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Aims Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. Methods and Results Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2•–) and hydrogen peroxide (H2O2) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. Conclusions TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. Significance and Impact of the Study These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.

Increasing CO2 levels in the atmosphere and the resulting negative impacts of climate change have compelled global efforts to achieve carbon neutrality or negativity. Most such efforts focus on carbon sequestration through chemical or physical approaches. Harnessing the power of synthetic biology to enhance the natural ability of carbon sequestration in plants, especially non-annuals, provides a biological approach to further reduce CO2 levels in the air. Here, we selected a photorespiration bypass pathway and tested its effectiveness on photosynthetic enhancement in a hybrid poplar, INRA717-IB4. The design includes an RNAi strategy to reduce the transportation of the photorespiration byproduct, glycolate, out of chloroplast and a shunt pathway to metabolize the retained glycolate back to CO2 for fixation through the Calvin-Benson cycle. Molecular and physiological data collected from two separate growth experiments indicate that transgenic plants expressing genes in the photorespiration bypass pathway have increased photosynthetic efficiency, leading to faster plant growth and elevated biomass production. One lead transgenic event accumulated 35%–53% more above-ground dry biomass over four months of growth in a controlled environment. Our results provide a proof of concept for engineering trees to help combat climate change.

To survive and replicate in the host, S. Typhimurium have evolved several metabolic pathways. The glyoxylate shunt is one such pathway that can utilize acetate for the synthesis of glucose and other biomolecules. This pathway is a bypass of the TCA cycle in which CO2 generating steps are omitted. Two enzymes involved in the glyoxylate cycle are isocitrate lyase (ICL) and malate synthase (MS). We determined the contribution of MS in the survival of S. Typhimurium under carbon limiting and oxidative stress conditions. The ms gene deletion strain (∆ms strain) grew normally in LB media but failed to grow in M9 minimal media supplemented with acetate as a sole carbon source. However, the ∆ms strain showed hypersensitivity (p ms strain has been significantly more susceptible to neutrophils. Interestingly, several folds induction of ms gene was observed following incubation of S. Typhimurium with neutrophils. Further, ∆ms strain showed defective colonization in poultry spleen and liver. In short, our data demonstrate that the MS contributes to the virulence of S. Typhimurium by aiding its survival under carbon starvation and oxidative stress conditions.

Trichophyton mentagrophytes secretes Metallocarboxypeptidase A and B of the M14 family as endoproteases and exoprotease. T. mentagrophytes produce Metalloprotease 3 and 4 which degrades the protein into the short peptides and amino acids. To understand the host fungal relationship and identification of such genes expressed during infection is utmost important. T. mentagrophytes encodes some proteins which are associated with the glyoxylate cycle. The glyoxylate cycle enzymes have been involving in virulence of dermatophytes and their up-regulation during dermatophytes growth on keratin. On comparing the expression level of virulence protease and non-protease genes, we observed, among exoprotease protease genes, Metallocarboxypeptidase B was strongly up regulated (134.6 fold high) followed by Metallocarboxypeptidase A (115.6 fold high) and Di-peptidyl-peptidases V (10.1 fold high), in dermatophytic patients as compared to ATCC strain. Furthermore, among endoprotease, Metalloprotease 4 was strongly up regulated (131.6 fold high) followed by Metalloprotease 3 (16.7 fold high), in clinical strains as compared to T. mentagrophytes ATCC strain. While among non-protease genes, Citrate Synthase was highly expressed (118 fold high), followed by Isocitrate Lyase (101.6 fold high) and Malate Synthase (52.9 fold high). All the studied virulence genes were considered the best suitable ones by geNorm, Best keeper, Norm Finder and Ref finder.

Significance The Calvin cycle is the most important carbon fixation pathway in the biosphere. However, its carboxylating enzyme Rubisco also accepts oxygen, thus producing 2-phosphoglycolate. Phosphoglycolate salvage pathways were extensively studied in photoautotrophs but remain uncharacterized in chemolithoautotrophs using the Calvin cycle. Here, we study phosphoglycolate salvage in the chemolithoautotrophic model bacterium Cupriavidus necator H16. We demonstrate that this bacterium mainly reassimilates 2-phosphoglycolate via the glycerate pathway. Upon disruption of this pathway, a secondary route, which we term the malate cycle, supports photorespiration by completely oxidizing 2-phosphoglycolate to CO2. While the malate cycle was not previously known to metabolize 2-phosphoglycolate in nature, a bioinformatic analysis suggests that it may support phosphoglycolate salvage in diverse chemoautotrophic bacteria., Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term “phosphoglycolate salvage.” Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

Background Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. Results The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. Conclusions The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.

The paper is the fundamental beginning of research series aimed at understanding the processes associated with high performance in higher animals. The research aim is to study correlation of dicarboxylic acid cycle with tricarboxylic acid cycle with establishment of activity and dislocation of enzymes, confirming the hypothesis of availability and active metabolic participation of peroxisome in highly productive animals. Research was conducted on the basis of the VNIIFBiP animal vivarium in 2019 with a group of piglets of the Irish Landrace breed (n = 10). After slaughter at the age of 210 days, the nuclear (with large tissue particles), mitochondrial and postmitochondrial fractions of the liver were studied with assessment of succinate dehydrogenase and activity of other dehydrogenes of the Krebs cycle. It was found that peroxisomes act as universal agents of communication and cooperation, and microtelets are able to generate various chemical signals that carry information, to control and arrange a number of mechanisms in the metabolic processes in the body. Despite the fact that the Krebs cycle dehydrogenases are considered mitochondrial enzymes, the experiment showed an increase in activity of priruvate dehydrogenase (P > 0.1), isocitrate dehydrogenase (0.1 > P > 0.05) and malate dehydrogenase (0.1 > P > 0.05), which, when comparing the mitochondrial and postmitochondrial fractions, indicates a higher activity of peroxisomal fractions. The peroxisome localization place is the postmitochondrial fraction, and the lower layer contains larger peroxisomes to a greater extent, while the upper layer contains smaller ones. It was found that indicator enzymes of glyoxylate cycle isocitratliase and malate synthase exhibit catalytic activity in the peroxisomal fraction of liver of highly productive pigs. The obtained data on functioning of key glyoxylate cycle enzymes and their intracellular compartmentalization in highly productive pigs allow learning more about the specifics of metabolism and its regulation processes. Application of this knowledge in practice opens up prospects for rationalizing the production of livestock products of increased quantity, improved quality with less feed, labor and financial resources spent.

Background and Purpose: Candida albicans is the fourth most common cause of nosocomial fungal infections across the world. The current drug regimens are suffering from such drawbacks as drug resistance, toxicity, and costliness; accordingly, they highlight the need for the discovery of novel drug agents. The metabolic adaptability under low-carbon conditions and expression of functional virulence traits mark the success of pathogens to cause infection. The metabolic pathways, such as glyoxylate cycle (GC), enable C. albicans to survive under glucose-deficient conditions prevalent in the hostile niche. Therefore, the key enzymes, namely isocitrate lyase (ICL) and malate synthase (MLS), represent attractive agents against C. albicans. Similarly, virulence traits, such as morphogenesis and biofilm formation, are the crucial determinants of C. albicans pathogenicity. Regarding this, the present study was conducted to uncover the role of vanillin (Van), a natural food flavoring agent, in inhibiting GC, yeast-to-hyphal transition, and biofilm formation in human fungal pathogen C. albicans. Materials and Methods: For the determination of hypersensitivity under low-glucose conditions, phenotypic susceptibility assay was utilized. In addition, enzyme activities were estimated based on crude extracts while in-silico binding was confirmed by molecular docking. The assessment of morphogenesis was accomplished using hyphalinducing media, and biofilm formation was estimated using calcofluor staining, MTT assay, and biomass measurement. Additionally, the in vivo efficacy of Van was demonstrated using Caenorhabditis elegans nematode model. Results: Based on the results, Van was found to be a potent GC inhibitor that phenocopied ICL1 deletion mutant and displayed hypersensitivity under low-carbon conditions. Accordingly, Van facilitated the inhibition of ICL and MLS activities in vitro. Molecular docking analyses revealed the in-silico binding affinity of Van with Icl1p and Mls1p. Those analyses were also confirmative of the binding of Van to the active sites of both proteins with better binding energy in comparison to their known inhibitors. Furthermore, Van led to the attenuation of such virulence traits as morphogenesis, biofilm formation, and cell adherence. Finally, the antifungal efficacy of Van was demonstrated by the enhanced survival of C. elegans with Candida infection. The results also confirmed negligible hemolytic activity on erythrocytes. Conclusion: As the findings of the present study indicated, Van is a persuasive natural compound that warrants further attention to exploit its anticandidal potential

Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl–CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.

Simple Summary In this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages. The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that thirty-six FAAs were identified, which increased drastically with fruit development until ripening. During the period of rapid fruit expansion, the aroma of XC changed from light to strong, and the contents of L-alanine and L-leucine were significantly higher than those of SX and LDB. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars. Abstract Amino acids are important component of fruit nutrition and quality. In this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages (S1–S6). The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that 36 FAAs were identified, and the total FAAs content ranged from 2601.0 to 9073.5 mg/kg, which increased drastically with fruit development until ripening. L-glutamic acid (Glu), L-alanine (Ala), L-arginine (Arg), γ-Aminobutyric acid (GABA), L-aspartic acid (Asp), L-leucine (Leu), hydroxyl-proline (Hypro), and L-serine (Ser) were the predominant FAAs (1619.9–7213.9 mg/kg) in pulp, accounting for 62.28–92.05% of the total amino acids. During the period of rapid fruit expansion (S2–S4), the aroma of XC changed from light to strong, and the contents of L-alanine (Ala) and L-leucine (Leu) were significantly higher than those of SX and LDB. Furthermore, a total of two 2-isopropyl malate synthase (IPMS), two 3-isopropyl malate dehydrogenase (IPMD), and 16 BCAA transferase (BCAT) genes were identified. The expression levels of DilBCAT1, -6, and -9 genes in XC were significantly higher than those in SX and LDB, while DilBCAT16 in XC was lower. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars.

Optimized NMR experiments are developed for isolating magnetization belonging to the I=1/2 manifolds of (13)CH(3) methyl groups in proteins, enabling the manipulation of the magnetization of a (13)CH(3) moiety as if it were an AX ((1)H-(13)C) spin-system. These experiments result in the same ‘simplification’ of a (13)CH(3) spin-system that would be obtained from the production of {(13)CHD(2)}-methyl-labeled protein samples. The sensitivity of I=1/2 manifold-selection experiments is a factor of approximately 2 less than that of the corresponding experiments acquired on {(13)CHD(2)}-labeled methyl groups. The methodology described here is primarily intended for small-to-medium sized proteins, where the losses in sensitivity associated with the isolation of I=1/2 manifold transitions can be tolerated. Several NMR applications that benefit from simplification of the (13)CH(3) (AX(3)) spin-systems are described, with an emphasis on the measurements of methyl (1)H-(13)C residual dipolar couplings in a {(13)CH(3)}-methyl-labeled deletion mutant of the human chaperone DNAJB6b, where modulation of NMR signal intensities due to evolution of methyl (1)H-(13)C scalar and dipolar couplings follows a simple cosine function characteristic of an AX ((1)H-(13)C) spin-system, significantly simplifying data analysis.

The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.

Polymalic acid (PMA) is a novel biopolymer produced by the polyextremotolerant fungus Aureobasidium pullulans. In this study, a GATA-family transcriptional factor, Gat1, which regulates nitrogen uptake and PMA biosynthesis, was investigated. PMA production increased to 11.2% in the mutant overexpressing gat1 but decreased to 49.1% of the PMA titre when gat1 was knocked out from the genome of A. pullulans. Comparative transcriptome analysis of wild-type and mutant strains (∆gat1 and OE::gat1) revealed that 23 common differentially expressed genes were related to oxidative phosphorylation, ribosome biogenesis, and nitrogen metabolism. Under nitrogen-limited stress, regardless of the preferred nitrogen (glutamine, Gln) or non-preferred nitrogen (proline, Pro), 70% of Gat1 in the cells was located in the nucleus-cytoplasm, which resulted in an increase in nitrogen uptake and PMA biosynthesis regulation. Quantitative RT-PCR revealed that glucosekinase (GLK) in the glycolytic pathway and malate synthase (MLS) in the glyoxylate shunt pathway may be cross-regulated by Gat1 and nitrogen concentration (Gln or Pro), Therefore, glk was overexpressed in mutant strain (OE::gat1), which resulted in an increased PMA titre and yield of 12.6% and 13.0% respectively. These findings indicate that Gat1 may play an important role in the dual regulation of the nitrogen and carbon metabolisms in PMA biosynthesis.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

This work inserts in the research field regarding the effects of altered gravity conditions on biological plant processes. Pinus pinea seeds germination was studied in simulated microgravity (2x10−3g) and hypergravity (20g) conditions. The effects of simulated gravity were evaluated monitoring the levels of the key enzymes, involved in the main metabolic pathway during germination process of lipid–rich seeds (oilseeds): isocitrate lyase and malate synthase for glyoxylate cycle, 3-hydroxyacyl-CoA dehydrogenase for beta-oxidation, isocitrate dehydrogenase for Krebs cycle, pyruvate kinase for glycolysis and glucose 6 phosphate dehydrogenase for pentose phosphate shunt. The simulated micro and hypergravity conditions were obtained by a Random Position Machine and a Hyperfuge, respectively. Results show that the levels of some tested enzymes, at different lag times of the germination process, have the same trend of controls (g = 1), but with significant differences from quantitative point of view. They are higher in microgravity conditions and lower in hypergravity ones, suggesting that, from a biochemical point of view, the germination process results accelerated in microgravity conditions and delayed in hypergravity ones. These biochemical results show a good correlation with morphological ones, obtained with the measurement of the length of the seeds sprouting radicle. These results give promising indications regarding the possibility to grow plant with lipid-rich seeds in spatial environment, to obtain food sources for astronauts during long term space missions and to reconstitute new atmosphere.

Summary • The ectomycorrhiza is a symbiotic organ formed between a filamentous fungus and a plant root, mainly tree roots. Root colonization involves significant shifts in gene expression resulting in metabolic and structural changes in the fungus, including growth toward the plant root, penetration and establishment of the symbiotic organ. • The preinfection stage of the association is crucial as changes that occur throughout mycorrhiza formation are set in motion. Using an in vitro system for identifying preinfection stage symbiosis-regulated genes from the Laccaria bicolor–Pinus resinosa interaction we have identified a malate synthase from L. bicolor (Lb-MS). • The glyoxylate pathway, of which malate synthase is an enzyme, acts as a tricarboxylic acid pathway bypass generating four-carbon compounds for biosynthesis. While it is anticipated that malate synthase would be a part of the genetic and metabolic machinery of any fungus, Lb-MS is of interest because it is symbiosis regulated. • Lb-MS is regulated through interaction between the fungus and the host, by glucose and by the presence of other carbon sources in the medium. Its proposed role in the symbiosis is in the utilization of two carbon compounds formed from catabolic processes in early interaction facilitating hyphal net growth.

The number and type of isoforms of superoxide dismutase (SOD) and their activities were compared in mitochondria and peroxisomes isolated from cotyledons of three different oilseed seedlings. Mitochondrial and peroxisomal isoforms of SOD could be distinguished in nondenaturing polyacrylamide gels by their differential sensitivities to KCN and/or H2O2. The type of SOD was not the same for each organelle in each of the three oilseed species. For example, a single Mn–SOD was found in cotton and cucumber mitochondria, whereas four CuZn–SODs were present in mitochondria from sunflower. At least one CuZn–SOD isoform was found in the peroxisomes of all three species. Cucumber peroxisomes contained both a CuZn–SOD and a Mn–SOD, cotton peroxisomes contained a single CuZn–SOD, whilst four separate CuZn–SODs, but no Mn–SOD were found in sunflower peroxisomes. Using antibodies against CuZn–SOD from watermelon peroxisomes or from chloroplasts of Equisetum, a single polypeptide of c. 16·5 kDa was detected on immunoblots of peroxisomal fractions from the three species. Post-embedment, electron-microscopic double immunogold-labelling showed that CuZn–SOD, with malate synthase used as marker enzyme of peroxisomes, was localized in the matrix of these organelles of all three species. These results suggest that CuZn–SOD is a characteristic matrix enzyme of peroxisomes in oilseed cotyledons.

Background and Purpose: The increment in fungal infections, particularly due to Candida species, is alarming due to the emergence of multidrug resistance (MDR). Hence, the identification of novel drug targets to circumvent the problem of MDR requires immediate attention. The metabolic pathway, such as glyoxylate cycle (GC), which utilizes key enzymes (isocitrate lyase [ICL] and malate synthase [MLS]), enables C. albicans to adapt under glucose-deficient conditions. This study uncovers the effect of GC disruption on the major MDR mechanisms of C. albicans as a human pathogenic fungus. Materials and Methods: For the purpose of the study, efflux pump activity was assessed by phenotypic susceptibilities in the presence of substrates rhodamine 6G (R6G) and Nile red, along with R6G extracellular concentration (527 nm). In addition, ergosterol content was estimated by the alcoholic potassium hydroxide hydrolysis method. The estimation of chitin was also accomplished by the absorbance (520 nm) of glucosamine released by acid hydrolysis. Results: The results revealed that the disruption of ICL enzyme gene (Δicl1) led to the impairment of the efflux activity of multidrug transporters belonging to the ATP-binding cassette superfamily. It was further shown that Δicl1 mutant exhibited diminished ergosterol and chitin contents. In addition, all abrogated phenotypes could be rescued in the reverting strain of Δicl1 mutant. Conclusion: Based on the findings, the disruption of GC affected efflux activity and the synthesis of ergosterol and chitin. The present study for the first time revealed that metabolic fitness was associated with functional drug efflux, ergosterol and chitin biosynthesis and validated GC as an antifungal target. However, further studies are needed to comprehend and exploit this therapeutic opportunity.

The composition and content of sugar play a pivotal role in goji berry (Lycium barbarum L.) fruits, determining fruit quality. Long-term exposure of goji berry to elevated CO2 (eCO2) was frequently demonstrated to reduce sugar content and secondary metabolites. In order to understand the regulatory mechanisms and improve the quality of fruit in the changing climate, it is essential to characterize sugar metabolism genes that respond to eCO2. The objectives of this study were to clone full-length cDNA of three sugar metabolism genes—LBGAE (Lycium barbarum UDP-glucuronate 4-epimerase), LBGALA (Lycium barbarum alpha-galactosidase), and LBMS (Lycium barbarum malate synthase)—that were previously identified responding to eCO2, and to analyze sequence characteristics and expression regulation patterns. Sugar metabolism enzymes regulated by these genes were also estimated along with various carbohydrates from goji berry fruits grown under ambient (400 μmol mol−1) and elevated (700 μmol mol−1) CO2 for 90 and 120 days. Homology-based sequence analysis revealed that the protein-contained functional domains are similar to sugar transport regulation and had a high sequence homology with other Solanaceae species. The sucrose metabolism-related enzyme’s activity varied significantly from ambient to eCO2 in 90-day and 120-day samples along with sugars. This study provides fundamental information on sugar metabolism genes to eCO2 in goji berry to enhance fruit quality to climate change.

Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3 % and 31.8 %, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1ΔGAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1Δkcs1ΔGAPmK. The level of SAM in Ymls1Δkcs1ΔGAPmK reached 2.89 g L−1 in a 250-mL flask and 8.86 g L−1 in a 10-L fermentation tank, increasing 30.2 % and 46.2 %, respectively, compared to those levels in Ymls1ΔGAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

Background Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. Methods In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. Results Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. Conclusion Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

The stramenopile alga Nannochloropsis evolved by secondary endosymbiosis of a red alga by a heterotrophic host cell and emerged as a promising organism for biotechnological applications, such as the production of polyunsaturated fatty acids and biodiesel. Peroxisomes play major roles in fatty acid metabolism but experimental analyses of peroxisome biogenesis and metabolism in Nannochloropsis are not reported yet. In fungi, animals, and land plants, soluble proteins of peroxisomes are targeted to the matrix by one of two peroxisome targeting signals (type 1, PTS1, or type 2, PTS2), which are generally conserved across kingdoms and allow the prediction of peroxisomal matrix proteins from nuclear genome sequences. Because diatoms lost the PTS2 pathway secondarily, we investigated its presence in the stramenopile sister group of diatoms, the Eustigmatophyceae, represented by Nannochloropsis. We detected a full-length gene of a putative PEX7 ortholog coding for the cytosolic receptor of PTS2 proteins and demonstrated its expression in Nannochloropsis gaditana. The search for predicted PTS2 cargo proteins in N. gaditana yielded several candidates. In vivo subcellular targeting analyses of representative fusion proteins in different plant expression systems demonstrated that two predicted PTS2 domains were indeed functional and sufficient to direct a reporter protein to peroxisomes. Peroxisome targeting of the predicted PTS2 cargo proteins was further confirmed in Nannochloropsis oceanica by confocal and transmission electron microscopy. Taken together, the results demonstrate for the first time that one group of stramenopile algae maintained the import pathway for PTS2 cargo proteins. To comprehensively map and model the metabolic capabilities of Nannochloropsis peroxisomes, in silico predictions needs to encompass both the PTS1 and the PTS2 matrix proteome.

A cDNA clone encoding malate synthase (MS; EC 4.1.3.2) was isolated from a 48-h postharvest asparagus (Asparagus officinalis L.) spear cDNA library using a MS clone from Brassica napus. The asparagus MS (AoMS1) cDNA hybridized to a 1.9-kb transcript that increased in abundance preferentially in spear-tip tissue during postharvest storage. The AoMS1 transcript also accumulated during natural foliar senescence of asparagus fern. The cDNA consists of 1960 nucleotides with an open reading frame of 1665 nucleotides or 555 amino acids, and encodes a deduced protein with a predicted Mr of 63 kDa and a pI of 8.1. The deduced amino acid sequence of AoMS1 showed high identity with the B. napus MS clone (77.2%) used to isolate it, and with MS from cucumber (77%). Genomic Southern analysis suggests that a single gene in asparagus encodes AoMS1. Controlled- atmosphere treatments aimed at reducing deterioration of harvested asparagus spears reduced the expression of AoMS1. The reduction was correlated with the reduced oxygen level, and reduced MS enzyme activity was also observed. Asparagus cell cultures were used to test the role of sugar status in regulating AoMS1 gene expression. In cultures without sucrose there was an accumulation of AoMS1 transcript that was absent in cultures containing sucrose.

Orchid (Orchidaceae) is one of the largest families in angiosperms and presents exceptional diversity in lifestyle. Their unique reproductive characteristics of orchid are attracted by scientist for centuries. One of the synapomorphies of orchid plants is that their seeds do not contain endosperm. Lipids are used as major energy storage in orchid seeds. However, regulation and mobilization of lipid usage during early seedling (protocorm) stage of orchid is not understood. In this study, we compared transcriptomes from developing Phalaenopsis aphrodite protocorms grown on 1/2-strength MS medium with sucrose. The expression of P. aphrodite MALATE SYNTHASE (PaMLS), involved in the glyoxylate cycle, was significantly decreased from 4 days after incubation (DAI) to 7 DAI. On real-time RT-PCR, both P. aphrodite ISOCITRATE LYASE (PaICL) and PaMLS were down-regulated during protocorm development and suppressed by sucrose treatment. In addition, several genes encoding transcription factors regulating PaMLS expression were identified. A gene encoding homeobox transcription factor (named PaHB5) was involved in positive regulation of PaMLS. This study showed that sucrose regulates the glyoxylate cycle during orchid protocorm development in asymbiotic germination and provides new insights into the transcription factors involved in the regulation of malate synthase expression.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Location of the proteases would affect on protease stability and photorespiratory bypass pathway, while it is unsolved. Potato rbcS signal peptide was analyzed and constructed into the protease for study of their localization site. The tartronate semialdehyde reductase (EcTSR) proteins could be accurately and efficiently located in chloroplast only when this signal peptide was extended to 80 amino acids. The signal peptide would help malate synthase (CmMS) locate to the surface of chloroplast, to form granules on the outer membrane of chloroplast. The whole spectrum scanning showed that these proteins could enter chloroplast. A signal peptide named PCS1 (Peptide of self-cleavage site 1) carrying a self-cleavage site was designed, and sixteen amino acids from the blue pigment precursor protein of chloroplast positioning signal of Silene pratensis were added to the C-terminal of PCS1. Transient expression, Western blot analysis and full-spectrum scanning showed that PCS1 could locate the EcTSR to the chloroplast, after the removal of the signal peptide.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Background Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations. Results Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low. Conclusion We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

Mycobacterium tuberculosis remains one of the most successful bacterial pathogens worldwide. The development of drug-resistant strains and the ability of the bacteria to persist in a latent form in the host are major problems for tuberculosis (TB) control. Glyoxylate shunt is a metabolic bypass of the Krebs cycle and is the key for M. tuberculosis to survive under latent conditions. Malate synthase (MtbMS) catalyzes the second step of the glyoxylate cycle and converts glyoxylate into malate. Phenyl-diketo acid (PDKA) is a potent inhibitor of MtbMS, and its efficacy is validated in a mouse model of TB. To identify novel PDKA analogs as anti-TB compounds, PDKA analogs that obeyed the Lipinski rules (n = 5473) were analyzed and docked with MtbMS structure in three sequential modes. These compounds were then assessed for ADMET parameters. Of the compounds examined, 19 were found to fit well for redocking studies. After optimization, four prospective inhibitors were identified, that along with the reference compound PDKA were subjected to 50 ns molecular dynamics simulation and binding-free energy analyses to evaluate the complex dynamics after ligand binding, the stability of the bound complexes, and the intermolecular interactions between the complexes. The MtbMS-PDKA complex showed the binding free energy of –57.16 kJ·mol−1. After a thorough analysis, our results suggested that three compounds which had binding-free energy of –127.96, −97.60, and –83.98 kJ·mol−1, with PubChem IDs 91937661, 14016246, and 126487337, respectively, have the potential to inhibit MtbMS and can be taken as lead compounds for drug discovery against TB. Communicated by Ramaswamy H. Sarma

International audience; Methyl groups are valuable probes for solution NMR, allowing the investigation of large protein complexes. Among the six methyl containing residues, threonine has one of the less hydrophobic side chain, and can reside both within the interior of a protein or on the protein surface. This article presents an efficient mixed chemical/enzymatic synthesis scheme, enabling the preparation of threonine with natural configuration on the two stereogenic centers together with an optimal introduction of 1H/2H and 12C/13C atoms at specific sites. Such specifically labelled amino acid can be efficiently incorporated in overexpressed proteins without scrambling or in combination with any other types of 13CH3 probes. Additionally we report application to the 82 kDa Malate Synthase G. Our findings demonstrate that structural meaningful long range nOes can be detected between threonine methyl probes and methionine and isoleucine methyl groups distant by 12 Å.

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles, however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.

Persistent infection by Mycobacterium tuberculosis requires the glyoxylate shunt. This is a bypass to the tricarboxylic acid cycle in which isocitrate lyase (ICL) and malate synthase (MS) catalyze the net incorporation of carbon during mycobacterial growth on acetate or fatty acids as the primary carbon source. To identify a potential antitubercular compound, we performed a structure-based screening of natural compounds from the ZINC database (n = 1 67 740) against the M tuberculosis MS (MtbMS) structure. The ligands were screened against MtbMS, and 354 ligands were found to have better docking score. These compounds were assessed for Lipinski and absorption, distribution, metabolism, excretion, and toxicity prediction where 15 compounds were found to fit well for redocking studies. After refinement by molecular docking and drug-likeness analysis, four potential inhibitors (ZINC1483899, ZINC1754310, ZINC2269664, and ZINC15729522) were identified. These four ligands with phenyl-diketo acid were further subjected to molecular dynamics simulation to compare the dynamics and stability of the protein structure after ligand binding. The binding energy analysis was calculated to determine the intermolecular interactions. Our results suggested that the four compounds had a binding free energy of -201.96, -242.02, -187.03, and -169.02 kJ·mol-1 , for compounds with IDs ZINC1483899, ZINC1754310, ZINC2269664, and ZINC15729522, respectively. We concluded that two compounds (ZINC1483899 and ZINC1754310) displayed considerable structural and pharmacological properties and could be probable drug candidates to fight against M tuberculosis parasites.

Human infection by Mycobacterium tuberculosis (Mtb) continues to be a global epidemic. Computer-aided drug design (CADD) methods are used to accelerate traditional drug discovery efforts. One noncovalent interaction that is being increasingly identified in biological systems but is neglected in CADD is the anion-π interaction. The study reported herein supports the conclusion that anion-π interactions play a central role in directing the binding of phenyl-diketo acid (PDKA) inhibitors to malate synthase (GlcB), an enzyme required for Mycobacterium tuberculosis virulence. Using density functional theory methods (M06-2X/6-31+G(d)), a GlcB active site template was developed for a predictive model through a comparative analysis of PDKA-bound GlcB crystal structures. The active site model includes the PDKA molecule and the protein determinants of the electrostatic, hydrogen-bonding, and anion-π interactions involved in binding. The predictive model accurately determines the Asp 633-PDKA structural position upon binding and precisely predicts the relative binding enthalpies of a series of 2-ortho halide-PDKAs to GlcB. A screening model was also developed to efficiently assess the propensity of each PDKA analog to participate in an anion-π interaction; this method is in good agreement with both the predictive model and the experimental binding enthalpies for the 2-ortho halide-PDKAs. With the screening and predictive models in hand, we have developed an efficient method for computationally screening and evaluating the binding enthalpy of variously substituted PDKA molecules. This study serves to illustrate the contribution of this overlooked interaction to binding affinity and demonstrates the importance of integrating anion-π interactions into structure-based CADD.

Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very little is known regarding the proteins containing multiple domains. It has been shown that the high stability of domains, in conjunction with inter-domain interactions, manifests as a frustrated energy landscape, causing complexity in the global folding pathway. However, multidomain proteins despite containing independently foldable, loosely cooperative sections can fold into native states with amazing speed and accuracy. To understand the complexity in mechanism, studies were conducted previously on the multidomain protein malate synthase G (MSG), an enzyme of the glyoxylate pathway with four distinct and adjacent domains. It was shown that the protein refolds to a functionally active intermediate state at a fast rate, which slowly produces the native state. Although experiments decoded the nature of the intermediate, a full description of the folding pathway was not elucidated. In this study, we use a battery of biophysical techniques to examine the protein's folding pathway. By using multiprobe kinetics studies and comparison with the equilibrium behavior of protein against urea, we demonstrate that the unfolded polypeptide undergoes conformational compaction to a misfolded intermediate within milliseconds of refolding. The misfolded product appears to be stabilized under moderate denaturant concentrations. Further folding of the protein produces a stable intermediate, which undergoes partial unfolding-assisted large segmental rearrangements to achieve the native state. This study reveals an evolved folding pathway of the multidomain protein MSG, which involves surpassing the multiple misfolding traps during refolding.

Malate is regarded as one of the key building block chemicals which can potentially be produced from biomass at a large scale. Although glucose has been extensively studied as the substrate for malate production, its high price and potential competition with food production are serious limiting factors. In this study, Escherichia coli was metabolically engineered to effectively produce malate from xylose, the second most abundant sugar component of lignocellulosic biomass. First, the biosynthetic route of malate was constructed by overexpressing D -tagatose 3-epimerase, L-fuculokinase, L-fuculose-phosphate aldolase, and aldehyde dehydrogenase A. Second, genes encoding malic enzyme, malate dehydrogenase, and fumarate hydratase were knocked out to eliminate malate consumption, resulting in a titer of 1.99 g/l malate and a yield of 0.47 g malate/g xylose. Third, glycolate oxidase and malate synthase were overexpressed to strengthen the conversion of glycolate to malate, which led to a titer of 4.33 g/l malate and a yield of 0.83 g malate/g xylose, reaching 93% of the theoretical yield. Finally, catalase HPII was overexpressed to decompose H2O2 and alleviate its toxicity, which improved cell growth and further boosted malate titer to 5.90 g/l with a yield of 0.80 g malate/g xylose. To the best of our knowledge, this is the first study to report efficient malate production from xylose as the carbon source.

Background: One of the main sunfl ower diseases is the white mold Sclerotinia sclerotiorum. The oxalic acid (OA), which is one of the main pathogenicity factors of this fungus, beside the direct toxicity on the host, has other functions such as the disruption of the cell wall and chelating out the calcium ions.Objectives: Regarding the importance of this disease, it is important to study the reactions of the plant against OA which is a nonspecifi c toxin of many other necrotrophic fungi.Materials and Methods: In this study, two susceptible and moderately resistant sunfl ower lines were inoculated with OA and samples at the fi rst leaf stage were collected within the intervals of 2, 6, 12 and 24 hours post inoculation. The expression of five genes related to tricarboxylic acid cycle, including citrate synthase, fumarase, iso-citrate lyase, malate synthase and malate dehydrogenase was studied under OA treatment.Results: Two hours after the inoculation, no signifi cant change was observed in the expression of the fi ve studied genes in the moderately resistant line. The iso-citrate lyase gene, which is related to glyoxylate cycle (a variation of the tricarboxylic acid cycle), showed no change in the moderately resistant line; however, it showed an increase in the susceptible line. The increase in fumarase gene expression in moderately resistant line was higher than the susceptible line. The result showed the activation of glyoxylate cycle and destruction of fatty acids in the susceptible line.Conclusions: Activation of glyoxylate cycle indicated induction of senescent symptoms by OA in susceptible line. Increasing in H2O2 leads to oxidative burst and cell death. Cell death has an apparent benefi t for development and growth of necrotrophic pathogens in the plant cells. The study of resistance mechanisms in response to the pathogen can be useful for breeding programs to provide lines with higher resistance to this pathogen.

The NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis. The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous s-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis, but also in Saccharomyces cerevisiae.

The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans. We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate.

Glyoxylate is an important chemical building block which is currently produced by chemical or electrochemical oxidation methods We herein described the achievement of microbial glyoxylate production from renewable biomass by metabolic engineering of Escherichia coli. The synthetic ribulose-1-phosphate pathway was expressed to produce glycolate from xylose. Additional genomic modifications including the inactivation of malate synthase genes aceB/glcB and glyoxylate carboligase gene gcl were performed to prevent the consumption of glyoxylate. The constructed strain was found to accumulate 0.13 g/L glyoxylate in shake flask cultivations. Further overexpression of glycolate oxidase and catalase significantly improved glyoxylate production to 0.74 g/L, which is the highest titer reported to date. These results demonstrate that microbial fermentation has a promising potential to manufacture glyoxylate from renewable feedstocks.

The ethylmalonyl-CoA pathway (EMCP) is an anaplerotic reaction sequence in the central carbon metabolism of numerous Proteo- and Actinobacteria. The pathway features several CoA-bound mono- and dicarboxylic acids that are of interest as platform chemicals for the chemical industry. The EMCP, however, is essential for growth on C1 and C2 carbon substrates and therefore cannot be simply interrupted to drain these intermediates. In this study, we aimed at reengineering central carbon metabolism of the Alphaproteobacterium Methylobacterium extorquens AM1 for the specific production of EMCP derivatives in the supernatant. Establishing a heterologous glyoxylate shunt in M. extorquens AM1 restored wild type-like growth in several EMCP knockout strains on defined minimal medium with acetate as carbon source. We further engineered one of these strains that carried a deletion of the gene encoding crotonyl-CoA carboxylase/reductase to demonstrate in a proof-of-concept the specific production of crotonic acid in the supernatant on a defined minimal medium. Our experiments demonstrate that it is in principle possible to further exploit the EMCP by establishing an alternative central carbon metabolic pathway in M. extorquens AM1, opening many possibilities for the biotechnological production of EMCP-derived compounds in future.

Malate synthase is a condensing enzyme responsible for conversion of glyoxylate to malate in the presence of acetyl-CoA. This reaction helps in bypassing the TCA cycle reactions involving carbon loss and leads to diverting some of the carbon skeletons to gluconeogenic events while rest can continue to provide TCA cycle intermediates. Malate synthase (GlcB) is encoded by MRA_1848 of Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a knockdown (KD) Mtb-Ra strain by down-regulating GlcB. The survival studies suggested increased susceptibility to oxidative and nitrosative stress as well as to rifampicin. The susceptibility profile was reversed in the presence of free radical scavengers. Also, KD showed reduced biofilm maturation, failed to enter persistent state, and showed reduced growth inside macrophages. The study of post-endocytosis events showed differences in late stage endosomal maturation behavior in macrophages infected with KD compared to WT. Increased iNOS, LAMP1 and cathepsin D expression was observed in macrophages infected with KD compared to WT.

Cyanobacteria are ubiquitous photoautotrophs that assimilate atmospheric CO2 as their main source of carbon. Several cyanobacteria are known to be facultative heterotrophs that are able to grow on diverse carbon sources. For selected strains, assimilation of organic acids and mixotrophic growth on acetate has been reported for decades. However, evidence for the existence of a functional glyoxylate shunt in cyanobacteria has long been contradictory and unclear. Genes coding for isocitrate lyase (ICL) and malate synthase were recently identified in two strains of the genus Cyanothece, and the existence of the complete glyoxylate shunt was verified in a strain of Chlorogloeopsis fritschii. Here, we report that the gene PCC7424_4054 of the strain Cyanothece sp. PCC 7424 encodes an enzymatically active protein that catalyses the reaction of ICL, an enzyme that is specific for the glyoxylate shunt. We demonstrate that ICL activity is induced under alternating day/night cycles and acetate-supplemented cultures exhibit enhanced growth. In contrast, growth under constant light did not result in any detectable ICL activity or enhanced growth of acetate-supplemented cultures. Furthermore, our results indicate that, despite the presence of a glyoxylate shunt, acetate does not support continued heterotrophic growth and cell proliferation. The functional validation of the ICL is supplemented with a bioinformatics analysis of enzymes that co-occur with the glyoxylate shunt. We hypothesize that the glyoxylate shunt in Cyanothece sp. PCC 7424, and possibly other nitrogen-fixing cyanobacteria, is an adaptation to a specific ecological niche and supports assimilation of nitrogen or organic compounds during the night phase.

Background: Resveratrol naturally occurring antioxidant in peanut (Legume: Arachis hypogaea) has phytochemical human health dietary effects associated with reduced inflammatory cancer risks. Its levels in peanut are ultra-low and variable (0 to 26 μg·g-1), which has made it difficult to market as a consistent high resveratrol produce. Objective: Understanding the regulation of resveratrol accumulation in peanut might lead to development of new techniques for optimizing and stabilizing its yield. Method: Peanuts were cultivated in horticultural field plots and treated with solutions of mineral salts (sulfate, potassium, phosphate, ammonium ion) that were optimized in stoichiometric (reactive) ratios. Peanut seed’s RNAs were subjected to Northern blot analysis for profiling the RNAs synthesized by glutamate dehydrogenase (GDH), and mRNAs encoding resveratrol synthase. The seed’s extracts were analyzed by GC-MS for determination of the resveratrol and fatty acid compositions. Result: Stoichiometric mixes of mineral ions induced the peanut GDH to synthesize some RNA that silenced the mRNAs encoding resveratrol synthase, phosphoglucomutase, isocitrate lyase, malate synthase, enolase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and phosphoglycerate mutase in the control, KN-, and NPKS-treated but not in the NPPK-treated peanut. These resulted to decreased resveratrol content (6.0 μg·g-1) in the control peanut but maximized it (1.15 mg·g-1) in the NPPK-treated peanut. Therefore, resveratrol accumulation was optimized by coupling of glycolysis and citric-glyoxylic acid cycles to resveratrol biosynthesis. Fatty acid content of control (55.6 g·kg-1) was higher than the NPKS-treated (48.5 g·kg-1) and NPPK-treated peanut (44.9 g·kg-1) meaning that malonyl-CoA intermediate in both fatty acid and stilbenoid pathways was diverted to support maximum resveratrol biosynthesis in the NPPK-treated peanut. Conclusion: The functional coupling of citric-glyoxylic acid cycles and glycolysis to optimize resveratrol biosynthesis may encourage development of horticultural technology specific for production of ultra-high resveratrol peanuts.

The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus. We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt. IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus. Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

Specialized metabolites are structurally diverse and cell- or tissue-specific molecules produced in restricted plant lineages. In contrast, primary metabolic pathways are highly conserved in plants and produce metabolites essential for all of life, such as amino acids and nucleotides. Substrate promiscuity - the capacity to accept non-native substrates - is a common characteristic of enzymes, and its impact is especially apparent in generating specialized metabolite variation. However, promiscuity only leads to metabolic diversity when alternative substrates are available; thus, enzyme cellular and subcellular localization directly influence chemical phenotypes. We review a variety of mechanisms that modulate substrate availability for promiscuous plant enzymes. We focus on examples where evolution led to modification of the 'cellular context' through changes in cell-type expression, subcellular relocalization, pathway sequestration, and cellular mixing via tissue damage. These varied mechanisms contributed to the emergence of structurally diverse plant specialized metabolites and inform future metabolic engineering approaches.

This research aimed to investigate the effects of progesterone, a mammalian steroid sex hormone, on the mitochondrial respiration in germinating maize seeds. For this purpose, maize seeds were divided into four different groups (control, 10−6, 10−8, and 10−10 mol·L−1 progesterone) and were grown in a germination cabinet in the dark at 24.5 ± 0.5 °C for 4 d. The changes in gene expression levels of citrate synthase (CS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6), which is involved in mitochondrial respiration, were studied in root and cotyledon tissues. Significant increases were recorded in the gene expression levels of all studied enzymes. In addition, progesterone applications stimulated activities of malate synthase (MS), isocitrate lyase (ICL), and alpha-amylase, which are important enzymes of the germination step. The changes in gene expression levels of mas1 and icl1 were found parallel to the rise in these enzymes’ activities. It was determined similar increases in root and coleoptile lengths and total soluble protein and total carbohydrate contents. The most remarkable changes were detected in 10−8 mol·L−1 progesterone-treated seedlings. These results clearly indicate that progesterone stimulates mitochondrial respiration by inducing biochemical and molecular parameters and thus accelerates seed germination thanks to the activation of other pathways related to mitochondrial respiration.