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those that do not will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
A coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein The term "phytase-specific probe", in the context of this method of invention, refers to probes that bind to nucleic acids encoding phytase polypeptides, or to complementary sequences thereof, to a detectably greater extent than to nucleic acids encoding other enzymes, or to complementary sequences thereof
In a strict sense, the terms "phytate", "phytic acid", and "phytin", may be differentiated as folllows "phytate" refers to an anionic form of phytic acid, "phytic acid" refers to inositol hexaphosphate, a compound that occurs naturally in plants, including particularly plant leaves, and that may serve as a substrate for the enzyme phytase, and
"phytin" refers to a salt of phytic acid, such as a calcium-magnesium salt of phytic acid It is understood, accordingly, that "phytate", "phytic acid", and "phytin" are chemically related and interconvertible forms having a shared chemical structure As used herein, therefore, "phytate", "phytic acid", and "phytin" are interchangeable terms in as much as they are highly related, similar, chemically interconvertible, and may all (either with or without said chemical interconversion) be subject to degredation by the novel phytase enzyme disclosed instantly Accordingly, where only one of the terms "phytate", "phytic acid", or "phytin" is used in the descnptions of the methods disclosed herein, it is understood to function as a representative term that further refers to any substrate of the enzyme phytase including "phytase", "phytic acid", and "phytin"
A "polynucleotide" is a molecule composed of 2 or more nucleotide bases or nucleotide base pairs
A molecule having a "pre-form" or a "pro-form" refers to a molecule that undergoes any combination of one or more covalent and noncovalent chemical modifications (e g glycosylation, proteolytic cleavage, dimeπzation or ohgomenzation, temperature-induced or pH-induced conformational change, association with a co-factor, etc ) en route to attain a more mamre molecular form having a property difference (e g an increase in activity) in comparison with the reference pro-form molecule When a precursor molecule in "pre-form" or in "pro-form" is able to undergo two or more chemical modification (e g two proteolytic cleavages, or a proteolytic cleavage and a change m glycosylation) en route to the production of a mature molecule, the term "pre- pro-form" may also be used in reference to the precursor molecule Accordingly, a pre- pro-enzyme is an enzyme in "pre -pro-form" Likewise, a pre-pro hormone is a hormone in "pre-pro-form".
As used herein, the term "reagent" includes phytase molecules of the instant invention. Preferably, such phytase molecules catalyze the hydrolysis of phytate to inositol and free phosphate with release of minerals from the phytic acid complex An exemplary phytase molecule is a phytase derived from Escherichia coli B This exemplary enzyme is shown in Figure 1. SEQ ID NO 2. Additionally, as used herein, the term "reagent" includes substrate reagents molecules of the instant invention, such as phytate molecules. Preferably, such phytate molecules are found in foodsmffs, potential foodstuffs, byproducts of foodsmffs (both in vitro byproducts and in vivo byproducts, e g ex vivo reaction products and animal excremental products), precursors of foodstuffs, and any other source of phytate.
"Recombinant" enzymes refer to enzymes produced by recombinant DNA techniques, i e , produced from cells transformed by an exogenous DNA construct encoding the desired enzyme "Synthetic" enzymes are those prepared by chemical synthesis
As known in the art "similarity" between two enzymes is determined by companng the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information)
The members of a pair of molecules (e g., an antibody-antigen pair or a nucleic acid pair) are said to "specifically bind" to each other if they bind to each other with greater affinity than to other, non-specific molecules. For example, an antibody raised against an antigen to which it binds more efficiently than to a non-specific protein can be described as specifically binding to the antigen. (Similarly, a nucleic acid probe can be described as specifically binding to a nucleic acid target if it forms a specific duplex with the target by base pairing interactions (see above).)
"Stringent hybridization conditions" means hybridization will occur only if there is at least 90%) identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences. See Sambrook et al, 1989, which is hereby incoφorated by reference in its entirety.
Also included in the invention are polypeptides having sequences that are "substantially identical" to the sequence of a phytase polypeptide, such as one of SEQ ID NO: l . A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence or sequences only by conservative amino acid substimtions, for example, substimtions of one amino acid for another of the same class (e.g., substimtion of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substimtion of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).
Additionally a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence or sequences or by one or more non-conservative substimtions, deletions, or insertions, particularly when such a substimtion occurs at a site that is not the active site the molecule, and provided that the polypeptide essentially retains its behavioural properties. For example, one or more amino acids can be deleted from a phytase polypeptide, resulting in modification of the strucmre of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for phytase biological activity can be removed. Such modifications can result in the development of smaller active phytase polypeptides.
The present invention provides a "substantially pure enzyme". The term
"substantially pure enzyme" is used herein to describe a molecule, such as a polypeptide (e g., a phytase polypeptide, or a fragment thereof) that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated. For example, a substantially pure molecule, such as a polypeptide. can be at least 60%, by dry weight, the molecule of interest. The purity of the polypeptides can be determined using standard methods including, e g , polyacrylamide gel electrophoresis (e g , SDS-PAGE), column chromatography (e g., high performance liquid chromatography (HPLC)), and amino-terminal ammo acid sequence analysis.
6. DETAILED DESCRIPTION OF THE INVENTION
6.1 - Novel Phytase
6.1.1 - Novel phytase - general overview: The present invention provides purified a recombinant phytase enzyme, shown in Figure 1. Additionally, the present invention provides isolated nucleic acid molecules (polynucleotides) which encode for the mature enzyme having the deduced amino acid sequence of Figure 1.
The phytase molecules of the instant invention (particularly the recombinant enzyme and the polynucleotides that encode it) are patentably novel with respect to their structures and with respect to their origin Additionally, the instant phytase molecules are patentably novel with respect to activity For example, using an assay (as descπbed in Food Chemicals Codex, 4th Ed.) the activity of the instant phytase enzyme was demonstrated to be far superior in comparison to a fungal (Aspergillus) phytase control. Specifically, a plurality of experiments showed the E. coli phytase to have an activity of about 4400 units/mg and the Aspergillus phytase to have an activity of about 105 units/mg This coπesponds to more than a 40-fold difference in activity.
6.1.2 - Phytase polypeptides: The present invention provides purified a recombinant enzyme that catalyzes the hydrolysis of phytate to inositol and free phosphate with release of minerals from the phytic acid complex. An exemplary purified enzyme is a phytase derived from Escherichia coli B. This exemplary enzyme is shown in Figure 1, SEQ ID NO-2
The enzymes of the present invention include, in addition to an enzyme of Figure 1 (in particular the mamre enzyme), polypeptides having sequences that are "substantially identical" to the sequence of a phytase polypeptide, such as one of SEQ ID 1
In one embodiment, the phytase enzyme of SEQ ID NO.2 of the present invention has a molecular weight of about 47,056 kilodaltons as measured by SDS-PAGE and infeπed from the nucleotide sequence of the gene. The pi is 6.70 The pH and temperature profile and stability data for this enzyme is presented in Figure 2 This purified enzyme may be used to catalyze the hydrolysis of phytate to inositol and free phosphate where desired. The phytase enzyme of the present invention has a high thermostabihty, thus it is particularly serviceable for raised temperature and/or pressure applications including, but not limited to, the preparation of fish foodstuff pellets that will not dissolve prematurely in water.
The phytase polypeptide included in the invention can have the amino acid sequences of the phytase shown in Figure 1 (SEQ ID NO:2). Phytase polypeptides. such as those isolated from E cob B , can be characterized by catalyzing the hydrolysis of phytate to inositol and free phosphate with the release of minerals from the phytic acid complex.
Other phytase polypeptides included in the invention are polypeptides having amino acid sequences that are at least about 50% identical to the amino acid sequence of a phytase polypeptide, such as any of the phytase in SEQ ID NO:2. The length of comparison in determining amino acid sequence homology can be, for example, at least 15 amino acids, and for example, at least 20, 25, or 35 amino acids
Homology or identity is often measured using sequence analysis software (e g ,
Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue. Madison, WI 53705) Such software matches similar sequences by assigning degrees of homology to vanous deletions, substitutions and other modifications. The terms "homology" and "identity" in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum coπespondence over a companson window or designated region as measured using any number of sequence compaπson algonthms or by manual alignment and visual inspection
For sequence companson, typically one sequence acts as a reference sequence, to which test sequences are compared, however a database of reference sequences can be used. When using a sequence companson algonthm, test and reference sequences are entered mto a computer, subsequence coordinates are designated, if necessary, and sequence algonthm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence companson algonthm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A "companson window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequence for companson are well-known in the art. Optimal alignment of sequences for companson can be conducted, eg , by the local homology algonthm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algonthm of Needleman & Wunsch, J. Mol. Biol 48'443, 1970, by the search for similanty method of person & Lipman, Proc. Nat'l. Acad. Sci. USA 85-2444, 1988, by computeπzed implementations of these algonthms (GAP, BESTFIT, FASTA. and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr , Madison, WI). or by manual alignment and visual inspection. Other algonthms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS. LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Frameahgn, Framesearch, DYNAMIC, FILTER, FSAP (Fπstensky Sequence Analysis Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local Content Program), MACAW (Multiple Alignment Construction & Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences. A number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (J. Roach, http://weber.u.Washιngton.edu/~roach/human_ genome_ progress 2.html) (Gibbs, 1995). At least twenty-one other genomes have already been sequenced, including, for example, M genitahum (Fraser et al, 1995), M. jannaschu (Bult et al, 1996), H. influenzae (Fleischmann et al, 1995), E. co (Blattner et al, 1997). and yeast (S cerevisiae) (Mewes et al , 1997), and D melanogaster (Adams et al, 2000). Significant progress has also been made in sequencing the genomes of model organism, such as mouse, C. elegans, and Arabadopsis sp. Several databases containing genomic information annotated with some functional information are maintained by different organization, and are accessible via the internet, for example, http://vvwvvtigr.org/tdb; http://www.genetics.wisc.edu; http://genome- www.stanford.edu/~ball; http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov; http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; and httpV/ www.genome.wi.mit.edu.
One example of a useful algonthm is BLAST and BLAST 2.0 algonthms, which are descnbed in Altschul et al. Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al, J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm nih gov/) This algonthm involves first identifying high sconng sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length m a database sequence. T is refeπed to as the neighborhood word score threshold (Altschul et al , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) For amino acid sequences, a sconng matnx is used to calculate the cumulative score. Extension of the word hits in each direction are halted when- the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-sconng residue alignments; or the end of either sequence is reached. The BLAST algonthm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a companson of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectations (E) of 10, and the BLOSUM62 sconng matnx (see Henikoff & Henikoff, Proc. Natl Acad. Sci. USA 89T0915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N= -4, and a companson of both strands.
The BLAST algonthm also performs a statistical analysis of the similaπty between two sequences (see, eg , Karhn & Altschul, Proc. Natl. Acad. Sci. USA 90:5873, 1993). One measure of similanty provided by BLAST algonthm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a references sequence if the smallest sum probability in a companson of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001 In one embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") In particular, five specific BLAST programs are used to perform the following task
(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database,
(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database,
(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database, (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands), and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database
The BLAST programs identify homologous sequences by identifying similar segments, which are refeπed to herein as "high-sconng segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database High-scoring segment pairs are preferably identified (i e , aligned) by means of a scoring matrix, many of which are known in the art Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al Science 256 1443-1445, 1992, Henikoff and Henikoff, Proteins J 49-61, 1993) Less preferably, the PAM or PAM250 matrices may also be used (see, e g , Schwartz and Dayhoff, eds , 1978, Matrices for Detecting Distance Relationships Atlas of Protein Sequence and Structure, Washington National Biomedical Research Foundation) BLAST programs are accessible through the U S National Library of Medicine, e g , at
Figure imgf000029_0001
The parameters used with the above algonthms may be adapted depending on the sequence length and degree of homology smdied In some embodiments, the parameters may be the default parameters used by the algonthms in the absence of instructions from the user.
The present invention further relates to an enzyme which has the deduced amino acid sequence of Figure 1 , as well as analogs, deπvatives, and fragments of such enzyme.
An analog, derivative, or fragment of the enzyme of Figure 1 may be (a) one in which one or more of the amino acid residues are substituted with an amino acid residue which is not encoded by the genetic code, or (b) one in which one or more of the amino acid residues includes a substiment group, or (c) one in which the mature enzyme is fused with another compound, such as a compound to increase the half-life of the enzyme (for example, polyethylene glycol), or (d) to provide a label or a tag, such as a 6xHιs tag or a green fluorescent protein tag, (e) one in which the additional amino acids are fused to the mature enzyme, such as a leader or secretory sequence or a sequence which is employed for purification of the mature enzyme or a proprotem sequence. Such analogs, denvatives, and fragments are deemed to be within the scope of those skilled in the art from the teachings herein.
A vanant, e.g., a "fragment", "analog" or "derivative" enzyme, and reference enzyme may differ in amino acid sequence by one or more substimtions, additions, deletions, fusions and truncations, which may be present in any combination.
Among prefeπed variants are those that vary from a reference by conservative amino acid substimtions. Such substimtions are those that substitute a given ammo acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substimtions are the replacements, one for another, among the aliphatic ammo acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substimtion between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr Thus, in a particular non-limiting exemplification, a substimtion can be comprised of a substimtion of one amino acid by another amino acid with a like property In another particular non-limiting exemplification, a substimtion can be comprised of a substimtion of an amino acid by an unlike amino acid, where the change is non-inhibitory or silent or improved with respect to at least one enzyme property
Additionally, in a non-limiting exemplification, an addition can be comprised of an addition either at the amino or the carboxy terminal of the protein or alternatively between the terminal sites, where the change is change is non-inhibitory or silent or improved with respect to at least one enzyme property
In another particular non-limiting exemplification, a change can be comprised of a plurality of modifications, including substimtions, additions, deletions, fusions and/or truncations, in the enzyme encoded by the reference polynucleotide (SEQ ID NO 1, such that, lπespective of the effects of the individual modifications, when taken together as a set, the effect of the modifications is non-inhibitory or silent or improved with respect to at least one enzyme property
Most highly prefeπed are variants which retain substantially the same biological function and activity as the reference polypeptide from which it varies
The term "variant" refers to polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of a phytase of the invention Vaπants can be produced by any number of means including methods such as, for example, eπor- prone PCR, shuffling, ohgonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, ligation reassembly, GSSM and any combination thereof as discussed more fully below 6.1.3 - Phytase polynucleotides: In accordance with an aspect of the present invention, there are provided isolated nucleic acid molecules (polynucleotides) which encode for the mamre enzyme having the deduced amino acid sequence of Figure 1
The polynucleotide encoding SEQ ID NO 2 was originally isolated from genomic
DNA recovered from Escherichia cob B as described below It contains an open reading frame encoding a protein of 432 amino acid residues
In accordance with another aspect of the present invention, there is provided an isolated polynucleotide encoding an exemplary enzyme of the present invention (SEQ ID NO 1) comprising the DNA of Figure 1
The present invention also relates to polynucleotides which differ from the reference polynucleotide such that the changes are silent changes, for example the changes do not alter the amino acid sequence encoded by the polynucleotide The present invention also relates to nucleotide changes which result in amino acid substitutions, additions, deletions, fusions and truncations in the enzyme encoded by the reference polynucleotide (SEQ ID NO 1) In a prefeπed aspect of the invention these enzymes retain about the same biological action as the enzyme encoded by the reference polynucleotide
The invention also provides isolated nucleic acid molecules that encode the phytase polypeptide described above For example, nucleic acids that encode SEQ ID NO 2 are included in the invention These nucleic acids can contain naturally occurring nucleotide sequences, or sequences that differ from those of the namrally occumng nucleic acids that encode phytases, but encode the same amino acids, due to the degeneracy of the genetic code The nucleic acids of the invention can contain DNA or RNA nucleotides, or combinations or modifications thereof Exemplary nucleic acids of the invention are shown in SEQ ID NO 1 The polynucleotide of the present invention may be in the form of DNA which DNA includes cDNA, genomic DNA, and synthetic DNA The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand The coding sequence which encodes the mamre enzyme may be identical to the coding sequences shown in Figure 1 and/or that of the deposited clone
(SEQ ID NO 1), or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mamre enzyme as the DNA of Figure 1 (e g , SEQ ID NO 1)
The polynucleotide which encodes for the mamre enzyme of Figure 1 (e g , SEQ
ID NO 2) may include, but is not limited to only the coding sequence for the mamre enzyme, the coding sequence for the mamre enzyme and additional coding sequence such as a leader sequence or a proprotem sequence, the coding sequence for the mature enzyme (and optionally additional coding sequence) and non-cod g sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mamre enzyme
The present invention further relates to vanants of the hereinabove descnbed polynucleotides which encode for fragments, analogs and derivatives of the enzyme having the deduced amino acid sequence of Figure 1 (e g , SEQ ID NO 2) The variant of the polynucleotide may be a naturally occumng allelic v ariant of the polynucleotide or a non-naturally occumng variant of the polynucleotide
Thus, the present invention includes polynucleotides encoding the same mamre enzyme as shown in Figure 1 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the enzyme of Figure 1 Such nucleotide variants include deletion variants, substimtion variants and addition or insertion variants
As hereinabove indicated, the polynucleotide may have a coding sequence which is a namrally occumng allelic variant of the coding sequence shown in Figure 1 As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substimtion, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded enzyme.
As discussed herein, variants can be produced by any number of means including methods such as, for example, eπor-prone PCR, shuffling, ohgonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis. cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site- specific mutagenesis, ligation reassembly, GSSM and any combination thereof.
In one aspect, a non-stochastic method termed synthetic ligation reassembly (SLR), that is somewhat related to stochastic shuffling, save that the nucleic acid building blocks are not shuffled or concatenated or chimeπzed randomly, but rather are assembled non- stochastically can be used to create variants.
The SLR method does not depend on the presence of a high level of homology between polynucleotides to be shuffled. The invention can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10100 different chimeras. Conceivably, SLR can even be used to generate libraries compnsed of over , QIOOO different progeny chimeras.
Thus, in one aspect, the invention provides a non-stochastic method of producing a set of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design, which method is comprised of the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
The mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders. Thus, in one aspect, the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends and. if more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s). In a one embodiment of the invention, the annealed building pieces are treated with an enzyme, such as a ligase (e g , T4 DNA ligase) to achieve covalent bonding of the building pieces.
In a another embodiment, the design of nucleic acid building blocks is obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules These progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, i e chimenzed or shuffled
In one exemplification, the invention provides for the chimeπzation of a family of related genes and their encoded family of related products In a particular exemplification, the encoded products are enzymes. Enzymes and polypeptides of the invention can be mutagenized in accordance with the methods descπbed herein.
Thus according to one aspect of the invention, the sequences of a plurality of progenitor nucleic acid templates are aligned in order to select one or more demarcation points, which demarcation points can be located at an area of homology The demarcation points can be used to delineate the boundaries of nucleic acid building blocks to be generated Thus, the demarcation points identified and selected in the progenitor molecules serve as potential chimeπzation points in the assembly of the progeny molecules
Typically a serviceable demarcation point is an area of homology (comprised of at least one homologous nucleotide base) shared by at least two progenitor templates, but the demarcation point can be an area of homology that is shared by at least half of the progenitor templates, at least two thirds of the progenitor templates, at least three fourths of the progenitor templates, and preferably at almost all of the progenitor templates Even more preferably still a serviceable demarcation point is an area of homology that is shared by all of the progenitor templates
In a one embodiment, the ligation reassembly process is performed exhaustively in order to generate an exhaustive library In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules At the same time, the assembly order (i e the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid) in each combination is by design (or non-stochastic) Because of the non-stochastic nature of the method, the possibility of unwanted side products is greatly reduced
In another embodiment, the method provides that, the ligation reassembly process is performed systematically, for example in order to generate a systematically compartmentalized library, with compartments that can be screened systematically, e g , one by one In other words the invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, an expenmental design can be achieved where specific sets of progeny products are made in each of several reaction vessels This allows a systematic examination and screening procedure to be performed Thus, it allows a potentially very large number of progeny molecules to be examined systematically in smaller groups
Because of its ability to perform chimeπzations in a manner that is highly flexible yet exhaustive and systematic as well, particularly when there is a low level of homology among the progenitor molecules, the instant invention provides for the generation of a library (or set) comprised of a large number of progeny molecules Because of the non- stochastic nature of the instant ligation reassembly invention, the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design In a particularly embodiment, such a generated library is comprised of greater than 103 to greater than 101000 different progeny molecular species In one aspect, a set of finalized chimeric nucleic acid molecules, produced as described is comprised of a polynucleotide encoding a polypeptide. According to one embodiment, this polynucleotide is a gene, which may be a man-made gene. According to another embodiment, this polynucleotide is a gene pathway, which may be a man-made gene pathway. The invention provides that one or more man-made genes generated by the invention may be incoφorated into a man-made gene pathway, such as pathway operable in a eukaryotic organism (including a plant).
In another exemphfaction, the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g , one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e g., by mutageneis) or in an in vivo process (e.g , by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.
Thus, according to another embodiment, the invention provides that a nucleic acid building block can be used to introduce an intron. Thus, the invention provides that functional introns may be introduced into a man-made gene of the invention. The invention also provides that functional introns may be introduced into a man-made gene pathway of the invention. Accordingly, the invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced ιntron(s).
Accordingly, the invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced ιntron(s). Preferably, the artificially introduced ιntron(s) are functional in one or more host cells for gene splicing much in the way that namrally-occumng introns serve functionally in gene splicing. The invention provides a process of producing man-made intron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing
A man-made genes produced using the invention can also serve as a substrate for recombination with another nucleic acid. Likewise, a man-made gene pathway produced using the invention can also serve as a substrate for recombination with another nucleic acid. In a prefeπed instance, the recombination is facilitated by, or occurs at, areas of homology between the man-made intron-containing gene and a nucleic acid with serves as a recombination partner. In a particularly prefeπed instance, the recombination partner may also be a nucleic acid generated by the invention, including a man-made gene or a man-made gene pathway. Recombination may be facilitated by or may occur at areas of homology that exist at the one (or more) artificially introduced ιntron(s) in the man-made gene.
The synthetic ligation reassembly method of the invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends. The two ligatable ends on each nucleic acid building block may be two blunt ends (i.e. each having an overhang of zero nucleotides), or preferably one blunt end and one overhang, or more preferably still two overhangs.
A serviceable overhang for this puφose may be a 3' overhang or a 5' overhang. Thus, a nucleic acid building block may have a 3' overhang or alternatively a 5' overhang or alternatively two 3' overhangs or alternatively two 5' overhangs The overall order in which the nucleic acid building blocks are assembled to form a finalized chimeric nucleic acid molecule is determined by puφoseful experimental design and is not random.
According to one prefeπed embodiment, a nucleic acid building block is generated by chemical synthesis of two single-stranded nucleic acids (also referred to as single- stranded ohgos) and contacting them so as to allow them to anneal to form a double- stranded nucleic acid building block A double-stranded nucleic acid building block can be of variable size The sizes of these building blocks can be small or large Prefeπed sizes for building block range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs) Other prefeπed size ranges are also provided, which have lower limits of from 1 bp to 10,000 bp (including every integer value in between), and upper limits of from 2 bp to 100, 000 bp (including every integer value in between)
Many methods exist by which a double-stranded nucleic acid building block can be generated that is serviceable for the invention, and these are known in the art and can be readily performed by the skilled artisan
According to one embodiment, a double-stranded nucleic acid building block is generated by first generating two single stranded nucleic acids and allowing them to anneal to form a double-stranded nucleic acid building block The two strands of a double-stranded nucleic acid building block may be complementary at every nucleotide apart from any that form an overhang, thus containing no mismatches, apart from any overhang(s) According to another embodiment, the two strands of a double-stranded nucleic acid building block are complementary at fewer than every nucleotide apart from any that form an overhang Thus, according to this embodiment, a double-stranded nucleic acid building block can be used to introduce codon degeneracy Preferably the codon degeneracy is introduced using the site-saturation mutagenesis described herein, using one or more N,N,G/T cassettes or alternatively using one or more N,N,N cassettes
The in vivo recombination method of the invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide
The approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA and growth hormone This approach may be used to generate proteins having altered specificity or activity The approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes Thus this approach may be used to generate genes having increased rates of expression. This approach may also be useful in the study of repetitive DNA sequences. Finally, this approach may be useful to mutate πbozymes or ap tamers.
In one aspect variants of the polynucleotides and polypeptides described herein are obtained by the use of repeated cycles of reductive reassortment, recombination and selection which allow for the directed molecular evolution of highly complex linear sequences, such as DNA, RNA or proteins thorough recombination.
In vivo shuffling of molecules is useful in providing variants and can be performed utilizing the natural property of cells to recombine multimers While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.
In a another embodiment, the invention includes a method for producing a hybnd polynucleotide from at least a first polynucleotide and a second polynucleotide. The invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell. The regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide. The term "hybrid polynucleotide", as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence withm a DNA molecule
The invention provides a means for generating hybrid polynucleotides which may encode biologically active hybrid polypeptides (e g , a hybrid phytase). In one aspect, the onginal polynucleotides encode biologically active polypeptides. The method of the invention produces new hybrid polypeptides by utilizing cellular processes which integrate the sequence of the original polynucleotides such that the resulting hybnd polynucleotide encodes a polypeptide demonstrating activities derived from the original biologically active polypeptides For example, the original polynucleotides may encode a particular enzyme from different microorganisms. An enzyme encoded by a first polynucleotide from one organism or variant may, for example, function effectively under a particular environmental condition, e g , high salinity An enzyme encoded by a second polynucleotide from a different organism or variant may function effectively under a different environmental condition, such as extremely high temperatures. A hybnd polynucleotide containing sequences from the first and second original polynucleotides may encode an enzyme which exhibits charactenstics of both enzymes encoded by the onginal polynucleotides. Thus, the enzyme encoded by the hybrid polynucleotide may function effectively under environmental conditions shared by each of the enzymes encoded by the first and second polynucleotides, e g , high salinity and extreme temperatures
Enzymes encoded by original polynucleotides include, but are not limited to, phytases A hybrid polypeptide resulting from the method of the invention may exhibit specialized enzyme activity not displayed in the original enzymes. For example, following recombination and/or reductive reassortment of polynucleotides encoding hydrolase activities, the resulting hybnd polypeptide encoded by a hybrid polynucleotide can be screened for specialized hydrolase activities obtained from each of the onginal enzymes, i e , the type of bond on which the hydrolase acts and the temperature at which the hydrolase functions Thus, for example, the hydrolase may be screened to ascertain those chemical functionalities which distinguish the hybrid hydrolase from the original hydrolyases, such as: (a) amide (peptide bonds), i.e., proteases; (b) ester bonds, i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, for example, the temperature, pH or salt concentration at which the hybrid polypeptide functions.
Sources of the original polynucleotides may be isolated from individual organisms
("isolates"), collections of organisms that have been grown in defined media ("enrichment cultures"), or, uncultivated organisms ("environmental samples"). The use of a culture-independent approach to derive polynucleotides encoding novel bioactivities from environmental samples is most preferable since it allows one to access untapped resources of biodiversity.
"Environmental libraries" are generated from environmental samples and represent the collective genomes of namrally occuπing organisms archived in cloning vectors that can be propagated in suitable prokaryotic hosts. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture. Additionally, a normalization of the environmental DNA present in these samples could allow more equal representation of the DNA from all of the species present in the original sample. This can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample which may be under-represented by several orders of magnimde compared to the dominant species.
For example, gene libraries generated from one or more uncultivated microorganisms are screened for an activity of interest. Potential pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries. Polynucleotides encoding activities of interest are isolated from such libraries and introduced into a host cell. The host cell is grown under conditions which promote recombination and/or reductive reassortment creating potentially active biomolecules with novel or enhanced activities. The microorganisms from which the polynucleotide may be prepared include prokaryotic microorganisms, such as Xanthobacter, Eubacteria and Archaebactena, and lower eukaryotic microorganisms such as fungi, some algae and protozoa Polynucleotides may be isolated from environmental samples in which case the nucleic acid may be recovered without culturmg of an organism or recovered from one or more cultured organisms In one aspect, such microorganisms may be extremophiles, such as hyperthermophiles, psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles Polynucleotides encoding enzymes isolated from extremophihc microorganisms are particularly prefeπed. Such enzymes may function at temperamres above 100°C in teπestnal hot springs and deep sea thermal vents, at temperamres below 0°C in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 1 1 in sewage sludge. For example, several esterases and lipases cloned and expressed from extremophihc organisms show high activity throughout a wide range of temperatures and pHs.
Polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell A suitable host cell is any cell which is capable of promoting recombination and or reductive reassortment The selected polynucleotides are preferably already in a vector which includes appropriate control sequences The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bactenal cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al, 1986)
As representative examples of appropriate hosts, there may be mentioned' bacterial cells, such as E cob, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma, adenoviruses; and plant cells. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein With particular references to various mammalian cell culmre systems that can be employed to express recombinant protein, examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, descnbed in "SV40-transformed simian cells support the replication of early SV40 mutants" (Gluzman, 1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscnbed sequences DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscnbed genetic elements
Host cells containing the polynucleotides of interest can be cultured in conventional nutπent media modified as appropπate for activating promoters, selecting transformants or amplifying genes The culmre conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinanly skilled artisan The clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the enhanced activity
In another aspect, methods can be used to generate novel polynucleotides encoding biochemical pathways from one or more operons or gene clusters or portions thereof For example, bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes The genes are clustered, in strucmres refeπed to as "gene clusters," on a single chromosome or immediately adjacent to one another and are transcnbed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster Thus, a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function An example of a biochemical pathway encoded by gene clusters are polyketides Polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (such as tetracychnes and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and vetennary products (monensin). Many polyketides (produced by polyketide synthases) are valuable as therapeutic agents. Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of an enormous variety of carbon chains differing in length and patterns of functionality and cyclization Polyketide synthase genes fall into gene clusters and at least one type (designated type I) of polyketide synthases have large size genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins
Gene cluster DNA can be isolated from different organisms and ligated into vectors, particularly vectors containing expression regulatory sequences which can control and regulate the production of a detectable protein or protein-related aπay activity from the ligated gene clusters. Use of vectors which have an exceptionally large capacity for exogenous DNA introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E cob. This f-factor of E. cob is a plasmid which affects high-frequency transfer of itself dunng conjugation and is ideal to achieve and stably propagate large DNA fragments, such as gene clusters from mixed microbial samples. Once ligated into an appropnate vector, two or more vectors containing different phytase gene clusters can be introduced into a suitable host cell Regions of partial sequence homology shared by the gene clusters will promote processes which result in sequence reorganization resulting in a hybrid gene cluster. The novel hybrid gene cluster can then be screened for enhanced activities not found in the original gene clusters.
Therefore, in a one embodiment, the invention relates to a method for producing a biologically active hybrid polypeptide and screening such a polypeptide for enhanced activity by.
1) introducing at least a first polynucleotide in operable linkage and a second polynucleotide in operable linkage, said at least first polynucleotide and second polynucleotide sharing at least one region of partial sequence homology, into a suitable host cell. 2) growing the host cell under conditions which promote sequence reorganization resulting in a hybrid polynucleotide in operable linkage,
3) expressing a hybrid polypeptide encoded by the hybrid polynucleotide,
4) screening the hybrid polypeptide under conditions which promote identification of enhanced biological activity; and
5) isolating the a polynucleotide encoding the hybnd polypeptide.
Methods for screening for various enzyme activities are known to those of skill in the art and are discussed throughout the present specification. Such methods may be employed when isolating the polypeptides and polynucleotides of the invention.
As representative examples of expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e g , vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus and yeast). Thus, for example, the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE vectors (Qiagen), pBluescπpt plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector may be used so long as they are replicable and viable in the host. Low copy number or high copy number vectors may be employed with the present invention.
A prefeπed type of vector for use in the present invention contains an f-factor ongin replication The f-factor (or fertility factor) in E cob is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself. A particularly prefeπed embodiment is to use cloning vectors, refeπed to as "fosmids" or bacterial artificial chromosome (BAC) vectors These are derived from E cob f-factor which is able to stably integrate large segments of genomic DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable "environmental DNA library "
Another type of vector for use in the present invention is a cosmid vector. Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA. Cloning into cosmid vectors is described in detail in "Molecular Cloning' A laboratory Manual" (Sambrook et al , 1989)
The DNA sequence in the expression vector is operatively linked to an appropπate expression control sequence(s) (promoter) to direct RNA synthesis. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector also contains a ribosome binding site for translation initiation and a transcnption terminator. The vector may also include appropriate sequences for amplifying expression. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracychne or ampicillin resistance in E. cob.
In vivo reassortment is focused on "inter-molecular" processes collectively refeπed to as "recombination" which in bacteria, is generally viewed as a "RecA-dependent" phenomenon. The invention can rely on recombination processes of a host cell to recombine and re-assort sequences, or the cells' ability to mediate reductive processes to decrease the complexity of quasi-repeated sequences in the cell by deletion. This process of "reductive reassortment" occurs by an "intra-molecular", RecA-independent process. Therefore, in another aspect of the invention, variant polynucleotides can be generated by the process of reductive reassortment. The method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector, and their subsequent introduction into an appropnate host cell The reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasi-repeated units. The reassortment process recombines and/or reduces the complexity and extent of the repeated sequences, and results in the production of novel molecular species. Various treatments may be applied to enhance the rate of reassortment. These could include treatment with ultra-violet light, or DNA damaging chemicals, and/or the use of host cell lines displaying enhanced levels of "genetic instability" Thus the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their own evolution.
Repeated or "quasi-repeated" sequences play a role in genetic instability. In the present invention, "quasi-repeats" are repeats that are not restncted to their ongmal unit strucmre. Quasi-repeated units can be presented as an aπay of sequences in a construct; consecutive units of similar sequences Once ligated, the junctions between the consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level. The deletion process the cell performs to reduce the complexity of the resulting construct operates between the quasi- repeated sequences. The quasi-repeated units provide a practically limitless repertoire of templates upon which slippage events can occur. The constructs containing the quasi- repeats thus effectively provide sufficient molecular elasticity that deletion (and potentially insertion) events can occur virtually anywhere within the quasi-repetitive units.
When the quasi-repeated sequences are all ligated in the same orientation, for instance head to tail or vice versa, the cell cannot distinguish individual units. Consequently, the reductive process can occur throughout the sequences. In contrast, when for example, the units are presented head to head, rather than head to tail, the inversion delineates the endpoints of the adjacent unit so that deletion formation will favor the loss of discrete units Thus, it is preferable with the present method that the sequences are in the same orientation Random orientation of quasi-repeated sequences will result in the loss of reassortment efficiency, while consistent onentation of the sequences will offer the highest efficiency However, while having fewer of the contiguous sequences in the same orientation decreases the efficiency, it may still provide sufficient elasticity for the effective recovery of novel molecules Constructs can be made with the quasi-repeated sequences in the same orientation to allow higher efficiency
Sequences can be assembled in a head to tail orientation using any of a variety of methods, including the following a) Primers that include a poly-A head and poly-T tail which when made single- stranded would provide onentation can be utilized This is accomplished by having the first few bases of the primers made from RNA and hence easily removed RNAseH b) Pnmers that include unique restriction cleavage sites can be utilized Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required c) The inner few bases of the primer could be thiolated and an exonuclease used to produce properly tailed molecules
The recovery of the re-assorted sequences relies on the identification of cloning vectors with a reduced RJ The re-assorted encoding sequences can then be recovered by amplification The products are re-cloned and expressed The recovery of cloning vectors with reduced RJ can be effected by
1 ) The use of vectors only stably maintained when the construct is reduced in complexity
2) The physical recovery of shortened vectors by physical procedures In this case, the cloning vector would be recovered using standard plasmid isolation procedures and size fractionated on either an agarose gel, or column with a low molecular weight cut off utilizing standard procedures 3) The recovery of vectors containing interrupted genes which can be selected when insert size decreases
4) The use of direct selection techniques with an expression vector and the appropriate selection.
Encoding sequences (for example, genes) from related organisms may demonstrate a high degree of homology and encode quite diverse protein products. These types of sequences are particularly useful in the present invention as quasi-repeats. However, while the examples illustrated below demonstrate the reassortment of nearly identical original encoding sequences (quasi-repeats). this process is not limited to such nearly identical repeats.
The following example demonstrates a method of the invention. Encoding nucleic acid sequences (quasi-repeats) derived from three (3) unique species are depicted. Each sequence encodes a protein with a distinct set of properties. Each of the sequences differs by a single or a few base pairs at a unique position in the sequence which are designated "A", "B" and "C". The quasi-repeated sequences are separately or collectively amplified and ligated into random assemblies such that all possible permutations and combinations are available in the population of ligated molecules. The number of quasi-repeat units can be controlled by the assembly conditions. The average number of quasi-repeated units in a construct is defined as the repetitive index (RJ)
Once formed, the constructs may, or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell. The cells are then propagated and "reductive reassortment" is effected. The rate of the reductive reassortment process may be stimulated by the introduction of DNA damage if desired Whether the reduction in RJ is mediated by deletion formation between repeated sequences by an "intra-molecular" mechanism, or mediated by recombination-like events through "mter-molecular" mechanisms is immaterial. The end result is a reassortment of the molecules into all possible combinations. Optionally, the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact, or catalyze a particular reaction (e g , such as catalyzing the hydrolysis of a haloalkane)
The polypeptides that are identified from such libraries can be used for therapeutic, diagnostic, research and related puφoses (e g , catalysts, solutes for increasing osmolaπty of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or selection
In another aspect, prior to or during recombination or reassortment, polynucleotides of the invention or polynucleotides generated by the method descnbed herein can be subjected to agents or processes which promote the introduction of mutations into the oπginal polynucleotides The introduction of such mutations would increase the diversity of resulting hybrid polynucleotides and polypeptides encoded therefrom The agents or processes which promote mutagenesis can include, but are not limited to (+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenme, see Sun and Hurley, 1992), an N-acelylated or deacetylated 4'-fluro-4-amιnobιphenyl adduct capable of inhibiting DNA synthesis (see. for example, van de Poll et al , 1992), or a N- acetylated or deacetylated 4-amιnobιphenyl adduct capable of inhibiting DNA synthesis (see also, van de Poll et al , 1992, pp 751-758), tnvalent chromium, a tnvalent chromium salt, a polycyclic aromatic hydrocarbon ("PAH") DNA adduct capable of inhibiting DNA replication, such as 7-bromomethyl-benz[<3]anthracene ("BMA"), tπs(2,3- dιbromopropyl)phosphate ("Tπs-BP"), 1 ,2-dιbromo-3-chloropropane ("DBCP"), 2- bromoacrolein (2BA), benzo[α]pyrene-7,8-dιhydrodιol-9-10-epoxιde ("BPDE"), a platιnum(II) halogen salt, N-hydroxy-2-amιno-3-methyhmιdazo[4,5 /]-quιnolιne ("N- hydroxy-IQ"), and N-hydroxy-2-amιno-l-methyl-6-phenylιmιdazo[4,5-J/]-pyπdιne ("N- hydroxy-PhlP") Especially prefeπed means for slowing or halting PCR amplification consist of UV light (+)-CC- 1065 and (^)-CC- 1065-(N3-Ademne) Particularly encompassed means are DNA adducts or polynucleotides comprising the DNA adducts from the polynucleotides or polynucleotides pool, which can be released or removed by a process including heating the solution comprising the polynucleotides prior to further processing
In another aspect the invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions according to the invention which provide for the production of hybrid or re-assorted polynucleotides.
The invention also provides for the use of proprietary codon primers (containing a degenerate N,N,N sequence) to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substimtions is represented at each amino acid position (gene site saturated mutagenesis (GSSM)). The ohgos used are comprised contiguously of a first homologous sequence, a degenerate N,N,N sequence, and preferably but not necessarily a second homologous sequence. The downstream progeny translational products from the use of such ohgos include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,N sequence includes codons for all 20 amino acids.
In one aspect, one such degenerate oligo (comprised of one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substimtions. In another aspect, at least two degenerate N,N,G/T cassettes are used - either in the same oligo or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substimtions. Thus, more than one N,N,G/T sequence can be contained in one oligo to introduce amino acid mutations at more than one site. This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s). In another aspect, ohgos serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and or substimtions. In a particular exemplification, it is possible to simultaneously mutagenize two or more contiguous amino acid positions using an oligo that contains contiguous N,N,G/T triplets, i e a degenerate (N,N.G'T)n sequence
In another aspect, the present invention provides for the use of degenerate cassettes having less degeneracy than the N,N,G/T sequence For example, it may be desirable in some instances to use (e g in an oligo) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet Alternatively, it may be desirable in some instances to use (e g , in an oligo) a degenerate N.N,N triplet sequence, or an N,N, G/C triplet sequence
It is appreciated, however, that the use of a degenerate triplet (such as N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instant invention is advantageous for several reasons In one aspect, this invention provides a means to systematically and fairly easily generate the substitution of the full range of possible amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide Thus, for a 100 amino acid polypeptide, the invention provides a way to systematically and fairly easily generate 2000 distinct species (/ e , 20 possible amino acids per position times 100 amino acid positions) It is appreciated that there is provided, through the use of an oligo containing a degenerate N,N,G/T or an N.N, G/C tnplet sequence, 32 individual sequences that code for 20 possible amino acids Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using one such oligo, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides In contrast, the use of a non-degenerate oligo in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel
This invention also provides for the use of nondegenerate ohgos, which can optionally be used in combination with degenerate primers disclosed It is appreciated that in some situations, it is advantageous to use nondegenerate ohgos to generate specific point mutations in a working polynucleotide This provides a means to generate specific silent point mutations, point mutations leading to coπesponding amino acid changes, and point mutations that cause the generation of stop codons and the coπesponding expression of polypeptide fragments
Thus, in one embodiment, each samration mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 amino acids are represented at the one specific amino acid position coπesponding to the codon position mutagenized in the parental polynucleotide. The 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g., cloned into a suitable E cob host using an expression vector) and subjected to expression screening. When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide), it can be sequenced to identify the coπespondingly favorable amino acid substimtion contained therein.
It is appreciated that upon mutagenizing each and every amino acid position in a parental polypeptide using samration mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substimtions For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined - 6 single point mutations (i e., 2 at each of three positions) and no change at any position.
In yet another aspect, site-saturation mutagenesis can be used together with shuffling, chimeπzation, recombination and other mutagenizing processes, along with screening. This invention provides for the use of any mutagenizing process(es), including samration mutagenesis, in an iterative manner In one exemplification, the iterative use of any mutagenizing process(es) is used in combination with screening. Thus, in a non-limiting exemplification, polynucleotides and polypeptides of the invention can be derived by samration mutagenesis in combination with additional mutagenization processes, such as process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment
In addition to performing mutagenesis along the entire sequence of a gene, mutagenesis can be used to replace each of any number of bases in a polynucleotide sequence, wherein the number of bases to be mutagenized is preferably every integer from 15 to 100,000 Thus, instead of mutagenizing every position along a molecule, one can subject every or a discrete number of bases (preferably a subset totaling from 15 to 100,000) to mutagenesis Preferably, a separate nucleotide is used for mutagenizing each position or group of positions along a polynucleotide sequence A group of 3 positions to be mutagenized may be a codon The mutations are preferably introduced using a mutagenic primer, containing a heterologous cassette, also refeπed to as a mutagenic cassette Prefeπed cassettes can have from 1 to 500 bases Each nucleotide position in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T, A C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E can be refeπed to as a designer oligo)
In a general sense, samration mutagenesis is comprised of mutagenizing a complete set of mutagenic cassettes (wherein each cassette is preferably about 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is preferably from about 15 to 100,000 bases in length) Thus, a group of mutations (ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized A grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of samration mutagenesis Such groupings are exemplified by deletions, additions, groupings of particular codons and groupings of particular nucleotide cassettes Defined sequences to be mutagenized include a whole gene, pathway, cDNA, an entire open reading frame (ORF), and entire promoter, enhancer, repressor/transactivator, origin of replication, intron, operator, or any polynucleotide functional group Generally, a "defined sequences" for this puφose may be any polynucleotide that a 15 base- polynucleotide sequence, and polynucleotide sequences of lengths between 15 bases and 15,000 bases (this invention specifically names every integer in between) Considerations in choosing groupings of codons include types of amino acids encoded by a degenerate mutagenic cassette
In a particularly prefeπed exemplification a grouping of mutations that can be introduced into a mutagenic cassette, this invention specifically provides for degenerate codon substitutions (using degenerate ohgos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each position, and a library of polypeptides encoded thereby
The present invention also includes polynucleotides, wherein the coding sequence for the mamre enzyme may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of an enzyme from a host cell, for example, a leader sequence which functions to control transport of an enzyme from the cell An enzyme having a leader sequence is an example of a preprotein and may have the leader sequence cleaved by the host cell to form the mamre form of the enzyme The polynucleotides may also encode for a proprotem which is exemplified by a mamre protein plus additional 5' amino acid residues An otherwise mamre protein having a prosequence is exemplified by a proprotem that is an inactive form of the protein Once the prosequence is cleaved an active mamre protein remains
Thus, for example, the polynucleotide of the present invention may encode for a mamre enzyme, or for an enzyme having a prosequence or for an enzyme having both a prosequence and a presequence (e g leader sequence) 6.1.4 - Methods of isolation: The coding sequences for the phytase enzymes of the present invention were identified by preparing E cob B genomic DNA, for example, and recovenng (via, for example, PCR amplification) from the genomic DNA, DNA encoding phytase activity Such methods for recovery are well-known in the art. One means, for example, comprises designing amplification primers to recover the coding sequence, amplifying the gene from the genomic DNA, subcloning the DNA into a vector, transforming the resulting construct into a host strain, and expressing the phytase enzyme for evaluation Such procedures are well known in the art and methods are provided, for example, in Sambrook et al , 1989, which is hereby incoφorated by reference in its entirety
In a prefeπed embodiment, the enzyme of the present invention, was isolated from an E.coli B genomic DNA by the following technique-
E.coli B genomic DNA was obtained comercially (Sigma Catalog # D-2001, St. Louis, New Jersey). The following pnmers were used to amplify the gene directly from the genomic DNA:
5' primer gtttctgaattcaaggaggaatttaaATGAAAGCGATCTTAATCCCATT (SEQ ID
NO:3); and
3' primer gtttctggatccTTACAAACTGCACGCCGGTAT (SEQ ID NO'4) Pfu polymerase was used according to manufacturers protocol (Stratagene Cloning
Systems, Inc., La Jolla, CA).
PCR product and pQE60 vector (Qiagen) were both digested with EcoRI and Bglll restnction endonucleases (New England Biolabs) according to manufacturers protocols. Ligation and transformation into, and expression in Ml 5 pREP4 host cells (Qiagen) yields c-term 6X-Hιs tagged protein.
6.1.5 - Determination of activity: The isolated nucleic acid sequences and other enzymes may then be measured for retention of biological activity characteristic to the enzyme of the present invention, for example, in an assay for detecting enzymatic phytase activity (Food Chemicals Codex, 4lh Ed.) Such enzymes include truncated forms of phytase, and variants such as deletion and insertion variants.
An in vitro example of such an assay is the following assay for the detection of phytase activity- Phytase activity can be measured by incubating 150μl of the enzyme preparation with 600μl of 2 mM sodium phytate in 100 mM Tris HCl buffer pH 7.5, supplemented with ImM CaCl2 for 30 minutes at 37°C. After incubation the reaction is stopped by adding 750μl of 5% tπchloroacetic acid. Phosphate released was measured against phosphate standard spectrophotometncally at 700nm after adding 1500μl of the color reagent (4 volumes of 1.5% ammonium molybdate in 5.5% sulfuπc acid and 1 volume of 2.7% feπous sulfate; Shimizu, 1992). One unit of enzyme activity is defined as the amount of enzyme required to liberate one μmol Pi per min under assay conditions. Specific activity can be expressed in units of enzyme activity per mg of protein.
The enzyme of the present invention has enzymatic activity with respect to the hydrolysis of phytate to inositol and free phosphate.
6.2 - Production of Novel Phvtase
6.2.1 - Methods of production - general overview: The enzymes and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The phytase polypeptide of the invention can be obtained using any of several standard methods. For example, phytase polypeptides can be produced in a standard recombinant expression system (see below), chemically synthesized (this approach may be limited to small phytase peptide fragments), or punfied from organisms in which they are namrally expressed. Serviceable recombinant expression methods include the use of mammalian hosts, microbial hosts, and plant hosts.
The recombinant expression of the instant phytase molecules may be achieved in combination with one or more additional molecules such as, for example, other enzymes. This approach is serviceable for producing combination products, such as a plant or plant part that contains the instant phytase molecules as well as one or more additional molecules - preferably said phytase molecules and said additional molecules are serviceable in a combination treatment The resulting recombinantly expresssed molecules may be used in homogenized and/or purified form or alternatively in relatively unpurified form (e g as consumable plant parts that are serviceable when admixed with other foodstuffs for catalyzing the degredation of phytate).
In sum, in a non-limiting embodiment, the present invention provides a recombinant enzyme expressed in a host. In another non-hmiting embodiment, the present invention provides a substantially pure phytase enzyme Thus, an enzyme of the present invention may be a recombinant enzyme, a natural enzyme, or a synthetic enzyme, preferably a recombinant enzyme.
6.2.2 - Recombinant expression: The present invention also relates to vectors which include polynucleotides of the present mvention, host cells which are genetically engineered with vectors of the invention, and the production of enzymes of the invention by recombinant techniques.
Host cells are genetically engineered (e g transduced or transformed or transfected) with the vectors containing the polynucleotides of this invention. Such vectors may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, a pπon, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, &/or selecting transformants or amplifying the genes of the present invention. The culmre conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan
The polynucleotides of the present invention may be employed for producing enzymes by recombinant techniques Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing an enzyme Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e g , denvatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors denved from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovims, fowl pox virus, and pseudorabies However, any other vector may be used as long as it is replicable and viable in the host
The appropπate DNA sequence may be inserted into the vector by a vanety of procedures In general, the DNA sequence is inserted into an appropπate restnction endonuclease sιte(s) by procedures known in the art Inclusive in this meaning is the use of blunt-ended molecules which could be generated by the use of restriction digestion as well as restriction digestion-independent means Alternatively, the insert may be incoφorated into a vector by so called "hgase-independent" means In a particular aspect, a "hgase-independent" means is exemplified by the use of topoisomerase-mediated ligation at room temperature, for example according to the commercially available kit termed TOPO-TA Cloning® (Invitrogen Coφoration, Carlsbad, CA) Alteranative enzymes, including isomers of topoisomerase as well as more distantly related recombination enzymes (e g recombinases), may also be serviceable for mediating this type of "hgase-independent" incoφoration In another particular aspect, a "hgase- independent" means is exemplified by the use host repair mechanisms Such procedures and others are deemed to be within the scope of those skilled in the art
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis As representative examples of such promoters, there may be mentioned an LTR or SV40 promoter, an E cob lac or trp, a phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator The vector may also include appropriate sequences for amplifying expression In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracychne or ampicilhn resistance in E cob.
The vector containing the appropnate DNA sequence as hereinabove descπbed, as well as an appropriate promoter or control sequence, may be employed to transform an appropnate host to permit the host to express the protein
As representative examples of appropnate hosts, there may be mentioned' bactenal cells, such as E cob, Streptomyces, Bacillus subtilis; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera 5/9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs compnsmg one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a prefeπed aspect of this embodiment, the construct further compnses regulatory sequences, including, for example, a promoter, operably linked to the sequence. One or more additional inserts may also be incoφorated that lead to expression of one or more aditional molecules, such as another phytase or a protease enzyme, preferably said one or more additional molecules are serviceable in combination with the instant phytase in a combination treatment.
Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. "Plasmids" are designated by a lower case p preceded and or followed by capital letters and or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
The following vectors are provided by way of example, Bactenal pQE70, pQE60, pQE-9 (Qiagen), pBluescπpt II (Stratagene), pTRC99a, pKK223-3, pDR540, pRIT2T (Pharmacia), Eukaryotic pXTl, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia) However, any other plasmids or other vectors may be used as long as they are replicable and viable in the host
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable markers Two appropriate vectors are pKK232-8 and pCM7 Particular named bactenal promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and tφ Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionem-I Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art
In a further embodiment, the present invention relates to host cells containing the above-descπbed constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, 1986).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the enzymes of the invention can be synthetically produced by conventional peptide synthesizers
Mamre proteins can be expressed in mammalian cells, yeast, bactena, or other cells under the control of appropriate promoters Cell-iree translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropπate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described (e.g Sambrook et al , 1989, the disclosure of which is hereby incoφorated by reference).
Transcription of the DNA encoding the enzymes of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication ongin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovims enhancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g. , the ampicilhn resistance gene of E. cob and 5. cerevisiae TRP1 gene, and a promoter derived from a highly- expressed gene to direct transcnption of a downstream structural sequence. Such promoters can be denved from operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), A-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropπate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated enzyme. Optionally, the heterologous sequence can encode a fusion enzyme including an N-terminal identification peptide imparting desired charactenstics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bactenal use are constructed by inserting a structural
DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will compnse one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host Suitable prokaryotic hosts for transformation include E cob, Bacillus subtilis, Salmonella typhunurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids compnsmg genetic elements of the well known cloning vector pBR322 (ATCC 37017) Such commercial vectors include, for example, pKK223- 3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e g , temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
Various mammalian cell culmre systems can also be employed to express recombinant protein Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, as described (Gluzman, 1981), and other cell lines capable of expressing a compatible vector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscnbed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscnbed genetic elements
The enzyme can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Protein refolding steps can be used, as necessary, in completing configuration of the mamre protein Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps
The enzymes of the present invention may be a namrally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) Depending upon the host employed in a recombinant production procedure, the enzymes of the present invention may be glycosylated or may be non-glycosylated Enzymes of the invention may or may not also include an initial methiomne amino acid residue
In a prefeπed embodiment, the enzyme of the present invention is a phytase enzyme which is stable to heat and is heat resistant and catalyzes the enzymatic hydrolysis of phytate, / e , the enzyme is able to renature and regain activity after a brief (. e , 5 to 30 seconds), or longer period, for example, minutes or hours, exposure to temperamres of up to about 50 °C or slightly above 50 °C
The present invention is further described with reference to the examples contained herein, however, it is to be understood that the present invention is not limited to such examples All parts or amounts, unless otherwise specified, are by weight
In one aspect of the invention, a method for producing an phytase enzyme, such as those shown in Figures 1. is provided The method includes growing a host cell which contains a polynucleotide encoding the enzyme (e g , SEQ ID NO 1), under conditions which allow the expression of the nucleic acid, and optionally isolating the enzyme encoded by the nucleic acid Methods of culturmg the host cell are described in the Examples and are known by those of skill in the art
6.2.3 - Use of transgenic plants and plant organs: In a particular embodiment, the present invention provides for the expression of phytase in transgenic plants or plant organs and methods for the production thereof DNA expression constructs are provided for the transformation of plants with a gene encoding phytase under the control of regulatory sequences which are capable of directing the expression of phytase These regulatory sequences include sequences capable of directing transcription in plants, either constitutively. or in stage and/or tissue specific manners
The manner of expression depends, in part, on the use of the plant or parts thereof The transgenic plants and plant organs provided by the present invention may be applied to a vanety of industrial processes either directly, e g in animal feeds or alternatively, the expressed phytase may be extracted and if desired, punfied before application Alternatively, the recombinant host plant or plant part may be used directly In a particular aspect, the present invention provides methods of catalyzing phytate-hydrolyzing reactions using seeds containing enhanced amounts of phytase The method involves contacting transgenic, non-wild type seeds, preferably in a ground or chewed form, with phytate- containing substrate and allowing the enzymes in the seeds to increase the rate of reaction By directly adding the seeds to a phytate-containing substrate, the invention provides a solution to the expensive and problematic process of extracting and purifying the enzyme In a particular - but by no means limiting - exemplification, the present invention also provides methods of treatment whereby an organism lacking a sufficient supply of an enzyme is administered the enzyme in the form of seeds containing enhanced amounts of the enzyme In a prefeπed embodiment, the timing of the administration of the enzyme to an organism is coordinated with the consumption of a phytate-containing foodstuff The expression of phytase in plants can be achieved by a variety of means Specifically, for example, technologies are available for transforming a large number of plant species, including dicotyledonous species (e g tobacco, potato, tomato. Petunia, Brassica) Additionally, for example, strategies for the expression of foreign genes in plants are available Additionally still, regulatory sequences from plant genes have been identified that are serviceable for the construction of chimeric genes that can be functionally expressed in plants and in plant cells (e g Klee et al , 1987, Clark et al , 1990, Smith et al , 1990)
The introduction of gene constucts mto plants can be achieved using several technologies including transformation with Agi obacterium tumejaciens or Agrobacterium thizogenes Non-limiting examples of plant tissues that can be transformed thusly include protoplasts, microspores or pollen, and explants such as leaves, stems, roots, hypocotyls, and cotyls Furthermore, DNA can be introduced directly into protoplasts and plant cells or tissues by microinjection, electnporation, particle bombardment, and direct DNA uptake
Proteins may be produced in plants by a variety of expression systems For instance, the use of a constitutive promoter such as the 35S promoter of Cauliflower Mosaic ims (Guilley et al , 1982) is serviceable for the accumulation of the expressed protein in virtually all organs of the transgenic plant Alternatively, the use of promoters that are highly tissue-specific and/or stage-specific are serviceable for this invention (Higgins, 1984, Shotwell, 1989) in order to bias expression towards desired tissues and/or towards a desired stage of development Further details relevant to the expression in plants of the phytase molecules of the instant invention are disclosed, for example, in USPN 5,770,413 (Van Ooijen et al ) and USPN 5,593,963 (Van Ooijen et al ), although these reference do not teach the inventive molecules of the instant application and instead teach the use of fungal phytases
In sum, it is relevant to this invention that a variety of means can be used to achieve the recombinant expression of phytase in a transgenic plant or plant part Such a transgenic plants and plant parts are serviceable as sources of recombinantly expressed phytase, which can be added directly to phytate-containing sources Alternatively, the recombinant plant-expressed phytase can be extracted away from the plant source and, if desired, purified prior to contacting the phytase substrate
6.2.4 - Examples of serviceable plants: Within the context of the present invention, plants to be selected include, but are not limited to crops producing edible flowers such as cauliflower (Brassica oleracea), artichoke (Cynara scolvmus), fruits such as apple (Malus, e g domesticus), banana (Musa, e g acuminata), bernes (such as the cuπant, Ribes, e g rubrum), cheπies (such as the sweet cherry, Prunus, e g avium), cucumber (Cucumis, e g sativus), grape (Vitis, e g vimfera), lemon (Citrus limon), melon (Cucumis melo), nuts (such as the walnut, Juglans, e g t egia, peanut, Arachis hypogeae), orange (Citrus, e g maxima), peach (Prunus, e g persica), pear (Pyra, e g communis), plum (Prunus, e g domestica), strawberry (Fragaπa, e g moschata), tomato (Lycopersicon, e g esculentum), leafs, such as alfalfa (Medicago, e g sativa), cabbages (e g Brassica oleracea), endive (Cichoreum, e g endivia), leek (Album, e g porrum), lettuce (Lactuca, e g sativa), spinach (Spinacia, e g oleraceae), tobacco (Nicotiana, e g tabacum), roots, such as aπowroot (Maranta, e g arundinacea), beet (Beta, e g vulgaris), caπot (Daucus, e g carota), cassava (Manihot, e g esculenta), turnip (Brassica, e g rapa), radish (Raphanus, e g sativus), yam (Dioscorea, e g esculenta), sweet potato (Ipomoea batatas) and seeds, such as bean (Phaseolus, e g vulgaris), pea (Pisum, e g sativum), soybean (Glycin, e g max), wheat (Tπticum, e g aestivum), barley (Hordeum, e g vulgaie), corn (Zea, e g mavs), rice (Oryza, e g sativa), rapeseed (Brassica napus), millet (Panicum L ), sunflower (Hebanthus annus), oats (Avena sativa), tubers, such as kohlrabi (Brassica, e g oleraceae), potato (Solanum, e g tuberosum) and the like
It is understood that additional plant as well as non-plant expression systems can be used within the context of this invention The choice of the plant species is primarily determined by the intended use of the plant or parts thereof and the amenability of the plant species to transformation 6.2.5 - Plant transformation methods: Several techniques are available for the introduction of the expression construct containing the phytase-encoding DNA sequence into the target plants Such techniques include but are not limited to transformation of protoplasts using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment (Potrykus, 1990) In addition to these so- called direct DNA transformation methods, transformation systems involving vectors are widely available, such as viral vectors (e g from the Cauliflower Mosaic Cirus (CaMV) and bacterial vectors (e g from the genus Agrobacteπum) (Potrykus, 1990) After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art (Horsch et al ,
1985) The choice of the transformation and/or regeneration techniques is not critical for this invention
6.2.6 - Methods for dicots: For dicots, a prefeπed embodiment of the present invention uses the principle of the binary vector system (Hoekema et al , 1983, EP
0120516 Schilperoort et al ) in which Agrobacteπum strains are used which contain a vir plasmid with the virulence genes and a compatible plasmid containing the gene construct to be transfeπed This vector can replicate in both E cob and in Agrobacteπum, and is derived from the binary vector Bin 19 (Bevan, 1984) which is altered in details that are not relevant for this invention The binary vectors as used in this example contain between the left- and right-border sequences of the T-DNA, an identical NPTII-gene coding for kanamycin resistance (Bevan, 1984) and a multiple cloning site to clone in the required gene constructs
6.2.7 - Methods for monocots: The transformation and regeneration of monocotyledonous crops is not a standard procedure However, recent scientific progress shows that in principle monocots are amenable to transformation and that fertile transgenic plants can be regenerated from transformed cells The development of reproducible tissue culmre systems for these crops, together with the powerful methods for introduction of genetic material into plant cells has facilitated transformation Presently the methods of choice for transformation of monocots are microprojectile bombardment of explants or suspension cells, and direct DNA uptake or electroporation of protoplasts For example, transgenic rice plants have been successfully obtained using the bacterial hph gene, encoding hygromycin resistance, as a selection marker The gene was introduced by electroporation (Shimamoto et al , 1993) Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin), into embryogemc cells of a maize suspension culmre by microparticle bombardment (Gordon- Kamm et al , 1990) The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee et al , 1989) Wheat plants have been regenerated from embryogemc suspension culmre by selecting only the aged compact and nodular embryogemc callus tissues for the establishment of the embryogemc suspension cultures (Vasil et al , 1972 Vasil et al , 1974) The combination with transformation systems for these crops enables the application of the present invention to monocots These methods may also be applied for the transformation and regeneration of dicots
6.2.8 - Methods for expression in plants: Expression of the phytase construct involves such details as transcription of the gene by plant polymerases, translation of mRNA, etc that are known to persons skilled in the art of recombinant DNA techniques Only details relevant for the proper understanding of this invention are discussed below Regulatory sequences which are known or are found to cause expression of phytase may be used in the present invention The choice of the regulatory sequences used depends on the target crop and/or target organ of interest Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized Such regulatory sequences are promoters active in directing transcription in plants, either constitutively or stage and or tissue specific, depending on the use of the plant or parts thereof These promoters include, but are not limited to promoters showing constitutive expression, such as the 35S promoter of Cauliflower Mosaic Virus (CaMV) (Guilley et al , 1982), those for leaf- specific expression, such as the promoter of the πbulose bisphosphate carboxylase small subunit gene (Coruzzi et al , 1984), those for root-specific expression, such as the promoter from the glutamin synthase gene (Tingey et al . 1987), those for seed-specific expression, such as the crucifeπn A promoter from Brassica napus (Ryan et al , 1989), those for tuber-specific expression, such as the class-I patatin promoter from potato (Koster-Topfer et al , 1989; Wenzler et al , 1989) or those for fruit-specific expression, such as the polygalacturonase (PG) promoter from tomato (Bird et al , 1988).
Other regulatory sequences such as terminator sequences and polyadenylation signals include any such sequence functioning as such in plants, the choice of which is within the level of the skilled artisan An example of such sequences is the 3' flanking region of the nopahne synthase (nos) gene of Agrobacterium tumefaciens (Bevan, supra). The regulatory sequences may also include enhancer sequences, such as found in the 35S promoter of CaMV, and mRNA stabilizing sequences such as the leader sequence of Alfalfa Mosaic Cirus (A1MV) RNA4 (Brederode et al , 1980) or any other sequences functioning in a like manner
The phytase should be expressed in an environment that allows for stability of the expressed protein. The choice of cellular compartments, such as cytosol, endoplasmic reticulum, vacuole, protein body or periplasmic space can be used in the present invention to create such a stable environment, depending on the biophysical parameters of the phytase. Such parameters include, but are not limited to pH-optimum, sensitivity to proteases or sensitivity to the molaπty of the prefeπed compartment.
To obtain expression in the cytoplasm of the cell, the expressed enzyme should not contain a secretory signal peptide or any other target sequence. For expression in chloroplasts and mitochondria the expressed enzyme should contain specific so-called transit peptide for import into these organelles. Targeting sequences that can be attached to the enzyme of interest in order to achieve this are known (Smeekens et al , 1990; van den Broeck et al , 1985, Wolter et al , 1988). If the activity of the enzyme is desired in the vacuoles a secretory signal peptide has to be present, as well as a specific targeting sequence that directs the enzyme to these vacuoles (Tague et al, 1990). The same is true for the protein bodies in seeds. The DNA sequence encoding the enzyme of interest should be modified in such a way that the enzyme can exert its action at the desired location in the cell
To achieve extracellular expression of the phytase, the expression construct of the present invention utilizes a secretory signal sequence Although signal sequences which are homologous (native) to the plant host species are prefeπed, heterologous signal sequences, i e those originating from other plant species or of microbial origin, may be used as well. Such signal sequences are known to those skilled in the art Appropriate signal sequences which may be used within the context of the present invention are disclosed in Blobel et al , 1979; Von Heijne, 1986, Garcia et al , 1987; Sijmons et al , 1990. Ng et al , 1994, and Powers et al , 1996).
All parts of the relevant DNA constructs (promoters, regulatory-, secretory-, stabilizing-, targeting-, or termination sequences) of the present invention may be modified, if desired, to affect their control characteristics using methods known to those skilled in the art. It is pointed out that plants containing phytase obtained via the present invention may be used to obtain plants or plant organs with yet higher phytase levels For example, it may be possible to obtain such plants or plant organs by the use of somoclonal variation techniques or by cross breeding techniques Such techniques are well known to those skilled in the art
6.2.9 - Dual expression of novel phytase & other molecules: In one embodiment, the instant invention provides a method (and products thereof) of achieving a highly efficient overexpression system for phytase and other molecules. In a prefeπed embodiment, the instant invention provides a method (and products thereof) of achieving a highly efficient overexpression system for phytase and pH 2.5 acid phosphatase in Tπchoderma. This system results in enzyme compositions that have particular utility in the animal feed industry Additional details regarding this approach are in the public literature and or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literature includes EP 0659215 (W0 9403612 Al) (Nevalainen et al ), although these reference do not teach the inventive molecules of the instant application.
6.2.10 - Soluble preparation of novel phytase & stabilized liquid formulations thereof: In one embodiment, the instant invention provides a method (and products thereof) of producing stabilized aqueous liquid formulations having phytase activity that exhibit increased resistance to heat inactivation of the enzyme activity and which retain their phytase activity during prolonged periods of storage. The liquid formulations are stabilized by means of the addition of urea and or a polyol such as sorbitol and glycerol as stabilizing agent. Also provided are feed preparations for monogastπc animals and methods for the production thereof that result from the use of such stabilized aqueous liquid formulations. Additional details regarding this approach are in the public literature and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes EP 0626010 (W0 9316175 Al) (Barendse et al ), although references in the publicly available literature do not teach the inventive molecules of the instant application.
6.3 - Use of Novel Phytase
6.3.1 - General uses, hydrolysis of phytate, & generation of inositol: In one embodiment, the instant invention provides a method of hydrolyzing phytate comprised of contacting the phytate with one or more of the novel phytase molecules disclosed herein Accordingly, the invention provides a method for catalyzing the hydrolysis of phytate to inositol and free phosphate with release of minerals from the phytic acid complex. The method includes contacting a phytate substrate with a degrading effective amount of an enzyme of the invention, such as the enzyme shown in SEQ ID NO:2 The term "degrading effective" amount refers to the amount of enzyme which is required to degrade at least 50% of the phytate, as compared to phytate not contacted with the enzyme. Preferably, at least 80%o of the phytate is degraded. In another embodiment, the invention provides a method for hydrolyzing phospho- mono-ester bonds in phytate The method includes administering an effective amount of phytase molecules of the invention (e g , SEQ ID NO 2), to yield inositol and free phosphate An "effective" amount refers to the amount of enzyme which is required to hydrolyze at least 50% of the phospho-mono-ester bonds, as compared to phytate not contacted with the enzyme Preferably, at least 80% of the bonds are hydrolyzed
In a particular aspect, when desired, the phytase molecules may be used in combination with other reagents, such as other catalysts, in order to effect chemical changes (e g hydrolysis) in the phytate molecules and/or in other molecules of the substrate source(s) According to this aspect, preferably the phytase molecules and the additional reagent(s) will not inhibit each other, more preferably the phytase molecules and the additional reagent(s) will have an overall additive effect, and more preferably still the phytase molecules and the additional reagent(s) will have an overall synergistic effect
Relevant sources of the substrate phytate molecules include foodsmffs, potential foodsmffs, byproducts of foodsmffs (both in vitro byproducts and in vivo byproducts, e g ex vivo reaction products and animal excremental products), precursors of foodsmffs, and any other matenal source of phytate
6.3.2 - Administration to organisms: In a non-limiting apsect, the recombinant phytase can be consumed by organisms and retains activity upon consumption In another exemplification, transgenic approches can be used to achieve expression of the recombinant phytase - preferably in a controlled fashion (methods are available for controlling expression of transgenic molecules in time-specific and tissue specific manners)
In a particular exemplification, the phytase activity in the source material (e g a transgenic plant source or a recombinant prokaryotic host) may be increased upon consumption, this increase in activity may occur, for example, upon conversion of a precursor phytase molecule in pro-form to a significantly more active enzyme in a more mamre form, where said conversion may result, for example, from the injestion and digestion of the phytase source Hydrolysis of the phytate substrate may occur at any time upon the contacting of the phytase with the phytate; for example, this may occur before injestion or after injestion or both before and after injestion of either the substrate or the enzyme or both It is additionally appreciated that the phytate substrate may be contacted with - in addition to the phytase - one or more additional reagents, such as another enzyme, which may be also be applied either directly or after purification from its source matenal.
It is appreciated that the phytase source mateπal(s) can be contacted directly with the phytate source mateπal(s); e g upon in vitro or in vivo grinding or chewing of either or both the phytase source(s) and the phytate source(s). Alternatively the phytase enzyme may be purified away from source matenal(s), or the phytate substrate may be purified away from source mateπal(s), or both the phytase enzyme and the phytate substrate may be purified away from source mateπal(s) pnor to the contacing of the phytase enzyme with the phytate substrate. It is appreciated that a combination of purified and unpunfied reagents - including enzyme(s) or substrates(s) or both - may be used.
It is appreciated that more than one source material may be used as a source of phytase activity This is serviceable as one way to achieve a timed release of reagent(s) from source mateπal(s), where release from different reagents from their source mateπals occur differentially, for example as injested source materials are digested in vivo or as source matenals are processed in in vitro applications. The use of more than one source material of phytase activity is also serviceable to obtain phytase activities under a range of conditions and fluctuations thereof, that may be encountered - such as a range of pH values, temperamres, salinities, and time intervals - for example during different processing steps of an application. The use of different source matenals is also serviceable in order to obtain different reagents, as exemplified by one or more forms or isomers of phytase and/or phytate &/or other materials It is appreciated that a single source material, such a trangemc plant species (or plant parts thereof), may be a source material of both phytase and phytate, and that enzymes and substrates may be differentially compartmentalized within said single source - e g secreted vs. non-secreted, differentially expressed &/or having differential abundances in different plant parts or organs or tissues or in subcellular compartments within the same plant part or organ or tissue. Purification of the phytase molecules contained therein may comprise isolating and/or further processing of one or more desirable plant parts or organs or tissues or subcellular compartments.
In a particular aspect, this invention provides a method of catalyzing in vivo and/or in vitro reactions using seeds containing enhanced amounts of enzymes. The method comprises adding transgenic, non-wild type seeds, preferably in a ground form, to a reaction mixmre and allowing the enzymes in the seeds to increase the rate of reaction. By directly adding the seeds to the reaction mixmre the method provides a solution to the more expensive and cumbersome process of extracting and purifying the enzyme.
Methods of treatment are also provided whereby an organism lacking a sufficient supply of an enzyme is administered the enzyme in the form of seeds from one or more plant species, preferably transgenic plant species, containing enhanced amounts of the enzyme. Additional details regarding this approach are in the public literature and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes USPN 5,543,576 (Van Ooijen et al) and USPN 5,714,474 (Van Ooijen et al ), although these reference do not teach the inventive molecules of the instant application and instead teach the use of fungal phytases.
In a particular non-limiting aspect, the instant phytase molecules are serviceable for generating recombinant digestive system life forms (or microbes or flora) and for the administration of said recombinant digestive system life forms to animals. Administration may be optionally performed alone or in combination with other enzymes &/or with other life forms that can provide enzymatic activity in a digestive system, where said other enzymes and said life forms may be may recombinant or otherwise. For example, administration may be performed in combination with xylanolytic bactena 6.3.3 - Steeping of cereals: In a non-limiting aspect, the present invention provides a method for steeping corn or sorghum kernels in warm water containing sulfur dioxide in the presence of an enzyme preparation comprising one or more phytin- degrading enzymes, preferably in such an amount that the phytin present in the com or sorghum is substantially degraded The enzyme preparation may compnse phytase and/or acid phosphatase and optionally other plant material degrading enzymes The steeping time may be 12 to 18 hours The steeping may be interrupted by an intermediate milling step, reducing the steeping time In a prefeπed embodiment, com or sorghum kernels are steeped in warm water containing sulfur dioxide in the presence of an enzyme preparation including one or more phytin-degradmg enzymes, such as phytase and acid phosphatases, to eliminate or greatly reduce phytic acid and the salts of phytic acid Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-limiting exemplification, such publicly available literamre includes USPN 4,914,029 (Caransa et al ) and EP 0321004 (Vaara et al ), although these reference do not teach the inventive molecules of the instant application
6.3.4 - Preparation of bread dough: In a non-hmiting aspect, the present invention provides a method to obtain a bread dough having desirable physical properties such as non-tackiness and elasticity and a bread product of superior quality such as a specific volume comprising adding phytase molecules to the bread dough In a prefeπed embodiment, phytase molecules of the instant invention are added to a working bread dough preparation that is subsequently formed and baked Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literamre includes JP 03076529 (Hara et al ), although this reference does not teach the inventive phytase molecules of the instant application
6.3.5 - Production of soybean-containing foodstuffs: In a non-limiting aspect, the present invention provides a method to produce improved soybean foodsmffs Soybeans are combined with phytase molecules of the instant invention to remove phytic acid from the soybeans, thus producing soybean foodsmffs that are improved in their supply of trace nutrients essential for consuming organisms and in its digestibility of proteins In a prefeπed embodiment, in the production of soybean milk, phytase molecules of the instant invention are added to or brought into contact with soybeans in order to reduce the phytic acid content In a non-limiting exemplification, the application process can be accelerated by agitating the soybean milk together with the enzyme under heating or by a conducting a mixing-type reaction in an agitation container using an immobilized enzyme Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-limiting exemplification, such publicly available literamre includes JP 59166049 (Kamikubo et al ), although this reference does not teach the inventive molecules of the instant application
6.3.6 - Production of liquid foodstuffs including sake: In one aspect, the instant invention provides a method of producing an admixture product for dπnking water or animal feed in fluid form, and which comprises using mineral mixtures and vitamin mixtures, and also novel phytase molecules of the instant invention In a prefeπed embodiment, there is achieved a coπectly dosed and composed mixmre of necessary nutrients for the consuming organism without any risk of precipitation and destruction of important minerals/vitamins, while at the same time optimum utilization is made of the phytin-bound phosphate in the feed Additional details regarding this approach are in the public literature and/or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literamre includes EP 0772978 (Bendixen et al ), although this reference does not teach the inventive molecules of the instant application
It is appreciated that the phytase molecules of the instant invention may also be used to produce other alcoholic and non-alcoholic drinkable foodsmffs (or dnnks) based on the use of molds &/or on grains &/or on other plants These drinkable foodsmffs mclude liquors, wines, mixed alcoholic drinks (e g wine coolers, other alcoholic coffees such as Irish coffees, etc ), beers, near-beers, juices, extracts, homogenates, and purees In a prefeπed exemplification, the instantly disclosed phytase molecules are used to generate transgenic versions of molds &/or grains &/or other plants serviceable for the production of such drinkable foodsmffs In another prefeπed exemplification, the instantly disclosed phytase molecules are used as additional ingredients in the manufacturing process &/or in the final content of such drinkable foodsmffs. Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan However - due to the novelty of the instant invention - references in the publicly available literamre do not teach the inventive molecules instantly disclosed
In another non-limiting exemplification, the present invention provides a means to obtain refined sake having a reduced amount of phytin and an increased content of inositol Such a sake may have - through direct &/or psychoge c effects - a preventive action on hepatic disease, arteriosclerosis, and other diseases. In a prefeπed embodiment, a sake is produced from rice Koji by multiplying a rice Koji mold having high phytase activity as a raw material. It is appreciated that the phytase molecules of the instant invention may be used to produce a serviceable mold with enhanced activity (preferably a transgenrc mold) &/or added exogenously to augment the effects of a Koji mold. The strain is added to boiled rice and Koji is produced by a conventional procedure In a prefeπed exemplification, the prepared Koji is used, the whole nee is prepared at two stages and Sake is produced at constant Sake temperamre of 15°C to give the objective refined Sake having a reduced amount of phytin and an increased amount of inositol. Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literamre includes JP 06153896 (Soga et al ) and JP 06070749 (Soga et al ), although these references do not teach the inventive molecules of the instant application
6.3.7 - Production of mineral absorbefacient: In a non-limiting aspect, the present invention provides a method to obtain an absorbefacient capable of promoting the absoφtion of minerals including ingested calcium without being digested by gastric juices or intestinal juices at a low cost In a prefeπed embodiment, said mineral absorbefacient contains a partial hydrolysate of phytic acid as an active ingredient Preferably, a partial hydrolyzate of the phytic acid is produced by hydrolyzing the phytic acid or its salts using novel phytase molecules of the instant invention The treatment with said phytase molecules may occur either alone &/or in a combination treatment (to inhibit or to augment the final effect), and is followed by inhibiting the hydrolysis within a range so as not to liberate all the phosphate radicals Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes JP 04270296 (Hoshino), although reference in the publicly available literamre do not teach the inventive molecules of the instant application.
6.3.8 - Use in combination with other phytases &/or acid phosphatases: In a non-hmiting aspect, the present invention provides a method (and products therefrom) to produce an enzyme composition having an additive or preferably a synergrstic phytate hydrolyzing activity; said composition comprises novel phytase molecules of the instant invention and one or more additional reagents to achieve a composition that is serviceable for a combination treatment In a prefeπed embodiment, the combination treatment of the present invention is achieved with the use of at least two phytases of different position specificity, i.e any combinations of 1-, 2-, 3-, 4-, 5-, and 6-phytases. By combining phytases of different position specificity an additive or synergistic effect is obtained. Compositions such as food and feed or food and feed additives comprising such phytases in combination are also included in this invention as are processes for their preparation. Additional details regarding this approach are in the public literature and/or are known to the skilled artisan. In a particular non-limiting exemplification, such publicly available literature includes W09 830681 (Ohmann et al ), although references in the publicly available literature do not teach the use of the inventive molecules of the instant application.
In another prefeπed embodiment, the combination treatment of the present invention is achieved with the use of an acid phosphatase having phytate hydrolyzing activity at a pH of 2 5, in a low ratio coπesponding to a pH 2.5 '5.0 activity profile of from about 0.1 1 0 to 10T , preferably of from about 0 5.1 0 to 5 1 , more preferably still of from about 0 8 1 0 to 3 1 , and more preferably still of from about 0 8 1 0 to 2 1 Said enzyme composition preferably displays a higher synergetic phytate hydrolyzing efficiency through thermal treatment Said enzyme composition is serviceable in the treatment of foodsmffs (drinkable and solid food, feed and fodder products) to improve phytate hydrolysis Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literamre includes USPN 5,554.399 (Vanderbeke et al ) and USPN 5,443,979 (Vanderbeke et al ), although these reference do not teach the use of the inventive molecules of the instant application, but rather teach the use of fungal (in particular Aspegillus) phytases
6.3.9 - Use in combination with enzymes that act on polysaccharides (e.g. xylanases) In a non-hmiting aspect, the present invention provides a method (and products therefrom) to produce composition comprised of the instant novel phytate-acting enzyme in combination with one or more additional enzymes that act on polysacchaπdes Such polysaccharides can be selected from the group consisting of arabinans, fmctans, fucans, galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan, caπageenan, galactocarolose, pectin, pectic acid, amylose, pullulan, glycogen, amylopectin, cellulose, carboxylmethylcellulose, hydroxypropylmethylcellulose, dextran, pustulan, chitin, agarose, keratan, chondroitin, dermatan, hyaluronic acid, alginic acid, and polysaccharides containing at least one aldose, ketose, acid or amine selected from the group consisting of erythrose, threose, πbose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythrulose, πbulose, xylulose, psicose, fructose, sorbose, tagatose, glucuromc acid, glucomc acid, glucaπc acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine and neuraminic acid
In a particular aspect, the present invention provides a method (and products therefrom) to produce composition having a synergistic phytate hydrolyzing activity comprising one or more novel phytase molecules of the instant invention, a cellulase (including preferably but not exclusively a xylanase), optionally a protease, and optionally one or more additonal reagents In prefeπed embodiments, such combination treatments are serviceable in the treatment of foodsmffs, wood products, such as paper products, and as cleansing solutions and solids
In one non-limiting exemplification, the instant phytase molecules are serviceable in combination with cellulosome components. It is known that cellulases of many cellulolytic bacteria are organized into discrete multienzyme complexes, called cellulosomes. The multiple subunits of cellulosomes are composed of numerous functional domains, which interact with each other and with the cellulosic substrate One of these subunits comprises a distinctive new class of noncatalytic scaffolding polypeptide, which selectively integrates the various cellulase and xylanase subunits into the cohesive complex. Intelligent application of cellulosome hybrids and chimeric constructs of cellulosomal domains should enable better use of cellulosic biomass and may offer a wide range of novel applications in research, medicine and industry.
In another non-hmitmg exemplification, the instant phytase molecules are serviceable - either alone or in combination treatments - in areas of biopulpmg and biobleaching where a reduction in the use of environmentally harmful chemicals traditionally used in the pulp and paper industry is desired Waste water treatment represents another vast application area where biological enzymes have been shown to be effective not only in colour removal but also in the bioconversion of potentially noxious substances into useful bioproducts.
In another non-hmiting exemplification, the instant phytase molecules are serviceable for generating life forms that can provide at least one enzymatic activity - either alone or in combination treatments - in the treatment of digestive systems of organisms. Particularly relevant organisms to be treated include non-ruminant organisms. Specifically, it is appreciated that this approach may be performed alone or in combination with other biological molecules (for example, xylanases) to generate a recombinant host that expresses a plurality of biological molecules. It is also appreciated that the administration of the instant phytase molecules &/or recombinant hosts expressing the instant phytase molecules may be performed either alone or in combination with other biological molecules, &/or life forms that can provide enzymatic activities in a digestive system - where said other enzymes and said life forms may be may recombinant or otherwise. For example, administration may be performed in combination with xylanolytic bacteria
For example, in addition to phytate, many organisms are also unable to adequately digest hemicelluloses Hemicelluloses or xylans are major components (35%) of plant materials For ruminant animals, about 50% of the dietary xylans are degraded, but only small amounts of xylans are degraded in the lower gut of nonrumrnant animals and humans. In the rumen, the major xylanolytic species are Butvrivibno fibrisolvens and Bacteroides rumimcola In the human colon, Bacteroides ovatus and Bacteroides fragilis subspecies "a" are major xylanolytic bacteria Xylans are chemically complex, and their degradation requires multiple enzymes. Expression of these enzymes by gut bacteria varies greatly among species. Butyπvibπo fibrisolvens makes extracellular xylanases but Bacteroides species have cell-bound xylanase activity. Biochemical characterization of xylanolytic enzymes from gut bacteria has not been done completely. A xylosidase gene has been cloned from B fibrosolvens 1 13. The data from DNA hybridizations using a xylanase gene cloned from B. fibrisolvens 49 indicate this gene may be present in other B. fibrisolvens strains A cloned xylanase from Bact. rumimcola was transfeπed to and highly expressed in Bact fragilis and Bact. umformis. Arabinosidase and xylosidase genes from Bact. ovatus have been cloned and both activities appear to be catalyzed by a single, bifunctional. novel enzyme
Accordingly, it is appreciated that the present phytase molecules are serviceable for 1) transfemng into a suitable host (such as Bact. fragilis or Bact. umformis), 2) achieving adequate expression in a resultant recombinant host; and 3) administering said recombinant host to organisms to improve the ability of the treated organisms to degrade phytate. Continued research in genetic and biochemical areas will provide knowledge and insights for manipulation of digestion at the gut level and improved understanding of colonic fiber digestion Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literamre includes USPN 5,624,678 (Bedford et al ). USPN 5,683,91 1 (Bodie et al ), USPN 5,720,971 (Beauchemin et al ), USPN 5,759,840 (Sung et al ), USPN 5,770,012 (Cooper), USPN 5,786.316 (Baeck et al ), USPN 5,817,500 (Hansen et al ), and journal articles (Jeffries, 1996, Prade, 1996, Bayer et al , 1994, Duarte et al , 1994, Hespell & Whitehead, 1990, Wong et al , 1988), although these reference do not teach the inventive phytase molecules of the instant application, nor do they all teach the addition of phytase molecules in the production of foodsmffs, wood products, such as paper products, and as cleansing solutions and solids In contrast, the instant invention teaches that phytase molecules - preferably the inventive phytase molecules of the instant application - may be added to the reagent(s) disclosed in order to obtain preparations having an additional phytase activity Preferably, said reagent(s) the additional phytase molecules and will not inhibit each other, more preferably said reagent(s) the additional phytase molecules will have an overall additive effect, and more preferably still said reagent(s) the additional phytase molecules will have an overall synergistic effect
6.3.10 - Use in combination with vitamin D: In a non-limiting aspect, the present invention provides a method (and products therefrom) for enhancement of phytate phosphorus utilization and treatment and prevention of tibial dyschondroplasia in animals, particularly poultry, by administering to animals a feed composition containing a hydroxylated vitamin D3 derivative The vitamin D3 derivative is preferably administered to animals in feed containing reduced levels of calcium and phosphorus for enhancement of phytate phosphorus utilization Accordingly, the vitamin D3 derivative is preferably administered in combination with novel phytase molecules of the instant invention for further enhancement of phytate phosphorus utilization Additional details regarding this approach are in the public literamre and/or are known to the skilled artrsan In a particular non-limiting exemplification, such publicly available literature includes USPN 5,516,525 (Edwards et al ) and USPN 5,366.736 (Edwards et al ), USPN 5,316,770 (Edwards et al ) although these reference do not teach the inventive molecules of the instant application 6.3.11 - Use in combination with lactic acid-producing bacteria: In a non- limiting aspect, the present invention provides a method (and products therefrom) to obtain foodstuff that 1) comprises phytin that is easily absorbed and utilized in a form of inositol in a body of an organism, 2) that is capable of reducing phosphorus in excrementary matter; and 3) that is accordingly useful for improving environmental pollution. Said foodstuff is comprised of an admixture of a phytin-contaimng grain, a lactic acid-producing microorganism, and a novel phytase molecule of the instant invention. In a prefeπed embodiment, said foodstuff is produced by compounding a phytin- containing grain (preferably, e.g rice bran) with an effective microbial group having an acidophilic property, producing lactic acid, without producing butyric acid, free from pathogenicity, and a phytase. Examples of an effective microbial group include e g Streptomyces sp. (ATCC 3004) belonging to the group of actinomyces and Lactobacillus sp (IFO 3070) belonging to the group of lactobacilh Further, a preferable amount of addition of an effective microbial group is 0.2 wt. % in terms of bactenal body weight based on a grain material. Furthermore, the amount of the addition of the phytase is preferably 1-2 wt. % based on the phytin in the grain material. Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-hmiting exemplification, such publicly available literamre includes JP 08205785 (Akahoπ et al ), although references in the publicly available literamre do not teach the inventive molecules of the instant application.
6.3.12 - Solubilization of proteins in combination with proteases: In a non- hmiting aspect, the present invention provides a method for improving the solubility of vegetable proteins. More specifically, the invention relates to methods for the solubilization of proteins in vegetable protein sources, which methods comprise treating the vegetable protein source with an efficient amount of one or more phytase enzymes - including phytase molecules of the instant invention - and treating the vegetable protein source with an efficient amount of one or more proteolytic enzymes. In another aspect, the invention provides animal feed additives compnsmg a phytase and one or more proteolytic enzymes Additional details regarding this approach are in the public literamre and/or are known to the skilled artisan. In a particular non-hmiting exemplification, such publicly available literamre includes EP 0756457 (WO 9528850 Al) (Nielsen and Knap), although references in the publicly available literamre do not teach the inventive molecules of the instant application
In a non-limiting aspect, the present invention provides a method of producing a plant protein preparation comprising dispersing vegetable protein source matenals in water at a pH in the range of 2 to 6 and admixing phytase molecules of the instant invention therein The acidic extract containing soluble protein is separated and dried to yield a solid protein of desirable character One or more proteases can also be used to improve the characteristics of the protein Additional details regarding this approach are in the public literamre and or are known to the skilled artisan In a particular non-limiting exemplification, such publicly available literamre includes USPN 3.966,971 (Morehouse et al ), although references in the publicly available literamre do not teach the inventive molecules of the instant application
6.3.13 - Triple treatment of compost using novel phytase, saponin, and chitosan: In a non-limiting aspect, the present invention provides a method (and products thereof) to activate inert phosphorus in soil and/or compost, to improve the utilization rate of a nitrogen compound, and to suppress propagation of pathogenic molds by adding three reagents, phytase, saponin and chitosan, to the compost In a non-limiting embodiment the method can comprise treating the compost by 1) adding phytase-contaimng microorganisms in media - preferably recombinant hosts that overexpress the novel phytase molecules of the instant invention - e g at 100 ml media/ 100 kg wet compost, 2) alternatively also adding a phytase-contaimng plant source - such as wheat bran - e g at 0 2 to 1 kg/100 kg wet compost, 3) adding a saponin-containing source - such as peat, mugworts and yucca plants - e g at 0 5 to 3 Og/kg , 4) adding chitosan-containing matenals - such as pulverized shells of shrimps, crabs, etc - e g at 100 to 300g/kg wet compost In another non-limiting embodiment, recombinant sources the three reagents, phytase. saponin. and chitosan, are used Additional details regarding this approach are m the public literamre and or are known to the skilled artisan In a particular non-hmiting exemplification, such publicly available literamre includes JP 07277865 (Toya Taisuke), although references in the publicly available literamre do not teach the inventive molecules of the instant application
6.3.14 - Use as hybridization probes & amplification templates: Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA or a genomic library to isolate the full length DNA and to isolate other DNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type have at least 10, preferably at least 15, and even more preferably at least 30 bases and may contain, for example, at least 50 or more bases. The probe may also be used to identify a DNA clone coπesponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns.
The present invention provides methods for identifying nucleic acid molecules that encode members of the phytase polypeptide family in addition to SEQ ID NO: l . In these methods, a sample, e.g., a nucleic acid library, such as a cDNA library, that contains a nucleic acid encoding a phytase polypeptide is screened with a phytase-specific probe, e g., a phytase-specific nucleic acid probe Phytase-specific nucleic acid probes are nucleic acid molecules (e.g , molecules containing DNA or RNA nucleotides, or combinations or modifications thereof) that specifically hybridize to nucleic acids encoding phytase polypeptides, or to complementary sequences thereof The term "phytase-specific probe," in the context of this method of invention, refers to probes that bind to nucleic acids encoding phytase polypeptides, or to complementary sequences thereof, to a detectably greater extent than to nucleic acids encoding other enzymes, or to complementary sequences thereof.
The invention facilitates production of phytase-specific nucleic acid probes. Methods for obtaining such probes can be designed based on the amino acid sequences shown in Figure 1 The probes, which can contain at least 12, e g ,at least 15, 25, 35, 50, 100, or 150 nucleotides, can be produced using any of several standard methods (see, e g., Ausubel et al , supra) For example preferably, the probes are generated using PCR amplification methods In these methods, primers are designed that coπespond to phytase- conserved sequences (see Figure 1), which can include phytase-specific amino acids, and the resulting PCR product is used as a probe to screen a nucleic acid library, such as a cDNA library
This invention can be used to isolate nucleic acid sequences substantially similar to the isolated nucleic acid molecule encoding an phytase enzyme disclosed in Figure 1 (SEQ ID NO 1) Isolated nucleic acid sequences are substantially similar if (I) they are capable of hybridizing under stnngent conditions, hereinafter described, to SEQ ID NO 1 , or (n) they encode a phytase polypeptide as set forth in SEQ ID NO 2 due to the degeneracy of the genetic code (e g Regenerate to SEQ ID NO 1 )
Degenerate DNA sequences encode the amino acid sequence of SEQ ID NO 2, but have vanations in the nucleotide coding sequences As used herein, "substantially similar" refers to the sequences having similar identity to the sequences of the instant invention The nucleotide sequences that are substantially similar can be identified by hybridization or by sequence comparison Enzyme sequences that are substantially similar can be identified by one or more of the following proteolytic digestion, gel electrophoresis and/or microsequencing
One means for isolating a nucleic acid molecule encoding a phytase enzyme is to probe a genomic gene library with a natural or artificially designed probe using art recognized procedures (see, e g , Ausubel et al , supra) It is appreciated to one skilled in the art that SEQ ID NO 1 , or fragments thereof (comprising at least 15 contiguous nucleotides), is a particularly useful probe Other particular useful probes for this puφose are hybndizable fragments to the sequences of SEQ ID NO 1 (/ e , comprising at least 15 contiguous nucleotides)
It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other animal sources or to screen such sources for related sequences.
With respect to nucleic acid sequences which hybridize to specific nucleic acid sequences disclosed herein, hybndization may be earned out under conditions of reduced stringency, medium stringency or even stringent conditions As an example of oligonucleotide hybridization, a polymer membrane containing immobilized denatured nucleic acid is first prehybπdized for 30 minutes at 45°C in a solution consisting of 0 9 M NaCI, 50 mM NaH:P04, pH 7 0, 5 0 mM Na2EDTA, 0.5% SDS, 10X Denhardt's. and 0 5 mg/mL polyπboadenyhc acid Approximately 2 X 10" cpm (specific activity 4-9 X 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution After 12- 16 hours of incubation, the membrane is washed for 30 minutes at room temperamre in IX SET (150 mM NaCI, 20 mM Tris hydrochloπde, pH 7 8, 1 mM Na,EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm-10°C for the oligonucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybndization signals.
The nucleic acid molecules of the invention can be used as templates in standard methods for production of phytase gene products (e g , phytase RNAs and phytase polypeptides ). In addition, the nucleic acid molecules that encode phytase polypeptides (and fragments thereof) and related nucleic acids - such as (1) nucleic acids containing sequences that are complementary to, or that hybridize to, nucleic acids encoding phytase polypeptides, or fragments thereof (e g , fragments containing at least 12, 15, 20, or 25 nucleotides), and (2) nucleic acids containing sequences that hybridize to sequences that are complementary to nucleic acids encoding phytase polypeptides, or fragments thereof (e g , fragments containing at least 12, 15, 20, or 25 nucleotides) - can be used in methods focused on their hybridization properties For example, as is descπbed in further detail herein, such nucleic acid molecules can be used in the following methods PCR methods for synthesizing phytase nucleic acids, methods for detecting the presence of a phytase nucleic acid in a sample, screening methods for identifying nucleic acids encoding new phytase family members Hybridization-based uses include Southern-type, Northern-type, RNA protection, and any hybridization procedure were a nucleic acid is used as a hybridization partner.
Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention. Accordingly, fragments or portions of the enzymes of the present invention may be employed for producing the coπesponding full-length enzyme by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length enzymes. Size separation of the cleaved fragments is generally performed using 8 percent polyacrylamide gel as descnbed in the literamre (e g by Goeddel et al , 1980)
6.3.15 - Use in Directed Evolution: This invention provides enzymes, as well as fragments, other derivatives, and analogs thereof, and the coπesponding nucleotides for use in directed evolution. The discovery and use of a plurality of templates as disclosed herein may significantly increase the potential yield of directed evolution in companson to the directed evolution of a single template protein. Hence, the need for discovery is based on the premise that nature provides a wealth of potentially unattainable or unpredictable feamres in distinct but related members of molecular groupings, and that the exploitation of these feamres may greatly facilitate directed evolution. Thus, in one aspect, related but distinct molecules may serve as unique starting templates for the directed evolution of a desired charactenstic In another aspect, they may serve as repositories of structure- function information including, but not limited to, a variety of consensus motifs. Both utilities help to obviate the logistically impractical task of at-once exploring an overly wide range of mutational permutations on any given molecule. For example, the full range of mutational permutations on a 100 amino acid protein includes over 10'30 possibilities (assuming there are 20 amino acid possibilities at each position), a number too large for practical consideration
Accordingly, particularly because of logistical and technical constraints, it is a desirable approach - in performing "directed evolution" - to discover and to make use of a plurality of related starting templates that have pre-evolved differences. These templates can then be subjected to a variety of mutagenic manipulations including, by way of non- hmiting exemplification, DNA mutagenesis and combinatorial enzyme development, an approach that is further elaborated in co-pending USPN 5,830,696 (Short et al ).
The enzyme activities of the novel molecules generated can then be screened by a vanety of methods including, by way of non-hmiting exemplification: a) molecular biopanning; b) recombinant clone screening, and c) extract screening.
6.3.16 - Use in antibody production: This invention provides enzymes, as well as fragments, other derivatives, and analogs thereof, and cells expressing them that can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimenc, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Vanous procedures known in the art may be used for the production of such antibodies and fragments
Antibodies generated against the enzymes coπesponding to a sequence of the present invention can be obtained by direct injection of the enzymes into an animal or by administering the enzymes to an animal, preferably a nonhuman The antibody so obtained will then bind the enzymes itself. In this manner, even a sequence encoding only a fragment of the enzymes can be used to generate antibodies binding the whole native enzymes. Such antibodies can then be used to isolate the enzyme from cells expressing that enzyme.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybndoma technique (Kohler and Milstein, 1975), the tπoma technique, the human B-cell hybndoma technique (Kozbor et al , 1983), and the EBV-hybπdoma technique to produce human monoclonal antibodies (Cole et al . 1985, pp 77-96). Techniques descπbed for the production of single chain antibodies (USPN 4,946 778 Ladner et al ) can be adapted to produce single chain antibodies to lmmunogenic enzyme products of this invention Also, transgenic mice may be used to express humanized antibodies to lmmunogenic enzyme products of this invention
Antibodies generated against the enzyme of the present invention may be used in screening for similar enzymes from other organisms and samples Such screening techniques are known in the art Antibodies may also be employed as a probe to screen gene libraries generated from this or other organisms to identify this or cross reactive activities
Isolation and purification of polypeptides produced in the systems described above can be earned out using conventional methods, appropriate for the particular system For example, preparative chromatography and immunological separations employing antibodies, such as monoclonal or polyclonal antibodies, can be used
As is mentioned above, antigens that can be used in producing phytase-specific antibodies include phytase polypeptides, e g , any of the phytase shown in Figures 1 polypeptide fragments The polypeptide or peptide used to immunize an animal can be obtained by standard recombinant, chemical synthetic, or purification methods As is well known in the art, in order to increase immunogenicity, an antigen can be conjugated to a earner protein Commonly used earners include keyhole limpet hemocyamn (KLH), thyroglobuhn, bovine serum albumin (BSA), and tetanus toxoid The coupled peptide is then used to immunize the animal (e g , a mouse, a rat, or a rabbit) In addition to such carriers, well known adjuvants can be administered with the antigen to facilitate induction of a strong immune response
Phytase-specific polvclonal and monoclonal antibodies can be purified, for example, by binding to and elution from, a matrix containing a phytase polypeptide, e g , the phytase polypeptide (or fragment thereof) to which the antibodies were raised
Additional methods for antibody purification and concentration are well known in the art and can be practiced with the phytase-specific antibodies of the invention (see. e.g , Cohgan et al , 1996)
Anti-idiotype antibodies coπesponding to phytase-specific antigens are also included in the invention, and can be produced using standard methods These antibodies are raised to phytase-specific antibodies, and thus mimic phytase-specific epitopes. This invention also includes additonal uses of fragments of the phytase polypeptides that retain at least one phytase-specific activity or epitope. Phytase activity can be assayed by examining the catalysis of phytate to inositol and free phosphate. Such fragments can easily be identified by companng the sequences of phytases found in Figure 1
In a non-limiting exemplification, a phytase polypeptide fragment containing, e g , at least 8-10 amino acids can be used as an immunogen in the production of phytase- specific antibodies. The fragment can contain, for example, an amino acid sequence that is conserved in phytases, and this amino acid sequence can contain amino acids that are conserved in phytases. In another non-limiting exemplification,, the above-described phytase fragments can be used in immunoassays, such as ELISAs, to detect the presence of phytase-specific antibodies in samples.
6.3.17 - Use in transgenics:
Various methods to make the transgenic animals of the subject invention can be employed. Generally speaking, three such methods may be employed. In one such method, an embryo at the pronuclear stage (a "one cell embryo") is harvested from a female and the transgene is microinjected into the embryo, in which case the transgene will be chromosomally integrated into both the germ cells and somatic cells of the resulting mature animal. In another such method, embryonic stem cells are isolated and the transgene incoφorated therein by electroporation, plasmid transfection or microinjection, followed by reintroduction of the stem cells into the embryo where they colonize and contribute to the germ line. Methods for microinjection of mammalian species is described in U.S. Pat. No. 4,873.191 In yet another such method, embryonic cells are infected with a retrovirus containing the transgene whereby the germ cells of the embryo have the transgene chromosomally integrated therein When the animals to be made transgenic are avian, because avian fertilized ova generally go through cell division for the first twenty hours in the oviduct, microinjection into the pronucleus of the fertilized egg is problematic due to the inaccessibility of the pronucleus. Therefore, of the methods to make transgenic animals described generally above, retrovims infection is prefeπed for avian species, for example as described in U.S Pat No 5,162,215. If micro-injection is to be used with avian species, however, a published procedure by Love et al , (Biotechnology, 12, Jan 1994) can be utilized whereby the embryo is obtained from a sacrificed hen approximately two and one-half hours after the laying of the previous laid egg, the transgene is microinjected into the cytoplasm of the germinal disc and the embryo is cultured in a host shell until matunty When the animals to be made transgenic are bovine or porcine, microinjection can be hampered by the opacity of the ova thereby making the nuclei difficult to identify by traditional differential interference-contrast microscopy. To overcome this problem, the ova can first be centrifuged to segregate the pronuclei for better visualization.
The "non-human animals" of the invention bovine, porcine, ovine and avian animals (e.g., cow, pig, sheep, chicken). The "transgenic non-human animals" of the invention are produced by introducing "transgenes" into the germhne of the non-human animal. Embryonal target cells at vanous developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. The use of zygotes as is target for gene transfer has a major advantage in that in most cases the injected DNA will be incoφorated into the host gene before the first cleavage (Bnnster et al, Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence, all cells of the transgenic non- human animal will carry the incoφorated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
The term "transgenic" is used to describe an animal which includes exogenous genetic material within all of its cells A "transgenic" animal can be produced by crossbreeding two chimenc animals which include exogenous genetic matenal within cells used in reproduction Twenty-five percent of the resulting offspring will be transgenic i e., animals which include the exogenous genetic material within all of their cells in both alleles, 50% of the resulting animals will include the exogenous genetic material withm one allele and 25% will include no exogenous genetic material
In the microinjection method useful in the practice of the subject invention, the transgene is digested and purified free from any vector DNA, e g , by gel electrophoresis It is prefeπed that the transgene include an operatively associated promoter which interacts with cellular proteins involved in transcription, ultimately resulting in constitutive expression Promoters useful in this regard include those from cytomegalovirus (CMV) , Moloney leukemia virus (MLV), and heφes virus, as well as those from the genes encoding metallothionin, skeletal actin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine kinase. Promoters for viral long terminal repeats (LTRs) such as Rous Sarcoma Virus can also be employed. When the animals to be made transgenic are avian, prefeπed promoters include those for the chicken β-globin gene, chicken lysozyme gene, and avian leukosis virus. Constructs useful in plasmid transfection of embryonic stem cells will employ additional regulatory elements well known in the art such as enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, and ribosome binding sites to permit translation
Retroviral infection can also be used to introduce transgene into a non-human animal, as described above The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R , Proc Natl Acad. Sci USA 73 1260- 1264, 1976). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida
(Hogan. et al (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y ) The viral vector system used to introduce the transgene is typically a replication-defective retro virus carrying the transgene (Jahner, et al , Proc Natl Acad. Sci USA 82 6927-6931, 1985, Van der Putten, et al , Proc. Natl. Acad Sci USA 82' 6148-6152, 1985) Transfection is easily and efficiently obtained by culmnng the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al, EMBO J. 6: 383-388, 1987). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (D. Jahner et al , Nature 298: 623-628, 1982). Most of the founders will be mosaic for the transgene since incoφoration occurs only in a subset of the cells which formed the transgenic nonhuman animal. Further, the founder may contain various retro viral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by lntrauteπne retroviral infection of the midgestation embryo (D. Jahner et al, supra).
A third type of target cell for transgene introduction is the embryonal stem cell (ES) ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al, Namre 292: 154-156, 1981 ; M. O. Bradley et al, Namre 309:255-258, 1984; Gossler, et al, Proc. Natl. Acad. Sci USA 83:9065-9069, 1986; and Robertson et al, Namre 322:445-448, 1986). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimenc animal. (For review see Jaenisch, R., Science 240: 1468-1474, 1988).
"Transformed" means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid molecule. "Heterologous" refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form pπmanly expressed in the cell.
"Transgene" means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i.e., either stably integrated or as a stable extrachromosomal element) which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Included within this definition is a transgene created by the providing of an RNA sequence which is transcnbed into DNA and then incoφorated into the genome The transgenes of the invention include DNA sequences which encode phytases or polypeptides having phytase activity, and include polynucleotides, which may be expressed in a transgenic non-human animal The term "transgenic" as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout. The term "gene knockout" as used herein, refers to the targeted disruption of a gene in vivo with complete loss of function that has been achieved by any transgenic technology familiar to those in the art In one embodiment, transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination As used herein, the term "transgenic" includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or "knocked out."
The transgene to be used the practice of the subject invention is a DNA sequence compnsmg a sequence coding for a phytase or a polypeptide having phytase activity. In a one embodiment, a polynucleotide having a sequence as set forth in SEQ ID NO 1 or a sequence encoding a polypeptide having a sequence as set forth in SEQ ID NO 2 is the transgene as the term is defined herein. Where appropriate. DNA sequences that encode proteins having phytase activity but differ in nucleic acid sequence due to the degeneracy of the genetic code may also be used herein, as may truncated forms, allelic vanants and interspecies homologues.
After an embryo has been microinjected, colonized with transfected embryonic stem cells or infected with a retrovims containing the transgene (except for practice of the subject invention in avian species which is addressed elsewhere herein) the embryo is implanted into the oviduct of a pseudopregnant female The consequent progeny are tested for incoφoration of the transgene by Southern blot analysis of blood or tissue samples using transgene specific probes. PCR is particularly useful in this regard Positive progeny (GO) are crossbred to produce offspring (Gl) which are analyzed for transgene expression by Northern blot analysis of tissue samples
Thus, the present invention includes methods for increasing the phosphorous uptake in the transgenic animal and/or decreasing the amount of polltant in the manure of the transgenic organism by about 15%, typically about 20%o, and more typically about 20% to about 50%
The animals contemplated for use in the practice of the subject invention are those animals generally regarded as domesticated animals including pets (e g., canines, felines, avian species etc.) and those useful for the processing of food stuffs, i e , avian such as meat bred and egg laying chicken and turkey, ovme such as lamb, bovine such as beef cattle and milk cows, piscine and porcine For puφoses of the subject invention, these animals are refeπed to as "transgenic" when such animal has had a heterologous DNA sequence, or one or more additional DNA sequences normally endogenous to the animal (collectively refeπed to herein as "transgenes") chromosomally integrated into the germ cells of the animal. The transgenic animal (including its progeny) will also have the transgene fortuitously integrated into the chromosomes of somatic cells.
6.3.18 - Uses in gene delivery:
In some instances it may be advantageous to deliver and express a phytase sequence of the invention locally (e g , within a particular tissue or cell type) For example, local expression of a phytase or digestive enzyme in the gut of an animal will assist in the digestion and uptake of, for example, phytate and phosporous, respectively The nucleic sequence may be directly delivered to the salivary glands, tissue and cells and/or to the epithelial cells lining the gut, for example Such delivery methods are known in the art and include electroporation, viral vectors and direct DNA uptake. Any polypeptide having phytase activity can be utilized in the methods of the invention (e g , those specficially described under this subsection 6 3 18, as well as those described in other sections of the invention) For example, a nucleic acid constructs of the present invention will comprise nucleic acid molecules in a form suitable for uptake into target cells within a host tissue The nucleic acids may be in the form of bare DNA or RNA molecules, where the molecules may comprise one or more structural genes, one or more regulatory genes, antisense strands, strands capable of tnplex formation, or the like Commonly, the nucleic acid construct will include at least one structural gene under the transcriptional and translational control of a suitable regulatory region More usually, nucleic acid constructs of the present invention will comprise nucleic acids incoφorated in a delivery vehicle to improve transfection efficiency, wherein the delivery vehicle will be dispersed within larger particles comprising a dried hydrophilic excipient material
One such delivery vehicles comprises viral vectors, such as retroviruses, adenoviruses, and adeno-associated viruses, which have been inactivated to prevent self- replication but which maintain the native viral ability to bind a target host cell, deliver genetic matenal into the cytoplasm of the target host cell, and promote expression of structural or other genes which have been incoφorated in the particle. Suitable retrovims vectors for mediated gene transfer are described in Kahn et al. (1992) CIRC. RES. 71- 1508-1517, the disclosure of which is incoφorated herein by reference. A suitable adenovims gene delivery is described in Rosenfeld et al (1991 ) SCIENCE 252 431-434, the disclosure of which is incoφorated herein by reference Both retroviral and adenovims delivery systems are described in Friedman (1989) SCIENCE 244 1275-1281 , the disclosure of which is also incoφorated herein by reference.
A second type of nucleic acid delivery vehicle comprises hposomal transfection vesicles, including both anionic and cationic hposomal constructs. The use of anionic hposomes requires that the nucleic acids be entrapped within the hposome. Cationic hposomes do not require nucleic acid entrapment and instead may be formed by simple mixing of the nucleic acids and hposomes. The cationic hposomes avidly bind to the negatively charged nucleic acid molecules, including both DNA and RNA, to yield complexes which give reasonable transfection efficiency in many cell types See, Farhood et al (1992) BIOCHEM BIOPHYS ACTA Hil 239-246, the disclosure of which is incoφorated herein by reference A particularly prefeπed material for forming hposomal vesicles is hpofectin which is composed of an equimolar mixmre of dioleylphosphatidyl ethanolamine (DOPE) and dioleyloxypropyl-tπethylammomum (DOTMA), as described in Feigner and Ringold (1989) NATURE 337 387-388, the disclosure of which is incoφorated herein by reference.
It is also possible to combine these two types of delivery systems. For example, Kahn et al (1992), supra . teaches that a retrovims vector may be combined in a cationic DEAE-dextran vesicle to further enhance transformation efficiency. It is also possible to incoφorate nuclear proteins into viral and or hposomal delivery vesicles to even further improve transfection efficiencies. See. Kaneda et al ( 1989) SCIENCE 243-375-378, the disclosure of which is incoφorated herein by reference
6.3.19 - Use in dietary aids: In another embodiment, a digestive aid containing an enzyme either as the sole active ingredient or in combination with one or more other agents and/or enzymes is provided (as descπbed in co-pending application U.S. Serial No , entitled
"Dietary Aids and Methods of Use Thereof," filed May 25, 2000, the disclosure of which is incoφorated herein by reference in its entirety) The use of enzymes and other agents in digestive aids of livestock or domesticated animals not only improves the animal's health and life expectancy but also assists in increasing the health of livestock and in the production of foodstuffs from livestock
Cuπently, some types of feed for livestock (e g , certain poultry feed) are highly supplemented with numerous minerals (e g , inorganic phosphorous), enzymes, growth factors, drugs, and other agents for delivery to the livestock. These supplements replace many of the calories and natural nutrients present in grain, for example
By reducing or eliminating the inorganic phosphorous supplement and other supplements (e g , trace mineral salts, growth factors, enzymes, antibiotics) from the feed itself, the feed would be able to carry more nutrient and energy Accordingly, the remaining diet would contain more usable energy For example, gram-oilseed meal diets generally contain about 3,200 kcal metabohzable energy per kilogram of diet, and mineral salts supply no metabohzable energy Removal of the unneeded minerals and substitution with grain would therefore increase the usable energy in the diet Thus, the invention can be differentiated over commonly used phytase containing feed For example, in one embodiment, a biocompatible material is used that is resistant to digestion by the gastrointestinal tract of an organism.
In many organisms, including, for example, poultry or birds such as, for example, chickens, turkeys, geese, ducks, paπots, peacocks, ostnches, pheasants, quail, pigeons, emu. kiwi, loons, cockatiel, cockatoo, canaries, penguins, flamingoes, and dove, the digestive tract includes a gizzard which stores and uses hard biocompatible objects (e g , rocks and shells from shell fish) to help in the digestion of seeds or other feed consumed by a bird. A typical digestive tract of this general family of organisms, includes the esophagus which contains a pouch, called a crop, where food is stored for a brief penod of time. From the crop, food moves down into the true stomach, or proventriculus, where hydrochloric acid and pepsin starts the process of digestion. Next, food moves mto the gizzard, which is oval shaped and thick walled with powerful muscles The chief function of the gizzard is to gnnd or crush food particles - a process which is aided by the bird swallowing small amounts of fine gravel or gnt From the gizzard, food moves into the duodenum The small intestine of birds is similar to mammals There are two blind pouches or ceca, about 4-6 inches in length at the junction of the small and large intestine. The large intestine is short, consisting mostly of the recmm about 3-4 inches in length. The rectum empties mto the cloaca and feces are excreted through the vent.
Hard, biocompatible objects consumed (or otherwise introduced) and presented in the gizzard provide a useful vector for delivery of various enzymatic, chemical, therapeutic and antibiotic agents. These hard substances have a life span of a few hours to a few days and are passed after a period of time Accordingly, the invention provides coated, impregnated (e g , impregnated matrix and membranes) modified dietary aids for deliver ' of useful digestive or therapeutic agents to an organism Such dietary aids include objects which are typically ingested by an organism to assist in digestion within the gizzard (e g , rocks or grit) The invention provides biocompatible objects that have coated thereon or impregnated therein agents useful as a digestive aid for an organism or for the delivery of a therapeutic or medicinal agent or chemical
In a first embodiment, the invention provides a dietary aid, having a biocompatible composition designed for release of an agent that assists in digestion, wherein the biocompatible composition is designed for oral consumption and release in the digestive tract (e g , the gizzard) of an organism "Biocompatible" means that the substance, upon contact with a host organism (e g , a bird), does not elicit a detnmental response sufficient to result in the rejection of the substance or to render the substance inoperable Such inoperabihty may occur, for example, by formation of a fibrotic structure around the substance limiting diffusion of impregnated agents to the host organism therein or a substance which results in an increase in mortality or morbidity in the organism due to toxicity or infection A biocompatible substance may be non-biodegradable or biodegradable In one embodiment, the biocompatible composition is resistant to degradation or digestion by the gastrointestinal tract In another embodiment, the biocompatible composition has the consistency of a rock or stone
A non-biodegradable material useful in the invention is one that allows attachment or impregnation of a dietary agent Such non-biodegradable materials include, for example, thermoplastics, such as acrylic, modacryhc, polyamide, polycarbonate, polyester, polyethylene, polypropylene, polystyrene, polysulfone, polyethersulfone, and polyvinyhdene fluonde Elastomers are also useful matenals and include, for example, polyamide, polyester, polyethylene, polypropylene, polystyrene, polyurethane, polyvinyl alcohol and sihcone (e g , sihcone based or containing silica) The invention provides that the biocompatible composition can contain a plurality of such materials, which can be, e g , admixed or layered to form blends, copolymers or combinations thereof
As used herein, a "biodegradable" material means that the composition will erode or degrade in vivo to form smaller chemical species Degradation may occur, for example, by enzymatic, chemical or physical processes Suitable biodegradable materials contemplated for use in the invention include poly(lactιde)s, poly(glycohde)s, poly(lactιc acιd)s, poly(glycohc acιd)s, polyanhydπdes, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides. polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate. Such materials can be admixed or layered to form blends, copolymers or combinations thereof
It is contemplated that a number different biocompatible substances may be ingested or otherwise provided to the same organism simultaneously, or in various combinations (e.g , one material before the other). In addition, the biocompatible substance may be designed for slow passage through the digestive tract. For example, large or fatty substances tend to move more slowly through the digestive tract, accordingly, a biocompatible material having a large size to prevent rapid passing in the digestive tract can be used Such large substances can be a combination of non- biodegradable and biodegradable substances. For example, a small non-biodegradable substance can be encompassed by a biodegradable substance such that over a penod of time the biodegradable portion will be degraded allowing the non-biodegradable portion to pass through the digestive trace. In addition, it is recognized that any number of flavonngs can be provided to the biocompatible substance to assist in consumption
Any number of agents alone or in combination with other agents can be coated on the biocompatible substance including polypeptides (e g , enzymes, antibodies, cytokines or therapeutic small molecules), and antibiotics, for example. Examples of particular useful agents are listed in Table 1 and 2, below. It is also contemplated that cells can be encapsulated into the biocompatible material of the invention and used to deliver the enzymes or therapeutics. For example, porous substances can be designed that have pores large enough for cells to grow in and through and that these porous materials can then be taken into the digestive tract. For example, the biocompatible substance can be compnsed of a plurality of microfloral environments (e g , different porosity, pH etc.) that provide support for a plurality of cell types. The cells can be genetically engineered to deliver a particular drug, enzyme or chemical to the organism. The cells can be eukaryotic or prokaryotic.
TABLE 1
Figure imgf000104_0001
Figure imgf000105_0001
Table 2. Therapeutic Formulations
Figure imgf000105_0002
Figure imgf000106_0001
Figure imgf000107_0001
Certain agents can be designed to become active or activated under certain conditions (e g , at certain pH's, in the presence of an activating agent etc.) In addition, it may be advantageous to use pro-enzymes in the compositions of the invention. For example, a pro-enzymes can be activated by a protease (e g., a salivary protease that is present in the digestive tract or is artificially introduced into the digestive tract of an organism). It is contemplated that the agents delivered by the biocompatible compositions of the invention can be activated or inactivated by the addition of an activating agent which may be ingested by, or otherwise delivered to, the organism. Another mechanism for control of the agent in the digestive tract is an environment sensitive agent that is activated in the proper digestive compartment. For example, an agent may be inactive at low pH but active at neutral pH. Accordingly, the agent would be inactive in the gut but active in the intestinal tract. Alternatively, the agent can become active in response to the presence of a microorganism specific factor (e g , microorganisms present in the intestine) In summary, the potential benefits of the present invention include, for example, ( 1) reduction in or possible elimination of the need for mineral supplements (e g , inorganic phosphorous supplements), enzymes, or therapeutic drugs for animal (including fish) from the daily feed or grain thereby increasing the amount of calories and nutrients present in the feed, and (2) increased health and growth of domestic and non-domestic animals including, for example, poultry, porcine, bovine, equine, canine, and feline animals.
A large number of enzymes can be used in the methods and compositions of the present invention These enzymes include enzymes necessary for proper digestion of consumed foods, or for proper metabolism, activation or derivation of chemicals, prodrugs or other agents or compounds delivered to the animal via the digestive tract. Examples of enzymes that can be delivered or incorporated into the compositions of the invention, include, for example, feed enhancing enzymes selected from the group consisting of α- galactosidases, β-galactosidases, in particular lactases, phytases, β-glucanases, in particular endo-β-l,4-glucanases and endo-β-l,3(4)-glucanases, cellulases, xylosidases, galactanases, in particular arabmogalactan endo-l,4-β-galactosιdases and arabinogalactan endo-l ,3-β-galactosιdases. endoglucanases, in particular endo-l ,2-β-glucanase, endo-1,3- u-glucanase, and endo-l ,3-β-glucanase, pectin degrading enzymes, in particular pectinases, pectinesterases, pectin lyases, polygalacturonases, arabinanases, rhamnogalacturonases, rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α- rhamnosidase. pectate lyases, and α-galacturomsidases, mannanases, β-mannosidases, mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases, arabinoxylanases and lipolytic enzymes such as lipases, phospholipases and cutinases. Phytases as set forth m
SEQ ID NO' 1 and 2 and in Table 3 below are preferred. The sequences descπbed in Table 3 are SEQ ID NO 1 and 2 having the amino acid substimtions and nucleotide substimtions as described therein. TABLE 3
Figure imgf000109_0001
The enzymes used in the invention can be modified to enhance their activity, delivery, activation and degradation Such modifications can be performed in vivo or in vitro and use methods and processes generally known in the art as descπbed more fully below Such methodology generally uses polynucleotide or polypeptide sequences that are either synthesized by automated machines or are cloned, expressed, or manipulated by recombinant DNA techniques
In a preferred embodiment, the enzyme used in the compositions (e g., a dietary aid) of the present invention is a phytase enzyme which is stable to heat and is heat resistant and catalyzes the enzymatic hydrolysis of phytate, 1 e , the enzyme is able to renature and regain activity after a brief (1 e , 5 to 30 seconds), or longer period, for example, minutes or hours, exposure to temperamres of above 50 CC
A "feed" and a "food," respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by an animal and a human being, respectively "Dietary Aid," as used herein, denotes, for example, a composition containing agents that provide a therapeutic or digestive agent to an animal or organism A "dietary aid," typically is not a source of caloπc intake for an organism, in other words, a dietary aid typically is not a source of energy for the organism, but rather is a composition which is taken in addition to typical "feed" or "food"
10. An agent or enzyme (e g , a phytase) may exert its effect in viti o or in vivo, i e before intake or in the stomach or gizzard of the organism, respectively Also a combined action is possible
Although any enzyme may be incorporated into a dietary aid, reference is made herein to phytase as an exemplification of the methods and compositions of the invention A dietary aid of the invention includes an enzyme (e g , a phytase) Generally, a dietary aid containing a phytase composition is liquid or dry
Liquid compositions need not contain anything more than the enzyme (e g a phytase), preferably in a highly purified form Usually, however, a stabilizer such as glycerol. sorbitol or mono propylen glycol is also added The liquid composition may also compnse other additives, such as salts, sugars, preservatives, pH-adjusting agents, proteins, phytate (a phytase substrate) Typical liquid compositions are aqueous or oil- based slurries The liquid compositions can be added to a biocompatible composition for slow release Preferably the enzyme is added to a dietary aid composition that is a biocompatible matenal (e g , biodegradable or non-biodegradable) and includes the addition of recombinant cells into, for example, porous microbeads
Dry compositions may be spray dried compositions, in which case the composition need not contain anything more than the enzyme in a dry form Usually, however, dry compositions are so-called granulates which may readily be mixed with a food or feed components, or more preferably, form a component of a pre-mix The particle size of the enzyme granulates preferably is compatible with that of the other components of the mixmre This provides a safe and convenient means of incorporating enzymes into animal feed Preferably the granulates are biocompatible and more preferably they biocompatible granulates are non-biodegradable
Agglomeration granulates coated by an enzyme can be prepared using agglomeration technique in a high shear mixer Absoφtion granulates are prepared by having cores of a carrier material to absorp/be coated by the enzyme Preferably the carrier material is a biocompatible non-biodegradable material that simulates the role of stones or grit in the gizzard of an animal Typical filler materials used in agglomeration techniques include salts, such as disodmm sulphate Other fillers are kaolin, talc, magnesium aluminium silicate and cellulose fibres Optionally, binders such as dextnns are also included in agglomeration granulates The carrier materials can be any biocompatible matenal including biodegradable and non-biodegradable materials (e g , rocks, stones, ceramics, various polymers) Optionally, the granulates are coated with a coating mixmre Such mixmre compnses coating agents, preferably hydrophobic coating agents, such as hydrogenated palm oil and beef tallow, and if desired other additives, such as calcium carbonate or kaolin
Additionally, the dietary aid compositions (e g , phytase dietary aid compositions) may contain other substituents such as colouring agents, aroma compounds, stabilizers, vitamins, minerals, other feed or food enhancing enzymes etc A typical additive usually compnses one or more compounds such as vitamins, minerals or feed enhancing enzymes and suitable earners and/or excipients
In a one embodiment, the dietary aid compositions of the invention additionally compnses an effective amount of one or more feed enhancing enzymes, in particular feed enhancing enzymes selected from the group consisting of α-galactosidases, β- galactosidases, in particular lactases, other phytases, β-glucanases, in particular endo-β- 1,4-glucanases and endo-β-l,3(4)-glucanases. cellulases, xylosidases, galactanases, in particular arabinogalactan endo-l ,4-β-galactosιdases and arabinogalactan endo-l,3-β- galactosidases, endoglucanases, in particular endo-l,2-β-glucanase, endo-l,3-α-glucanase, and endo-l,3-β-glucanase, pectin degrading enzymes, in particular pectinases, pectinesterases, pectin lyases, polygalacturonases, arabinanases, rhamnogalacturonases, rhamnogalacmronan acetyl esterases, rhamnogalacturonan-α-rhamnosidase, pectate lyases, and α-galacmronisidases. mannanases, β-mannosidases, mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases, arabinoxylanases and lipolytic enzymes such as lipases, phospholipases and cutinases The animal dietary aid of the invention is supplemented to the mono-gastπc animal before or simultaneously with the diet. In one embodiment, the dietary aid of the invention is supplemented to the mono-gastric animal simultaneously with the diet. In another embodiment, the dietary aid is added to the diet in the form of a granulate or a stabilized liquid
An effective amount of an enzyme in a dietary aid of the invention is from about 10-20,000; preferably from about 10 to 15,000, more preferably from about 10 to 10,000, in particular from about 100 to 5,000, especially from about 100 to about 2,000 FYT/kg dietary aid
Examples of other specific uses of the phytase of the invention is in soy processing and in the manufacture of inositol or derivatives thereof.
The invention also relates to a method for reducing phytate levels in animal manure, wherein the animal is fed a dietary aid containing an effective amount of the phytase of the invention. As stated in the beginning of the present application one important effect thereof is to reduce the phosphate pollution of the environment.
In another embodiment, the dietary aid is a magnetic carrier. For example, a magnetic carrier containing an enzyme (e.g , a phytase) distributed in, on or through a magnetic carrier (e g., a porous magnetic bead), can be distributed over an area high in phytate and collected by magnets after a period of time. Such distribution and recollection of beads reduces additional pollution and allows for reuse of the beads. In addition, use of such magnetic beads in vivo allows for the localization of the dietary aid to a point in the digestive tract where, for example, phytase activity can be carried out. For example, a dietary aid of the invention containing digestive enzymes (e g , a phytase) can be localized to the gizzard of the animal by juxtapositionmg a magnet next to the gizzard of the animal after the animal consumes a dietary aid of magnetic earners. The magnet can be removed after a period of time allowing the dietary aid to pass through the digestive tract. In addition, the magnetic carriers are suitable for removal from the organism after sacrificing or to aid in collection
When the dietary aid is a porous particle, such particles are typically impregnated by a substance with which it is desired to release slowly to form a slow release particle Such slow release particles may be prepared not only by impregnating the porous particles with the substance it is desired to release, but also by first dissolving the desired substance in the first dispersion phase. In this case, slow release particles prepared by the method in which the substance to be released is first dissolved in the first dispersion phase are also within the scope and spirit of the invention The porous hollow particles may, for example, be impregnated by a slow release substance such as a medicine, agπcultural chemical or enzyme. In particular, when porous hollow particles impregnated by an enzyme are made of a biodegradable polymers, the particles themselves may be used as an agπcultural chemical or fertilizer, and they have no adverse effect on the environment. In one embodiment the porous particles are magnetic in namre.
The porous hollow particles may be used as a bioreactor support, in particular an enzyme support Therefore, it is advantageous to prepare the dietary aid utilizing a method of a slow release, for instance by encapsulating the enzyme of agent in a microvesicle, such as a hposome, from which the dose is released over the course of several days, preferably between about 3 to 20 days. Alternatively, the agent (e g , an enzyme) can be formulated for slow release, such as incorporation into a slow release polymer from which the dosage of agent (e g , enzyme) is slowly released over the course of several days, for example from 2 to 30 days and can range up to the life of the animal
As is known in the art, hposomes are generally derived from phosphohpids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium Any non-toxic, physiologically acceptable and metabohzable lipid capable of forming liposomes can be used. The present compositions in hposome form can contain stabilizers, preservatives, excipients, and the like in addition to the agent The preferred lipids are the phosphohpids and the phosphatidyl chohnes (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. ( 1976), p. 33 et seq.
Also within the scope of the invention is the use of a phytase of the invention during the preparation of food or feed preparations or additives, i e , the phytase excerts its phytase activity during the manufacture only and is not active in the final food or feed product. This aspect is relevant for instance in dough making and baking. Accordingly, phytase or recombinant yeast expressing phytase can be impregnated in, on or through a magnetic earners, distributed in the dough or food medium, and retπeved by magnets.
The dietary aid of the invention may be administered alone to animals in an biocompatible (e.g , a biodegradable or non-biodegradable) earner or in combination with other digestion additive agents. The dietary aid of the invention thereof can be readily administered as a top dressing or by mixing them directly into animal feed or provided separate from the feed, by separate oral dosage, by injection or by transdermal means or in combination with other growth related edible compounds, the proportions of each of the compounds in the combination being dependent upon the particular organism or problem being addressed and the degree of response desired. It should be understood that the specific dietary dosage administered in any given case will be adjusted m accordance with the specific compounds being administered, the problem to be treated, the condition of the subject and the other relevant facts that may modify the activity of the effective ingredient or the response of the subject, as is well known by those skilled in the art. In general, either a single daily dose or divided daily dosages may be employed, as is well known in the art.
If administered separately from the animal feed, forms of the dietary aid can be prepared by combining them with non-toxic pharmaceutically acceptable edible earners to make either immediate release or slow release formulations, as is well known in the art Such edible carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol If a solid earner is used the dosage form of the compounds may be tablets, capsules, powders, troches or lozenges or top dressing as micro-dispersable forms. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeuticaUy valuable substances.
Thus, a significant advantages of the invention include for example, 1) ease of manufacture of the active ingredient loaded biocompatible compositions; 2) versatility as it relates to the class of polymers and/or active ingredients which may be utilized; 3) higher yields and loading efficiencies; and 4) the provision of sustained release formulations that release active, intact active agents in vivo, thus providing for controlled release of an active agent over an extended penod of time. In addition, another advantage is due to the local delivery of the agent with in the digestive tract (e g., the gizzard) of the organism. As used herein the phrase "contained within" denotes a method for formulating an agent into a composition useful for controlled release, over an extended period of time of the agent.
In the sustained-release or slow release compositions of the invention, an effective amount of an agent (e g , an enzyme or antibiotic) will be utilized. As used herein, sustained release or slow release refers to the gradual release of an agent from a biocompatible material, over an extended period of time. The sustained release can be continuous or discontinuous, linear or non-linear, and this can be accomplished using one or more biodegradable or non-biodegradable compositions, drug loadings, selection of excipients, or other modifications. However, it is to be recognized that it may be desirable to provide for a "fast" release composition, that provides for rapid release once consumed by the organism. It is also to be understood that by "release" does not necessarily mean that the agent is released from the biocompatible earner Rather in one embodiment, the slow release encompasses slow activation or continual activation of an agent present on the biocompatible composition For example, a phytase need not be released from the biocompatible composition to be effective. In this embodiment, the phytase is immobilized on the biocompatible composition.
The animal feed may be any protein-containing organic meal normally employed to meet the dietary requirements of animals. Many of such protein-containing meals are typically pnmaπly composed of com, soybean meal or a corn soybean meal mix. For example, typical commercially available products fed to fowl include Egg Maker Complete, a poultry feed product of Land O'Lakes AG Services, as well as Country Game & Turkey Grower a product of Agwa, Inc. (see also The Emu Farmer's Handbook by Phillip Minnaar and Mana Minnaar) Both of these commercially available products are typical examples of animal feeds with which the present dietary aid and/or the enzyme phytase may be incoφorated to reduce or eliminate the amount of supplemental phosphorus, zinc, manganese and iron intake required in such compositions.
The present invention is applicable to the diet of numerous animals, which herein is defined as including mammals (including humans), fowl and fish. In particular, the diet may be employed with commercially significant mammals such as pigs, cattle, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), fur-bearing animals such as mink and fox, and zoo animals such as monkeys and apes, as well as domestic mammals such as cats and dogs Typical commercially significant avian species include chickens, turkeys, ducks, geese, pheasants, emu, ostrich, loons, kiwi, doves, parrots, cockatiel, cockatoo, canaries, penguins, flamingoes, and quail. Commercially farmed fish such as trout would also benefit from the dietary aids disclosed herein. Other fish that can benefit include, for example, fish (especially in an aquarium or acquaculture environment, e.g , tropical fish), goldfish and other ornamental caφ, catfish, trout, salmon, shark, ray, flounder, sole, tilapia, medaka, guppy, molly, platyfish, swordtail, zebrafish, and loach.
Unless otherwise stated, transformation was performed as descnbed in the method of Sambrook, Fπtsch and Maniatus, 1989 The following examples are intended to illustrate, but not to limit, the invention. While the procedures descπbed in the examples are typical of those that can be used to carry out certain aspects of the invention, other procedures known to those skilled in the art can also be used
EXAMPLE 1 ISOLATION. BACTERIAL EXPRESSION. AND PURIFICATION OF PHYTASE
E cob B genomic DNA was obtained from Sigma (Catalog # D-2001), St Louis, New Jersey
The following pnmers were used to PCR amplify the gene directly from the genomic DNA
5' pnmer gtttctgaattcaaggaggaatttaaATGAAAGCGATCTTAATCCCATT
(SEQ ID NO 3), and
3 ' pnmer gtttctggatccTTACAAACTGCACGCCGGTAT (SEQ ID NO 4)
Pfu polymerase in the PCR reaction, and amplification was performed according to manufacmrers protocol (Stratagene Cloning Systems, Ine , La Jolla, CA)
PCR product was purified and punfied product and pQE60 vector (Qiagen) were both digested with EcoRI and Bglll restriction endonucleases (New England Biolabs) according to manufacmrers protocols Overnight ligations were performed using standard protocols to yield pQE60
The amplified sequences were inserted in frame with the sequence encoding for the RBS The ligation mixmre was then used to transform the E cob strain M15/pREP4 (Qiagen, Ine ) by electroporation M15/pREP4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan1) Plasmid DNA was isolated and confirmed by restπction analysis Clones containing the desired constructs were grown overnight (O/N) in liquid culmre in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) The O/N culmre was used to inoculate a large culmre at a ratio of 1 100 to 1 250 The cells were grown to an optical density 600 (O D 600) of between 0 4 and 0 6 IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM IPTG induces by inactivating the lad repressor clearing the P/O leading to increased gene expression Cells were grown an extra 3 to 4 hours Cells were then harvested by centrifugation
The primer sequences set out above may also be employed to isolate the target gene from the deposited material by hybndization techniques described above
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described It is to be understood that, while the invention has been descπbed with reference to the above detailed descnption, the foregoing description is intended to illustrate, but not to limit, the scope of the invention Other aspects, advantages, and modifications of the invention are within the scope of the following claims All publications, patent applications, patents, and other references mentioned herein are incoφorated by reference in their entirety
This invention also provides for the isolation and use of phytase molecules (nucleic acids and phytase enzymes encoded thereby) from all other strains of E coli (whether vimlent or non-virulent, including K12, W, C), as well as all bacteria These include all known species and strains belonging to
Thermotogales
Green Nonsulfur Bacteria
Cyanobacteπa & chloroplasts
Low G+C Gram-Positive Bacteria Fusobacteπa
High G+C Gram-Positive Bacteria
Gytophaga/Flexibacter/Bacteroides group
Fibrobacteπa
Spnochaetes Planctomyces/Chlamydia group
Puφle bacteria (Proteobacteπa), including the following subdivisions Delta & Epsilon, including
Desulfuromonas acetoxidans Desulfosarcina vaπabihs
Bdellovibπo stolpn Nannocystis exedens Stigmatella aurantiaca
Myxococcus xanthus
Desulfovibπo desulfuπcans
Thiovulum sp Campylobacterjejuni
Wohnella succinogenes
Hehcobacter pylori Alpha, including
Methylobacteπum extorquens Beijeπnckia indica
Hyphomicrobium vulgare
Rhodomicrobium vanmeli
Agrobacteπum mmefaciens
B cella abortus Rochahmaea qmntana
Rhodopseudomonas marina subsp agilis
Zea mays - mitochondrion
Rickettsia rickettsn
Ehrhchia πsticii Wolbachia pipientis
Anaplasma marginale
Erythrobacter longus
Rhodospiπllum salexigens
Rhodobacter capsulatus Azospinllum hpofemm
Rhodospiπllum rubrum Gamma, including
Ectothiorhodospira shaposhmkovu
Chromatium vinosum Methylomonas methamca
Cardiobacteπum hominis
Xanthomonas maltophiha
Coxiella burnetn
Legionella pneumophila subsp pneumophila Oceanospiπllum hnum
Acinetobacter calcoaceticus
Pseudomonas aeruginosa
Haemophilus lnfluenzae
Vibrio parahaemolyticus Proteus vulgaris
Erwinia carotovora
Echeπchia coli, including
Beta, including Eikenella corrodens
Neisseπa gonorrhoeae Vitreoscilla stercoraπa Chromobacteπum violaceum Alcahgenes faecahs Rubπvivax gelatinosus Pseudomonas testosterom Nitrosomonas europae Spirillum volutans
Such phytase molecules can be isolated from these bacteria by know methods, including library screening methods, e.g. expression screening, hybridization methods, PCR (e.g see Sammbrook, 1989).
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Claims

What is claimed is
1 A method for improving the nutritional value of a phytate-containing foodsmff comprising" contacting said phytate-containing foodsmff with a substantially pure phytase enzyme having an amino acid sequence selected from the group consisting of SEQ ID NO:2, such that said substantially pure phytase enzyme catalyzes the liberation of inorganic phosphate from the phytate in said phytate-containing foodstuff.
2. The method of claim 1, wherein said substantially pure phytase enzyme is produced by a recombinant expression system comprising a phytase-encoding nucleic acid having a nucleotide sequence selected from the group consisting of a) SEQ ID NO" 1, and b) SEQ ID NO" 1 where T can also be U; wherein the expression of the phytase-encoding nucleic acid leads to the production of said substantially pure phytase enzyme.
3. The method of claim 1, wherein the liberation of the inorganic phosphate from the phytate in said phytate-containing foodsmff occurs prior to the ingestion of said phytate-containing foodsmff by a recipient organism.
4. The method of claim 1, wherein the liberation of the inorganic phosphate from the phytate in said phytate-containing foodsmff occurs after the ingestion of said phytate-containing foodsmff by a recipient organism.
5. The method of claim 1, wherein the liberation of the inorganic phosphate from the phytate in said phytate-containing foodsmff occurs in part pπor to and in part after the ingestion of said phytate-containing foodsmff by a recipient organism.
6 A recombinant expression system for use in the method of claim 2, wherein said recombinant expression system is contained in a host cell and expresses a nucleotide sequence encoding a phytase enzyme having an amino acid sequence as set forth in SEQ ID NO 2, and wherein the nucleotide sequence encoding said enzyme is operably linked to a transcription control sequence operable in said host cell.
7. A vector which comprises the expression system according to claim 6.
8. The expression system of claim 6, wherein the control sequence comprises a constitutive promoter.
9. The expression system of claim 6, wherein the control sequence compnses a tissue- specific promoter.
10. The expression system of claim 6. wherein said host cell is a prokaryotic cell.
1 1. The expression system of claim 6, wherein said host cell is a eukaryotic cell.
12. The expression system of claim 6, wherein said host cell is a plant cell
13. The expression system of claim 6, wherein said nucleotide sequence is preceded by a polynucleotide sequence encoding a signal peptide operably linked to said nucleotide sequence.
14 The expression system of claim 13, wherein said signal peptide is the PR protein PR-S signal peptide from tobacco.
15 A prokaryotic cell modified to contain the expression system of claim 6
16. A eukaryotic cell modified to contain the expression system of claim 6.
17 A plant cell, plant part or plant modified to contain the expression system of claim 6.
18 A method to produce an animal feed containing a microbial phytase compnsmg a) culmnng the plant cell, plant part or plant of claim 17 under conditions wherein said nucleotide sequence is expressed; and b) converting said plant cells, plant parts or plants into a composition suitable for animal feed.
19 A feed composition for animals which compnses the plant seeds, plant cells, plant parts or plants of claim 17 in admixture with a phytate-containing foodsmff
20 A method to treat a human or an animal able to benefit from digestive enhancement by the activity of an exogenous phytase enzyme, which method comprises admimsteπng to said human or animal an amount of plant seeds, plant cells, plant parts or plants of a transgenic plant, wherein said transgenic plant is modified to contain an expression system which expresses a nucleotide sequence encoding a phytase enzyme in an amount effective to provide phytase activity in said human's or animal's digestive tract
21. A transgenic non-human organism whose genome compnses a heterologous nucleic acid sequence encoding a polypeptide having phytase activity, wherein said transgene results in expression of a phytase polypeptide.
22. A method of generating a variant compnsmg obtaining a nucleic acid compnsmg a sequence as set forth in SEQ ID NO: 1, sequences substantially identical thereto, sequences complementary thereto, fragments compnsmg at least 30 consecutive nucleotides thereof, and fragments comprising at least 30 consecutive nucleotides of the sequences complementary to SEQ ID NO 1 ; and modifying one or more nucleotides in said sequence to another nucleotide, deleting one or more nucleotides in said sequence, or adding one or more nucleotides to said sequence.
23. The method of claim 22, wherein the modifications are introduced by a method selected from the group consisting of error-prone PCR, shuffling, ohgonucleotide- directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, ligation reassembly, GSSM and any combination thereof.
24 The method of claim 22, wherein the modifications are introduced by error-prone PCR.
25. The method of claim 22, wherein the modifications are introduced by shuffling
26. The method of claim 22, wherein the modifications are introduced by ohgonucleotide-directed mutagenesis.
27. The method of claim 22, wherein the modifications are introduced by assembly PCR.
28. The method of claim 22, wherein the modifications are introduced by sexual PCR mutagenesis.
29. The method of claim 22, wherein the modifications are introduced by in vivo mutagenesis.
30 The method of claim 22, wherein the modifications are introduced by cassette mutagenesis
31 The method of claim 22, wherein the modifications are introduced by recursive ensemble mutagenesis.
32 The method of claim 22, wherein the modifications are introduced by exponential ensemble mutagenesis.
33. The method of claim 22, wherein the modifications are introduced by site-specific mutagenesis.
34 A computer readable medium having stored thereon a nucleic acid sequence as set forth in SEQ ID NO. l, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO 2, and sequences substantially identical thereto.
35. A computer system compnsmg a processor and a data storage device wherein said data storage device has stored thereon a nucleic acid sequence as set forth m SEQ ID NO: 1, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO"2 and sequences substantially identical thereto.
36. The computer system of claim 35, further compnsmg a sequence companson algonthm and a data storage device having at least one reference sequence stored thereon.
37. The computer system of claim 36, wherein the sequence companson algonthm compnses a computer program which indicates polymoφhisms.
38. The computer system of claim 35. further compnsmg an identifier which identifies feamres in said sequence. 39 A method for companng a first sequence to a reference sequence or a database of sequences wherein said first sequence is a nucleic acid sequence as set forth in SEQ ID NO 1, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO"2, and sequences substantially identical thereto compnsmg reading the first sequence and the reference sequence or database of sequences through use of a computer program which compares sequences; and determining differences between the first sequence and the reference sequence or database of sequences with the computer program.
40 The method of claim 39, wherein determining differences between the first sequence and the reference sequence compnses identifying polymoφhisms
PCT/US2000/014846 1999-05-25 2000-05-25 Recombinant bacterial phytases and uses thereof WO2000071728A1 (en)

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CA002374532A CA2374532A1 (en) 1999-05-25 2000-05-25 Recombinant bacterial phytases and uses thereof
KR1020017015109A KR20020018672A (en) 1999-05-25 2000-05-25 Recombinant bacterial phytases and uses thereof
EP00937932A EP1180152A1 (en) 1999-05-25 2000-05-25 Recombinant bacterial phytases and uses thereof
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US7232677B2 (en) 2007-06-19
US20010055788A1 (en) 2001-12-27
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US20080089879A1 (en) 2008-04-17
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US6183740B1 (en) 2001-02-06
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