Note: Descriptions are shown in the official language in which they were submitted.
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Thermostable phytases in
feed preparation and plant expression
Technical Field
This application relates to thermostable phytases, viz.
their use in processes for the production of animal feed, and
their expression in plants.
Background art
io WO 91/14782 describes transgenic tobacco and rapeseed
plants expressing a phytase derived from Aspergillus ficuum NRRL
3135. The transgenic tobacco seeds are fed to broilers.
US 5,829,779 describes in standard fashion how to produce
transgenic alfalfa expressing the same A. ficuum phytase, and
the preparation of a phytase-containing concentrate which can be
used per se as an animal feed supplement.
EP 0 556 883 B1 describes a method for preparing feed
pellets based on an extrusion technique. The addition of
temperature sensitive agents, one example of which is phytase,
2o takes place after extrusion of the feed pellets, and the
sensitive agents are loaded onto the pellets under reduced
pressure.
As acknowledged in EP 0 556 883 B1 the loss of activity of
heat-sensitive substances during feed preparation processes is a
well-known problem. The above EP-patent proposes to solve this
problem by adding these substances under reduced pressure
subsequent to the extrusion process. This solution, however,
requires a liquid form of the sensitive substance, as well as
the installation of additional expensive process equipment.
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The present invention provides an improved process for
preparing animal. feed, as well as improved phytase-expressing
transgenic plants.
Summary of the Invention
The present invention provides a process of preparing an
animal feed, which process comprises an agglomeration of feed
ingredients, wherein a thermostable phytase is added before or
during the agglomeration.
Also provided is a transgenic plant or part thereof which
comprises a DNA-construct encoding a thermostable phytase.
The transgenic plant or part thereof, e.g. seeds or
leaves, may be used in the feed preparation process of the
invention, to thereby provide - in a preferred embodiment - at
the same time a nutrient (feed ingredient) and the. feed additive
phytase.
Brief description of the Figures
In the detailed description of the invention below,
2o reference is made to the drawings, of which
Fig. 1 is a differential scanning calorimetry (DSC) chart
of consensus phytase-1 and consensus phytase-10;
Fig. 2 a DSC of consensus phytase-10-thermo-QSOT and
consensus phytase-10-thermo-QSOT-K91A;
Fig. 3 a DSC of consensus phytase-1-thermo[8]-Q50T and
consensus phytase-1-thermo[8]-QSOT-K91A;
Fig. 4 a DSC of the phytase from A. fumigatus ATCC 13073
and of its a.-mutant: and
Fig. 5 shows the design of the consensus-phytase-1 amino
3o acid sequence;
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Fig. 6 an alignment and the basidiomycete consensus
sequence of five Basidiomycete phytases;
Fig. 7 the design of the consensus-phytase-10 amino acid
sequence;
Fig. 8 an alignment for the design of consensus-phytase-11
(all Basidiomycete phytases were used as independent
sequences using an assigned vote weight of 0.2 for
each Basidiomycete sequence; still further the amino
acid sequence of A. niger T213 was used);
1o Fig. 9 the DNA and amino acid sequence of consensus-
phytase-1-thermo(8)-Q50T-K91A;
Fig. 10 the DNA and amino acid sequence of Consensus-
phytase-10-thermo(3)-Q50T-K91A;
Fig. 11 the DNA and amino acid sequence of A. fumigatus ATCC
13073 a-mutant; and
Fig. 12 the DNA and amino acid sequence of Consensus-
phytase-7 which comprises the following mutations
as compared to Consensus-phytase-1: S89D, S92G,
A94K, D164S, P201S, G203A, G205S, H212P, G224A,
2o D226T, E255T, D256E, V258T, P265S, Q292H, G300K,
Y305H, A314T, S364G, M365I, A397S, S398A, G404A, and
A405S.
Detailed description of the invention
In the present context a "feed" or an "animal feed" means
any natural or artificial diet, meal or the like intended or
suitable for being eaten, taken in, digested, by an animal. Food
for human beings is included in the above definition of feed.
"Animals" include all animals, be it polygastric animals
(ruminants); or monogastric animals such as human beings,
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PCT/DK99/00154
poultry, swine and fish. Preferred animals are the mono-gastric
animals, in particular pigs and broilers.
The concept of "feed ingredients" includes the raw
materials from which a feed is to be, or is, produced; or the
intended, or actual, component parts of a feed. Feed ingredients
for non-human animals are usually, and preferably, selected from
amongst the following non-exclusive list:
plant derived products
such as seeds, grains, leaves, roots, tubers, flowers,
1o pods, husks - and they may take the form of flakes, cakes,
grits, flour, and the like;
animal derived products
such as fish meal, milk powder, bone extract, meat
extract, blood extract and the like;
additives
such as minerals, vitamins, aroma compounds, and feed
enhancing enzymes.
Phytic acid or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen
phosphate (or for short myo-inositol hexakisphosphate) is the
2o primary source of inositol and the primary storage form of
phosphate in plant seeds and grains. In the seeds of legumes it
accounts for about 70~ of the phosphate content. Seeds, cereal
grains and legumes are important feed ingredients.
Phytic acid, or its salts phytates - said terms being,
unless otherwise indicated, in the present context used
synonymously or at random - is an anti-nutritional factor. This
is partly due to its binding of nutritionally essential ions
such as calcium, trace minerals such as mangane, and also
proteins (by electrostatic interaction). And partly due to the
3o fact that the phosphorous thereof is not nutritionally available
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either, since phytic acid and its salts, phytates, are often not
metabolized.
This leads to a need of supplementing food and feed
preparations with e.g. inorganic phosphate.
5 The non-metabolizable phytic acid phosphorous passes
through the gastrointestinal tract of such animals and is_
excreted with the manure, resulting in an undesirable phosphate
pollution of the environment resulting e.g. in eutrophication of
the water environment and extensive growth of algae.
1o Phytic acid is degradable by phytases. In the present
context a "phytase" is an polypeptide or enzyme which exhibits
phytase activity, viz. which catalyzes the hydrolysis of phytate
(myo-inositol hexakisphosphate) to (1) myo-inositol and/or (2)
mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3)
inorganic phosphate.
The production of phytases by plants as well as by
microorganisms has been reported. Amongst the microorganisms,
phytase producing bacteria as well as phytase producing fungi
are known.
2o There are several descriptions of phytase producing
filamentous fungi belonging to the fungal phylum of Ascomycota
(ascomycetes). In particular, there are several references to
phytase producing ascomycetes of the Aspergillus genus such as
Aspergillus terreus (Yamada et al., 1986, Agric. Biol. Chem.
322:1275-1282). Also, the cloning and expression of the phytase
gene from Aspergillus niger var. awamori has been described
(Piddington et al., 1993, Gene 133:55-62). EP 0420358 describes
the cloning and expression of a phytase of Aspergillus ficuum
(niger). EP 0684313 describes the cloning and expression of
3o phytases of the ascomycetes Aspergillus niger, Myceliophthora
thermophila, Aspergillus terreus. Still further, some partial
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sequences of phytases of Aspergillus nidulans, Talaromyces
thermophilus, Aspergillus fumigatus and another strain of
Aspergillus terreus are given.
The cloning and expression of a phytase of Thermomyces
lanuginosus is described in WO 97/35017.
WO 98/28409 describes the cloning and expression o~
several basidiomycete phytases, e.g. from Peniophora lycii,
Agrocybe pediades, Paxillus involutus and Trametes pubescens.
According to the Enzyme nomenclature database ExPASy (a
to repository of information relative to the nomenclature of
enzymes primarily based on the recommendations of the
Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (IUBMB) describing each type
of characterized enzyme for which an EC (Enzyme Commission)
number has been provided), two different types of phytases are
presently known: A so-called 3-phytase (myo-inositol
hexaphosphate 3-phosphohydrolase, EC 3.1.3.8) and a so-called 6-
phytase (myo-inositol hexaphosphate 6-phosphohydrolase, EC
3.1.3.26). The 3-phytase hydrolyses first the ester bond at a 3-
2o position, whereas the 6-phytase hydrolyzes first an ester bond
at the 6-position of phytic acid. Both of these types of
phytases are included in the above definition of phytase.
Many assays of phytase activity are known, and any of
these can be used for the purpose of the present invention.
Preferred phytase assays are included in the examples.
The concept of "agglomeration" is defined as a process in
which various components are mixed under the influence of heat.
The resulting product is preferably an "agglomerate" or
conglomerate in which the components adhere to each other while
3o forming a product of a satisfactory physical stability. The
formation of dust from such agglomerate is an indication of its
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physical stability - the less dust being formed, the better. A
suitable assay for dust formation from agglomerates is ASAE
standard S 269-1. A satisfactory agglomerate has below 200,
preferably below 15~, more preferably below 10g, even more
preferably below 6% dust.
"Under the influence of heat" means that the temperature
is at least 65°C, as measured on the product at the outlet of
the agglomeration unit. More preferred temperatures are at least
70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or even at
l0 least 130°C.
A preferred agglomeration process is operated at an
increased pressure. The pressure is typically due to a
compacting of the ingredients, optionally in combination with a
reduction of the cross-sectional or throughput area. Preferably,
by properly adjusting process parameters such as temperature and
pressure, the resulting shear forces and shear velocities are of
such magnitude, that the starch- and protein-containing feed
ingredients become fluid.
"Increased pressure" means increased as compared to normal
2o atmospheric pressure, and the maximum pressure as measured
within the agglomeration unit.
The addition of water vapour or steam is often included in
agglomeration, but not as an absolute requirement.
Agglomeration includes, but is not limited to, the well
known processes called extrusion, expansion (or pressure
conditioning) and pelleting (or pellet pressing).
Extrusion is i.a. described at pp. 149-153 of a handbook
which is available on request from the Danish Company Sprout-
Matador, Glentevej 5-7, DK-6705 Esbjerg 0 or Niels Finsensvej 4,
3o DK-7100 Vejle ("Handbog i Pilleteringsteknik 1996"). However, in
the agglomeration process of the invention, the following
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process steps mentioned in the above handbook are entirely
optional:
(i) pre-treating the feed ingredients in a cascade mixer;
(ii) cutting the product leaving the nozzle-section into pieces
(iii) of a desired size;
(iv) acclimatizing or conditioning it; -
(v) coating it;
(vi) drying it;
(vii) cooling it.
to The process of expansion (pressure conditioning) is i.a.
described in the same handbook at pp. 61-66. Also for expansion,
the above process steps (i)-(vi), in particular steps (i) and
(vi), are entirely optional steps.
This is so also for the following process steps:
(ii' ) comminuting the product (using e.g. a blade granulator as
shown at p. 65);
(vii) pelleting the product (using e.g. a pellet press as shown
at p. 62);
The process of pelleting is i.a. described in the same
2o handbook at pp. 71-107. Also here, steps (i)-(vii) above are
entirely optional steps. These steps are i.a. described in more
detail at pp. 29-70 of the above handbook.
In a preferred agglomeration process of the invention, one
or more of the above mentioned further process steps (i)-(vii)
are included.
A particularly preferred further step is step (i).
In a most preferred embodiment, the feed-ingredients are
pre-heated in a first step (a) to a temperature of at least
45°C, preferably at least 50, 55, 60, 65, 70, 75, 80 °C; and
3o then heated in a second step (b) to a temperature of at least
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65°C, preferably 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, or even at least 130°C.
The addition of thermostable phytase takes place before or
during step (a) and/or before or during step (b).
Water is preferably added in step (a). More preferably,
heated steam is added during the mixing of the ingredients
(steps (a) and/or (b) ) .
Process step (a) is preferably performed in a cascade
mixer (see the above cited handbook p. 44).
1o A "thermostable" phytase is a phytase which has a Tm
(melting temperature) as measured on purified phytase protein by
Differential Scanning Calorimetry (DSC) of at least 65°C,
preferably using for the DSC a constant heating rate, more
preferably of 10°C/min. In preferred embodiments, the Tm is at
least 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75°C. Preferably,
the Tm is equal to or lower than 150°C, more preferably equal to
or lower than 145, 140, 135, 130, 125, 120, 115 or 110°C.
Accordingly, preferred intervals of Tm are: 65-150°C, 66-
150°C,
- (etc.) - 75-150°C; 65-195°C, 66-145°C, - (etc.) - 75-
145°C;
65-140°C, - (etc.) - 75-140°C; - (etc.) - 65-110°C, 66-
110°C, -
(etc.) - 75-110°C.
Particularly preferred ranges for Tm are the following:
between 65 and 110°C; between 70 and 110°C; between 70 and
100°C; between 75 and 95°C, or between 80 and 90°C.
In Example 3 below, the measurement of Tm by DSC is
described, and the Tm's of a number of phytases are shown.
The optimum temperatures are also indicated, since - in
the alternative - a thermostable phytase can be defined as a
phytase having a temperature-optimum of at least 60°C.
3o Preferably, the optimum temperature is determined on the
substrate phytate at pH 5.5, or on the substrate phytic acid at
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pH 5Ø Preferred units are FYT, FTU or the units of Example 3.
The phytase assay of Example 3 is most preferred.
In preferred embodiments, the optimum temperature is at
least 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C. Preferably,
5 the optimum temperature is equal to or lower than 140°C, more
preferably equal to or lower than 135, 130, 125, 120, 115, 110,-
105 or 100°C. Accordingly, preferred intervals of optimum
temperature are: 60-140°C, 61-140°C, - (etc.) - 70-140°C;
60-
135°C, 61-135°C, - (etc.) - 70-135°C; 60-130°C, -
(etc.) - 70-
10 130°C; - (etc.) - 60-100°C, 61-100°C, - (etc.) - 70-
100°C.
Preferred phytases of the present invention exhibit a
degree of similarity or homology, preferably identity, to the
complete amino acid sequence of either .of the phytases mentioned
below under (iii) - preferably to the complete amino acid
sequence of Consensus-phytase-10-thermo-Q50T-K91A - of at least
48~, preferably at least 50, 52, 55, 60, 62, 65, 67, 70, 73, 75,
77, 80, 82, 85, 88, 90, 92, 95, 98 or 99~.
The degree of similarity or homology, alternatively
identity, can be determined using any alignment programme known
2o in the art . A preferred alignment programme is GAP provided in
the GCG version 8 program package (Program Manual for the
Wisconsin Package, Version 8, August 1994, Genetics Computer
Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (see
also Needleman, S.B. and Wunsch, C.D., (1970), Journal of
Molecular Biology, 48, 443-453). Using GAP with the following
settings for polypeptide sequence comparison: GAP weight of
3.000 and GAP lengthweight of 0.100.
A multiple sequence alignment can be made using the
program Pileup (Program Manual for the Wisconsin Package,
3o Version 8, August 1994, Genetics Computer Group, 575 Science
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Drive, Madison, Wisconsin, USA 53711), with a GapWeight of
3.000 and a GapLengthWeight of 0.100.
Using the program GAP, some selected phytases exhibit the
following percentage similarity (identity in brackets) to the
Consensus-phytase-10-thermo(3)-Q50T-K91A amino acid sequence:
A. fumigatus ATCC-13073 a-mutant 86.7% (81.80
Basidiomycet consensus 64.1% (49.0%)
Consensus-phytase-1 98.7% (97.9%)
1oConsensus-phytase-10 96.6% (94.40)
Consensus-phytase-1-thermo(8)-Q50T-K91A 97.4% (95.5%)
Consensus-phytase-11 96.5% (94.2%)
Consensus-phytase-12 92.5% (89.9%)
Consensus-phytase-7 95.5% (93.4%)
A "purified" phytase is essentially free of other non-
phytase polypeptides, e.g. at least about 20% pure, preferably
at least about 40% pure, more preferably about 60% pure, even
more preferably about 80% pure, most preferably
about 90% pure,
2oand even most preferably about 95% pure,
as determined by SDS-
PAGE.
Preferred thermostable phytases are the so-called
consensus phytases of EP 98113176.6 (EP 0897985),
viz.
(i) any thermostable phytase which is obtainable by the
25processes described therein;
(ii) a phytase comprising the amino acid equence shown in Fig.
s
2 thereof or any variant or mutein thereof,
preferred
muteins being those comprising the substitutions Q50L;
Q50T; Q50G; Q50T-Y51N or Q50L-Y51N.
30
Other preferred thermostable phytases are
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(iii) a thermostable phytase which comprises at least one of the
following amino acid sequence (some of which are shown in
Figs. 5-12 herein), preferably the following phytases:
Consensus-phytase-1 (or simply Consensus phytase);
Consensus-phytase-1-thermo(3); Consensus-phytase-1-Q50T;
basidiomycete-consensus (or simply Basidio); Consensus.
phytase-10 (or Fcp 10); Consensus-phytase-11 (or Consensus
Seq. 11); Consensus-phytase-1-thermo(8)-Q50T-K91A;
Consensus-phytase-1-thermo(8)-Q50T; Consensus-phytase-1-
1o thermo(8); Consensus-phytase-10-thermo(3)-Q50T-K91A;
Consensus-phytase-10-thermo(3)-Q50T (sometimes, "(3)" is
deleted from this expression); Aspergillus fumigatus
ATCC 13073 phytase a-mutant; Aspergillus fumigatus
ATCC 13073 phytase a-mutant plus the mutations E59A,
15 S126N, R329H, S364T, G404A; Aspergillus fumigatus
ATCC 13073 phytase a-mutant plus the mutations E59A, K68A,
S126N, R329H, S364T, G404A; Consensus-phytase-7;
Consensus-phytase-12.
(iv) as well as thermostable variants and muteins of the
2o phytases of (iv) and (v), in particular those comprising
one or more of the following substitutions: Q50L,T,G;
Q50L-Y51N; Q50T-Y51N.
The term "plant" is intended to include not only whole
25 plants as such, but also plant parts or organs, such as leaves,
seeds or grains, stem, root, tubers, flowers, callus, fruits
etc.; tissues, cells, protoplats etc.; as well as any
combinations or sub-combinations thereof. Plant tissue cultures
and plant cell lines as well as plant protoplasts are
3o specifically included herein.
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The term "transgenic plant" is a plant as defined above,
which has been genetically modified, as well as its progeny and
propagating material thereof having retained the genetical
modification. Preferably, the transgenic plant comprises at
least one specific gene introduced into an ancestral plant by
recombinant gene technology. The term is not confined to a
single plant variety.
The invention relates to a transgenic plant which
comprises a DNA-construct encoding a thermostable phytase.
1o In a preferred embodiment the transgenic plant is a plant
grouping which is characterized in that it comprises a DNA-
construct encoding a thermostable phytase. The members of this
plant grouping may very well possess individuality, but are
clearly distinguishable from other varieties by their common
characteristic feature of the the thermostable phytase DNA-
construct.
Accordingly, the present teaching is applicable to more
than one plant variety. No naturally occuring plant varieties
are included amongst the plants of the invention.
2o In another preferred embodiment the invention relates to a
transgenic plant variety or a variant thereof a transgenic
plant species, a transgenic plant genus, a transgenic plant
family, and/or a transgenic plant order. More preferably, plant
varieties as such are disclaimed.
Any thermostable phytase may be used in the present
invention, e.g. any wild-type phytases, genetically engineered
phytases, consensus phytases, phytase muteins, and/or phytase
variants. Genetically engineered phytases include, but are not
limited to, phytases prepared by site-directed mutagenesis, gene
3o shuffling, random mutagenesis, etc.
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The nucleotide sequence encoding a wild-type thermostable
phytase may be of any origin, including mammalian, plant and
microbial origin and may be isolated from these sources by
conventional methods. Preferably, the nucleotide sequence is
derived from a microorganism, such as a fungus, e.g. a yeast or
a filamentous fungus, or a bacterium. The DNA sequence encoding-
a thermostable phytase may be isolated from the cell producing
it, using various methods well known in the art (see e.g. WO
98/28409 and EP 0897985).
1o The nucleotide sequence encoding a thermostable
genetically engineered or consensus phytase, including muteins
and variants thereof, may be prepared in any way, e.g. as
described in Example 3 hereof and in EP 0897985.
In order to accomplish expression of the thermostable
phytase in a plant of the invention the nucleotide sequence
encoding the phytase is inserted into an expression construct
containing regulatory elements or sequences capable of directing
the expression of the nucleotide sequence and, if necessary or
desired, to direct secretion of the gene product or targetting
of the gene product to the seeds of the plant.
In order for transcription to occur the nucleotide
sequence encoding the thermostable phytase is operably linked to
a suitable promoter capable of mediating transcription in the
plant in question. The promoter may be an inducible promoter or
a constitutive promoter. Typically, an inducible promoter
mediates transcription in a tissue-specific or growth-stage
specific manner, whereas a constitutive promoter provides for
sustained transcription in all cell tissues. An example of a
suitable constitutive promoter useful for the present invention
3o is the cauliflower mosaic virus 35 S promoter. Transcription
initiation sequences from the tumor-inducing plasmid (Ti) of
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Agrobacterium such as the octopine synthase, nopaline synthase,
or mannopine synthase initiator, are further examples of
preferred constitutive promoters.
Examples of suitable inducible promoters include a seed
5 specific promoter such as the promoter expressing alpha-amylase
in wheat seeds (see Stefanov et al, Acta Biologica Hungarica_
Vol. 42, No. 4 pp. 323-330 (1991), a promoter of the gene
encoding a rice seed storage protein such as glutelin, prolamin,
globulin or albumin (Wu et al., Plant and Cell Physiology Vol.
10 39, No. 8 pp. 885-889 (1998)), a Vicia faba promoter from the
legumin B4 and the unknown seed protein gene from Vicia faba
described by Conrad U. et al, Journal of Plant Physiology Vol.
152, No. 6 pp. 708-711 (1998), the storage protein napA promoter
from Brassica napus, or any other seed specific promoter known
15 in the art, eg as described in WO 91/14772.
In order to increase the expression of the thermostable
phytase it is desirable that a promoter enhancer element is
used. For instance, the promoter enhancer may be an intron which
is placed between the promoter and the amylase gene. The intron
2o may be one derived from a monocot or a dicot. For instance, the
intron may be the first intron from the rice Waxy (Wx) gene (Li
et al., Plant Science Vol. 108, No. 2, pp. 181-190 (1995)), the
first intron from the maize Ubil (Ubiquitin) gene (Vain et al.,
Plant Cell Reports Vol. 15, No. 7 pp. 489-494 (1996)) or the
first intron from the Act1 (actin) gene. As an example of a
dicot intron the chsA intron (Vain et al. op cit.) is mentioned.
Also, a seed specific enhancer may be used for increasing the
expression of the thermostable phytase in seeds. An example of a
seed specific enhancer is the one derived from the beta-
3o phaseolin gene encoding the major seed storage protein of bean
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(Phaseolus vulgaris) disclosed by Vandergeest and Hall, Plant
Molecular Biology Vol. 32, No. 4, pp. 579-588 (1996).
Also, the expression construct preferably contains a
terminator sequence to signal transcription termination of the
thermostable phytase gene such as the rbcS2' and the nos3'
terminators.
To facilitate selection of successfully transformed
plants, the expression construct should also preferably include
one or more selectable markers, e.g. an antibiotic resistance
1o selection marker or a selection marker providing resistance to a
herbicide. One widely used selection marker is the neomycin
phosphotransferase gene (NPTII) which provides kanamycin
resistance. Examples of other suitable markers include a marker
providing a measurable enzyme activity, e.g. dihydrofolate
reductase, luciferase, and b-glucoronidase (GUS).
Phosphinothricin acetyl transferase may be used as a selection
marker in combination with the herbicide basta or bialaphos.
The transgenic plant of the invention may be prepared by
methods known in the art. The transformation method used will
2o depend on the plant species to be transformed and can be
selected from any of the transformation methods known in the art
such as Agrobacterium mediated transformation (Zambryski et al.,
EMBO Journal 2, pp 2143-2150, 1993), particle bombardment,
electroporation (Fromm et al. 1986, Nature 319, pp 791-793), and
virus mediated transformation. For transformation of monocots
particle bombardment (ie biolistic transformation) of
embryogenic cell lines or cultured embryos are preferred. Below,
references are listed, which disclose various methods for
transforming various plants: Rice (Cristou et al. 1991,
3o Bio/Technology 9, pp. 957-962), Maize (cordon-Kamm et al. 1990,
Plant Cell 2, pp. 603-618), Oat (Somers et al. 1992,
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Bio/Technology 10, pp 1589-1594), Wheat (Vasil et al. 1991,
Bio/Technology 10, pp. 667-674, Weeks et al. 1993, Plant
Physiology 102, pp. 1077-1084) and Barley (Wan and Lemaux 1994,
Plant Physiology 102, pp. 37-48, review Vasil 1994, Plant Mol.
Biol. 25, pp 925-937).
More specifically, Agrobacterium mediated transformation
is conveniently achieved as follows:
A vector system carrying the thermostable phytase is
constructed. The vector system may comprise of one vector, but
1o it can comprise of two vectors . In the case of two vectors the
vector system is referred to as a binary vector system (Gynheung
An et al.(1980), Binary Vectors, Plant Molecular Biology Manual
A3, 1-19 ) .
An Agrobacterium based plant transformation vector
consists of replication origins) for both E.coli and
Agrobacterium and a bacterial selection marker. A right and
preferably also a left border from the Ti plasmid from
Agrobacterium tumefaciens or from the Ri plasmid from
Agrobacterium rhizogenes is nessesary for the transformation of
2o the plant. Between the borders the expression construct is
placed which contains the thermostable phytase gene and
appropriate regulatory sequences such as promotor and terminator
sequences. Additionally, a selection gene e.g. the neomycin
phosphotransferase type II (NPTII) gene from transposon Tn5 and
a reporter gene such as the GUS (betha-glucuronidase) gene is
cloned between the borders. A disarmed Agrobacterium strain
harboring a helper plasmid containing the virulens genes is
transformed with the above vector. The transformed Agrobacterium
strain is then used for plant transformation.
3o The invention also relates to a method of preparing a
transgenic plant capable of expressing a thermostable phytase,
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PCT/D K99/00154
said method comprising the steps of (i) isolating a nucleotide
sequence encoding a thermostable phytase; (ii) inserting the
nucleotide sequence of (i) in an expression construct capable of
mediating the expression of the nucleotide sequence in a
selected host plant; and (iii) transforming the selected host
plant with the expression construct.
The above method in which "at least one" replaces "a,"
when used in relation to the thermostable phytase, is also
within this invention.
1o This method is an essentially non-biological method.
Any plant may be a selected host plant. More specifically,
the plant can be dicotyledonous or monocotyledonous, for short a
dicot or a monocot. Of primary interest are such plants which
are potential food or feed components. These plants may comprise
phytic acid. Examples of monocot plants are grasses, such as
meadow grass (blue grass, Poa), forage grass such as festuca,
lolium, temperate grass, such as Agrostis, and cereals, e.g.
wheat, oats, rye, barley, rice, sorghum and maize (corn).
Examples of dicot plants are legumes, such as lupins, pea,
2o bean and soybean, and cruciferous (family Brassicaceae), such as
cauliflower, oil seed rape and the closely related model
organism Arabidopsis thaliana.
Of particular interest are monocotyledonous plants, in
particular crops or cereal plants such as wheat (Triticum, e.g.
aestivum), barley (Hardeum, e.g. vulgare), oats, rye, rice,
sorghum and corn (Zea, e.g. mat's).
Of further particular interest are dicotyledonous plants,
such as those mentioned above.
In a preferred embodiment, the ancestral plant or host
3o plant is per se a desired feed ingredient.
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Examples
Example 1
FYT-assay - for analyzing phytase enzyme preparations
The phytase activity can be measured using the following assay:
10 ul diluted enzyme samples (diluted in 0.1 M sodium acetate,
0.01 % Tween20, pH 5.5) are added into 250 ul 5 mM sodium
phytate (Sigma) in 0.1 M sodium acetate, 0.01 o Tween20, pH 5.5
(pH adjusted after dissolving the sodium phytate: the substrate
is preheated) and incubated for 30 minutes at 37°C. The reaction
1o is stopped by adding 250 ul 10 % TCA and free phosphate is
measured by adding 500 ul 7.3 g FeS04 in 100 ml molybdate
reagent (2.5 g (NH4) 6MO,O~4.4H20 in 8 ml HzS04 diluted to 250 ml) .
The absorbance at 750 nm is measured on 200 ul samples in 96
well microtiter plates. Substrate and enzyme blanks are
included. A phosphate standard curve is also included (0-2 mM
phosphate). 1 FYT equals the amount of enzyme that releases 1
umol phosphate/min at the given conditions. This assay is
preferred for phytase enzyme preparations (when not in admixture
with other feed ingredients).
Example 2
FTU assay - for analyzing phytase in admixture with feed ingre-
dients
One FTU is defined as the amount of enzym, which at stan-
dard conditions (37°C, pH 5,5; reaction time 60 minutes and
start concentration of phytic acid 5 mM) releases phosphate
equivalent to 1 ~mol phosphate per minute.
1 FTU = 1 FYT
The FTU assay is preferred for phytase activity measure-
3o ments on animal feed premixes and the like complex compositions.
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Reagents lst~bstrates
Fx~Ya--~~~n buffer for feed etc.
This buffer is also used for preparation of POQ-standards
and further dilution of premix samples.
5 0 22 M aceta~A h»ffPr with Tween 20 nH 5.5 _
g sodium acetate trihydrate (MW = 136,08 g/mol) e.g.
Merck Art 46267 per liter and 0,1 g Tween 20 e.g. Merck Art
22184 pr. liter are weighed out.
The sodium acetate is dissolved in demineralised water.
10 Tween 20 is added, and pH adjusted to 5,50 ~ 0,05 with
acetic acid.
Add demineralised water to total volume.
0,22 M acetate buffer with Tween 20, EDTA, P093-og BSA.
15 30 g sodium acetate trihydrate e.g. Merck Art 6267 per li-
ter.
0,1 g Tween 20 e.g. Merck Art 22184 per liter.
30 g EDTA f.eks. Merck Art 8418 pr. liter.
20 g Na2HP0,, 2Hz0 e.g. Merck Art 6580 per liter.
20 0,5 g BSA (Bovine Serum Albumine, e.g. Sigma Art A-9647
per liter.
The ingredients are dissolved in demineralised water, and
pH is adjusted to 5,50 ~ 0,05 with acetic acid.
Add demineralised water to total volume.
25 BSA is not stable, and must therefore be added the same
day the buffer is used.
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50 mM PO~stock solution
0,681 g KH2P04 (MW = 136,09 g/mol) e.g. Merck Art 4873 is
weighed out and dissolved in 100 ml 0,22 M sodium acetat with
Tween, pH 5,5.
Storage stability: 1 week in refrigerator.
~~ M acetate buffer pH 5 5 without Tween
This buffer is used for production of phytic acid sub-
strate) .
150 g sodium acetate trihydrate (MW = 136,08) e.g. Merck
1o Art 6267 is weighed out and dissolved in demineralised water,
and pH is adjusted with acetic acid to 5,50 ~ 0,05.
Add demineralised water to 5000 ml.
Storage stability: 1 week at room temperature.
~5 The volume of phytic acid is calculated with allowance for
the water content of the used batch.
If the water content is e.g. 8,4 % the following is ob-
tained:
2 0 0005 mol / 1 x 923,8 g l mol _ 504 / 1
~1= 0,084 g
Phytic acid (Na-salt) (MW = 923,8 g/mol) e.g. Sigma P-8810
is weighed out and dissolved in 0,22 M acetate buffer (without
tween). Addition of (diluted) acetic acid increases the disso-
lution speed.
25 pH is adjusted to 5,50 t 0,05 with acetic acid.
Add 0,22 M acetate buffer to total volume.
2i 7 % nitric acid solution
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For stop solution.
1 part concentrated (65%) nitric acid is mixed into 2
parts demineralised water.
Mo~y~~darP reagen
For stop solution.
100 g ammonium heptamolybdate tetrahydrate (NHQ) 6Mo.,029, 4H20
e.g. Merck Art 1182 is dissolved in demineralised water. 10 ml
2 5 % NH3 i s added .
Add demineralised water to 1 liter.
~ 2a % Ammonium vanadate
Bought from fra Bie & Berntsen.
Molybdat/vanadat stop solution
1 part vanadate solution (0,24 % ammonium vanadate) + 1
part molybdate solution are mixed. 2 parts 21,7 % nitric acid
solution are added.
The solution is prepared not more than 2 hours before use,
and the bottle is wrapped in tinfoil.
Frozen samples are defrosted in a refrigerator overnight.
2o Sample size for feed samples: At least 70 g, preferably
100 g.
Choose a solution volume which allows addition of buffer
corresponding to 10 times the sample weight, e.g. 100 g is dis-
solved in 1000 ml 0,22 M acetate buffer with Tween, see enclo-
sure 1. Round up to nearest solution volume.
If the sample size is approx. 100 g all the sample is
ground in a coffee grinder and subsequently placed in tared
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beakers. The sample weight is noted. It is not necessary to
grind not-pelleted samples. If a sample is too big to handle, it
is sample split into parts of approx. 100 g.
Magnets are placed in the beakers and 0,22 M acetate
buffer with Tween is added.
The samples are extracted for 90 minutes.
After extraction the samples rest for 30 minuts to allow
for the feed to sediment. A 5 ml sample is withdrawn with a pi-
pette. The sample is taken 2 - 5 cm under the surface of the so-
lotion and placed in a centrifuge glass, which is covered by a
lid.
The samples are centrifuged for 10 minutes at 4000 rpm.
Choose a solution volume which allows addition of buffer
corresponding to 10 times the sample weight. Round up to nearest
solution volume.
If the samples have been weighed (50 - 100 g) all of the
sample is placed in tared beakers. The sample weight is noted.
If a sample is too big to handle, it is split into parts of ap-
2o prox. 100 g.
Magnets are placed in the beakers and 0,22 M acetate
buffer with Tween, EDTA og PO,'- is added.
The samples are extracted for 60 minutes.
After extraction the samples rest for 30 minutes to allow
for the premix to sediment. A 5 ml sample is withdrawn with a
pipette. The sample is taken 2 - 5 cm under the surface of the
solution and placed in a centrifuge glass, which is covered by a
lid.
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The samples are centrifuged for 10 minutes at 4000 rpm.
Extracts of feed samples are analysed directly.
Extracts of premix are diluted to approx. 1,5 FTU/g (A4ls
(main sample) < 1,0 ).
0,22 M acetate buffer with Tween 20 is used for the dilu-
tion.
wra; n Samples
2 x 100 ml of the supernatant from the extracted and cen-
lo trifuged samples are placed in marked glass test tubes and a
magnet is placed in each tube.
When all samples are ready they are placed on a water bath
with stirring. Temperature: 37 °C.
3,0 ml substrate is added.
Incubation for exactly 60 minutes after addition of sub-
strate.
The samples are taken off the water bath and 2,0 ml stop
solution is added (exactly 60 minutes after addition of sub-
strate).
2o The samples are stirred for 1 minute or longer.
Feed samples are centrifuged for 10 minutes at 4000 rpm
(It is not necessary to centrifuge premix samples).
100 ml of the supernatant from the extracted and centri-
fuged samples are placed in marked glass test tubes, and a mag-
net is placed in each tube.
2,0 ml stop solution is added to the samples.
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3,0 ml substrate is added to the samples.
The samples are incubated for 60 minutes at room tempera-
ture.
The feed samples are centrifuged for 10 minutes at 4000
5 rpm (it is not necessary to centrifuge premix samples).
2 x 100 ml are taken from each of the 8 standards and also
4 x 100 ml 0,22 M acetate buffer (reagent blind).
A415 is measured on all samples .
10 CALCULATION
FTU/g = ~Cmol P043- / (min * g (sample) )
C g sample is weighed out (after grinding).
15 100 ~.1 is taken from the extracted and centrifuged sample.
Po93- standard curve is linear.
From the regression curve for the P093- standard the actual con-
2o centration of the sample is found (concentration in mM):
[P043-] - (x - b) / a x = A,15 a = slbpe b = in-
tercept with y-axis
25 ~Cmol P04'-/min = ( [PO43-] (mM) x Vol (liter) x 1000 ~mol/mmol
/t
t = incubation time in minutes.
Vol = sample volume in liter = 0,0001 liter
1000 - conversion factor from mmol to ~.mol
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FTU / gpr0"~ _ { ( x - b ) x Vol x 10 0 0 x Fp } / t a x t x C
C = gram sample weighed out
FP= Relation between the sample taken out and the total sample
(after extraction). Example: 0,100 ml taken from 1000 ml -~ Fp =
1000/0,100 - 10000.
Reduced expression with insertion of the following values:
l0 t = 60
Vo1 = 0,0001 1
FP = 10000
FTU /gsa,np~e = { (X - b) x 0, 0001 X 1000 x 10000} / ( a x 60 X C}
Example 3
Determination of optimum temperature and melting point Tm of
various phytases
The thermostability of various phytases has been
determined, viz. the melting temperature, Tm, and/or the optimum
2o temperature.
The phytase of Aspergillus niger NRRL 3135 was prepared as
described in EP 0420358 and van Hartingsveldt et al (Gene, 127,
87-94, 1993).
The phytases of Aspergillus fumigatus ATCC 13073,
Aspergillus terreus 9A-1, Aspergillus terreus CBS 116.46,
Aspergillus nidulans, Myceliophthora thermophila, and
Talaromyces thermophilus were prepared as described in
EP-0897985 and the references therein.
Consensus-phytase-1 (Fig. 5) and Consensus-phytase-1-Q50T
3o are shown in and were prepared as described in EP 0897985.
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Consensus-phytase-10 was derived and prepared according to
the teachings of EP-0897985 (Examples 1-2 and 3-7,
respectively), however adding to the alignment at Fig. 1 thereof
the phytase sequence of Thermomyces lanuginosa (Berka et al,
Appl. Environ. Microbiol. 64, 4423-4427, 1998) and a
basidiomycete consensus sequence (derivation described below).,
omitting the sequence of A.niger T213, and assigning a vote
weight of 0.5 for the remaining A.niger phytase sequences. The
derivation of the sequence of Consensus-phytase-10 is shown in
1o Fig . 7 .
The basidiomycete consensus sequence was also derived
according to the principles of EP-0897985, viz. from the five
basidiomycete phytases of WO 98/28409, starting with the first
amino acid residue of the mature phytases (excluding signal
peptide). A vote weight of 0.5 was assigned to the two Paxillus
phytases, all other genes were used with a vote weight of 1.0 -
see Fig. 6.
The muteins Consensus-phytase-10-thermo, Consensus
phytase-10-thermo-Q50T-K91A (Fig. 10) and Consensus-phytase-10
2o thermo-Q50T were prepared from consensus-phytase-10, in analogy
to Examples 5-8 of EP-0897985, by introducing the three back-
mutations K94A, V158I and A396S ("thermo(3)" or "thermo") and,
where applicable, also the mutations Q50T or Q50T-K91A.
The muteins Consensus-phytase-1-thermo(8), Consensus
phytase-1-thermo(8)-Q50T-K91A (Fig. 9) and Consensus-phytase-1
thermo(8)-Q50T, were prepared from consensus-phytase-1, in
analogy to Example 8 of EP-0897985, by introducing the eight
mutations E58A, D197N, E267D, R291I, R329H, S364T, A379K and
G404A ("thermo(8)") and, where applicable, also the mutations
3o Q50T or Q50T-K91A.
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Consensus-phytase-1-thermo(3) was prepared from consensus-
phytase-1 by introduction of the three mutations K94A, V158I and
A396S.
An Aspergillus fumigatus so-called a-mutant (with the
mutations Q51(27)T, F55Y, V100I, F114Y, A243L, S265P, N294D) and
the further muteins thereof shown in Table 1 were prepared as~
generally described above. The position numbering refers to Fig.
11 hereof, except for the number in parentheses which refers to
the numbering used in EP 0897010.
1o DNA constructs encoding the above thermostable phytases
can be prepared e.g. according to the teachings of EP 0897985.
For expression thereof in plants, reference is made to the
present description.
In order to determine the unfolding temperature or melting
~5 temperature, Tm, of a phytase, differential scanning calorimetry
was applied as previously published by Brugger et al (1997):
"Thermal denaturation of phytases and pH 2.5 acid phosphatase
studied by differential scanning calorimetry," in The
Biochemistry of phytate and phytase (eds. Rasmussen, S.K; Raboy,
2o V.: Dalbmge, H. and Loewus, F.; Kluwer Academic Publishers).
Homogenous or purified phytase solutions of 50-60 mg/ml of
protein are prepared, and extensively dialyzed against 10 mM
sodium acetate, pH 5Ø A constant heating rate of 10°C/min is
applied up to 90-95°C.
25 The results of Tm determinations on the above phytases are
shown in Table 1 below: for selected phytases also in Figs. 1-4.
In Table 1 below, the optimum temperature of various
phytases is also indicated. For this determination, phytase
activity was determined basically as described by Mitchell et al
30 (Microbiology 143, 245-252, 1997): The activity was measured in
an assay mixture containing 0.5% phytic acid (~ 5 mM) in 200 mM
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sodium acetate, pH 5Ø After 15 min of incubation at 37°C, the
reaction was stopped by addition of an equal volume of 15%
trichloroacetic acid. The liberated phosphate was quantified by
mixing 100 ~,1 of the assay mixture with 9001 HZO and 1 ml of
0.6 M HZSOq, 2% ascorbic acid and 0.5% ammonium molybdate.
Standard solutions of potassium phosphate were used as.
reference. One unit of enzyme activity was defined as the amount
of enzyme that releases 1 ~mol phosphate per minute at 37°C. The
protein concentration was determined using the enzyme extinction
1o coefficient at 280 nm calculated according to Pace et al
(Prot.Sci. 4, 2411-2423, 1995): Consensus phytase, 1.101;
consensus phytase 7, 1.068; consensus phytase 10, 1.039.
For determination of the temperature optimum, enzyme
(100,1) and substrate solution (1001) were pre-incubated for 5
min at the given temperature. The reaction was started by
addition of the substrate solution to the enzyme. After 15 min
incubation, the reaction was stopped with trichloroacetic acid
and the amount of phosphate released was determined. Phytase-
activity-versus-temperature is plotted, and the temperature
optimum is determined as that temperature at which the acitivity
reaches its maximum value.
Table 1
Temperature optimum and Tm for various phytases
Phytase Optimum temperature Tm (°C)
(°C)
Aspergillus niger 55 63.3
NRRL 3135
Aspergillus 55 ~'w
fumigatus ATCC 13073
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Aspergillus terreus 49 57.5
9A-1
Aspergillus terreus 45 58.5
CBS 116.46
Aspergillus nidulans 45 55.7
Myceliophthora 55 - '
thermophiia
Talaromyces 45 -
thermophilus
Consensus-phytase- 82 89.3
10-thermo-Q50T-K91A
Consensus-phytase- 82 ' 88.6
10-thermo-Q50T
Consensus-phytase-10 80 85.4
Consensus-phytase-1- - 85'7
thermo(8)-Q50T-K91A
Consensus-phytase-1- 78 84'7
thermo(8)-Q50T
Consensus-phytase-1- 81
thermo(8)
Consensus-phytase-1- 78 84'7
thermo(8)-Q50T-K91A
Consensus-phytase-1- 75
thermo(3)
Consensus-phytase-1- - 78'9
Q50T
Consensus-phytase-1 71 78.1
Aspergillus 63 -
fumigatus a,-mutant,
plus mutations E59A,
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S126N, R329H, S364T,
G404A
Aspergillus 63 -
fumigatus - as
above, plus mutation
K68A
Aspergillus
fumigatus a-mutant
(Q51(27)T, F55Y,
V100I, F114Y, A293L,
S265P, N294D)
