Optimization of Growth and Carotenoid Production by Haloferax mediterranei Using Response Surface Methodology.

Haloferax mediterranei produces C50 carotenoids that have strong antioxidant properties. The response surface methodology (RSM) tool helps to accurately analyze the most suitable conditions to maximize C50 carotenoids production by haloarchaea. The effects of temperature (15⁻50 °C), pH (4-10), and salinity (5⁻28% NaCl (w/v)) on the growth and carotenoid content of H. mediterranei were analyzed using the RSM approach. Growth was determined by measuring the turbidity at 600 nm. To determine the carotenoid content, harvested cells were lysed by freeze/thawing, then re-suspended in acetone and the total carotenoid content determined by measuring the absorbance at 494 nm. The analysis of carotenoids was performed by an HPLC system coupled with mass spectrometry. The results indicated the theoretical optimal conditions of 36.51 or 36.81 °C, pH of 8.20 or 8.96, and 15.01% or 12.03% (w/v) salinity for the growth of haloarchaea (OD600 = 12.5 ± 0.64) and production of total carotenoids (3.34 ± 0.29 mg/L), respectively. These conditions were validated experimentally for growth (OD600 = 13.72 ± 0.98) and carotenoid production (3.74 ± 0.20 mg/L). The carotenoid profile showed four isomers of bacterioruberin (89.13%). Our findings suggest that the RSM approach is highly useful for determining optimal conditions for large-scale production of bacterioruberin by haloarchaea.

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea.

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.

Rapid detection of haloarchaeal carotenoids via liquid-liquid microextraction enabled direct TLC MALDI-MS.

For the first time, we demonstrate the use of TiO2 nanoparticles (NPs) for enhancing the carotenoid production by the extremophilic haloarchea, Haloferax mediterranei. TiO2 NPs at optimal concentration of 375 mg/L results in a 95% increase in the production of carotenoid pigment compared to the control (no TiO2 NPs). The carotenoid pigments extracted from TiO2 NPs treated H. mediterranei cells, were separated using thin layer chromatography (TLC). The separated carotenoid spots were subjected directly for MALDI MS detection. To limit the sample diffusion during matrix addition on TLC plates, a simple bordering mode was exercised. Using this method we were able to detect the pigments successfully using MALDI-MS, directly from TLC plates after separation. In addition, we also applied the Pt NPs capped with ODT via Liquid-liquid microextraction (LLME) for extracting the pigment molecules from the halobacteria in MALDI-MS. These novel NP approaches possess numerous advantages such as; rapidity, ease in synthesis, high sensitivity and low cost.

Multiple propionyl coenzyme A-supplying pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Haloferax mediterranei.

Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of (13)C positional enrichment in 3HV. Notably, (13)C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ΔphaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction.

[Identification of the phaB genes and analysis of the PHBV precursor supplying pathway in Haloferax mediterranei].

OBJECTIVE: Identification and characterization of the genes involved in precursor supplying for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in the haloarchaeon Haloferax mediterranei. METHODS: By using BLAST (Basic Local Alignment Search Tool) search methodology, we obtained five genes (phaB1, phaB2, phaJ1, phaJ2 and phaJ3) that were possibly involved in the 3-hydroxyacyl-CoA precursor supplying for PHBV biosynthesis in H. mediterranei. Firstly, we proved that these five genes were all transcribed under the PHBV-accumulating condition in H. mediterranei. Then, we knocked out these genes individually or in combination, by double-crossover homologous recombination, resulting in the following mutants: deltaphaB1, deltaphaB2, AphaJ1, deltaphaJ2, deltaphaJ3, deltaphaB1phaB2, deltaphaJ1phaJ2 and deltaphaJ1phaJ2phaJ3. Finally, we performed the complementation analysis of the deltaphaB1phaB2 strain, with the phaB1 and phaB2 genes, respectively. RESULTS: Whenever the three phaJ genes were knocked out individually or in combination, there was no obvious influence on PHBV accumulation in H. mediterranei. Knockout of phaB1 also did not affect the PHBV accumulation obviously. However, when phaB2 was knocked out, the yield of PHBV and the fraction of the 3-HV monomer decreased significantly. Notably, when the phaB1 and phaB2 were knocked out in combination, the CONCLUSIONS: The PHBV-specific acetoacetyl-CoA reductases mutant deltaphaB1phaB2 no longer produced PHBV. (PhaB) involved in the precursor supplying for PHBV biosynthesis are encoded by phaB1 and phaB2 in H. mediterranei.

GvpD-induced breakdown of the transcriptional activator GvpE of halophilic archaea requires a functional p-loop and an arginine-rich region of GvpD.

The two proteins involved in the regulation of gas vesicle formation in Haloferax mediterranei, mcGvpE (activator) and mcGvpD (repressive function), are able to interact in vitro. It was also found that the respective proteins cGvpE and cGvpD of Halobacterium salinarum and the heterologous pairs mcGvpD-cGvpE and cGvpD-mcGvpE were able to interact. Previously constructed mcGvpD mutants with alterations in regions affecting the repressive function of GvpD (p-loop motif or the two arginine-rich regions bR1 and bR2) were tested for their ability to interact with GvpE, and all still bound GvpE. Even a deletion of or near the p-loop motif in GvpD did not affect this ability to interact. Further deletion variants lacking larger N- or C-terminal portions of mcGvpD yielded that neither the N-terminal region with the p-loop motif nor the C-terminal portion were important for the binding of GvpE, and suggested that the central portion is involved in GvpE binding. The GvpD protein also induces a reduction in the amount of GvpE in Haloferax volcanii transformants expressing both genes under fdx promoter control on a single plasmid. Such DE(ex) transformants contain GvpD, but no detectable GvpE, whereas large amounts of GvpE are found in DeltaDE(ex) transformants that have incurred a deletion within the gvpD gene. A similar reduction was observed in D(ex)+E(ex) transformants harbouring both reading frames under fdx promoter control on two different plasmids. GvpD wild-type and also GvpD mutants were tested, and a significant reduction in the amount of GvpE was obtained in the case of GvpD wild-type and the super-repressor mutant GvpD(3-AAA). In contrast, transformants harbouring GvpD mutants with alterations in the p-loop motif or the bR1 region still contained GvpE. Since the amount of gvpE transcript was not reduced, the reduction occurred at the protein level. These results underlined that a functional p-loop and the arginine-rich region bR1 of GvpD were required for the GvpD-mediated reduction in the amount of GvpE.

NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway.

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe-2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).