Ectoine Biosynthesis Research Articles

High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli

BackgroundRecently, the compatible solute 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) has attracted considerable interest due to its great potential as a protecting agent. To overcome the drawbacks of high salinity in the traditional bioprocess of ectoine using halophilic bacteria, various attempts have been made to engineer ectoine biosynthesis in nonhalophilic bacteria. Unfortunately, the yields of ectoine in these producers are still low and hardly meet the demands of large scale production. In this paper, the whole-cell biocatalytic process using aspartate and glycerol as substrates was tried for high production of ectoine in nonhalophilic bacteria.ResultsThe ectoine genes ectABC from the halophilic bacterium Halomonas elongata were successfully introduced into Escherichia coli K-12 strain BW25113 under the arabinose-inducible promoter. To our delight, a large amount of ectoine was synthesized and excreted into the medium during the course of whole-cell biocatalysis, when using aspartate and glycerol as the direct substrates. At the low cell density of 5 OD/mL in flask, under the optimal conditions (100 mM sodium phosphate buffer (pH 7.0), 100 mM sodium aspartate, 100 mM KCl and 100 mM glycerol), the concentration of extracellular ectoine was increased to 2.67 mg/mL. At the high cell density of 20 OD/mL in fermentor, a maximum titre of 25.1 g/L ectoine was achieved in 24 h. Meanwhile, the biomass productivity of ectoine is as high as 4048 mg per gram dry cell weight (g DCW)−1, which is the highest value ever reported. Furthermore, it was demonstrated that the same batch of cells could be used for at least three rounds. Finally, a total yield of 63.4 g ectoine was obtained using one litre cells.ConclusionUsing aspartate and glycerol as the direct substrates, high production of ectoine was achieved by the whole-cell biocatalysis in recombinant E. coli. Multiple rounds of whole-cell biocatalysis were established to further improve the production of ectoine. Our study herein provided a feasible biosynthesis process of ectoine with potential applications in large-scale industrial production.

The complete genome sequence of the rumen methanogen Methanobacterium formicicum BRM9.

Methanobacterium formicicum BRM9 was isolated from the rumen of a New Zealand Friesan cow grazing a ryegrass/clover pasture, and its genome has been sequenced to provide information on the phylogenetic diversity of rumen methanogens with a view to developing technologies for methane mitigation. The 2.45 Mb BRM9 chromosome has an average G + C content of 41%, and encodes 2,352 protein-coding genes. The genes involved in methanogenesis are comparable to those found in other members of the Methanobacteriaceae with the exception that there is no [Fe]-hydrogenase dehydrogenase (Hmd) which links the methenyl-H4MPT reduction directly with the oxidation of H2. Compared to the rumen Methanobrevibacter strains, BRM9 has a much larger complement of genes involved in determining oxidative stress response, signal transduction and nitrogen fixation. BRM9 also has genes for the biosynthesis of the compatible solute ectoine that has not been reported to be produced by methanogens. The BRM9 genome has a prophage and two CRISPR repeat regions. Comparison to the genomes of other Methanobacterium strains shows a core genome of ~1,350 coding sequences and 190 strain-specific genes in BRM9, most of which are hypothetical proteins or prophage related.

Biosynthesis of ectoine by the methanotrophic bacterial consortium isolated from Bogdanka coalmine (Poland)

Ectoine belongs to the family of compatible solutes, which are known to contribute mainly to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition, the cell’s essential functions are maintained under difficult conditions like high salinity, heat, or aridity stress. Biosynthesis of ectoine has been found in halophilic and halotolerant microorganisms. We showed that the methanotrophic bacterial consortium (MBC) isolated from coalbed rocks from coalmine Bogdanka (Poland) and resistant to extreme environmental conditions (low content of moisture) was able to synthesize ectoine. MBC was cultured in mineral nitrate mineral salts medium supplied with NaCl at atmospheric air enriched with 10% of methane. The levels of methanotrophic activity were determined by the gas chromatography technique (943.05 ± 30.73 − 94.14 ± 0.85 μM CH4 gDW−1 day−1) and the biomass concentration of MBC was evaluated based on OD600, as well as biosynthesis of ectoine in relation to the salinity (0–5% NaCl) of the medium. The levels of ectoine tested by NIR measurements ranged from 1.33 ± 0.10 mg gDW−1 to 0.42 ± 0.08 mg gDW−1 depending on the salinity of the solution. In addition, we identified MBC based on the pmoA gene.

The hydroxyectoine gene cluster of the non-halophilic acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic α-Proteobacterium which thrives in acidic, metal-rich environments (e.g. acid mine drainage). Recently, an ectABCDask gene cluster for biosynthesis of the compatible solutes ectoine and hydroxyectoine was detected in the genome sequence of A. cryptum JF-5. We were able to demonstrate that the type strain A. cryptum DSM 2389(T) is capable of synthesizing the compatible solute hydroxyectoine in response to moderate osmotic stress caused by sodium chloride and aluminium sulphate, respectively. Furthermore, we used the A. cryptum JF-5 sequence to amplify the ectABCDask gene cluster from strain DSM 2389(T) and achieved heterologous expression of the gene cluster in Escherichia coli. Hence, we could for the first time prove metabolic functionality of the genes responsible for hydroxyectoine biosynthesis in the acidophile A. cryptum. In addition, we present information on specific enzyme activity of A. cryptum DSM 2389(T) ectoine synthase (EctC) in vitro. In contrast to EctCs from halophilic microorganisms, the A. cryptum enzyme exhibits a higher isoelectric point, thus a lower acidity, and has maximum specific activity in the absence of sodium chloride.

Identification and characterization of ectoine biosynthesis genes and heterologous expression of the ectABC gene cluster from Halomonas sp. QHL1, a moderately halophilic bacterium isolated from Qinghai Lake

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ(70)-dependent promoter and a δ(38)-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine

BackgroundThe stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems.ResultsHere, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L-1 day-1 under growth conditions that did not rely on the use of high-salinity media.ConclusionsThe present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.

Genomes of “Spiribacter”, a streamlined, successful halophilic bacterium

BackgroundThalassosaline waters produced by the concentration of seawater are widespread and common extreme aquatic habitats. Their salinity varies from that of sea water (ca. 3.5%) to saturation for NaCl (ca. 37%). Obviously the microbiota varies dramatically throughout this range. Recent metagenomic analysis of intermediate salinity waters (19%) indicated the presence of an abundant and yet undescribed gamma-proteobacterium. Two strains belonging to this group have been isolated from saltern ponds of intermediate salinity in two Spanish salterns and were named “Spiribacter”.ResultsThe genomes of two isolates of “Spiribacter” have been fully sequenced and assembled. The analysis of metagenomic datasets indicates that microbes of this genus are widespread worldwide in medium salinity habitats representing the first ecologically defined moderate halophile. The genomes indicate that the two isolates belong to different species within the same genus. Both genomes are streamlined with high coding densities, have few regulatory mechanisms and no motility or chemotactic behavior. Metabolically they are heterotrophs with a subgroup II xanthorhodopsin as an additional energy source when light is available.ConclusionsThis is the first bacterium that has been proven by culture independent approaches to be prevalent in hypersaline habitats of intermediate salinity (half a way between the sea and NaCl saturation). Predictions from the proteome and analysis of transporter genes, together with a complete ectoine biosynthesis gene cluster are consistent with these microbes having the salt-out-organic-compatible solutes type of osmoregulation. All these features are also consistent with a well-adapted fully planktonic microbe while other halophiles with more complex genomes such as Salinibacter ruber might have particle associated microniches.

Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes

Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes

Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica

In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ(70)-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.

The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae

Vibrio cholerae inhabits aquatic environments and colonizes the human digestive tract to cause the disease cholera. In these environments, V. cholerae copes with fluctuations in salinity and osmolarity by producing and transporting small, organic, highly soluble molecules called compatible solutes, which counteract extracellular osmotic pressure. Currently, it is unclear how V. cholerae regulates the expression of genes important for the biosynthesis or transport of compatible solutes in response to changing salinity or osmolarity conditions. Through a genome-wide transcriptional analysis of the salinity response of V. cholerae, we identified a transcriptional regulator we name CosR for compatible solute regulator. The expression of cosR is regulated by ionic strength and not osmolarity. A transcriptome analysis of a ΔcosR mutant revealed that CosR represses genes involved in ectoine biosynthesis and compatible solute transport in a salinity-dependent manner. When grown in salinities similar to estuarine environments, CosR activates biofilm formation and represses motility independently of its function as an ectoine regulator. This is the first study to characterize a compatible solute regulator in V. cholerae and couples the regulation of osmotic tolerance with biofilm formation and motility.

Use of Raman spectroscopy for identification of compatible solutes in halophilic bacteria

We explored the use of Raman spectroscopy to detect organic osmotic solutes as biomarkers in the moderately halophilic heterotrophic bacterium Halomonas elongata grown in complex medium (accumulation of glycine betaine) and in defined medium with glucose as carbon source (biosynthesis of ectoine), and in the anoxygenic phototrophic Ectothiorhodospira marismortui known to synthesize glycine betaine in combination with minor amounts of trehalose and N-α-carbamoyl glutamineamide. We tested different methods of preparation of the material: lyophilization, two-phase extraction of water-soluble molecules, and perchlorate extraction. Raman signals of glycine betaine and ectoine were detected; perchlorate extraction followed by desalting the extract on an ion retardation column gave the best results. Lyophilized cells of E. marismortui showed strong signals of carotenoid pigments, and glycine betaine could be detected only after perchlorate extraction and desalting. The data presented show that Raman spectroscopy is a suitable tool to assess the mode of osmotic adaptation used by halophilic microorganisms.

The PII protein GlnK is a pleiotropic regulator for morphological differentiation and secondary metabolism in Streptomyces coelicolor.

GlnK is an important nitrogen sensor protein in Streptomyces coelicolor. Deletion of glnK results in a medium-dependent failure of aerial mycelium and spore formation and loss of antibiotic production. Thus, GlnK is not only a regulator of nitrogen metabolism but also of morphological differentiation and secondary metabolite production. Through a comparative transcriptomic approach between the S. coelicolor wild-type and a S. coelicolor glnK mutant strain, 142 genes were identified that are differentially regulated in both strains. Among these are genes of the ram and rag operon, which are involved in S. coelicolor morphogenesis, as well as genes involved in gas vesicle biosynthesis and ectoine biosynthesis. Surprisingly, no relevant nitrogen genes were found to be differentially regulated, revealing that GlnK is not an important nitrogen sensor under the tested conditions.

Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

Regulatory aspects of ectoine biosynthesis in halophilic bacteria

In this review, the current concepts of metabolic and genetic regulation of the synthesis of ectoine and hydroxyectoine, widely distributed osmoprotectors of halophilic bacteria, are analyzed and generalized. Due to their water-retaining properties, these compounds can be used as multifunctional bioprotectants. The relevance of deciphering ectoine biosynthesis regulation at the levels of enzyme activity and gene transcription in halophiles is related to the search and creation of novel, more efficient producers by deliberate genetic-engineering construction. In spite of the conservativeness of the ectoine biosynthesis pathway in relation to genes and enzymes, indicating the horizontal transfer of ect genes, different enzyme properties and mechanisms of transcriptional regulation of the ectoine operon were revealed in various halophilic bacteria. The models of transcriptional regulation of the genes of ectoine biosynthesis in Chromohalobacter salexigens and Methylomicrobium alcaliphilum are discussed.

Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus "Nitrosopumilus maritimus" strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

Problem statement: The cyclic imino acid ectoine is a widely distributed compatible solute synthesizing by halophilic and halotolerant bacteria to prevent osmotic stress at high external salinity. This water-keeping compound is used in a variety of commercial cosmetics and therapeutic products. Approach: Development of integrated, predictive functional model of the metabolic and regulatory netwoks of ectoine-producing microbes is an active area of research. In this article we present a brief overview of the current knowledge on genetic and biochemical aspects of ectoine biosynthesis in aerobic halophilic and halotolerant bacteria utilizing C1 compounds (methylotrophs). Although enzymology and genetics of the ectoine biosynthesis in methylotrophs are similar to other halophilic bacteria, the regulatory patterns are different. In all methylotrophic bacteria studied, the genes coding for specific enzymes of ectoine biosynthesis: Diaminobutyric Acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA) and ectoine synthase (EctC) are organized into ectABC or ectABC-ask, whith is linked to gene encoding Aspartokinase isozyme (Ask). Results: Remarkably, the methylotrophic bacteria possessing a four-gene cluster showed higher halotolerance and accumulated more ectoine than bacteria with a cluster composed of three genes. The DABA acetyltransferases from three methylotrophic species have been comparatively characterized. The properties of the enzymes correlate with eco-physiological and metabolic particularities of the host. Some elements of the regulatory system governing the ectoine pathway operation have been revealed in both methane and methanol utilizing bacteria. In Methylomicrobium alcaliphilum transcription of the ectABC-ask operon is initiated from two σ70-like promoters and controlled by the EctR, a MarR-type negative regulator. EctR orthologs were identified in genomes of several heterotrophic halophilic bacteria. Here we present genomic data indicating that similar regulatory system may occur in diverse halophilic and halotolerant bacteria. Conclusion: Currently available data suggest that in methylotrophic bacteria the ectoine biosynthesis pathways are evolutionary well conserved, particularly with respect to the genes and enzymes involved. However, some differences in the ect-gene cluster organization and regulation could be observed.

Functional characterization, secondary structure prediction and analysis of ectoine biosynthesis genes from Bacillus halodurans: an osmolyte involved in stress tolerance

An open reading frame encoding the ectoine biosynthesis genes was cloned from the Bacillus halodurans genome. An expression plasmid containing the operon was introduced into Escherichia coli cells, and the recombinant ectoine was quantified. The secondary structure of ectoine biosynthesis proteins were predicted and was quite similar to that of reported proteins from eubacteria.

Metabolomic Characterization of the Salt Stress Response in Streptomyces coelicolor

The humicolous actinomycete Streptomyces coelicolor routinely adapts to a wide variety of habitats and rapidly changing environments. Upon salt stress, the organism is also known to increase the levels of various compatible solutes. Here we report the results of the first high-resolution metabolomics time series analysis of various strains of S. coelicolor exposed to salt stress: the wild type, mutants with progressive knockouts of the ectoine biosynthesis pathway, and two stress regulator mutants (with disruptions of the sigB and osaB genes). Samples were taken from cultures at 0, 4, 8, and 24 h after salt stress treatment and analyzed by liquid chromatography-mass spectrometry with an LTQ Orbitrap XL mass spectrometer. The results suggest that a large fraction of amino acids is upregulated in response to the salt stress, as are proline/glycine-containing di- and tripeptides. Additionally we found that 5'-methylthioadenosine, a known inhibitor of polyamine biosynthesis, is downregulated upon salt stress. Strikingly, no major differences between the wild-type cultures and the two stress regulator mutants were found, indicating a considerable robustness of the metabolomic response to salt stress, compared to the more volatile changes in transcript abundance reported earlier.

EctR—A novel transcriptional regulator of ectoine biosynthesis genes in the haloalcaliphilic methylotrophic bacterium Methylophaga alcalica

EctR—A novel transcriptional regulator of ectoine biosynthesis genes in the haloalcaliphilic methylotrophic bacterium Methylophaga alcalica

Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

Genes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z have been shown to be organized into an ectABC-ask operon. Transcription of the ect operon is initiated from two promoters, ectAp(1) and ectAp(2) (ectAp(1)p(2)), similar to the sigma(70)-dependent promoters of Escherichia coli. Upstream of the gene cluster, an open reading frame (ectR1) encoding a MarR-like transcriptional regulator was identified. Investigation of the influence of EctR1 on the activity of the ectAp(1)p(2) promoters in wild-type M. alcaliphilum 20Z and ectR1 mutant strains suggested that EctR1 is a negative regulator of the ectABC-ask operon. Purified recombinant EctR1-His(6) specifically binds as a homodimer to the putative -10 motif of the ectAp(1) promoter. The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp. Transcription of the ectR1 gene is initiated from a single sigma(70)-like promoter. The location of the EctR1 binding site between the transcriptional and translational start sites of the ectR1 gene suggests that EctR1 may regulate its own expression. The data presented suggest that in Methylomicrobium alcaliphilum 20Z, EctR1-mediated control of the transcription of the ect genes is not the single mechanism for the regulation of ectoine biosynthesis.