Soybean phytase and nucleic acid encoding the same

Details Soybean phytase and nucleic acid encoding the same Soybean phytase and nucleic acid encoding the same

1999-05-14

2001-10-16

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S410000, C435S069100, C800S295000

Reexamination Certificate

active

06303766

ABSTRACT:

1. FIELD OF THE INVENTION
The invention relates to nucleic acids encoding the soybean phytase protein, the soybean phytase per se, and method for obtaining expression of the soybean phytase in appropriate hosts.
2. BACKGROUND OF THE INVENTION
2.1 Phytate
Phosphorous is an essential dietary requirement for animal growth.
Abundant stores of phosphorous are present in plant seeds in the form of phytate (myo-inositol hexakisphosphate). Phytate serves as the storage form of phosphate and myo-inositol for germination and seedling growth. Phytate is synthesized during seed development and comprises 1-3% of the total dry weight of seeds (Reddy et al.,
Phytates in Cereals and Legumes
, CRC Press, Inc., Boca Raton, Fla., 1989). In cereals, phytate is deposited in aleurone particles. It is found in protein bodies in legume cotyledons. A biochemical and ultrastructural analysis of soybean seeds showed the presence of phytate in protein bodies, with a significant amount deposited with globoid inclusions (Prattley and Stanley,
J. Food Biochem
. 6:243-253. 1982).
The soybean [
Glycine max
(L.)] has long been recognized as a valuable component of medicine, food and animal feed. Soybean is an important crop worldwide as both a protein and oil source. Soybean is processed to yield soybean oil and soybean meal by a hexane extraction process. Because of its high nutritional value, wide availability, low cost and consistency of composition, soybean meal has displaced most of the animal proteins (meat meal, tankage, and fish meal) and has become a vital component of poultry and swine feeds. Together, poultry and swine feeds account for over two thirds of soybean meal utilization in the United States. Phytate accumulates in soybean during seed development, where it serves a phosphorous storage function. Phytate is normally utilized after germination of the soybean seedling, when it is broken down by newly-synthesized phytase.
Phytate is poorly utilized by humans and other monogastric animals. Because non-ruminant animals lack the enzymes necessary for phytate degradation, phytate-bound phosphorous passes through the gastrointestinal tract undigested, making it unavailable to non-ruminants. Phytate is considered an anti-nutritional factor because it chelates essential minerals such as zinc, manganese, iron, calcium and potassium and forms indigestible protein-phytate complexes (Erdman and Forbes,
J. Am. Oil Chem. Soc
. 58:489-495 (1981); Zhu et al.,
Anim. Feed Sci. Techn
. 27:341-351 (1990); Simons et al.,
Brit. J. Nutr
. 64:525-540 (1990)). Plant phytate is present predominantly as the calcium-magnesium-potassium salt called phytin. These complex forming properties of phytate are responsible for lowered mineral and protein bioavailability in animal diets containing high phytate levels (Reddy et al., supra).
To meet dietary requirements, inorganic phosphorous is routinely added to swine and poultry rations. Phosphorous supplementation increases feed costs and contributes to the phosphorous content of animal waste. Moreover, non-utilized phytate is excreted by the animals contributes to environmental pollution in areas of intensive animal production. Annually in the United States, swine and poultry generate over 20 million tons of manure containing over 300,000 tons of waste phosphorous (Cromwell and Coffey, Altech Conference Proceedings, 1991). Typically, manure is applied as fertilizer to pastures and croplands, causing an increase in soil phosphorous, eventually resulting in water contamination. Since phosphorous is the limiting nutrient for aquatic plant life growth, increases in water phosphorous content results in eutrophication and decreased water quality. Increased regulatory scrutiny of animal waste disposal has fostered interest in finding solution for decreasing phosphorous output.
2.2 Phytase
Phytases are enzymes that sequentially remove phosphates from the phytate molecule. Phytase (E.C. 3.1.3.8) catalyzes the removal of orthophosphate from various myo-inositol phosphates. Phytases have been characterized from a variety of fungi, bacteria, plants and animals. Fungal phytases are in the general class of acid phosphatases. Numerous studies have shown that supplementation of poultry and livestock feed with microbial phytase improves phosphorus bioavailability (Nelson et al.,
Poultry Sci
. 47:1372-1374,1968; Swick and Ivey,
Feed Management
43:8-17, 1992). Commercially available phytase preparations are derived primarily from the fungus
Aspergillus niger
(originally designated
A. ficuum
) because of high levels of production of this extracellular enzyme (Shieh and Ware,
Appl. Microbiol
. 16:1348-1351, 1968; Ullah, supra). An engineered form of the
A. niger
phytase is now available as a supplement (Natuphos®, BASF). The beneficial effects of supplementation have been demonstrated by the addition of microbial phytase to diets of poultry and swine (Cromwell et al.,
J. Anim. Sci
. 73 449-456, 1995; Denbow et al.,
Poultry Sci
. 74: 1831-1842, 1995; Potter et al.,
Poultry Sci
. 74: 813-820, 1995; Ravindran et al.,
Poultry Sci
. 74:1820-1830,1995; Yi et al.,
Poultry Sci
. 75:240-249, 1996).
Phytase supplementation reduces the need for inorganic phosphorous and lowers the percentage of dietary phosphorous that is excreted (Yi et al.,
Poultry Science
75:240-249, 1996; Cromwell et al.,
J. Animal Science
73:449-456, 1995). However, commercial phytase supplements are expensive. Moreover, phytase must be added to the feed after pelleting to avoid heat denaturation of the enzyme at the high pelleting temperature.
While phytase occurs naturally in soybean, there is little enzyme activity until seed germination. Hence, phytase is not present in substantial levels in soybean meal. The phytate in soy meal is not utilized by poultry and swine, and passes undigested into manure. The desired timing of phytase expression is during seed development, which would lead to phytase accumulation in the soybean, and soybean meal with reduced phytate.
There have been numerous reports of the isolation and cloning and expression of phytase DNA of non-plant origin. Such sources include ruminal microorganisms (WO 97/48812);
Escherichia coli
B (U.S. Pat. No. 5,876,997);
Thermomyces lanuginosus
(U.S. Pat. No. 5,866,118);
Aspergillus ficum
(U.S. Pat. No. 5,863,533);
Schwanniomyces occidentalis
(U.S. Pat. No. 5,840,562);
Aspergillus niger
var. ALKO243 (U.S. Pat. No. 5,834,286);
Schwanniomyces occidentalis
(U.S. Pat. No. 5,830,732);
Bacillus subtilis
(WO 98/06856);
Myceliopthora thermophila
and
Aspergillus terreus
(EP 0 684 313);
Peniophora lycii
(WO 98/28408). U.S. Pat. No. 5,780,292 discloses methods for the overexpression of recombinant
Aspergillus niger
phytase in the filamentous fungus
Trichoderma reesei
. U.S. Pat. No. 5,593,963 discloses methods for expression of foreign phytase genes in plants, preferably microbial phytase genes. A heat stable phytase from the fungus
Aspergillus fumigatus
has been cloned and characterized (Pasamontes et al.,
Appl. Environ. Microbiol
. (1997) 63(5):1696-700).
The cloning and sequencing of the phytase from
Aspergillus niger
is described in U.S. Pat. No. 5,436,156 to van Gorcom et al. The gene has been introduced into tobacco (Pen et al.,
Bio/Technology
11:811-814, (1993); Verwood et al.,
Plant Physiol
. 109:1199-1205 (1995)). While feeding trials have demonstrated that transgenic tobacco seeds expressing
A. niger
were effective as a dietary phytase source in poultry feed, tobacco seeds are expensive to produce and are not normally used as a feedstuff.
Fungal phytase has been expressed in soybean. A study was conducted comparing milled soybean seeds expressing fungal phytase with the commercial Natuphos® preparation as supplements for poultry feeding (Denbow et al.,
Poultry Sci
. 77:878-881, 1998). As measured by body weight gain, phosphorus utilization and phosphorus excretion, the recombinant fungal phytase produced in transgenic soybean was just as effective as Natuphos® as a dietary supplement. However, milled, full-fat soy

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