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
2000-10-16
2003-12-16
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, C800S285000, C800S289000, C800S306000, C800S312000, C800S313000, C800S317200, C800S317300, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000, C435S069100, C435S069800, C435S101000, C435S320100, C435S468000, C435S193000, C536S023200, C536S023600
Reexamination Certificate
active
06664444
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to transgenic plants presenting a modified inulin producing profile, to a method for producing said plants, to a method for modifying and controlling the inulin producing profile of plants and to a method for producing inulin from said transgenic plants.
Furthermore, the present invention relates to novel 1-SST and 1-FFT enzyme encoding DNA sequences, to novel recombinant DNA constructs and recombinant genes derived thereof, to novel combinations of expressible 1-SST and 1-FFT enzyme encoding genes, as well as to novel polypeptides or fragments thereof presenting 1-SST and/or 1-FFT activity, and to antibodies capable of binding to them.
BACKGROUND AND PRIOR ART
Inulin is a fructan type carbohydrate polymer which occurs as a polydisperse composition in many plants and can also be produced by certain bacteria and fungi. Inulin from plant origin consists of a polydisperse composition of mainly linear chains composed of fructose units, mostly terminating in one glucose unit, which are linked to each other through &bgr;(2-1) fructosyl-fructose linkages.
Inulin can be generally represented, depending from the terminal carbohydrate unit, by the formulae GF
n
and F
m
, wherein G and F respectively represent a glucose unit and a fructose unit, n is an integer representing the number of fructose units linked to the terminal glucose unit, and m is an integer representing the number of fructose units linked to each other in the polyfructose chain.
The number of saccharide units (fructose and glucose units) in one molecule, i.e. the values n+1 and m in the above formulae, are commonly referred to as the degree of polymerisation, represented by DP. Often also the parameter (number) average degree of polymerisation, represented by ({overscore (DP)}) is used, which is the value {overscore (DP)}
n
calculated, after complete hydrolysis and considering that in native inulins the F
m
fraction is negligible, as follows:
DP
_
n
=
total
%
F
total
%
G
+
1
In the equation % refers to weight percent (wt %). Furthermore, in this calculation the saccharides glucose (G), fructose (F) and saccharose (GF) which are present in the polydisperse polysaccharide, should not be taken into account. The average degree of polymerisation is thus the ({overscore (DP)}
n
) of inulin, herein interchangeably referred to in short as ({overscore (DP)}) inulin or ({overscore (DP)}) (De Leenheer, 1996).
The polysaccharide chains of native inulin from plant origin generally have a degree of polymerisation (DP) ranging from 3 to about 100, whereas the ({overscore (DP)}) of the native inulin largely depends from the plant source, the growth phase of the plant, the harvesting time and the storage conditions. The ({overscore (DP)}) of isolated inulin largely depends on the ({overscore (DP)}) of the native inulin and on the process conditions used for the extraction, purification and isolation of the inulin from the plant or plant parts.
By native inulin or crude inulin is meant herein inulin that has been extracted from plants or parts of plants, without applying any process to increase or decrease the ({overscore (DP)}), while taking precautions to inhibit the plant's own hydrolase activity and to avoid hydrolysis. The ({overscore (DP)}) of native inulin thus essentially corresponds to the ({overscore (DP)}) of the inulin as present in the plant or plant parts.
The isolated inulin obtained from plants or plant parts through conventional manufacturing techniques, commonly including extraction, purification and isolation, without any process to modify the ({overscore (DP)}) of the native inulin, is termed herein, interchangeably, standard ({overscore (DP)}) grade inulin or standard grade inulin. As a consequence of the manufacturing process, the ({overscore (DP)}) of standard grade inulin is usually about 1 to 1.5 lower than the ({overscore (DP)}) of the native inulin.
Inulin molecules with a DP≦10 are commonly termed oligofructose, inulo-oligosaccharides or fructo-oligosaccharides (in short FOS). Both, inulin chains with a DP≦10 and inulin chains with a DP>10, are embraced herein by the term inulin.
By inulin profile is meant the relative composition of the polydisperse inulin as formed by the individual components including glucose, fructose, sucrose and individual inulin chains, including the distribution pattern of the polyfructose (inulin) chains.
Linear inulin is common in a specific plant family, the Asteraceae, including the plant species Jerusalem artichoke (
Helianthus tuberosus
) and chicory (
Cichorium intybus
). Inulin is commonly stored in tap roots (chicory) or in tubers (Jerusalem artichoke) and acts as a storage reserve for regrowth of the sprout after the winter period.
Accordingly, typical sources for the production of inulin at industrial scale are roots of chicory and, on a much smaller scale, tubers of Jerusalem artichoke, in which inulin can be present in concentrations of about 14% to 18% by weight on fresh weight. Inulin can be readily extracted from these plant parts, purified and optionally fractionated in order to remove impurities, monosaccharides, disaccharides and undesired oligosaccharides, as for example described in PCT patent application WO 96/01849.
Conventional processing of roots of chicory yields a standard grade inulin, containing about 8 wt % of mono- and di-saccharides (including glucose, fructose and sucrose) and a polydisperse mixture of inulin molecules with a DP ranging from 3 to about 60 and a ({overscore (DP)}) of about 10. The DP of the inulin molecules of standard grade inulin from Jerusalem Artichoke tubers ranges from 3 to about 40 whereas the ({overscore (DP)}) is about 7.
It is known that in Asteraceaous plants, including Jerusalem artichoke and chicory, inulin molecules are synthesised by the concerted action of two enzymes:sucrose:sucrose 1-fructosyltransferase (in short 1-SST enzyme or 1-SST, used interchangeably) and fructan:fructan 1-fructosyltransferase (in short 1-FFT enzyme or 1-FFT, used interchangeably) (Koops and Jonker, 1994 and 1996). Both 1-SST and 1-FFT are active during the period of inulin synthesis and accumulation:
1-SST catalyses the initial reaction of inulin biosynthesis, the conversion of sucrose into the smallest inulin molecule, the trisaccharide kestose (GFF), according to:
GF+GF→GFF+G (1)
1-FFT catalyses the redistribution of terminal fructosyl units (−F) between inulin molecules, which results in a stepwise increase in chain length, according to:
GFF
n
+GFF
m
→GFF
n−1
+GFF
m+
1, (2)
(wherein n and m are integers >0)
Some examples of this type of reaction are
GFF+GFF→GFFF+GF (2a)
GFFF+GFFF→GFFFF+GFF (2b)
GFFFF+GFFFF→GFFFFF+GFFF (2c)
An essential difference between the 1-SST enzyme and the 1-FFT enzyme is that the 1-FFT enzyme cannot catalyse reaction (1). In contrast, the 1-SST enzyme, next to reaction (1), can catalyse reactions of type (2) yielding inulin molecules with a low DP (catalysis by known 1-SST enzymes being able to yield inulin molecules with a DP up to about 5).
Accordingly, in plants both the 1-SST and the 1-FFT enzymes are contributing to inulin synthesis and the profile of native inulin is determined, inter alia, by sucrose supply, expression of the 1-SST enzyme encoding genes and 1-FFT enzyme encoding genes and the kinetic properties and relative activity of the 1-SST and 1-FFT enzymes which may be controlled by the relative expression of the 1-SST and the 1-FFT enzyme encoding genes.
Inulin is an edible, water soluble polydisperse polysaccharide composition which is used in the manufacture of many food and feed products, drinks and non-food products. In food, feed and drinks, inulin can be used, inter alia, as a bulking agent as well as a total or partial substitute for sugar and/or fat. Furthermore, inulin can be added to food, feed and drink
De Leenheer Lena
Koops Andries Jurriaan
Sevenier Robert
Van Tunen Arjen Johannes
Fox David T.
Hayes & Soloway P.C.
Tiense Suikerraffinaderij N.V.
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