Biochemical Properties of Ectoine Hydroxylases from Extremophiles and Their Wider Taxonomic Distribution among Microorganisms

Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo-form, thereby revealing that the iron-free structure exists already in a pre-set configuration to incorporate the iron catalyst. Collectively, our work defines the taxonomic distribution and salient biochemical properties of the ectoine hydroxylase protein family and contributes to the understanding of its structure.     

Genome Sequence of the Moderately Halotolerant, Arsenite-Oxidizing Bacterium Pseudomonas stutzeri TS44

We present the draft genome sequence of Pseudomonas stutzeri TS44, a moderately halotolerant, arsenite-oxidizing bacterium isolated from arsenic-contaminated soil. The genome contains genes for arsenite oxidation, arsenic resistance, and ectoine/hydroxyectoine biosynthesis. The genome information will be useful for exploring adaptation of P. stutzeri TS44 to an arsenic-contaminated environment.     

Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190(T) and evaluate the suitability of P. stutzeri DSM5190(T) for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.     

The biosynthesis of ectoine

Abstract The biosynthetic pathway of the novel compatible solute ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was studied in the two extremely halophilic eubacteria Ectothiorhodospira halochloris and Halomonas elongata . The pathway starts with the phosphorylation of l -aspartate and shares its first two enzymatic steps with the biosynthesis of amino acids of the aspartate family: aspartokinase and l -aspartate-β-semialdehyde dehydrogenase. Evidence is presented for the presence of the enzymes l -diaminobutyric acid transaminase and l -diaminobutyric acid acetyl transferase and for the new enzyme the ring-forming ectoine synthase.     

Regulation of aspartate-derived amino acid biosynthesis in the yeastSaccharomyces cerevisiae

The activity of three enzymes, aspartokinase, homoserine dehydrogenase, and homoserine kinase, has been studied in the industrial strainSaccharomyces cerevisiae IFI256 and in the mutants derived from it that are able to overproduce methionine and/or threonine. Most of the mutants showed alteration of the kinetic properties of the enzymes aspartokinase, which was less inhibited by threonine and increased its affinity for aspartate, and homoserine dehydrogenase and homoserine kinase, which both lost affinity for homoserine. Furthermore, they showed in vitro specific activities for aspartokinase and homoserine kinase that were higher than those of the wild type, resulting in accumulation of aspartate, homoserine, threonine, and/or methionine/S-adenosyl-methionine (Ado-Met). Together with an increase in the specific activity of both aspartokinase and homoserine kinase, there was a considerable and parallel increase in methionine and threonine concentration in the mutants. Those which produced the maximal concentration of these amino acids underwent minimal aspartokinase inhibition by threonine. This supports previous data that identify aspartokinase as the main agent in the regulation of the biosynthetic pathway of these amino acids. The homoserine kinase in the mutants showed inhibition by methionine together with a lack or a reduction of the inhibition by threonine that the wild type undergoes, which finding suggests an important role for this enzyme in methionine and threonine regulation. Finally, homoserine dehydrogenase displayed very similar specific activity in the mutants and the wild type in spite of the changes observed in amino acid concentrations; this points to a minor role for this enzyme in amino acid regulation.     

Ectoine-induced proteins in Sinorhizobium meliloti include an Ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.     

Regulation of lysine biosynthesis in Escherichia coli K12.

A general survey of the regulation in lysine biosynthesis in Escherichia coli K12 is presented. No polygenic operon exists for the genes that code for enzymes of the lysine biosynthetic pathway. Lysyl-tRNA is not directly involved as a co-repressor in the pathway. Different regulation mechanisms must exist for the different enzymes. In the case of the last enzyme, diaminopimelate decarboxylase, its synthesis is induced in vivo by the lysine-sensitive aspartokinase under its non-inhibited allosteric conformation.     

Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis

Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, l-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the l-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus.     

Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow

Abstract Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).     

Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production

Deregulation of allosteric inhibition of enzymes is a challenge for strain engineering and has been achieved so far primarily by random mutation and trial-and-error. In this work, we used aspartokinase, an important allosteric enzyme for industrial amino acids production, to demonstrate a predictive approach that combines protein dynamics and evolution for a rational reengineering of enzyme allostery. Molecular dynamic simulation of aspartokinase III (AK3) from Escherichia coli and statistical coupling analysis of protein sequences of the aspartokinase family allowed to identify a cluster of residues which are correlated during protein motion and coupled during the evolution. This cluster of residues forms an interconnected network mediating the allosteric regulation, including most of the previously reported positions mutated in feedback insensitive AK3 mutants. Beyond these mutation positions, we have successfully constructed another twelve targeted mutations of AK3 desensitized toward lysine inhibition. Six threonine-insensitive mutants of aspartokinase I-homoserine dehydrogenase I (AK1-HD1) were also created based on the predictions. The proposed approach can be widely applied for the deregulation of other allosteric enzymes.     

Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor.     

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.     

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.     

新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定

研究旨在克隆新的四氢嘧啶合成基因簇,并对其功能进行鉴定,为应用于四氢嘧啶的生产奠定基础。从新喀里多尼亚弧菌CGJ02-2中克隆获得四氢嘧啶合成基因簇ectABC,ectABC与表达载体pBAD连接后转化至大肠杆菌BW25113中,通过L-阿拉伯糖诱导表达。采用SDS-PAGE和液质联用鉴定重组表达蛋白,利用全细胞催化合成四氢嘧啶,通过高分辨质谱鉴定四氢嘧啶,并从天冬氨酸浓度、KCl浓度、温度和pH 4个方面优化催化条件。结果表明,来自新喀里多尼亚弧菌CGJ02-2基因组的ectABC大小为2 235 bp。SDS-PAGE显示表达产物中有3个重组蛋白产生,液质联用鉴定表明其分子量分别与ectA、ectB、ectC的理论分子量一致。高分辨质谱分析发现全细胞催化上清中有四氢嘧啶产生。优化后的最适全细胞催化条件为：天冬氨酸浓度100 mmol·L^-1,KCl浓度100 mmol·L^-1,温度30℃,pH 7.0,最优条件下产量为1.11 mg·mL^-1。研究从弧菌中克隆了四氢嘧啶合成基因簇ectABC,并在大肠杆菌BW2511...     

Ectoines as compatible solutes and carbon and energy sources for the halophilic bacterium Chromohalobacter salexigens

AIMS: To investigate the catabolism of ectoine and hydroxyectoine, which are the major compatible solutes synthesized by Chromohalobacter salexigens. METHODS AND RESULTS: Growth curves performed in M63 minimal medium with low (0.75 mol l(-1) NaCl), optimal (1.5 mol l(-1) NaCl) or high (2.5 mol l(-1) NaCl) salinity revealed that betaine and ectoines were used as substrate for growth at optimal and high salt. Ectoine transport was maximal at optimal salinity, and showed 3- and 1.5-fold lower values at low and high salinity respectively. The salt-sensitive ectA mutant CHR62 showed an ectoine transport rate 6.8-fold higher than that of the wild type. Incubation of C. salexigens in a mixture of glucose and ectoine resulted in a biphasic growth pattern. However, CO(2) production due to ectoine catabolism was lower, but not completely abolished, in the presence of glucose. When used as the sole carbon source, glycine betaine effectively inhibited ectoine and hydroxyectoine synthesis at any salinity. CONCLUSIONS: The catabolic pathways for ectoine and hydroxyectoine in C. salexigens operate at optimal and high (although less efficiently) salinity. Endogenous ectoine(s) may repress its own transport. Ectoine utilization was only partially repressed by glucose. Betaine, when used as carbon source, suppresses synthesis of ectoines even under high osmolarity conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a previous step to the subsequent isolation and manipulation of the catabolic genes, so as to generate strains with enhanced production of ectoine and hydroxyectoine.     

Amino acid biosynthesis: new architectures in allosteric enzymes.

This review focuses on the allosteric controls in the Aspartate-derived and the branched-chain amino acid biosynthetic pathways examined both from kinetic and structural points of view. The objective is to show the differences that exist among the plant and microbial worlds concerning the allosteric regulation of these pathways and to unveil the structural bases of this diversity. Indeed, crystallographic structures of enzymes from these pathways have been determined in bacteria, fungi and plants, providing a wonderful opportunity to obtain insight into the acquisition and modulation of allosteric controls in the course of evolution. This will be examined using two enzymes, threonine synthase and the ACT domain containing enzyme aspartate kinase. In a last part, as many enzymes in these pathways display regulatory domains containing the conserved ACT module, the organization of ACT domains in this kind of allosteric enzymes will be reviewed, providing explanations for the variety of allosteric effectors and type of controls observed.