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The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid—a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2‐fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary‐complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic‐semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria‐host co‐evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts. [ABSTRACT FROM AUTHOR]

Lysine content is a criterion of the nutritional quality of rice. Understanding the process of lysine biosynthesis in early-flowering superior grain (SG) and late-flowering inferior grain (IG) of rice would advance breeding and cultivation to improve nutritional quality. However, little information is available on differences in lysine anabolism between SG and IG and the underlying mechanism, and whether and how irrigation regimes affect lysine anabolism in these grains. A japonica rice cultivar was grown in the field and two irrigation regimes, continuous flooding (CF) and wetting alternating with partial drying (WAPD), were imposed from heading to the mature stage. Lysine content and activities of key enzymes of lysine biosynthesis, and levels of brassinosteroids (BRs) were lower in the IG than in the SG at the early grain-filling stage but higher at middle and late grain-filling stages. WAPD increased activities of these key enzymes, BR levels, and contents of lysine and total amino acids in IG, but not SG relative to CF. Application of 2,4-epibrassinolide to rice panicles in CF during early grain filling reproduced the effects of WAPD, but neither treatment altered the activities of enzymes responsible for lysine catabolism in either SG or IG. WAPD and elevated BR levels during grain filling increased lysine biosynthesis in IG. Improvement in lysine biosynthesis in rice should focus on IG.

Fresh and dried Cordyceps sinensis are widely used by the public for medicinal and health purposes. However, the differences between them have not been examined. In this study, fresh wild and artificial C. sinensis (WFC and AFC) were dried to obtain dried wild and artificial C. sinensis (WDC and ADC). Non-targeted GC-MS was used to analyze the metabolic profile characteristics of the four groups of samples. The results showed that air drying significantly altered the composition and content of C. sinensis, mainly in the form of higher abundance of organic acids and derivatives and lower abundance of lipids and lipid-like molecules in fresh C. sinensis. Hierarchical cluster analysis (HCA) and quantitative analyses showed that air drying increased the abundance of Valine, Zinniol, Urocanate, Vulpinic acid, and Uridine 5'-diphosphate, and decreased Xanthotoxol, Vitexin-4-o-glucoside, Val-trp, and Wogonin. These differentially accumulated metabolites (DAMs) were also shown to be potential biomarkers for C. sinensis. KEGG enrichment analysis identified lysine biosynthesis as the most significantly enriched pathway. Annotation of these DAMs to lysine biosynthesis revealed that citrate cycle and pyruvate metabolism entered lysine biosynthesis via 2-oxohlutarate and Homocitrate, respectively, resulting in significant enrichment of L-saccharopine and L-lysine content was significantly higher. Alanine, aspartate, and Glutamate metabolism synthesized more L-aspartate to promote L-lysine synthesis. Thus, high levels of L-lysine result in lysine degradation and pymolysine, which are the most active metabolic pathways during the drying of fresh C. sinensis and indirectly lead to differences in metabolic profiles. [ABSTRACT FROM AUTHOR]

Flammulina filiformis, the most produced edible mushroom species in China, is rich in lysine. Further enhancing its lysine biosynthesis is vital for improving its quality in industrialized cultivation. Citric acid induction significantly increases both the biomass and growth rate of F. filiformis hyphae, as well as the lysine content. The genes encoding enzymes in the lysine biosynthesis pathway were detected under the optimal induction, revealing that the expression levels of hcs, hac, and hah were 2.67, 1.97, and 1.90 times greater, respectively, relative to the control, whereas no significant difference was seen for hdh, aat, sr, and shd, and the expression of aar decreased. Furthermore, the transcriptional levels of Ampk, GCN2, GCN4, and TOR were found significantly upregulated, with the most upregulated, Ampk, reaching a level 42.68 times greater than that of the control, while the phosphorylation of AMPK rose by nearly 54%. In AMPK-silencing strains under the optimal induction, however, the phosphorylation increment dropped to about 16% and the lysine content remained at the same level as in the WT. Thus, AMPK is presented as the critical intermediary in citric acid's regulation of lysine biosynthesis in F. filiformis. [ABSTRACT FROM AUTHOR]

ABSTRACT The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.

Fresh and dried Cordyceps sinensis are widely used by the public for medicinal and health purposes. However, the differences between them have not been examined. In this study, fresh wild and artificial C. sinensis (WFC and AFC) were dried to obtain dried wild and artificial C. sinensis (WDC and ADC). Non-targeted GC-MS was used to analyze the metabolic profile characteristics of the four groups of samples. The results showed that air drying significantly altered the composition and content of C. sinensis, mainly in the form of higher abundance of organic acids and derivatives and lower abundance of lipids and lipid-like molecules in fresh C. sinensis. Hierarchical cluster analysis (HCA) and quantitative analyses showed that air drying increased the abundance of Valine, Zinniol, Urocanate, Vulpinic acid, and Uridine 5’-diphosphate, and decreased Xanthotoxol, Vitexin-4-o-glucoside, Val-trp, and Wogonin. These differentially accumulated metabolites (DAMs) were also shown to be potential biomarkers for C. sinensis. KEGG enrichment analysis identified lysine biosynthesis as the most significantly enriched pathway. Annotation of these DAMs to lysine biosynthesis revealed that citrate cycle and pyruvate metabolism entered lysine biosynthesis via 2-oxohlutarate and Homocitrate, respectively, resulting in significant enrichment of L-saccharopine and L-lysine content was significantly higher. Alanine, aspartate, and Glutamate metabolism synthesized more L-aspartate to promote L-lysine synthesis. Thus, high levels of L-lysine result in lysine degradation and pymolysine, which are the most active metabolic pathways during the drying of fresh C. sinensis and indirectly lead to differences in metabolic profiles.

Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine‐biosynthetic pathway converting pyruvate and l‐aspartate‐β‐semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)‐2‐bromopropionate inactivates the enzyme in a pseudo‐first‐order process. An initial velocity pattern indicates that (S)‐2‐bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)‐2‐bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml−1 DHDPS. The crystals diffracted to 2.15 Å resolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active‐site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half‐reaction of the ping‐pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)‐2‐bromopropionate indicates the covalent adduct formed from (S)‐2‐bromopropionate leads to a rotation of about 180° of the β–δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate. [ABSTRACT FROM AUTHOR]

The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthesis of diaminopimelate and lysine to facilitate peptidoglycan and protein synthesis. Dihydrodipicolinate synthase catalyzes the condensation of L-aspartate 4-semialdehyde and pyruvate to synthesize L-2,3-dihydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are presented. Protein crystals were grown in conditions consisting of 20%(w/v) PEG 4000, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.10 Å resolution. They belonged to space group P212121, with unit-cell parameters a = 79.96, b = 106.33, c = 136.25 Å. The final R values were Rr.i.m. = 0.098, Rwork = 0.183, Rfree = 0.233.

L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes ( malAP ) from Bacillus subtilis , to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E . coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene ( gdhA ) and assimilated nitrate reductase genes ( BsnasBC ) from B . subtilis , along with nitrite reductase genes ( EcnirBD ) from E . coli , in strain E . coli XC3. This allowed the construction of E . coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E . coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata , and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysC S301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Background: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO., Results: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM., Conclusion: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO., (© 2024. The Author(s).)

Core histones in the nucleosome are subject to a wide variety of posttranslational modifications (PTMs), such as methylation, phosphorylation, ubiquitylation, and acetylation, all of which are crucial in shaping the structure of the chromatin and the expression of the target genes. A putative histone methyltransferase LaeA/Lae1, which is conserved in numerous filamentous fungi, functions as a global regulator of fungal growth, virulence, secondary metabolite formation, and the production of extracellular glycoside hydrolases (GHs). LaeA's direct histone targets, however, were not yet recognized. Previous research has shown that LaeA interacts with core histone H2B. Using S-adenosyl-L-methionine (SAM) as a methyl group donor and recombinant human histone H2B as the substrate, it was found that Penicillium oxalicum LaeA can transfer the methyl groups to the C-terminal lysine (K) 108 and K116 residues in vitro. The H2BK108 and H2BK116 sites on recombinant histone correspond to P. oxalicum H2BK122 and H2BK130, respectively. H2B K122A and H2B K130A , two mutants with histone H2B K122 or K130 mutation to alanine (A), were constructed in P. oxalicum. The mutants H2B K122A and H2B K130A demonstrated altered asexual development and decreased extracellular GH production, consistent with the findings of the laeA gene deletion strain (ΔlaeA). The transcriptome data showed that when compared to wild-type (WT) of P. oxalicum, 38 of the 47 differentially expressed (fold change ≥ 2, FDR ≤ 0.05) genes that encode extracellular GHs showed the same expression pattern in the three mutants ΔlaeA, H2B K122A , and H2B K130A . The four secondary metabolic gene clusters that considerably decreased expression in ΔlaeA also significantly decreased in H2B K122A or H2B K130A . The chromatin of promotor regions of the key cellulolytic genes cel7A/cbh1 and cel7B/eg1 compacted in the ΔlaeA, H2B K122A , and H2B K130A mutants, according to the results of chromatin accessibility real-time PCR (CHART-PCR). The chromatin accessibility index dropped. The histone binding pocket of the LaeA-methyltransf_23 domain is compatible with particular histone H2B peptides, providing appropriate electrostatic and steric compatibility to stabilize these peptides, according to molecular docking. The findings of the study demonstrate that H2BK122 and H2BK130, which are histone targets of P. oxalicum LaeA in vitro, are crucial for fungal conidiation, the expression of gene clusters encoding secondary metabolites, and the production of extracellular GHs., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Brucellosis, caused by intracellular gram-negative pathogens of the genus Brucella, continues to be one of the most pandemic zoonotic diseases in most countries. At present, the therapeutic treatment of brucellosis relies on a combination of multiple antibiotics that involves a long course of treatment, easy relapse, and high side effects from the use of certain antibiotics (such as streptomycin). Thus, the need to identify novel drugs or targets to control this disease is urgent. Diaminopimelate decarboxylase (DAPDC), a key enzyme involved in the bacterial diaminopimelate (DAP) biosynthetic pathway, was suggested to be a promising anti-Brucella target in our previous study. In this work, the biological activity of Brucella melitensis DAPDC was characterized, and a library of 1,591 compounds was screened for inhibitors of DAPDC. The results of a high-throughput screening (HTS) assay showed that 24 compounds inhibited DAPDC activity. In a further in vitro bacterial inhibition experiment, five compounds exhibited anti-Brucella activity (SID3, SID4, SID14, SID15, and SID20). These results suggested that the identified compounds can be used as potent molecules against brucellosis and that the application ranges of these approved drugs can be expanded in the future.

Brucellosis, caused by intracellular gram-negative pathogens of the genus Brucella , continues to be one of the most pandemic zoonotic diseases in most countries. At present, the therapeutic treatment of brucellosis relies on a combination of multiple antibiotics that involves a long course of treatment, easy relapse, and high side effects from the use of certain antibiotics (such as streptomycin). Thus, the need to identify novel drugs or targets to control this disease is urgent. Diaminopimelate decarboxylase (DAPDC), a key enzyme involved in the bacterial diaminopimelate (DAP) biosynthetic pathway, was suggested to be a promising anti- Brucella target in our previous study. In this work, the biological activity of Brucella melitensis DAPDC was characterized, and a library of 1,591 compounds was screened for inhibitors of DAPDC. The results of a high-throughput screening (HTS) assay showed that 24 compounds inhibited DAPDC activity. In a further in vitro bacterial inhibition experiment, five compounds exhibited anti- Brucella activity (SID3, SID4, SID14, SID15, and SID20). These results suggested that the identified compounds can be used as potent molecules against brucellosis and that the application ranges of these approved drugs can be expanded in the future. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. Databases: PDB ID: 6P90. [ABSTRACT FROM AUTHOR]

The hyperthermophilic archaeon, Sulfolobus, synthesizes lysine via the α‐aminoadipate pathway; however, the gene encoding homocitrate synthase, the enzyme responsible for the first and committed step of the pathway, has not yet been identified. In the present study, we identified saci_1304 as the gene encoding a novel type of homocitrate synthase fused with a Regulation of Amino acid Metabolism (RAM) domain at the C terminus in Sulfolobus acidocaldarius. Enzymatic characterization revealed that Sulfolobus homocitrate synthase was inhibited by lysine; however, the mutant enzyme lacking the RAM domain was insensitive to inhibition by lysine. The present results indicated that the RAM domain is responsible for enzyme inhibition. [ABSTRACT FROM AUTHOR]

Fresh and dried Cordyceps sinensis are widely used by the public for medicinal and health purposes. However, the differences between them have not been examined. In this study, fresh wild and artificial C. sinensis (WFC and AFC) were dried to obtain dried wild and artificial C. sinensis (WDC and ADC). Non-targeted GC-MS was used to analyze the metabolic profile characteristics of the four groups of samples. The results showed that air drying significantly altered the composition and content of C. sinensis , mainly in the form of higher abundance of organic acids and derivatives and lower abundance of lipids and lipid-like molecules in fresh C. sinensis . Hierarchical cluster analysis (HCA) and quantitative analyses showed that air drying increased the abundance of Valine, Zinniol, Urocanate, Vulpinic acid, and Uridine 5'-diphosphate, and decreased Xanthotoxol, Vitexin-4-o-glucoside, Val-trp, and Wogonin. These differentially accumulated metabolites (DAMs) were also shown to be potential biomarkers for C. sinensis . KEGG enrichment analysis identified lysine biosynthesis as the most significantly enriched pathway. Annotation of these DAMs to lysine biosynthesis revealed that citrate cycle and pyruvate metabolism entered lysine biosynthesis via 2-oxohlutarate and Homocitrate, respectively, resulting in significant enrichment of L-saccharopine and L-lysine content was significantly higher. Alanine, aspartate, and Glutamate metabolism synthesized more L-aspartate to promote L-lysine synthesis. Thus, high levels of L-lysine result in lysine degradation and pymolysine, which are the most active metabolic pathways during the drying of fresh C. sinensis and indirectly lead to differences in metabolic profiles., Competing Interests: The authors declare no conflicts of interest.

Fungi uniquely synthesize lysine through the α-aminoadipate pathway. The saccharopine reductase ScLys9 catalyzes the formation of saccharopine from ɑ-aminoadipate 6-semialdehyde, the seventh step in the lysine biosynthesis pathway in Saccharomyces cerevisiae. Here, we characterized the functions of TrLys9, an ortholog of S. cerevisiae ScLys9 in the industrial filamentous fungus Trichoderma reesei. Transcriptional level analysis indicated that TrLYS9 expression was higher in the conidial stage than in other stages. Disruption of TrLYS9 led to lysine auxotrophy. Phenotype analysis of the ΔTrlys9 mutant showed that TrLYS9 was involved in fungal development including vegetative growth, conidiation, and conidial germination and lysine biosynthesis. Cellulase production was also impaired in the ΔTrlys9 mutant due to the failure of conidial germination in liquid cellulase-inducing medium. Defects in radial growth and asexual development of the ΔTrlys9 mutant were fully recovered when exogenous lysine was added to the medium. These results imply that TrLys9 is involved in fungal development and lysine biosynthesis in T. reesei.

Thermus thermophilus biosynthesizes lysine via α-aminoadipate as an intermediate using the amino-group carrier protein, LysW, to transfer the attached α-aminoadipate and its derivatives to biosynthetic enzymes. A gene named lysV, which encodes a hypothetical protein similar to LysW, is present in the lysine biosynthetic gene cluster. Although the knockout of lysV did not affect lysine auxotrophy, lysV homologs are conserved in the lysine biosynthetic gene clusters of microorganisms belonging to the phylum Deinococcus-Thermus, suggesting a functional role for LysV in lysine biosynthesis. Pulldown assays and crosslinking experiments detected interactions between LysV and all of the biosynthetic enzymes requiring LysW for reactions, and the activities of most of all these enzymes were affected by LysV. These results suggest that LysV modulates the lysine biosynthesis through protein-protein interactions.

Lysine (Lys) connects the mitochondrial electron transport chain to amino acid catabolism and the tricarboxylic acid cycle. However, our understanding of how a deficiency in Lys biosynthesis impacts plant metabolism and growth remains limited. Here, we used a previously characterized Arabidopsis mutant (dapat) with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase to investigate the physiological and metabolic impacts of impaired Lys biosynthesis. Despite displaying similar stomatal conductance and internal CO2concentration, we observed reduced photosynthesis and growth in the dapat mutant. Surprisingly, whilst we did not find differences in dark respiration between genotypes, a lower storage and consumption of starch and sugars was observed in dapat plants. We found higher protein turnover but no differences in total amino acids during a diurnal cycle in dapat plants. Transcriptional and two-dimensional (isoelectric focalization/SDS-PAGE) proteome analyses revealed alterations in the abundance of several transcripts and proteins associated with photosynthesis and photorespiration coupled with a high glycine/serine ratio and increased levels of stress-responsive amino acids. Taken together, our findings demonstrate that biochemical alterations rather than stomatal limitations are responsible for the decreased photosynthesis and growth of the dapat mutant, which we hypothesize mimics stress conditions associated with impairments in the Lys biosynthesis pathway. [ABSTRACT FROM AUTHOR]

Abstract 10-deacetyl-bacatin III (10-DAB) is a natural plant-derived taxane diterpene, whose antimicrobial activity against phytopathogens remains unknown. In this study, we demonstrated the antimicrobial effect of 10-DAB on plant-pathogenic oomycetes. Our results revealed that 10-DAB exhibited significant antimicrobial activities against test oomycetes, especially against Phytophthora capsici , with a median effective concentration (EC 50) of 1.46 μg/mL, but had no effect on test fungi. Under 10-DAB treatment, mycelia of P. capsici were contorted with an increased number of top branches, and the production and germination of zoospores were inhibited and delayed, respectively. In addition, 10-DAB had favorable protective and curative activities with control efficacies of 63.90% and 74.81% at 200 μg/mL on detached pepper leaves. Furthermore, 10-DAB caused a significant decrease in soluble protein, lysine, and α, ε-diaminopimelic acid content of P. capsici , which suggested that 10-DAB inhibited the lysine biosynthesis. On the contrary, treatment with exogenous lysine effectively counteracted 10-DAB's inhibition activity on P. capsici. Moreover, relative expression of four key lysine biosynthesis-related genes of P. capsici were decreased upon 10-DAB treatment. Taken together, our findings suggest a lysine biosynthesis inhibiting-dependent antimicrobial activity of 10-DAB against P. capsici , which contributes to accelerating the application of 10-DAB for successful management of phytophthora blight disease in agricultural production. Graphical abstract Unlabelled Image Highlights • 10-DAB inhibits growth of P. capsici in vitro as well as infection in planta. • The inhibitory effect of 10-DAB against P. capsici is dependent on impeding lysine biosynthesis. • Expression levels of genes involved in lysine biosynthesis were decreased upon 10-DAB treatment. [ABSTRACT FROM AUTHOR]

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Neisseria gonorrhoeae , an obligate human pathogen, is a leading cause of communicable diseases globally. Due to rapid development of drug resistance, the rate of successfully curing gonococcal infections is rapidly decreasing. Hence, research is being directed toward finding alternative drugs or drug targets to help eradicate these infections. 4-Hydroxy-tetrahydrodipicolinate reductase (DapB), an important enzyme in the meso -diaminopimelate pathway, is a promising target for the development of new antibiotics. This manuscript describes the first structure of DapB from N. gonorrhoeae determined at 1.85 Å. This enzyme uses NAD(P)H as cofactor. Details of the interactions of the enzyme with its cofactors and a substrate analog/inhibitor are discussed. A large scale bioinformatics analysis of DapBs' sequences is also described. [ABSTRACT FROM AUTHOR]

The class I aldolase dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the diaminopimelate (DAP) lysine biosynthesis pathway in bacteria, archaea and plants. Despite the existence, in databases, of numerous fungal sequences annotated as DHDPS, its presence in fungi has been the subject of contradictory claims. We report the characterization of DHDPS candidates from fungi. Firstly, the putative DHDPS from Coccidioides immitis (PDB ID: 3QFE ) was shown to have negligible enzyme activity. Sequence analysis of 3QFE showed that three out of the seven amino acid residues critical for DHDPS activity are absent; however, exact matches to catalytic residues from two other class I aldolases, 2-keto-3-deoxygluconate aldolase (KDGA), and 4-hydroxy-2-oxoglutarate aldolase (HOGA), were identified. The presence of both KDGA and HOGA activity in 3QFE was confirmed in vitro using enzyme assays, the first report of such dual activity. Subsequent analyses of all publically available fungal sequences revealed that no entry contains all seven residues important for DHDPS function. The candidate with the highest number of identities (6 of 7), KIW77228 from Fonsecaea pedrosoi , was shown to have trace DHDPS activity in vitro , partially restored by substitution of the seventh critical residue, and to be incapable of complementing DHDPS-deficient E. coli cells. Combined with the presence of all seven sequences for the alternative α-aminoadipate (AAA) lysine biosynthesis pathway in C. immitis and F. pedrosoi , we believe that DHDPS and the DAP pathway are absent in fungi, and further, that robust informed methods for annotating genes need to be implemented. [ABSTRACT FROM AUTHOR]

Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the pathway for the biosynthesis of L-lysine in most bacteria and plants. The substrates for the enzyme are pyruvate and L-aspartate-β-semialdehyde (ASA). The product of the reaction was originally proposed to be 2,3-dihydrodipicolinate (DHDP), but has now generally been assumed to be (4 S )-4-hydroxy-2,3,4,5-tetrahydro-(2 S )-dipicolinate (HTPA). ASA is unstable at high pH and it is proposed that ASA reacts with itself. At high pH ASA also reacts with Tris buffer and both reactions are largely reversible at low pH. It is proposed that the basic un-protonated form of the amine of Tris or the α-amine of ASA reacts with the aldehyde functional group of ASA to generate an imine product. Proton NMR spectra of ASA done at different pH values shows new NMR peaks at high pH, but not at low pH, confirming the presence of reaction products for ASA at high pH. The enzymatic product of the DHDPS reaction was examined at low pH by proton NMR starting with either 3 h-pyruvate or 3 d-pyruvate and identical NMR spectra were obtained with four new NMR peaks observed at 1.5, 2.3, 3.9 and 4.1 ppm in both cases. The NMR results were most consistent with DHDP as the reaction product. The UV -spectral studies of the DHDPS reaction shows the formation of an initial product with a broad spectral peak at 254 nM. The DHDPS reaction product was further examined by reduction of the enzymatic reaction components with borohydride followed by GC-MS analysis of the mixture. Three peaks were found at 88, 119 and 169  m/z , consistent with pyruvate, homoserine (reduction product of ASA), and the reduction product of DHDP (1,2,3,6-tetrahydropyridine-2,6-dicarboxylate). There was no indication for a peak associated with the reduced form of HTPA. [ABSTRACT FROM AUTHOR]

Aspartate β‐semialdehyde dehydrogenase (ASADH) is an enzyme involved in the diaminopimelate pathway of lysine biosynthesis. It is essential for the viability of many pathogenic bacteria and therefore has been the subject of considerable research for the generation of novel antibiotic compounds. This manuscript describes the first structure of ASADH from Francisella tularensis, the causative agent of tularemia and a potential bioterrorism agent. The structure was determined at 2.45 Å resolution and has a similar biological assembly to other bacterial homologs. ASADH is known to be dimeric in bacteria and have extensive interchain contacts, which are thought to create a half‐sites reactivity enzyme. ASADH from higher organisms shows a tetrameric oligomerization, which also has implications for both reactivity and regulation. This work analyzes the apo form of F. tularensis ASADH, as well as the binding of the enzyme to its cofactor NADP+. [ABSTRACT FROM AUTHOR]

Engineering of enzymes and pathways is generally required for the development of efficient strains for bioproduction processes. To this end, quantitative and reliable data of intracellular metabolites are highly desired, but often not available, especially for conditions more close to industrial applications, i.e. at high cell density and product concentration. Here, we investigated the intracellular metabolite profiles of an engineered l-lysine-producing Corynebacterium glutamicum strain and the corresponding wild-type strain to assess the impacts of deregulation of product inhibition of the key enzymes aspartate kinase and phosphoenolpyruvate carboxylase and to identify potentials for their further improvement. A bioreactor system with automated fast-sampling, filtration and on-filter quenching of the metabolism was used for a more reliable determination of intracellular metabolites in batch cultures with optical cell density (OD660) up to 40. The l-lysine-producing strain showed substantially different metabolite profiles in the amino acid metabolism, including increased intracellular pool sizes in the l-lysine-, l-homoserine- and l-threonine pathways and decreased intracellular pool sizes for all other determined amino acids. By comparing data of in vitro inhibition of the engineered enzymes and determined intracellular concentrations of the inhibitors it was found that the inferred in vivo activities of these enzymes are still significantly below their in vitro maximums. This work demonstrates the usefulness of metabolic analysis for assessing the impact of engineered enzymes and identifying targets for further strain development. [ABSTRACT FROM AUTHOR]

The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The Δ argW , Δ argX , Δ argB , and Δ argD mutant strains display an arginine auxotrophic phenotype, while the Δ dapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the Δ dapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea., Competing Interests: The authors declare no conflict of interest.

In bacteria, the second committed step in the diaminopimelate/lysine anabolic pathways is catalyzed by the enzyme dihydrodipicolinate reductase (DapB). DapB catalyzes the reduction of dihydrodipicolinate to yield tetrahydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of DapB from the human-pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are reported. Protein crystals were grown in conditions consisting of 5%( w/ v) PEG 4000, 200 m M sodium acetate, 100 m M sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.3 Å resolution. They belonged to space group P4322, with unit-cell parameters a = 109.38, b = 109.38, c = 176.95 Å. Rr.i.m. was 0.11, Rwork was 0.177 and Rfree was 0.208. The three-dimensional structural features of the enzymes show that DapB from B. henselae is a tetramer consisting of four identical polypeptides. In addition, the substrate NADP+ was found to be bound to one monomer, which resulted in a closed conformational change in the N-terminal domain. [ABSTRACT FROM AUTHOR]

ABSTRACT Evolution of transcriptional control is essential for organisms to cope with diversification into a spectrum of environments, including environments with limited nutrients. Lysine biosynthesis in fungi occurs in eight enzymatic steps. In Saccharomyces cerevisiae, amino acid starvation elicits the induction of LYS gene expression, mediated by the master regulator Gcn4 and the pathway-specific transcriptional regulator Lys14. Here, we have shown that the activation of LYS gene expression in the human fungal pathogen Candida albicans is predominantly controlled by Gcn4 under amino acid starvation conditions. Multiple lines of study showed that the four C. albicansLYS14-like genes have no role in the regulation of lysine biosynthesis. Whereas Gcn4 is dispensable for the growth of S. cerevisiae under lysine deprivation conditions, it is an essential regulator required for the growth of C. albicans under these conditions, as gcn4 deletion caused lysine auxotrophy. Gcn4 is required for the induction of increased LYS2 and LYS9 mRNA but not for the induction of increased LYS4 mRNA. Under lysine or isoleucine-valine deprivation conditions, Gcn4 recruitment to LYS2 and LYS9 promoters was induced in C. albicans. Indeed, in contrast to the S. cerevisiaeLYS gene promoters, all LYS gene promoters in C. albicans harbored a Gcn4 binding site but not all harbored the S. cerevisiae Lys14 binding site, indicating the evolutionary divergence of cis-regulatory motifs. Thus, the transcriptional rewiring of the lysine biosynthetic pathway in C. albicans involves not only neofunctionalization of the four LYS14-like genes but the attendant strengthening of control by Gcn4, indicating a coordinated response with a much broader scope for control of amino acid biosynthesis in this human pathogen. IMPORTANCE Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candida albicans. We show that although both Saccharomyces cerevisiae and C. albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C. albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S. cerevisiae pathway-specific regulator Lys14.

Flammulina filiformis , the most produced edible mushroom species in China, is rich in lysine. Further enhancing its lysine biosynthesis is vital for improving its quality in industrialized cultivation. Citric acid induction significantly increases both the biomass and growth rate of F. filiformis hyphae, as well as the lysine content. The genes encoding enzymes in the lysine biosynthesis pathway were detected under the optimal induction, revealing that the expression levels of hcs , hac , and hah were 2.67, 1.97, and 1.90 times greater, respectively, relative to the control, whereas no significant difference was seen for hdh , aat , sr , and shd , and the expression of aar decreased. Furthermore, the transcriptional levels of Ampk , GCN2 , GCN4 , and TOR were found significantly upregulated, with the most upregulated, Ampk , reaching a level 42.68 times greater than that of the control, while the phosphorylation of AMPK rose by nearly 54%. In AMPK-silencing strains under the optimal induction, however, the phosphorylation increment dropped to about 16% and the lysine content remained at the same level as in the WT. Thus, AMPK is presented as the critical intermediary in citric acid's regulation of lysine biosynthesis in F. filiformis .

This work reports the 13 C-assisted metabolic flux analysis of Haladaptatus paucihalophilus , a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus . In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1- 13 C]pyruvate and [2- 13 C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus , and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. [ABSTRACT FROM AUTHOR]

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 K M m e s o - DAP of 0.97 mM and 1.62 mM and a k cat 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 K M m e s o - DAP of 0.68 mM and a k cat 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. [ABSTRACT FROM AUTHOR]

Amino acid biosynthesis forms part of an integrated stress response against oxidants in Saccharomyces cerevisiae and higher eukaryotes. Here we show an essential protective role of the l-lysine biosynthesis pathway in response to the oxidative stress condition induced by the lipid oxidant-linoleic acid hydroperoxide (LoaOOH), by means of transcriptomic profiling and phenotypic analysis, and using the deletion mutant dal80∆ and lysine auxotroph lys1∆. A comprehensive up-regulation of lysine biosynthetic genes ( LYS1, LYS2, LYS4, LYS9, LYS12, LYS20 and LYS21) was revealed in dal80Δ following the oxidant challenge. The lysine auxotroph ( lys1∆) exhibited a significant decrease in growth compared with that of BY4743 upon exposure to LoaOOH, albeit with the sufficient provision of lysine in the medium. Furthermore, the growth of wild type BY4743 exposed to LoaOOH was also greatly reduced in lysine-deficient conditions, despite a full complement of lysine biosynthetic genes. Amino acid analysis of LoaOOH-treated yeast showed that the level of cellular lysine remained unchanged throughout oxidant challenge, suggesting that the induced lysine biosynthesis leads to a steady-state metabolism as compared to the untreated yeast cells. Together, these findings demonstrate that lysine availability and its biosynthesis pathway play an important role in protecting the cell from lipid peroxide-induced oxidative stress, which is directly related to understanding environmental stress and industrial yeast management in brewing, wine making and baking. [ABSTRACT FROM AUTHOR]

Grain amaranths, edible C4 dicots, produce pseudo-cereals high in lysine. Lysine being one of the most limiting essential amino acids in cereals and C4 photosynthesis being one of the most sought-after phenotypes in protein-rich legume crops, the genome of one of the grain amaranths is likely to play a critical role in crop research. We have sequenced the genome and transcriptome of Amaranthus hypochondriacus, a diploid (2n = 32) belonging to the order Caryophyllales with an estimated genome size of 466 Mb. Of the 411 linkage single-nucleotide polymorphisms (SNPs) reported for grain amaranths, 355 SNPs (86%) are represented in the scaffolds and 74% of the 8.6 billion bases of the sequenced transcriptome map to the genomic scaffolds. The genome of A. hypochondriacus, codes for at least 24,829 proteins, shares the paleohexaploidy event with species under the superorders Rosids and Asterids, harbours 1 SNP in 1,000 bases, and contains 13.76% of repeat elements. Annotation of all the genes in the lysine biosynthetic pathway using comparative genomics and expression analysis offers insights into the high-lysine phenotype. As the first grain species under Caryophyllales and the first C4 dicot genome reported, the work presented here will be beneficial in improving crops and in expanding our understanding of angiosperm evolution. [ABSTRACT FROM PUBLISHER]

Dihydrodipicolinate synthase (DHDPS, E.C.4.2.1.52) catalyzes the first committed step in the lysine biosynthetic pathway: the condensation of ( S)-aspartate semialdehyde and pyruvate to form (4 S)-4-hydroxy-2,3,4,5-tetrahydro-(2 S)-dipicolinic acid. Since ( S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, we report the crystal structure of DHDPS from a hyperthermophilic bacterium Aquifex aeolicus (AqDHDPS). l-Lysine is used as an important animal feed additive where the production is at the level of 1.5 million tons per year. The biotechnological manufacture of lysine has been going for more than 50 years which includes over synthesis and reverse engineering of DHDPS. AqDHDPS revealed a unique disulfide linkage which is not conserved in the homologues of AqDHDPS. In silico mutation of C139A and intermolecular ion-pair residues and the subsequent molecular dynamics simulation of the mutants showed that these residues are critical for the stability of AqDHDPS tetramer. MD simulations of AqDHDPS at three different temperatures (303, 363 and 393 K) revealed that the molecule is stable at 363 K. Thus, this structural and in silico study of AqDHDPS likely provides additional details towards the rational and structure-based design of hyper- l-lysine producing bacterial strains. [ABSTRACT FROM AUTHOR]