Haloferax Research Articles

Dead Sea water as a sustainable source for the production of microbial bioplastics polyhydroxyalkanoates by halophiles

The Dead Sea is a unique salt-saturated water body. This work utilized the Dead Sea water (DSW) as a medium instead of fresh water for the biosynthesis of polyhydroxyalkanoates (PHAs) by Haloferax mediterranei. The intracellular content of PHAs reached a maximum (6.35 %) at 40 % DSW. An artificial DSW media that mimics the DSW composition was prepared and used for PHAs production by H. mediterranei. The cell dry mass (CDM) and PHAs comcentration (32.61 g L−1 and 4.56 g L−1, respectively) were significantly higher than the results obtained from the reported H. mediterranei highly saline medium. The PHAs production was scaled-up to fed-batch utilizing no-cost date fruit waste and resulted in CDM and PHA concentration of 46.89 g L−1 and 12.77 g L−1, respectively under non-sterile conditions. H. mediterranei accumulated poly-3-(hydroxybutyrate-co-9 %-hydroxyvalerate) with molar mass 916.0 kDa and melting point at 143.2 °C. These results indicate the production of a high-quality biopolymer.

Long-term effects of cycle time and volume exchange ratio on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from food waste digestate by Haloferax mediterranei cultivated in sequencing batch reactors for 450 days

Food wastedigestatewas fed into a sequencing batch reactor (SBR) forHaloferax mediterranei(HM) to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This SBR was operated uninterruptedly for 450 days to test its stability, during which the cycle time and volume exchange ratiowere varied to understand their impacts on the PHBV fermentation performance under ranged organic loading rates (OLR). Results showed that 1) PHBV productivity was proportional to OLR of food wastedigestate; 2) substrate and product inhibitions were two limiting factors constraining substrate utilization and PHBV yields; 3) PHBV titer was dependent on the hydraulic retention time of the SBR while a volume exchange ratio lower than 0.5 is unfavorable due to the product inhibitor accumulation. This study for the first time demonstrated that the long-term stability of food waste-fed PHBV production byHMand revealed that inhibition effects could be barriers in SBR limiting the full-scale application of the technology.

Bio-adsorption of Fe (II) by dry biomass of metal-tolerant haloarchaeon Haloferax alexandrinus GUSF-1.

Mining-associated activities result in iron pollution exceeding the acceptable limit of 0.3 mg L- 1 and are rampant in estuarine soil and water bodies that harbor halophilic microorganisms. Biotechnologies are underway to unveil the concentrations and recover the metals that skip existing physico-chemical methods. Concerning this, the present study describes for the first time the development of a bio-adsorption batch system using dried cells of Haloferax alexandrinus GUSF-1 for Fe (II) from saline water under microaerophilic conditions. A maximum of 99.5% Fe (II) was adsorbed at pH 6.0, 30 ºC in 3h with 92% efficiency over three adsorption-desorption cycles with saturation and pseudo-second-order kinetics and heterogeneity of Freundlich model having KF of 1.38 mg g- 1 with the n value of 0.96. Adsorbed Fe (II) by the cells was detected by scanning electron microscopy. The involvement of the carboxyl, amino, hydroxyl, and phosphate groups of the cells in interaction with the metal ions was detected by infrared spectroscopy. Conclusively, the study is the first report of whole dried cells mediated metal adsorption by the haloarcheon Haloferax alexandrinus GUSF-1 which acts as promising candidate for metal clean-up strategy and bioremediation in hypersaline ecosystems.

ECO-FRIENDLY BIONANOCOMPOSITE BASED ON BIOSYNTHESIZED POLY(3-HYDROXYBUTYRATE-CO-3-HYDROXYVALERATE) REINFORCED WITH Ni-CuO NANOPARTICLES FOR REMOVAL OF BRILLIANT GREEN

Ni-doped Copper oxide (Ni-CuO) nanoparticles have been synthesized from Arabic Gum. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) polyester was biosynthesized by the microorganism Haloferax mediterranei utilizing date waste as carbon source. The produced Ni-doped copper oxide (Ni-CuO) nanoparticles have been incorporated with different percentages into the PHBV matrix. The produced bionanocomposites were achieved with different percentages of the nanoparticles: 1%, 3%, 5% and 10%, and were referred to as PHBV/Ni-CuO(1,3,5,10%). FTIR, TGA, XRD, SEM and EDX techniques have been used to study and characterize the synthesized bionanocoposites. In addition, the prepared nanocomposites were studied for their efficiency as solid phase adsorbents for Brilliant Green (B.G.) dye from water resources under different conditions. The prepared nanocomposites were found to be very efficient and promising solid phase adsorbent materials to treat water samples for the purpose of dye removal. The percentage of the removed dye increased from 45.6% to 97.7% as the PHBV/Ni-CuO(10%) nanocomposite amount increased from 10 mg to 70 mg per 25 mL of water sample. The dye removal percentage reached an equilibrium in 90 min. Natural water samples from three different sources have been tested against the Ni-CuO/PHBV(3%) nanocomposite as solid adsorbent for the B.G. removal, and the results showed >90% dye removal in all cases under the optimum experimental conditions.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei Using Candy Industry Waste as Raw Materials.

The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical properties of biotechnological and biomedical interest and can be used as an alternative to plastics made from chemical synthesis (which are not environmentally friendly). The versatile metabolism of H. mediterranei makes the use of waste as a carbon source for cellular growth and PHA synthesis possible. In this work, cellular growth and the production and characterization of PHBV using two different types of confectionery waste were analyzed and compared with cellular growth and PHBV synthesis in a standard culture media with glucose of analytical grade as a carbon source. The PHBV granules produced were analyzed by TEM and the biopolymer was isolated and characterized by GC-MS, FTIR NMR, and DSC. The results reveal that H. mediterranei can use these two residues (R1 and R2) for pure PHBV production, achieving 0.256 and 0.983 g PHBV/L, respectively, which are among the highest yields so far described using for the first-time waste from the candy industry. Thus, a circular economy-based process has been designed to optimize the upscaling of PHBV production by using haloarchaea as cell factories and valorizing confectionery waste.

Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis.

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance.IMPORTANCEDenitrification, a fundamental process within the nitrogen cycle, has been subject to extensive investigation due to its close association with anthropogenic activities, and its contribution to the global warming issue, mainly through the release of N2O emissions. Although our comprehension of denitrification and its implications is generally well established, most studies have been conducted in non-extreme environments with mesophilic microorganisms. Consequently, there is a significant knowledge gap concerning extremophilic denitrifiers, particularly those inhabiting hypersaline environments. The significance of this research was to delve into the process of haloarchaeal denitrification, utilizing the complete denitrifier haloarchaeon Haloferax mediterranei as a model organism. This research led to the analysis of the metabolic state of this microorganism under denitrifying conditions and the identification of regulatory signals and genes encoding proteins potentially involved in this pathway, serving as a valuable resource for future molecular studies.

Food wastes for bioproduct production and potential strategies for high feedstock variability

Unavoidable food wastes could be an important feedstock for industrial biotechnology, while their valorization could provide added value for the food processor. However, despite their abundance and low costs, the heterogeneous/mixed nature of these food wastes produced by food processors and consumers leads to a high degree of variability in carbon and nitrogen content, as well as specific substrates, in food waste hydrolysate. This has limited their use for bioproduct synthesis. These wastes are often instead used in anaerobic digestion and mixed microbial culture, creating a significant knowledge gap in their use for higher value biochemical production via pure and single microbial culture. To directly investigate this knowledge gap, various waste streams produced by a single food processor were enzymatically hydrolyzed and characterized, and the degree of variability with regard to substrates, carbon, and nitrogen was quantified. The impact of hydrolysate variability on the viability and performance of polyhydroxyalkanoates biopolymers production using bacteria (Cupriavidus necator) and archaea (Haloferax mediterranei) as well as sophorolipids biosurfactants production with the yeast (Starmerella bombicola) was then elucidated at laboratory-scale. After which, strategies implemented during this experimental proof-of-concept study, and beyond, for improved industrial-scale valorization which addresses the high variability of food waste hydrolysate were discussed in-depth, including media standardization and high non-selective microbial organisms growth-associated product synthesis. The insights provided would be beneficial for future endeavors aiming to utilize food wastes as feedstocks for industrial biotechnology.

Bioplastic Production from Agri-Food Waste through the Use of Haloferax mediterranei: A Comprehensive Initial Overview.

The research on bioplastics (both biobased and biodegradable) is steadily growing and discovering environmentally friendly substitutes for conventional plastic. This review highlights the significance of bioplastics, analyzing, for the first time, the state of the art concerning the use of agri-food waste as an alternative substrate for biopolymer generation using Haloferax mediterranei. H. mediterranei is a highly researched strain able to produce polyhydroxybutyrate (PHB) since it can grow and produce bioplastic in high-salinity environments without requiring sterilization. Extensive research has been conducted on the genes and pathways responsible for PHB production using H. mediterranei to find out how fermentation parameters can be regulated to enhance cell growth and increase PHB accumulation. This review focuses on the current advancements in utilizing food waste as a substitute for costly substrates to reduce feedstock expenses. Specifically, it examines the production of biomass and the recovery of PHB from agri-food waste. Furthermore, it emphasizes the characterization of PHB and the significance of hydroxyvalerate (HV) abundance in the formation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer. The downstream processing options are described, and the crucial factors associated with industrial scale-up are assessed, including substrates, bioreactors, process parameters, and bioplastic extraction and purification. Additionally, the economic implications of various options are discussed.

Production of Polyhydroxyalkanoates for Biodegradable Food Packaging Applications Using Haloferax mediterranei and Agrifood Wastes.

Polyhydroxyalkanoates (PHAs) are high-value biodegradable polyesters with thermoplastic properties used in the manufacturing of different products such as packaging films. PHAs have gained much attention from researchers and industry because of their biobased nature and appropriate features, similar to conventional synthetic plastics. This review aims to discuss some of the recent solutions to challenges associated with PHA production. The implementation of a cost-effective process is presented by following different strategies, such as the use of inexpensive carbon sources, the selection of high-producing microorganisms, and the functionalization of the final materials to make them suitable for food packaging applications, among others. Research efforts are needed to improve the economic viability of PHA production at a large scale. Haloferax mediterranei is a promising producer of PHAs due to its ability to grow in non-sterile conditions and the possibility of using seawater to prepare the growth medium. Additionally, downstream processing for PHA extraction can be simplified by treating the H. mediterranei cells with pure water. Further research should focus on the optimization of the recycling conditions for the effluents and on the economic viability of the side streams reutilization and desalinization as an integrated part of PHA biotechnological production.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Development of a genetic system for Haloferax gibbonsii LR2-5, model host for haloarchaeal viruses.

Species from all domains of life are infected by viruses. In some environments, viruses outnumber their microbial hosts by a factor of 10, and viruses are the most important predators of microorganisms. While much has been discovered about the infection mechanisms of bacterial and eukaryotic viruses, archaeal viruses remain understudied. Good model systems are needed to study their virus-host interactions in detail. The salt-loving archaeon Haloferax gibbonsii LR2-5 has been shown to be infected by a variety of different viruses and, thus, is an excellent model to study archaeal viruses. By establishing a genetic system, we have significantly expanded the toolbox for this model organism, which will fuel our understanding of infection strategies of the underexplored archaeal viruses.

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through β-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

Deepening the knowledge of universal stress proteins in Haloferax mediterranei

Haloarchaea, like many other microorganisms, have developed defense mechanisms such as universal stress proteins (USPs) to cope with environmental stresses affecting microbial growth. Despite the wide distribution of these proteins in Archaea, their biochemical characteristics still need to be discovered, and there needs to be more knowledge about them focusing on halophilic Archaea. Therefore, elucidating the role of USPs would provide valuable information to improve future biotechnological applications. Accordingly, transcriptional expression of the 37 annotated USPs in the Haloferax mediterranei genome has been examined under different stress conditions. From a global perspective, finding a clear tendency between particular USPs and specific stress conditions was not possible. Contrary, data analysis indicates that there is a recruitment mechanism of proteins with a similar sequence able to modulate the H. mediterranei growth, accelerating or slowing it, depending on their number. In fact, only three of these USPs were expressed in all the tested conditions, pointing to the cell needing a set of USPs to cope with stress conditions. After analysis of the RNA-Seq data, three differentially expressed USPs were selected and homologously overexpressed. According to the growth data, the overexpression of USPs induces a gain of tolerance in response to stress, as a rule. Therefore, this is the only work that studies all the USPs in an archaeon. It represents a significant first base to continue advancing, not only in this important family of stress proteins but also in the field of biotechnology and, at an industrial level, to improve applications such as designing microorganisms resistant to stress situations.Key points• Expression of Haloferax mediterranei USPs has been analyzed in stress conditions.• RNA-seq analysis reveals that most of the USPs in H. mediterranei are downregulated.• Homologous overexpression of USPs results in more stress-tolerant strains.

Carotenoid production by Haloferax mediterranei using starch residues from the candy industry as a carbon source

Carotenoids are pigments attracting the attention of several industries due to their antioxidant, biological and coloring properties. Low-cost substrates, such as agro-industrial wastes, are being investigated as a viable option to reduce microbial production costs in processes in which microorganisms such as haloarchaea are used as cell factories to produce marketed compounds like carotenoids. They can grow on various agro-industrial wastes and produce the C50 carotenoid bacterioruberin (BR), which is an extraordinary antioxidant compound with anticancer properties. In this study, the haloarchaeon Haloferax mediterranei is grown in the presence of starch residues from thecandy industry to induce the production of carotenoids. Cells grew successfully with this industrial waste (max. O.D. 600 nm = 27.75 ± 0.09). Biomass production increased in the presence of higher quantities of starch up to 17.3 ± 0.2 mg/ml of cell culture. The maximum BR concentration was 97.39 ± 1.86 µg/ml. The total amount of BRs synthesized increased when cells grew with increasing concentrations of the industrial starch. The relative percentages of all-trans-BR, 5-cis-BR and a double isomeric BR rose, whereas 9-cis-BR and 13-cis-BR levels decreased.Herein, haloarchaeal growth and carotenoid production can be enhanced using industrial waste products as the starch residues selected for this experiment which were provided by a candy company.

Analysis of Lsm Protein-Mediated Regulation in the Haloarchaeon Haloferax mediterranei.

The Sm protein superfamily includes Sm, like-Sm (Lsm), and Hfq found in the Eukarya, Archaea, and Bacteria domains. Archaeal Lsm proteins have been shown to bind sRNAs and are probably involved in various cellular processes, suggesting a similar function in regulating sRNAs by Hfq in bacteria. Moreover, archaeal Lsm proteins probably represent the ancestral Lsm domain from which eukaryotic Sm proteins have evolved. In this work, Haloferax mediterranei was used as a model organism because it has been widely used to investigate the nitrogen cycle and its regulation in Haloarchaea. Predicting this protein's secondary and tertiary structures has resulted in a three-dimensional model like the solved Lsm protein structure of Archaeoglobus fulgidus. To obtain information on the oligomerization state of the protein, homologous overexpression and purification by means of molecular exclusion chromatography have been performed. The results show that this protein can form hexameric complexes, which can aggregate into 6 or 12 hexameric rings depending on the NaCl concentration and without RNA. In addition, the study of transcriptional expression via microarrays has allowed us to obtain the target genes regulated by the Lsm protein under nutritional stress conditions: nitrogen or carbon starvation. Microarray analysis has shown the first universal stress proteins (USP) in this microorganism that mediate survival in situations of nitrogen deficiency.

Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T

BackgroundThe extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance.ResultsThe growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM–EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data.ConclusionsOur results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals.

Study on the correlation between the dominant microflora and the main flavor substances in the fermentation process of cigar tobacco leaves

The flavor of cigar tobacco leaf determines the quality of finished cigar tobacco, and the enhancement of flavor generally relies on microbial fermentation. In this paper, the correlation between the dominant microorganisms and the main flavor substances of cigar tobacco leaves during fermentation and the correlation between the two were investigated to reveal the correlation between microorganisms and flavor and the metabolic pathways of microorganisms affecting the flavor substances. During the fermentation process, the main flavors of cigar tobacco leaves were sweet, light and grassy, with hexanal, 2,6-dimethylpyridine, nonanal, phenylacetaldehyde, naphthalene, and methyl benzoate as the main constituents, and the key microorganisms Haloferax mediterranei, Haloterrigena limicola, Candidatus Thorarchaeota archaeon SMTZ-45, the genera Methyloversatilis, Sphingomonas, Thauera, Pseudomonas, Penicillium, and Aspergillus. Correlation analysis revealed that fungi were negatively correlated with the main aroma and inhibited the main flavor substances, while bacteria were positively correlated with Benzoic acid, methyl ester in the main flavor substances, which was conducive to the accumulation of green aroma. Functional analysis revealed that the dominant bacterial population was producing aroma by metabolizing glycoside hydrolases and glycosyltransferases, performing amino acid metabolism, carbohydrate metabolism and film transport metabolism. The present study showed that the bacterial and fungal dominant microorganisms during the fermentation of cigar tobacco were influencing the production and degradation of the main flavor substances through the enzyme metabolism by the occurrence of the Merad reaction.

Neighboring inteins interfere with one another's homing capacity.

Inteins are mobile genetic elements that invade conserved genes across all domains of life and viruses. In some instances, a single gene will have several intein insertion sites. In Haloarchaea, the minichromosome maintenance (MCM) protein at the core of replicative DNA helicase contains four intein insertion sites within close proximity, where two of these sites (MCM-a and MCM-d) are more likely to be invaded. A haloarchaeon that harbors both MCM-a and MCM-d inteins, Haloferax mediterranei, was studied in vivo to determine intein invasion dynamics and the interactions between neighboring inteins. Additionally, invasion frequencies and the conservation of insertion site sequences in 129 Haloferacales mcm homologs were analyzed to assess intein distribution across the order. We show that the inteins at MCM-a and MCM-d recognize and cleave their respective target sites and, in the event that only one empty intein invasion site is present, readily initiate homing (i.e. single homing). However, when two inteins are present co-homing into an intein-free target sequence is much less effective. The two inteins are more effective when invading alleles that already contain an intein at one of the two sites. Our in vivo and computational studies also support that having a proline in place of a serine as the first C-terminal extein residue of the MCM-d insertion site prevents successful intein splicing, but does not stop recognition of the insertion site by the intein's homing endonuclease.

Unveiling the mechanism of static magnetic field effects on carbon flux into polyhydroxyalkanoate synthesis by Haloferax mediterranei

The high cost of polyhydroxyalkanoate (PHA) production significantly limits the large-scale industrial production of PHA. In this regard, applying static magnetic field (SMF) to increase PHA productivity could be a novel approach. The study explored the biological magnetic effects of H. mediterranei for PHA production. A magnetic window for PHA synthesis was found under 110 mT SMF, at which the PHA volumetric productivity increased by 27.28% compared with the control group. The carbon flux in the pathway from acetyl-CoA to the PHA synthesis increased while the flux to the TCA cycle decreased. Combined with transcriptome and proteome analysis, the key genes pdhA2, Paaj, and AccA in the PHA synthesis pathway were up-regulated, while gltA in the TCA cycle was down-regulated under 110 mT SMF. This study revealed the mechanism of SMF enhancement on PHA synthesis by H. mediterranei, which provides an effective solution to increasing PHA volumetric productivity.

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.