Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-02-01
2001-06-19
Fredman, Jeffrey (Department: 1655)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S288000, C800S295000, C800S278000, C436S074000, C436S169000, C436S169000
Reexamination Certificate
active
06248938
ABSTRACT:
FIELD OF THE INVENTION
The invention is drawn to value-added animal feed compositions and additives containing unprocessed or minimally processed matter from transgenic alfalfa which expresses exogenous phytase activity in concentrations nutritionally significant in monogastric animals. The invention is further drawn to novel uses of the animal feed compositions.
BIBLIOGRAPHY
Complete bibliographic citations of the references described herein can be found in the Bibliography section, immediately preceding the claims.
DESCRIPTION OF THE PRIOR ART
Livestock production, especially large-scale commercial livestock production for human consumption, requires the use of vast amounts of nutritionally balanced animal feed. Because of the large amounts of feed required to sustain commercial livestock production, world-wide research efforts have been made to develop feedstock additives which maximize the bioavailability of nutritionally important elements and compounds found in common animal feedstocks.
In the early 1950's, for instance, it was speculated that the dietary requirements of egg-laying fowl might be met by utilizing protein-rich extracts from green plants. Hughes and Eyles (1953) describe a feeding trial with laying hens which used dietary protein extracted from the leaves of green plants. The authors hypothesized that dietary protein could be extracted from green plants in an economical fashion, thereby easing the shortage and lowering the cost of high protein feed in Great Britain.
In more recent years, with the development of sophisticated methods of genetic manipulation, transgenic plants which express nutritionally important compounds have been developed. However, in order to effectively utilize transgenic plants which express exogenous proteins, the transgenic plants must be more economical to use than the feedstocks or feedstock additives they are designed to replace.
Therefore, it is necessary to maximize the expression of the exogenous protein while simultaneously stabilizing the beneficial activity of the protein. Additionally, the exogenous expressed protein ideally should be utilizable with very little or no post-harvest processing of the transgenic plant material. If the exogenous protein is expressed in only small quantities, or if the transgenic plant material must be extensively processed prior to use, or if the exogenous protein lacks sufficient stability in the harvested plant material, the slim profit margins encountered in commercial feed production will dictate against switching to the use of transgenic plant material. In short, because alternative sources of nutrients continue to be relatively cheap and widely available, the positive economics of producing nutritionally important feed additives in transgenic plants remains marginal unless the above criteria are present.
The remarkable progress in recombinant plant genetics has greatly spurred new investigations into the economics of manufacturing, isolating, and using exogenous proteins expressed in transgenic herbage plants such as alfalfa. In effect, valuable recombinant protein products, which are now produced by fermentation using transgenic microorganisms, might be economically produced using transgenic plants rather than native or recombinant microbes.
Austin et. al. (1994) studied the production of industrial enzymes in transgenic alfalfa, a report of which appeared in the
Annals of the New York Academy of Sciences.
These investigators researched the feasibility of producing industrially important enzymes using alfalfa plants as “factories.” The focus of this study was whether, using genetic engineering technology, cloned genes for valuable enzymes could be expressed and economically harvested from plant hosts. The concept is economically attractive because, assuming the heterologous gene can be stably incorporated, many herbage plants are perennial, hardy crops, which are capable of more than one harvest per year. In the case of alfalfa specifically, in temperate climates such as those found in the midwestern United States, alfalfa does not require irrigation and is capable of three or more harvests a year. Moreover, since alfalfa is leguminous, it grows well without nitrogen fertilizer.
The Austin et al. paper noted above used a reporter gene, &bgr;-glucuronidase (GUS), as a model system. The analysis concluded (as noted generally above) that the concentration of the desired value-added product (in this case, GUS) in the transgenic plant is most critical variable for economic viability. Analogous field studies for transgenic alfalfa which expresses &agr;-amylase and manganese-dependent lignin peroxidase have also been reported by Austin et al. (1995).
An enzyme group of particular interest is the phytases. Phytases, more properly referred to as myo-inositol hexaphosphate phosphohydrolases, are a family of enzymes which catalyze the step-wise removal of inorganic orthophosphate from phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate). The economic interest in phytase is due to its ability to increase the bio-availability of inorganic phosphorous in phytate-containing non-ruminant animal feeds. Currently, feed for non-ruminant animals must be supplemented with inorganic phosphorous because these animal cannot utilize the phosphorous present as phytate.
While phytase occurs widely in both plants and microorganisms, the enzyme has been extensively studied mostly from the filamentous fungi, particularly the Aspergilli, notably
A. ficuum,
and
A. nidulans.
For an excellent review of phytases and their action on phytic acid see Gibson, D. M. and Ullah, A. B. J. (1990), incorporated herein by reference.
Regarding the nucleotide sequences which encode phytase, several such sequences have been identified, sequenced, and cloned into various heterologous hosts. For instance, Van Gorcom et al., U.S. Pat. No. 5,436,156, incorporated herein by reference in its entirety, describe the isolation and cloning from
A. ficuum
of a DNA sequence coding for phytase. The isolated nucleotide sequence was successfully cloned and inserted into a vector capable of transforming a microbial expression host. Specifically, the nucleotide sequence was first cloned using the bacteriophage lambda AFD01, and further sub-cloned into pAN 8-1 and pUC19. The construct was then used to transform various types of filamentous fungi. (See also, EP 0 420 358 A1, to the same inventor.)
Ehrlich et al. (1993), describe the cloning and sequencing of a gene for a second type of phytase, designated PhyB. This phytase was isolated from
A. niger
NRRL 3135, and had a pH optimum of 2.5. PhyB was found to have different properties from the previously known phytase PhyA, which has a pH optimum of 5.0.
European Patent Application EP 0 449 375 A2 (Pen et al.) describes the expression of phytase in tobacco seeds and rapeseeds.
Likewise, Verwoerd et al. (1995) describe the production and accumulation of phytase in the leaves of tobacco plants transformed with a phytase-coding gene of
A. niger.
This paper describes the constitutive expression of a phytase cDNA from transgenic tobacco plants. The exogenous phytase enzyme was secreted into the extracellular fluid at concentrations approximately 90 times higher than that in the total leaf extract. The phytase produced by the transgenic tobacco plants was compared to native Aspergillus phytase and found to have identical activities. During plant maturation, it was found that the phytase produced in the tobacco remained biologically active and accumulated in amounts up to 14.4% of the total soluble protein found in the tobacco.
As noted briefly, above, the economic interest in phytase is due to its ability to increase the bio-availability of inorganic phosphorous in phytate-containing animal feeds. The increase in intensive, large-scale livestock production has resulted in increased environmental problems, specifically eutrophication, due to the tremendous amount of manure produced in such enterprises. Phosphorous, an essential nutrient for both ruminants and non-ruminants, is necessarily ad
Austin-Phillips Sandra
Cook Mark
Koegel Richard G.
Straub Richard J.
DeWitt Ross & Stevens S.C.
Einsmann Juliet C.
Fredman Jeffrey
Leone, Esq. Joseph T.
Wisconsin Alumni Research Foundation
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