Solution-state methyl NMR spectroscopy of large non-deuterated proteins enabled by deep neural networks.

Methyl-TROSY nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterising large biomolecules in solution. However, preparing samples for these experiments is demanding and entails deuteration, limiting its use. Here we demonstrate that NMR spectra recorded on protonated, uniformly 13C labelled samples can be processed using deep neural networks to yield spectra that are of similar quality to typical deuterated methyl-TROSY spectra, potentially providing information for proteins that cannot be produced in bacterial systems. We validate the methodology experimentally on three proteins with molecular weights in the range 42-360 kDa. We further demonstrate the applicability of our methodology to 3D NOESY spectra of Escherichia coli Malate Synthase G (81 kDa), where observed NOE cross-peaks are in good agreement with the available structure. The method represents an advance in the field of using deep learning to analyse complex magnetic resonance data and could have an impact on the study of large biomolecules in years to come.

Sodium and lithium exert differential effects on the central carbon metabolism of Debaryomyces hansenii through the glyoxylate shunt regulation.

Debaryomyces hansenii is a halotolerant/halophilic yeast usually found in salty environments. The yeast accumulated sodium at high concentrations, which improved growth in salty media. In contrast, lithium was toxic even at low concentrations and its presence prevented cell proliferation. To analyse the responses to both cations, metabolite levels, enzymatic activities and gene expression were determined, showing that NaCl and LiCl trigger different cellular responses. At high concentrations of NaCl (0.5 or 1.5 M) cells accumulated higher amounts of the intermediate metabolites glyoxylate and malate and, at the same time, the levels of intracellular oxoglutarate decreased. Additionally, 0.5 M NaCl increased the activity of the enzymes isocitrate lyase and malate synthase involved in the synthesis of glyoxylate and malate respectively and decreased the activity of isocitrate dehydrogenase. Moreover, transcription of the genes coding for isocitrate lyase and malate synthase was activated by NaCl. Also, cells accumulated phosphate upon NaCl exposure. None of these effects was provoked when LiCl (0.1 or 0.3 M) was used instead of NaCl. Lithium induced accumulation of higher amounts of oxoglutarate and decreased the concentrations of glyoxylate and malate to non-detectable levels. Cells incubated with lithium also showed higher activity of the isocitrate dehydrogenase and neither increased isocitrate lyase and malate synthase activities nor the transcription of the corresponding genes. In summary, we show that sodium, but not lithium, up regulates the shunt of the glyoxylic acid in D. hansenii and we propose that this is an important metabolic adaptation to thrive in salty environments.

Ectopic expression of rice malate synthase in Arabidopsis revealed its roles in salt stress responses.

Expression of rice malate synthase (OsMS), one of the two key genes in the glyoxylate cycle, is highly upregulated under salt stress. In this study, we aimed to investigate the role of OsMS in salt stress responses using the Arabidopsis T-DNA insertional mutant line of malate synthase (AtMS), an OsMS orthologous gene, for ectopic expression. Germination of the Atms mutant under salt stress was lower than that of Arabidopsis Col-0 wildtype (WT); furthermore, the two Atms mutant lines ectopically expressing OsMS reversed the salt-sensitive phenotype. Atms mutants salt-treated for 3 days exhibited higher electrolyte leakage, higher Na+/K+ ratio, lower expression of stress-responsive genes, and lower soluble sugar content than WT and the two OsMS-expressing Atms mutant lines. Consistently, Atms mutants salt-treated for 3 days followed by a 5-day recovery period displayed greater fresh-weight reduction. Notably, leaf greenness and chlorophyll and total carotenoid contents were higher in the Atms mutant lines than in the WT under stress. OsMS-expressing Atms mutants exhibited a change in pigment content closer to that of WT. During dark-induced senescence, an Atms mutant, Aticl, mutant (the other key gene in the glyoxylate cycle), and three double mutant lines of Atms and Aticl exhibited lower decreases in leaf greenness than the WT and OsMS-expressing Atms mutant lines. Furthermore, SAG12 expression levels in the Atms mutant, Aticl mutant, and three double mutant lines were lower than those in OsMS-expressing Atms mutant lines. Altogether, our data indicate that OsMS likely plays a key role in salt stress responses, possibly through the induction of leaf senescence.

Malate synthase contributes to the survival of Salmonella Typhimurium against nutrient and oxidative stress conditions.

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 < 0.05) to hypochlorite. Further, ∆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.

Mechanistic assessment of tolerance to iron deficiency mediated by Trichoderma harzianum in soybean roots.

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 (H2 O2 ) 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.

Structure-based discovery of phenyl-diketo acids derivatives as Mycobacterium tuberculosis malate synthase inhibitors.

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.

Characterisation and structural analysis of glyoxylate cycle enzymes of Teladorsagia circumcincta.

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min-1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01µM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.

Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.

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.

Expression regulation of MALATE SYNTHASE involved in glyoxylate cycle during protocorm development in Phalaenopsis aphrodite (Orchidaceae).

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.

Is Autophagy Involved in Pepper Fruit Ripening?

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.

Location, location! cellular relocalization primes specialized metabolic diversification.

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.

Optimized NMR Experiments for the Isolation of I=1/2 Manifold Transitions in Methyl Groups of Proteins.

Optimized NMR experiments are developed for isolating magnetization belonging to the I=1/2 manifolds of 13 CH3 methyl groups in proteins, enabling the manipulation of the magnetization of a 13 CH3 moiety as if it were an AX (1 H-13 C) spin-system. These experiments result in the same 'simplification' of a 13 CH3 spin-system that would be obtained from the production of {13 CHD2 }-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 CHD2 }-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 CH3 (AX3 ) spin-systems are described, with an emphasis on the measurements of methyl 1 H-13 C residual dipolar couplings in a {13 CH3 }-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.

Effect of Target Gene Silencing on Calcite Single Crystal Formation by Thermophilic Bacterium Geobacillus thermoglucosidasius NY05.

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.

Overproduction of recombinant E. coli malate synthase enhances Chlamydomonas reinhardtii biomass by upregulating heterotrophic metabolism.

High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic Chlamydomonas reinhardtii was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (MDH4) and acetyl-CoA synthetase (ACS3), isocitrate lyase (ICL1) and malate synthase (MAS1), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic Fv/Fm was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass.

A step forward: Compatible and dual-inducible expression vectors for gene co-expression in Corynebacterium glutamicum.

The Gram-positive bacterium Corynebacterium glutamicum represents a promising platform for the production of amino acids, organic acids, and other bio-products. However, the availability of only few expression vectors limits its use for production purposes, using metabolic engineering approaches when co-expression of several target genes is desired. To widen the scope for co-expression, the pCG1/p15A and pBL1/colE1 replicons were employed to construct the two differentially-inducible and compatible expression vectors pRG_Duet1 and pRG_Duet2. To functionally validate these newly constructed expression vectors, target genes for easily measurable enzymes were cloned and over-expression of these genes was investigated using respective enzyme assays. Furthermore, functionality and co-existence of the pCG1-based C. glutamicum - E. coli shuttle vector pRG_Duet1 were confirmed with pBL1-based expression vectors pRG_Duet2 and pEKEx2, using co-transformation and enzyme assays. The novel shuttle expression vectors pRG_Duet1 and pRG_Duet2 are attractive additions to the existing set of vectors for co-expression studies and metabolic engineering of C. glutamicum.

Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle.

Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5'-triphosphate (ATP)-dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.

Anion-π Interactions in Computer-Aided Drug Design: Modeling the Inhibition of Malate Synthase by Phenyl-Diketo Acids.

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.

Spontaneous refolding of the large multidomain protein malate synthase G proceeds through misfolding traps.

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.

Enhanced polymalic acid production from the glyoxylate shunt pathway under exogenous alcohol stress.

Polymalic acid (PMA) is a water-soluble biopolymer produced by the yeast-like fungus Aureobasidium pullulans. In this study, the physiological response of A. pullulans against exogenous alcohols stress was investigated. Interestingly, ethanol stress was an effective inducer of enhanced PMA yield, although cell growth was slightly inhibited. The stress-responsive gene malate synthase (mls), which is involved in the glyoxylate shunt, was identified and was found to be regulated by exogenous ethanol stress. Therefore, an engineered strain, YJ-MLS, was constructed by overexpressing the endogenous mls gene, which increased the PMA titer by 16.2% compared with the wild-type strain. Following addition of 1% (v/v) of ethanol, a high PMA titer of 40.0 ±â€¯0.38 g/L was obtained using batch fermentation with the mutant YJ-MLS in a 5-L fermentor, with a strongest PMA productivity of 0.56 g/L h. This study was the interesting report to show strengthening of the carbon metabolic flow from the glyoxylate shunt for PMA synthesis, and also provided a new sight for re-recognizing the regulatory behavior of alcohol stress in eukaryotic microbes.

Lack of glyoxylate shunt dysregulates iron homeostasis in Pseudomonas aeruginosa.

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5' RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.