Ectoine hyperproduction by engineered Halomonas bluephagenesis.

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.

Metabolic engineering combined with enzyme engineering for overproduction of ectoine in Escherichia coli.

Ectoine, a natural protective agent, is naturally synthesized at low titers by some extreme environment microorganisms that are usually difficult to culture. There is a need for an efficient and eco-friendly ectoine production process. In this study, Escherichia coli BL21(DE3) with the ectABC gene cluster from Halomonas venusta achieved 1.7 g/L ectoine. After optimizing the expression plasmid, 2.1 g/L ectoine was achieved. Besides, the aspartate kinase mutant LysCT311I from Corynebacterium glutamicum and aspartate semialdehyde dehydrogenase from Halomonas elongata were overexpressed to increase precursors supply. Furthermore, the rate-limiting enzyme EctB was semirationally engineered, and the E407D mutation enhanced ectoine production by 13.8 %. To improve acetyl-CoA supply, the non-oxidative glycolysis pathway was introduced. Overall, the optimized strain ECT9-5 produced 67.1 g/L ectoine by fed-batch fermentation with a 0.3 g/g of glucose and the kinetic model resulted in a good fit.

Highly efficient production of ectoine via an optimized combination of precursor metabolic modules in Escherichia coli BL21.

Ectoine is an osmotic pressure protectant observed in various microorganisms and is widely used in cosmetics and pharmaceuticals. The market value of ectoine has increased considerably with social progress, resulting in high demand for ectoine production technology. Herein, a microbial cell factory in Escherichia coli that produces ectoine at high titers is described as developing efficient and environmentally friendly bio-based ectoine production technology. The ectoine biosynthetic pathway of Halomonas hydrothermalis was introduced into E. coli BL21 (DE3). Subsequent overexpression of precursor metabolic modules, including aspartate branching, pyruvate-oxoacetate, and glutamate biosynthesis pathways, resulted in the final strain, E. coli BCT08, which produced ectoine at a titer of 36.58 g/L during 30 h of fermentation. Sugar feeding speed optimization improved the ectoine titer to 131.8 g/L after 96 h of cultivation. This represents a remarkable achievement in ectoine production from glucose under low-salt conditions and has vast potential for industrial applications.

Chronic BMAA exposure combined with TDP-43 mutation elicits motor neuron dysfunction phenotypes in mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with an average age-of-onset of ∼60 years and is usually fatal within 2-5 years of diagnosis. Mouse models based upon single gene mutations do not recapitulate all ALS pathological features. Environmental insults may also contribute to ALS, and ß-N-methylamino-L-alanine (BMAA) is an environmental toxin linked with an increased risk of developing ALS. BMAA, along with cycasin, are hypothesized to be the cause of the Guam-ALS epicenter of the 1950s. We developed a multihit model based on low expression of a dominant familial ALS TDP-43 mutation (Q331K) and chronic low-dose BMAA exposure. Our two-hit mouse model displayed a motor phenotype absent from either lesion alone. By LC/MS analysis, free BMAA was confirmed at trace levels in brain, and were as high as 405 ng/mL (free) and 208 ng/mL (protein-bound) in liver. Elevated BMAA levels in liver were associated with dysregulation of the unfolded protein response (UPR) pathway. Our data represent initial steps towards an ALS mouse model resulting from combined genetic and environmental insult.

Microbial conversion of carbon dioxide and hydrogen into the fine chemicals hydroxyectoine and ectoine.

This study explores a novel conversion of CO2 into the chemicals hydroxyectoine and ectoine, which are compounds with high retail values in the pharmaceutical industry. Firstly, 11 species of microbes able to use CO2 and H2 and that have the genes for ectoines synthesis (ectABCD) were identified through literature search and genomic mining. Laboratory tests were then conducted to ascertain the capacity of these microbes to produce ectoines from CO2. Results showed that the most promising bacteria for CO2 to ectoines bioconversion areHydrogenovibrio marinus, Rhodococcus opacus, and Hydrogenibacillus schlegelii.Upon salinity and H2/CO2/O2 ratio optimization,H. marinus accumulated 85 mg of ectoine g biomass-1. Interestingly, R.opacusand H. schlegelii mainly produced hydroxyectoine (53 and 62 mg g biomass-1), which has a higher commercial value. Overall, these results constitute the first proof of a novel valorization platform of CO2 and lay the foundation for a new economic niche aimed at CO2 recircularization into pharmaceuticals.

Guilty by association: Importers, exporters and MscS-type mechanosensitive channels encoded in biosynthetic gene clusters for the stress-protectant ectoine.

Ectoine and its derivative hydroxyectoine are widely synthesized or imported by bacteria to fend off the detrimental effects of high osmolarity on cellular hydration and growth. Genes that are connected to a particular physiological process are often found in the same genomic context. We exploited this feature in a comprehensive bioinformatical analysis of 1103 ectoine biosynthetic gene clusters from Bacteria and Archaea through which we identified 415 ect operons that colocalize with genes encoding potential osmolyte transporters. These belong to various importer families. Focusing on the complex ect gene clusters of the alpha-proteobacteria Hyphomonas neptunium and Novoshingobium sp. LH128, we analysed several transporters with respect to their substrate specificities through physiological, molecular and modelling approaches. Accordingly, we identified an MFS-type uptake system specific for ectoines (EctU) and a novel SSS-type ectoine/hydroxyectoine importer (EctI) with a broader substrate profile for osmostress protectants. Furthermore, some ect gene clusters encode a MscS/YbdG-type mechanosensitive channel protein, whose functionality was assessed through down-shock assays. Moreover, our analysis identified the gene for the first putative ectoine/hydroxyectoine-specific efflux system (EctX), a member of the MFS superfamily. Our findings make substantial contributions to the understanding of the ecophysiology of ectoines, key players in microbial osmostress adjustment systems.

A mouse model of type B cystinuria due to spontaneous mutation in FVB/NJcl mice.

Cystinuria is an autosomal metabolic disorder caused by mutations in the SLC3A1 and SLC7A9 genes, encoding the amino acid transporter proteins rBAT and b0,+AT, respectively. Based on the causative gene, cystinuria is classified into 3 types: type A (SLC3A1), type B (SLC7A9), and type AB (SLC3A1 and SLC7A9). Patients with cystinuria exhibit hyperexcretion of cystine and dibasic amino acids in the urine and develop cystine crystals due to its low solubility in the urine, often resulting in calculus formation. In this study, we present an inbred strain FVB/NJcl mice affected with cystinuria. In the affected mouse kidney, Slc7a9 expression was completely abolished because of a large sequence deletion in the promoter region of the Slc7a9 mutant allele. Slc7a9-deficient mice with FVB/NJcl genetic background developed cystine calculi in the bladder with high penetrance, as compared to the previously reported mouse models of cystinuria. This model may be useful to understand the determinants of crystal aggregation, affecting calculus formation.

Isolation of an ectoine-producing Sinobaca sp. and identification of genes that are involved in ectoine biosynthesis.

A moderate halophilic bacterium that could accumulate ectoine and hydroxyectoine was isolated from soil near a salt mine and was identified as a Sinobaca sp. (designed strain H24) according to 16S rRNA gene sequence analysis. The bacterium grew well in the presence of 1-2 M NaCl, while growth in a medium that contained 2 M NaCl led to higher accumulation of ectoines. The yields of ectoine and hydroxyectoine by Sinobaca sp. H24 reached 11.27 mg/l and 1.34 mg/l, respectively, when cultured in the following medium: NaCl (2 M), peptone (5 g/l), yeast extract (1 g/l), NH4Cl (0.02 M), KH2PO4 (1 M), K2HPO4 (0.1 M), and glycerol (1% w/v). Genes that are involved in ectoine biosynthesis of Sinobaca sp. H24 were also identified, and their sequences were determined by a metagenomics approach. The results demonstrated that Sinobaca sp. H24 possesses ectoine metabolism genes for both ectoine biosynthesis (ectA, ectB, ectC, and ectD) and ectoine degradation (doeA). Genes that are related to ectoine biosynthesis, such as lysC and asd, were also characterized. The identification and characterization results for ectoine/hydroxyectoine biosynthesis genes are in agreement with the physiology of Sinobaca sp. H24 as a potential candidate for ectoine production for industrial applications. This report established for the first time the accumulation of ectoine/hydroxyectoine in Sinobaca sp. and characterized the genes that are involved in ectoine/hydroxyectoine biosynthesis in Sinobaca sp. H24.

Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

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

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

Streptomyces acidicola sp. nov., isolated from a peat swamp forest in Thailand.

A novel actinobacterium, designated strain K1PN6T, was isolated from soil sample collected in Kantulee peat swamp forest, Surat Thani province, Thailand. The morphological, chemotaxonomic, and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. Based on 16S rRNA gene sequence analysis, strain K1PN6T showed highest similarity to Streptomyces phyllanthi PA1-07T (98.6 %), Streptomyces spongiae Sp080513SC-24T (98.3%) and Streptomyces adustus WH-9T (98.3%). The G + C content of the genomic DNA was 70.3 mol%. Digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain K1PN6T with S. phyllanthi TISTR 2346T (33.7 and 89.1%), S. spongiae NBRC 106415T (38.6 and 90.6%) and S. adustus NBRC 109810T (26.0 and 86.2%) were below the thresholds of 70 and 95-96% for prokaryotic conspecific assignation. Chemotaxonomic data revealed that strain K1PN6T possessed MK-9(H8) (45%) and MK-9(H6) (34%) as the predominant menaquinones. It contained LL-diaminopimelic acid as the diagnostic diamino acid and galactose, glucose, mannose, and ribose as whole-cell sugars. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified aminolipids, an unidentified phospholipid, and glycophospholipid. The predominant cellular fatty acids (>10%) were iso-C16:0, C16:0, anteiso-C15:0, and iso-C14:0. On the basis of these genotypic and phenotypic data, strain K1PN6T should be designated as a representative of a novel species of the genus Streptomyces, for which the name Streptomyces acidicola sp. nov. is proposed with the type strain K1PN6T (=TBRC 11341T=NBRC 114304T).

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications.

The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.

Brevibacterium linens RS16 confers salt tolerance to Oryza sativa genotypes by regulating antioxidant defense and H+ ATPase activity.

Soil salinity is one of the major limitations that affects both plant and its soil environment, leading to reduced agricultural production. Evaluation of stress severity by plant physical and biochemical characteristics is an established way to study plant-salt stress interaction, but the halotolerant properties of plant growth promoting bacteria (PGPB) along with plant growth promotion is less studied till date. The aim of the present study was to elucidate the strategy, used by ACC deaminase-containing halotolerant Brevibacterium linens RS16 to confer salt stress tolerance in moderately salt-tolerant (FL478) and salt-sensitive (IR29) rice (Oryza sativa L.) cultivars. The plants were exposed to salt stress using 0, 50, and 100 mM of NaCl with and without bacteria. Plant physiological and biochemical characteristics were estimated after 1, 5, 10 days of stress application. H+ ATPase activity and the presence of hydroxyectoine gene (ectD) that is responsible for compatible solute accumulation were also analyzed in bacteria. The height and dry mass of bacteria inoculated plants significantly increased compared to salt-stressed plants, and the differences increased in time dependent manner. Bacteria priming reduced the plant antioxidant enzyme activity, lipid peroxidation and it also regulated the salt accumulation by modulating vacuolar H+ ATPase activity. ATPase activity and presence of hydroxyectoine gene in RS16 might have played a vital role in providing salt tolerance in bacteria inoculated rice cultivars. We conclude that dual benefits provided by the halotolerant plant growth promoting bacteria (PGPB) can provide a major way to improve rice yields in saline soil.

Diversity of Bacillus-like bacterial community in the rhizospheric and non-rhizospheric soil of halophytes (Salsola stocksii and Atriplex amnicola), and characterization of osmoregulatory genes in halophilic Bacilli.

Salinity is one of the major abiotic stresses; a total of 3% of the world's land mass is affected by salinity. Approximately 6.3 million hectares of land in Pakistan is affected by salinity to varying degrees, and most of the areas are arid to semiarid with low annual precipitation. The aim of the present study is to identify and characterize Bacillus and Bacillus-derived bacterial genera from the rhizospheric and non-rhizospheric soil samples from the Khewra Salt Mine, Pakistan, by using culture-independent and -dependent methods. Seven Bacillus-like bacterial genera, Bacillus, Halobacillus, Virgibacillus, Brevibacillus, Paenibacillus, Tumebacillus, and Lysinibacillus, were detected by using pyrosequencing analysis, whereas only four genera, Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus, were identified by culture-dependent methods. Most of the Bacillus-like isolates identified in this study were moderately halophilic, alkaliphilic, and mesophilic bacteria and were considered a good source of hydrolytic enzymes because of their ability to degrade proteins, carbohydrates, and lipids. Eight Bacillus-like strains from the genera Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus showed positive results for the presence of ectABC gene cluster (ectoine), six strains could synthesize betaine from choline, and six strains tested positive for the synthesis of proline from either glutamate or ornithine by using proline dehydrogenase enzyme.

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.

Ectoine and 5-hydroxyectoine accumulation in the halophile Virgibacillus halodenitrificans PDB-F2 in response to salt stress.

The moderately halophilic bacterium Virgibacillus halodenitrificans PDB-F2 copes with salinity by synthesizing or taking up compatible solutes. The main compatible solutes in this strain were ectoine and hydroxyectoine, as determined by (1)H nuclear magnetic resonance spectroscopy ((1)H-NMR). A high-performance liquid chromatography (HPLC) analysis showed that ectoine was the major solute that was synthesized in response to elevated salinity, while hydroxyectoine was a minor solute. However, the hydroxyectoine/ectoine ratio increased from 0.04 at 3 % NaCl to 0.45 at 15 % NaCl in the late exponential growth phase. A cluster of ectoine biosynthesis genes was identified, including three genes in the order of ectA, ectB, and ectC. The hydroxyectoine biosynthesis gene ectD was not part of the ectABC gene cluster. Reverse transcription-quantitative polymerase chain reactions (RT-qPCR) showed that the expression of the ect genes was salinity dependent. The expression of ectABC reached a maximum at 12 % NaCl, while ectD expression increased up to 15 % NaCl. Ectoine and hydroxyectoine production was growth phase dependent. The hydroxyectoine/ectoine ratio increased from 0.018 in the early exponential phase to 0.11 in the stationary phase at 5 % NaCl. Hydroxyectoine biosynthesis started much later than ectoine biosynthesis after osmotic shock, and the temporal expression of the ect genes differed under these conditions, with the ectABC genes being expressed first, followed by ectD gene. Increased culture salinity triggered ectoine or hydroxyectoine uptake when they were added to the medium. Hydroxyectoine was accumulated preferentially when both ectoine and hydroxyectoine were provided exogenously.

Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli.

Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called "bacterial milking" efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l-threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR, a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8g/g DCW and 0.84gL(-)(1)h(-)(1) respectively. The overall ectoine yield was 0.11g/g of glucose.

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).

Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis.

Advanced high-throughput screening methods for small molecules may have important applications in the metabolic engineering of the biosynthetic pathways of these molecules. Ectoine is an excellent osmoprotectant that has been widely used in cosmetics. In this study, the Escherichia coli regulatory protein AraC was engineered to recognize ectoine as its non-natural effector and to activate transcription upon ectoine binding. As an endogenous reporter of ectoine, the mutated AraC protein was successfully incorporated into high-throughput screening of ectoine hyper-producing strains. The ectoine biosynthetic cluster from Halomonas elongata was cloned into E. coli. By engineering the rate-limiting enzyme L-2,4-diaminobutyric acid (DABA) aminotransferase (EctB), ectoine production and the specific activity of the EctB mutant were increased. Thus, these results demonstrated the effectiveness of engineering regulatory proteins into sensitive and rapid screening tools for small molecules and highlighted the importance and efficacy of directed evolution strategies applied to the engineering of genetic components for yield improvement in the biosynthesis of small molecules.

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-ß-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.