Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis.

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients.

Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory.

Ectoine and hydroxyectoine are widely synthesized by members of the Bacteria and a few members of the Archaea as potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacterium Pseudomonas stutzeri by transferring it into Escherichia coli, an enterobacterium that does not produce ectoines naturally. Using ect-lacZ reporter fusions, we found that the heterologous ect promoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the -10, -35, and spacer regions of the sigma-70-type ect promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the ect promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters of E. coli afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set of ect promoter mutants, we engineered an E. coli chassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.IMPORTANCE Ectoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited the ect promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact that E. coli does not synthesize ectoines naturally, the ect promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation of ect transcription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of the ect promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.

Feeding on compatible solutes: A substrate-induced pathway for uptake and catabolism of ectoines and its genetic control by EnuR.

Ectoine and 5-hydroxyectoine are widely synthesized microbial osmostress protectants. They are also versatile nutrients but their catabolism and the genetic regulation of the corresponding genes are incompletely understood. Using the marine bacterium Ruegeria pomeroyi DSS-3, we investigated the utilization of ectoines and propose a seven steps comprising catabolic route that entails an initial conversion of 5-hydroxyectoine to ectoine, the opening of the ectoine ring, and the subsequent degradation of this intermediate to l-aspartate. The catabolic genes are co-transcribed with three genes encoding a 5-hydroxyectoine/ectoine-specific TRAP transporter. A chromosomal deletion of this entire gene cluster abolishes the utilization of ectoines as carbon and nitrogen sources. The presence of ectoines in the growth medium triggers enhanced expression of the importer and catabolic operon, a process dependent on a substrate-inducible promoter that precedes this gene cluster. EnuR, a member of the MocR/GabR-type transcriptional regulators, controls the activity of this promoter and functions as a repressor. EnuR contains a covalently bound pyridoxal-5'-phosphate, and we suggest that this co-factor is critical for the substrate-mediated induction of the 5-hydroxyectoine/ectoine import and catabolic genes. Bioinformatics showed that ectoine consumers are restricted to the Proteobacteria and that EnuR is likely a central regulator for most ectoine/5-hydroxyectoine catabolic genes.

EctD-mediated biotransformation of the chemical chaperone ectoine into hydroxyectoine and its mechanosensitive channel-independent excretion.

BACKGROUND: Ectoine and its derivative 5-hydroxyectoine are cytoprotectants widely synthesized by microorganisms as a defense against the detrimental effects of high osmolarity on cellular physiology and growth. Both ectoines possess the ability to preserve the functionality of proteins, macromolecular complexes, and even entire cells, attributes that led to their description as chemical chaperones. As a consequence, there is growing interest in using ectoines for biotechnological purposes, in skin care, and in medical applications. 5-Hydroxyectoine is synthesized from ectoine through a region- and stereo-specific hydroxylation reaction mediated by the EctD enzyme, a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenases. This chemical modification endows the newly formed 5-hydroxyectoine with either superior or different stress- protecting and stabilizing properties. Microorganisms producing 5-hydroxyectoine typically contain a mixture of both ectoines. We aimed to establish a recombinant microbial cell factory where 5-hydroxyectoine is (i) produced in highly purified form, and (ii) secreted into the growth medium. RESULTS: We used an Escherichia coli strain (FF4169) defective in the synthesis of the osmostress protectant trehalose as the chassis for our recombinant cell factory. We expressed in this strain a plasmid-encoded ectD gene from Pseudomonas stutzeri A1501 under the control of the anhydrotetracycline-inducible tet promoter. We chose the ectoine hydroxylase from P. stutzeri A1501 for our cell factory after a careful comparison of the in vivo performance of seven different EctD proteins. In the final set-up of the cell factory, ectoine was provided to salt-stressed cultures of strain FF4169 (pMP41; ectD (+)). Ectoine was imported into the cells via the osmotically inducible ProP and ProU transport systems, intracellularly converted to 5-hydroxyectoine, which was then almost quantitatively secreted into the growth medium. Experiments with an E. coli mutant lacking all currently known mechanosensitive channels (MscL, MscS, MscK, MscM) revealed that the release of 5-hydroxyectoine under osmotic steady-state conditions occurred independently of these microbial safety valves. In shake-flask experiments, 2.13 g l(-1) ectoine (15 mM) was completely converted into 5-hydroxyectoine within 24 h. CONCLUSIONS: We describe here a recombinant E. coli cell factory for the production and secretion of the chemical chaperone 5-hydroxyectoine free from contaminating ectoine.

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.

Strangers in the archaeal world: osmostress-responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus.

Ectoine and hydroxyectoine are compatible solutes widely synthesized by members of the Bacteria to cope with high osmolarity surroundings. Inspection of 557 archaeal genomes revealed that only 12 strains affiliated with the Nitrosopumilus, Methanothrix or Methanobacterium genera harbour ectoine/hydroxyectoine gene clusters. Phylogenetic considerations suggest that these Archaea have acquired these genes through horizontal gene transfer events. Using the Thaumarchaeon 'Candidatus Nitrosopumilus maritimus' as an example, we demonstrate that the transcription of its ectABCD genes is osmotically induced and functional since it leads to the production of both ectoine and hydroxyectoine. The ectoine synthase and the ectoine hydroxylase were biochemically characterized, and their properties resemble those of their counterparts from Bacteria. Transcriptional analysis of osmotically stressed 'Ca. N. maritimus' cells demonstrated that they possess an ectoine/hydroxyectoine gene cluster (hyp-ectABCD-mscS) different from those recognized previously since it contains a gene for an MscS-type mechanosensitive channel. Complementation experiments with an Escherichia coli mutant lacking all known mechanosensitive channel proteins demonstrated that the (Nm)MscS protein is functional. Hence, 'Ca. N. maritimus' cells cope with high salinity not only through enhanced synthesis of osmostress-protective ectoines but they already prepare themselves simultaneously for an eventually occurring osmotic down-shock by enhancing the production of a safety-valve (NmMscS).

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

BACKGROUND: The 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. RESULTS: Here, 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. CONCLUSIONS: The 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.

Succinyl-CoA:3-sulfinopropionate CoA-transferase from Variovorax paradoxus strain TBEA6, a novel member of the class III coenzyme A (CoA)-transferase family.

The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act(TBEA6) to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. Act(TBEA6) was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 µmol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 µmol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acd(TBEA6), located downstream of act(TBEA6).

Proline utilization by Bacillus subtilis: uptake and catabolism.

L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the Δ(1)-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.

A specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501.

The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. l-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. l-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from l-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an askgene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_askgenes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. colistrain carrying the plasmid-based ectABCD_askgene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCDgene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis.

Aerobic degradation of mercaptosuccinate by the gram-negative bacterium Variovorax paradoxus strain B4.

The Gram-negative bacterium Variovorax paradoxus strain B4 was isolated from soil under mesophilic and aerobic conditions to elucidate the so far unknown catabolism of mercaptosuccinate (MS). During growth with MS this strain released significant amounts of sulfate into the medium. Tn5::mob-induced mutagenesis was successfully employed and yielded nine independent mutants incapable of using MS as a carbon source. In six of these mutants, Tn5::mob insertions were mapped in a putative gene encoding a molybdenum (Mo) cofactor biosynthesis protein (moeA). In two further mutants the Tn5::mob insertion was mapped in the gene coding for a putative molybdopterin (MPT) oxidoreductase. In contrast to the wild type, these eight mutants also showed no growth on taurine. In another mutant a gene putatively encoding a 3-hydroxyacyl-coenzyme A dehydrogenase (paaH2) was disrupted by transposon insertion. Upon subcellular fractionation of wild-type cells cultivated with MS as sole carbon and sulfur source, MPT oxidoreductase activity was detected in only the cytoplasmic fraction. Cells grown with succinate, taurine, or gluconate as a sole carbon source exhibited no activity or much lower activity. MPT oxidoreductase activity in the cytoplasmic fraction of the Tn5::mob-induced mutant Icr6 was 3-fold lower in comparison to the wild type. Therefore, a new pathway for MS catabolism in V. paradoxus strain B4 is proposed: (i) MPT oxidoreductase catalyzes the conversion of MS first into sulfinosuccinate (a putative organo-sulfur compound composed of succinate and a sulfino group) and then into sulfosuccinate by successive transfer of oxygen atoms, (ii) sulfosuccinate is cleaved into oxaloacetate and sulfite, and (iii) sulfite is oxidized to sulfate.