Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TRANSGENIC PLANT OR PLANTS WITH A NATURALLY HIGH WATER CONTENT
OVERPRODUCING AT LEAST TWO AMINO ACIDS OF THE ASPARTATE FAMILY
DESCRIPTION
Summary of the invention
( A chimeric double gene construct comprising a nucleic acid
sequence encoding an enzyme having aspartate kinase (AK) activity and a
nucleic acid sequence encoding an enzyme having dihydrodipicolinate
synthase (DHPS) activity is provided. This construct is capable of
differential expression of the two genes resulting in an increased level
of both lysine and threonine more than 5-fold the wild type level of each
amino acid in a plant or parts thereof. This construct also renders
increase in the methionine level possible. This expression is now
possible without incurring the previous problems associated with high
accumulation of lysine or combined expression of both AK and DHPS genes
in a plant. The expression regulation should be such that expression
occurs in such a way that both lysine and threonine are produced to a
comparable extent without damaging the plant i.e. without causing a
negative abberations in the phenotype compared to wild type plants. The
construct resulting in lysine and threonine increase also provides an
increase fn the methionine level.
Background to the invention
Human and monogastric animals cannot synthesize 9 out of 20
amino acids and therefore need to obtain these essential amino
acids from
their diet. The diet of human beings and livestock is based
largely on
plant material. Among the essential amino acids needed for animal
and
human nutrition are lysine and threonine. These essential amino
acids are
often only present in low concentrations in crop plants. Therefore.
synthetic amino acids are usually added as supplements to grain-based
and
other vegetable based diets, in order to increase the nutritional
value
of feed.
Various attempts have been made in the past to increase the
levels of free threonine and lysine in plants by classical breeding
and
by mutant selection) but with little success. Also it has been
attempted
to increase both lysine and threonine simultaneously in the
same
transgenic plant by molecular genetic modification. Increase
of free
lysine proved to operate at the expense of the accumulation
of free
threonine (Shaul and Galili) 1993; Falco et al.) 1995). To date
no
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description of increased methionine level has been given.
Biosynthesis of aspartate-family amino acids.
The essential amino acids lysine) threonine and methionine are
synthesized from aspartate by a complex pathway which is similar for
bacteria and higher plants (fig. 1). The aspartate family pathway has
been characterized in detail in Escherichia colt by isolation of enzymes
involved in the pathway, which were later also purified from higher
plants (Bryan) 1980) Umbarger, 198).
The rate of synthesis of the aspartate-family amino acids is
regulated primarily by a complex process of feedback inhibition of the
activity of some key enzymes in the pathway by the relevant amino acid
end product. The first enzymatic activity in the pathway that is common
to all of the aspartate-family amino acids, aspartate kinase (AK)
activity, is feedback inhibited by both lysine and threonine. In
addition) lysine also inhibits the activity of the enzyme
dihydrodipicolinate synthase (DHPS), the first enzyme of the pathway
after the branch point that leads to the synthesis of lysine. Threonine
inhibits the activity of homoserine dehydrogenase (HSD)) the first enzyme
involved in the biosynthesis of threonine (Matthews et al., 1989).
The enzyme aspartate kinase (AK) catalyses the phosphorylation
of aspartate to form 3-aspartyl phosphate, with the accompanying
hydrolysis of ATP. Both in E. coti and in plants several different AK
isoenzymes have been identified which are differentially inhibited either
by lysine or by threonine. AK-III, the product of the E. eoti lysC locus
has been shown to consist of two identical subunits as a homodimer
(Cassan et al. , 1986; Richaud et al. , 193) . The E. coZi LysC gene has
been cloned and sequenced (Cassan et al., 1986).
The product of AK activity) 3-aspartyl phosphate) is converted
in the next enzymatic step to 3-aspartic semialdehyde (3-ASA)) which
serves as a common substrate for the synthes-is of both lysine and
threonine. The enzyme dihydrodipicolinate synthase (DHPS) catalyses the
first reaction that is unique to lysine biosynthesis, the condensation of
3-a.spartate semialdehyde with pyruvate to form 2,3-dihydrodipicolinate.
In E. eoZi this enzyme is encoded by the dapA locus and appears to
consist of four identical subunits as a homotetramer (Shedlarski and
Gilvarg) 190). The E. coti dapA gene has been cloned and sequenced
(Richaud et al., 1986). In plants DHPS enzyme also appears to be a single
enzyme comparable to the E. toll enzyme. Among the major regulatory
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enzymes of the aspartate family pathway in plants) DHPS is the most
- sensitive to feedback inhibition by its end product (I5o of DHPS
for
lysine ranges between 10 and 50 pM). Plant DHPS is about 10-fold
more
sensitive to lysine inhibition than are plant AKs that are sensitive
to
lysine (ISo,between 100 and 700 u1H). Plant DHPS is about 100-fold
more
sensitive to lysine inhibition than E. cots DHPS (I5o is about 1
mM)
(Yugari and Gilvarg, 1962; Galili, 1995)
Homoserine dehydrogenase (HSD) catalyses the first reaction
that is specific for the synthesis of threonine) methionine and
_.. 10 isoleucine. Higher plants generally possess at least two forms of
HSD: a
threonine-sensitive form and an insensitive form (Bryan) 1980) Lea
et
al.) 1985).
Several lines of evidence have indicated that in plants AK is
the rate-limiting enzyme for threonine synthesis, while DHPS is
the mayor
rate-limiting enzyme for lysine synthesis. Mutants of several plant
species possessing feedback-insensitive AK isozyme were found to
overproduce free threonine, but exhibited only a slight increase
in the --
level of lysine (Bright et al.. 1982; Cattoir-Reynaerts et al..
1983;
Dotson et al.) 1990; Frankard et al.) 1992). On the other hand)
a
feedback-insensitive DHPS mutant tobacco plant overproduced lysine
{Negrutiu et al.) 1984). Similar results have been reported with
transgenic plants expressing feedback-insensitive DHPS or AK from
E. eotf
(Glassman. 1992; Perl et al.) 1992; Shaul and Galili, 1992 a and
b).
Transgenic plants that expressed the E. cott AK overproduced threonine
and exhibited only a slight increase in the level of lysine. Studies
of
transgenic plants have also demonstrated that AK and DHPS are not
only
regulated by feedback inhibition, but that the levels of these enzymes
also limit the rate of production of threonine and lysine. In transgenic
plants a significant positive correlation was detected between the
levels
of the bacterial AK and DHPS enzymes and the levels of free threonine
and
lysine, respectively (Shaul and Galili, 1992 a and b).
However, transgenic plants expressing both a feedback-
insensitive AK and a feedback-insensitive DHPS (Shaul and Galili)
1993)
contain free lysine levels that far exceed those in plants expressing
only the introduced insensitive AK-or DHPS. This lysine increase
is also
accompanied by a significant reduction in threonine accumulation
as
compared with transgenic plants expressing the insensitive AK only.
This
means that when DHPS becomes deregulated the branch leading to lysine
synthesis competes strongly with the other branch of the pathway
and a
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considerable amount of 3-aspartic semialdehyde is thus converted into
lysine at the expense of threonine. The same results were obtained by
crossing a lysine overproducing mutant with a threonine overproducing
mutant characterized by an altered regulation of respectively DHPS and AK
(Frankard et al.) 1992).
European patent application No. 485.970 discloses a method of
increasing the level of both free threonine and lysine by introducing
into plants cells a first chimeric gene comprising a DNA sequence coding
for an enzyme having AK activity and a second chimeric gene comprising a
DNA sequence coding for an enzyme having DHPS activity. Both chimeric
genes further comprise a DNA sequence enabling expression of the enzymes
in the plant cells and subsequent targeting of the enzymes to the
chloroplast.
European patent application No. EP 9390$395 describes two
isolated DNA fragments comprising a fragment encoding AK insensitive to
inhibition by lysine and a second fragment encoding DHPS which is at
least 20-fold less sensitive to inhibition by lysine than plant DHPS. It
is claimed that the lysine-insensitive AK causes a higher than normal
threonine production and that the DHPS causes a higher than normal lysine
production in transformed plants.
However, while it is shown that transgenic plants expressing
feedback-insensitive DHPS from E. cots overproduce lysine and that
transgenic plants expressing the E. coZi AK overproduce threonine
(Glassman, 1992; Perl et al., 1992; Shaul and Galili, 1992 a and b))
combining these two genes in transgenic plants never resulted in a
comparable increase of both threonine and lysine. Shaul and Galili (1993)
obtained transgenic plants expressing both feedback-insensitive AK and
feedback-insensitive DHPS by crossing transgenic plants that expressed
each of these enzymes individually. These plants were shown to contain
free lysine levels that far exceeded those in plants expressing only the
insensitive DHPS. The lysine increase however was accompanied by a
___ significant reduction in threonine accumulation as compared with .plants
expressing the insensitive AK only.
The same results were found when feedback-insensitive bacterial
DHPS and AK enzymes encoded by the Coryrtebacteriiun dapA gene and a mutant
E. cots lysC gene, respectively) were expressed together in transgenic -
canola and soybean seeds. Several hundred-fold increases in free lysine
in transgenic seed was observed, whereas the accumulation of excess
threonine that was seen in transgenic seed expressing feedback-
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insensitive AK alone was prevented by the co-expression of DHPS (Falco et
al.. 1995). -
Furthermore) abnormal phenotypes have been observed in
transgenic plants in which the free lysine concentration is increased in
the whole plant to more than 10-fold the concentration found in
untransformed plants (Shaul and Galili, 1992; Glassman, 1992; Frankard et
al.) 1992).
In European patent application No. EP 435970 the expression of
both feedback-insensitive AK and DHPS genes was driven by the same
constitutive CaMV35S promoter. Also in the experiments of Galili and
Shaul (1993) and of Falco et al. (1995) in which both feedback-
insensitive AK and DHPS were co-expressed in transformed plants) the same
promoter was used to drive expression of the two genes.
The problems that are encountered when using genetic
engineering technology to increase both free lysine and threonine in
plants are reported by Falco et al. (1995). They also showed that using
the same seed-specific promoter to drive both feedback-insensitive AK and
DHPS expression only resulted in increasing the lysine content of canola
and soybean seeds. Furthermore, they showed that seeds from transformants
accumulating the highest levels of lysine had an abnormal appearance and
germinated poorly (Falco et al. (1995).
The following hypothesis is offered on the basis of the
findings from the prior articles. The ratio between lysine and threonine
synthesis in plants is regulated by at least two factors: First, the
availability of 3-ASA which is the common substrate for the two key-
enzymes specific for threonine and lysine synthesis (respectively
homoserine dehydrogenase and DHPS). Second, the competition between these
two key-enzymes for 3-ASA as a common substrate. The level of 3-ASA seems
to be determined by the activity of AK. When feedback-insensitive AK is
overexpressed in transgenic plants, the higher 3-ASA concentration can be
channeled into the threonine synthesis branch which may also result in an
increase of methionine. However, when DHPS becomes feedback-insensitive
the branch leading to lysine synthesis competes strongly with the other _.__
branch of the pathway and a considerable amount of 3-ASA is thus
converted into lysine at the expense of threonine synthesis. In order to
increase the amount of both free lysine and threonine in transgenic
plants to desirable levels) the .timing and the level of expression of
each introduced feedback-insensitive gene should be highly regulated. The
production of methionine another amino acid of the aspartate pathway is
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also concomitantly increased.
- According to the invention the expression of a nucleic acid
sequence encoding an enzyme having aspartate kinase (AK) activity and a
nucleic acid sequence encoding an enzyme having dihydrodipicolinate
synthase (DHPS) activity is regulated such that the expression of the two
sequences is different in strength and such that the expression of the AK
encoding sequence occurs at a higher rate than that of the DHPS encoding
sequence. This can be achieved by using different strength promoters or
even by using the same promoters but additionally providing the DHPS
__._ 10 regulating promoter with an enhancer. The object of the invention is
to
generate plants that overproduce both threonine and lysine to a
comparable extent. Overproduction of methionine is also an objective.
This can occur through using special sequences according to the invention
with recombinant DNA technology techniques that are well known in the art
such as by transformation of plant cells.
Preferably the expression of the DHPS and AK encoding sequences
is regulated at different stages of plant development) so that the plant
has a chance to develop and mature without interference from overproduced
free amino acid) in particular overproduced lysine. This can be achieved
in a number of manners.
Firstly by regulating at least the DHPS encoding sequence with
an inducible promoter which inducible promoter can be induced after the
AK encoding sequence has begun to be expressed. The AK encoding sequence
may naturally also be regulated by an inducible promoter, however with
the restriction that a stronger expressing system is provided for the AK
encoding sequence than for the DHPS encoding sequence and the restriction
that the expression of DHPS occurs at a later stage than that of the AK
encoding sequence. In particular it is preferred to induce the DHPS
encoding sequence when the plant has reached a sufficient degree of
maturity not to be negatively influenced by overproduction of lysine.
Secondly the expression at different stages can be achieved in
an alternative embodiment of the invention by regulating expression with
promoters that are associated with cell types or organs and drive natural
expression at different stages of plant development. Suitable examples of
promoters for the DHPS encoding sequence are promoters associated with
processes that occur late in plant development or even upon maturity. In
particular tuber or fruit associated promoters are suited for regulating
expression of the DHPS encoding sequence. The AK encoding sequence may
also be regulated by such promoters but with the restriction that a
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stronger expressing system is provided for the AK encoding sequence than
for the DHPS encoding sequence. Preferably in such an embodiment also the
DHPS encoding sequence is expressed later than the AK encoding sequence
for example due to the use of an inducible promoter.
A very strong constitutive promoter can be used to drive
feedback-insensitive AK expression throughout the whole plant during all
stages of organ development, resulting in a higher 3-ASA concentration
which can be channeled into the threonine synthesis branch and will lead
to threonine and methionine overproduction. In combination therewith the
feedback-insensitive DHPS enzyme can be regulated such that it is only
expressed at a lower level and at a later stage e.g. in specific plant
organs. The expression of the AK encoding sequence can be constitutive
but it may also be restricted to specific organs.
The AK encoding sequence may be expressed in the same organs as
the DHPS encoding sequence) however the encoding sequences must be
differentially expressed. Because the expression of DHPS is at a later
stage and at a lower level, the branch leading to lysine synthesis
competes only weakly and during a well defined period of development with
the other branch of the pathway and both threonine and lysine will be
overproduced. Methionine also a component of the aspartate pathway is
also overproduced.
It is preferable when very high levels of lysine are to be
achieved, e.g. tenfold or more higher than normal that the DHPS encoding
sequence is expressed in specifically targeted organs to prevent negative
characteristics of the phenotype from appearing in the plant. Such
targeted plant organs are organs that develop later on in plant
maturation such as tubers, seed, fruit and flowers. Because lysine is
only overproduced in the specifically targeted plant organs like tubers,
the rest of the plant will develop normally without showing aberrant
phenotype.
The expression of the DHPS encoding sequence is preferably
carried out in specific plant organs with a high water content. This also
offers the advantage that minimum damage to phenotype will occur. Such
organs comprise tubers) fruit, flowers or leaves. Preferably water rich
organs comprise more than '~Ox water, preferably more than 80x. In addition
the expression of the DHPS encoding sequence in a plant organ with a high
water content provides the required amino acids readily dissolved in a
water fraction which renders it easily accessible for harvesting. For
this reason it is also preferable for the AK encoding gene also to be at
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least expressed in a plant organ with a high water content. Most
preferably the two genes should be expressed with an area of overlap of
expression being the plant organ with a high water content in which the
DHPS encoding sequence is expressed. Preferably the water rich organ will
be a readily harvestable organ such as tuber, flower, fruit, root or
leaves.
Such an organ can either be used as such as source of nutrition
comprising a high content of at least two amino acids of the aspartate
family selected from methionine, lysine and threonine or can be subjected
to an extraction process for obtaining a water fraction) said water
fraction comprising a high content of at least two amino acids of the
aspartate family selected from methionine, lysine and threonine.
Naturally the plant as such or a part of plant comprising the water rich
organ can be harvested and used as such e.g. as food or food supplement
and/or be subjected to a water extraction process. Such water fraction
obtained from a plant or plant organ according to the invention can
subsequently also be used as such for nutritional purposes. Numerous
simple economical extraction processes are known e.g. from fruit or
vegetable juice production. It thus becomes possible to provide fruit or
vegetable juices in a manner known per se, said fruit or vegetable juices
however being of considerable additional nutritional value due to the
high presence of at least two amino acids of the aspartate family
selected from methionine, lysine and threonine.
Plants or parts of plants e.g. fruit, flowers, tubers, roots or
leaves according to the invention as such are particularly suited for
vegetarians due to their automatic supplementation of two essential amino
acids. In addition the water fraction from plants or parts of plants
comprising the water rich organs expressing the combination of genes
according to the invention provides an easily obtainable source with a
high content of at least two amino acids of the aspartate family selected
from methionine, lysine and threonine from which the over produced amino
acids can readily be obtained in large amounts. The increased levels of
methionine result in an easily obtainable source of methionine for the
first time. It now becomes possible to obtain methionine, lysine and
threonine in a simple manner.
The present invention relates to a chimeric double gene
construct comprising
(a) a nucleic acid sequence encoding an enzyme having aspartate kinase
(AK) activity)
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(b) a nucleic acid sequence encoding an enzyme having dihydrodipicolinate
synthase (DHPS) activity,
(c) an expression regulating sequence for the encoding sequence {a)
operably linked to (a),
(d) an expression regulating sequence for the encoding sequence (b)
operably linked to (b)) said expression regulating sequences (c) and (d)
being such that the expression of the two sequences is different in
strength and such .that the expression of the AK encoding sequence (a)
occurs at a higher rate than that of the DHPS encoding sequence (b) i.e.
{c) is stronger than (d). In addition the sequences (a) and (b) of the
chimeric double gene construct must also each comprise a nucleic acid
sequence (e) coding for a transit peptide which is involved in the
translocation of protein from the cytosol into the plastids (Van den
Broek et al.) 1985; Schreier et al., 1985) as the organelle in which
lysine and most of the threonine biosynthesis takes place in higher
plants is the plastid. This nucleic acid sequence encoding a chloroplast
transit peptide fused to the encoding sequences of (a) and (b), will on
expression produce a fused AK/transit peptide or DHPS/transit peptide
chimeric protein in the cytoplasm of the transformed plant cell, which
will be transported to the plastids) where increased production of both
threonine and lysine is thereby obtained. Nucleic acid sequences coding
for any kind of plastid transit peptide can be used in this invention. A
suitable example is provided by the DNA sequences derived from the
ferredoxin gene coding for targeting of proteins into the chloroplast
stroma (Smeekens et al.) 1985a) and sequences from the plastocyanine gene
coding for targeting of proteins into the chloroplast lumen (Smeekens et
al.) 1985b). The preferred DNA sequence coding for plasmid targeting of
AK and DHPS encodes the transit peptide originating from the pea rbcS-3A
gene (figure 2; Fluhr et al., 1986). The 3' end of the nucleic acid
sequence coding for the transit peptide is fused to the sequence encoding
AK or DHPS.
The sequences encoding AK and DHPS (e) and (b) must in turn
also be fused to a transcription termination DNA signal (g) and (h)
respectively. This termination signal comprises a 3' transcription
termination and a mRNA polyadenylation signal. Termination signals
present at the 3' flanking region of any cloned gene can be used, e.g.
from the pea rbcS gene) the bean phaseoline gene) or the nopaline
synthase gene derived from the Ti plasmid of Agrobacterium tumefaciens.
The preferred terminator sequence originates from the 3' flanking region
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of the octopine synthase gene from the Ti plasmid of Agrobactertum
tumefactens (figure 2; Greve et al.) 1983).
- The gene construct according to the invention preferably
comprises as expression regulating sequence (d) for nucleic acid sequence
(b) a regulating sequence enabling expression of DHPS at a later stage
during development compared to the expression of nucleic acid sequence
{e). In an alternative embodiment the expression regulating sequence (d)
is a sequence that does not lead to constitutive expression. Preferably
the sequence leads to expression in one or more specific plant organs
e.g. a tuber, flower, fruit, root or stem. A preference exists for the
sequence to regulate expression in water rich organs.
The expression regulating sequence (c) for nucleic acid
sequence (a} can be a sequence that enables high expression of the
encoding sequence (a) at an early stage during plant and/or plant organ
development. The expression regulating sequence (c) for nucleic acid
sequence (a) can be a sequence that enables high expression of the
encoding sequence (a) in all plant cells or plant organs. The expression
regulating sequence (c} is suitably a constitutive promoter. Such a
constitutive promoter can be the cauliflower mosaic virus (CaMV) 35S
promoter.
The expression regulating sequence (c) can comprise not only a
promoter but also an expression enhancer sequence. If this is the case
the expression regulating sequence (d) can comprise the same promoter or
a promoter of the same strength as that of expression regulating sequence
(c) as the expression level of encoding sequences (a) and (b) will be
different.
The expression regulating sequences in particular the promoters
are to be found in the 5'region of each of the encoding sequences (a) and
(b). At the 3' end of the promoter a short nucleic acid sequence
representing 5' mRNA non-translated sequence may be added, which enhances
translation of the mRNA transcribed from the chimeric genes. An example
is the omega sequence derived from the coat protein gene of the tobacco
mosaic virus (Gallie et al.) 1987)) of the alfalfa mosaic virus
translational enhancer (Brederode et al., 1980).
A suitable enhanced promoter for expression regulating sequence
(c} to drive expression of the AK enzyme encoding sequence (a) according
to the invention is the enhanced cauliflower mosaic virus (CaMV) 35S
promoter (Benfey et al., 1990; Pen et al.) 1992). In this promoter the
enhancer sequence is duplicated which turns it into a strong promoter
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which drives expression constitutively throughout the whole plant in all
stages of development. Any other promoter which drives constitutive
expression at a high level can be used.
The promoters for expression regulating sequences _(c) and (d)
may originate from either mono- or dicotyledonous plants. They are
preferably tissue specific but can also be non-tissue specific promoters.
The same tissue-specific promoter in expression regulating sequence (d)
that regulates DHPS (b) expression can be used to drive feedback
insensitive AK expression (a) when fused to an enhancer.
A suitable organ specific regulating sequence (d) to drive
expression of the DHPS enzyme encoding sequence (b) according to the
invention is the tuber-specific class I patatin promoter from SoZartum
tuberosum (Mignery et al., 1988; Wenzler et al., 1989}. Other tuber-
specific promoters can be used to drive the DHPS expression like the
proteinase inhibitor II promoter from potato (Keil et al.) 1989)) or the
cathepsin D inhibitor potato promoter (Herbers et al., 1994). Also other
tissue-specific promoters can be used like the fruit-specific promoter
from tomato polygalacturonidase gene (Grierson et al.) 1986), the seed-
specific phaseoline promoter from bean (Sengupta-Gopalan, 1985) or the
root specific CaMV 35S promoter subdomains (Benfey et al. 1990).
The expression regulating sequences (c) and (d) can both
-- comprise inducible promoters. Preferably the induction stimulus will be
different for the two promoters in order to allow differential expression
of the encoding sequences to which they are operably linked. Other
examples of inducible promoters are the light inducible promoter derived
from the pea rbcS gene (Coruzzi et al., 1984) and the actine promoter
from rice (McElroy et al.) 1990).
The nucleic acid encoding sequences (a) and (b) may be derived
from any suitable source, e.g. from different plant cells or from
bacteria) or may be a synthetic encoding sequence. In a preferred
embodiment, such a sequence (a) encodes an enzyme that is less sensitive
to inhibition by lysine and such a sequence (b) encodes an enzyme that is
less susceptible to inhibition by threonine than the corresponding wild
type plant enzymes. Suitably such sequences are derived from endogenous
organisms. The preferable exogenous sources are bacteria) e.g. E. coZi. A
suitable nucleic acid sequence (a) encoding AK activity is the DNA
sequence of E. cotf mutant LysC gene coding for the isoenzyme AK-III.
which is less sensitive to lysine than the plant enzyme (Boy et al. 19'79;
Cassan et al. ( 1983) . A suitable nucleic acid sequence (b) coding for a
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DHPS enzyme is the dapA gene from E. colt (Richaud et al.) 1983). The
resulting expression product is less sensitive to lysine inhibition than
the plant DHPS enzyme. Naturally sequences encoding mutated plant enzymes
which are less sensitive to feedback inhibition are also suitable
embodiments of sequences that can be used.
By way of example the chimeric double gene construct can thus
consist of two encoding sequences encoding AK and DHPS driven by
different promoters ) both present in the same binary vector ( figure 2 ) .
The AK encoding part of the chimeric gene construct has the strong
{enhanced) cauliflower mosaic virus 35S promoter in the 5' region linked
to the 5' end of the omega DNA sequence; the omega DNA sequence is linked
to the 5' end of the DNA sequence coding for the chloroplast transit
peptide derived from the pea rbcS-3A gene, which is linked to the 5' end
of the coding sequence of the mutant E. colt lysC allele coding for a
desensitized AK-III, which is linked to the 5' end of the octopine
synthase terminator sequence.
The DHPS encoding part of the chimeric gene construct has
basically the same structure, but the strong constitutive promoter of the
AK gene construct is replaced by a weaker tuber-specific potato patatin
{class I) promoter and the lysC gene coding for desensitized AK-III is
replaced by the E. colt dapA gene coding for DHPS.
The invention also relates to an expression vector comprising a
chimeric double gene construct of the invention in any of the embodiments
described.
Transgenic plants containing said double gene construct in
their cells also fa3.1 within the scope of the invention as do parts of
such transgenic plants.
Preferred plants are those with a high water content either in
the plant as such or plant parts with high water content: in the tubers.
leaves, stems, fruit, roots or flowers. Suitable plants are potato
plants) sugar beet plants) grass and plants that are used to provide
Fruit or vegetables for fruit juice or vegetable juice. Examples of
suitable fruit are apple) orange, grapefruit, grape, tomato, mango, ~--
banana) melon, strawberry, cherry) kiwi. Examples of suitable vegetables
are potato and sugarbeet. Any other fruit or vegetable comprising a
similar amount of water is considered suitable.
Preferably the. plant or plant part will be derived from a plant
or plant part normally considered acceptable for consumption in order to
produce lysine and threonine destined for consumption. For economical
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reasons the plants that provide the highest amount of water fraction per
acreage per unit of time at the most economical price for obtaining the
water fraction are preferred. Naturally this will vary per country due to
geographical and climatic influences. The plant can be a plant already
commercially grown or can be a type of plant not commercial hitherto that
can become commercially interesting upon incorporation of the gene
construct according to the invention.
Parts of plants comprising water rich organs capable of
expressing the double gene construct according to the invention form a
preferred embodiment for plant parts as are plant cells that can develop
into such water rich organs for plant cells. Preferred plant parts are
also the water rich organs as such. Suitable organs are flowers) tubers
and fruit and examples have already been discussed elsewhere in this
document.
Plants or plant cells according to the invention produce
concomitantly high levels of threonine and lysine. Plants according to
the invention or plant cells according to the invention overproduce
methionine. Preferably concomitantly at least two amino acids of the
aspartate family are overproduced. Preferably in plants or plant parts
according to the invention the lysine is produced only in specific organs
with a high water content. For plants, plant parts or plant cells
according -to the invention expressing more than ten fold the amount of
free lysine than the wild type preferably the DHPS encoding sequence is
only expressed in particular late developing organs such as tubers, seed,
fruit and flowers. Plant tissue derived from transgenic plants or plant
cells according to the invention also falls within the scope of the
invention.
In another embodiment) the invention provides transgenic plant
cel3s containing or transformed by an expression vector according to the
invention.
Both chimeric gene constructs can be subcloned into expression
vectors, such as the Ti plasmids of Agrobactertum tumefaciens, the
. preferred plasmid being the binary vector pBINPLUS (Van Engelen et al.,
1995).
The expression vector in which the double gene construct is
cloned, is then introduced into plant cells. The introduction can be
realised using any kind of transformation protocol capable of
transferring DNA to either monocytoledonous or dicotyledonous plant
cells. For example: transformation of plant cells by direct DNA transfer
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via electroporation (Dekeyser et al., 1990), via PEG precipitation
(Hayashimoto et al.. 1990) or via particle bombardment {cordon-Kann et
al.) 1990) and DNA transfer to plant cells via infection with
Agrobacterium. The preferred method is via infection of plant cells with
Agrobactertum tume,~aciens (Horsch et al.) 1985; Visser.1991). The
methodology used here in the Example for potato or sugar beet can be used
to improve crop plants of the type discussed above like tomato and apple.
Transformed plants are then selected by resistance to kanamycin
or other antibiotics like hygromycin. Selection for plant-cells or plants
that overproduce any of threonine) lysine and methionine may also be
performed by adding any of threonine, lysine and methionine to the plant
growth media.
Plants containing in their cells both the AK and DHPS
components of the gene construct according to the invention can also be
obtained by crossing a transgenic plant containing only the AK gene
construct (a) with a transgenic plant containing only the DHPS gene
construct (b) with the corresponding expression regulating sequences (c)
and (d) respectively. The separate gene constructs may optionally include
the termination sequences and/or transit sequences as described above for
the chimeric double gene construct.
The examples illustrate the invention but are not to considered
w v -xhe only embodiments. The content of the cited prior art is incorporated
by reference.
Example 1
Construction of the chimeric double gene construct for exD~ession of AK
and DHPS.
DNA isolation) subcloning) restriction analysis and DNA
sequence analysis was performed using standard methods (Sambrook et al.,
1989; Ausubel et al.) 1994).
The chimeric gene containing the lysC gene was constructed by
fusing the enhanced CaMV35S promoter to the chimeric lysC gene. This
chimeric lysC construct contains the DNA fragment of the mutant lysC
allele which is fused at the 5' end to the Q DNA sequence from the coat
protein of tobacco mosaic virus (Gallie et al., 1989). Downstream of the
lysC sequences the termination signal of the octopine synthase gene from
Agrobacterium tumefac~ens is inserted. (Greve et al., 1983). A DNA
fragment containing the pea rbcS-3A transit peptide coding sequence
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(Fluhr et al.. 1986) was inserted between the il DNA and the lysC coding
sequence. The chimeric lysC gene construct was cloned as a BamHI/Spel
fragment in pBluescript (Stratagen). The enhanced CaMV35S promoter
(Benfey et al. 1990) was ligated as a SmaI/BamHI fragment in front of the
lysC chimeric gene digested with BamHI/Xbal in the Xbal/SmaI fragment of
the binary vector pBINPLUS (Van Engelen et al., 1995) (pAAP30; figure
2A).
The chimeric gene containing the DapA gene was constructed by
fusing the enhanced CaMV35S promoter to the chimeric DapA gene. This
chimeric DapA construct contains the DNA fragment of the E. cola DapA
gene (Richaud et al. 1986) which is fused at the 5' end to the Q DNA
sequence from the coat protein of tobacco mosaic virus (Gallie et al.,
1989). Downstream of the DapA sequences the termination signal of the
octopine synthase gene from Agrobacterium tumefacferes is inserted (Grave
et ai., 1983). A DNA fragment containing the pea rbcS-3A transit peptide
coding sequence (Fluhr et al., 1986) was inserted between the i2 DNA and
the DapA coding sequence. The chimeric DapA gene construct was cloned as _
a BamHI/SpeI fragment in pBluescript (Stratagen}. The patatin promoter
(Wenzler et al.) 1989) was ligated as a blunt (HindIII filled in)/BamHI
fragment in front of the DapA chimeric gene digested with SmaI/BamHI
(pAAP3l; figure 2B).
The double gene construct was realized by ligating the patatin
dapA chimeric gene as a Kpnl/Sacl fragment from pAAP31 into the binary
vector with enh. CaMV35S-LysC (pAAP30} digested with Saci/Kpnl (pAAP50;
figure 2C).
Example 2
Introduction of the ch~~,er.j~~enes into potato
2.1 Transformation of potato plants
The binary vector pAAP50 was used for freeze-thaw
transformation of Agrobacterium tumefaciens strain AGLO (HSfgen and
Willmitzer, 1988). Transformed AGLO was subsequently used for inoculation
of diploid potato (Sotanum tuberosum, variety Kardal) stem explants as
described by Visser (1991). After shoot and root regeneration on
kanamycin-containing media plants were put in soil and transferred to the
greenhouse. Plants regenerated (on kanamycin-free media) from stem
explants treated with the Agrobacterium strain AGLO lacking a binary
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16
vector served as a control.
2.2 In vitro tuber formation
In order to induce in vitro tuberization) nodal cuttings (about
4-5 cm long) of transformed potato plantlets were placed vertically in
solid Murashige and Skoog (1962) medium supplemented with 10~ (w/v)
sucrose, 5 uM BAP. The cultures were maintained at 19°C in the dark.
After 14 days microtubers of 4 mm in diameter were harvested and analyzed
for free lysine and threonine concept) and for AK and DHPS activity.
Example 3
r n' m
potato plant
Tissue {0.5-1.0 gram) was homogenized with mortar and pestle in
2 ml 50 mM Pi-buffer (pH 'j.0) containing 1 mM dithiothreitol. Nor-leucine
was added as an internal standard. Free amino acids were partly purified
by extraction with 5 ml of a water: chloroform: methanol mixture (3:5:12).
Water phase was collected and the remaining re-extracted twice. After
concentration by lyophilization to 3 ml, a 20 ul sample was used in a 6
aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization
reaction. Derivitized amino acids were analyzed by DHPS on a reversed
phase C18 column according to the manufactures instructions {Waters
AccQ.Tag system).
Example 4
Analysis of AK activity in transeenic potato plants
Microtubers, leaves or mature tubers were harvested and
homogenized with mortar and pestle in an equal volume of cold 20 mM
--- potassium-phosphate buffer, pH 7.0, containing 30 mM f3-mercaptoethanol)
0.1 mM L-lysine, 0.1 mM L-threonine) 1 mM phenylmethylsulfonylfluoride
and 0.5 ug/ml leupeptin. Following 5 min of centrifugation (16,000 g.
4°C)) the supernatant was collected and its protein concentration
determined. Equal amount of protein were then tested for AK activity as
described by Black and Wright (1955). The assay mixture (final volume
of -0.25 ml) contained 300 ug of protein, 100 mM tris-HC1) pH 8.0, 200 mM
NH20H) neutralised prior to use with KOH) 10 mM ATPm 5 mM MgCl2) and 30 mM
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1'7
L-aspartate. The control assays contained all the ingredients except L-
aspartate. The reactions were performed at 3'7°C for 1 h and then
terminated by addition of 0.25 ml of a 1:1:1 solution of 10 (w/v) FeCl3
in 0.1 N HC1) 3N HC1 and.l2x TCA mixed prior to use. Following 5 min on
ice, the reaction mixture was centrifuged (5 min 16,000 g, 4°C and the
light absorbance of the supernatant at 490 nm was measured. For each
sample, the non-specific activity obtained in the absence of L-aspartate
was substracted from that obtained in its presence.
io Example 5
Analysis of DHPS activity in trans~genic potato tubers
Microtubers -ar mature tubers were-~-homogenized with mortar and
pestle in an equal volume of cold. 100 mM tris-HC1 pH 7.5 containing 2 mM
EDTA, 1.4x sodium ascorbate) 1 mM phenylmethylsulphonilfluoride and 0.5
pg/ml leupeptin. Following 5 min centrifugation (16.000 g~at 4°C) the
supernatant was collected. DHPS activity was measured using the 0-
aminobenzaldehyde (0-ABA) method of Yugari and Gilvarg (1965).
Example 6
Construction of a double eene construct for transformatir,r, of sugar beet
byr di rect sane transfer
In order to introduce the chimeric double construct into sugar
beet, the chimeric genes had to be inserted into a plasmid vector
specific for direct- gene transformation of sugar beet. The plasmid pPGS
( Hall et al . , 1996 ) containing a pat gene which encodes phosphinothricin
acetyl transferase was digested with Hind III, filled in and digested
with Sacl. The binary vector pAAP50 (figure 2B) was digested with Xbal)
filled in and digested with Sacl. The blunt/Sacl fragment containing the
double construct was isolated and ligated to the blunt/Sacl fragment of
the pPGS plasmid (pAAP60; figure 2D).
Example 7
Introducti on of the chimer~.c ee-n_Ps into sugar beet
Shoot cultures of a diploid sugar beet (Beta vutgarts L.)
breeding line (Bv-NF) Hall et al.) 1993) were used for transformation)
according to a protocol which was described before (Hall et al. , 1996) .
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In this protocol a polyethylene glycol-mediated DNA transfer technique is
used which is applied to protoplast populations enriched specifically for
a cell type derived from stomatal guard cells. Stably transformed cells
were selected on medium supplemented with 200 pl/1 bialaphos for 4
weeks. Bialaphos resistant calli were subsequently subcuitured on non-
selective medium) and after shoot and root generation plants were put in
soil and transferred to the greenhouse.
Example 8
~,~,ysis of free lysine. threonine and methionine in transgenic sue~g~
beet plants
Free amino acid levels were analysed in leaf and tap root tissue from
transgenic sugar beet plants according to the protocol described in
example 3.
Figure description
fig. 1: A diagram of the aspartate family biosynthetic pathway. Only
the major key enzymes snd their products are indicated. Curved
arrows represent feedback inhibition by the end product amino
acids. DHPS, dihydrodipicolinate synthase; HDS, homoserine
dehydrogenase; TDH, threonine dehydratase.
fig. 2A = construct pAAP30
fig. 2B = construct pAAP31
fig. 2C = construct pAAP50
fig. 2D = construct pAAP60
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