Halophilic Halomonas Research Articles

Metabolic engineering of Halomonas bluephagenesis for the production of ethylene glycol and glycolate from xylose

Halophilic Halomonas bluephagenesis, a natural producer of poly-3-hydroxybutyrate (PHB), was metabolically engineered to synthesize ethylene glycol and glycolate from xylose. Xylose utilization was achieved by overexpressing either the xylonate pathway or the ribulose-1-phosphate pathway. The key genes encoding for xylonate dehydratase and 2-keto-3-deoxy-xylonate aldolase in the xylonate pathway were screened. With further overexpressing aldehyde reductase gene yjgB, ethylene glycol accumulation was improved to 0.91g/L, accompanied with 1.48g/L of PHB accumulation. The disruption of native glycolate oxidase was found to be essential for glycolate production, and the defective recombinant strain produced 0.80g/L glycolate with 1.14g/L PHB in shake flask cultures. These results indicated that H. bluephagenesis has the potential to produce diverse metabolic chemicals from xylose.

Microbial assemblages and associated biogeochemical processes in Lake Bonney, a permanently ice-covered lake in the McMurdo Dry Valleys, Antarctica

BackgroundLake Bonney, which is divided into a west lobe (WLB) and an east lobe (ELB), is a perennially ice-covered lake located in the McMurdo Dry Valleys of Antarctica. Despite previous reports on the microbial community dynamics of ice-covered lakes in this region, there is a paucity of information on the relationship between microbial genomic diversity and associated nutrient cycling. Here, we applied gene- and genome-centric approaches to investigate the microbial ecology and reconstruct microbial metabolic potential along the depth gradient in Lake Bonney.ResultsLake Bonney is strongly chemically stratified with three distinct redox zones, yielding different microbial niches. Our genome enabled approach revealed that in the sunlit and relatively freshwater epilimnion, oxygenic photosynthetic production by the cyanobacterium Pseudanabaena and a diversity of protists and microalgae may provide new organic carbon to the environment. CO-oxidizing bacteria, such as Acidimicrobiales, Nanopelagicales, and Burkholderiaceae were also prominent in the epilimnion and their ability to oxidize carbon monoxide to carbon dioxide may serve as a supplementary energy conservation strategy. In the more saline metalimnion of ELB, an accumulation of inorganic nitrogen and phosphorus supports photosynthesis despite relatively low light levels. Conversely, in WLB the release of organic rich subglacial discharge from Taylor Glacier into WLB would be implicated in the possible high abundance of heterotrophs supported by increased potential for glycolysis, beta-oxidation, and glycoside hydrolase and may contribute to the growth of iron reducers in the dark and extremely saline hypolimnion of WLB. The suboxic and subzero temperature zones beneath the metalimnia in both lobes supported microorganisms capable of utilizing reduced nitrogens and sulfurs as electron donors. Heterotrophs, including nitrate reducing sulfur oxidizing bacteria, such as Acidimicrobiales (MAG72) and Salinisphaeraceae (MAG109), and denitrifying bacteria, such as Gracilimonas (MAG7), Acidimicrobiales (MAG72) and Salinisphaeraceae (MAG109), dominated the hypolimnion of WLB, whereas the environmental harshness of the hypolimnion of ELB was supported by the relatively low in metabolic potential, as well as the abundance of halophile Halomonas and endospore-forming Virgibacillus.ConclusionsThe vertical distribution of microbially driven C, N and S cycling genes/pathways in Lake Bonney reveals the importance of geochemical gradients to microbial diversity and biogeochemical cycles with the vertical water column.

Engineering EPS in Halomonas bluephagenesis leads to self-flocculation and filamentation for convenient downstream processing

The halophile Halomonas bluephagenesis has established itself as a cost-effective chassis in industrial bioproduction. However, downstream processing (DSP) expense is a persistent challenge. This study uses genetic knockout of exopolysaccharides (EPS-KO) in six industrially relevant H. bluephagenesis strains, each producing distinct polyhydroxyalkanoate (PHA), to evaluate their impact on reducing DSP costs. Remarkably, EPS-KO cells exhibited an unprecedented filamentous morphology, reaching lengths over 50 times (10–75 μm) that of standard cells. This unexpected behavior initially posed a challenge to industrial applicability due to rapid cell lysis under fermentation agitation. Systematic media supplementation of salt, carbon source, nitrogen, and essential minerals significantly influenced morphology, and tailored nutrient schemes that mitigated filamentation while maintaining production levels were designed. Scaled production in a 5000 L bioreactor demonstrated 85 % PHA and 96 g/L cell dry weight after 40 h fermentation, consistent with non-KO cells. During fermentation, EPS-KO enhanced cell-wall penetrability, reducing the need for high-cost γ-butyrolactone by 40 %. Additionally, a new innovative 5000 L FlipFlow bioreactor design cut rotation speed by 50 % and air input by 25 %. During DSP, centrifugation time decreased tenfold due to rapid cell self-flocculation (15–30 min), and the water and enzyme required for PHA purification was reduced by 30 % and 35 %, respectively. This study highlights the use of EPS-KO cells, a controlled media strategy, and low shear force FlipFlow bioreactor to successfully reduce DSP costs without sacrificing productivity. These novel findings represent a notable advancement in cost-saving strategies while presenting the intriguing discovery of media-controlled filamentous cell behavior.

Halophilic Plant Growth-Promoting Rhizobacteria as Producers of Antifungal Metabolites under Salt Stress

Salinity is one of the main factors causing soil deterioration, making it unsuitable for agriculture. It is well documented that the application of halotolerant and halophilic plant growth-promoting bacteria (PGPR: plant growth-promoting rhizobacteria) with biological control activities as an inoculant of cultivated plants offers a biological alternative to the use of agrochemicals, particularly when subjected to salt stress. From this perspective, 70 bacterial strains were isolated from saline soils (sebkha) in arid and semi-arid areas of Eastern Algeria. Three isolates were selected based on their ability to produce bioactive molecules allowing them to promote plant growth, such as hydrolytic enzymes, indole acetic acid (auxin-phytohormone), HCN, NH3, etc. Two of these isolates belonged to the genus Serratia and the third was a halophilic Halomonas bacteria. These bacteria were identified based on their 16S rDNA sequences. Antagonism tests against phytopathogenic fungi were carried out. The identification of the antifungal molecules produced by these bacteria was determined using high-performance liquid chromatography. These bacteria can inhibit mycelial development against phytopathogenic fungi with rates reaching 80.67% against Botrytis cinerea, 76.22% against Aspergillus niger, and 66.67% against Fusarium culmorum for Serratia sp. The strain Halomonas sp. inhibited mycelial growth through the production of volatile substances of Aspergillus niger at 71.29%, Aspergillus flavus at 75.49%, and Penicillium glabrum at a rate of 72.22%. The identification of the antifungal molecules produced by these three bacteria using HPLC revealed that they were polyphenols, which makes these strains the first rhizobacteria capable of producing phenolic compounds. Finally, pot tests to determine the effectiveness of these strains in promoting wheat growth under salinity stress (125 mM, 150 mM, and 200 mM) was carried out. The results revealed that a consortium of two isolates (Serratia sp. and Halomonas sp.) performed best at 125 mM. However, at higher concentrations, it was the halophilic bacteria Halomonas sp. that gave the best result. In all cases, there was a significant improvement in the growth of wheat seedlings inoculated with the bacteria compared to non-inoculated controls.

Enhanced poly(3-hydroxybutyrateco-3-hydroxyvalerate) production from high-concentration propionate by a novel halophile Halomonas sp. YJ01: Detoxification of the 2-methylcitrate cycle

As a carbon substrate, propionate can be used to synthesize poly(3-hydroxybutyrateco-3-hydroxyvalerate) [PHBV] biopolymer, but high concentrations can inhibit PHBV production. Therefore, novel PHBV producers that can utilize high propionate concentrations are needed. Here, a novel halophile, Halomonas sp. YJ01 was applied to PHBV production via a propionate-dependent pathway, and optimal culture growth conditions were determined. The maximum poly(3-hydroxybutyrate) [PHB] content and yield in the presence of glucose were 89.5 wt% and 5.7 g/L, respectively. This strain utilizes propionate and volatile fatty acids (VFAs) for PHBV accumulation. Multiple genes related to polyhydroxyalkanoate (PHA) synthesis were identified using whole-genome annotation. The PHBV yield and 3HV fraction obtained by strain YJ01 utilizing 15 g/L propionate were 0.86 g/L and 29 mol%, respectively, but in cultures with glucose-propionate, it decreased its copolymer dry weight. This indicates that propionyl-CoA was converted to pyruvate through the 2-methylcitrate cycle (2MCC), which reduced propionate detoxification for the strain.

Structure elucidation {single X-ray crystal diffraction studies, Hirshfeld surface analysis, DFT} and antibacterial studies of sulfonamide functionalized Schiff base copper (II) and zinc (II) complexes

Microbial infections tremendously affect the human health and can be cured by antimicrobial agents. In this regard, the copper and zinc complexes of a sulfonamide functionalized Schiff base have been synthesized. The molecular structures confirm that the ligand has acted as N,O- chelator and stabilized the metal complexes by forming six membered ring system. The square planar and octahedral geometries have been assigned to the synthesized complexes on the basis of various spectral analyses. The crystal structure data has been utilized to calculate UV-visible, FT-IR, NMR, HOMO-LUMO energies, molecular electrostatic potential and frontier molecular orbitals using density function theory. The relative stability of the complexes was predicted using global chemical reactivity. The synthesized metal complexes were screened against different bacterial strains. It was concluded that both compounds were effective against almost all microbes. The compound 2 is more potent as compared to 1 with highest activity against Halomonas halophila (22.4 ± 1.2 mm; MIC 79 ± 5 µg). The compounds were further evaluated in terms of docking studies to find out their antimicrobial studies which confirmed that complexes have better antibacterial trends.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Biosynthesis, characterization and optimization of TiO2 nanoparticles by novel marine halophilic Halomonas sp. RAM2: application of natural dye-sensitized solar cells

BackgroundMetal oxide nanoparticles (NPs) are becoming valuable due to their novel applications. The green synthesis of TiO2 NPs is more popular as a flexible and eco-friendly method compared to traditional chemical synthesis methods. TiO2 NPs are the most commonly used semiconductor in dye-sensitized solar cells (DSSCs).ResultsThe biogenic TiO2 NPs were produced extracellularly by the marine halophilic bacterium Halomonas sp. RAM2. Response surface methodology (RSM) was used to optimize the biosynthesis process, resulting in a starting TiO2 concentration of 0.031 M and a pH of 5 for 92 min (⁓15 nm). TiO2 NPs were well-characterized after the calcination process at different temperatures of 500, 600, 700 and 800 °C. Anatase TiO2 NPs (calcined at 500 °C) with a smaller surface area and a wider bandgap were nominated for use in natural dye-sensitized solar cells (NDSSCs). The natural dye used as a photosensitizer is a mixture of three carotenoids extracted from the marine bacterium Kocuria sp. RAM1. NDSSCs were evaluated under standard illumination. After optimization of the counter electrode, NDSSCBio(10) (10 layers) demonstrated the highest photoelectric conversion efficiency (η) of 0.44%, which was almost as good as NDSSCP25 (0.55%).ConclusionThe obtained results confirmed the successful green synthesis of TiO2 NPs and suggested a novel use in combination with bacterial carotenoids in DSSC fabrication, which represents an initial step for further efficiency enhancement studies.

Biopolymer production from biomass produced by Nordic microalgae grown in wastewater

Biomass from four different Nordic microalgal species, grown in BG-11 medium or synthetic wastewater (SWW), was explored as inexpensive carbohydrate-rich feedstock for polyhydroxybutyrate (PHB) production via microbial fermentation. Thermochemical pre-treatment (acid treatment followed by autoclavation) with 2% hydrochloric acid or 1% sulphuric acid (v/v) was used to maximize sugar yield prior to fermentation. Pre-treatment resulted in ∼5-fold higher sugar yield compared to the control. The sugar-rich hydrolysate was used as carbon source for the PHB-producing extremophilic bacterium Halomonas halophila. Maximal PHB production was achieved with hydrolysate of Chlorococcum sp. (MC-1) grown on BG-11 medium (0.27 ± 0.05 g PHB/ g DW), followed by hydrolysate derived from Desmodesmus sp. (RUC-2) grown on SWW (0.24 ± 0.05 g PHB/ g DW). Nordic microalgal biomass grown on wastewater therefore can be used as cheap feedstock for sustainable bioplastic production. This research highlights the potential of Nordic microalgae to develop a biobased economy.

Inhibition of Cyclopropane Fatty Acid Synthesis in the Membrane of Halophilic Halomonas socia CKY01 by Kanamycin

Inhibition of Cyclopropane Fatty Acid Synthesis in the Membrane of Halophilic Halomonas socia CKY01 by Kanamycin

Tunisian hypersaline sediments to set up suitable halotolerant microbial bioanodes for electrostimulated biodegradation of thiabendazole

This study investigated for the first time the performance of microbial halotolerant bioanodes designed from two Tunisian Hypersaline Sediments (THS) for simultaneous electrostimulated biodegradation of synthetic fruit packaging wastewater containing thiabendazole (TBZ), and recovery of an anodic current signal. Halotolerant bioanodes formation has been conducted on 6 cm2 carbon felt electrodes polarized at −0.1Vvs Saturated Calomel Electrode (SCE), inoculated with 80% (v:v) of synthetic wastewater containing 50 ppm of irradiated or not irradiated TBZ and 20% (v:v) of THS for a period of 7 days. Microbial bioanodes, and the corresponding anolytes, i.e., synthetic wastewater, were studied comparatively by electrochemical, microscopic, spectroscopic, molecular and microbial ecology tools. Despite the low maximum current densities recorded in the 50 ppm TBZ runs (3.66 mA/m2), more than 80% of the TBZ was degraded when non-irradiated TBZ (nTBZ) was used as the sole carbon energy by the microorganisms. Nevertheless, the degradation in the presence of irradiated TBZ (iTBZ) was greatly reduced by increasing the irradiation dose with maximum current density of 0.95 mA/m2 and a degradation rate less than 50% of iTBZ. In addition, chemical changes were observed in TBZ as a result of gamma irradiation and bioelectrochemical degradation. FT-IR and UV-Vis techniques confirmed the degradation of TBZ structural bonds producing novel functional groups. Culture-dependent approach and 16S ribosomal RNA sequencing demonstrated that bacterial community of halotolerant bioanodes formed with nTBZ were dominated by Proteobacteria (75%) and Firmicutes (25%). At species level, enrichment of Halomonas smyrnensis, Halomonas halophila, Halomonas salina, Halomonasor ganivorans and Halomonas koreensis on carbon felt electrodes were correlated with maximal current production and nTBZ degradation. As a result, THS halotolerant bacteria, and specifically those from Chott El Djerid (CJ) site certainly have well established application for the electrostimulated microbial biodegradation of fungicide in the real fruit and vegetable processing industries.

Mineralogy of Bioprecipitate Evolution over Induction Times Mediated by Halophilic Bacteria under Various Mg/Ca Molar Ratios

In microbial mineralization experiments, the induction time of mineral precipitation is ambiguous, and this may lead to difficulties in reproducing and confirming the test results. To explore the link between induction time and microbially mediated carbonate precipitation, we report here the mineralogy and morphology of carbonate precipitates induced by the halophilic Halomonas utahensis WMS2 bacterium in media with various Mg/Ca molar ratios over a range of induction times. The results show that the biominerals are formed in an alkaline environment affected by ammonia secreted by H. utahensis WMS2 bacteria. The content of dissolved inorganic carbon increased as a result of carbonic anhydrase catalyzing the hydration of carbon dioxide to release bicarbonate and carbonate ions. The X-ray diffraction (XRD) results show that the phase of mineral precipitated gradually changes from an unstable Mg-rich calcite to metastable monohydrocalcite and then to stable hydromagnesite with an increase in the Mg2+ ion concentration and induction time. The scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FTIR) results show that minerals mostly change from single particles/crystallites to aggregations under the action of the microorganisms at different Mg2+ ion concentrations and induction times. Our experiments demonstrate that the carbonate minerals produced in the presence of microbes change significantly with the induction time, in addition to the influence of the hydrochemical factors; this indicates that the induction time is significant in determining the mineralogy of biominerals.

Baseline proteomics characterisation of the emerging host biomanufacturing organism Halomonas bluephagenesis

Despite its greener credentials, biomanufacturing remains financially uncompetitive compared with the higher carbon emitting, hydrocarbon-based chemical industry. Replacing traditional chassis such as E. coli with novel robust organisms, are a route to cost reduction for biomanufacturing. Extremophile bacteria such as the halophilic Halomonas bluephagenesis TD01 exemplify this potential by thriving in environments inherently inimical to other organisms, so reducing sterilisation costs. Novel chassis are inevitably less well annotated than established organisms. Rapid characterisation along with community data sharing will facilitate adoption of such organisms for biomanufacturing. The data record comprises a newly sequenced genome for the organism and evidence via LC-MS based proteomics for expression of 1160 proteins (30% of the proteome) including baseline quantification of 1063 proteins (27% of the proteome), and a spectral library enabling re-use for targeted LC-MS proteomics assays. Protein data are annotated with KEGG Orthology, enabling rapid matching of quantitative data to pathways of interest to biomanufacturing.

Antibacterial Properties of Several Lichen Extracts against Two Moderately Halophilic Bacteria from Salted Sheepskins

It is well known that possible undesirable defects in finished leathers can result from microbial activities on the salted raw hides/skins during storage. The traditionally used salt curing method can control bacterial activities on the raw stock, but it does not seem possible to completely eliminate microbial attacks. Moderately halophilic bacteria can cause serious damage to hides/skins. From this point of view, potential procedures such as applying new antibacterialagents in the leather industry should be considered. Since some lichen species have been indicated for their promising biological efficacies in the literature; most researchers have focused on their potencies in various fields including leather industry from ancient times. In this study, the bacterial growth of proteolytic and lipolytic Gram-negative moderately halophilic bacteria, Chromohalobacter canadensis (YN6) and Halomonas eurihalina (BL5), from saltedsheepskin samples were tested with the extracts of Usnea sp., Plastismatia glauca, Ramalina farinacea, Evernia divaricata, Bryoria capillaris, Hypogymnia tubulosa, Pseudevernia furfuracea and Lobaria pulmonaria. Some of these lichen species were found to be successful to inhibit the bacterial growth of C. canadensis (YN6) and H. eurihalina (BL5). In conclusion, lichen extracts may be utilized in stacked raw hides/skins in tanneries or warehouses to control moderately halophilic bacteria which causes several defects on leather.

Whole genome sequencing of the halophilic Halomonas qaidamensis XH36, a novel species strain with high ectoine production.

Here, we report the whole genome of a novel halophilic Halomonas species strain XH36 with high ectoine production potential. The genome was 3,818,310bp in size with a GC content of 51.97%, and contained 3533 genes, 61 tRNAs and 18 rRNAs. The phylogenetic analysis using the 16s rRNA genes, the UBCGs and the TYGS database indicated that XH36 belongs to a novel Halomonas species, which we named as Halomonas qaidamensis. Osmoadaptation related genes including Na(+) and K(+) transport and compatible solute accumulation were both present in the XH36 genome, the latter of which mainly contained ectoine, 5-hydroxyectoine and betaine. HPLC validation studies showed that H. qaidamensis XH36 accumulated ectoine to cope with salt stress, and the content of ectoine could be as high as 315mg/g CDW under 3mol/l NaCl. Our results show that XH36 is a new promising industrial strain for ectoine production, and the genomic analysis will guide us to better understand its salt-induced osmoadaptation mechanisms, and provide theoretical references for future application research of ectoine.

Study of osmoadaptation mechanisms of halophilic Halomonas alkaliphila XH26 under salt stress by transcriptome and ectoine analysis.

Halophilic bacteria such as the genus Halomonas are promising candidates in diverse industrial, agricultural and biomedical applications. Here, we successfully isolated a halophilic Halomonas alkaliphila strain XH26 from Xiaochaidan Salt Lake, and studied its osmoadaptation strategies using transcriptome and ectoine analysis. Divergent mechanisms were involved in osmoadaptation at different salinities in H. alkaliphila XH26. At moderate salinity (6% NaCl), increased transcriptions of ABC transporters related to iron (III), phosphate, phosphonate, monosaccharide and oligosaccharide import were observed. At high salinity (15% NaCl), transcriptions of flagellum assembly and cell motility were significantly inhibited. The transcriptional levels of ABC transporter genes related to iron (III) and iron3+-hydroxamate import, glycine betaine and putrescine uptake, and cytochrome biogenesis and assembly were significantly up-regulated. Ectoine synthesis and accumulation was significantly increased under salt stress, and the increased transcriptional expressions of ectoine synthesis genes ectB and ectC may play a key role in high salinity induced osmoadaptation. At extreme high salinity (18% NaCl), 5-hydroxyectoine and ectoine worked together to maintain cell survival. Together these results give valuable insights into the osmoadaptation mechanisms of H. alkaliphila XH26, and provide useful information for further engineering this specific strain for increased ectoine synthesis and related applications.

Theoretical and Experimental Investigations of N‐ and O‐Alkylated Sulfonamides: Density Functional Theory, Hirshfeld Surface Analysis, and Molecular Docking Studies

AbstractThe current article is based on the synthesis of new N‐ and O‐alkylated sulfonamides; 4‐{[Ethyl‐(naphthalene‐2‐sulfonyl)‐amino]‐methyl}‐cyclohexanecarboxylic acid ethyl ester (IV), 4‐[(2‐nitrobenzene‐1‐sulfonylamino)‐methyl]‐cyclohexanecarboxylic acid ethyl ester (V) and 4‐[(Cyclohexa‐1,5‐dienesulfonylamino)‐methyl]‐cyclohexanecarboxylic acid methyl ester (VI). These compounds were characterized by FT‐IR spectroscopy, ESI‐MS spectrometry, and X‐ray crystallography. Density functional theory (DFT) of sulfonamide esters was carried out through the Gaussian‐09 program to optimize structures. Hirshfeld surface analysis has been performed to understand the intermolecular interactions. Biological evaluation of all compounds was done to check their application for antibacterial (Halomonas halophila, Shigella sonnei, Bacillus subtilis, Chromohalobacter salexigens, Staphylococcus aureus Escherichia coli, Serratia marcescens, Chromohalobacter israelensis, Nesseria gonorrhoeae, Klebsiella pneumonia, and Halomonas salina), anti‐fungal (Aspergillus niger), enzyme inhibition activity (Acetylcholine esterase and butyrylcholine esterase) and anti‐oxidant activity (DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) scavenging method, were carried out. Molecular docking was performed to check the interaction modes of these sulfonamide esters.

Sustainable use of agro-industrial wastes as potential feedstocks for exopolysaccharide production by selected Halomonas strains.

Large quantities of waste biomass are generated annually worldwide by many industries and are vastly underutilized. However, these wastes contain sugars and other dissolved organic matter and therefore can be exploited to produce microbial biopolymers. In this study, four selected Halomonas strains, namely, Halomonas caseinilytica K1, Halomonas elongata K4, Halomonas smyrnensis S3, and Halomonas halophila S4, were investigated for the production of exopolysaccharides (EPS) using low-cost agro-industrial wastes as the sole carbon source: cheese whey, grape pomace, and glycerol. Interestingly, both yield and monosaccharide composition of EPS were affected by the carbon source. Glucose, mannose, galactose, and rhamnose were the predominant monomers, but their relative molar ratio was different. Similarly, the average molecular weight of the synthesized EPS was affected, ranging from 54.5 to 4480kDa. The highest EPS concentration (446mg/L) was obtained for H. caseinilytica K1 grown on cheese whey that produced an EPS composed mostly of galactose, rhamnose, glucose, and mannose, with lower contents of galacturonic acid, ribose, and arabinose and with a molecular weight of 54.5kDa. Henceforth, the ability of Halomonas strains to use cost-effective substrates, especially cheese whey, is a promising approach for the production of EPS with distinct physicochemical properties suitable for various applications.

Engineering the permeability of Halomonas bluephagenesis enhanced its chassis properties

Bacterial outer membrane (OM), an asymmetric lipid bilayer functioning as a self-protective barrier with reduced permeability for Gram-negative bacteria, yet wasting nutrients and energy to synthesize, has not been studied for its effect on bioproduction. Here we construct several OM-defected halophile Halomonas bluephagenesis strains to investigate the effects of OM on bioproduction. We achieve enhanced chassis properties of H. bluephagenesis based on positive cellular properties among several OM-defected strains. The OM-defected H. bluephagenesis WZY09 demonstrates better adaptation to lower salinity, increasing 28%, 30% and 12% on dry cell mass (DCM), poly(3-hydroxybutyrate) (PHB) accumulation and glucose to PHB conversion rate, respectively, including enlarged cell sizes and 21-folds reduced endotoxin. Interestingly, a poly(3-hydroxybutyrate-co-21mol%4-hydroxybutyrate) (P(3HB-co-21mol%4HB)) is produced by H. bluephagenesis WZY09 derivate WZY249, increasing 60% and 260% on polyhydroxyalkanoate (PHA) production and 4HB content, respectively. Furthermore, increased electroporation efficiency, more sensitive isopropyl β-D-1-thio-galactopyranoside (IPTG) induction, better oxygen uptake, enhanced antibiotics sensitivity and ectoine secretion due to better membrane permeability are observed if OM defected, demonstrating significant OM defection impacts for further metabolic engineering, synthetic biology studies and industrial applications.

Thirty years of metabolic engineering for biosynthesis of polyhydroxyalkanoates

Polyhydroxyalkanoate (PHA) is a family of biodegradable polyesters synthesized by microorganisms. It has various monomer structures and physical properties with broad application prospects. However, its large-scale production is still hindered by the high cost. In the past 30 years, metabolic engineering approach has been used to tune the metabolic flux, engineer and introduce pathways. The efficiency of PHA synthesis by microorganisms has been significantly improved, and the diversity of PHA monomer, structure and substrate have also been enriched. Meanwhile, by changing cell morphology and PHA particle size, more efficient downstream production process has achieved and PHA production costs have been reduced. In recent years, "Next generation industrial biotechnology" (NGIB) based on extremophiles, especially halophilic Halomonas spp., has been rapidly developed. NGIB has achieved the opening and continuous production of PHA, which simplifies the production process and saves energy and fresh water. Combined with metabolic engineering, Halomonas spp. can be transformed into low-cost production platform of numerous PHA. It is expected to improve the market competitiveness and promote the commercialization of PHA.