Archaeon Haloferax Mediterranei Research Articles

Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography.

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.

Utilizing the crop waste of date palm fruit to biosynthesize polyhydroxyalkanoate bioplastics with favorable properties

Polyhydroxyalkanoate (PHA), a family of biodegradable and renewable biopolymers that could potentially play a significant role in bioeconomy. In this study we investigated the potential of date waste (DW) biomass as feedstock to produce PHA by the halophilic archaeon Haloferax mediterranei. The concentration of essential trace elements for H. mediterranei cells during growth and PHA biopolymer accumulation was optimized. A maximum cell dry mass of (CDM) (12.8 g L−1) and PHA concentration of (3.20 g L−1) were achieved in DW extract media that was not supplemented with trace elements, indicating that DW is a promising source for trace elements. The cultivation was scaled-up to fed-batch bioreactor fermentations under non-sterile conditions and resulted in CDM and PHA content of 18.0 g L−1 and 25%, respectively. The produced PHA was confirmed to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high 3-hydroxyvalerate (3 HV) content of 18.0 mol%. This 3 HV molar percent was achieved without the addition of expensive precursors. The PHBV is of high molecular weight (746.0 kDa) and narrow polydispersity (PDI = 1.5), and displayed reduced melting at 148.1 °C. The X-ray diffraction (XRD) analysis showed that the PHBV has amorphous nature which increases the degradation rates and workability of the biopolymer. The isotopic ratio 13C/12C (δ 13C) for PHBV was found to be – 19.1‰, which indicated that H. mediterranei prefers lighter bonds to break and uses the lighter atoms for the biosynthesis of PHBV.

Optimization of nitrogen source supply for enhanced biosynthesis and quality of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by extremely halophilic archaeon Haloferax mediterranei.

The extreme halophilic archaeon, Haloferax mediterranei can accumulate polyhydroxyalkanoate (PHA) from different renewable resources. To enhance the biosynthesis and quality of PHA, H. mediterranei cultivation media was optimized at different C/N ratios using glucose as the main carbon source. Three sets of media (yeast extract [YE], NH4Cl and combination of YE and NH4Cl) were prepared at different nitrogen concentrations to achieve C/N ratios of 9, 20, and 35, respectively. The media containing YE (organic nitrogen source) produced a higher growth rate of H. mediterranei than NH4Cl (inorganic source) at all tested C/N ratios. The highest PHA accumulation (18.4% PHA/cell dry mass) was achieved in a media that combined YE with NH4Cl at a C/N ratio of 20. Analysis of the produced polymers revealed the production of poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBHV) with different 3‐hydroxyvalerate (3HV) content. The polymers produced from YE and the combined media have greater 3HV content (10 mol%) than those polymers recovered from NH4Cl (1.5 mol%). Resultingly, PHBHV from YE and the combined media displayed reduced melting points at 144°C. The nitrogen type/concentration was found to also have an impact on the molecular weights and polydispersity indices of the produced biopolymers. Furthermore, the tensile strengths were found to vary with the best tensile strength (14.4 MPa) being recorded for the polymer recovered from YE at C/N = 9. Interestingly, the tensile strength of PHBHV was significantly higher than petroleum‐based polyethylene (13.5 MPa), making it a much more suitable bioplastic for industrial application.

New proposal of nitrogen metabolism regulation by small RNAs in the extreme halophilic archaeon Haloferax mediterranei.

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.

Amylolytic Activities Excreted by the Halophilic Archaeon Haloferax mediterranei to Assimilate Available Starch Depend on the Nitrogen Source

This work was supported by project BIO2013-42921-P from the Spanish Ministry of Economy and Competitiveness.

Optimized bioreactor setup for scale-up studies of extreme halophilic cultures

Adaptation to highly saline conditions required halophilic organisms to develop unique features. The mounting interest in those features has resulted in a number of potential applications for moderate and extreme halophiles. However, industrial exploitation has been reported only for few instances. With the aim to bring concepts from laboratory to pilot-scale a customized bioreactor for halophilic cultures was developed. The setup combined a bubble column reactor (BCR) with a membrane module for cell retention. The BCR was compared to a conventional stirred tank reactor (STR) in terms of physiological and hydro-dynamical parameters. The results showed that the BCR is preferable in terms of energy efficiency. The BCR reached a maximum kLa of 84h−1 at ambient pressure, which equals an oxygen transfer rate (OTR) of 6mmol/(L*h) in medium with 150 g/L NaCl. To reach this mass transfer a STR required more than 3-fold the amount of energy. Cultivation of the extreme halophilic archaeon Haloferax mediterranei showed suitability of the BCR for continuous halophilic processes. To reduce cost and ecological impact of this process a highly saline industrial waste stream from a chemical production process was used as source for NaCl. The BCR presented in this study allows continuous and competitive cultivation of halophilic organisms at high volumetric rates and high biomass productivities as required for large scale industrial applications.

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Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77kDa and optimum activity at 55°C, pH 7.5 and 1.5M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.

Analysis of the Extracellular Polysaccharide-based Structures Produced by a Halophylic Archaeon

This paper investigates the components distribution and structure of an extracellular polysaccharide (EPS) produced by the halophilic archaeon Haloferax mediterranei in the presence or absence of the archaeon by using optical microscopy. The results show that EPS at the end of cultivation is arranged as a discontinuous biofilm and the cells are embedded in this biofilm. EPS and cells cannot be separated through centrifugation, cells remaining attached to the polysaccharide; the biofilm in the supernatant has a similar aspect as in the cultivation broth. The biofilm formed from the supernatant by drying at room temperature is very rich in salts from the cultivation broth (around 200 g/l from which 125 g/l NaCl) and consists on salt crystals which incorporate or are embedded by a film of EPS, distributed discontinuously and having some globular components. In the absence of salts (removed through dialysis), the biofilm formed in the aqueous environment is discontinuous and consists on globular polysaccharides connected by linear EPS which entraps cells remained after centrifugation.

Comparison of four phaC genes from Haloferax mediterranei and their function in different PHBV copolymer biosyntheses in Haloarcula hispanica

BackgroundThe halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, PhaCHme and PhaEHme, has been identified in this strain, and shown to account for the PHBV biosynthesis.ResultsWith the aid of the genome sequence of Hfx. mediterranei CGMCC 1.2087, three additional phaC genes (designated phaC1, phaC2, and phaC3) were identified, which encoded putative PhaCs. Like PhaCHme (54.8 kDa), PhaC1 (49.7 kDa) and PhaC3 (62.5 kDa) possessed the conserved motifs of type III PHA synthase, which was not observed in PhaC2 (40.4 kDa). Furthermore, the longer C terminus found in the other three PhaCs was also absent in PhaC2. Reverse transcription PCR (RT-PCR) revealed that, among the four genes, only phaCHme was transcribed under PHA-accumulating conditions in the wild-type strain. However, heterologous coexpression of phaEHme with each phaC gene in Haloarcula hispanica PHB-1 showed that all PhaCs, except PhaC2, could lead to PHBV accumulation with various 3HV fractions. The three kinds of copolymers were characterized using gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Their thermal properties changed with the variations in monomer composition as well as the different molecular weights (Mw), thus might meet various application requirements.ConclusionWe discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties.

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

Polyhydroxyalkanoate (PHA) Biosynthesis from Whey Lactose

AbstractSummary: The potential of three different microbial wild type strains as polyhydroxyalkanoate (PHA) producers from whey lactose is compared. Homopolyester and co‐polyester biosynthesis was investigated by the archaeon Haloferax mediterranei and the eubacterial strains Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava. H. mediterranei accumulated 50 wt.‐% of poly‐3‐(hydroxybutyrate‐co‐6%‐hydroxyvalerate) in cell dry mass from hydrolyzed whey without addition of 3‐hydroxyvalerate (3HV) precursors (specific productivity qp: 2.9 mg/g h). Using P. hydrogenovora, the final percentage of poly‐3‐hydroxybutyrate (PHB) amounted to 12 wt.‐% (qp: 0.03 g/g h); co‐feeding of valeric acid resulted in the production of 12 wt.‐%. P‐3(HB‐co‐21%‐HV) (qp: 0.02 g/g h). With H. pseudoflava, it was possible to reach 40 wt.‐% P‐3 (HB‐co‐5%‐HV) on not‐hydrolyzed whey lactose plus valeric acid as 3HV precursor (qp: 9.1 mg/g h); on hydrolyzed whey lactose without addition of valeric acid, the strain produced 30 wt.‐% of PHB (qp: 0.16 g/g h). The characterization of the isolated biopolyesters completes the study.

Potential of Various Archae‐ and Eubacterial Strains as Industrial Polyhydroxyalkanoate Producers from Whey

Three different microbial wild-type strains are compared with respect to their potential as industrial scale polyhydroxyalkanoate (PHA) producers from the feed stock whey lactose. The halophilic archaeon Haloferax mediterranei as well as two eubacterial strains (Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava) are investigated. H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-8%-hydroxyvalerate) from hydrolyzed whey without addition of 3-hydroxyvalerate (3HV) precursors (specific productivity q(p): 9.1 mg x g(-1) x h(-1)). Using P. hydrogenovora, the final percentage of poly-3-hydroxybutyrate (PHB) amounted to 12 wt.-% (q(p): 2.9 mg x g(-1) x h(-1)). With H. pseudoflava, it was possible to reach 40 wt.-% P-3(HB-co-5%-HV) on non-hydrolyzed whey lactose plus addition of valeric acid as 3HV precursor (q(p): 12.5 mg x g(-1) x h(-1)). A detailed characterization of the isolated biopolyesters and an evaluation with regard to the economic feasibility completes the study.

Assessment of formal and low structured kinetic modeling of polyhydroxyalkanoate synthesis from complex substrates

A formal kinetic mathematical model for poly-(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolyester synthesis from glucose and galactose derived from whey permeate supplemented with gamma-butyrolactone by the archaeon Haloferax mediterranei was created. Further, a low structured mathematical model for poly-3-hydroxybutyrate synthesis from whey permeate by Pseudomonas hydrogenovora was developed. In both cases, biosyntheses for obtaining the experimental data used for compiling the models were performed via fed-batch cultivations. The model developed for H. mediterranei consists of 10 differential and 11 algebraic equations, including 27 kinetic constants. The model compiled for P. hydrogenovora encompasses 10 differential and 3 algebraic equations, including 36 kinetic constants. Both models were solved by Runge-Kuta variable step numerical integration with Monte Carlo parameter optimization procedure. Difficulties arising from the modeling of redirection of metabolic fluxes from biomass growth toward polyhydroxyalkanoate synthesis and byproducts are discussed.

A new d-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new d-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although α-ketoisoleucine was the most favourable of all α-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.

Enzymatic extruded starch as a carbon source for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Haloferax mediterranei

The extremely halophilic Archaeon Haloferax mediterranei can accumulate polyhydroxyalkanoate (PHA) on a medium containing high salt concentration. The available carbon source for the cell growth and PHA production was the native cornstarch being treated by an enzymatic reactive extrusion process. The extrusion process was processed at a 25 mm single-screw extruder with α-amylase quantities at 1–5 g/100 g wet weight of cornstarch, screw speed at 39–78 rpm and temperatures controlled at 55 °C for zone 1, 65 °C for zone 2, 85 °C for zone 3 and 90 °C for the die. By using an extruded starch/yeast extract ratio (ES/YE ratio) of 1/1.7 g/g in the feeding medium to maintain starch and organic nitrogen concentrations during the pH-stat fed-batch fermentation, the PHA content could reach 50.8% (w/w) of dry cell. From the nuclear magnetic resonance ( 1H NMR) spectrum, the PHA was confirmed to be a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer with a composition of 10.4 mol% of HV unit. The glass transition temperature of this copolymer was −1.2 °C as measured by a differential scanning calorimeter. On the same thermogram, two melting peaks were found at 129.1 and 144.0 °C, respectively; which were much lower than those of the polyhydroxybutyrate (PHB) homopolymer.

Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei

The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.

Identification and transcriptional analysis of nitrate assimilation genes in the halophilic archaeon Haloferax mediterranei

Sequencing a 6,720-bp segment of the extreme halophilic archaeon Haloferax mediterranei genome has revealed the genomic organization of the putative structural genes for nitrate assimilation. We report a gene operon containing nasABC and nasD gene. nasA encodes an assimilatory nitrate reductase, nasB codes for a membrane protein with similarity to the NarK transporter, nasC encodes a protein with similarity to MobA; and nasD codes for an assimilatory ferredoxin-dependent nitrite reductase. Reverse transcription-PCR and primer extension experiments have demonstrated the existence of one polycistronic messenger nasABC and one monocistronic nasD initiated from a different promoter. The gene order and the grouping in two adjacent transcriptional units constitutes a novel organization of nas genes. The promoter regions harbor direct palindromes reminiscent of target sites for binding of a hypotetical regulatory protein(s). Transcription of the nasABC and nasD regions was found to be repressed by the presence of ammonium as nitrogen source.

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w0=[H2O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP+ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.