High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation.

BACKGROUND: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products. RESULTS: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane. CONCLUSIONS: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass.

BACKGROUND: Ectoine as an amino acid derivative is widely applied in many fields, such as the food industry, cosmetic manufacturing, biologics, and therapeutic agent. Large-scale production of ectoine is mainly restricted by the cost of fermentation substrates (e.g., carbon sources) and sterilization. RESULTS: In this study, Halomonas cupida J9 was shown to be capable of synthesizing ectoine using xylose as the sole carbon source. A pathway was proposed in H. cupida J9 that synergistically utilizes both WBG xylose metabolism and EMP glucose metabolism for the synthesis of ectoine. Transcriptome analysis indicated that expression of ectoine biosynthesis module was enhanced under salt stress. Ectoine production by H. cupida J9 was enhanced by improving the expression of ectoine biosynthesis module, increasing the intracellular supply of the precursor oxaloacetate, and utilizing urea as the nitrogen source. The constructed J9U-P8EC achieved a record ectoine production of 4.12 g/L after 60 h of xylose fermentation. Finally, unsterile production of ectoine by J9U-P8EC from either a glucose-xylose mixture or corn straw hydrolysate was demonstrated, with an output of 8.55 g/L and 1.30 g/L of ectoine, respectively. CONCLUSIONS: This study created a promising H. cupida J9-based cell factory for low-cost production of ectoine. Our results highlight the potential of J9U-P8EC to utilize lignocellulose-rich agriculture waste for open production of ectoine.

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions.

Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: • Hydroxyectoine production in H. salifodinae correlates with the salinity of medium • Transcriptomic analysis reveals the limiting factors for hydroxyectoine production • The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.

The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine.

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

Biochemistry of plants N-heterocyclic non-protein amino acids.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N-heterocyclic non-protein amino acids from plants are described. These N-heterocyclic non-protein amino acids are composed of ß-substituted alanines and include mimosine, ß-pyrazol-1-yl-L-alanine, willardiine, isowillardiine, and lathyrine. These ß-substituted alanines consisted of an N-heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N-heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained.

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum.

Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result in an increase in cytoplasmic sodium concentration in S. salinarum. This, together with the decrease in the catalytic activities of MDH and ICDH at high salt concentration, demonstrates that S. salinarum employs the salt-out strategy for haloadaptation.

Ectoines as novel anti-inflammatory and tissue protective lead compounds with special focus on inflammatory bowel disease and lung inflammation.

The compatible solute ectoine is one of the most abundant and powerful cytoprotectant in the microbial world. Due to its unique ability to stabilize biological membranes and macromolecules it has been successfully commercialized as ingredient of various over-the-counter drugs, achieving primarily epithelial protection. While trying to elucidate the mechanism of its cell protective properties in in-vitro studies, a significant anti-inflammatory effect was documented for the small molecule. The tissue protective potential of ectoine considerably improved organ quality during preservation. In addition, ectoine and derivatives have been demonstrated to significantly decrease inflammatory cytokine production, thereby alleviating the inflammatory response following organ transplantation, and launching new therapeutic options for pathologies such as Inflammatory Bowel Disease (IBD) and Chronic Obstructive Pulmonary Disease (COPD). In this review, we aim to summarize the knowledge of this fairly nascent field of the anti-inflammatory potential of diverse ectoines. We also point out that this promising field faces challenges in its biochemical and molecular substantiations, including defining the molecular mechanisms of the observed effects and their regulation. However, based on their potent cytoprotective, anti-inflammatory, and non-toxic properties we believe that ectoines represent promising candidates for risk free interventions in inflammatory pathologies with steeply increasing demands for new therapeutics.

Microbial production of ectoine and hydroxyectoine as high-value chemicals.

Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a "bacterial milking" process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina. However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.

Promoter engineering for high ectoine production in a lower saline medium by Halomonas hydrothermalis Y2.

OBJECTIVES: For the stress from fermenters, downstream processing equipment, and wastewater treatment to be alleviated, lowering salt-dependence in the ectoine synthesis process is of great significance in the moderately halotolerant Halomonas hydrothermalis Y2. RESULTS: In H. hydrothermalis Y2, the σ70- and σ38-controlled promoters of ectA are predicted to be involved in the osmotic regulation of ectoine synthesis. By substituting the ectA promoter with a promoter P265 that identified in the outer membrane pore protein E of H. hydrothermalis Y2, the salt dependence of ectoine synthesis was significantly decreased. In the 500-ml flask containing various NaCl contents, the engineered strain (p/Y2/△ectD/△doeA) showed a remarkably enhanced ability in ectoine synthesis, especially under lower saline stress. After a 36-h fed-batch fermentation in the 1-l fermenter, p/Y2/△ectD/△doeA synthesized 11.5 g ectoine l-1 in the presence of 60 g NaCl-1 l, with a high 0.32 g ectoine l-1 h-1 productivity, a specific productivity of 512.2 mg ectoine per g cell dry weight (CDW)-1, and an excretion ratio of 67 % ectoine. CONCLUSIONS: As no impaired growth was observed in strain p/Y2/△ectD/△doeA while ectoine synthesis was increased, this promoter engineering strategy provides a practical protocol for lowering the salt-dependence of ectoine synthesis in this moderately halotolerant strain.

Metabolic Pathway Construction and Optimization of Escherichia coli for High-Level Ectoine Production.

Ectoine is widely produced by various bacteria as a natural cell protectant against environment stress, e.g., osmotic and temperature stress. Its protective properties therefore exhibit high commercial value, especially in agriculture, medicine, cosmetics, and biotechnology. Here, we successfully constructed an engineered Escherichia coli for the heterologous production of ectoine. Firstly, the ectABC genes from Halomonas elongata were introduced into E. coli MG1655 to produce ectoine without high osmolarity. Subsequently, lysA gene was deleted to weaken the competitive L-lysine biosynthesis pathway and ectoine bioconversion was further optimized, leading to an increase of ectoine titer by 16.85-fold. Finally, at the low cell density of 5 OD600/mL in Erlenmeyer flask, the concentration of extracellular ectoine was increased to 3.05 mg/mL. At the high cell density of 15 OD600/mL, 12.7 g/L of ectoine was achieved in 24 h and the overall yield is 1.27 g/g glycerol and sodium aspartate. Our study herein provides a feasible and valuable biosynthesis pathway of ectoine with a potential for large-scale industrial production using simple and cheap feedstocks.

Optimizing ectoine biosynthesis using response surface methodology and osmoprotectant applications.

PURPOSE: Numerous applications of compatible salts (osmolytes) as ectoine in food and pharmaceutical industries have been intensively increased nowadays. Decreasing the cost of industrial production of ectoine using low-cost cultivation media and improving the yield through modeling procedures are the main scopes of the present study. METHODS: Three statistical design experiments have been successfully applied for screening the parameters affecting the production process, studying the relations among parameters and optimizing the production using response surface methodology. RESULTS: A novel semi-synthetic medium based on hydrolyzed corn gluten meal has been developed to cultivate moderate halophilic bacterial strains; Vibrio sp. CS1 and Salinivibrio costicola SH3, and support ectoine synthesis under salinity stress. Two regression equations describe the production process in the new medium have been formulated for each bacterial strain. Response surface optimizer of the central composite model predicts the maximum ectoine production is achieved at incubation time; 63.7 h, pH; 7.47 and salinity; 7.27% for Vibrio sp. CS1 whereas these variables should be adjusted at 56.95 h, 7.089 and 10.34%; on the same order regarding Salinivibrio costicola SH3. In application studies, 50 µg ectoine decreases RBCs hemolysis due to streptolysin O toxin by 21.7% within ten minutes. In addition, 2% ectoine succeeds to increase the viability of lactic acid bacteria in Yogurt as a classic example of functional food during the storage period (7 days). CONCLUSION: The present study emphasizes on modeling the process of ectoine production by halophilic bacteria as well as its activity as a cryoprotectant agent.

Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness.IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.

Ectoine biosynthesis genes from the deep sea halophilic eubacteria, Bacillus clausii NIOT-DSB04: Its molecular and biochemical characterization.

Ectoine, the most prominent osmolyte in nature, is a vital compatible solute present in halophilic bacterium. It protects the cellular biomolecules of the halophilic bacteria and retains their intrinsic function from extreme circumstances. In the current research, ectoine biosynthesis gene cluster (ectABC) in Bacillus clausii NIOT-DSB04 was expressed heterologically in E. coli M15 (pREP4). RP-HPLC resolved several fractions of the purified recombinant product, one of which had been confirmed as ectoine. The recombinant ectoine was further characterized by 1H and 13C Nuclear Magnetic Resonance. The purified recombinant ectoine was also authenticated by FT-IR studies with the existence of ester carbonyl and C-H group. In IPTG induced E. coli M15 transgenic cells, the enzymatic activity of the ectA, B and C genes were found to be higher than that of uninduced cells.

High ectoine production by an engineered Halomonas hydrothermalis Y2 in a reduced salinity medium.

BACKGROUND: As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications. RESULTS: The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L-1 NaCl in a 500-mL flask, the double mutant of Y2/ΔectD/ΔdoeA synthesized 3.13 g L-1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L-1), and also exceeds the production of Y2/ΔectD (2.21 g L-1). The remarkably enhanced accumulation of ectoine by Y2/ΔectD/ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na+/H+ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ΔectD/ΔdoeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L-1 or 500 mg (g DCW) -1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L-1 NaCl stress, a maximum 10.5 g L-1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)-1 and a yield of 0.21 g g-1 monosodium glutamate. CONCLUSION: The remarkably enhanced production of ectoine by Y2/ΔectD/ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na+/H+ antiporters in their halophilic producers and thus lowering the medium salinity.

Betaine accumulation suppresses the de-novo synthesis of ectoine at a low osmotic concentration in Halomonas sp SBS 10, a bacterium with broad salinity tolerance.

The study aims to find out osmoadaptive mechanism used to overcome the salinity stress by Halomonas sp SBS 10 isolated from the saltern crystallizer ponds of the Sambhar Salt Lake and its taxonomic position using neighbor-joining algorithm. The strain SBS 10 was tested for accumulation of two major compatable solutes betaine and ectoine and was observed that osmoprotection in the strain SBS 10 is achieved by the accumulation of betaine or by the de-novo synthesis of betaine or ectoine. Amount of endogenous content of the betaine and ectoine per milligram of cell biomass was estimated to be 581 µg, 587 µg, 588 µg, 617 µg, and 761 µg for betaine and 1.52 µg, 2.74 µg, 3.14 µg, 3.50 µg, and 52.67 µg for ectoine, when exposed to 5, 10, 15, 20 and 25% of NaCl concentration. Results obtained from HPLC analysis showed that the betaine accumulation suppresses the de-novo synthesis of ectoine partially at low NaCl concentration in the growth medium. However, at a high NaCl concentration, the ectoine concentration increases abruptly as compared to the betaine. This indicates that the ectoine accumulation is transcriptionally up-regulated by the salinity stress. Phylogenetic analysis based on the neighbor-joining algorithm included the strain SBS 10 in the genus Halomonas of the family Halomonadaceae belonging to the class Gammaproteobacteria. Most closely related type strain was found to be Halomonas gudaonensis SL014B-69T (98.2% similarity). Ultrastructure characteristics showed the strain to be non-spore forming rod, 0.3-0.4 × 0.75-1.65 µm in size and motile with the help of peritrichous flagella.

Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory.

Ectoine and hydroxyectoine are widely synthesized by members of the Bacteria and a few members of the Archaea as potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacterium Pseudomonas stutzeri by transferring it into Escherichia coli, an enterobacterium that does not produce ectoines naturally. Using ect-lacZ reporter fusions, we found that the heterologous ect promoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the -10, -35, and spacer regions of the sigma-70-type ect promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the ect promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters of E. coli afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set of ect promoter mutants, we engineered an E. coli chassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.IMPORTANCE Ectoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited the ect promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact that E. coli does not synthesize ectoines naturally, the ect promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation of ect transcription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of the ect promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.

Insights into metabolic osmoadaptation of the ectoines-producer bacterium Chromohalobacter salexigens through a high-quality genome scale metabolic model.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. RESULTS: We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. CONCLUSIONS: Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.

Understanding the interplay of carbon and nitrogen supply for ectoines production and metabolic overflow in high density cultures of Chromohalobacter salexigens.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. RESULTS: Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect - mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L-1) and volumetric ectoine yields (up to 4.21 g L-1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes. CONCLUSIONS: Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.

Determining the mode of action of anti-mycobacterial C17 diyne natural products using expression profiling: evidence for fatty acid biosynthesis inhibition.

BACKGROUND: The treatment of microbial infections is becoming increasingly challenging because of limited therapeutic options and the growing number of pathogenic strains that are resistant to current antibiotics. There is an urgent need to identify molecules with novel modes of action to facilitate the development of new and more effective therapeutic agents. The anti-mycobacterial activity of the C17 diyne natural products falcarinol and panaxydol has been described previously; however, their mode of action remains largely undetermined in microbes. Gene expression profiling was therefore used to determine the transcriptomic response of Mycobacterium smegmatis upon treatment with falcarinol and panaxydol to better characterize the mode of action of these C17 diynes. RESULTS: Our analyses identified 704 and 907 transcripts that were differentially expressed in M. smegmatis after treatment with falcarinol and panaxydol respectively. Principal component analysis suggested that the C17 diynes exhibit a mode of action that is distinct to commonly used antimycobacterial drugs. Functional enrichment analysis and pathway enrichment analysis revealed that cell processes such as ectoine biosynthesis and cyclopropane-fatty-acyl-phospholipid synthesis were responsive to falcarinol and panaxydol treatment at the transcriptome level in M. smegmatis. The modes of action of the two C17 diynes were also predicted through Prediction of Activity Spectra of Substances (PASS). Based upon convergence of these three independent analyses, we hypothesize that the C17 diynes inhibit fatty acid biosynthesis, specifically phospholipid synthesis, in mycobacteria. CONCLUSION: Based on transcriptomic responses, it is suggested that the C17 diynes act differently than other anti-mycobacterial compounds in M. smegmatis, and do so by inhibiting phospholipid biosynthesis.

Strangers in the archaeal world: osmostress-responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus.

Ectoine and hydroxyectoine are compatible solutes widely synthesized by members of the Bacteria to cope with high osmolarity surroundings. Inspection of 557 archaeal genomes revealed that only 12 strains affiliated with the Nitrosopumilus, Methanothrix or Methanobacterium genera harbour ectoine/hydroxyectoine gene clusters. Phylogenetic considerations suggest that these Archaea have acquired these genes through horizontal gene transfer events. Using the Thaumarchaeon 'Candidatus Nitrosopumilus maritimus' as an example, we demonstrate that the transcription of its ectABCD genes is osmotically induced and functional since it leads to the production of both ectoine and hydroxyectoine. The ectoine synthase and the ectoine hydroxylase were biochemically characterized, and their properties resemble those of their counterparts from Bacteria. Transcriptional analysis of osmotically stressed 'Ca. N. maritimus' cells demonstrated that they possess an ectoine/hydroxyectoine gene cluster (hyp-ectABCD-mscS) different from those recognized previously since it contains a gene for an MscS-type mechanosensitive channel. Complementation experiments with an Escherichia coli mutant lacking all known mechanosensitive channel proteins demonstrated that the (Nm)MscS protein is functional. Hence, 'Ca. N. maritimus' cells cope with high salinity not only through enhanced synthesis of osmostress-protective ectoines but they already prepare themselves simultaneously for an eventually occurring osmotic down-shock by enhancing the production of a safety-valve (NmMscS).