169
Production of modified polymeric carbohydrates
Arnd G Heyer*, James R Lloyd† and Jens Kossmann‡
The cloning of a gene responsible for the phosphorylation of
glucans has made it possible to genetically engineer the
phosphorylation level of starches in higher plants. Through the
manipulation of starch synthase activity, it is now also possible to
genetically tailor the chain-length distribution in the amylopectin.
Both findings will lead to the development of novel starches
utilized as a renewable resource. The production of fructans on a
large scale can also be envisioned for the near future.
Addresses
Max-Planck-Institute of Molecular Plant Physiology, Karl-Liebknecht-Strasse
24–25, D-14476 Golm, Germany
*e-mail: [email protected]
†e-mail: [email protected]
‡e-mail: [email protected]
Current Opinion in Biotechnology 1999, 10:169–174
http://biomednet.com/elecref/0958166901000169
© Elsevier Science Ltd ISSN 0958-1669
Abbreviations
1-SST sucrose:sucrose fructosyltransferase
6-SFT sucrose:fructan 6-fructosyltransferase
FFT
fructan:fructan fructosyltransferase
GBSS granule-bound SS
SS
starch synthase
Introduction
Starch and fructans are both polymeric carbohydrates in
plants, for which the biosynthesis is sufficiently understood
to allow the bioengineering of their properties, or to engineer crops to produce polysaccharides not normally present.
Annually, 20–30 × 106 tons of starch are isolated to serve a
wide range of industrial applications, such as the coating
of textiles and paper, or as a thickening or gelling agent in
the food industry [1]. Most of the starch is isolated from
corn, but cassava, potato and wheat are also sources of
starch. Potato starch is produced mainly in countries of the
European Union. For a wide range of applications starch
is treated chemically or physically in order to adapt its
properties to optimally serve its purpose. It is a major challenge to engineer the biosynthesis of starch to make these
treatments obsolete. One of the first crop plants amenable
to genetic transformation was the potato. As it is also a
major source of isolated starch, most of the advances with
respect to the production of modified polymers will be
discussed in the context of this species.
Fructans are of growing interest as functional food ingredients because they are beneficial for human health [2••]. As
human enzymes cannot digest fructans, they reach the colon
and serve as a substrate for enterobacterial growth. Inulincontaining diets selectively stimulate bifidobacteria and
make them the predominant species [3,4]. Consequently, an
increased fecal content of short-chain fatty acids and a
decreased concentration of of tumor-promoting substances,
such as ammonia, is observed [5–7]. Fructans are normally
isolated from crop plants with low agronomic value, such as
the Jerusalem artichoke (Helianthus tuberosus) and chicory.
There are, however, other fructan producers which are
important in human nutrition (e.g. wheat, onion, garlic,
banana, artichoke, asparagus; for review, see [8]). The average daily consumption is estimated to be 1–4 g in the United
States and 3–11 g in Europe [2••]. In order to make the production of fructans economically more feasible, the ability to
transfer the biochemical capacity for the synthesis of fructans to plants with higher agronomic value is of major
interest. The crop of predominant interest is the sugar beet,
because the major storage compound of this species is
sucrose, the direct precursor for fructan biosynthesis.
This review will cover the progress made in the fields of
bioengineering starch and fructan metabolism in higher
plants from the end of 1997 until the end of 1998.
Starch: structure and biosynthesis
Starch is deposited as granular material in the plastidic compartment of plant cells, for example, in the amyloplast of
plant storage organs. Starch is constituted of 20–30% of the
essentially linear polymer amylose, in which glucose is
polymerized via α-1,4-glycosidic linkages. To a lesser
extent, α-1,6-glycosidic linkages (branchpoints) occur
(0.1%). 70–80% of the starch is accounted for by amylopectin, which has a higher molecular weight than amylose
(107–108 Da in contrast to 105–106) and is more frequently
branched (4–5%). In contrast to amylose, amylopectin is
semi-crystalline in nature, which is probably due to the fact
that the branchpoints do not occur randomly, but rather the
branches are arranged in clusters allowing the formation of
α-helices [9]. Another unique feature of amylopectin is the
presence of covalently linked phosphate monoesters.
These can be linked either to the C3- or C6-position of the
glucose monomers, and occur to a higher extent in starch
from tuberous species, especially in potato starch [10].
The initial step in starch synthesis is the conversion of glucose-1-phosphate to ADP-glucose by the enzyme
ADP-glucose pyrophosphorylase. ADP-glucose serves as a
substrate for the starch synthases. The starch synthases
(SSs) catalyze the chain elongation through transferring the
glucose moiety from ADP-glucose to α-1,4-glucans. At least
four isozymes are known and, depending on plant species,
different isozymes contribute different amounts to the
incorporation of glucose into starch. One isoform, the granule-bound SS (GBSS), however, is known to be responsible
for the synthesis of amylose. Numerous mutants have been
described that accumulate an amylose-free starch due to a
mutation in the respective gene. The branchpoints into
starch are introduced by branching enzymes. For one isoform (branching enzyme A) a range of mutations have been
170
Plant biotechnology
Figure 1
Graminan
Levan (phlein)
6-SFT
6-FFT ?
6-SFT
G-F 2- 1F
G-F 2
F6
F6
6-SFT
6-SST
1-FFT
6-SFT
1-SST
2 G-F
2 1
Inulin
G-F - F
G-F
Enzymology of fructan synthesis in plants.
Sucrose (G-F) is a substrate for 1-kestose
(G-F2-1F) and 6-kestose (G-F2-6F) synthesis.
The former is catalyzed by 1-SST [39••]. 6-SFT
catalyzes 6-kestose synthesis in barley [19]
and can introduce branches into longer chains,
but the chain elongating activity that produces
levan, a putative fructan:fructan 6fructosyltransferase (6-FFT), has not yet been
isolated. For species accumulating unbranched
levan (e.g. Poa ampla), a 6-SST is postulated
[15]. 1-kestose is a substrate for inulin
synthesis, as well as for the fructan neoseries,
which requires the enzyme fructan:fructan
6-glucose-fructosyltransferase (6-SFT) [17].
Chain elongation for inulin and the inulin
neoseries is catalyzed by fructan:fructan
1-fructosyltransferase (1-FFT) [45•].
6G-FFT
G-F
F 2- 6G-F
6-FFT ?
F 2- 6G-F
F6
1-FFT
Levan neoseries
F 2- 1F 2- 6G-F
Inulin neoseries
Current Opinion in Biotechnology
described resulting in the synthesis of high-amylose-containing starch. The role of the other isozyme (branching
enzyme B) has yet to be determined. The crystalline nature
of amylopectin is probably dependent on the action of
debranching enzymes. In one model of discontinuous synthesis of amylopectin, these are needed to remove
excessive branchpoints introduced by the branching
enzymes [9,11,12]. Certain aspects of starch synthesis are
still unclear. These relate to the questions on how chain
elongation is initiated, how granule formation is triggered,
and how the incorporation of phosphate monoesters occurs.
Fructans: structure and biosynthesis
Fructans are a diverse group of polysaccharides that contain one or more β-linked fructose units. In the most
prominent structural types, inulin and levan, the fructose
chain emerges from the fructose part of a sucrose molecule,
proceeding via β-2,1- and β-2,6-linkages, respectively.
Besides inulin and levan, the so-called neo-kestose series
has been described where chain elongation occurs at the
glucose portion of sucrose or in both directions [13].
Branches occur in all types of fructans, but are more frequent in fructans of Gramineae [14,15].
According to a model of fructan synthesis proposed by
Edelman and Jefford in 1968 [16], biosynthesis of fructans
starts from two molecules of sucrose. One of them is the
acceptor of a fructosyl residue that is transferred from the
other by the action of the enzyme sucrose:sucrose fructosyltransferase (1-SST). The trisaccharide 1-kestose that is
produced by 1-SST serves — like all higher fructans — as
donor and acceptor of fructosyl residues for the second
enzyme, the fructan:fructan fructosyltransferase (FFT) in
the production of inulin. Levan production is initiated by
the enzyme sucrose:fructan 6-fructosyltransferase (6-SFT)
that transfers a fructose residue from sucrose preferentially to the F6 position of 1-kestose, but can also use sucrose
as the fructosyl acceptor [17]. Because 1-kestose is the preferred fructosyl acceptor for 6-SFT, the enzyme 1-SST is
believed to be important in the biosynthesis of levan as
well as in inulin accumulation [18].
The first report of the isolation of a plant fructosyltransferase gene was on 6-SFT from barley [19]. This was
followed by the cloning of fructan:fructan 6-glucose-fructosyltransferase from onion [20], which catalyzes the first step
in the production of neo-kestose type fructans typical for
species of the genus Allium. The current knowledge on the
enzymology of fructan synthesis is outlined in Figure 1.
Manipulation of the amylose content in
potato starch
The presence or absence of amylose greatly influences the
physico-chemical properties of starch [21,22]. Mutants
Production of modified polymeric carbohydrates Heyer, Lloyd and Kossman
lacking amylose have been described for a long time for
species such as maize, rice and sorghum [23,24]. Other
components of starch, such as the covalently linked phosphate, which is found in extremely high levels in potato
starch, also determine its properties and uses. The production of potato starch exclusively composed of amylopectin,
therefore, was regarded as being of extreme value.
Amylose-free potatoes were developed using both mutation induction and genetic engineering [25,26]. In both
cases, GBSS activity is largely abolished, similar to mutants
of other plant species lacking amylose. In the Netherlands,
600 ha of these plants were grown in 1996; this area was
increased to 2000 ha in 1997 [27•]. In the near future, the
amylose-free potato starch will be introduced into commercialization. Along with the performance of these
genotypes in breeding programs, the physico-chemical
properties of these starches have now been intensively
characterized [27•,28•,29]. The most prominent change
was observed with respect to the clarity of starch pastes
after storage at low temperatures. In contrast to pastes
formed using amylose-containing potato starch, these
pastes stay clear for prolonged storage periods, indicating
that they could serve as a thickening agent in food applications, where turbidity is unwanted.
In another approach it was intended to increase the amylose content of potato starch by downregulating the
expression of the major branching enzyme isozyme
(branching enzyme B) [30•]. Surprisingly, no effects on the
amylose content were observed. A 50–100% increase of the
phosphorous content was observed, however, which might
be useful for specific applications of potato starch, such as
in the wet end during papermaking.
The in vitro synthesis of amylose
The in vitro synthesis of amylose with isolated starch granules has only been achieved when high concentrations of
malto-oligosaccharides were added to the incubation
medium with ADP-glucose [31]. In a novel approach using
starch granules devoid of amylose, but containing GBSS
and hence having the capacity to synthesize starch, it was
possible to achieve the synthesis of amylose after prolonged incubation periods with labeled ADP-glucose
without the addition of primer molecules [32••]. The
authors propose that longer linear glucans are first generated by the elongation of the reducing ends of the
amylopectin and released subsequently by hydrolysis. The
enzyme responsible for hydrolysis is unknown. If the
mechanism underlying the cleavage of amylose from amylopectin becomes understood, this would enable novel
technologies for the manipulation of starch structure.
The determination of chain-length distribution
in the amylopectin
For two mutants, dull1 in maize and rugosus5 in pea, it was
possible to show that the mutation is associated with the
inactivation of one isozyme of SS [33•,34•,35]. This is the
first time that mutations for SS isozymes other than GBSS
171
have been identified in higher plants. In the case of dull1,
a novel isozyme of SS is affected, whereas in the case of
rugosus5 the SS II isozyme is absent. Both mutations cause
similar effects on the starch content and structure. The
starch content is greatly lowered, leading to a wrinkled
seeded phenotype in the case of the pea mutant, and the
apparent amylose content of the starch seems to be
increased. Most interestingly, the amylopectin structure
also is greatly affected, as a shift in the chain-length distribution is observed. In both of the mutants, a shift from
intermediate-size glucans towards shorter and extra-long
chains is observed. These findings are surprising, because
different classes of starch synthase are affected in each of
the mutants. It is possible, therefore, to speculate that the
different starch synthases interact in the production of
intermediate-size glucans. If this interaction is disturbed
by the absence of one isozyme, the production of these
glucans is largely arrested.
The industrial use of these polysaccharides with altered structure is hindered by the decreased starch yield. This might be
overcome by the generation of transgenic potato plants producing starch with similar modifications, which was achieved
by the simultaneous reduction of the expression of two
isozymes of SS [36•]. In this case, no drastic reduction of the
starch content in the transgenic plant is observed.
The incorporation of phosphate monoesters
into starch
The presence of relatively high amounts of phosphate
monoesters in potato starch was described almost thirty
years ago [37]. The biochemical mechanism underlying
the phosphorylation of starch still remains elusive; however, it was possible to isolate a novel cDNA from potato
encoding a 160 kDa protein that is probably responsible for
the phosphorylation of starch [38••]. Antisense suppression
of this protein leads to a drastic reduction of the phosphorylation level of potato starch. Conversely, it is possible to
stimulate the phosphorylation of glycogen, a polysaccharide very similar to amylopectin, if this protein is expressed
in Escherichia coli. This protein seems to influence the
phosphorylation of α-1,4-glucans at both the C3- and C6position of the glucose monomers, because both are
equally affected in the transgenic plants.
It now seems possible, therefore, to achieve the phosphorylation of cereal starches through the ectopic expression
of this protein. This is of economic and ecological importance, because maize starch is often chemically
phosphorylated to make it applicable for the paper industry. The respective gene is not unique to solanaceous
genomes, but seems to be universally present in higher
plant genomes, as corresponding sequences can be found
in databases from Arabidopsis and rice. The different phosphorylation levels of the starches derived from varying
storage organs has to be explained, therefore, either by the
expression levels of this protein, or by the substrate availability of the putative phosphoryl-donor.
172
Plant biotechnology
Production of fructans in potato
The first 1-SST gene sequence was published by
Hellwege et al. [39•] and was isolated from the inulin-producing artichoke (Cynara scolymus). As the SST-catalyzed
reaction that yields 1-kestose and glucose from two molecules of sucrose is essentially irreversible, this enzyme
could be the controlling factor in fructan synthesis.
Expression of the 1-SST gene in potato led to the accumulation of 1-kestose and nystose (glucose–fructose3) and
an overall increased content of soluble sugars. It could be
demonstrated that the enzyme is capable of kestose production even at low substrate concentrations. This is an
important requirement for the production of fructans in
vegetables and other edible plants under the objective of
raising their nutritional value.
Production of fructans in sugar beet
A major advance in agricultural fructan production was the
transformation of sugar beet, this time with a 1-SST gene
form Jerusalem artichoke (Helianthus tuberosus) as reported
by Sévenier et al. [40••]. In sugar beet, vacuolar sucrose concentrations between 350 and 600 mM are ideal
preconditions for high-yield fructan production.
Nevertheless, a conversion of more than 90% of sucrose
into fructan is remarkable.
The authors claim that the fructo-oligosacchrides produced in the so-called fructan beet could replace sucrose as
a sweetener, but detailed studies show that the sweetening
ability of the trisaccharide kestose is only 31% as compared
to sucrose and decreases with increasing chain length [41].
Besides, the caloric value of fructans is not negligible. Due
to bacterial fermentation and resorption of fermentation
products, the available energy content of fructans is about
4.13 kJ/g (1 kcal/g) reaching 40% of the value for free hexoses [42]. It is therefore unlikely that fructans can compete
with sweeteners such as maltitol (the disaccharide α-1,4glucosylsorbitol), but longer chain fructans could serve as
bulking agents in combination with sweeteners or as a fat
replacement [43].
The expression of both SST and FFT activities that
should lead to high yields of inulin in an agronomically
important crop plant might be even more attractive than
short chain fructan synthesis in sugar beet as reported by
Sévenier et al. [40••]. Expression of both activities in
transgenic plants was reported by van der Meer et al. [44•].
Interestingly, the expression of SST and FFT in petunia
did not yield the same fructan pattern as found in the
endogenous system, Jerusalem artichoke. There are two
possible explanations for this finding. One would imply
that the model of fructan synthesis as proposed for
Jerusalem artichoke by Edelman and Jefford [16] was
wrong and additional enzymes are needed to reach the
complete set of fructans. Alternatively, the difference
could be due to FFT substrate concentrations in the
transgenic plants. It has been shown that Jerusalem arti-
choke FFT prefers short chain fructans as acceptors of
fructosyl residues and synthesizes longer chains only
when threshold concentrations of precursors are achieved.
Hellwege et al. [45•] demonstrated that FFT enzymes of
different species show characteristic differences in their
substrate affinities when expressed in heterologous systems. It can be speculated, therefore, that expression of
the artichoke FFT in the ‘fructan beet’ described by
Sévenier et al. [40••] might be the most promising
approach towards agricultural fructan production.
Engineering fructan synthesis in
fructan-storing crops
Approaches to improve quality and/or quantity of fructan
by expressing heterologous fructosyltransferases in fructan-producing species have already been demonstrated by
Vijn et al. [46] and Sprenger et al. [47]. In the first case, the
previously mentioned 6-glucose-fructosyltransferase of
onion was expressed in chicory, thereby leading to the production of neo-kestose-type fructans in parallel to inulin
[46]. In the second, the barley 6-SFT was expressed in
chicory and led to the synthesis of a branched fructan typical for Graminean species [47].
Conclusions
In the past two years, large amounts of genetically modified potatoes producing an amylose-free starch have been
produced. This will be the first example of genetically
engineered starch with superior quality over traditional
starches entering the markets. It is foreseeable that
starches with other alterations will follow in the next few
years. Examples will be starches with an altered amylopectin chain length distribution or a modified
phosphate content, as it is possible now to specifically
engineer these traits. It can also be envisioned that a
broad range of novel starches will be produced through
combining the downregulation or overexpression of several genes.
It is now also possible to produce fructans in crops with
superior agronomic performance. After additional engineering, this will allow the utilization of fructans as a food
ingredient on a larger scale. The cloning of further genes
will enable the production of a larger diversity of fructans
with different structures, such as a FFT catalyzing the
polymerization of α-2,6-linked fructose units.
Furthermore, the cloning of a fructanexohydrolase will
lead to the engineering of fructan-storing crops to produce fructans with an increased chain length.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Lillford PJ, Morrison A: Structure/function relationship of starches
in food. In Starch Structure and Functionality. Edited by Frazier PJ,
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2. Roberfroid MB, Delzenne NM: Dietary fructans. Annu Rev Nutr
•• 1998, 18:117-143.
This excellent review on the physiological effects and potential health benefits of fructans gives a critical overview of what is known on the fermentation
of inulin by bacteria in the large intestine and the effects on the host of production of short-chain fatty acids by these bacteria. Physiological consequences on mineral absorption, lipid metabolism and blood glucose and
insulin are summarized, and the potentials in risk reduction of diseases are
discussed. It distinguishes between inulin, oligofructoses produced by
hydrolysis of inulin, and synthetic fructans.
3.
Gibson GR, Beatty ER, Wang X, Cummings JH: Selective
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Roberfroid MB, Vanloo JAE, Gibson GR: The bifidogenic nature of
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Characterization of fructan oligomers from species of the genus
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27.
•
Visser RGF, Suurs LCJM, Bruinenberg PM, Bleeker I, Jacobsen E:
Comparison between amylose-free and amylose-containing
potato starches. Starch/Staerke 1997, 49:438-443.
In this paper, the authors analyze the chemical properties of amylose-free
compared to amylose-containing potato starch. They are able to show
that next to the lack of amylose no other changes occur to the starch, indicating that the superior properties of potato starch, such as the low lipid
or the high phosphate content, are maintained within the genetically modified starch.
28. Visser RGF, Suurs LCJM, Steeneken PAM, Jacobsen E: Some
•
physicochemical properties of amylose-free potato starch.
Starch/Staerke 1997, 49:443-448.
The authors analyze the physico-chemical properties of amylose-free compared to amylose-containing potato starch. It becomes evident that the environmental benefits will be high if the amylose-free starch is introduced into
the market. Because it displays a reduced viscosity and a increased gel stability and clarity, derivatisations which are normally needed to achieve this
become obsolete.
29. Heeres P, Jacobsen E, Visser RGF: Behaviour of genetically
modified amylose free potato clones as progenitors in a breeding
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30. Safford R, Jobling SA, Sidebottom CM, Westcott RJ, Cooke D,
•
Tober KJ, Strongitharm BH, Russell AL, Gidley MJ: Consequences of
antisense RNA inhibition of starch branching enzyme activity on
properties of potato starch. Carbohyd Polym 1998, 35:155-168.
The almost complete suppression of branching enzyme activity down to
less than 5% of wild-type levels in transgenic potato tubers is described.
Surprisingly, no changes in the amylose content of the starches derived
from these transgenic lines are found when compared to wild-type starch.
Differences in the gelatinization properties (an increase of up to 5°C in the
peak temperature and viscosity onset temperature) of the different starches as determined by differential scanning calorimetry are reported. The
authors speculate that these changes are correlated with the branching
pattern of the starch that results in changes of double helix lengths.
Conversely, it is also possible that the increased phosphate content measured in the genetically modified starches results in the observed elevation
of the gelatinization temperature.
31. Denyer K, Clarke B, Hylton C, Tatge H, Smith AM: The elongation of
amylose and amylopectin chains in isolated starch granules.
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•• Amylose is synthesized in vitro by extension and cleavage from
amylopectin. J Biol Chem 1998, 273:22232-22240.
For the first time, amylose is synthesized in vitro without the addition of
high amounts of maltodextrins. A model that amylose is synthesized by
GBSS attached to amylopectin and released by subsequent hydrolysis is
proposed. It is speculated that GBSS might harbor a second catalytic
(hydrolytic) activity, or that the branching enzymes catalyze an intramolecular transglycosylation, or that a hitherto unknown endoamylase entrapped
in starch granules releases the amylose from amylopectin. If the model is
correct, the first possibility seems most probable because a knockout
mutation should abolish both activities of GBSS. This would explain why
a mutation in an amylose-releasing activity has never been identified.
33. Craig J, Lloyd JR, Tomlinson K, Barber L, Edwards A, Wang TL,
•
Martin C, Hedley CL, Smith AM: Mutations in the gene encoding
starch synthase II profoundly alter amylopectin structure in pea
embryos. Plant Cell 1998, 10:413-426.
The authors conclusively demonstrate that the rugosus5 gene from pea
encodes the major starch synthase expressed in pea embryos. Biochemical
studies indicate that the protein is not detectable in some rugosus5 mutants.
Furthermore, one mutated allele was cloned that carries a basepair substitution which introduces a stop codon into the open reading frame. The analysis of the starch from the mutated plants reveals that the effects are similar
to those observed in dull1 mutants from corn.
174
Plant biotechnology
34. Gao M, Wanat J, Stinard PS, James MG, Myers AM: Characterization
•
of dull1, a maize gene coding for a novel starch synthase. Plant
Cell 1998, 10:399-412.
A portion of the dull1 locus from maize was cloned by transposon tagging
and a nearly full-length cDNA was isolated and subsequently sequenced.
Sequence alignments indicate that dull1 encodes a novel class of starch
synthase with a predicted molecular mass of 188 kDa. The high molecular
mass is unique among the starch synthases known so far.
35. Taylor CB: Synthesizing starch: roles for rugosus5 and dull1. Plant
Cell 1998, 10:311-314.
36. Lloyd JR, Landschütze V, Kossmann J: Simultaneous antisense
•
inhibition of two starch synthase isoforms in potato tubers leads
to accumulation of grossly modified amylopectin. Biochem J
1999, in press.
The authors describe the effects on the chain length distribution of the amylopectin if starch synthase (SS) II and SS III are reduced solely or simultaneously. A shift from longer chains to shorter chains is observed if SS II and SS
III are suppressed on their own. The effects are only moderate if SS II is
reduced in activity and are more pronounced if SS III is reduced, which correlates with the amount of activity they each contribute to the total starch synthase activity in potato tubers. This is the first report, however, on a change in
starch structure that was observed in plants with reduced activity of SS II. If
SS II and SS III are suppressed simultaneously, the effects are similar to other
mutants with a defect in a starch synthase isozyme [33•, 34•,35]. An increase
in extra long chains is observed and a even more pronounced shift towards
chains of the shortest fraction in the amylopectin is observed.
37.
Hizukuri S, Tabata S, Nikuni Z: Studies on starch phosphate. Part 1.
Estimation of glucose-6-phospate residues in starch and the
presence of other bound phosphate(s). Starch/Staerke 1970,
10:338-343.
38. Lorberth R, Ritte G, Willmitzer L, Kossmann J: Inhibition of a starch
•• granule-bound protein leads to modified starch and repression of
cold sweetening. Nat Biotechnol 1998, 16:473-477.
The authors describe the cloning of a novel cDNA that was isolated on the
basis that its product binds to potato starch granules. Using antisense suppression, they are able to show that this protein is probably responsible for
the phosphorylation of starch. Intriguingly, there were side effects observed
in the potato plants with a lowered level of starch phosphorylation. The transgenic plants display a starch excess phenotype in leaves, as they do not
degrade their starch even after prolonged periods of darkness. Similarly, the
degradation of starch and the accumulation of reducing sugars during cold
storage of potato tubers are largely eliminated. Both effects could be interpreted as non-degradability of the non-phosphorylated starch in vegetative
plant organs; however, experiments have to be undertaken to prove this
assumption. These observations are both of agronomic and economic importance. The accumulation of starch in green photosynthesizing organs of
plants makes them more suitable as a fodder crop for ruminants, because it
ultimately increases the C:N ratio of the substrate and prevents `bloat´. The
accumulation of reducing sugars in potato tubers is a long-standing problem
in the potato processing industry, as it causes the Maillard reaction to occur
leading to an undesired dark coloring of fried products.
39. Hellwege EM, Gritscher D, Willmitzer L, Heyer AG: Transgenic
•
potato tubers accumulate high levels of 1-kestose and nystose —
functional identification of a sucrose sucrose 1fructosyltransferase of artichoke (Cynara scolymus) blossom
discs. Plant J 1997, 12:1057-1065.
This paper gives the first report of cloning and heterologous expression of a
sucrose:sucrose1-fructosyltransferase (1-SST) cDNA. The cDNA was
cloned from an artichoke blossom disk library by homology to the
sucrose:fructan 6-fructosyltransferase from barley that was cloned by
Sprenger and co-workers in 1995 [19]. The cDNA was expressed transiently in tobacco protoplasts and also in transgenic potato. As a character-
istic difference, the enzyme produced only 1-kestose in vitro, but also the
next higher homologs nystose and fructosyl-nystose in vivo. In contrast to
experiences with bacterial fructosyltransferases, expression of the artichoke
1-SST gene in potato under the control of the constitutive cauliflower mosaic virus 35S promoter did not cause a visible phenotype and did not influence tuber yield.
40. Sévenier R, Hall RD, van der Meer IM, Hakkert HJC, van Tunen AJ,
•• Koops AJ: High level fructan accumulation in a transgenic sugar
beet. Nat Biotechnol 1998, 16:843-846.
This paper describes the transformation of sugar beet with a cDNA of 1-SST
from Jerusalem artichoke. Sugar beet is highly recalcitrant to genetic modification, and therefore a special protocol for the transformation of protoplasts
has been developed. Expression of the 1-SST cDNA under the control of the
constitutive cauliflower mosaic virus 35S promoter leads to a nearly complete conversion of sucrose into short chain oligofructoses. The plants show
no visible phenotype and have no yield reduction. The high conversion rate
for sucrose is accompanied by an eightfold increase in glucose, which might
raise problems in isolation and purification of fructan.
41. Yun JW: Fructooligosaccharides — occurrence, preparation, and
application. Enzyme Microb Technol 1996, 19:107-117.
42. Roberfroid M, Gibson GR, Delzenne N: The biochemistry of
oligofructose, an approach to calculate its caloric value. Nutr Rev
1993, 51:137-146.
43. Rapaille A, Gonze M, Vanderschueren F: Formulating sugar-free
chocolate products with maltitol. Food Technol 1995, 49:51-54.
44. van der Meer IM, Koops AJ, Hakkert JC, van Tunen AJ: Cloning of the
•
fructan biosynthesis pathway of Jerusalem artichoke. Plant J
1998, 15:489-500.
The paper describes transgenic petunia plants that express both fructosyltransferases from the Jerusalem artichoke that are believed to be necessary
for inulin production: 1-SST and 1-FFT. Only in senescent leaves, fructans
up to GF25 could be detected, whereas the maximal degree of polymerization of inulin is about 50 hexose units in Jerusalem artichoke. Additionally, the
ratios of the fructan levels ranging from GF2 to GF9 were different in the
petunia as compared to Helianthus tuberosus.
45. Hellwege EM, Raap M, Gritscher D, Willmitzer L, Heyer AG:
•
Differences in chain length distribution of inulin from Cynara
scolymus and Helianthus tuberosus are reflected in a transient
plant expression system using the respective 1-FFT cDNAs. FEBS
Lett 1998, 427:25-28.
The authors compared two fructan:fructan 1-fructosyltransferase (1-FFT)
cDNAs from different species by transient expression in tobacco protoplasts. They expressed an FFT cDNA of Jerusalem artichoke and artichoke and
found that the latter enzyme was poorly active on 1-kestose as substrate as
compared to the Jerusalem artichoke counterpart. Nevertheless, it produced
longer chain fructans than the other when incubated with a mixture of medium length fructo-oligosaccharides (GF2 to GF4). The results can explain the
difference in fructan patterns found among different species of the
Asteraceae. It is also obvious that the 1-FFT of artichoke offers advantages
for the production of fructans in transgenic plants because of the high mean
degree of polymerization of inulin produced by this enzyme.
46. Vijn I, Vandijken A, Sprenger N, Van Dun K, Weisbeek P, Wiemken A,
Smeekens S: Fructan of the inulin neoseries is synthesized in
transgenic chicory plants (Cichorium intybus l) harbouring onion
(Allium cepa l) fructan-fructan 6g-fructosyltransferase. Plant J
1997, 11:387-398.
47.
Sprenger N, Schellenbaum L, van Dun K, Boller T, Wiemken A:
Fructan synthesis in transgenic tobacco and chicory plants
expressing barley sucrose-fructan 6-fructosyltransferase. FEBS
Lett 1997, 400:355-358.