Heterologous expression of sulfur: quinone oxidoreductase (Sqr) to improve Thioalkalivibrio versutus D301 desulfurization performance.

AIMS: Heterologous expression of sulfur: quinone oxidoreductase (Sqr) from Halomonas mongoliensis JS01, which is responsible for oxidizing sulfide to elemental sulfur, in Thioalkalivibrio versutus (T. versutus) D301 improves desulfurization. METHODS AND RESULTS: We expressed sqr in T. versutus D301 by conjugative transfer and then assayed its desulfurization capacity in an airlift reactor and analyzed its transcriptome at -380 mV ORP. Our findings demonstrate that the D301-sqr+ strain, utilizing sodium sulfide as a sulfur source under optimal ORP conditions (-380 mV), achieved an elemental sulfur yield of 95%. This represents an 8% increase over the T. versutus D301. Moreover, the sodium sulfide utilization rate for D301-sqr+ showed a marked improvement [0.741 vs. 0.651 mmol∙(l·h)-1], with a concurrent increase in the rate of elemental sulfur production when compared to the T. versutus D301 (0.716 vs. 0.518 mmol ∙(l·h)-1). Transcriptome analysis revealed that the flavocytochrome c (fcc) and the sox system were differentially transcriptionally down-regulated in D301-sqr+ compared with the T. versutus D301. CONCLUSIONS: Heterologous expression of the gene sqr altered the transcription of related genes in T. versutus D301 sulfur oxidation pathway, increasing the yield of elemental sulfur and the rate of sulfur oxidation, and making D301-sqr+ more potential for industrial applications.

Bioremediation of uranium enriched coal fly ash based on microbially induced calcite precipitation.

Coal fly ash (CFA) is an essential raw material in brickmaking industry worldwide. There are some coal mines with a relatively high content of uranium (U) in the Xinjiang region of China that are yet understudied. The CFA from these coal mines poses substantial environmental risks due to the concentrated uranium amount after coal burning. In this paper, we demonstrated a calcifying ureolytic bacterium Halomonas sp. SBC20 for its biocementation of U in CFA based on microbially induced calcite precipitation (MICP). Rectangle-shaped CFA bricks were made from CFA using bacterial cells, and an electric testing machine tested their compressive strength. U distribution pattern and immobility against rainfall runoff were carefully examined by a five-stage U sequential extraction method and a leaching column test. The microstructural changes in CFA bricks were characterized by FTIR and SEM-EDS methods. The results showed that the compressive strength of CFA bricks after being cultivated by bacterial cells increased considerably compared to control specimens. U mobility was significantly decreased in the exchangeable fraction, while the U content was markedly increased in the carbonate-bound fraction after biocementation. Much less U was released in the leaching column test after the treatment with bacterial cells. The FTIR and SEM-EDX methods confirmed the formation of carbonate precipitates and the incorporation of U into the calcite surfaces, obstructing the release of U into the surrounding environments. The technology provides an effective and economical treatment of U-contaminated CFA, which comes from coal mines with high uranium content in the Xinjiang region, even globally.

Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered Halomonas elongata.

Halomonas elongata OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible ectABC operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing H. elongata GOP-Gad (ΔectABC::mCherry-HopGadmut) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted gabT, which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the H. elongata GOP-Gad genome. The resulting strain H. elongata ZN3 (ΔectABC::mCherry-HopGadmut ΔgabT) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from H. elongata GOP-Gad's 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a gabT-deletion mutant of H. elongata OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an ectB gene of the ectABC operon, shares sequence similarity with GABA-AT, a complementation analysis of the gabT and the ectB genes was performed in the H. elongata ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in H. elongata ZN3 and establish DABA-AT's aminotransferase activity toward GABA in vivo. IMPORTANCE: In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing H. elongata ZN3 strain by deleting the gabT gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first in vivo evidence for GABA aminotransferase activity of an ectB-encoded DABA-AT, confirming a longstanding speculation based on the reported in vitro GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the ectB gene is upregulated.

Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata.

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

Combined alkali-photocatalytic stimulation enables click microbial domestication for boosted ammonia nitrogen removal.

Microbe-driven ammonia nitrogen removal plays a crucial role in the nitrogen cycle and wastewater treatment. However, the rational methods and mechanisms for boosting nitrogen conversion through microbial domestication are still limited. Herein, a combined alkali-photocatalytic stimulation strategy was developed to activate the Halomonas shizuishanensis DWK9 for efficient ammonia nitrogen removal. The strain DWK9 selected from saline-alkaline soil in Northwestern China possessed strong resistance to stress of saline-alkaline environment and free radicals, and was abundant in nitrogen conversion genes, thus is an ideal model for advanced microbial domestication. Bacterial in the combined alkali-photocatalytic stimulation group achieved a high ammonia nitrogen conversion rate of 67.5 %, 10 times outperforming the non-stimulated and single alkali/photocatalytic stimulation control groups. Morphology analysis revealed that the bacteria in the alkali-photocatalytic stimulated group formed a favorable structure for bioelectric transfer. Remarkably, the domesticated bacteria demonstrated improved electrochemical properties, including increased current capacity and lower overpotentials and impedance. Prokaryotic transcription genetic analysis together with qPCR analysis showed upregulation of denitrification-related metabolic pathway genes. A novel FAD dependent and NAD(P)H independent energy mode has been proposed. The universality and effectiveness of the as-developed combined alkali-photocatalytic microbial domestication strategy were further validated through indicator fish survival experiments. This work provides unprecedented degrees of freedom for the exploration of rational microbial engineering for optimized and controllable biogeochemical conversion.

[One-pot synthesis of itaconic acid by engineered Halomonas bluephagenesis TDZI-08].

Itaconic acid (IA) is one of the twelve high value-added platform compounds applied in various fields including coatings, adhesives, plastics, resins, and biofuels. In this study, we established a one-pot catalytic synthesis system for IA from citric acid based on the engineered salt-tolerant bacterial strain Halomonas bluephagenesis TDZI-08 after investigating factors that hindered the process and optimizing the carbon source, nitrogen source, inducer addition time, and surfactant dosage. The open, non-sterile, one-pot synthesis with TDZI-08 in a 5 L fermenter achieved the highest IA titer of 40.50 g/L, with a catalytic yield of 0.68 g IA/g citric acid during the catalytic stage and a total yield of 0.42 g IA/g (citric acid+gluconic acid). The one-pot synthesis system established in this study is simple and does not need sterilization or aseptic operations. The findings indicate the potential of H. bluephagenesis for industrial production of IA.

Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass.

BACKGROUND: Ectoine as an amino acid derivative is widely applied in many fields, such as the food industry, cosmetic manufacturing, biologics, and therapeutic agent. Large-scale production of ectoine is mainly restricted by the cost of fermentation substrates (e.g., carbon sources) and sterilization. RESULTS: In this study, Halomonas cupida J9 was shown to be capable of synthesizing ectoine using xylose as the sole carbon source. A pathway was proposed in H. cupida J9 that synergistically utilizes both WBG xylose metabolism and EMP glucose metabolism for the synthesis of ectoine. Transcriptome analysis indicated that expression of ectoine biosynthesis module was enhanced under salt stress. Ectoine production by H. cupida J9 was enhanced by improving the expression of ectoine biosynthesis module, increasing the intracellular supply of the precursor oxaloacetate, and utilizing urea as the nitrogen source. The constructed J9U-P8EC achieved a record ectoine production of 4.12 g/L after 60 h of xylose fermentation. Finally, unsterile production of ectoine by J9U-P8EC from either a glucose-xylose mixture or corn straw hydrolysate was demonstrated, with an output of 8.55 g/L and 1.30 g/L of ectoine, respectively. CONCLUSIONS: This study created a promising H. cupida J9-based cell factory for low-cost production of ectoine. Our results highlight the potential of J9U-P8EC to utilize lignocellulose-rich agriculture waste for open production of ectoine.

Expression of an engineered salt-inducible proline biosynthetic operon in a glutamic acid over-producing mutant, Halomonas elongata GOP, confers increased proline yield due to enhanced growth under high-salinity conditions.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata Glutamic acid Over-Producing, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

Isolation and characterization of a Halomonas species for non-axenic growth-associated production of bio-polyesters from sustainable feedstocks.

Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01." Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strain's exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to ~60% of its biomass (dry wt/wt) in poly(3-hydroxybutyrate), while reaching a doubling time of 1.7 h at 30°C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but also facilitate the release of intracellular products through osmolysis. The development of a minimal medium also allowed the estimation of maximum polyhydroxybutyrate production rates, which were projected to exceed 5 g/h. Finally, also, the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCE: The urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing, especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

Molecular identification of Halomonas AZ07 and its multifunctional enzymatic activities to degrade Pyropia yezoensis under high-temperature condition.

BACKGROUND: Pyropia yezoensis a commercially important red seaweed species, is susceptible to various microorganisms infections, among which bacterial infections are the most prominent ones. Pyropia yezoensis is often affected by harmful bacterial communities under high temperatures that can lead to its degradation and economic losses. The current study aimed to explore Pyropia yezoensis-associated microbiota and further identify potential isolates, which can degrade Pyropia yezoensis under high-temperature conditions. METHODS AND RESULTS: The 16S rRNA gene sequencing was used to identify the agarolytic bacterial species. The results showed that Chromohalobacter sp. strain AZ6, Pseudoalteromonas sp. strain AZ, Psychrobacter sp. strain AZ3, Vibrio sp. strain AZ, and Halomonas sp. strain AZ07 exhibited algicidal properties as these strains were more abundant at high temperature (25 °C). Among the five isolated strains, the potential isolate Halomonas sp. strain AZ07 showed high production of agarolytic enzymes, including lipase, protease, cellulase, and amylase. This study confirmed that the isolated strain could produce these four different enzymes. The strain Halomonas AZ07 was co-treated with Pyropia yezoensis cells under two different temperature environments, including 13 °C and 25 °C. The degradation of Pyropia yezoensis occurred at the optimum temperature of 25 °C and effectively degraded their cell wall, proteins, lipids, and carbohydrates. CONCLUSION: The successful cultivation of Pyropia yezoensis in coastal farm environments is dependent on specific temperature and environmental factors, and lower temperatures have been observed to be particularly beneficial for the survival and growth of Pyropia yezoensis. The temperature below 13 °C was confirmed to be the best niche for the symbiotic relationship of microbiota associated with Pyropia yezoensis for its growth, development, and production.

A nuclease domain fused to the Snf2 helicase confers antiphage defence in coral-associated Halomonas meridiana.

The coral reef microbiome plays a vital role in the health and resilience of reefs. Previous studies have examined phage therapy for coral pathogens and for modifying the coral reef microbiome, but defence systems against coral-associated bacteria have received limited attention. Phage defence systems play a crucial role in helping bacteria fight phage infections. In this study, we characterized a new defence system, Hma (HmaA-HmaB-HmaC), in the coral-associated Halomonas meridiana derived from the scleractinian coral Galaxea fascicularis. The Swi2/Snf2 helicase HmaA with a C-terminal nuclease domain exhibits antiviral activity against Escherichia phage T4. Mutation analysis revealed the nickase activity of the nuclease domain (belonging to PDD/EXK superfamily) of HmaA is essential in phage defence. Additionally, HmaA homologues are present in ~1000 bacterial and archaeal genomes. The high frequency of HmaA helicase in Halomonas strains indicates the widespread presence of these phage defence systems, while the insertion of defence genes in the hma region confirms the existence of a defence gene insertion hotspot. These findings offer insights into the diversity of phage defence systems in coral-associated bacteria and these diverse defence systems can be further applied into designing probiotics with high-phage resistance.

Determination of the safety of Halomonas sp. KM-1-derived d-ß-hydroxybutyric acid and its fermentation-derived impurities in mice and Japanese adults.

The use of halophilic bacteria in industrial chemical and food production has received great interest because of the unique properties of these bacteria; however, their safety remains under investigation. Halomonas sp. KM-1 intracellularly stores poly-D-ß-hydroxybutyric acid under aerobic conditions and successively secretes D-ß-hydroxybutyric acid (D-BHB) under microaerobic conditions. Therefore, we tested the safety of Halomonas sp. KM-1-derived D-BHB and the impurities generated during D-BHB manufacturing at a 100-fold increased concentration in acute tests using mice and daily intake of 16.0 g D-BHB in Japanese adults for 12 weeks. In the mice test, there were no abnormalities in the body weights or health of mice fed the purified D-BHB or its impurities. In the Japanese adult test, blood parameters and body condition showed no medically problematic fluctuations. These findings indicate that Halomonas sp. KM-1 is safe and can be used for commercial production of D-BHB and its derivatives.

A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM.

Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions.

Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: • Hydroxyectoine production in H. salifodinae correlates with the salinity of medium • Transcriptomic analysis reveals the limiting factors for hydroxyectoine production • The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.

Characterization of the surface-active exopolysaccharide produced by Halomonas sp TGOS-10: Understanding its role in the formation of marine oil snow.

In this study, we characterize the exopolymer produced by Halomonas sp. strain TGOS-10 -one of the organisms found enriched in sea surface oil slicks during the Deepwater Horizon oil spill. The polymer was produced during the early stationary phase of growth in Zobell's 2216 marine medium amended with glucose. Chemical and proton NMR analysis showed it to be a relatively monodisperse, high-molecular-mass (6,440,000 g/mol) glycoprotein composed largely of protein (46.6% of total dry weight of polymer). The monosaccharide composition of the polymer is typical to that of other marine bacterial exopolymers which are generally rich in hexoses, with the notable exception that it contained mannose (commonly found in yeast) as a major monosaccharide. The polymer was found to act as an oil dispersant based on its ability to effectively emulsify pure and complex oils into stable oil emulsions-a function we suspect to be conferred by the high protein content and high ratio of total hydrophobic nonpolar to polar amino acids (52.7:11.2) of the polymer. The polymer's chemical composition, which is akin to that of other marine exopolymers also having a high protein-to-carbohydrate (P/C) content, and which have been shown to effect the rapid and non-ionic aggregation of marine gels, appears indicative of effecting marine oil snow (MOS) formation. We previously reported the strain capable of utilising aromatic hydrocarbons when supplied as single carbon sources. However, here we did not detect biodegradation of these chemicals within a complex (surrogate Macondo) oil, suggesting that the observed enrichment of this organism during the Deepwater Horizon spill may be explained by factors related to substrate availability and competition within the complex and dynamic microbial communities that were continuously evolving during that spill.

Active prophages in coral-associated Halomonas capable of lateral transduction.

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.

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.

Metabolic Engineering of Corynebacterium glutamicum for Highly Efficient Production of Ectoine.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

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.

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.