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Methionine is an essential amino acid in mammals and a precursor for vital metabolites required for the survival of all organisms. Consequently, its inclusion is required in diverse applications, such as food, feed, and pharmaceuticals. Although amino acids and other metabolites are commonly produced through microbial fermentation, high-yield biosynthesis of L-methionine remains a significant challenge due to the strict cellular regulation of the biosynthesis pathway. As a result, methionine is produced primarily synthetically, resulting in a racemic mixture of D,L-methionine. This study explores methionine bio-production in E. coli by replacing its native trans-sulfurylation pathway with the more common direct-sulfurylation pathway used by other bacteria. To this end, we generated a methionine auxotroph E. coli strain (MG1655) by simultaneously deleting metA and metB genes and complementing them with metX and metY from different bacteria. Complementation of the genetically modified E. coli with metX/metY from Cyclobacterium marinum or Deinococcus geothermalis, together with the deletion of the global repressor metJ and overexpression of the transporter yjeH, resulted in a substantial increase of up to 126 and 160-fold methionine relative to the wild-type strain, respectively, and accumulation of up to 700 mg/L using minimal MOPS medium and 2 ml culture. Our findings provide a method to study methionine biosynthesis and a chassis for enhancing L-methionine production by fermentation., (© 2023. The Author(s).)

The sulfur-containing amino acid methionine belongs to the group of essential amino acids, meaning that humans and animals must consume it in their diets. However, plant seeds have low levels of methionine, limiting their nutritional potential. For this reason, efforts have been made over the years to increase methionine levels in seeds. Here, we summarize these efforts and focus particularly on those utilizing diverse genetic and molecular tools. Four main approaches are described: (i) expression of methionine-rich storage proteins in a seed-specific manner to incorporate more soluble methionine into the protein fraction; (ii) reduction of methionine-poor storage proteins inside the seeds to reinforce the accumulation of methionine-rich proteins; (iii) silencing methionine catabolic enzymes; and (iv) up-regulation of key biosynthetic enzymes participating in methionine synthesis. We focus on the biosynthetic genes that operate de novo in seeds and that belong to the sulfur assimilation and aspartate family pathways, as well as genes from the methionine-specific pathway. We also include those enzymes that operate in non-seed tissues that contribute to the accumulation of methionine in seeds, such as S-methylmethionine enzymes. Finally, we discuss the biotechnological potential of these manipulations to increase methionine content in plant seeds and their effect on seed germination., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ-SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild-type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress-related metabolites. Despite this, FA plants were more sensitive to short- and long-term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non-stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non-stress and stress conditions is important for enabling the plants to cope with stress conditions., (© 2017 Scandinavian Plant Physiology Society.)

Key Message: Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine synthesis in seeds. In addition, mutual effects exist between the S -methylmethionine cycle and the aspartate family pathway in seeds. Methionine, a sulfur-containing amino acid, is a key metabolite in plant cells. The previous lines of evidence proposed that the S-methylmethionine (SMM) cycle contributes to methionine synthesis in seeds where methionine that is produced in non-seed tissues is converted to SMM and then transported via the phloem into the seeds. However, the relative regulatory roles of the S-methyltransferases operating within this cycle in seeds are yet to be fully understood. In the current study, we generated transgenic Arabidopsis seeds with altered expression of three HOMOCYSTEINE S-METHYLTRANSFERASEs (HMTs) and METHIONINE S-METHYLTRANSFERASE (MMT), and profiled them for transcript and metabolic changes. The results revealed that AtHMT1 and AtHMT3, but not AtHMT2 and AtMMT, are the predominant enzymes operating in seeds as altered expression of these two genes affected the levels of methionine and SMM in transgenic seeds. Their manipulations resulted in adapted expression level of genes participating in methionine synthesis through the SMM and aspartate family pathways. Taken together, our findings provide new insights into the regulatory roles of the SMM cycle and the mutual effects existing between the two methionine biosynthesis pathways, highlighting the complexity of the metabolism of methionine and SMM in seeds.

The sulfur-containing essential amino acid methionine controls the level of important metabolites and processes in plants. In addition, methionine levels limit the nutritional quality of many crop plants. The level of methionine is regulated mainly by cystathionine γ-synthase (CGS), the first enzyme committed to its biosynthesis. Within our efforts to reveal factors that regulate CGS and methionine content in plants, we have analyzed how light regulates the transcript and protein level of Arabidopsis CGS (AtCGS). The expression of AtCGS is up-regulated in the light and reduced in the dark, independent of the diurnal cycle. Using tobacco plants overexpressing AtCGS, we have found that the light sensitive motives of the AtCGS gene are found within the coding sequence of AtCGS and not in its promoter, terminator or the untranslated regions of the gene. Sucrose can partially mimic the effect of light in dark grown plants while the addition of nitrogen and sulfur sources does not have any effect. The kinetics of the changes in the expression level of AtCGS suggest that its level can be maintained during extended darkness, or even when the sucrose content is reduced, such as during abiotic stresses. The up-regulation of AtCGS by light is in agreement with previous studies showing that other enzymes regulating the level of the carbon/amino skeleton and the sulfur group of Met, are up-regulated by light. The results indicate that light and dark participate in the regulation of the carbon/amino skeleton flux in the synthesis of amino acids of the aspartate family.

Soybean seeds provide an excellent source of protein for human and livestock nutrition. However, their nutritional quality is hampered by a low concentration of the essential sulfur amino acid, methionine (Met). In order to study factors that regulate Met synthesis in soybean seeds, this study used the Met-insensitive form of Arabidopsis cystathionine γ-synthase (AtD-CGS), which is the first committed enzyme of Met biosynthesis. This gene was expressed under the control of a seed-specific promoter, legumin B4, and used to transform the soybean cultivar Zigongdongdou (ZD). In three transgenic lines that exhibited the highest expression level of AtD-CGS, the level of soluble Met increased significantly in developing green seeds (3.8-7-fold). These seeds also showed high levels of other amino acids. This phenomenon was more prominent in two transgenic lines, ZD24 and ZD91. The total Met content, which including Met incorporated into proteins, significantly increased in the mature dry seeds of these two transgenic lines by 1.8- and 2.3-fold, respectively. This elevation was accompanied by a higher content of other protein-incorporated amino acids, which led to significantly higher total protein content in the seeds of these two lines. However, in a third transgenic line, ZD01, the level of total Met and the level of other amino acids did not increase significantly in the mature dry seeds. This line also showed no significant change in protein levels. This suggests a positive connection between high Met content and the synthesis of other amino acids that enable the synthesis of more seed proteins.

Methionine and threonine are two essential amino acids, the levels of which limit the nutritional quality of plants. Both amino acids diverge from the same branch of the aspartate family biosynthesis pathway; therefore, their biosynthesis pathways compete for the same carbon/amino substrate. To further elucidate the regulation of methionine biosynthesis and seek ways of increasing the levels of these two amino acids, we crossed transgenic tobacco plants overexpressing the bacterial feedback-insensitive aspartate kinase (bAK), containing a significantly higher threonine level, with plants overexpressing Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co-expressing bAK and the full-length AtCGS (F-AtCGS) have significantly higher methionine and threonine levels compared with the levels found in wild-type plants, but the methionine level does not increase beyond that found in plants expressing F-AtCGS alone. This finding can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the transcript level of AtCGS. To test this assumption, plants expressing bAK were crossed with plants expressing two mutated forms of AtCGS in which the domains responsible for the feedback regulation have been deleted. Indeed, significantly higher methionine contents and its metabolites levels accumulated in the newly produced plants, and the levels of threonine were also significantly higher than in the wild-type plants. The transcript level of the two mutated forms of AtCGS significantly increased when there was a high content of threonine in the plants, suggesting that threonine modulates, probably indirectly, the transcript level of AtCGS.

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.

Cystathionine gamma-synthase (CGS), the first enzyme of methionine biosynthesis in higher plants, plays an important role in the biosynthesis pathway and in regulating methionine metabolism in plant cells. In response to methionine, the expression of this enzyme is regulated via amino acid sequences located in its N-terminal. Here, using reverse transcription PCR and ribonuclease protection analysis, we demonstrate that, in addition to the full-length CGS transcript, a deleted form exists in Arabidopsis. The deleted transcript of CGS that lacks 90 or 87 nt located internally in the regulatory N-terminal region of CGS maintains the reading frame of the protein. Its association with polyribosomes indicates that this deleted form of CGS is translated. In order to study the function of this deleted form of CGS, we overexpressed it in transgenic tobacco plants. We found that the transgenic plants engineered to express only the deleted form of CGS accumulated methionine to a much higher level than those that expressed the full-length CGS. Furthermore, in vitro feeding experiments revealed that the deleted form of CGS is not subject to feedback regulation by methionine, as reported for the full-length transcript. Therefore, although most likely produced from the full-length CGS, the transcript of the deleted form is insensitive to methionine application and its expression may be important for maintaining methionine metabolism even in the presence of a high level of methionine.

The sulfur-containing amino acid methionine is a nutritionally important essential amino acid and is the precursor of several metabolites that regulate plant growth and responses to the environment. Methionine production is largely regulated by cystathionine gamma-synthase, the first specific enzyme for its synthesis. This enzyme competes in a complex manner with threonine synthase, the last enzyme in threonine biosynthesis, for their common substrate O-phosphohomoserine. New genetic and molecular data suggest that methionine synthesis and catabolism are coordinately regulated by novel post-transcriptional and post-translational mechanisms that are associated with a regulatory part within the N-terminal part of cystathionine gamma-synthase.

Cystathionine gamma-synthase (CGS) is a key enzyme of Met biosynthesis in bacteria and plants. Aligning the amino acid sequences revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme. However, this region is not essential for the catalytic activity of this enzyme, as deduced from the complementation test of an Escherichia coli CGS mutant. To determine the function of this N-terminal region, we overexpressed full-length Arabidopsis CGS and its truncated version that lacks the N-terminal region in transgenic tobacco (Nicotiana tabacum) plants. Transgenic plants expressing both types of CGS had a significant higher level of Met, S-methyl-Met, and Met content in their proteins. However, although plants expressing full-length CGS showed the same phenotype and developmental pattern as wild-type plants, those expressing the truncated CGS showed a severely abnormal phenotype. These abnormal plants also emitted high levels of Met catabolic products, dimethyl sulfide and carbon disulfide. The level of ethylene, the Met-derived hormone, was 40 times higher than in wild-type plants. Since the alien CGS was expressed at comparable levels in both types of transgenic plants, we further suggest that post-translational modification(s) occurs in this N-terminal region, which regulate CGS and/or Met metabolism. More specifically, since the absence of the N-terminal region leads to an impaired Met metabolism, the results further suggest that this region plays a role in protecting plants from a high level of Met catabolic products such as ethylene.

ABSTRACT Cysteine is the precursor for the biosynthesis of glutathione, a key stress‐protective metabolite, and methionine, which is imperative for cell growth and protein synthesis. The exact mechanism that governs the routing of cysteine toward glutathione or methionine during stresses remains unclear. Our study reveals that under oxidative stress, methionine and glutathione compete for cysteine and that the increased oxidized glutathione (GSSG) levels under stress hinder methionine biosynthesis. Moreover, we find that inhibition occurs as GSSG binds to and accelerates the degradation of cystathionine γ‐synthase, a key enzyme in the methionine synthesis pathway. Consequently, this leads to a reduction in the flux toward methionine‐derived metabolites and redirects cysteine utilization toward glutathione, thereby enhancing plant protection. Our study suggests a novel regulatory feedback loop involving glutathione, methionine, and cysteine, shedding light on the plant stress response and the adaptive rerouting of cysteine. These findings offer new insights into the intricate balance of growth and protection in plants and its impact on their nutritional value due to low methionine levels under stress. [ABSTRACT FROM AUTHOR]

The sulfur-containing essential amino acid, methionine, is a key metabolite in plant cells since it is used as a precursor for the synthesis of vital metabolites. The transcript level of methionine's catabolic enzyme, methionine g-lyase (MGL), accumulates in the seeds to a high level compared to other organs. The aim of this study was to reveal the role of MGL during seed development and germination. Using [13C]S-methylmethionine (SMM), the mobile form of methionine that is used to feed flower stalks of wild-type (WT) plants, revealed that the contents of [13C] methionine in seeds were significantly reduced when the plants underwent heat and osmotic stresses. Moreover, the levels of [13C]isoleucine, a product of MGL, significantly increased. Also, using the MGL promoter and gene fused to the GUS reporter gene, it was demonstrated that the heat stress significantly increased the protein level in the seeds. Therefore, we can conclude that MGL became active under stresses apparently to produce isoleucine, which is used as an osmoprotectant and an energy source. Transgenic Arabidopsis thaliana RNAi seeds with targeted repression of AtMGL during the late developmental stages of seeds show that the seeds did not accumulate methionine when they were grown under standard growth conditions, unlike the mgl-2, a knockout mutant, which showed a three-fold higher level of methionine. Also, when the RNAi plants developed under mid-heat stress, the level of methionine significantly increased while the content of isoleucine decreased compared to the control seeds, which strengthened the assumption that MGL is active under stress. The germination efficiency of the RNAi lines and mgl seeds were similar to their controls. However, the seeds that developed during heat or salt stress showed significantly lower germination efficiency compared to the control seeds. This implies that MGL is important to maintain the ability of the seeds to germinate. The RNAi lines and mgl seeds that developed under regular conditions, but germinated during salt or osmotic stress, exhibited a lower germination rate, suggesting an essential role of MGL also during this process. The results of this study show the important role of AtMGL in seeds under stresses. [ABSTRACT FROM AUTHOR]

Summary: The synthesis of essential amino acids in plants is pivotal for their viability and growth, and these cellular pathways are therefore targeted for the discovery of new molecules for weed control. Herein, we describe the discovery and design of small molecule inhibitors of cystathionine gamma‐synthase, a key enzyme in the biosynthesis of methionine. Based on in silico screening and filtering of a large molecular database followed by the in vitro selection of molecules, we identified small molecules capable of binding the target enzyme. Molecular modelling of the interaction and direct biophysical binding enabled us to explore a focussed chemical expansion set of molecules characterized by an active phenyl‐benzamide chemical group. These molecules are bio‐active and efficiently inhibit the viability of BY‐2 tobacco cells and seedlings growth of Arabidopsis thaliana on agar plates. [ABSTRACT FROM AUTHOR]

The low level of methionine, an essential sulfur-containing amino acid, limits the nutritional quality of seeds. Two main factors can control the level of protein-bound methionine: the level of free methionine that limits protein accumulation and the methionine residues inside the storage proteins. To reveal the main limiting factor, we generated transgenic Arabidopsis thaliana seed-specific plants expressing the methionine-rich sunflower seed storage (SSA) protein (A1/A2). The contents of protein-bound methionine in the water-soluble protein fraction that includes the SSA in A1/A2 were 5.3- and 10.5-fold, respectively, compared to control, an empty vector (EV). This suggests that free methionine can support this accumulation. To elucidate if the level of free methionine could be increased further in the protein-bound methionine, these lines were crossed with previously characterized plants having higher levels of free methionine in seeds (called SSE). The progenies of the crosses (A1S, A2S) exhibited the highest level of protein-bound methionine, but this level did not differ significantly from A2, suggesting that all the methionine residues of A2 were filled with methionine. It also suggests that the content of methionine residues in the storage proteins is the main limiting factor. The results also proposed that the storage proteins can change their content in response to high levels of free methionine or SSA. This was assumed since the water-soluble protein fraction was highest in A1S/A2S as well as in SSE compared to EV and A1/A2. By using these seeds, we also aimed at gaining more knowledge about the link between high free methionine and the levels of metabolites that usually accumulate during abiotic stresses. This putative connection was derived from a previous analysis of SSE. The results of metabolic profiling showed that the levels of 29 and 20 out of the 56 metabolites were significantly higher in SSE and A1, respectively, that had higher level of free methionine, compared A1S/A2S, which had lower free methionine levels. This suggests a strong link between high free methionine and the accumulation of stress-associated metabolites. [ABSTRACT FROM AUTHOR]

Methionine and threonine are two essential amino acids, the levels of which limit the nutritional quality of plants. Both amino acids diverge from the same branch of the aspartate family biosynthesis pathway; therefore, their biosynthesis pathways compete for the same carbon/amino substrate. To further elucidate the regulation of methionine biosynthesis and seek ways of increasing the levels of these two amino acids, we crossed transgenic tobacco plants overexpressing the bacterial feedback-insensitive aspartate kinase (bAK), containing a significantly higher threonine level, with plants overexpressing Arabidopsis cystathionine γ-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co-expressing bAK and the full-length AtCGS (F-AtCGS) have significantly higher methionine and threonine levels compared with the levels found in wild-type plants, but the methionine level does not increase beyond that found in plants expressing F-AtCGS alone. This finding can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the transcript level of AtCGS. To test this assumption, plants expressing bAK were crossed with plants expressing two mutated forms of AtCGS in which the domains responsible for the feedback regulation have been deleted. Indeed, significantly higher methionine contents and its metabolites levels accumulated in the newly produced plants, and the levels of threonine were also significantly higher than in the wild-type plants. The transcript level of the two mutated forms of AtCGS significantly increased when there was a high content of threonine in the plants, suggesting that threonine modulates, probably indirectly, the transcript level of AtCGS. [ABSTRACT FROM AUTHOR]

Cystathionine γ-synthase (CGS), the first enzyme of methionine biosynthesis in higher plants, plays an important role in the biosynthesis pathway and in regulating methionine metabolism in plant cells. In response to methionine, the expression of this enzyme is regulated via amino acid sequences located in its N-terminal. Here, using reverse transcription PCR and ribonuclease protection analysis, we demonstrate that, in addition to the full-length CGS transcript, a deleted form exists in Arabidopsis. The deleted transcript of CGS that lacks 90 or 87 nt located internally in the regulatory N-terminal region of CGS maintains the reading frame of the protein. Its association with polyribosomes indicates that this deleted form of CGS is translated. In order to study the function of this deleted form of CGS, we overexpressed it in transgenic tobacco plants. We found that the transgenic plants engineered to express only the deleted form of CGS accumulated methionine to a much higher level than those that expressed the full-length CGS. Furthermore, in vitro feeding experiments revealed that the deleted form of CGS is not subject to feedback regulation by methionine, as reported for the full-length transcript. Therefore, although most likely produced from the full-length CGS, the transcript of the deleted form is insensitive to methionine application and its expression may be important for maintaining methionine metabolism even in the presence of a high level of methionine. [ABSTRACT FROM AUTHOR]