Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli.

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.

Combined Hybridization and Evaluation of High-Lysine Rice: Nutritional and Physicochemical Qualities and Field Performance.

Rice, as a major food crop, provides necessary energy and nutrition for humans and livestock. However, its nutritional value is affected by lysine. Using point mutation, we previously obtained AK2 (aspartokinase) and DHDPS1 (dihydrodipicolinate synthase) genes insensitive to lysine feedback inhibition and constructed transgenic lines AK2-52 and DHDPS1-22, which show increased lysine synthesis, as well as Ri-12, which shows decreased lysine degradation by inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) activity. In this study, further transgenic lines were hybridized and evaluated. The lysine content of mature seeds from pyramid lines PRD and PRA increased 32.5- and 29.8-fold, respectively, compared with the wild-type, while the three-gene pyramiding line PRDA had a moderate lysine content. The total lysine, total free lysine, and total protein contents of PRD and PRA also increased and had no obvious impact on the physical and chemical quality, seed appearance, and main agronomic traits. Meanwhile, comparative analysis with polygenic polymeric lines GR containing bacterial AK (lysC) and DHDPS (dapA) genes revealed differences in the way bacterial and endogenous rice AK and DHDPS regulate lysine biosynthesis. These results provide a reference for further evaluation and commercialization of high-lysine transgenic rice.

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1.

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.

Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila.

Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.

The Metabolite Saccharopine Impairs Neuronal Development by Inhibiting the Neurotrophic Function of Glucose-6-Phosphate Isomerase.

Mutations in the Aminoadipate-Semialdehyde Synthase (AASS) gene encoding α-aminoadipic semialdehyde synthase lead to hyperlysinemia-I, a benign metabolic variant without clinical significance, and hyperlysinemia-II with developmental delay and intellectual disability. Although both forms of hyperlysinemia display biochemical phenotypes of questionable clinical significance, an association between neurologic disorder and a pronounced biochemical abnormality remains a challenging clinical question. Here, we report that Aass mutant male and female mice carrying the R65Q mutation in α-ketoglutarate reductase (LKR) domain have an elevated cerebral lysine level and a normal brain development, whereas the Aass mutant mice carrying the G489E mutation in saccharopine dehydrogenase (SDH) domain exhibit elevations of both cerebral lysine and saccharopine levels and a smaller brain with defective neuronal development. Mechanistically, the accumulated saccharopine, but not lysine, leads to impaired neuronal development by inhibiting the neurotrophic effect of glucose-6-phosphate isomerase (GPI). While extracellular supplementation of GPI restores defective neuronal development caused by G498E mutation in SDH of Aass. Altogether, our findings not only unravel the requirement for saccharopine degradation in neuronal development, but also provide the mechanistic insights for understanding the neurometabolic disorder of hyperlysinemia-II.SIGNIFICANCE STATEMENT The association between neurologic disorder and a pronounced biochemical abnormality in hyperlysinemia remains a challenging clinical question. Here, we report that mice carrying the R65Q mutation in lysine α-ketoglutarate reductase (LKR) domain of aminoadipate-semialdehyde synthase (AASS) have an elevated cerebral lysine levels and a normal brain development, whereas those carrying the G489E mutation in saccharopine dehydrogenase (SDH) domain of AASS exhibit an elevation of both cerebral lysine and saccharopine and a small brain with defective neuronal development. Furthermore, saccharopine impairs neuronal development by inhibiting the neurotrophic effect of glucose-6-phosphate isomerase (GPI). These findings demonstrate saccharopine degradation is essential for neuronal development.

LYSINE KETOGLUTARATE REDUCTASE TRANS-SPLICING RELATED 1 is involved in temperature-dependent root growth in rice.

Root length is an important root parameter directly related to the uptake of water and nutrients. However, the molecular mechanisms controlling root length are still not fully understood. Here, we isolated a short-root mutant of rice, dice2 (defective in cell elongation 2). The cell length and meristem size of the roots were decreased in dice2, but the root function in terms of mineral element uptake, root cell width, and root anatomy were hardly altered compared with wild-type (WT) rice. The root growth defect in dice2 could be partially rescued by high temperature. Map-based cloning combined with a complementation test revealed that the short-root phenotype was caused by a nonsense mutation in a gene which was annotated to encode Lysine Ketoglutarate Reductase Trans-Splicing related 1 (OsLKRT1). OsLKRT1, encoding a cytosol-localized protein, was expressed in all cells of the root tip and elongation region as well as the shoot. RNA-seq analysis showed that there was no difference between dice2 and the WT in the expression level of genes involved in root development identified so far. These results indicate that OsLKRT1 is involved in a novel pathway required for root cell elongation in rice, although its exact role remains to be further investigated.

New Therapeutic Candidates for the Treatment of Malassezia pachydermatis -Associated Infections.

The opportunistic pathogen Malassezia pachydermatis causes bloodstream infections in preterm infants or individuals with immunodeficiency disorders and has been associated with a broad spectrum of diseases in animals such as seborrheic dermatitis, external otitis and fungemia. The current approaches to treat these infections are failing as a consequence of their adverse effects, changes in susceptibility and antifungal resistance. Thus, the identification of novel therapeutic targets against M. pachydermatis infections are highly relevant. Here, Gene Essentiality Analysis and Flux Variability Analysis was applied to a previously reported M. pachydermatis metabolic network to identify enzymes that, when absent, negatively affect biomass production. Three novel therapeutic targets (i.e., homoserine dehydrogenase (MpHSD), homocitrate synthase (MpHCS) and saccharopine dehydrogenase (MpSDH)) were identified that are absent in humans. Notably, L-lysine was shown to be an inhibitor of the enzymatic activity of MpHCS and MpSDH at concentrations of 1 mM and 75 mM, respectively, while L-threonine (1 mM) inhibited MpHSD. Interestingly, L- lysine was also shown to inhibit M. pachydermatis growth during in vitro assays with reference strains and canine isolates, while it had a negligible cytotoxic activity on HEKa cells. Together, our findings form the bases for the development of novel treatments against M. pachydermatis infections.

Effects of rice feeding on growth performance and protein (amino acids) metabolism in weanling piglets.

We investigated the effects of rice feeding on growth performance and protein (amino acids) metabolism of weanling piglets. In all, 16 weanling piglets with an average initial weight of 7.5 kg were divided into two groups. One group was fed a corn-soybean meal-based diet, and the other was fed a rice-soybean meal diet, containing around 46% of corn or rice, respectively. A two-week growth trial was conducted. The average daily gain (p = .025) and feed efficiency (p = .011) in rice-fed piglets were significantly higher than those in corn-fed piglets. Liver lysine-ketoglutarate reductase activity tended to be lower (p = .073) in rice-fed piglets than in corn-fed piglets. Plasma urea nitrogen concentration in rice-fed piglets was significantly lower than that in corn-fed piglets. Plasma glucose and insulin concentrations were significantly higher in rice-fed piglets than in corn-fed piglets. Plasma-free valine, isoleucine, and tryptophan concentrations were significantly higher in rice-fed piglets than in corn-fed piglets. In contrast, plasma histidine concentration was significantly lower in rice-fed piglets than in corn-fed piglets. Overall, these results show that rice feeding improves the growth performance and affects the protein (amino acids) metabolism in weanling piglets.

Overexpression of the saccharopine dehydrogenase gene improves lysine biosynthesis in Flammulina velutipes.

Saccharopine dehydrogenase (EC 1.5.1.7) regulates the last step of fungal lysine biosynthesis. The gene (Fvsdh) encoding saccharopine dehydrogenase was identified and cloned from the whole genome of Flammulina velutipes. The genomic DNA of Fvsdh is 1257 bp, comprising three introns and four exons. The full-length complementary DNA of Fvsdh comprises 1107 bp with a deduced amino acid sequence of 368 residues. A 1,000-bp promoter sequence containing the TATA box, CAAT box, and several putative cis-acting elements was also identified. The results of tissue expression analysis showed that the expression level of the Fvsdh gene was higher in the pileus than in the stipe whether in the elongation or maturation stage. Further research showed that the lysine contents were 3.03 and 2.95 mg/g in maturation-pileus and elongation-pileus, respectively. In contrast, the lysine contents were 2.49 and 2.07 mg/g in elongation-stipe and maturation-stipe, respectively. To study the function of Fvsdh, we overexpressed Fvsdh in F. velutipes and found that Fvsdh gene expression was increased from 1.1- to 3-fold in randomly selected transgenic strains. The lysine contents were also increased from 1.12- to 1.3-fold in these five transformants, except for strain T3, in which the lysine contents were the same as the control. These results indicate that the expression of the Fvsdh gene can affect the lysine content of F. velutipes.

The lysine catabolite saccharopine impairs development by disrupting mitochondrial homeostasis.

Amino acid catabolism is frequently executed in mitochondria; however, it is largely unknown how aberrant amino acid metabolism affects mitochondria. Here we report the requirement for mitochondrial saccharopine degradation in mitochondrial homeostasis and animal development. In Caenorhbditis elegans, mutations in the saccharopine dehydrogenase (SDH) domain of the bi-functional enzyme α-aminoadipic semialdehyde synthase AASS-1 greatly elevate the lysine catabolic intermediate saccharopine, which causes mitochondrial damage by disrupting mitochondrial dynamics, leading to reduced adult animal growth. In mice, failure of mitochondrial saccharopine oxidation causes lethal mitochondrial damage in the liver, leading to postnatal developmental retardation and death. Importantly, genetic inactivation of genes that raise the mitochondrial saccharopine precursors lysine and α-ketoglutarate strongly suppresses SDH mutation-induced saccharopine accumulation and mitochondrial abnormalities in C. elegans Thus, adequate saccharopine catabolism is essential for mitochondrial homeostasis. Our study provides mechanistic and therapeutic insights for understanding and treating hyperlysinemia II (saccharopinuria), an aminoacidopathy with severe developmental defects.

Metabolomic Analysis Reveals That the Drosophila melanogaster Gene lysine Influences Diverse Aspects of Metabolism.

The fruit fly Drosophila melanogaster has emerged as a powerful model for investigating the molecular mechanisms that regulate animal metabolism. However, a major limitation of these studies is that many metabolic assays are tedious, dedicated to analyzing a single molecule, and rely on indirect measurements. As a result, Drosophila geneticists commonly use candidate gene approaches, which, while important, bias studies toward known metabolic regulators. In an effort to expand the scope of Drosophila metabolic studies, we used the classic mutant lysine (lys) to demonstrate how a modern metabolomics approach can be used to conduct forward genetic studies. Using an inexpensive and well-established gas chromatography-mass spectrometry-based method, we genetically mapped and molecularly characterized lys by using free lysine levels as a phenotypic readout. Our efforts revealed that lys encodes the Drosophila homolog of Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase, which is required for the enzymatic degradation of lysine. Furthermore, this approach also allowed us to simultaneously survey a large swathe of intermediate metabolism, thus demonstrating that Drosophila lysine catabolism is complex and capable of influencing seemingly unrelated metabolic pathways. Overall, our study highlights how a combination of Drosophila forward genetics and metabolomics can be used for unbiased studies of animal metabolism, and demonstrates that a single enzymatic step is intricately connected to diverse aspects of metabolism.

Effect of dietary lysine restriction and arginine supplementation in two patients with pyridoxine-dependent epilepsy.

Pyridoxine-Dependent Epilepsy (PDE) is a recessive disorder caused by deficiency of α-aminoadipic semialdehyde dehydrogenase in the catabolic pathway of lysine. It is characterized by intractable seizures controlled by the administration of pharmacological doses of vitamin B6. Despite seizure control with pyridoxine, intellectual disability and developmental delays are still observed in some patients with PDE, likely due to the accumulation of toxic intermediates in the lysine catabolic pathway: alpha-aminoadipic semialdehyde (AASA), delta-1-piperideine-6-carboxylate (P6C), and pipecolic acid. Here we evaluate biochemical and clinical parameters in two PDE patients treated with a lysine-restricted diet and arginine supplementation (100-150mg/kg), aimed at reducing the levels of PDE biomarkers. Lysine restriction resulted in decreased accumulation of PDE biomarkers and improved development. Plasma lysine but not plasma arginine, directly correlated with plasma levels of AASA-P6C (p<0.001, r(2)=0.640) and pipecolic acid (p<0.01, r(2)=0.484). In addition, plasma threonine strongly correlated with the levels of AASA-P6C (p<0.0001, r(2)=0.732) and pipecolic acid (p<0.005, r(2)=0.527), suggesting extreme sensitivity of threonine catabolism to pyridoxine availability. Our results further support the use of dietary therapies in combination with pyridoxine for the treatment of PDE.

Proteins involved in biophoton emission and flooding-stress responses in soybean under light and dark conditions.

To know the molecular systems basically flooding conditions in soybean, biophoton emission measurements and proteomic analyses were carried out for flooding-stressed roots under light and dark conditions. Photon emission was analyzed using a photon counter. Gel-free quantitative proteomics were performed to identify significant changes proteins using the nano LC-MS along with SIEVE software. Biophoton emissions were significantly increased in both light and dark conditions after flooding stress, but gradually decreased with continued flooding exposure compared to the control plants. Among the 120 significantly identified proteins in the roots of soybean plants, 73 and 19 proteins were decreased and increased in the light condition, respectively, and 4 and 24 proteins were increased and decreased, respectively, in the dark condition. The proteins were mainly functionally grouped into cell organization, protein degradation/synthesis, and glycolysis. The highly abundant lactate/malate dehydrogenase proteins were decreased in flooding-stressed roots exposed to light, whereas the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme was increased in both light and dark conditions. Notably, however, specific enzyme assays revealed that the activities of these enzymes and biophoton emission were sharply increased after 3 days of flooding stress. This finding suggests that the source of biophoton emission in roots might involve the chemical excitation of electron or proton through enzymatic or non-enzymatic oxidation and reduction reactions. Moreover, the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme may play important roles in responses in flooding stress of soybean under the light condition and as a contributing factor to biophoton emission.

The Importance of the MM Environment and the Selection of the QM Method in QM/MM Calculations: Applications to Enzymatic Reactions.

In this chapter, we discuss the influence of an anisotropic protein environment on the reaction mechanisms of saccharopine reductase and uroporphyrinogen decarboxylase, respectively, via the use of a quantum mechanical and molecular mechanical (QM/MM) approach. In addition, we discuss the importance of selecting a suitable DFT functional to be used in a QM/MM study of a key intermediate in the mechanism of 8R-lipoxygenase, a nonheme iron enzyme. In the case of saccharopine reductase, while the enzyme utilizes a substrate-assisted catalytic pathway, it was found that only through treating the polarizing effect of the active site, via the use of an electronic embedding formalism, was agreement with experimental kinetic data obtained. Similarly, in the case of uroporphyrinogen decarboxylase, the effect of the protein environment on the catalytic mechanism was found to be such that the calculated rate-limiting barrier is in good agreement with related experimentally determined values for the first decarboxylation of the substrate. For 8R-lipoxygenase, it was found that the geometries and energies of the multicentered open-shell intermediate complexes formed during the mechanism are quite sensitive to the choice of the density functional theory method. Thus, while density functional theory has become the method of choice in QM/MM studies, care must be taken in the selection of a particular high-level method.

Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.

Saccharopine reductase catalyzes the reductive amination of l-α-aminoadipate-δ-semialdehyde with l-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid-base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid-base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue.

Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops.

Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes.

Pathways of Amino Acid Degradation in Nilaparvata lugens (Stål) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH).

Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.

An optimized coupled assay for quantifying diaminopimelate decarboxylase activity.

Diaminopimelate decarboxylase (DAPDC) catalyzes the conversion of meso-DAP to lysine and carbon dioxide in the final step of the diaminopimelate (DAP) pathway in plants and bacteria. Given its absence in humans, DAPDC is a promising antibacterial target, particularly considering the rise in drug-resistant strains from pathogens such as Escherichia coli and Mycobacterium tuberculosis. Here, we report the optimization of a simple quantitative assay for measuring DAPDC catalytic activity using saccharopine dehydrogenase (SDH) as the coupling enzyme. Our results show that SDH has optimal activity at 37 °C, pH 8.0, and in Tris buffer. These conditions were subsequently employed to quantitate the enzyme kinetic properties of DAPDC from three bacterial species. We show that DAPDC from E. coli and M. tuberculosis have [Formula: see text] of 0.97 mM and 1.62 mM and a kcat of 55 s(-1) and 28 s(-1), respectively, which agree well with previous studies using more labor-intensive assays. We subsequently employed the optimized coupled assay to show for the first time that DAPDC from Bacillus anthracis possesses a [Formula: see text] of 0.68 mM and a kcat of 58 s(-1). This optimized coupled assay offers excellent scope to be employed in high throughput drug discovery screens targeting DAPDC from bacterial pathogens.

The saccharopine pathway in seed development and stress response of maize.

Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into α-aminoadipic semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into aminoadipic acid (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). Here, we show that LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing endosperm. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD(+) in the micromolar range. In the developing endosperm, the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles, the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in the stressed tissues, the LKR/SDH protein is not produced. In the developing seeds, the saccharopine pathway is used for pipecolic acid synthesis although proline may play a major role in abiotic stress response. The results indicate that the saccharopine pathway in maize seed development and stress responses significantly differ from that observed for dicot plants.

RNA interference-aided knockdown of a putative saccharopine dehydrogenase leads to abnormal ecdysis in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control.