Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.     

Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase

The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.     

Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts

A major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein-bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.     

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.     

Seed-specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco

In order to study the regulation of threonine and methionine synthesis in plant seeds, tobacco plants were transformed with a chimeric gene containing the coding DNA sequence of a mutant lysC gene from Escherichia coli fused to a promoter from a phaseolin seed storage protein gene. The bacterial mutant lysC gene codes for aspartate kinase (AK) which is desensitized to feedback inhibition by lysine and threonine. Increased AK activity, compared with control non-transformed plants, was detected in seeds but not in leaves, roots and flowers of the transgenic plants. This expression was accompanied by a significant increase in the levels of free threonine and methionine in the seed. The level of these amino acids also correlated positively with the levels of the bacterial enzyme. No alteration in plant phenotype and 'average seed weight' was observed in any of the transgenic plants, indicating that plant growth and seed development were normal. This study demonstrates, for the first time, that the threonine and methionine biosynthetic pathways are active in plant seeds. Thus, targeting of the production of favorable biosynthetic enzymes to plant seeds may represent a desirable molecular approach for production of crop plants with a more balanced nutritional quality.     

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.     

Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T₀) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T₁ generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0–9.5-fold increase for DHPS. T₁ seeds of DHPS transformants showed the same changes in free amino acids as observed in T₀ seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.     

Metabolic engineering of a complex biochemical pathway: The lysine and threonine biosynthesis as an example

The nutritional quality of crop plants is determined by their content in essential amino acids provided in food for humans or in feed for monogastric animals. Amino acid composition of crop–based diets can be improved via manipulation of the properties of key enzymes of amino acid biosynthetic pathways by mutation and transformation. We focused on the aspartate-derived amino acid pathway producing four essential amino acids: lysine, threonine, isoleucine and methionine. Genes encoding aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) that operate as key genes of the aspartate pathway have been cloned from Arabidopsis. Genetic and molecular studies revealed that at least five different ak genes are represented. Some of them were characterized in terms of gene and promoter structure, developmental expression and regulatory properties. In the case of dhdps, two quite identical genes have been identified and characterized at expression level. Mutated genes encoding a fully feedback-insensitive form of the DHDPS enzyme were obtained from Nicotiana sylvestris and Arabidopsis. Several chimeric constructs harbouring this mutated allele under the control of constitutive or seed-specific promoters were transferred via Agrobacterium or biolistics in various plant species. In all cases, lines with significant increase of free lysine content were obtained in vegetative organs, but the impact of the transgene in seeds is limited due to the presence of an active catabolic enzyme, lysine ketoreductase. These results show that, although dealing with a complex, highly regulated pathway, the overexpression of a single gene encoding a feedback-insensitive form of the key enzyme DHDPS exerts a significant effect on the carbon flux through the aspartate pathway towards lysine production.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Selection and characterization of a feedback-insensitive tissue culture of maize

Tissue culture selection techniques were used to isolate a maize (Zea mays L.) variant D33, in which the aspartate family pathway was less sensitive to feedback inhibition by lysine. D33 was recovered by successively subculturing cultures originally derived from immature embryos on MS medium containing growth-inhibitory levels of lysine+threonine. The ability of D33 to grow vigorously on lysine+ threonine medium was retained after growth for 12 months on nonselection medium. New cultures initiated from shoot tissues of plants regenerated from D33 also were resistant to lysine+threonine inhibition. The K_i of aspartokinase for its feedback inhibitor, lysine, was about 9-fold higher in D33 than for the enzyme from unselected cultures. The free pools of lysine, threonine, isoleucine and methionine were increased 2–9-fold in D33 cultures. This was consistent with the observed change in feedback regulation of aspartokinase, the first enzyme common to the biosynthesis of these amino acids in the aspartate pathway. The accumulated evidence including the stability of resistance in the cultures, the resistance of cultures initiated from regenerated plants, the altered feedback regulation, and the increased free amino acids, indicates a mutational origin for these traits in line D33.Abbreviation LT lysine+threonine in equimolar concentration Paper No. 10880, Scientific Journal Series, Minnesota Agricultural Expertment Station     

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes)

Summary Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.Abbreviations AK aspartate kinase (EC 2.7.2.4) - DHPS dihydrodipicolinate synthase (EC 4.2.1.52) - AEC S-(2-aminoethyl)L-cysteine     

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

Lysine and threonine overproducer mutants in Nicotiana sylvestris, characterized by an altered regulation of, respectively, dihydrodipicolinate synthase and aspartate kinase activities, were crossed to assess the effects of the simultaneous presence of these genes on the biosynthesis of aspartate-derived amino acids. The monogenic dominant behavior of both resistance traits was confirmed, and their loci were found to be unlinked. Study of the inhibition properties of dihydrodipicolinate synthase and aspartate kinase activities in RAEC-1 × RLT 70 confirmed the heterozygote state of both mutations, because only half of their lysine-sensitive activity could still be inhibited by this negative effector. Analysis of the free amino acid pool during the growth of the double mutant revealed a major free lysine overproduction reaching up to 50% of the total pool, whereas the other aspartate-derived amino acids remained equally or even less abundant than in the wild type. An abnormal phenotype was clearly associated with such high levels of lysine accumulation, which points out the possible role of this amino acid in the developmental features of the plant. Comparison of the amino acid content, free and total (free + protein-bound), between the wild type, the two mutants, and the double mutant obtained by crossing them brings new insights on the regulation of the aspartate pathway, and on its implications in relationship to plant nutritional value improvement.     

High threonine producer mutant ofNicotiana sylvestris (Spegg. and Comes)

Summary Mutagenesis and the subsequent selection of mesophyll diploid protoplasts ofNicotiana sylvestris on growth inhibitory concentrations of lysine plus threonine has led to the isolation of an LT-resistant mutant. Regeneration of this line (RLT 70) and analysis of its descendants demonstrated the dominant monogenic nuclear character of the resistance gene, further namedak-LT1. When the inhibition properties of aspartate kinase were examined in the homozygous mutant, lysine-sensitive activity could no longer be detected. In comparison, 70%–80% of the wild-type enzyme activity was usually inhibited by lysine, and the rest by threonine. Evidence for the existence of at least two AK isoenzymes was obtained by ion-exchange chromatography, where two peaks of activity could be detected: the first one to be eluted is lysine sensitive, and the second one threonine sensitive. One consequence of the altered regulation of AK in the mutant was the enhanced production of soluble threonine. Threonine accumulation was observed to occur throughout the life cycle of the mutant plant as well as in its different organs. In particular, leaves exhibited a 45-fold increment of soluble threonine, which corresponds to a 13-fold increase in total threonine: almost one-third of the total amino acids was free and proteinbound threonine. In RLT 70 seeds, 20% of the free amino acid pool was in the form of threonine (70-fold accumulation compared to the wild type), and total threonine content was increased five fold. As a general rule, the other amino acids were also more abundant in RLT 70 seeds, such that the total of amino acids present was between two to four times higher, but in contrast with the situation encountered in leaves, this was also due to a higher protein-bound amino acid content.     

Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine-methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. Reduction in SAMS expression level leads most probably to the reduction of SAM found in plants that feed with lysine. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. Elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality.     

Increasing lysine levels in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp) seeds through genetic engineering

In higher plants the essential amino acids lysine, threonine, methionine and isoleucine are synthesised through a branched pathway starting from aspartate. The key enzyme of lysine biosynthesis in this pathway—dihydrodipicolinate synthase (DHDPS)—is feedback-inhibited by lysine. The dhdps-r1 gene from a mutant Nicotiana sylvestris, which encodes a DHDPS enzyme insensitive to feedback inhibition, was used to improve the lysine content in pigeonpea seeds. The dhdps-r1 coding region driven by a phaseolin or an Arabidopsis 2S2 promoter was successfully overexpressed in the seeds of pigeonpea by using Agrobacterium transformation and particle bombardment. In 11 lines analysed, a 2- to 6-fold enhanced DHDPS activity in immature seeds at a late stage of maturation was found in comparison to wild type. The overexpression of dhdps-r1 led to an enhanced content of free lysine in the seeds of pigeonpea from 1.6 to 8.5 times compared with wild type. However, this was not reflected in an increase in total seed lysine content. This might be explained by a temporal discrepancy between maximal expression of dhdps-r1 and the rate of amino acid incorporation into storage proteins. Assays of the lysine degradative enzyme lysine-ketoglutarate reductase in these seeds showed no co-ordinated regulation of lysine biosynthesis and catabolism during seed maturation. All transgenic plants were fertile and produced morphologically normal seeds.     

Characterization of monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the endosperm

A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional aspartate kinase, the nature of the monofunctional aspartate kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast aspartate kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2-Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.