Note: Descriptions are shown in the official language in which they were submitted.
CA 02257621 1998-12-08
WO 97/47806 PCT/LTS96/10190
SUBSTTrUTES FOR MODIFIED STARCH TN PAPER MANUFACTURE
Field of the Invention
The present invention involves the field of paper
manufacture. Specifically, the present invention provides
sources alternative to modified starch in paper manufacture.
I« Background of the Invention
There are three major phases in paper manufacture where
starch is used as an ingredient. The first is the "wet end"
where cellulose fibers are mixed with starch in a slurry,
and the slurry is forced through a narrow opening onto a
15 wire belt. Water is rapidly removed as the forming sheet
travels the length of the belt. After a distance of
typically five to fifteen meters on the belt, the sheet has
had enough water removed from it so that it can support its
own weight. The sheet travels through a number of foils and
20 rolls wherein more water is removed. It is dried to about
llo moisture.
The second phase in paper manufacturing involving
starch is the "sizing step". Here, the paper goes through a
sizing press where a starch slurry is applied to the sheet.
25 The sheet again goes through a series of foils and rolls.
It is dried on rollers and can be taken off the press as a
finished product.
The third step involves coating the paper with a
mixture of starch and a thermoplastic molecule. On certain
30 lines, this occurs after the sizing step. The nascent roll
can also be removed and reinstalled onto a different press
for coating. A typical coating device has two blades that
run the width of the paper. The blades apply the coating
material onto two rolling drums. The paper passes between
35 the drums and the coating material, comprising starch and
the thermoplastic moiety, comes off the drums onto the
paper. After the paper leaves the drums, it goes through a
1
CA 02257621 1998-12-08
WO 97/47806 PCT/US96/10190
number of dryers. When the paper is dry, it goes onto a
"soft calendar" comprising two drums, one made of a hard
density fabric and the other a heated steel drum. The paper
passes between the two drums and the heated steel drum is
sufficiently hot to melt thermoplastic components of the
coating mix providing a hard gloss finish on the paper.
The cellulosic wood pulp fibers, typically used in the
above process, are anionic in nature. The addition of a
cationic starch to the "wet end" slurry acts as an adhesive
l0 by cross linking the pulp fibers through salt linkages.
Thus a cross linked polymeric network is made, comprising
the starch and cellulose fibers. Typically, the cationic
starches used in the "wet end" are tertiary or quaternary
amines . These amino groups are added to the starch by wet
millers.
Surface sizing starches are used to impart both
strength and smooth finish to the sheet after it leaves the
"wet end". Such starches also prepare the sheet to receive
the various coatings. In cheaper grades of paper and in
fiberboard manufacture, sizing starches are used simply as
unmodified corn starch. For high grades of paper,
chemically-modified starches are used. This is important
for the application of a smooth, uniform high quality
surface to the paper.
There is a tendency for starches to retrograde i.e. re-
form high ordered structures (both helices and crystallites)
in an otherwise gelatinous starch slurry. Deposition of
retrograded starch onto high quality paper causes regional
inconsistencies on the paper and is unacceptable.
Furthermore, retrograded starch in the sizing press may
necessitate shutting the line down to clear the apparatus.
The starch most often used for sizing applications is a
starch having a covalently attached neutral adduct, for
instance hydroxyethyl starch. This is prepared by the
reaction of ethylene oxide with starch after it is isolated
at the wet milling plant. The function of the hydroxyethyl
(or similar) adduct is independent of its chemical nature:
2
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WO 97/47806 PCT/U596110190
rather, it serves to provide steric hindrance, inhibiting
the formation of high ordered structures. This steric
hindrance is critical to decrease retrogradation. The
periodic protuberance afforded by the adduct disrupts the
formation of higher ordered structures that leads to
retrogradation.
Speed is of paramount importance in paper
manufacturing. Limiting in press speed is starch
consistency. Presses often run below their full capacity
speeds. Depending on the application, starch slurries are
between 3-150 (usually 5-60) solids. An increase in solids
would necessarily result in a decrease in the amount of
water that would have to be removed from a paper sheet being
manufactured. This would allow the press to work at higher
speeds .
Hydroxethylated starch also forms higher ordered
structures as the temperature decreases or the concentration
increases. The formation of the higher ordered structures
on the surface of the paper is required. After application
to the sheet the starch reforms some of these higher ordered
structures and creates a uniform surface that imparts
structural strength and facilitates the acceptance of inks
and dyes. However, the higher ordered structures should not
form in the slurry nor on the application device because
this necessitates shutting down the production line to clear
off retrograded starch.
The function of the hydroxyethyl group is to lower the
temperature and/or raise the concentration of starch at
which retrogradation occurs. As the processing lines have
already been optimized for a particular temperature of the
starch slurry, a decrease in the tendency to retrograde
would allow for a higher carbohydrate content in the slurry.
The mixture applied to the paper sheet in the coating
process contains hydroxethylated starch and thermoplastic
molecules. The most prevalent thermoplastic molecules used
are latexes, such as styrene butadiene. The function of the
hydroxethyl starch is as indicated above. The function of
3
CA 02257621 1998-12-08
WO 97/47806 PCT/US96/10190
the thermoplastic molecule is to form a high gloss finish on
the paper. This causes an increased ability to take inks
and dyes and improves the resolution, in general, on the
printed sheet.
Based on the foregoing, there exists a need, in paper
manufacturing, for modified starch substitutes which are
functionally similar to modified starch. There is a further
need to provide substitutes for modified starch which are
less prone to retrogradation. There is a further need to
to provide methods of manufacturing paper which are faster than
current methods and allow presses to run closer to their
full capacity speed. There is a further need to provide
methods of manufacturing paper that are environmentally
friendly and do not involve input materials that require
chemical processing.
It is therefore an object of the present invention to
provide substitutes for modified starch which are less prone
to retrogradation when used in paper manufacture.
It is a further object of the present invention to
provide methods of manufacturing paper which are faster and
more efficient than existing methods.
It is a further object of the present invention to
provide substitutes for starch in paper manufacturing that
do not require costly chemical modification as does starch.
It is a further object of the present invention to
provide methods for manufacturing paper that are more
environmentally-friendly than existing methods.
It is a further object of the present invention to
provide substitutes for thermoplastic molecules currently
used in the coating step during paper manufacture.
SZJMMP~RY OF THE INVENTION
The present invention provides glucans which can be
used as substitutes for modified starch and/or latex in
paper manufacturing. The present glucans are produced by
glucosyltransferase B ("GTF B") enzymes of the species
Streptococcus mutans, and are functionally similar to the
4
CA 02257621 2002-11-13
modified starch currently used in paper manufacturing. The present glucans
also
exhibit similar physical properties to the thermoplastic molecules currently
used in
the coating step of paper manufacturing.
The present invention also provides methods of manufacturing paper
utilizing the present glucans, input materials that are produced biologically.
Thus,
the present methods are more cost-effective and environmentally-friendly than
current methods which require input materials producing chemical effluents.
According to one aspect of the present invention there is provided a
method of manufacturing paper comprising adding a glucan isolated from a host
10 transformed with a gene encoding a glucosyltransferase B (GTF B) enzyme,
wild-
type or mutant, obtained from Streptococcus mutans, to one or more steps of
(A)
wet ending, (B) sizing, and (C) coating in paper manufacturing, wherein
modified
starch is used. In a further embodiment, the glucan is added to the coating
step.
According to another aspect of the present invention there is provided a
15 method of imparting gloss on paper during a manufacturing process
comprising
adding a glucan to a coating step, wherein the glucan is synthesized by a
Streptococcus mutans glucosyltransferase B enzyme, wild-type or mutant.
According to another aspect of the present invention there is provided an
isolated nucleic acid comprising a member selected from the group consisting
of:
20 (a) a polynucleotide which encodes a Streptoccoccus mutans
glucosyltransferase B polypeptide having changes at positions
Y 169A/Y 170A/Y 171 A;
(b) a polynucleotide complementary to a polynucleotide of (a).
According to a still further aspect of the present invention there is provided
25 an expression cassette comprising a transit sequence and at least one
Streptococcus mutans glucosyltransferase B nucleic acid operably linked to a
CA 02257621 2002-11-13
promoter, wherein the transit sequence directs the nucleic acid to an
amyloplast or
vacuole. In another embodiment, the promoter is a 22 kDa zero, opaque 2, gamma
zero, or waxy promoter. The invention further provides vectors, host cells,
and
transgenic plant cells comprising the expression cassette described above.
5 According to a still further aspect of the present invention there is
provided
an expression cassette comprising a transit sequence and at least one
Streptococcus mutans nucleic acid operably linked to a promoter, wherein the
transit sequence directs the nucleic acid to an amyloplast or vacuole, and
wherein
said nucleic acid comprises a polynucleotide which encodes a
glucosyltransferase
10 B polypeptide having changes at positions selected from the group
consisting of
1448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K;
D567T/D571K; D567T/D571K/K1014T;
1448V/D457N/D567T/D571K/K779Q/K1014T; Y169A/Y170A/Y171A and
K779Q.
15 According to a still further aspect of the present invention there is
provided
a method for producing a glucan in a plant comprising:
(a) transforming a plant cell with the expression cassette described
above;
(b) growing the plant cell under plant growing conditions to produce a
20 regenerated plant; and
(c) expressing the nucleic acid for a time sufficient to produce a glucan
in the regenerated plant.
According to a still further aspect of the present invention there is provided
an isolated protein comprising a polypeptide encoded by Streptococcus mutans
25 glucosyltransferase B nucleic acid having changes at positions
Y169A/Y 170A/Y 171 A. The invention further provides ribonucleic acid
sequences encoding the protein described above.
5a
CA 02257621 2002-11-13
DETAILED DESCRIPTION OF THE INVENTION
As used herein, "glucan" means a glucose polymer having linkages that
are a ( 1--~3), a ( 1--~6), and branching a ( 1-~3, 6).
As used herein, "amyloplast" means starch accumulating organelle in plant
storage tissue.
As used herein, "vacuole" means the cellular compartment bounded by the
tonoplast membrane.
Streptococcus mutans is a species that is endogenous to the oral cavity and
colonizes tooth enamel. See e.g. Kuramitsu, "Characterization of Extracellular
Glucosyl Transferase Activity of Streptococcus-mutans." Infect. Immun.; Vol.
12(4); pp. 738-749; (1975); and Yamashita, et al., "Role of the Streptococcus-
Mutans-gtf Genes in Caries Induction on the Specific-Pathogen-Free Rat Model,"
Infect. Immun.; Vol. 61(9); pp. 3811-3817; (1993). Streptococcus mutans
species
secrete glucosyltransferase B ("GTF B") enzymes which utilize dietary sucrose
to
make a variety of extracellular glucans. See e.g. Kametaka, et al.,
"Purification
and Characterization of Glucosyltransferase from Streptococcus-mutans OMZ176
with Chromatofocusing," Microbios; Vol. 51(206); pp. 29-36; (19?8).
Both soluble and insoluble glucans are synthesized, and the proteins
responsible have been isolated and characterized. See e.g. Aoki, et al.,
"Cloning
of a
Sb
CA 02257621 2000-10-18
Strepr_ococcus-mutans Glucosyltransferase Gene Coding for
Insoluble Gluca ~ Synthesis, " infect . Immun. ; Vol . 53 (3) ;
pp. 58?-594; !1986); Shimamura, et al., "Identification of
Amino Acid Residues in Streptococcus Mutans
Glucosyl~ransferases In~luencing the Structure of the Glucan
Produced," J. Bacteriol.; Vol. 176(16): pp. 4845-50; (1994);
and Kametaka, et al., "Purification and Characterization of
Glucosyltransferase from Streptococcus-mutans OMZ176 with
Chromatofocusing," Microbios; Vol. 51(206); pp. 29-36;
( 1987).
The proteins involved are large (~I55 kDa) and catalyze
the group transfer of the glucosyl portion of sucrose to an
acceptor glucan via x ( 1-~3) and ac ( 1-~6) linkages . See
e.g., Wenham, et al., "Regulation of Glucosyl Transferase
' IS and Fructosyl Transferase Synthesis by Continuous Cultures
of Streptococcus-mutans," J. Gen. Microbiol.; Vol. 114 (Part
I); pp. .117-124; (1979); Fu, et al., "Maltodextrin
Acceptor Reactions of Streptococcus-mutans 6715
glucosyltransferases," Carbohydr. Res.; Vol. 217; pp. 210-
2I1; (1991); and Bhattacharjee, et al., "Formation of Alpha
- ( 1--~6 ) , Alpha - ( I~3 ) , and Alpha ( 1~2 ) Glycosidic Linkages
by Dextransucrase from Streptococcus Sanguis in Acceptor-
Dependent Reactions," Carbohydr. Res.; Vol. 242; pp. 191-
201;(1993).
The genes involved in glucan synthesis have been
isolated and sequenced. See Shimamura, et al., cited
hereinabove and Russet, et al., "Expression of a Gene for
Glucan-binding Protein from Streptococcus-mutans in
Escherichia-coli," J. Gen. Microbiol.; Vol. 131(2); pp.
295-300; (1985); Russell et al., "Characterization of
Glucosyltransferase Expressed from a Streptococcus-Sobrinus
Gene Cloned in Escherichia-coli," J. Gen. Microbiol.; Vol.
133(4); pp. 935-944; (1987); and Shiroza, et al., "Sequence
Analysis of the GTF B Gene from Streptococcus mutans," J.
Bacteriol.; Vol. 169(9); pp. 4263-4270; (1987).
6
CA 02257621 1998-12-08
WO 97/47806 PCT/US96/10190
The structures of the various glucans produced by GTF
enzymes are quite heterogeneous with respect to the
proportions of oc ( 1--~3 ) , oc ( 1-~6 ) and ac ( 1-~3, 6 ) branches
Y present in any given glucan. Transformation of genes which
encode naturally occurring GTF B and GTF B mutant proteins
into plants, such as maize, provides amyloplasts and
vacuoles with novel compositions.
GTF B enzyme activity incorporated into the amyloplast
and/or vacuole leads to the accumulation of starch and
to glucan in the same amyloplast and/or vacuole.
Retrogradation occurs as portions of starch molecules
interact and subsequently form inter- or intra-chain
helices. In a mixture of starch and glucans, the frequency
of starch-starch interactions, that lead to helix formation,
is diminished. A paste made from the mixed polymers is less
prone to retrogradation as a result. This is especially
true in the starch accumulation mutants envisioned as
transformation targets where the relative proportion of
starch is reduced.
2~ Glucans produced in maize amyloplasts and/or vacuoles
by the transgenic GTF B enzymes can function in paper
processing without chemical modification, as required of
starch. The polymer solution consequently has altered
rheological properties and is less prone to retrogradation
compared to starch. The glucans are branched and irregular
and able to supplant modified starches with comparable or
superior efficacy. They do not require any costly chemical
modification as does starch. For coating applications, the
present glucans exhibit thermoplastic properties in addition
to the above advantages.
The wild type GTF and mutants thereof useful in
producing glucans according to the present invention are
provided below. The following code is employed:
7
CA 02257621 1998-12-08
WO 97/47806 PCT/US96/10190
Amino Acid One-letter Symbol
Alanine A
Asparagine N
Aspartic Acid D
Glutamine Q
Glutamic Acid E
Isoleucine I
Lysine K
Threonine T
Tyrosine ~ Y
Valine V
The nomenclature used to identify the mutant GTF B
enzymes used to produce the present glucans is as follows:
the number refers to the amino acid position in the
polypeptide chain; the first letter refers to the amino acid
in the wild type enzyme; the second letter refers to the
amino acid in the mutated enzyme; and enzymes with multiple
mutations have each mutation separated by /.
The mutant GTF B enzyme used to produce glucans for
paper coating is preferably selected from the group
consisting of I448V; D457N; D567T; K1014T; D457N/D567T;
D457N/D571K; D567T/D571K; D567T/D571K/K1014T;
I448V/D457N/D567T/D571K/K779Q/ K1014T; and
Y169A/Y170A/Y171A.
The mutant GTF B enzyme used to produce glucans for
paper coating is more preferably selected from the group
consisting of I448V; K1014T;D567T/D571K/K1014T;
I448V/D457N/D567T/D571K/K779Q/ K1014T; and
Y169A/Y170A/Y171A.
3o The mutant GTF B enzyme used to produce glucans for
paper coating is even more preferably selected from the
group consisting of K1014T;
I448V/D457N/D567T/D571K/K779Q/K1014T; and Y169A/Y170A/Y171A.
The mutant GTF B enzyme used to produce glucans for
paper coating is most preferably I448V/D45?N/D567T/
D571K/K779Q/K1014T; or Y169A/Y170A/Y171A.
8
CA 02257621 2000-10-18
T::e :~L=a:~t GTF B enzyme used to , produce glucans for
paper sizing is preferably selected from the group
cc~s~st=-:g oLI448V; D457N; Duo'%T; K779Q; KI014T;
D457~I/D567T; D457N/D571K; D567T/D571K and
D567T/DSiIK/K1014T.
The mutant GTF B enzyme used to produce glucans for
paper sizing is more preferably selected from the group
consisting of I448V; D457N: K779Q; D567T/D571K; and
D567T/D571K/K1014T.
1o The mutant GTF B enzyme used to produce glucans for
paper sizing is most preferably I448V.
The glucans of the present invention are preferably
produced in transgenic maize, potato, cassava, sweet potato,
rye, barley, wheat, sorghum, oats, millet, triticale,
' IS sugarcane or rice. More preferably, the present glucans are
produced in maize, potato, sugarcane, cassava or sweet
potato. Even more preferably, the present. glucans are
produced in maize or potato. Most preferably, the present
glucans are produced in maize.
20 In a highly preferred embodiment of the present
invention, maize lines deficient in starch biosynthesis are
transformed with mutant GTF B genes. Such lines may be
natural 1y occurring maize mutants ( i . a . she, bt2, btl ) or
transgenic maize engineered so as to accumulate low amounts
25 of starch in the endosperm when compared to wild type maize.
See e.g. Miiller-Rober, et al., "Inhibition of the ADP-
glucose Pyrophosphorylase in Transgenic Potatoes Leads to
Sugar-Storing Tubers and Influences Tuber Formation and
Expression of Tuber Storage Protein Genes," The EMBO
30 Journal; Vol. 11(4); pp. 1229-1238; (1992); and Creech,
"Carbohydrate Synthesis in Maize," Advances in Agronomy;
VoI. 20; pp. 275-322; (1968).
The production of the present glucans is performed
3S according to methods of transformation that are well known
in the art, and thus constitute no part of this invention.
The compounds of the present invention are synthesized by
9
CA 02257621 1998-12-08
WO 97/47806 PCT/C1S96/10190
insertion of an expression cassette containing a synthetic
gene which, when transcribed and translated, yields a GTF
enzyme that produces the desired glucan. Such empty
expression cassettes, providing appropriate regulatory
sequences for plant expression of the desired sequence, are
also well-known, and the nucleotide sequence for the
synthetic gene, either RNA or DNA, can readily be derived
from the amino acid sequence for the protein using standard
texts and the references provided. The above-mentioned
to synthetic genes preferably employ plant-preferred codons to
enhance expression of the desired protein.
The following description further exemplifies the
compositions of this invention and the methods of making and
using them. However, it will be understood that other
methods, known by those of ordinary skill in the art to be
equivalent, can also be employed.
The genes which code for the present enzyme or mutants
can be inserted into an appropriate expression cassette and
introduced into cells of a plant species. Thus, an
especially preferred embodiment of this method involves
inserting into the genome of the plant a DNA sequence coding
for a mutant or wild type gene in proper reading frame,
together with transcription promoter and initiator sequences
active in the plant. Transcription and translation of the
DNA sequence under control of the regulatory sequences
causes expression of the protein sequence at levels which
provide an elevated amount of the protein in the tissues of
the plant.
Synthetic DNA sequences can then be prepared which code
for the appropriate sequence of amino acids of a GTF B
protein, and this synthetic DNA sequence can be inserted
into an appropriate plant expression cassette.
Likewise, numerous plant expression cassettes and
vectors are well known in the art. By the term "expression
cassette" is meant a complete set of control sequences
including promoter, initiation, and termination sequences
which function in a plant cell when they flank a structural
to
CA 02257621 1998-12-08
WO 97/47806 PCT/US96/10190
gene in the proper reading frame. Expression cassettes
frequently and preferably contain an assortment of
restriction sites suitable for cleavage and insertion of any
desired structural gene. It is important that the cloned
gene have a start codon in the correct reading frame for the
structural sequence.
By the term "vector" herein is meant a DNA sequence
which is able to replicate and express a foreign gene in a
host cell. Typically, the vector has one or more restriction
l0 endonuclease recognition sites which may be cut in a
predictable fashion by use of the appropriate enzyme such
vectors are preferably constructed to include additional
structural gene sequences imparting antibiotic or herbicide
resistance, which then serve as markers to identify and
separate transformed cells. Preferred markers/selection
agents include kanamycin, chlorosulfuron, phosphonothricin,
hygromycin and methotrexate. A cell in which the foreign
genetic material in a vector is functionally expressed has
been "transformed" by the vector and is referred to as a
"transformant".
A particularly preferred vector is a plasmid, by which
is meant a circular double-stranded DNA molecule which is
not a part of the chromosomes of the cell.
As mentioned above, both genomic DNA and cDNA encoding
the gene of interest may be used in this invention. The gene
of interest may also be constructed partially from a cDNA
clone and partially from a genomic clone. When the gene of
interest has been isolated, genetic constructs are made
which contain the necessary regulatory sequences to provide
for efficient expression of the gene in the host cell.
According to this invention, the genetic construct will
contain (a) a genetic sequence coding for the protein or
trait of interest and (b) one or more regulatory sequences
operably linked on either side of the structural gene of
interest. Typically, the regulatory sequences will be
selected from the group comprising of promoters and
11
CA 02257621 1998-12-08
WO 97/47806 PCT/US96110190
terminators. The regulatory sequences may be from autologous
or heterologous sources.
The expression cassette comprising the structural gene
for a mutant of this invention operably linked to the
desired control sequences can be ligated into a suitable
cloning vector. In general, plasmid or viral (bacteriophage)
vectors containing replication and control sequences derived
from species compatible with the host cell are used. The
cloning vector will typically carry a replication origin, as
to well as specific genes that are capable of providing
phenotypic selection markers in transformed host cells.
Typically, genes conferring resistance to antibiotics or
selected herbicides are used. After the genetic material is
introduced into the target cells, successfully transformed
cells and/or colonies of cells can be isolated by selection
on the basis of these markers.
Typically, an intermediate host cell will be used in
the practice of this invention to increase the copy number
of the cloning vector. With an increased copy number, the
vector containing the gene of interest can be isolated in
significant quantities for introduction into the desired
plant cells. Host cells that can be used in the practice of
this invention include prokaryotes, including bacterial
hosts such as E. coli, S. typhimurium, and Serratia
marcescens. Eukaryotic hosts such as yeast or filamentous
fungi may also be used in this invention. Since these hosts
are also microorganisms, it will be essential to ensure that
plant promoters which do not cause expression of the protein
in bacteria are used in the vector.
The isolated cloning vector will then be introduced
into the plant cell using any convenient technique,
including electroporation (in protoplasts), retroviruses,
bombardment, and microinjection into cells from
monocotyledonous or dicotyledonous plants in cell or tissue
culture to provide transformed plant cells containing as
foreign DNA at least one copy of the DNA sequence of the
plant expression cassette. Using known techniques,
12
CA 02257621 2002-02-26
WO 971J7806 PCT!LVS961 (0190
pr:W :G L j(5 Cart ~e t-f?(j~'r'r' i'~3~- .°(~ i3f'C: (:. ' _ .
'i(° ~ 1 _>;~tao ~l3 l r.',:~ ='
Carl :tee rE'gen2Cai.ed tai f:lt=:i; W~lC)i ? % c?r; , 1 r? Y:~ .~r,~-, ~
Srl~~~.~~rl C3ri l
and exp~eJj (_'(o <~ene f ~a,. ~:~ F)~-of F 3 r1 at;;E'Or :lnq (c ch.~s
'_nVOntlol,. t'~C~_~rCln~ly, d t:lEStl~'J ~:~rE'tt=.'Cre~:~l
E'ILLhE'~~~.titf.'!':~ O~ t.~.~=
present invention is a t ransf-orme~i mare plaa.t, tt:e cei~s of
which coma-n as foreign DNt,~ ac4 !_easr one cony of the DNP.
sequence of an expressic>n ca:,seLte of a GTE B rretant_
It will also be appreciated (>y those of ordinary skil'
that the plant vectors provided herein can be incorporated
to into Agrobacterium tumefaciens, which can thin be used co
transfer the vector into susceptible plant cells, primarily
from dicotyledonous species. Thus, this invention provides a
method for introducing GTF t3 in Agrobacterium tumefaciens-
susceptible dicotyledonous plant:. in wt:ich the expressicn
15 cassette is introduced in to the c(:ll.s by infecting the cells
with Agrobacterium tumet:aciens, a plasmid of which has been
modified to include a plant: expres"ion cassette of this
invention.
For example, the potat._.o plant can (>e transformed via
2o Agrobacterium tumefacien:, to pr_oczrcce the present gl»~ans.
The transformation cas>ette compri se~~ a patatin promoter,
followed by the relevant:. GTF E3 coding sequence and the
neomycin phosphotransfer_-a se polyadenylation si.te/terminat:or .
See e.g. Utsumi, et al.d "Expressiort and Accumulation for
25 Normal and Modified Sc>ybean Glycinins in Potato Tubers,"
Plant Science; Vol. 102'(:0 ; pp. 181-188; (1994) ; (Limerick),
The
transgenic cassette is placed into a transformation vector.
For example, BIN19, or derivatives thereof, are useful when
3o transforming via Agrobacterium tuntefaciens. See e.g.
Visser, et al., "Transfoz-mat:lon Of Homozygous Diploid Potato
with an Agrobacterium-tumefaciens Binary Vector System by
Adventitious Shoot Regeneration on Leaf and Stem Segments,"
Plant Mol. Biol.; ~'ol. 12(3); pp. 329-338; (1989.
For maize transformation vectors, the promoters include
any promoter whose expression is specific and limited to
I 3 _.._'.._....
CA 02257621 2000-10-18
endosp°=~- c°1's. Included are those encoding either 22 lc~a
'ei~. ocaque2, gamma zero and waxy. These lead into the GTF
ge:'-E aid are foilcwed by the endogenous terminator or the
n°t°=cge===o~.=s PIIvII terminator. The GTF B protein are
directed .o .he maize endosperm amylcplast using a suitable
trar:sic sequence. Transit sequences useful in directing
the enzyme into the amyloplast for accumulation within the
amyloplast include but are not limited to ribulose
biophosp::ate carboxylase small subur.it, waxy, brittle-1, and
to chlorophyll P.B binding protein. The transit sequences are
juxtaposed between the promoter and the GTF B coding
sequence and fused in translational reading frame with the
GTF B moiety.
Transit sequences useful in directing the enzyme into
' 15 the vacuole for accumulation within the vacuole are well-
known in the art. For vacuolar targeting, see e.g. Ebskamp,
et al., "Accumulation of Fructose Polymers in Transgenic
Tobacco," Bio / technology; Vol. 12; pp. 272-275; (1994).
2o For maize transformation and regeneration see e.g.
Armstrong, C., "Regeneration of Plants from Somatic Cell
Cultures: Applications for in vitro Genetic Manipulation,"
The Maize Handbook; Freeling, et al., eds, pp. 663-671;
( 1994). '
25 Once a given plant is transformed, the glucarls
synthesized can be isolated, by standard methods, known to
one skilled in the art. The glucan thus obtained in the
transgenic plant can be substituted for modified starches
and utilized in the sizing and/or coating steps. For
30 formulations useful in the coating step, see e.g. Heiser, et
al., "Starch Formations," Starch and Starch Products _in
Paper Coating; Kearney, et al., eds., pp. 147-162; (1990) ;
Tappi Press.
35 In both sizing and coating, the present glucans are
utilized in an amount of from about 4 to about 15 weight
percent, more preferably from about 5 to about 12 weight
t .t
CA 02257621 2000-10-18
percent, also preferably from about 6 to about 8 weight
percent. Weight percent is defined as grams of molecule per
100 ml solution.
The present glucans are used to replace the starch
and/or latex molecules completely, or a starch-glucan or a
latex-glucan mixture is used in the slurry. In t he sizing
application, the glucan:starch ratio ranges from about 10:90
to about 100:0; more preferably from about 40:60 to about
100:0; more preferably still from about 60:40 to about
100:0; most preferably about 100:0.
In the coating application, the glucan:starch ratio
ranges from about 10:90 to about 100:0; more preferably from
about 40:60 to about 100:0; more preferably still from
about 60:40 to about 100:0; most preferably about 100:0. In the
coating application, the glucan:latex ratio ranges from
about 10:90 to about 100:0; more preferably from about 40:60
to about 100:0; more preferably still from about 60:40 to
about 100:0; most preferably about 100:0.
All publications cited in this application are
indicative of the level of skill of those skilled in the art
to which this invention pertains. Variations on the
above embodiments are within the ability of one of the
ordinary skill in the art, and such variations do not
depart from the scope of the present invention as
described in the following claims.
