Effect of Halomonas titanicae on fluctuating water-line corrosion of EH40 steel.

The fluctuating water-line corrosion of EH40 steel in sterile and biotic media was investigated with a wire beam electrode. When the coupons were partially immersed in the sterile medium, the position of the low water-line acted as the cathodic zone and the area below the low water-line constantly served as the main anodic zone. The thin electrolyte layers with uneven thickness promoted the galvanic current of the region below the low water-line. Different from the sterile environment, the metabolism of Halomonas titanica with oxygen as the final electron acceptor reduced the dissolved oxygen concentration, which resulted in the position of the low water-line acting as the anodic zone.

Unlocking growth potential in Halomonas bluephagenesis for enhanced PHA production with sulfate ions.

The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132 g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124 g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101 g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.

Localization and symbiotic status of probiotics in the coral holobiont.

Corals establish symbiotic relationships with microorganisms, especially endosymbiotic photosynthetic algae. Although other microbes have been commonly detected in coral tissues, their identity and beneficial functions for their host are unclear. Here, we confirm the beneficial outcomes of the inoculation of bacteria selected as probiotics and use fluorescence in situ hybridization (FISH) to define their localization in the coral Pocillopora damicornis. Our results show the first evidence of the inherent presence of Halomonas sp. and Cobetia sp. in native coral tissues, even before their inoculation. Furthermore, the relative enrichment of these coral tissue-associated bacteria through their inoculation in corals correlates with health improvements, such as increases in photosynthetic potential, and productivity. Our study suggests the symbiotic status of Halomonas sp. and Cobetia sp. in corals by indicating their localization within coral gastrodermis and epidermis and correlating their increased relative abundance through active inoculation with beneficial outcomes for the holobiont. This knowledge is crucial to facilitate the screening and application of probiotics that may not be transient members of the coral microbiome. IMPORTANCE: Despite the promising results indicating the beneficial outcomes associated with the application of probiotics in corals and some scarce knowledge regarding the identity of bacterial cells found within the coral tissue, the correlation between these two aspects is still missing. This gap limits our understanding of the actual diversity of coral-associated bacteria and whether these symbionts are beneficial. Some researchers, for example, have been suggesting that probiotic screening should only focus on the very few known tissue-associated bacteria, such as Endozoicomonas sp., assuming that the currently tested probiotics are not tissue-associated. Here, we provide specific FISH probes for Halomonas sp. and Cobetia sp., expand our knowledge of the identity of coral-associated bacteria and confirm the probiotic status of the tested probiotics. The presence of these beneficial microorganisms for corals (BMCs) inside host tissues and gastric cavities also supports the notion that direct interactions with the host may underpin their probiotic role. This is a new breakthrough; these results argue against the possibility that the positive effects of BMCs are due to factors that are not related to a direct symbiotic interaction, for example, that the host simply feeds on inoculated bacteria or that the bacteria change the water quality.

Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation.

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Ectoine hyperproduction by engineered Halomonas bluephagenesis.

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.

Production of polyhydroxyalkanoates by engineered Halomonas bluephagenesis using starch as a carbon source.

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.

Proteomic insights into the response of Halomonas sp. MNB13 to excess Mn(â ¡) and the role of H2S in Mn(â ¡) resistance.

Halomonas spp. are moderately halophilic bacteria with the ability to tolerate various heavy metals. However, the role of basic cellular metabolism, particularly amino acid metabolism, has not been investigated in Halomonas spp. under excess Mn(Ⅱ). The strain Halomonas sp. MNB13 was isolated from a deep-sea ferromanganese nodule and can tolerate 80 mM Mn(Ⅱ). To comprehensively explore the mechanisms underlying its resistance to excess Mn(Ⅱ), we conducted a comparative proteome analysis. The data revealed that both 10 mM and 50 mM Mn(Ⅱ) significantly up-regulated the expression of proteins involved in Mn(Ⅱ) transport (MntE), oxidative stress response (alkyl hydroperoxide reductase and the Suf system), and amino acid metabolism (arginine, cysteine, methionine, and phenylalanine). We further investigated the role of cysteine metabolism in Mn(Ⅱ) resistance by examining the function of its downstream product, H2S. Consistent with the up-regulation of cysteine desulfurase, we detected an elevated level of H2S in Halomonas sp. MNB13 cells under Mn(Ⅱ) stress, along with increased intracellular levels of H2O2 and O2•-. Upon exogenous addition of H2S, we observed a significant restoration of the growth of Halomonas sp. MNB13. Moreover, we identified decreased intracellular levels of H2O2 and O2•- in MNB13 cells, which coincided with a decreased formation of Mn-oxides during cultivation. In contrast, in cultures containing NaHS, the residual Mn(Ⅱ) levels were higher than in cultures without NaHS. Therefore, H2S improves Mn(Ⅱ) tolerance by eliminating intracellular reactive oxygen species rather than decreasing Mn(Ⅱ) concentration in solution. Our findings indicate that cysteine metabolism, particularly the intermediate H2S, plays a pivotal role in Mn(Ⅱ) resistance by mitigating the damage caused by reactive oxygen species. These findings provide new insights into the amino acid mechanisms associated with Mn(Ⅱ) resistance in bacteria.

Structure of the O-polysaccharide from the moderately halophilic bacterium Halomonas fontilapidosi KR26.

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-ß-d-Galp-(1→.

Halomonas rhizosphaerae sp. nov. and Halomonas kalidii sp. nov., two novel moderate halophilic phenolic acid-degrading species isolated from saline soil.

Four vanillic acid-degrading bacterial strains, named LR5S13T, LR5S20, and M4R5S39T and LN1S58, were isolated from Kalidium cuspidatum rhizosphere and bulk soils, respectively. Phylogenetic analysis based on 16S rRNA gene as well as core genome revealed that LR5S13T and LR5S20 clustered closely with each other and with Halomonas ventosae Al12T, and that the two strains shared the highest similarities (both 99.3 %) with H. ventosae Al12T, in contrast, M4R5S39T and LN1S58 clustered together and with Halomonas heilongjiangensis 9-2T, and the two strains shared the highest similarities (99.4 and 99.2 %, respectively) with H. heilongjiangensis 9-2T. The average nucleotides identities based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) values of strains LR5S13T to LR5S20, and M4R5S39T to LN1S58, were both higher than the threshold values for delineation of a species. The ANIb and dDDH values of the four strains to their closely relatives were lower than the threshold values. All four strains take phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol as the major polar lipids, Summed Feature 8, Summed Feature 3, and C16:0 as the major fatty acids. Based on the phylogenetic and phenotypic results, the four strains should be classified as two novel Halomonas species. Therefore, Halomonas rhizosphaerae sp. nov. (type strain LR5S13T = KCTC 8016T = CGMCC 1.62049T) and Halomonas kalidii (type strain M4R5S39T = KCTC 8015T = CGMCC 1.62047T) are proposed. The geographical distribution analysis based on 16S rRNA gene revealed that the two novel species are widely distributed across the globe, specifically in highly saline habits, especially in Central and Eastern Asia.

Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov., isolated from French cheese rinds.

Eight Gram-stain-negative bacterial strains were isolated from cheese rinds sampled in France. On the basis of 16S rRNA gene sequence analysis, all isolates were assigned to the genus Halomonas. Phylogenetic investigations, including 16S rRNA gene studies, multilocus sequence analysis, reconstruction of a pan-genome phylogenetic tree with the concatenated core-genome content and average nucleotide identity (ANI) calculations, revealed that they constituted three novel and well-supported clusters. The closest relative species, determined using the whole-genome sequences of the strains, were Halomonas zhanjiangensis for two groups of cheese strains, sharing 82.4 and 93.1 % ANI, and another cluster sharing 92.2 % ANI with the Halomonas profundi type strain. The strains isolated herein differed from the previously described species by ANI values <95 % and several biochemical, enzymatic and colony characteristics. The results of phenotypic, phylogenetic and chemotaxonomic analyses indicated that the isolates belonged to three novel Halomonas species, for which the names Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov. are proposed, with isolates FME63T (=DSM 113315T=CIRM-BIA2430T=CIP 111880T=LMG 32013T), FME64T (=DSM 113316T=CIRM-BIA2431T=CIP 111877T=LMG 32015T) and FME66T (=DSM 113318T=CIRM-BIA2433T=CIP 111876T=LMG 32014T) as type strains, respectively.

Nonsterile microbial production of chemicals based on Halomonas spp.

The use of extremophile organisms such as Halomomas spp. can eliminate the need for fermentation sterilization, significantly reducing process costs. Microbial fermentation is considered a pivotal strategy to reduce reliance on fossil fuel resources; however, sustainable processes continue to incur higher costs than their chemical industry counterparts. Most organisms require equipment sterilization to prevent contamination, a practice that introduces complexity and financial strain. Fermentations involving extremophile organisms can eliminate the sterilization process, relying instead on conditions that are conductive solely to the growth of the desired organism. This review discusses current challenges in pilot- and industrial-scale bioproduction when using the extremophile bacteria Halomomas spp. under nonsterile conditions.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance.

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Identification and analysis of a clinically isolated strain of Halomonas based on whole-genome sequencing and comparative genomics.

OBJECTIVE: The aim of this study was to identify the species of a Halomonas strain isolated from a neonatal blood sample and to understand the potential pathogenicity and characteristic genes of the strain. METHODS: The genomic DNA of strain 18071143 (identified as Halomonas by matrix-assisted laser desorption-ionization time of flight-mass spectrometry and the 16S ribosomal RNA (rRNA) gene sequence) was sequenced using Nanopore PromethION platforms. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated using the complete genome sequences of the strain. Comparative genomic analyses were performed on strain 18071143 and 3 strains of Halomonas (Halomonas stevensii S18214, Halomonas hamiltonii KCTC 22154, and Halomonas johnsoniae KCTC 22157) that were associated with human infections and had high genomic similarity to strain 18071143. RESULTS: Phylogenetic, ANI, and dDDH similarity analyses based on genome sequence indicated that strain 18071143 belonged to the species H stevensii. Similarities exist between strain 18071143 and the other 3 Halomonas strains in terms of gene structure and protein function. Nonetheless, strain 18071143 has greater potential for DNA replication, recombination, repair, and horizontal transfer. CONCLUSION: Whole-genome sequencing holds great promise for accurate strain identification in clinical microbiology. In addition, the results of this study provide data for understanding Halomonas from the perspective of pathogenic bacteria.

Efficient polyhydroxybutyrate production using acetate by engineered Halomonas sp. JJY01 harboring acetyl-CoA acetyltransferase.

Polyhydroxybutyrate (PHB) is a well-known biodegradable bioplastic synthesized by microorganisms and can be produced from volatile fatty acids (VFAs). Among VFAs acetate can be utilized by Halomonas sp. YLGW01 for growth and PHB production. In this study, Halomonas sp. JJY01 was developed through introducing acetyl-CoA acetyltransferase (atoAD) with LacIq-Ptrc promoter into Halomonas sp. YLGW01. The effect of expression of atoAD on acetate was investigated by comparison with acetate consumption and PHB production. Shake-flask study showed that Halomonas sp. JJY01 increased acetate consumption rate, PHB yield and PHB production (0.27 g/L/h, 0.075 g/g, 0.72 g/L) compared to the wild type strain (0.17 g/L/h, 0.016 g/g, 0.11 g/L). In 10 L fermenter scale fed-batch fermentation, the growth of Halomonas sp. JJY01 resulted in higher acetate consumption rate, PHB yield and PHB titer (0.55 g/L/h, 0.091 g/g, 4.6 g/L) than wild type strain (0.35 g/L/h, 0.067 h/h, 2.9 g/L). These findings demonstrate enhanced acetate utilization and PHB production through the introduction of atoAD in Halomonas strains.

Metabolic engineering of high-salinity-induced biosynthesis of γ-aminobutyric acid improves salt-stress tolerance in a glutamic acid-overproducing mutant of an ectoine-deficient Halomonas elongata.

A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.

Halomonas alkaliantarctica as a platform for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from biodiesel-derived glycerol.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by a wide range of microorganisms, including extremophiles. These unique microorganisms have gained interest in PHA production due to their ability to utilise low-cost carbon sources under extreme conditions. In this study, Halomonas alkaliantarctica was examined with regards to its potential to produce PHAs using crude glycerol from biodiesel industry as the only carbon source. We found that cell dry mass concentration was not dependent on the applying substrate concentration. Furthermore, our data confirmed that the analysed halophile was capable of metabolising crude glycerol into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer within 24 h of the cultivation without addition of any precursors. Moreover, crude glycerol concentration affects the repeat units content in the purified PHAs copolymers and their thermal properties. Nevertheless, a differential scanning calorimetric and thermogravimetric analysis showed that the analysed biopolyesters have properties suitable for various applications. Overall, this study described a promising approach for the valorisation of crude glycerol as a future strategy of industrial waste management to produce high value microbial biopolymers.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.