Ectoine Synthesis Research Articles

Application of Biotechnology and Chiral Technology Methods in the Production of Ectoine Enantiomers

Natural ectoine, (+)-(4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, is an extremely important small biomolecule belonging to the class of osmolytic/osmoprotective compounds. It stabilizes biomacromolecules such as DNA and proteins and protects them from denaturation by heat, dehydration, and UV radiation. The rapidly growing interest in this compound resulted in currently exclusive biotechnological production, while a chemical process along with enantioseparation as an alternative has not yet been established. An improved chemical synthesis of racemic ectoine starting from γ-butyrolactone in very good yield is described. Regioselective monoacetylation is achieved by the complexation of a copper(II)-ion with two molecules of 2,4-diamonobutyric acid in the key synthetic step. The racemic ectoine was synthesized with the aim of being successfully enantioseparated for the first time by high-performance liquid chromatography (HPLC) using a teicoplanin-based Chiral-T column in different solvent systems. The presence of (+)-ectoine was determined and quantified using an HPLC protocol on the Synergy Polar-RP column in fermentation broths inoculated with different strains of Streptomyces sp. bacteria isolated from the Adriatic Sea and grown on different NaCl concentrations.

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.

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.

Isolation, Identification and Survival Strategy of <i>Dietzia maris</i> MX2 Halotolerant Strain from the Yakshinskoe Mineral Salts Deposit

Halophilic and halotolerant microorganisms have a high biotechnological potential. They are producers of biologically active substances, stress-protective agents, hydrolytic enzymes, and are used for environmental bioremediation. At the same time, the characterization of novel halotolerant bacteria and the disclosure of their salt tolerance strategy are topical fundamental problems. In the present work, a new strain MX2 was isolated from the salt well brine of the Yakshinskoe potassium-magnesium salt deposit. The isolate is represented by aerobic gram-positive non-motile bacteria that do not produce spores. The cell morphology varies from cocci to short rods that are capable of producing V-shaped forms. Colonies on the surface of agar nutrient medium were circular with an entire edge and raised center, glistening and orange. Bacteria of strain MX2 are halotolerant microorganisms capable of growing at NaCl concentrations up to 9%. Strain MX2 was sequenced. Its size was estimated at 3747717 b. p., the number of protein-coding genes — 3562. Strain MX2 was identified as belonging to the species Dietzia maris based on analysis of 16S rRNA, gyrB, rpoB, recA, ppk gene sequences and using time-of-flight mass spectrometry (MALDI-TOF-MS). D. maris MX2 has complete metabolic pathways for the synthesis of ectoine, hydroxyectoine, and trehalose, as well as transport systems for ectoine, hydroxyectoine, trehalose, glycerol, glycerol-3-phosphate, L-proline, and glycine-betaine. Thus, to ensure the osmotic balance, D. maris MX2 uses the strategy of accumulating compatible organic solutes.

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.

Denitrificimonas halotolerans sp. nov., a novel species isolated from UASB sludge treating landfill leachate.

A Gram-stain-negative, facultative anaerobe, rod-shaped strain JX-1T was isolated from UASB sludge treating landfill leachate in Wuhan, China. The isolate is capable of growing under conditions of pH 6.0-11.0 (optimum, pH 7.0-8.0), temperature 4-42℃ (optimum, 20-30℃), 0-8.0% (w/v) NaCl (optimum, 5.0%), and ammonia nitrogen concentration of 200-5000mg/L (optimum, 500mg/L) on LB plates. The microorganism can utilize malic acid, D-galactose, L-rhamnose, inosine, and L-glutamic acid as carbon sources, but does not reduce nitrates and nitrites. The major fatty acids are C18:1ω7c/C18:1ω6c, iso-C15:0, and anteiso-C15:0. The respiratory quinones are Q9 (91.92%) and Q8 (8.08%). Polar lipids include aminolipid, aminophospholipid, diphosphatidylglycerol, glycolipid, phosphatidylethanolamine, phosphatidylglycerol, and phospholipid. Compared with other strains, strain JX-1T and Denitrificimonas caeni HY-14T have the highest values in terms of 16S rRNA gene sequence similarity (96.79%), average nucleotide identity (ANI; 76.06%), and average amino acid identity (AAI; 78.89%). Its digital DNA-DNA hybridization (dDDH) result is 20.3%. The genome of strain JX-1T, with a size of 2.78Mb and 46.12mol% G + C content, lacks genes for denitrification and dissimilatory nitrate reduction to ammonium (DNRA), but contains genes for ectoine synthesis as a secondary metabolite. The results of this polyphasic study allow genotypic and phenotypic differentiation of the analysed strain from the closest related species and confirm that the strain represents a novel species within the genus Denitrificimonas, for which the name Denitrificimonas halotolerans sp. nov. is proposed with JX-1T (= MCCC 1K08958T = KCTC 8395T) as the type strain.

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.

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.

The synthesis of ectoine enhance the assimilation of ammonia nitrogen in hypersaline wastewater by the salt-tolerant assimilation bacteria sludge

In contrast to nitrification-denitrification microorganisms that convert ammonia nitrogen in hypersaline wastewater into nitrogen for discharge, this research utilizes sludge enriched with salt-tolerant assimilation bacteria (STAB) to assimilate organic matter and ammonia nitrogen in hypersaline wastewater into ectoine - a biomass with high economic value and resistance to external osmotic pressure. The study investigates the relationship between the synthesis of ectoine and nitrogen removal efficiency of STAB sludge in three sequencing batch reactors (SBR) operated at different salinities (50, 75, and 100 g/L) and organic matter concentrations. The research reveals that, under low concentration carbon sources (TOC/N = 4, NH4+-N = 60 mg/L), the ammonia nitrogen removal efficiency of SBR reactors increased by 14.51 % and 17.25 % within 5 d and 2 d, respectively, when salinity increased from 50 g/L to 75 g/L and 100 g/L. Under high concentration carbon sources (TOC/N = 8, NH4+-N = 60 mg/L), the ammonia nitrogen removal efficiency of STAB sludge in the three reactors stabilized at 80.20 %, 76.71 %, and 72.87 %, and the total nitrogen removal efficiency was finally stabilized at 80.47 %, 73.15 %, and 65.53 %, respectively. The nitrogen removal performance by ammonium-assimilating of STAB sludge is more sustainable under low salinity, while it is more short-term explosive under high salinity. Moreover, the intracellular ectoine concentration of STAB sludge was found to be related to this behavior. Empirical formulas confirm that STAB sludge synthesizes ectoine from nutrients in wastewater through assimilation, and intracellular ectoine has a threshold defect (150 mg/gVss). The ectoine metabolism pathways of STAB sludge was constructed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The ammonia nitrogen in sewage is converted into glutamic acid under the action of assimilation genes. It then undergoes a tricarboxylic acid cycle to synthesize the crucial precursor of ectoine - aspartic acid. Subsequently, ectoine is produced through ectoine synthase. The findings suggest that when the synthesis of intracellular ectoine reaches saturation, it inhibits the continuous nitrogen removal performance of STAB sludge under high salinity. STAB sludge does not actively release ectoine through channels under stable external osmotic pressure.

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.

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.

Metabolic engineering of Halomonas campaniensis strain XH26 to remove competing pathways to enhance ectoine production

Ectoine has gained considerable attention as a high-value chemical with significant application potential and market demand. This study aimed to increase ectoine yields by blocking the metabolic shunt pathway of l-aspartate-4-semialdehyde, the precursor substrate in ectoine synthesis. The homoserine dehydrogenase encoded by hom in H. campaniensis strain XH26 is responsible for the metabolic shunt of l-aspartate-4-semialdehyde to glycine. CRISPR/Cas9 technology was used to seamlessly knockout hom, blocking the metabolic shunt pathway to increase ectoine yields. The ectoine yield of XH26/Δhom was 351.13 mg (g CDW)−1 after 48 h of incubation in 500 mL shake flasks using optimal medium with 1.5 mol L−1 NaCl, which was significantly higher than the 239.18 mg (g CDW)−1 of the wild-type strain. Additionally, the absence of the ectoine metabolic shunt pathway affects betaine synthesis, and thus the betaine yields of XH26/Δhom was 19.98 mg (g CDW)−1, considerably lower than the 69.58 mg (g CDW)−1 of the wild-type strain. Batch fermentation parameters were optimized, and the wild-type strain and XH26/Δhom were fermented in 3 L fermenters, resulting in a high ectoine yield of 587.09 mg (g CDW)−1 for the defective strain, which was significantly greater than the ectoine yield of 385.03 mg (g CDW)−1 of the wild-type strain. This study showed that blocking the metabolic shunt of synthetic substrates effectively increases ectoine production, and a reduction in the competitively compatible solute betaine appears to promote increased ectoine synthesis.

Tandem mass tag-based quantitative proteomics reveals osmotic adaptation mechanisms in Alkalicoccus halolimnae BZ-SZ-XJ29T , a halophilic bacterium with a broad salinity range for optimal growth.

The moderate halophilic bacterium Alkalicoccus halolimnae BZ-SZ-XJ29T exhibits optimum growth over a wide range of NaCl concentrations (8.3-12.3%, w/v; 1.42-2.1 mol L-1 ). However, its adaptive mechanisms to cope with high salt-induced osmotic stress remain unclear. Using TMT-based quantitative proteomics, the cellular proteome was assessed under low (4% NaCl, 0.68 mol L-1 NaCl, control (CK) group), moderate (8% NaCl, 1.37 mol L-1 NaCl), high (12% NaCl, 2.05 mol L-1 NaCl), and extremely high (16% NaCl, 2.74 mol L-1 NaCl) salinity conditions. Digital droplet PCR confirmed the transcription of candidate genes related to salinity. A. halolimnae utilized distinct adaptation strategies to cope with different salinity conditions. Mechanisms such as accumulating different amounts and types of compatible solutes (i.e., ectoine, glycine betaine, glutamate, and glutamine) and the uptake of glycine betaine and glutamate were employed to cope with osmotic stress. Ectoine synthesis and accumulation were critical to the salt adaptation of A. halolimnae. The expression of EctA, EctB, and EctC, as well as the intracellular accumulation of ectoine, significantly and consistently increased with increasing salinity. Glycine betaine and glutamate concentrations remained constant under the four NaCl concentrations. The total content of glutamine and glutamate maintained a dynamic balance and, when exposed to different salinities, may play a role in low salinity-induced osmoadaptation. Moreover, cellular metabolism was severely affected at high salt concentrations, but the synthesis of amino acids, carbohydrate metabolism, and membrane transport related to haloadptation was preserved to maintain cytoplasmic concentration at high salinity. These findings provide insights into the osmoadaptation mechanisms of moderate halophiles and can serve as a theoretical underpinning for industrial production and application of compatible solutes.

Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches.

The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

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

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

Metabolic Engineering of <i>Escherichia coli</i> for Production of Ectoine

Ectoine, a cyclic amino acid derivative is produced by some moderately halophilic microorganisms. It is widely used in the field of medicine and cosmetics; however its high cost, low yield and complicated production process restrict its use in an industry. The present study was carried out to construct an engineered Escherichia coli for the heterologous ectoine production. Firstly, the ectoine synthesis cluster (ectABC genes) from Halomonas elongata were introduced into E. coli for the ectoine production in heterologous system. Secondly, the effect of deletion for lysA and thrA on producing ectoine was investigated. Thirdly, the bioconversion conditions for synthesizing ectoine were optimized. In this study, nine strains of E.coli BW25113 were constructed, and an engineering strain ABC (∆lysA)/BW25113 with ectoine synthesis ability was screened out. An improved yield of ectoine was obtained with optimized conditions like 100 mmol/L sodium aspartate, 100 mmol/L glycerol, 80 mmol/L glucose and 120 mmol/L KCl at temperature 30°C, and the pH 7.0. A yield 2.08 g/L of ectoine was obtained. After preliminary optimization, the ABC (∆lysA)/BW25113 catalytic synthesis of ectoine increased by 2 times, indicating that optimized reaction conditions can increase the yield of ectoine. Here, we successfully constructed an engineered Escherichia coli for the heterologous production of ectoine. Our study herein provides a feasible and valuable biosynthesis pathway of ectoine with a potential for large-scale industrial production using simple and cheap feedstocks.

Identification and characterization of an ectoine biosynthesis gene cluster from Aestuariispira ectoiniformans sp. nov., isolated from seawater

An ectoine-producing bacterium, designated SWCN16T, was isolated from seawater and could be grown in a medium containing up to 12 % NaCl. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SWCN16T belonged to the genus Aestuariispira, class Alphaproteobacteria, and shared the highest 16S rRNA gene sequence similarity of 96.8% with Aestuariispira insulae CECT 8488T. The phenotypic, chemotaxonomic, and genotypic characteristics findings of this study suggested that strain SWCN16T represented a novel species of the genus Aestuariispira. We propose the name Aestuariispira ectoiniformans sp. nov. for this species. Whole-genome sequencing analysis of the isolate revealed a putative ectABC gene cluster for ectoine biosynthesis. These genes were found to be functional using ectoine synthesis testing and S16-ectBAC cells, which were pET21a-ectBAC-transformed E. coli BL21 cells. We found that S16-ectBAC synthesized about 1.67 g/L extracellular ectoine and about 0.59 g/L intracellular ectoine via bioconversion at optimum conditions.

Metabolic engineering of Escherichia coli for efficient osmotic stress‐free production of compatible solute hydroxyectoine

Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/yield, the presence of the byproduct ectoine, and the requirement of high salinity. Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based auto-regulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.

Ectoine Production Using Novel Heterologous EctABCS. salarius from Marine Bacterium Salinicola salarius

Ectoine, a heterocyclic amino acid produced by various bacteria, was widely used in the fields of cosmetics and medicine. In this study, a novel ectoine synthesis cluster from marine bacterium Salinicola salarius 1A01339 was firstly introduced into Escherichia coli BL21(DE3) for heterologous production of ectoine. The bioinformatic analysis proved the function of these ectoine synthesis enzymes, and showed the highest identities of 83.3–87.7% with enzymes from other microorganisms. Using the whole-cell biocatalytic method, 3.28 g/L ectoine was synthesized and excreted into the medium with the substrate of 200 mM sodium aspartate at 25 °C, pH 6.5 in flask-level. Further bioconversion was performed in the fermentor system at the high cell density of 20 OD/mL, and the concentration of extracellular ectoine was increased to 22.5 g/L in 24 h (equivalent to the specific productivity of 0.94 g/L·h), achieving over 6 times of production compared with that in flasks. Significantly, the recombinant strain demonstrated a lower catalytic temperature with the optimum of 25 °C, and a stronger tolerance to the substrate aspartate of 300 mM. These results might provide a compelling case for ectoine synthesis as well as potential applications in large-scale industrial production.

Revealing the salinity adaptation mechanism in halotolerant bacterium Egicoccus halophilus EGI 80432T by physiological analysis and comparative transcriptomics.

Egicoccus halophilus EGI 80432T, a halotolerant bacterium isolated from a saline-alkaline soil, belongs to a member of the class Nitriliruptoria, which exhibits high adaptability to salt environments. At present, the detailed knowledge of the salinity adaptation strategies of Nitriliruptoria was limited except for one research by using comparative genomics analysis. Here, we investigated the salinity adaptation mechanism of E. halophilus EGI 80432T by comparative physiological and transcriptomic analyses. The results of physiological analyses showed that trehalose and glutamate were accumulated by salt stress and showed the maximum at moderate salinity condition. Furthermore, the contents of histidine, threonine, proline, and ectoine were increased with increasing salt concentration. We found that both 0% and 9% NaCl conditions resulted in increased expressions of genes involved in carbohydrate and energy metabolisms, but negatively affected the Na+ efflux, iron, and molybdate transport. Moreover, the high salt condition led to enhancement of transcription of genes required for the synthesis of compatible solutes, e.g., glutamate, histidine, threonine, proline, and ectoine, which agree with the results of physiological analyses. The above results revealed that E. halophilus EGI 80432T increased inorganic ions uptake and accumulated trehalose and glutamate in response to moderate salinity condition, while the salinity adaptation strategy was changed from a "salt-in-cytoplasm" strategy to a "compatible solute" strategy under high salinity condition. The findings in this study would promote further studies in salt tolerance molecular mechanism of Nitriliruptoria and provide a theoretical support for E. halophilus EGI 80432T's application in ecological restoration.Key Points• Salt stress affected gene expressions responsible for carbohydrate and energy metabolisms of E. halophilus EGI 8042T.• E. halophilus EGI 80432T significantly accumulated compatible solutes under salt stress.• E. halophilus EGI 80432T adopted a "compatible solute" strategy to withstand high salt stress.