Ectoine Production Research Articles

Methylotuvimicrobium alcaliphilum 20Z based ectoine production from various industrial CH4 sources: Cultivation optimization and comprehensive analysis

Methanotroph-based CH4 conversion technology is being widely studied due to its advantages of mild operating conditions and environmental friendliness. In this study, operating strategies were investigated for industrial CH4 sources in order to widely disseminate methanotroph-based CH4 conversion technology using the promising strain, Methylotuvimicrobium alcaliphilum 20Z. The optimal O2/CH4 ratio for extremely different CH4 concentrations was explored based on kinetic-based simulation for biological reaction, and experimentally proven using fermenter-scale experiments. As a result, when high CH4 concentration is used, ectoine production rate can be maximized (33.8 mg/L·h) at an O2/CH4 ratio of 1 while favoring the “production of metabolites”. On the other hand, in the low CH4 concentration case, when changing from the general O2/CH4 ratio of 4 to the optimal O2/CH4 ratio (i.e., 1), although the improvement in ectoine concentration was not noticeable (3.9 to 7.5 mg/L·h), the CH4 conversion rate rapidly decreased (50 to 20 %), suggesting that the focus in the low concentration case should be on “removal of CH4”. In the case of biogas, the rapid pH drop caused by the large amount of CO2 in the feed is offset by a buffer solution, and the concentration of Na+ ions increases during the overall reaction time. Therefore, the production of metaboiltes can be maximized while simultaneously controlling pH and Na+ ion through continuous operation with a high dilution rate (19.8 mg/L·h). Furthermore, major performance indicators for each industrial gas were derived through comprehensive comparative analysis, through which the “dual-functions” of methanotrophs could be emphasized. This study provides a practical and strategic approach for methanotrophs that can flexibly respond to diverse industrial CH4 sources.

Methane capture, utilization, and sequestration technology based on biological ectoine production using Methylotuvimicrobium alcaliphilum 20Z

Methanotroph-based CH4 conversion technology is a promising alternative to conventional CH4 utilization methods that rely on energy-intensive CO2 capture processes. In this study, we introduce a novel CH4 capture, utilization, and sequestration (MCUS) technology as a proof-of-concept for eco-friendly CH4 conversion that is applicable to various methanotroph strains. We validated this hypothesis by evaluating ectoine production in Methylotuvimicrobium alcaliphilum 20Z. Cultivation experiments were conducted using pure O2 pulse injection to facilitate the separation of the CO2 generated during CH4 oxidation by concentrating it. To verify the MCUS concept systematically, we performed a model-based process analysis. The operational strategies for the reactors were developed using experiment-based kinetics and reactor models, followed by the design of a rigorous process flow diagram. Economic and environmental impact analyses were conducted to compare MCUS with air-based cultures and existing oxidative CH4 conversion technologies. The key advantage of MCUS is its significantly lower CO2 emissions (0.71 kg-CO2 eq/kg-CH4) than oxidative CH4 utilization (>2.75 kg-CO2 eq/kg-CH4). Moreover, the net present value per CH4 molecule was estimated at 0.7 $/kg-CH4, substantially higher than existing oxidative conversion methods (0.03 $/kg-CH4). Additionally, compared with air-based cultivation, the MCUS process exhibits superior economic and environmental performances. We used methanotrophs for CH4 “capture” and “utilization,” effectively leveraging “sequestration” to convert emissions into pure CO2. The proposed MCUS technology holds promise for practical applications of methanotrophs in producing various high-value-added products, emphasizing eco-friendly approaches.

Effect of operating condition on the properties of ectoine particles for improving the purification process

Ectoine, which is produced from methane by methane-oxidizing bacteria, has attracted attention because of its many useful functions in living organisms. It is known that the purification process using alcohol-based solvents is a limiting step in the ectoine production process because of the precipitation of fine particles. This study investigated quantitatively the effects of operating conditions (cooling rate and solvent) on the properties of ectoine particles for improving the efficiency of the ectoine purification process. The experimental results showed that the properties of ectoine particles were significantly influenced by operating condition such as the cooling rate and solvent of feed solution. Furthermore, it was revealed that the critical supersaturation ratio at the nucleation using methanol was approximately ten times higher than using water. Consequently, it was found that the high critical supersaturation ratio at the nucleation induces the precipitation the fine particles that have high filtration resistance. In conclusion, we succeeded in finding the relationship between the operating conditions and properties of ectoine particles.

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

BackgroundEctoine 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.ResultsIn 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.ConclusionsThis 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.

The tropical marine actinomycete Nocardiopsis dassonvillei NCIM 5124 as novel source of ectoine: Genomic and transcriptomic insights

Since ectoine is a high-value product, overviewing strategies for identifying novel microbial sources becomes relevant. In the current study, by following a genome mining approach, the ectoine biosynthetic cluster in a tropical marine strain of Nocardiopsis dassonvillei (NCIM 5124) was located and compared with related organisms. Transcriptome analysis of Control and Test samples (with 0 and 5% NaCl, respectively) was carried out to understand salt induced stress response at the molecular level. There were 4950 differentially expressed genes with 25 transcripts being significantly upregulated in Test samples. NaCl induced upregulation of the ectoine biosynthesis cluster and some other genes (stress response, chaperone/Clp protease, cytoplasm, ribonucleoprotein and protein biosynthesis). The production of ectoine as a stress response molecule was experimentally validated via LCMS analysis. The investigation sheds light on the responses exhibited by this actinomycete in coping up with salt stress and provides a foundation for understanding salt induced molecular interactions.

Sugar beet molasses: a sweet solution for ectoine production by Nesterenkonia sp.

Ectoine, a biologically significant compound, was successfully produced by a strain of bacteria capable of utilizing sucrose. In a ground-breaking approach, we harnessed the potential of sugar beet molasses, a by-product rich in sucrose, amino acid, and vitamins, as a growth medium for this purpose. Through meticulous investigation, we identified the ideal conditions for maximizing ectoine synthesis. This remarkable milestone was reached by introducing only 1g of (NH₄)₂SO₄ and 5mL of molasses per liter, maintaining a pH level of 8.0, upholding a 7.5% NaCl concentration, employing agitation at 120rpm, and sustaining a temperature of 30°C. This study marks a pioneering endeavour as it represents the first instance where molasses has been effectively employed to produce ectoine through the cultivation of Nesterenkonia sp. We showcased the production of 75.56g of the valuable compound ectoine utilizing 1 L of waste molasses with this specific bacterial strain. These findings hold tremendous promise, not only in terms of resource utilization but also for the potential applications of ectoine in various biological contexts.

Optimization of methane gas-liquid mass transfer during biogas-based ectoine production in bubble column bioreactors

Nowadays, the utilization of biogas for energy generation is hindered by the declining production costs of solar and wind power. A shift towards the valorization of biogas into ectoine, a highly valuable bioproduct priced at 1000 €⸱kg−1, offers a novel approach to fostering a more competitive biogas market while contributing to carbon neutrality. This study evaluated the optimization of CH4 gas-liquid mass transfer in 10 L bubble column bioreactors for CH4 conversion into ectoine and hydroxyectoine using a mixed methanotrophic culture. The influence of the empty bed residence time (EBRTs of 27, 54, and 104 min) at different membrane diffuser pore sizes (0.3 and 0.6 mm) was investigated. Despite achieving CH4 elimination capacities (CH4-ECs) of 10–12 g⸱m−3⸱h−1, an EBRT of 104 min mediated CH4 limitation within the cultivation broth, resulting in a negligible biomass growth. Reducing the EBRT to 54 min entailed CH4-ECs of 21–24 g⸱m−3⸱h−1, concomitant to a significant increase in biomass growth (up to 0.17 g⸱L⸱d−1) and reaching maximum ectoine and hydroxyectoine accumulation of 79 and 13 mg⸱gVSS−1, respectively. Conversely, process operation at an EBRT of 27 min lead to microbial inhibition, resulting in a reduced biomass growth of 0.09 g⸱L⸱d−1 and an ectoine content of 47 mg⸱gVSS−1. While the influence of diffuser pore size was less pronounced compared to EBRT, the optimal process performance was observed with a diffuser pore size of 0.6 mm.

Efficiently manufacturing ectoine via metabolic engineering and protein engineering of L-2,4-diaminobutyrate transaminase

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.

Advances in ectoine biosynthesis and biochemical characteristics of key enzymes

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.

Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation

BackgroundMethane 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.ResultsIn 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.ConclusionsThis 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.

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.

Ectoine production from a novel bacterial strain and high-purity purification with a cost-effective and single-step method

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.

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

BackgroundThe 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.ResultsThis 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.ConclusionsThis 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.

Biotechnological production of ectoine: current status and prospects.

Ectoine is an important natural secondary metabolite in halophilic microorganisms. It protects cells against environmental stressors, such as salinity, freezing, drying, and high temperatures. Ectoine is widely used in medical, cosmetic, and other industries. Due to the commercial market demand of ectoine, halophilic microorganisms are the primary method for producing ectoine, which is produced using the industrial fermentation process "bacterial milking." The method has some limitations, such as the high salt concentration fermentation, which is highly corrosive to the equipment, and this also increases the difficulty of downstream purification and causes high production costs. The ectoine synthesis gene cluster has been successfully heterologously expressed in industrial microorganisms, and the yield of ectoine was significantly increased and the cost was reduced. This review aims to summarize and update microbial production of ectoine using different microorganisms, environments, and metabolic engineering and fermentation strategies and provides important reference for the development and application of ectoine.

Metabolic engineering combined with enzyme engineering for overproduction of ectoine in Escherichia coli

Ectoine, a natural protective agent, is naturally synthesized at low titers by some extreme environment microorganisms that are usually difficult to culture. There is a need for an efficient and eco-friendly ectoine production process. In this study, Escherichia coli BL21(DE3) with the ectABC gene cluster from Halomonas venusta achieved 1.7 g/L ectoine. After optimizing the expression plasmid, 2.1 g/L ectoine was achieved. Besides, the aspartate kinase mutant LysCT311I from Corynebacterium glutamicum and aspartate semialdehyde dehydrogenase from Halomonas elongata were overexpressed to increase precursors supply. Furthermore, the rate-limiting enzyme EctB was semirationally engineered, and the E407D mutation enhanced ectoine production by 13.8 %. To improve acetyl-CoA supply, the non-oxidative glycolysis pathway was introduced. Overall, the optimized strain ECT9-5 produced 67.1 g/L ectoine by fed-batch fermentation with a 0.3 g/g of glucose and the kinetic model resulted in a good fit.

Highly efficient production of ectoine via an optimized combination of precursor metabolic modules in Escherichia coli BL21

Ectoine is an osmotic pressure protectant observed in various microorganisms and is widely used in cosmetics and pharmaceuticals. The market value of ectoine has increased considerably with social progress, resulting in high demand for ectoine production technology. Herein, a microbial cell factory in Escherichia coli that produces ectoine at high titers is described as developing efficient and environmentally friendly bio-based ectoine production technology. The ectoine biosynthetic pathway of Halomonas hydrothermalis was introduced into E. coli BL21 (DE3). Subsequent overexpression of precursor metabolic modules, including aspartate branching, pyruvate-oxoacetate, and glutamate biosynthesis pathways, resulted in the final strain, E. coli BCT08, which produced ectoine at a titer of 36.58 g/L during 30 h of fermentation. Sugar feeding speed optimization improved the ectoine titer to 131.8 g/L after 96 h of cultivation. This represents a remarkable achievement in ectoine production from glucose under low-salt conditions and has vast potential for industrial applications.

Ectoines production from biogas in pilot bubble column bioreactors and their subsequent extraction via bio-milking

Despite the potential of biogas from waste/wastewater treatment as a renewable energy source, the presence of pollutants and the rapid decrease in the levelized cost of solar and wind power constrain the use of biogas for energy generation. Biogas conversion into ectoine, one of the most valuable bioproducts (1000 €/kg), constitutes a new strategy to promote a competitive biogas market. The potential for a stand-alone 20 L bubble column bioreactor operating at 6% NaCl and two 10 L interconnected bioreactors (at 0 and 6% NaCl, respectively) for ectoine production from biogas was comparatively assessed. The stand-alone reactor supported the best process performance due to its highest robustness and efficiency for ectoine accumulation (20–52 mgectoine/gVSS) and CH4 degradation (up to 84%). The increase in N availability and internal gas recirculation did not enhance ectoine synthesis. However, a 2-fold increase in the internal gas recirculation resulted in an approximately 1.3-fold increase in CH4 removal efficiency. Finally, the recovery of ectoine through bacterial bio-milking resulted in efficiencies of >70% without any negative impact of methanotrophic cell recycling to the bioreactors on CH4 biodegradation or ectoine synthesis.

Complete genome sequence of Marinobacter shengliensis D49 harboring ectABC genes for ectoine synthesis.

A complete genome sequence of Marinobacter shengliensis D49 in the class Gamma-proteobacteria was isolated from activated sludge treating landfill leachate. The genome encodes the functional genes for the biosynthesis of ectoine (ectABC), a compatible solute for cosmetics. Deciphering the genome helps pave the way for ectoine production by the isolate.