Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.

Genomes of Halomonas species have been studied using the BV-BRC Bioinformatics tool for the presence of CDS, non-CDS, AMR genes, VF genes, transporters, drug targets, GC content, and GC skew from outside to the center of the circular view, followed by phylogenetic analysis of unique 1, 4, 5, 6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (THMP) gene clusters for relatedness within the genus Halomonas. Protein structure and chemical structure of 1, 4, 5, 6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (THMP) encoded by the UspA gene in Halomonas strains and amino acid sequence of the novel UspA gene have been predicted by computational method.

Ectoine, so-called tetrahydropyrimidine, is an important osmotic adjustment solute and widely applied in cosmetics and protein protectant. Some attempts have been made to improve the ectoine productivity. However, the strains with both high ectoine production capacity and high glucose conversion were still absent so far. Aim to construct a strain for efficiently producing ectoine, ectoine synthetic gene cluster ectABC from Pseudomonas stutzeri was overexpressed in E. coli BL21 (DE3). The ection production was improved by 382 % (ectoine titer increased from 1.73 g/L to 8.33 g/L) after the rational design of rate-limiting enzyme L-2,4-diaminobutyrate transaminase EctBps (protein engineering) combined with the metabolic engineering that focused on the enrichment and conversion of precursors. The final strain YW20 was applied to overproduce ectoine in fed-batch fermentation and yield 68.9 g/L of ectoine with 0.88 g/L/h of space-time yield and the highest glucose conversion reported [34 % (g/g)]. From the fermentation broth, ectoine was purified with 99.7 % purity and 79.8 % yield. This study successfully provided an engineered strain as well as an efficient method for the industrial bio-synthesis and preparation of ectoine.

Chinese hamster ovary (CHO) cells represent the most preferential host cell system for therapeutic monoclonal antibody (mAb) production. Enhancing mAb production in CHO cells can be achieved by adding chemical compounds that regulate the cell cycle and cell survival pathways. This study investigated the impact of ectoine supplementation on mAb production in CHO cells. The results showed that adding ectoine at a concentration of 100 mM on the 3rd day of cultivation improved mAb production by improving cell viability and extending the culture duration. RNA sequencing analysis revealed differentially expressed genes associated with cell cycle regulation, cell proliferation, and cellular homeostasis, in particular promotion of cell cycle arrest, which was then confirmed by flow cytometry analysis. Ectoine-treated CHO cells exhibited an increase in the number of cells in the G0/G1 phase. In addition, the cell diameter was also increased. These findings support the hypothesis that ectoine enhances mAb production in CHO cells through mechanisms involving cell cycle arrest and cellular homeostasis. Overall, this study highlights the potential of ectoine as a promising supplementation strategy to enhance mAb production not only in CHO cells but also in other cell lines.

Compatible solutes are highly water-soluble organic osmolytes produced by microorganisms to adapt to extreme environments, such as high salinity and osmotic pressure. Among these, ectoine plays a crucial role in repairing and protecting nucleic acids, protein, biofilms, and cells. As a result, it has found widespread applications in cosmetics, biological agents, the enzyme industry, medicine, and other fields. Currently, the market value of ectoine is around US$ 1 000/kg, with a global demand reaching 15 000 tons per year. Although halophilic bacteria serve as the natural source of ectoine synthesis, its production in high-salinity media presents challenges such as equipment corrosion and high cost for industrial production. Advancements in functional genomics, systems biology, and synthetic biology have paved the way for the development of high-yielding cell factories through metabolic engineering, leading to significant progress. For example, engineered Escherichia coli achieved a maximum ectoine titer of 131.8 g/L, with a productivity of 1.37 g/(L·h). This review aims to explore the biosynthetic pathway, biochemical characteristics of key enzymes, and the biosynthesis of ectoine, shedding light on current research status and offering insights for industrial-scale ectoine production.

BACKGROUND: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products.
RESULTS: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane.
CONCLUSIONS: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

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.

This study marks the exploration into the production of ectoine, a valuable compound with significant potential as an antioxidant, osmoprotectant, anti-inflammatory agent, and stabilizer of cell membranes, proteins, and DNA integrity. Our focus centred on investigating the presence of ectoine and optimizing its production by the novel ectoine producer bacterial strain, Piscibacillus halophilus. For the optimization of ectoine production the effects of carbon and nitrogen sources, salt, pH, agitation and incubation period were optimized by one-factor-at-a-time. We started with an initial ectoine content of 46.92 mg/L, and through a series of optimization processes, we achieved a remarkable increase, resulting in an ectoine content of 1498.2 mg/L. The bacterial species P. halophilus achieved its highest ectoine production after 48 h of incubation, with conditions set at 10 % (w/v) salinity, pH of 7.50, and an agitation speed of 160 rpm. These precise conditions were found to be the most favourable for maximizing ectoine production by this strain. Besides, we have achieved successful purification of ectoine from the crude extract through a streamlined single-step process. This purification method has delivered an exceptional level of purity, surpassing 99.15 %, and an impressive yield of over 99 %. Importantly, we accomplished this using readily available and cost-effective strong acids (HCl) and strong bases (NaOH) to arrange pH gradients. The use of acid and base in the purification process of ectoine reflects an innovative and sustainable methodology.

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.

OBJECTIVE: To explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal epithelial cell survival and barrier from hyperosmotic stress.
METHODS: Primary human corneal epithelial cells (HCECs) were established from donor limbus. The confluent cultures in isosmolar medium were switched to hyperosmotic media (400-500 mOsM), with or without ectoine or rhIL-37 for different time periods. Cell viability and proliferation were evaluated by MTT or WST assay. The integrity of barrier proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, and immunostaining with confocal microscopy.
RESULTS: HCECs survived well in 450mOsM but partially damaged in 500mOsM medium. Ectoine well protected HCEC survival and proliferation at 500mOsM. The integrity of epithelial barrier was significantly disrupted in HCECs exposed to 450mOsM, as shown by 2D and 3D confocal immunofluorescent images of tight junction proteins ZO-1 and occludin. Ectoine at 5-20 mM well protected these barrier proteins under hyperosmotic stress. The expression of TNF-α, IL-1β, IL-6 and IL-8 were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5-40 mM. Cathepsin S, which was stimulated by hyperosmolarity, directly disrupted epithelial barrier. Interestingly, anti-inflammatory cytokine IL-37 was suppressed by hyperosmolarity, but restored by ectoine at mRNA and protein levels. Furthermore, rhIL-37 suppressed cathepsin S and rescued cell survival and barrier in HCECs exposed to hyperosmolarity.
CONCLUSIONS: Our findings demonstrate that ectoine protects HCEC survival and barrier from hyperosmotic stress by promoting IL-37. This provides new insight into pathogenesis and therapeutic potential for dry eye disease.