Lysine Synthesis Research Articles

A Structural Code for Discriminating between Transcription Signals Revealed by the Feast/Famine Regulatory Protein DM1 in Complex with Ligands

Feast/famine regulatory proteins (FFRPs) comprise the largest group of archaeal transcription factors. Crystal structures of an FFRP, DM1 from Pyrococcus, were determined in complex with isoleucine, which increases the association state of DM1 to form octamers, and with selenomethionine, which decreases it to maintain dimers under some conditions. Asp39 and Thr/Ser at 69-71 were identified as being important for interaction with the ligand main chain. By analyzing residues surrounding the ligand side chain, partner ligands were identified for various FFRPs from Pyrococcus, e.g., lysine facilitates homo-octamerization of FL11, and arginine facilitates hetero-octamerization of FL11 and DM1. Transcription of the fl11 gene and lysine synthesis are regulated by shifting the equilibrium between association states of FL11 and by shifting the equilibrium toward association with DM1, in response to amino acid availability. With FFRPs also appearing in eubacteria, the origin of such regulation can be traced back to the common ancestor of all extant organisms, serving as a prototype of transcription regulations, now highly diverged.

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.

A phenomenological model to represent the kinetics of growth by Corynebacterium glutamicum for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum.

BackgroundQuantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates.ResultsQuantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes.ConclusionWe have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at different phases of fermentation. The methodology was also used to determine the feasible solution space for a given set of substrate uptake rates under specific optimization criteria. Such an approach can be used to determine the optimality of the accumulation rates of any metabolite in a given network.

Theoretical analysis of amino acid-producing Escherichia coli using a stoichiometric model and multivariate linear regression

This work demonstrates a novel computational approach combining flux balance modeling with statistical methods to identify correlations among fluxes in a metabolic network, providing insight as to how the fluxes should be redirected to achieve maximum product yield. The procedure is demonstrated using the example of amino acid production from an industrial Escherichia coli production strain and a hypothetical engineered strain overexpressing two heterologous genes. Regression analysis based on a random sampling of 5,000 points within the feasible solution space of the E. coli stoichiometric network suggested that increased activity of the glyoxylate cycle or PEP carboxylase and elimination of malic enzyme will improve lysine and arginine synthesis.

Synthesis and characterization of Poly(L-lysine-co-L-proline) as a non-viral gene delivery vector

Gene therapy, the expression of genetic materials with therapeutic activity, has been considered as an encouraging approach to heal life threatening diseases with genetic deficiency. Since direct delivery of the DNA in the absence of a carrier experiences rapid degradation by nuclease and generally exhibits poor cellular uptake, it is one of the main challenges facing today’s gene therapy to establish safe and efficient vectors for delivering therapeutic genes to specific cells. Gene delivery systems investigated so far include viral and non-viral vectors. Viral vectors are intensively used in clinical applications because they efficiently integrate their genetic information into the host chromosomes; however, toxic immunological reactions and recombination events into virulent products still remain as major drawbacks. Non-viral vectors, including liposomes and cationic polymers, display nonimmunogenicity, low acute toxicity, and flexibility to design a carrier with well-defined structures and chemical properties, while they have major problems in transfection efficiency which is limited as only few percentage of that by viral vectors. Among non-viral vectors, poly(L-lysine) (PLL) has been widely used due to the reasonable efficiency and biocompatible nature of peptide bonds in the backbones. However, transfection efficiency of PLL is not so high enough that modification of PLL, such as introduction of targeting ligands or endosomal escape moieties, has been investigated for the use of PLL in vivo. Proline is the only amino acid with a cyclic structure containing a secondary α-amino group and this structural feature gives unique stereochemical and biological properties to the peptides containing proline residue. Water soluble prolinerich peptides have been reported as naturally occurring cellpermeant peptides, which are able to break the cell membrane and deliver the attached carriers into the cells without causing lethal membrane disruption. In this communication, we report the synthesis of lysine and proline based amino acid copolymers. Characterization of the copolymer and the effect of proline content on the formation of copolymer/DNA complex, in vitro cytotoxicity and transfection efficiency were investigated to examine the possibility of the synthesized copolymers as a polymeric gene delivery carrier.

Synthesis and absorption of intestinal microbial lysine in humans and non-ruminant animals and impact on human estimated average requirement of dietary lysine

While there are reports on the nature of synthesis and absorption of intestinal microbial lysine in humans and non-ruminant animals, there are few efforts to quantify microbial amino acid absorption in human subjects. We review the available information on the synthesis of microbial lysine and the quantification of its absorption and utilization by the human host and monogastric model animals. In addition, we explore the impact of microbial lysine on the current estimated average requirement of dietary lysine. It is still uncertain whether microbial amino acids are absorbed primarily from the small or the large intestine in humans. In the pig, the majority of microbial lysine is absorbed in the small intestine. It appears that microbial lysine contribution is responsive to the nutritional status of the host. Estimates for microbial lysine contribution in adult humans on adequate or low protein diets range from 12 to 68 mg/kg per day. It is unlikely that these estimates represent net values because of methodological concerns related to the 15N tracer methodology used. We conclude that microbial lysine contributes to the lysine homeostasis in humans and other non-ruminant mammals. Microbial lysine utilization by the host is a continuous process and occurs both with low, adequate, and high protein intakes, and under protein-free and low lysine dietary conditions in growing and adult individuals. We also conclude that the estimated average lysine requirement for humans already considers lysine contributed by the intestinal microbiota.

Inactivation of Homocitrate Synthase Causes Lysine Auxotrophy in Copper/Zinc-containing Superoxide Dismutase-deficient Yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe lacking copper/zinc-containing superoxide dismutase (CuZn-SOD) is auxotrophic for lysine and sulfurous amino acids under aerobic growth conditions. A multicopy suppressor gene (phx1+) that restored the growth of CuZn-SOD-deficient cells on minimal medium was isolated. It encodes a putative DNA-binding protein with a conserved homeobox domain. Overproduction of Phx1 increased the amount of several proteins, and one of those turned out to be a putative homocitrate synthase (HCS) encoded by the lys4+ gene in S. pombe as judged by mass spectrometric analysis. Consistent with this observation, overexpression of the lys4+ gene increased HCS enzyme activity and was sufficient to suppress the lysine requirement of the CuZn-SOD-deficient cells. Enzyme activity and Western blot analyses revealed that the activity and protein level of HCS were dramatically reduced upon depletion of CuZn-SOD. Treatment of exponentially growing S. pombe cells with paraquat, a superoxide generator, caused a decrease in the amount of Lys4 protein as expected. These results led us to conclude that HCS, the first enzyme in the alpha-aminoadipate-mediated pathway for lysine synthesis common in fungi and some bacteria, is a labile target of oxidative stress caused by CuZn-SOD depletion and that its synthesis is positively regulated by the putative transcriptional regulator Phx1.

Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase

Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + alpha-ketoglutarate) of the novel alpha-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-alpha-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.

Profiling the Expression of Genes Controlling Rice Grain Quality

Rice provides a staple source of energy, protein and other nutrients to half of the world population. Over 90 of the rice seeds consists of starch and protein by dry weight. The quantity and property of starch and protein thus play a dominant role in the yield and quality of rice. The amylase content of starch is a determining factor in the eating and cooking quality while the amount and essential amino acids balance of storage proteins affect the nutritional quality of rice. In China, the super-hybrid rice currently under the last phase of development has a 35 yield advantage over the best inbred rice varieties. However, its grain quality needs further improvement. This study reported the expression patterns of 44 genes participating in starch, storage protein, and lysine synthesis in the developing rice grain. Field grown rice cultivar 9311, the paternal line of an elite super-hybrid rice LYP9 with its draft genomic sequence released, was used as plant material. Results revealed diverse yet coordinated expression profiles of the genes involved in the three pathways which lead to the final composition and property of starch, protein and lysine that determine the quality of rice, providing useful information for rice quality improvement.

Are high-lysine cereal crops still a challenge?

The essential amino acids lysine and threonine are synthesized in higher plants via a pathway starting with aspartate that also leads to the formation of methionine and isoleucine. Lysine is one of most limiting amino acids in plants consumed by humans and livestock. Recent genetic, molecular, and biochemical evidence suggests that lysine synthesis and catabolism are regulated by complex mechanisms. Early kinetic studies utilizing mutants and transgenic plants that over-accumulate lysine have indicated that the major step for the regulation of lysine biosynthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, recent strong evidence indicates that lysine catabolism is also subject to control, particularly in cereal seeds. The challenge of producing crops with a high-lysine concentration in the seeds appeared to be in sight a few years ago. However, apart from the quality protein maize lines currently commercially available, the release of high-lysine crops has not yet occurred. We are left with the question, is the production of high-lysine crops still a challenge?

Biosynthesis of lysine in plants: evidence for a variant of the known bacterial pathways

With the aim of elucidating how plants synthesize lysine, extracts prepared from corn, tobacco, Chlamydomonas and soybean were tested and found to lack detectable amounts of N-alpha-acyl-L,L-diaminopimelate deacylase or N-succinyl-alpha-amino-epsilon-ketopimelate-glutamate aminotransaminase, two key enzymes in the central part of the bacterial pathway for lysine biosynthesis. Corn extracts missing two key enzymes still carried out the overall synthesis of lysine when provided with dihydrodipicolinate. An analysis of available plant DNA sequences was performed to test the veracity of the negative biochemical findings. Orthologs of dihydrodipicolinate reductase and diaminopimelate epimerase (enzymes on each side of the central pathway) were readily found in the Arabidopsis thaliana genome. Orthologs of the known enzymes needed to convert tetrahydrodipicolinate to diaminopimelic acid (DAP) were not detected in Arabidopsis or in the plant DNA sequence databases. The biochemical and reinforcing bioinformatics results provide evidence that plants may use a novel variant of the bacterial pathways for lysine biosynthesis.

Recent Topics in Rumen Microbiology with Particular Reference to Animal Production in Japan

Microbes with which ruminants have established symbiotic relationships include bacteria, protozoa and fungi. Microbiological studies in Japan have emphasized the ecology of the ruminal system including the physiological and biochemical interrelationships between individual microbes. The difference in the distribution of various types of microbes in the rumen was studied first and the importance of the attachment of microbes to solid feed substrates was clarified. The existence of substrate-binding factors in bacteria has also been demonstrated. Many genes encoding fibrolytic enzymes have been cloned and characterized from ruminal microbes to improve the digestion of fiber. Some highly fibrolytic bacteria were produced and the survival of recombinant bacteria in the rumen was studied. The manipulation of ruminal bacteria has not been successful and is still at a challenging stage. Both microbial genetics and ecological approaches are needed to improve ruminant productivity through microbial manipulation. As for the study of microbial protein and amino acid metabolism, a particular emphasis was given to the role of ciliate protozoa. The synthesis of lysine from diaminopimelate by protozoa was demonstrated. The metabolic pathways for many kinds of amino acids were well studied, and it was shown that protozoa actively decompose feed protein and excrete amino acids as metabolic products. Therefore, protozoa tended to reduce the microbial amino acid flow to the intestine. A specific role for pipecolic acid in the physiology and behavior of the host animal was postulated. Protozoal function is still an important problem and deserves further research. Concerning the metabolism of carbohydrate, the reduction of ruminal methane production has been studied because methane is viewed as a pollutant greenhouse gas. Some possible methods of reducing methane production with the least adverse effect have been developed. In this review, some recent topics in ruminal microbiology are described.

Lysine synthesis control in Corynebacterium glutamicum RC 115 in mixed substrate (glucose-acetate) medium

The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated. It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield. In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters. A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition. Therefore it was shown that acetate in low concentrations can be used as a glucose co-substrate to increase the cell specific rate of lysine synthesis and lysine yield by C. glutamicum RC 115. Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucose-acetate media with an increased concentration of acetate.

Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds.

To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway. Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained approximately 12-fold or approximately 5-fold higher levels, respectively, of seed free Lys than wild-type plants. However, the combination of these two traits caused a synergistic approximately 80-fold increase in seed free Lys level. The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn. This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds. Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to approximately 38-fold in the various transgenic and knockout plants. Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism.

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.

De novo synthesis of amino acids by the ruminal anaerobic fungi, Piromyces communis and Neocallimastix frontalis.

Anaerobic fungi are an important component of the cellulolytic ruminal microflora. Ammonia alone as N source supports growth, but amino acid mixtures are stimulatory. In order to evaluate the extent of de novo synthesis of individual amino acids in Piromyces communis and Neocallimastix frontalis, isotope enrichment in amino acids was determined during growth on (15)NH(4)Cl in different media. Most cell N (0.78 and 0.63 for P. communis and N. frontalis, respectively) and amino acid N (0.73 and 0.59) continued to be formed de novo from ammonia when 1 g l(-1) trypticase was added to the medium; this concentration approximates the peak concentration of peptides in the rumen after feeding. Higher peptide/amino acid concentrations decreased de novo synthesis. Lysine was exceptional, in that its synthesis decreased much more than other amino acids when Trypticase or amino acids were added to the medium, suggesting that lysine synthesis might limit fungal growth in the rumen.

De novo synthesis of amino acids by the ruminal anaerobic fungi, Piromyces communis and Neocallimastix frontalis

Anaerobic fungi are an important component of the cellulolytic ruminal microflora. Ammonia alone as N source supports growth, but amino acid mixtures are stimulatory. In order to evaluate the extent of de novo synthesis of individual amino acids in Piromyces communis and Neocallimastix frontalis, isotope enrichment in amino acids was determined during growth on 15NH 4Cl in different media. Most cell N (0.78 and 0.63 for P. communis and N. frontalis, respectively) and amino acid N (0.73 and 0.59) continued to be formed de novo from ammonia when 1 g l −1 trypticase was added to the medium; this concentration approximates the peak concentration of peptides in the rumen after feeding. Higher peptide/amino acid concentrations decreased de novo synthesis. Lysine was exceptional, in that its synthesis decreased much more than other amino acids when Trypticase or amino acids were added to the medium, suggesting that lysine synthesis might limit fungal growth in the rumen.

New insights into the regulation and functional significance of lysine metabolism in plants.

Lysine is one of the most limiting essential amino acids in vegetative foods consumed by humans and livestock. In addition to serving as a building block of proteins, lysine is also a precursor for glutamate, an important signaling amino acid that regulates plant growth and responses to the environment. Recent genetic, molecular, and biochemical evidence suggests that lysine synthesis and catabolism are regulated by novel concerted mechanisms. These include intracellular compartmentalization of enzymes and metabolites, complex transcriptional and posttranscriptional controls of genes encoding enzymes in lysine metabolism during plant growth and development, as well as interactions between different metabolic fluxes. The recent advances in our understanding of the regulation of lysine metabolism in plants may also prove valuable for future production of high-lysine crops.

Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum

Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.