Ectoine Production Research Articles

Enhanced ectoines production by carbon dioxide capture: A step further towards circular economy

Recycling of greenhouse gases to produce industrially valuable products has become one of the big pillars to achieve circularity. This study demonstrates for the first time the feasibility of producing ectoine with Halomonas stevensii with CO2 as the added carbon source, and CO2 and glucose. Initially, CO2 alone was fed to continuous reactors, adding thiosulphate as the energy source. Maximum CO2 elimination capacities of 24.2 mg CO2 L−1 h−1 were obtained, and ectoine contents up to 7.3% (g ectoine⸱g biomass−1). To enhance ectoine production, CO2 conversion coupled with discontinuous glucose addition was implemented. The amendment of 0.5 g L−1 of glucose at the beginning of reactor operation enhanced CO2 removal to 37.1 mg CO2 L−1 h−1 and increased ectoine contents up to 22%. Our results represent the proof of concept for a CO2 biotransformation platform to produce ectoines, so far unexplored. This can foster the development of more sustainable microbial processes for the production of ectoines helping in the abatement of CO2.

Advances in the microbial production of the compatible solute ectoine: a review

Ectoine is an amino acid derivative and an important natural product in halophilic microorganisms. It plays an important role in protecting cells and stabilizing biological macromolecules, and can be widely used in biomedical fields such as drug preparation adjuvants, organ transplantation and preservation, skin wound repair and cosmetics. Due to the medical value and commercial market demand of ectoine, this article summarized the recent advances in the microbial production of ectoine, including the mutation and breeding of hyper-producing strains, construction of genetically and metabolically engineered strains, optimization of fermentation processes, and extraction and purification processes. The application of multi-omics technologies and computational biology to develop an ectoine producing cell factory was prospected, with the aim to provide a reference for ectoine overproduction.

Whole genome sequencing of the halophilic Halomonas qaidamensis XH36, a novel species strain with high ectoine production.

Here, we report the whole genome of a novel halophilic Halomonas species strain XH36 with high ectoine production potential. The genome was 3,818,310bp in size with a GC content of 51.97%, and contained 3533 genes, 61 tRNAs and 18 rRNAs. The phylogenetic analysis using the 16s rRNA genes, the UBCGs and the TYGS database indicated that XH36 belongs to a novel Halomonas species, which we named as Halomonas qaidamensis. Osmoadaptation related genes including Na(+) and K(+) transport and compatible solute accumulation were both present in the XH36 genome, the latter of which mainly contained ectoine, 5-hydroxyectoine and betaine. HPLC validation studies showed that H. qaidamensis XH36 accumulated ectoine to cope with salt stress, and the content of ectoine could be as high as 315mg/g CDW under 3mol/l NaCl. Our results show that XH36 is a new promising industrial strain for ectoine production, and the genomic analysis will guide us to better understand its salt-induced osmoadaptation mechanisms, and provide theoretical references for future application research of ectoine.

Metabolic Engineering of Escherichia coli for Ectoine Production With a Fermentation Strategy of Supplementing the Amino Donor

Ectoine, an osmotic pressure-compensated solute, is used in the food, agriculture, medicine, and cosmetics industries due to its ability to protect macromolecules. In this study, an ectoine-producing variant of Escherichia coli, ET08, was genetically constructed by introducing the ectABC gene cluster and eliminating metabolic pathways involving lysine and pyruvate. Medium optimization enhanced ectoine production from 1.87 to 10.2 g/L. Analysis of the transcriptional levels revealed that supplementation with ammonium sulfate enhanced the metabolic flux towards the biosynthesis of ectoine. Furthermore, by optimizing the copy number of ectA, ectB, and ectC, the recombinant E. coli ET11 (ectA:ectB:ectC = 1:2:1) produced 12.9 g/L ectoine in the shake flask and 53.2 g/L ectoine in a fed-batch fermenter, representing the highest ectoine titer produced by E. coli, which has great industrial prospects.

Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

BackgroundEctoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains.ResultsWe constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date.ConclusionsEctoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production.Graphical

High-Yield Ectoine Production in Engineered Corynebacterium Glutamicum by Fine Metabolic Regulation Via Plug-In Repressor Library

High-Yield Ectoine Production in Engineered Corynebacterium Glutamicum by Fine Metabolic Regulation Via Plug-In Repressor Library

Ectoine Production from Biogas in Waste Treatment Facilities: A Techno-Economic and Sensitivity Analysis.

The capacity of haloalkaliphilic methanotrophic bacteria to synthesize ectoine from CH4-biogas represents an opportunity for waste treatment plants to improve their economic revenues and align their processes to the incoming circular economy directives. A techno-economic and sensitivity analysis for the bioconversion of biogas into 10 t ectoine·y–1 was conducted in two stages: (I) bioconversion of CH4 into ectoine in a bubble column bioreactor and (II) ectoine purification via ion exchange chromatography. The techno-economic analysis showed high investment (4.2 M€) and operational costs (1.4 M€·y–1). However, the high margin between the ectoine market value (600–1000 €·kg–1) and the estimated ectoine production costs (214 €·kg–1) resulted in a high profitability for the process, with a net present value evaluated at 20 years (NPV20) of 33.6 M€. The cost sensitivity analysis conducted revealed a great influence of equipment and consumable costs on the ectoine production costs. In contrast to alternative biogas valorization into heat and electricity or into low added-value bioproducts, biogas bioconversion into ectoine exhibited high robustness toward changes in energy, water, transportation, and labor costs. The worst- and best-case scenarios evaluated showed ectoine break-even prices ranging from 158 to 275 €·kg–1, ∼3–6 times lower than the current industrial ectoine market value.

Three Novel Bacteria Associated with Two Centric Diatom Species from the Mediterranean Sea, Thalassiosira rotula and Skeletonema marinoi.

Diatoms are a successful group of microalgae at the base of the marine food web. For hundreds of millions of years, they have shared common habitats with bacteria, which favored the onset of interactions at different levels, potentially driving the synthesis of biologically active molecules. To unveil their presence, we sequenced the genomes of bacteria associated with the centric diatom Thalassiosira rotula from the Gulf of Naples. Annotation of the metagenome and its analysis allowed the reconstruction of three bacterial genomes that belong to currently undescribed species. Their investigation showed the existence of novel gene clusters coding for new polyketide molecules, antibiotics, antibiotic-resistance genes and an ectoine production pathway. Real-time PCR was used to investigate the association of these bacteria with three different diatom clones and revealed their preference for T. rotula FE80 and Skeletonema marinoi FE7, but not S. marinoi FE60 from the North Adriatic Sea. Additionally, we demonstrate that although all three bacteria could be detected in the culture supernatant (free-living), their number is up to 45 times higher in the cell associated fraction, suggesting a close association between these bacteria and their host. We demonstrate that axenic cultures of T. rotula are unable to grow in medium with low salinity (<28 ppt NaCl) whereas xenic cultures can tolerate up to 40 ppt NaCl with concomitant ectoine production, likely by the associated bacteria.

Metabolic Engineering of &lt;i&gt;Escherichia coli&lt;/i&gt; 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.

Engineering Escherichia coli for high-yield production of ectoine

Ectoine is a natural macromolecule protector and synthesized by some extremophiles. It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics. To meet its growing commercial demands, we engineered Escherichia coli strains for the high-yield production of ectoine. The ectABC gene cluster from the native ectoine producer Halomonas elongata was introduced into different Escherichia coli (E. Coil) strains via plasmids and 0.8 g L-1 of ectoine was produced in flask cultures by engineered E. coli BL21 (DE3). Subsequently, we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA, ectB, and ectC, which increased the ectoine yield to 1.6 g L-1. After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant (G1A, C932T) and the H. elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor, the titer of ectoine reached to 5.5 g L-1 in flask cultures. Finally, the engineered strain produced 60.7 g L-1 ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.

Elucidating the key environmental parameters during the production of ectoines from biogas by mixed methanotrophic consortia

Anaerobic digestion (AD) is a robust biotechnology for the valorisation of organic waste into biogas. However, the rapid decrease in renewable electricity prices requires alternative uses of biogas. In this context, the engineering of innovative platforms for the bio-production of chemicals from CH4 has recently emerged. The extremolyte and osmoprotectant ectoine, with a market price of ~1000€/Kg, is the industrial flagship of CH4-based bio-chemicals. This work aimed at optimizing the accumulation of ectoines using mixed microbial consortia enriched from saline environments (a salt lagoon and a salt river) and activated sludge, and biogas as feedstock. The influence of NaCl (0, 3, 6, 9 and 12 %) and Na2WO4 (0, 35 and 70 μg L−1) concentrations and incubation temperature (15, 25 and 35 °C) on the stoichiometry and kinetics of the methanotrophic consortia was investigated. Consortia enriched from activated sludge at 15 °C accumulated the highest yields of ectoine and hydroxyectoine at 6 % NaCl (105.0 ± 27.2 and 24.2 ± 5.4 mgextremolyte gbiomass−1, respectively). The consortia enriched from the salt lagoon accumulated the highest yield of ectoine and hydroxyectoine at 9 % NaCl (56.6 ± 2.5 and 51.0 ± 2.0 mgextremolyte gbiomass−1, respectively) at 25 °C. The supplementation of tungsten to the cultivation medium did not impact on the accumulation of ectoines in any of the consortia. A molecular characterization of the enrichments revealed a relative abundance of ectoine-accumulating methanotrophs of 7–16 %, with Methylomicrobium buryatense and Methylomicrobium japanense as the main players in the bioconversion of methane into ectoine.

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.

Beyond the farm: Making edible protein from CO2 via hybrid bioinorganic electrosynthesis

Beyond the farm: Making edible protein from CO2 via hybrid bioinorganic electrosynthesis

Metabolic engineering of Escherichia coli for efficient ectoine production

Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry, cosmetics, and biomedicine. In this study, rational engineering strategies have been implemented in Escherichia coli to efficiently produce ectoine. First, the synthetic pathway of ectoine was constructed in E. coli MG1655 by introducing an artificial thermal switch system harboring the ectABC cluster from Halomonas elongate, and the resulting strain produced 1.95 g/L ectoine. Second, crr encoding the glucose-specific enzyme II domain A of phosphotransferase system and iclR encoding the glyoxylate shunt transcriptional repressor were deleted in E. coli for enhancing the oxaloacetate supply, leading to the increasement of the ectoine titer to 9.09 g/L. Third, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was removed from the genome to weaken the competitive pathway; simultaneously, an endogenous feedback-resistant lysC was overexpressed to complement the enzymatic activity deficiency of the aspartate kinase, leading to 30.36% increase of ectoine titer. Next, the expression of phosphoenolpyruvate carboxylase was modulated with varying gradient strength promoters to accelerate the biosynthesis efficiency of ectoine. Finally, aspDH encoding aspartate dehydrogenase from Pseudomonas aeruginosa PAO1 was overexpressed to further improve the biosynthesis of ectoine. The final strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 produced 30.37 g/L ectoine after 36-h fed-batch fermentation with a yield of 0.132 g/g glucose and a productivity of 0.844 g/(L h).

Accumulation of Ectoines By Halophilic Bacteria Isolated from Fermented Shrimp Paste: An Adaptation Mechanism to Salinity, Temperature, and pH Stress.

Shrimp paste is a traditional fermented food produced by many Asian countries. Bacteria play important roles in the shrimp paste fermentation process. In order to survive under the low water activity (Aw) conditions caused by the high salt concentration, the bacteria need to employ a special adaptation strategy. This study found that most halophilic bacteria isolated from shrimp paste accumulated ectoines (ectoine and hydroxyectoine) as protective osmotic agents. Five isolated bacteria, including three high ectoine producers and two high hydroxyectoine producers, were selected for further study. Based on their morphological and biochemical characteristics and 16S rRNA gene sequences, the five strains were classified into three genera: Salinivibrio (strains M7 and M316), Salimicrobium (strains M31 and M69), and Vibrio (strain M92). The accumulation of ectoines by Salimicrobium species is reported here for the first time. The effects of salinity, incubation temperature, and initial pH on the growth rate and accumulation of ectoines by the five strains were investigated. The results revealed that the bacterial growth rate was inhibited while the accumulation of ectoines by the five selected strains was triggered by an increase in the external salinity, incubation temperature, or initial pH. In addition, a high concentration of ectoine only (21.2 wt%) was produced by strain M316 at the optimum salinity and temperature, and under pressure of a high initial pH value. To the best of our knowledge, this is the first report demonstrating that the production of ectoines by bacterial strains can be enhanced by increasing the pH of the culture medium to induce pH stress. This finding suggests a new ectoine producer and fermentation strategy that may help to improve the production of ectoines in the future.

Methane in wastewater treatment plants: status, characteristics, and bioconversion feasibility by methane oxidizing bacteria for high value-added chemicals production and wastewater treatment

Methane is a type of renewable fuel that can generate many types of high value-added chemicals, however, besides heat and power production, there is little methane utilization in most of the wastewater treatment plants (WWTPs) all round the world currently. In this review, the status of methane production performance from WWTPs was firstly investigated. Subsequently, based on the identification and classification of methane oxidizing bacteria (MOB), the key enzymes and metabolic pathway of MOB were presented in depth. Then the production, extraction and purification process of high value-added chemicals, including methanol, ectoine, biofuel, bioplastic, methane protein and extracellular polysaccharides, were introduced in detail, which was conducive to understand the bioconversion process of methane. Finally, the use of methane in wastewater treatment process, including nitrogen removal, emerging contaminants removal as well as resource recovery was extensively explored. These findings could provide guidance in the development of sustainable economy and environment, and facilitate biological methane conversion by using MOB in further attempts.

Enhancing ectoine production by recombinant Escherichia coli through step-wise fermentation optimization strategy based on kinetic analysis.

In this study, the recombinant ectoine-producing Escherichia coli ET01 was constructed by introducing the ectABC operon from Halomonas venusta ZH. To further improve ectoine production, the regulation of the fermentation process was systematically investigated. First, the effects of the initial glucose concentrations and glucose feeding mode on ectoine production were analyzed. Using a combination of pH-feedback feeding and glucose-controlled feeding, the ectoine titer reached 25.5g/L, representing an 8.8-fold increase over standard batch culture. Then, the effects of dissolved oxygen (DO) levels (50, 40, 30, or 20%) on ectoine production were studied, and a DO control strategy was developed based on the fermentation kinetics. When the final optimized two-stage fermentation strategy was used, the ectoine titer reached 47.8g/L, which was the highest level of ectoine produced by E. coli fermentation. The fermentation regulation strategy developed in this study might be useful for scaling up the commercial production of ectoine.

Chelativorans alearense sp. nov., A Novel Bacterial Species Isolated From Soil in Alear, China.

A novel Gram-strain-negative, rod-shaped, non-flagellated, non-gliding, beige-pigmented and aerobic bacterium, designated strain UJN715T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alear city, located in Xinjiang province, PR China. Growth optimally occurred at 37°C, pH 6.5-7.5, and 0-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain UJN715T belonged to the genus Chelativorans, with the highest sequence similarity to Chelativorans multitrophicus DSM 9103T (97.7%). Genome sequencing revealed a genome size of 5 702 301bp and a G + C content of 64.1mol%. The ANI, POCP and the dDDH between strain UJN715T and C. multitrophicus DSM 9103T were 76.2%, 49.3%, and 20.5%, respectively. The prediction result of secondary metabolites based on genome showed that the strain UJN715T contained one cluster of ectoine production, one cluster of non-ribosomal peptide synthetase (NRPS), one cluster of type I polyketide synthases (TIPKS), one cluster of bacteriocin, one cluster of TfuA-related, one cluster of N-acetylglutaminylglutamine amide (NAGGN) production, one cluster of terpene production, two clusters of homoserine lactone (Hserlactone) production. The major respiratory quinone was Q-10. The major fatty acids were iso-C17:0, C18:0 and C19:0 cyclo ω8c and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phospholipids, unknown lipids, diphosphatidylglycerol, aminoglycolipid, unidentified aminophospholipids. On the basis of these data, strain UJN715T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans alearense sp. nov. is proposed. The type strain is UJN715T (= KCTC 72856T = CCTCC AB2019378T).

Promoter engineering for high ectoine production in a lower saline medium by Halomonas hydrothermalis Y2.

For the stress from fermenters, downstream processing equipment, and wastewater treatment to be alleviated, lowering salt-dependence in the ectoine synthesis process is of great significance in the moderately halotolerant Halomonas hydrothermalis Y2. In H. hydrothermalis Y2, the σ70- and σ38-controlled promoters of ectA are predicted to be involved in the osmotic regulation of ectoine synthesis. By substituting the ectA promoter with a promoter P265 that identified in the outer membrane pore protein E of H. hydrothermalis Y2, the salt dependence of ectoine synthesis was significantly decreased. In the 500-ml flask containing various NaCl contents, the engineered strain (p/Y2/△ectD/△doeA) showed a remarkably enhanced ability in ectoine synthesis, especially under lower saline stress. After a 36-h fed-batch fermentation in the 1-l fermenter, p/Y2/△ectD/△doeA synthesized 11.5g ectoine l-1 in the presence of 60g NaCl-1l, with a high 0.32g ectoine l-1 h-1 productivity, a specific productivity of 512.2mg ectoine per g cell dry weight (CDW)-1, and an excretion ratio of 67 % ectoine. As no impaired growth was observed in strain p/Y2/△ectD/△doeA while ectoine synthesis was increased, this promoter engineering strategy provides a practical protocol for lowering the salt-dependence of ectoine synthesis in this moderately halotolerant strain.

Chelativorans xinjiangense sp. nov., a novel bacterial species isolated from soil in Xinjiang, China

A novel Gram-strain-negative, beige-pigmented, aerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated strain lm93T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alar city, located in Xinjiang province, China. Growth optimally occurred at 30°C, pH 6.5-7.5, and 0-2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain lm93T belonged to the genus Chelativorans, with highest sequence similarity to Chelativorans multitrophicus DSM 9103T (96.9%). Genome sequencing revealed a genome size of 5 689 708bp and a G + C content of 64.3mol%. The ANI, POCP and the dDDH between strain lm93T and C. multitrophicus DSM 9103T were 76.4%, 54.8% and 0.8%, respectively. The prediction result of secondary metabolites based on genome showed that the strain lm93T contained one cluster of bacteriocin, one cluster of terpene production, two clusters of ectoine production, one cluster of non-ribosomal peptide synthetase, one cluster of type I polyketide synthases, three clusters of homoserine lactone production, one cluster of N-acetylglutaminylglutamine amide production and one cluster of phosphonate production. The major respiratory quinone was Q-10. The major fatty acids were C19:0 cyclo ω8c, iso-C17:0 and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, aminoglycolipid, three unknown lipids and diphosphatidylglycerol. On the basis of these data, strain lm93T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans xinjiangense sp. nov. is proposed. The type strain is lm93T (= KCTC 72857T = CCTCC AB2019376T).