Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
2000-12-06
2003-10-28
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S286000, C800S320100, C800S263000, C435S069100, C435S101000, C435S210000, C435S320100, C435S412000, C435S419000, C435S468000
Reexamination Certificate
active
06639126
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the production of modified polyglucans through the alteration of the polyglucan biosynthesis pathway.
BACKGROUND OF THE INVENTION
Starch constitutes 65-75% of the corn kernel and is the main source of energy for livestock and poultry fed corn-based feed rations. Energy availability from corn is limited to a certain degree by endosperm matrix factors that prevent the release of intact starch granules during digestion. Protein and fiber characteristics may be manipulated to facilitate the release of starch granules, thereby enhancing energy availability.
Energy availability from corn is also determined by starch and oil content, starch structure (amylose:amylopectin ratio), and interactions among these different factors. Degradation characteristics of isolated starch are largely determined by the polyglucan structure. Waxy starch (all amylopectin), once gelatinized, is more rapidly digested than normal starch (70-75% amylopectin, 25-30% amylose). High amylose (70%) starch is more slowly and less expensively digested. Highly branched polysaccharides such as phytoglycogen are soluble and very rapidly digested. The enhanced in vitro digestibility of isolated starch from waxy corn over isolated starch from normal corn does not always translate to improved digestibility of ground corn (e.g., Ertl and Dale (1997)
Appl. Poul. Res
. 6:432-435), which is possibly caused by component interactions.
Starch can be converted into simple sugars by an enzymatic process carried out in two stages: the liquefaction of starch and the saccharification of the liquefied starch. See, for example, Manners (1985) “Structural Analysis of Starch Components by Debranching Enzymes,” in
New Approaches to Research on Cereal Carbohydrates
, ed. Hill (Amsterdam), pp. 45-54; and Enevoldsen (1985) “Aspects of the Fine Structure of Starch”, in
New Approaches to Research on Cereal Carbohydrates
, ed. Hill (Amsterdam), pp. 55-60.
Amylopectin is a branched glucose polymer that is a major constituent of plant starch granules and the primary determinant of their structural and physical properties. The spatial positioning of &agr;(1→6) glycosidic bonds, i.e., branch linkages, is a critical aspect of the three dimensional structure of amylopectin. Branch linkages are introduced by the actions of starch branching enzymes (BEs) and are hydrolyzed by the, actions of starch debranching enzymes (DBEs). See, for example, Preiss (1996)
Starch Synthesis in Sinks and Sources
(Marcell Dekker, Inc., New York), pp. 63-96; Smith et al. (1997)
Annu. Rev. Plant Physiol. Plant Mol. Biol
. 48:67-87. Mutations that result in DBE deficiencies, such as the sugary1 (su1) mutations of maize and rice (James et al. (1995)
Plant Cell
7:417-429; Nakamura et al. (1996)
Physiol. Plant
. 97:491-498; Pan et al. (1984)
Plant Physiol
. 74:324-328; Rahman et al. (1998)
Plant Physiol
. 117:425-435), alter the number and spatial distribution of branches in amylopectin. DBEs, therefore, are believed to be involved in branch pattern determination, possibly providing an editing function (Ball. et al. (1996)
Cell
86:349-352).
The two classes of DBEs that have been identified in plants and are distinguishable by their substrate specificity (Doehlert et al. (1991)
J. Plant Physiol
. 138:566-572; Lee et al. (1971)
Arc. Biochem. Biophys
. 143:365-374; and Lee et al. (1971) “Glycogen and Starch Debranching Enzymes,”
The Enzymes
, Vol. 3, ed. Boyer (Academic Press, New York), pp. 191-234. Isoamylases cleave &agr;(1→6) branch linkages in amylopectin and glycogen but do not hydrolyze the chemically identical bonds in pullulan, an &agr;(1→6)-linked maltotriose polymer. In contrast, pullulanases, also referred to as R-enzymes or limit-dextrinases (Manners (1997)
J. Appl. Glycosci
. 44:83-85), readily hydrolyze &agr;(1→6) linkages of pullulan or amylopectin, but have little activity toward glycogen. Biochemical fractionation experiments identified both isoamylase and pullulanase activities in developing maize kernels during the starch biosynthetic period (Doehlert et al. (1991)
J. Plant Physiol
. 138:566-572; Pan et al. (1984)
Plant Physiol
. 74:324-328), but the specific functions of these two DBEs in polyglucan biosynthesis have not yet been established.
The primary sequences of a pullulanase from rice and maize endosperm are known from cloned cDNAs. Rice R-enzyme (RE) was purified biochemically and characterized as a pullulanase-type DBE, and the cDNA coding for RE was cloned (Nakamura et al. (1996)
Planta
199:209-218; Toguri (1991)
J. Plant Physiol
. 137:541-546). A maize pullulanase, ZPU1, has also been cloned (Beatty et al. (1999)
Plant Physiol
. 119:255-266). In addition, a maize cDNA identified from a cloned fragment of the su1 gene codes for a protein similar to bacterial isoamylases (James et al. (1995)
Plant Cell
7:417-429). The su1 gene product, SU1, functions as an isoarnylase-type DBE and is present in developing maize endosperm during the time that starch is synthesized (Rahman et al. (1998)
Plant Physiol
. 117:425-435).
Expression of the isoamylase- and pullulanase-type DBEs of maize seemingly is coordinately controlled. Even though the su1 gene codes for an isoamylase (Rahman et al. (1998)
Plant Physiol
. 117:425-435), previous studies have demonstrated a reduction in the activity of a pullulanase-type DBE in su1—mutant endosperms. (Pan et al. (1984)
Plant Physiol
. 74:324-328). Consistent with these data, a protein related immunologically to rice RE is present in nonmutant maize kernels at 20 days after pollination (DAP) but deficient in su1—mutant kernels of the same age (Rahman et al. (1998)
Plant Physiol
. 117:425-435). Thus, su1—mutations apparently result in the deficiency of two distinct DBEs. In rice, the su1 mutation controlling RE expression maps to a chromosomal location that is distinct from the gene that codes for RE (Nakamura et al. (1996)
Planta
199:209-218). Accordingly, coordinated control of the amount of isoamylase and pullulanase protein (and activity) is seemingly operative in rice as well (Kubo et al. (1999)
Plant Physiol
. 121:399-409).
Mutations in su1 increase phytoglycogen content and produce several advantageous physical characteristics of polyglucan. For example, the accumulation of phytoglycogen in su1 mutants is associated with smaller and more numerous starch granules. In addition, a polyglucan containing a high phytoglycogen content has a reduced temperature of gelatinization compared to that of waxy or normal starch (Wang et al. (1992)
Cereal Chem
. 69:328-334). The reduced gelatinization temperature increases starch solubility after processing (grinding, pelleting, steam flaking) at temperatures below the gelatinization temperature of normal starch. The smaller granule size and reduced temperature of gelatinization may both contribute to the high digestibility of starch from sugary1 mutant corn. See, for example, Fuwa et al. (1979)
J. Nutr. Sci. Vitaminol
. 25:103-114 and Fuwa et al. (1979)
Cereal Chem
54:230-237.
The relative importance of SU1 and ZPU1 in polyglucan debranching and the production of phytoglycogen is unclear, since protein levels and debranching activities of both enzymes are reduced in the su1 mutant. However, in rice the su1 mutation is primarily associated with a reduction in pullulanase activity and the reduction in the ratio of debranching to branching enzyme activities (Nakamura et al. (1997)
Plant J
. 12:143-153). This phenotype suggests that the reduction in pullulanase activity is important to the rice su1 phenotype (Kubo et al. (1999)
Plant Physiol
. 121:399-409).
The present invention combines the altered expression of a pullulanase debranching enzyme, preferably with various other alterations in the polyglucan biosynthesis pathway, to produce modified polyglucan having optimized energy availability for different classes of livestock and optimized adduct modification of glucan production for food and industrial use.
SUMMARY OF INVENTION
Methods and compositions are provided to modify the
Sewalt Vincent J. H.
Singletary George W.
Alston & Bird LLP
Fox David T.
Pioneer Hi-Bred International , Inc.
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