SK65299A3 - A chimeric double gene construct, expression vector, transgenic plant or its parts with a naturally high water content overproducing at least two amino acids of the aspartate family and method to obtain such a plant - Google Patents

Abstract

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.

Description

Chimeric construction of two genes, an expression vector, a transgenic plant or parts thereof with a naturally high water content that overproduce at least two amino acids of the aspartate family and a method for obtaining such a plant

Technical field

The invention relates to the chimeric construction of two genes, which makes it possible to achieve increased production of lysine and threonine and an increased amount of methionine without damaging the plant in which the construction is expressed.

BACKGROUND OF THE INVENTION

People and animals with a single stomach are unable to synthesize 9 of the 20 amino acids and therefore need a dietary source of these essential amino acids. Food for humans and breeding animals is largely based on plant material. Essential amino acids that are an indispensable part of human and animal nutrition include lysine and threonine. However, in plant crops, these amino acids often occur at low concentrations. Therefore, synthetic amino acids are often added to foods based on grain and vegetables to increase their nutritional value.

In the past, they have attempted to increase the amount of free threonine and lysine in plants by classical breeding methods and by selection of mutants, but not much success has been achieved.

Also, scientists have attempted to simultaneously increase threonine and lysine production in one transgenic plant by molecular gene modification. An increase in the amount of free lysine has been shown to cause an increase in free threonine accumulation (Shaul, O. and Galili, G. (1993) Plant Mol. Biol. 23: 759-768; Falco SC et al., (1995), Bio / Technology 13 : 577-582.

To date, increased production of methionine has not been described.

Disclosed is a chimeric construction of two genes comprising a nucleic acid sequence encoding an enzyme having aspartic kinase (AK) activity and a nucleic acid sequence that encodes an enzyme having dihydrodipicolinate synthase (DHPS) activity. This construct is capable of producing differential expression of the two genes, resulting in an increase in the amount of both lysine and threonine by more than 5-fold compared to the amount of each said amino acid in the wild-type plant or in part thereof. This construction also makes it possible to increase the possible amount of methionine. Said expression is now possible without creating earlier problems associated with high lysine accumulation or combined expression of both AK and DHPS genes in the plant. Expression is regulated so that lysine and threonine are produced to a comparable extent without damaging the plant, i.e., there is no negative phenotype aberration compared to wild-type plants. A construction that provides increased amounts of lysine and threonine also makes it possible to achieve increased amounts of methionine.

Biosynthesis of amino acids of the aspartate family.

Essential amino acids such as lysine, threonine and methionine are synthesized from aspartate in a complex manner that is similar for bacteria and higher plants (Fig. 1). The synthesis of the aspartate family has been characterized in detail in Escherichia coli by isolation of enzymes involved in said pathway. These enzymes were later isolated from higher plants (Bryan, JK (1980), The Biochemistry of Plants (ed. BJ Miflin) Vol. 5: 403-452, Academic Press, NY; Umbarger, HE (1978) Ann. Rev. Biochem. 47: 533-606).

The rate of amino acid synthesis of the aspartate family is regulated primarily by a complex process of inhibiting the activity of some of the feedback enzymes by the relevant amino acid end product. First, the enzymatic activity that is common to all amino acids of the aspartate family, which is aspartate kinase (AK) activity, is inhibited by lysine and threonine feedback. In addition, lysine also inhibits the activity of the enzyme dihydrodipicolinate synthase (DHPS), which is the first enzyme of the branching pathway that leads to lysine synthesis. Threonine inhibits the activity of homoserine dehydrogenase (HSD), the first enzyme involved in threonine biosynthesis (Matthews, B. F. et al., (1989) Plant Physiol. 91: 1569-1574).

The enzyme aspartate kinase (AK) catalyzes the phosphorylation of aspartate to form 3-aspartyl phosphate, thereby hydrolyzing ΑΤΡ. Several different AK enzymes have been identified in E. coli and in plants that differentially inhibit either lysine or threonine. AK-III, a product of the lysC locus in E. coli, has been shown to consist of two of the same subunits as the homodimer (Cassan,

M., et al. (1986) J. Biol. Chem. 261: 1052-1057. Richaud, C. et al., (1973) Eur. J. Biochem. 40: 619-629). The LysC gene from E. coli was cloned and sequenced (Cassan, M., et al. (1986), J. Biol., Chem. 261: 1052-1057).

The product of the activity of AK, which is 3-aspartyl phosphate, is converted in the next enzymatic step to 3-aspartate semialdehyde (3-ASA), which serves as a conventional substrate for the synthesis of lysine and threonine. The enzyme dihydrodipicolinate synthase (DHPS) catalyzes the first reaction, which is unique in biosynthesis, by condensing 3-aspartate semialdehyde with pyruvate to form 2,3dihydrodipicolinate. In E. coli, this enzyme is encoded by the dapA locus and appears to contain four identical subunits as the homotetramer (Shedlarski, JG and Gilvarg, C. (1970) J. Biol. Chem. 245: 1362-1373). The E. coli dapA gene was cloned and sequenced (Richaud, F. et al., (1986) J. Bacteriol. 166: 297-300). The DHPS enzyme in plants is the only enzyme comparable to that of E. coli. For the major regulatory enzymes of the aspartate family pathway in plants, DHPS is most sensitive to feedback inhibition by its end product (I ₅₀ DHPS for lysine ranges between 10 and 50 μΜ). Plant DHPS is compared in plant IF (I _50, in the case of lysine the range from 100 to 700 μΜ) about 10-fold more sensitive to inhibition by lysine. Plant DHPS is approximately 100 times more sensitive to lysine inhibition than DHPS of E. coli (I ₅₀ is approximately 1 mM) (Yugari, Aby and Gilvard, C. (1962) Biochem. Biophys. Acta 62: 612-614; Galili G., (1995) The Plant Cell 7: 899906).

Homoserine dehydrogenase (HSD) catalyzes the first reaction, which is specific for the synthesis of threonine, methionine and isoleucine. Higher plants generally have at least

two forms HSD: form, which is a sensitive to threonine and form, which on it sensitive not is a (Bryan, J. K. (1980) , The Biochemistry of Plants (Ed. B. J. Miflin) Vol. 5: 403-452

Academic Press, N.Y .; Lea, P.J. et al. (1985) Chemistry and Biochemistry of the Amino Acids (edited by G. C. Barrett): 197-226, London: Chapman and Hall.

Several evidence shows that plant AK is an enzyme that limits the rate of threonine synthesis, while DHPS is a major enzyme that limits the rate of lysine synthesis. Mutants of several plant species that show a feedback-insensitive AK isozyme have been found to overproduce free threonine and show only a slightly increased amount of lysine (Bright,

S. W. J. et al., (1982) Nature 299: 278-279; Cattoir-Reynaerts

A. et al., (1983) Biochem. Physiol. Pflanzen 178: 81-90; Dotson, S. B. et al., (1990) Planta 182: 546-552; Frankard, V. et al. (1992) Plant Physiol. 99: 1285-1293). On the other hand, feedback-insensitive DHPS, a tobacco plant mutant, showed excessive lysine production (Negrutiu, I. et al., (1984) Theor. Appl. Genet. 6: 11-20). Similar results were recorded with transgenic plants that express feedback-insensitive DHPS or AK from E. coli (Glassman, KF (1992) Biosynthesis and Mol. Regul. Of Amino Acids in Plants (ed. BK Singh et al.): 217- 228, Peri, A. et al., (1992) Plant Mol Biol 19: 815-823, Shaul, O, and Galili, G. (1992a) Plant J. 2: 203-209; and Galili,

G. (1992b) Plan. Physiol. 100: 1157-1163). Transgenic plants that express E. coli AK produce excessively threonine and show only a slight increase in lysine. Transgenic plant studies also show that AK and DHPS are not only regulated by feedback inhibition, but that a number of these enzymes also limit the rate of threonine and lysine production. Substantially positive correlation in transgenic plants was detected between the amount of bacterial enzymes AK and DHPS and the amount of free threonine and lysine (Shaul, O. and Galili, G. (1992a) Plant J. 2: 203-209;

Shaul, O. and Galili, G. (1992b) Plant. Physiol. 100: 11571163).

Transgenic plants that express both feedback-insensitive AK and feedback-insensitive DHPS (Shaul, 0. and Galili, G. (1993) Plant Mol. Biol. 23: 759-768) contain an amount of free lysine that is much higher than that found in plants that express only the introduced insensitive AK or DHPS. This increase in lysine is also accompanied by a substantial reduction in threonine accumulation, which is compared to transgenic plants that express only insensitive AK. That is, when DHPS becomes unregulated, the branch that leads to lysine synthesis strongly competes with other branches of the synthesis pathway and a substantial amount of 3-aspartate semialdehyde is thus converted to lysine at the expense of threonine. The same results were obtained by crossing a lysine overproducing mutant with a threonine overproducing mutant characterized by altered DHPS and AK regulation (Frankard, V. et al. (1992) Plant Physiol. 99: 1285-1293).

European patent application no. 485 970 discloses a method for increasing free threonine and lysine. The method comprises introducing a first chimeric gene into plant cells, wherein the gene comprises a DNA sequence encoding an enzyme having AK activity, and introducing a second chimeric gene comprising a DNA sequence encoding an enzyme having DHPS activity. Both chimeric genes further contain a DNA sequence that allows the expression of enzymes in plant cells and subsequent targeting of the enzymes to the chloroplast.

European patent application no. No. 93908395 discloses two isolated DNA fragments that contain a fragment encoding AK that is not susceptible to lysine inhibition and a second fragment encoding DHPS that is at least 20-fold less susceptible to lysine inhibition compared to plant DHPS. The claims include the fact that lysine sensitive AK causes threonine production higher than normal and that DHPS causes lysine production higher than normal in transformed plants.

However, it has been shown that transgenic plants expressing feedback-insensitive DHPS that originate from E.

coli, overproduce lysine and that transgenic plants expressing AK from E. coli overproduce threonine (Glassman,

K.F. (1992) Biosynthesis and Mol. Regul. Of Amino Acids in Plants (ed., B. K. Singh et al.): 217-228; Perí, A. et al. , (1992) Plant Mol. Biol. 19: 815-823; Shaul, O. and Galili, G. (1992a) Plant J. 2: 203-209; Shaul, O. and Galili, G. (1992b) Plant. Physiol. 100: 1157-1163), then combining these two genes in transgenic plants never resulted in a comparable increase in the amount of threonine and lysine. In Shaul, 0 and Galili, G. (1993) Plant Mol. Biol.

23: 759-768, it is reported that the authors obtained transgenic plants that express both feedback insensitive AK and feedback insensitive DHPS by crossing transgenic plants that express each of these enzymes individually. These plants have been shown to contain an amount of free lysine that does not by far exceed the amount in plants that express only insensitive DHPS. However, the increase in lysine is accompanied by a substantial reduction in threonine accumulation compared to plants that express only insensitive AK.

The same results were obtained when the feedback insensitive bacterial enzymes DHPS and AK, which are encoded by the Corynebacterium dapA gene and the E. coli mutant lysC gene, were co-expressed in the transgenic canola and soybean seeds. In the seeds, a several fold increase in the amount of free lysine was observed, while the accumulation of excess threonine in transgenic seeds that express feedback-insensitive AK alone was prevented by expression with

DHPS (Falco S. C. et al., (1995), Bio / Technology 13: 577-582).

Furthermore, in transgenic plants, where the concentration of free lysine increased throughout the plant relative to the concentration of lysine in plants, an abnormal phenotype more than 10-fold untransformed occurred (Glassman, K. F.

(1992) Biosynthesis and Mol. Regul. Of Amino Acids in Plants (eds. B.K. Singh et al.: 217-228; Shaul, O. and Galili, G.

(1992a) Plant J. 2: 203-209; Frankard, V. et al. (1992) Plant

Physiol. 99: 1285-1293).

In European patent application no. EP 435970 describes that expression of both feedback-insensitive AK and DHPS genes was driven by the same constitutive CaMV35s promoter. Also in the experiments described in Flaco S. C. et al., (1995), Bio / Technology 13: 577-582; Shaul, O. and Galili, G. (1993) Plant Mol. Biol. 23: 759-768, feedback-insensitive AK and DHPS were co-expressed in transformed plants, and the expression of the two genes was controlled by the same promoter.

Problems arising from the use of gene manipulation technology to increase the amount of free lysine and threonine in plants are described in Falco S. C. et al., (1995) Bio / Technology 13: 577-582. It has also been shown that the use of the same seed-specific promoter in controlling the expression of feedback-insensitive AK and DHPS only resulted in an increase in lysine content in canola and soybean seeds. It is further described that the seeds of transformants which accumulate the highest amount of lysine exhibit an abnormal appearance

and little germination (described in (1995), Bio / Technology 13: 577-582). Falco S. C. et al., SUMMARY OF THE INVENTION The following hypothesis is submitted on the the findings listed above Articles. ratio between synthesis

lysine and threonine in plants are regulated by at least two factors: The first factor is the availability of 3-ASA, which is a common substrate for two key enzymes specific for threonine and lysine synthesis (respectively, homoserine dehydrogenase and DHPS). The second factor is the competition between the two key enzymes in the case of 3-ASA, which is a common substrate for them. The amount of 3-ASA appears to be determined by AK activity. If feedback-insensitive AK is overexpressed in transgenic plants, a higher concentration of 3-ASA can lead to the threonine synthesis branch, which may also lead to an increase in the amount of methionine. If DHPS becomes feedback insensitive, then the branch that leads to lysine synthesis competes strongly with another strand of the pathway and a substantial amount of 3-ASA is converted to lysine at the expense of threonine synthesis. In order to increase the amount of free threonine and lysine in the transgenic plants to the desired amount, the timing and expression level of each introduced feedback insensitive gene must be strictly regulated. Production of an amino acid other than the methionine of the aspartate pathway will also increase.

(·

With respect to the invention, the expression of a nucleic acid sequence encoding an enzyme having an aspartic kinase (AK) activity and a nucleic acid sequence encoding an enzyme having an activity of dihydrodipicoline synthesis (DHPS) is regulated such that the expression of the two sequences differs in potency and coding DHPS. It can vary in strength or even

AK is stronger than the sequence achieved by using a promoter using the same promoters, wherein the DHPS regulatory promoter comprises an enhancer. The invention also describes the formation of plants that overproduce threonine and lysine to a comparable extent. Excessive methionine production is described below. This can be achieved using special sequences of the invention by recombinant DNA methods well known in the art, such as plant cell transformation.

It is preferred that expression of the DHPS and AK coding sequences is regulated at various stages of plant development such that the plant has a chance to develop and mature without interfering with overproduced free amino acid, particularly overproduced lysine. This can be achieved in a number of ways.

The first method is to regulate the DHPS coding sequence by an inducible promoter that can be induced after the AK coding sequence has been expressed. The AK coding sequence can also be naturally regulated by an inducible promoter. However, there is a limitation that a stronger expression system is used for the AK coding sequence and that DHPS expression occurs at a later stage than the AK coding sequence. In particular, it is preferred to induce the DHPS coding sequence when the plant has reached a sufficient degree of maturity and will therefore not be negatively affected by excessive lysine production.

A second way to achieve expression at different stages is to regulate expression by promoters that are associated with cell types or organs and direct natural expression at different stages of plant development. Suitable examples of promoters for the DHPS coding sequence are those associated with processes that occur at later stages of plant development or even at the stage of maturity. Promoters associated with fetuses and tubers are useful in regulating the expression of the DHPS coding sequence. The AK coding sequence can also be regulated by the aforementioned promoters, or is limited by using a stronger expression system for the AK coding sequence than the DHPS coding sequence. It is also preferred an embodiment of the invention wherein the DHPS coding sequence is expressed later than the AK coding sequence, which may be accomplished using an inducible promoter.

In order to control the feedback-insensitive AK expression throughout the plant and at all stages of organ development, a very strong constitutive promoter can be used, resulting in a higher concentration of 3-ASA, which leads to the threonine synthesis branch and causes methionine. This procedure is insensitive to later overproduction of threonine and further combined with DHPS may express development, for example, in specific organs

Expression of the AK coding sequence may be constitutive, but may also be limited to a specific brain enzyme stage that feedback only weakly and in the plant.

authorities.

The AK coding sequence may be expressed in the same organs as the DHPS coding sequence, but both coding sequences must be expressed separately. Since DHPS expression occurs later and is weaker, the branch that leads to lysine synthesis is subject to competition with other branches of the synthesis course only weakly and only during a well defined development period. Therefore, both lysine and threonine are overproduced. Methionine, which participates in the aspartate course, is also overproduced.

It is preferred, in order to achieve a very high amount of lysine, for example ten times or more than normal, that the DHPS coding sequence be expressed in specifically targeted organs, thereby avoiding negative manifestations of the plant phenotype. Such target organs that develop later in the plant at the time of ripening are tubers, seeds, fruits and flowers. Since lysine is overproduced only in specifically targeted organs of plants, such as tubers, the remainder of the plant develops normally without the appearance of another phenotype.

Preferably, the expression of the DHPS coding sequence occurs in specific plant organs with a high water content. This is also advantageous because there is minimal phenotype damage. Such organs include tubers, fruits, flowers and leaves. It is preferred that these organs contain more than 70% water, more preferably the water content is higher than 80%. The fact that the DHPS coding sequence is expressed in specific bodies with high water content also has the advantage that the desired amino acids are readily soluble in the aqueous fractions and are therefore readily available after harvest. Therefore, it is preferable that the gene encoding AK also express in plant organs with a high water content. Most preferably, the two genes are expressed with an overlap region of expression found in a high water plant organ in which the DHPS coding sequence is expressed. It is preferred that the harvesting of the water-rich organ is readily practicable. Such organs are, for example, tubers, flowers, roots or leaves.

The organ may be used as a source of nutrients having a high content of at least two amino acids of the aspartate family selected from the group consisting of methionine, lysine and threonine, or may be subjected to extraction to obtain an aqueous fraction. Said aqueous fraction contains a high content of at least two amino acids of the aspartate family selected from the group consisting of methionine, lysine and threonine. The plant itself or a portion thereof containing a water-rich organ may be harvested and used as a food or food supplement and / or processed in a water extraction process. Such an aqueous fraction obtained from the plant or plant organ of the invention may be substantially used as a source of nutrients. A number of simple, economically advantageous extraction processes are known, for example in the production of fruit or vegetable juices. Thus, fruit or vegetable juices can be produced in a manner well known in the art. However, these fruit or vegetable juices have a substantially high nutritional value due to the presence of a high content of at least two amino acids of the aspartate family selected from the group consisting of methionine, lysine and threonine.

Plants or parts of plants, for example, fruits, flowers, tubers, roots or leaves of the invention as such are particularly suitable for vegetarians, thereby ensuring the automatic administration of the two essential amino acids. In addition, an aqueous fraction of plants or parts thereof containing water-rich organs expressing a combination of genes of the invention is an easily obtainable source with a high content of at least two aspartate family amino acids selected from the group consisting of methionine, lysine and threonine; amino acids produced in excess are readily obtainable. The increased amount of methionine is primarily an easily obtainable source of methionine. It is now easy to obtain methionine, lysine and threonine.

The invention relates to a chimeric construction of two genes comprising:

(a) a nucleic acid sequence encoding an enzyme having an aspartic kinase (AK) activity, (b) a nucleic acid sequence encoding an enzyme having a dihydrodipicolinate synthase (DHPS) activity, (c) a sequence that regulates the expression of the coding sequence (d) described in (a) a sequence operably linked to said sequence that regulates the expression of the coding sequence of the sequence shown in (c) and (d) are those that regulate expression that the expression of the two sequences differs operably in (b) and the expression of the coding sequence (AK) (a) occurs at a higher rate compared to the DHPS coding sequence (b).

That is, (c) is stronger than (d). In addition, the sequences of (a) and (b) of the chimeric double gene construct must each contain a nucleic acid sequence (e) encoding a transit peptide involved in translocating the protein from cytosol to plastids (Van den Broek, G. et al., (1985) Nature 313: 358-363; Schreier, PH et al., (1985) EMBO J. 4: 25-32), which is an organelle in which the synthesis of lysine and most of the threonine occurs in plants. This nucleic acid sequence encoding the chloroplast transit sequences (a) and (b) of the transformed plant peptide fused to the coding will produce a fused chimeric protein in the cytoplasm of the cell

AK / transit peptide or DHPS / transit peptide that will be transported to plastids where increased production of both threonine and lysine is obtained. A nucleic acid sequence encoding any plastid transit peptide can be used according to the invention. Suitable examples are DNA sequences derived from the feredoxin gene that encode protein targeting into the chloroplast tree (Smeekens, S. et al., (1985a) Nucl. Acids Res. 13: 3179-3194) and sequences from the plastocyanine gene that encode protein targeting into the chloroplast tree. chloroplast lumen (Smeekens, S. et al., (1985b) Nature 317; 456-458).

A preferred DNA sequence encoding the targeting of AK and HDPS to plastids encodes a transit peptide that originates from the pea gene rbcS-3A (Fig. 2; Fluhr, R. et al., (1986) EMBO J. 5:

2063-2071). The 3'-end of a nucleic acid sequence encoding a transit peptide is fused to a sequence encoding AK, or

DHPS.

Conversely, sequences encoding AK and DHPS (a) and (b) must also be fused to a DNA transcription termination signal (g) and (h). This termination signal contains a 3 'transcription termination signal and mRNA polyadenylation. The signals present in the region adjacent to the cloned gene, e.g. from the pea rbcS gene, from the termination

3 ¹ -terminal bean phaseolin gene, or the nopaline synthase gene derived from Ti plasmid of Agrobacterium tumefaciens. Preferably, the termination sequence originates from the region flanking the 3 ¹ -terminus of the octopine synthase gene from Agrobacterium tumefaciens Ti plasmid (Figure 2; Greve, HD et al., (1983))

J. Mol. Appl. Genet. 1: 499-511).

It is preferred that the gene construct of the invention comprises a sequence that regulates expression (d) for the nucleic acid sequence (b), wherein the regulatory sequence allows DHPS expression at a later stage during development as compared to expression of the nucleic acid sequence (a). In an alternative embodiment of the invention, the sequence that regulates expression (d) is a sequence that does not result in constitutive expression. Preferably, a sequence that results in expression in one or more specific plant organs is used. Examples are tubers, flowers, fruits, roots or stems. There are sequence preferences that regulate expression in water-rich organs.

The sequence that regulates expression (c) for the nucleic acid sequence (a) may be a sequence that is capable of strongly expressing the coding sequence (a) at an early stage in the development of the plant and / or the plant species. The sequence that regulates expression (c) for the nucleic acid sequence (a) may be a sequence that is capable of expressing strongly the coding sequence (a) in all plant cells or plant organs. A sequence that regulates expression (c) is a suitable constitutive promoter. Such a constitutive promoter may be a cauliflower mosaic virus (CaMV) 35S promoter.

A sequence that regulates expression (c) may contain not only a promoter but also a sequence that enhances expression. If this is the case of a sequence that regulates expression (d), it may contain the same promoter or a promoter with the same potency as the sequence that regulates expression (c), the expression levels of coding sequences (a) and (b) being different.

Sequences that regulate expression, particularly promoters, can be located in the 5'-region of each of the coding sequences (a) and (b). They can be added to the 3'-end of the promoter short nucleic acid sequence, representing the 5'-mRNA untranslated sequence. Translation of mRNA transcribed from chimeric genes will be enhanced. An example is the omega sequence derived from the tobacco mosaic virus coating protein gene (Gallie, D. R. et al., (1987) Nucl. Acids Res. 15:

3257-3273) or mosaic virus alpha-alpha translational enhancers (Brederode, F. T. et al., (1980) Nucl. Acids Res. 8: 2213-2223).

A suitable enhancer promoter for the expression control sequence (c) that directs the expression of the AK (a) coding sequence of the invention is the cauliflower mosaic virus (CaMV) 35S promoter (Benfey, PN et al., (1990) EMBO J. 9: Pen, J. et al (1992)

Bio / Technol. 10: 292-296). In this promoter, the enhancer sequence is twice, making it a potent promoter that drives expression constitutively throughout the plant at all stages of development. Any other promoter that drives strong constitutive expression can be used.

Promoters for sequences that regulate expression of (c) and (d) may originate from either monocotyledonous or dicotyledonous plants. It is preferred that tissue-specific promoters be used, but need not be tissue-specific promoters. To control the feedback-insensitive AK (a) expression, the same tissue-specific promoter can be used in an expression control sequence (d) that regulates DHPS (b) expression when fused to an enhancer.

A suitable organ-specific regulatory sequence (d) for controlling expression of the DHPS coding sequence (b) of the invention is a tuber-specific class I patetin promoter derived from Solanum tuberosum (Mignery, CA et al. (1988) Gene 62: 27-). 44; Wenzler, HC et al., (1989) Plant Mol. Biol. 12: 41-50). Other tuber-specific promoters may be used to control DHPS enzyme expression. It is, for example, the promoter of the proteinase inhibitor II for potatoes (Keil, M. et al., (1989) EMBO J. 8: 13231330) or the cathepsin D inhibitor from potatoes (Herbers, K. et al., (1994) Plant Mol. Biol., 26: 73-83). Other tissue-specific promoters, such as the fruit-specific promoter, derived from the tomato polygalacturonidase gene may also be used (Grierson, D. et al., (1986) Nucl. Acids Res. 14:

8595-8603), a seed-specific phazeolin promoter derived from beans (Sengupta-Gopalan, C. (1985)

Proc. Natl. Acad. Sci. USA 82: 3320-3324) or the root-specific subdomain of the CaMV 35S promoter (Benfey, P. N. et al., (1990) EMBO J. 9: 1685-1696).

Sequences that regulate expression of (c) and (d) may both contain inducible promoters. It is preferred that the inducing stimuli of the two promoters differ so that different expression of the two coding sequences to which it is operably linked may occur. Other examples of inducible promoters are the weak inducible promoter obtained from the pea rbcS gene (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1679) and the actin rice promoter (McElroy, D. et al., (1990) The Plan Cell 2: 163-171).

The coding sequence of nucleic acids (a) and (b) may be derived from any source, for example, from various plant cells or bacteria, or may be a synthetic coding sequence. In a preferred embodiment of the invention, such sequence (a) encodes an enzyme that is less susceptible to lysine inhibition and said sequence (b) encodes an enzyme that is less susceptible to threonine inhibition compared to the corresponding wild-type plant enzymes. Suitably, such sequences are obtained from endogenous organisms. Preferred exogenous sources are bacteria, for example E. coli. A suitable nucleic acid sequence (a) encoding AK activity is the DNA sequence of the E. coli mutant LysC gene that encodes the AK-III isoenzyme. This enzyme is less susceptible to lysine compared to the plant enzyme (Boy, E. et al. (1979) Biochimie 61: 1151-1160; Cassan, M. et al. (1986), J. Biol. Chem. 261: 1052-1057). A suitable nucleic acid sequence (b) encoding DHPS is the dapA gene from E. coli (Richaud, C. et al., (1973) Eur. J. Biochem. 40: 619-629). The resulting expression product is less sensitive to lysine inhibition compared to the plant enzyme DHPS. Sequences encoding mutated plant enzymes that are less sensitive to feedback inhibition are also a suitable embodiment of the invention to sequences that can be used.

Thus, the chimeric construction of two genes may comprise two coding sequences that encode AK and DHPS and which are under the control of different promoters. Both are on the same binary vector (Fig. 2). The AK coding portion of the chimeric gene construct has a 35S cauliflower mosaic virus promoter at the 5'-region associated with the 5'-end of the omega DNA sequence; the omega DNA sequence is linked to the 5'-end of the DNA sequence encoding the chloroplast transit peptide obtained from the pea rbcS-3A gene, which is linked to the 5'-end of the E. coli mutant lysC allele coding sequence that encodes insensitive AK-III; which is connected to ^one 5 -terminal sequence of the octopine synthase terminator.

Part of the chimeric gene construct encoding DHPS has the same structure, with the strong constitutive promoter of the AK gene construct being replaced by the weaker tuber-specific potato-derived promoter and the lysC gene that encodes the insensitive AK-III gene is replaced by the E. coli dapA bacterium coding DHPS.

The invention also provides an expression vector comprising the chimeric construction of the two genes of the invention in any embodiment.

Transgenic plants containing the two genes in their cells are also within the scope of the invention.

Preferred plants are those that have a high water content or parts thereof have a high water content: for example, tubers, leaves, stems, fruits, roots or flowers. Suitable plants include potato, sugar beet, grasses and plants that provide fruit or vegetables for making fruit or vegetable juice. Examples of suitable fruits are apples, oranges, grapefruits, vines, tomatoes, mangoes, bananas, melon, strawberries, cherries and kiwi. Examples of suitable vegetables are potatoes and sugar beets. Any fruit or vegetable that contains a similar amount of water is considered appropriate.

Preference is given to plants or parts thereof derived from plants or plant parts which are normally acceptable for consumption in order to produce lysine and threonine for consumption. For economic reasons, plants that provide the highest amount of water fraction per area per unit of time at the lowest price are preferred to obtain an aqueous fraction. This factor will vary by country depending on climatic and geographical impacts. A plant that is normally grown or not a commonly grown plant can be used, but which becomes of interest after the introduction of the gene construct of the invention.

Plant parts which contain organs rich in genes according to the invention in cells which have a high water content.

capable of expressing the construction of the two form a preferred embodiment of plant parts such as plant develop into plant cell organs

Preferred plant parts are also high water organs per se. Suitable organs include flowers, tubers and fruits.

water and are of the invention if they can

The plants and plant cells of the invention produce high levels of threonine and lysine as a concomitant phenomenon. The plants of the invention or plant cells of the invention produce excessive amounts of methionine. Overproduction of at least two amino acids of the aspartate family is preferred. In the plants or plant parts according to the invention, it is preferred that lysine is produced only in specific bodies with a high water content. In the case of plants, parts thereof or plant cells according to the invention which express more than ten times the amount of free lysine compared to the wild type, it is preferred that the DHPS coding sequence is expressed only in organs that develop in late growth, which are tubers , seeds, fruits and flowers. Plant tissue obtained from the transgenic plants or plant cells of the invention is also within the scope of the invention.

In another embodiment of the invention, transgenic plant cells are described which comprise or are transformed with an expression vector of the invention.

Both of these chimeric gene constructs can be subcloned into expression vectors, such as Ti plasmids of Agrobacterium tumefaciens. A preferred plasmid is the binary vector pBINPLUS (Van Engelen, F.A. et al., (1985) Transgenic Research 4: 288-290).

The expression vector into which the construction of the two genes was cloned is then introduced into plant cells. Said cell transformation can be performed using various protocols that allow the transfer of DNA into cells of monocotyledons or dicotyledons. Transformation of plant cells by direct DNA transfer can be accomplished, for example, by electroporation (Dekeyser, RA et al., (1990) The Plant Cell 2: 591-602), precipitation using PEG (Hayashimoto, A. et al., (1990) Plant Physiol. 93: 857-863) or particle bombardment (Gordon-Kann, WJ et al., (1990) The Plant Cell 2: 603-618). Transfer of DNA to plant cells may be by infection with Agrobacterium. A preferred method is to infect plant cells with Agrobacterium tumefaciens (Horsch, RB et al., (1985) Science 227: 12291231; Visser, RGF (1991) Plant Tissue Culture Manual B5 (ed. K. Lindsey): 1-9, Kluwer Acad). Publishers, The Netherlands). The methods used in the examples for potato and sugar beet plants can be adapted for the above-mentioned plants, such as tomatoes and apple trees.

The transformed plants are then isolated based on resistance to kanamycin or other antibiotics such as hygromycin. Selection of plant cells or plants that overproduce threonine, lysine and methionine can also be performed by adding threonine, lysine and methionine to the plant growth medium.

Plants which contain AK and DHPS components of the gene construct of the invention in their cells can also be obtained by crossing a transgenic plant that contains only the AK (a) gene construct with a transgenic plant that contains only the DHPS (b) gene construct with corresponding sequences which regulate expression of (c) and (d). Separate gene constructs may include termination sequences and / or transit sequences as described above, chimeric constructs of two genes.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph of the aspartate family biosynthetic pathway. Only the major key enzymes and their products are shown in the figure. Curved arrows represent feedback inhibition of the final amino acid product. DHPS stands for dihydrodipicolinate synthase; HDS is homoserine dehydrogenase; TDH is a threonine dehydratase.

Fig. 2a shows the construction of pAAP30.

Fig. 2b shows the construction of pAAP31.

Fig. 2c shows the construction of pAAP50.

Fig. 2d shows the construction of pAAP60.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Construction of chimeric construction of two genes for expression of AK and DHPS

DNA isolation, subcloning, restriction analysis, and DNA sequence analysis were performed using standard methods (Sambrook, J. et al., (1989) Molecular Cloning. A laboratory manual, Cold Spring Laboratory Press; Ausubel. FM et al., (1994) ) Current Protocols in Molecular Biology, John Wiley &

A chimeric gene containing the lysC gene was constructed by fusing the amplified CaMV35S promoter to the chimeric lysC gene. This chimeric lysC construct contains a DNA fragment of the mutant lysC allele that fused to the 5'-end of the DNA sequence of the omega tobacco mosaic virus envelope protein (Gallie, D. R. et al., (1987) Nucl. Acids Res. 15: 3257-3273). The octopine synthase gene termination signal from Agrobacterium tumefaciens was inserted downstream of the lysC sequence (Greve, H. D. et al., (1983) J. Mol. Appl. Genet. 1: 499-511). A DNA fragment containing the sequence encoding the pea transit peptide rbcS-3A was inserted between the omega DNA and the lysC coding sequence (Fluhr, R. et al., (1986) EMBO J. 5: 2063-2071). The chimeric construction of the lysC gene was cloned as a BamHI / SpeI fragment into plasmid pBluescript (Stratagen). The amplified CaMV35S promoter (Benfey, PN et al., (1990) EMBO J. 9: 1685-1696) was ligated as a SmaI / BamHI fragment upstream of the BamHI / XbaI chimeric lysC gene in the XbaI / SmaI fragment of the binary vector pBINPLUS (Van Engelen, FA et al., (1995) Transgenic Research 4: 288-290) (pAAP30; Figure 2a).

The Dap-containing chimeric gene was constructed by fusing the amplified CaMV35S promoter to the DapA chimeric gene (Tat Van Engelen, F. A. et al., (1995) Transgenic Research 4: 288-290). This chimeric DapA construct contains a DNA fragment of the E. coli DapA gene (Richaud, F. et al., (1986) J. Bacteriol. 166: 297-300), which fused to the 5'-end of the omega DNA sequence that originated from tobacco mosaic virus envelope protein (Gallie, DR et al., (1987) Nucl. Acids Res. 15: 3257-3273). The octopine synthase gene termination signal from Agrobacterium tumefaciens (Greve, H. D. et al., (1983) J. Mol. Appl. Genet. 1: 499-511) was inserted downstream of the DapA sequence. A DNA fragment containing the sequence encoding the pea rbcS-3A transit peptide was inserted between the omega DNA and the DapA coding sequence. The chimeric construction of the DapA gene was cloned as a BamHI / SpeI fragment into plasmid pBluescript (Stratagen). The patatin promoter was ligated as a blunt-ended HindIII / BamHI fragment upstream of the chimeric DapA gene digested with the restriction enzymes SmaI / BamHI (pAAP31; Figure 2b).

Two genes were constructed by ligating the chimeric patatin dapA gene as a KpnI / SacI fragment from pAA31 into a binary vector with enhanced CaMV35S-LysC (pAAP30) digested with SacI / KpnI restriction enzymes (pAAP50; Figure 2c).

Example 2

Introduction of chimeric genes into potato plants

Transformation of potato plants

The binary vector pAAP50 was used for the freeze-thaw transformation of Agrobacterium tumefaciens AGLO strain (Hofgen, R. and Willmitzer, L. (1988) Nucl. Acids. Res. 16: 9877). The transformed AGLO was then used to inoculate diploid potato stem expats, antates (Solanum tuberosum, Kardal variety) as described in Visser, RGF (1991) Plan Tissue Culture Manual B5 (ed. K. Lindsey): 1-9 Kluwer Acad. Publishers, The Netherlands. To regenerate the shoots and roots on the kanamycin-containing medium, the plants were placed in the soil and transferred to a greenhouse. Plants regenerated from stem explants (on kanamycin-free medium) lacking the binary vector and to which the Agrobacterium AGLO strain is applied serve as a control.

2.2 In vitro tuber formation

In order to induce tuber formation in vitro, sections containing nodus (approximately 4 to 5 cm long) of transformed plants were placed vertically in solid medium supplemented with 10% (w / v) sucrose, 5 μΜ BAP (Murashige, T.). and Skoog, F. (1962) Physiol. Plant 15: 473-497). The cultures were kept in the dark at 19 ° C. After 14 days, small tubers with a diameter of 4 mm were harvested to determine the content of free lysine and threonine and the activity of AK and DHPS.

Example 3

Analysis of free lysine, threonine and methionine content in transgenic potato plants

The tissue (0.5-1.0 g) was homogenized in a plate with a ml of 50 mM Pi-buffer (pH 7.0) containing 1 mM dithiothreitol. Leucine was not added as an internal standard. The free amino acids were partially purified by extraction with 5 mL of water: chloroform: methanol phase and the residue was lyophilized twice to a volume of 3 mL (3: 5: 12). The water was re-extracted. After concentration, a 20 μΐ sample was used for the 6 aminoquinolyl- N -hydroxysuccinimidylcarbamate (AQC) derivatization reaction. Derivatized amino acids were analyzed by DHPS on a C18 reverse phase column according to the manufacturer's instructions (Waters AccQ.Tag system).

Example 4

Analysis of AK activity in transgenic potato plants

Micro-tubers, leaves or ripened tubers were harvested and homogenized in a platter with an equal volume of cold 20 mM potassium phosphate buffer pH 7.0 containing 30 mM β-mercaptoethanol, 0.1 mM L-lysine, 0.1 mM L- threonine, 1 mM phenylmethylsulfonyl fluoride and 0.5 µg / ml leupeptin. Centrifuge for 5 minutes (16000 g, at 4 ° C). The supernatant was collected and the protein concentration was determined. AK activity was assayed at the same amount of protein as described by Black, S. et al. (1955) J. Biol. Chem. 213: 27-38. The test mixture (final volume 0.25 ml) contained 300 µg protein, 100 mM tris-HCl, pH 8.0, 200 mM NH ₂ OH, which was neutralized with KOH, 10 mM ATPm, 5 mM MgCl ₂ and 30 mM before use. L-aspartate. The control test contained all ingredients except L-aspartate. The reactions were run at 37 ° C for 1 hour and then quenched by the addition of 0.25 mL of a 1: 1: 1 solution of 10% (w / v) FeCl ₃ in 0.1 N HCl, 3 N HCl and 12% TCA. , which was mixed just before use. The reaction mixture is allowed to stand on ice for 5 minutes and centrifuged (for 5 minutes, at 16000 g, at 4 ° C) and the light absorbance of the supernatant is determined at 490 nm. For each sample, the non-specific activity obtained in the absence of L-aspartate was subtracted from the value obtained in the presence of L-aspartate.

Example 5

Analysis of DHPS activity in transgenic potato tubers

Micro-tubers and mature tubers were homogenized in a bowl with a spot in an equivalent volume of cold 100 mM Tris-HCl pH

7.5 containing 2 mM EDTA, 1.4% sodium ascorbate, 1 mM phenylmethylsulfonyl chloride and 0.5 µg / ml leupeptin. Following a 5 minute centrifugation (16000 g at 4 ° C), the supernatant was collected. DHPS activity was measured using the 0 aminobenzaldehyde (O-ABA) method described in

Yugari, Aby and Gilvard, C. (1965) J. Biol. Chem. 240: 47104716.

Example 6

Preparation of construction of two genes for sugar beet transformation by direct gene transfer

In order to introduce the chimeric construct into sugar beet, the chimeric genes were incorporated into a plasmid vector that is specific for the direct transformation of genes into sugar beet. Plasmid pPG5 (described in Hal, RD et al., (1996), Nature Biotechnol. 14: 1133-1138) containing the pat gene that encodes phosphinothricin acetyltransferase was digested with the restriction enzyme HindIII, filled in and further digested with the restriction enzyme. suction. The binary vector pAAP50 (Fig. 2b) was digested with the restriction enzyme XbaI, filled in and further digested with the restriction enzyme SacI. The blunt-ended SacI fragment containing the construct was isolated and ligated into the blunt-ended SacI plasmid pPG5 fragment (pAAP60; Fig. 2d).

Example 7

Introduction of chimeric genes into sugar beet plants

In transformation according to the protocol described by Halí, R. D. et al., (1996) Nature Biotechnol. 14: 1133-1138, shoot cultures of the diploid sugar beet breeding line (Bv-NF, Hali, R. D. et al. (1993) Plant CellRep. 12: 339-342) were used. According to the protocol, a polyethylene glycol-mediated DNA transfection method is used, which is applied to populations of protoplasts specifically enriched with cell-type derived from dental cells. Stably transformed cells were selected on medium supplemented with 200 μΐ / ml biaphalos for 4 weeks. The biaphalos resistant kali were subsequently subcultured on a medium that does not serve for selection, and after the shoots and leaf regeneration, the plants were placed in the soil and transferred to a greenhouse.

Example 8

Analysis of free lysine, threonine and methionine in transgenic sugar beet plants

In the tissues of leaves and roots of transgenic sugar beet plants, the amount of free amino acids was analyzed according to the protocol described above in Example 3.

Claims (28)

PATENT CLAIMS A chimeric construction of two genes which, when introduced into a plant cell, is capable of producing at least two amino acids through the aspartate family pathway, in particular lysine and threonine and / or methionine, said construction comprising: (a) a nucleic acid sequence encoding an enzyme having an aspartic kinase (AK) activity, (b) a nucleic acid sequence encoding an enzyme having a dihydropicolinate synthase (DHPS) activity, (c) (d) an expression control sequence encoding an operably linked one with (a) an expression control sequence encoding operably linked to (b), sequence (a), sequence (b), (e) a nucleic acid sequence encoding a chloroplast transit peptide that is involved in translocating the expression product a), ( f) a chloroplastic encoding nucleic acid sequence the transit peptide that is included in the expression product b), to translocation (G) sequence nucleic acid, which provides termination signal transcription DNA in case transcription (a) (H) sequence nucleic acid, which provides termination signal transcription DNA in case transcription (b) wherein the expression regulating sequences (c) and (d) are such that the expression of the AK coding sequence (a) occurs to a greater extent than the expression of the DHPS coding sequence (b), further regulating the sequences of later sequence (c) and (d) expressing sequence (b) at the plant development stage as expression (a) occurs, and / or sequence (c) comprises a promoter sequence that allows expression in all plant cells or plant organs, and sequence (d) comprises a promoter sequence that is specific to authorities. The gene construct of claim 1, wherein the sequence (d) allows expression in one or more water-rich plant organs, which is a tuber, a flower, a stem, a fetus, a root or a leaf. The gene construct according to claim 2, wherein the sequence (d) comprises a tuber-specific class I patatin promoter derived from Solanum tuberosum, a potato proteinase inhibitor II promoter, a potato cathepsin D inhibitor promoter, or a fruit specific promoter. that comes from the tomato polygalacturonidase gene. The gene construct of any one of claims 1 to 3, wherein sequence (c) comprises a promoter and an expression enhancer sequence, such as an omega sequence derived from the tobacco mosaic virus envelope protein gene or a mosaic virus alpha-alpha translation enhancer, and wherein sequence (c) and (d) may contain the same promoter. by sequence The gene construct of claims 1 to 3, wherein the promoter, in particular the 35S promoter (CaMV). 6. The gene construct of claim 1, wherein the sequence of any one of the preceding (c) comprises a constitutive cauliflower mosaic virus of any one of the preceding (c) and (d) comprising inducible promoters and wherein the induction stimulus differs for the two promoters. The gene construct of claim 6, wherein the inducible promoters are selected from light-inducible promoters. Gene construct according to any one of the preceding claims, wherein the sequence (e) is a nucleic acid sequence obtained from a feredoxin gene encoding a protein targeting to a chloroplast tree, a sequence from a plastocyanin gene encoding a protein targeting to a chloroplast lumen or a transit peptide sequence originating from the pea gene rbcS-3A. The gene construct according to any one of the preceding claims, wherein the coding sequences (a) and (b) have been fused to a DNA transcription termination signal (g), respectively. (h), wherein the termination signals (g) and (h) comprise a transcription termination signal and a mRNA polyadenylation signal. Gene construct according to any one of the preceding claims, wherein the sequence (g) and / or (h) comprises termination signals that are located in the 3'-flanking region of the pea rbcS gene, the bean phazeolin gene, the nopaline synthase gene obtained from Ti Agrobacterium plasmid. tumefaciens, or preferably an octopine synthase gene, which originates from the plasmid Ti A. tumefaciens. The gene construct of any one of the preceding claims, wherein sequence (a) encodes an enzyme that is less sensitive to lysine inhibition and sequence (b) encodes an enzyme that is less susceptible to threonine inhibition as compared to corresponding wild-type plant enzymes. The gene construct according to any one of the preceding claims, wherein the nucleic acid sequences (a) and (b) are obtained from bacteria, wherein in particular (a) is a mutant E. coli LysC gene encoding the AK-III isoenzyme and (b) is a gene dapA from E. coli. The gene construct of any one of claims 1 to 11, wherein the coding nucleic acid sequence of (a) and / or (b) encodes a mutated plant enzyme that is less sensitive to feedback inhibition. 14. An expression vector comprising the chimeric construction of two genes according to any one of the preceding claims. 15. A transgenic plant comprising the construction of two genes according to any one of claims 1 to 13 or the expression vector according to claim 14. 16. The transgenic plant of claim 15, wherein said transgenic plant is capable of providing said two gene construction or elements thereof to the progeny of the plant. Transgenic plant according to claim 15 or 16, characterized in that it has the ability to produce threonine and lysine in an amount of more than five times higher than the production of a wild-type plant under the same culture conditions, preferably the production is more than seven times higher. Transgenic plant according to any one of claims 15 to 17, characterized in that it has the ability to produce methionine in an amount that exceeds that of a wild-type plant under the same culture conditions. Transgenic plant according to any one of claims 15 to 18, characterized in that in a water-rich organ, in particular in a tuber, stem, fruit, root, flower or leaf as compared to. A wild-type plant under the same culture conditions overproduces threonine and lysine. 20. The transgenic plant of claim 19, wherein the water content of said tuber, flower, stem, root, fruit of the plant is at least 80% under normal culture conditions of a non-transgenic plant. Transgenic plant according to any one of claims 15 to 20, characterized in that it is selected from the group comprising potato, sugar beet or grass plants. A plant part according to any one of claims 15 to 21, characterized in that it comprises at least a cell. Part according to claim 22, characterized in that it is obtained from a tuber, a fruit, a root, a stem, a seed or a flower or is a tuber, a fruit, a stem, a seed, a flower or a leaf. An aqueous fraction, which is obtainable by extraction from a plant according to any one of claims 15 to 21 or in part according to claim 22 or 23. 25. A method of obtaining a plant capable of overproducing at least lysine and threonine, the method comprising: introducing the construction of two genes according to any one of claims 1 to 13 or elements (a) to (h) or a vector according to claim 14 in a manner known to introduce nucleic acid sequences into a plant cell, followed by culturing the plant cell on tissue or part of the plant or to a mature plant under conditions where regulatory sequences (c) and (d) are activated. 26. The method of claim 25, wherein the gene construct has been introduced into the plant cell by transformation. 27. The method of claim 25, wherein two plants that cross subfragments of said gene construct are crossed. The method of any one of claims 25 to 27, comprising selecting a portion of the plant or tissue obtained and growing the plant from said tissue or tissue or organs derived from said plant.