ABSTRACT
Ectoine, so-called tetrahydropyrimidine, is an important osmotic adjustment solute and widely applied in cosmetics and protein protectant. Some attempts have been made to improve the ectoine productivity. However, the strains with both high ectoine production capacity and high glucose conversion were still absent so far. Aim to construct a strain for efficiently producing ectoine, ectoine synthetic gene cluster ectABC from Pseudomonas stutzeri was overexpressed in E. coli BL21 (DE3). The ection production was improved by 382 % (ectoine titer increased from 1.73 g/L to 8.33 g/L) after the rational design of rate-limiting enzyme L-2,4-diaminobutyrate transaminase EctBps (protein engineering) combined with the metabolic engineering that focused on the enrichment and conversion of precursors. The final strain YW20 was applied to overproduce ectoine in fed-batch fermentation and yield 68.9 g/L of ectoine with 0.88 g/L/h of space-time yield and the highest glucose conversion reported [34 % (g/g)]. From the fermentation broth, ectoine was purified with 99.7 % purity and 79.8 % yield. This study successfully provided an engineered strain as well as an efficient method for the industrial bio-synthesis and preparation of ectoine.
Subject(s)
Amino Acids, Diamino , Escherichia coli , Metabolic Engineering , Protein Engineering , Transaminases , Metabolic Engineering/methods , Amino Acids, Diamino/biosynthesis , Amino Acids, Diamino/metabolism , Amino Acids, Diamino/genetics , Transaminases/genetics , Transaminases/metabolism , Protein Engineering/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Pseudomonas stutzeri/genetics , Pseudomonas stutzeri/enzymology , Glucose/metabolism , Multigene FamilyABSTRACT
Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: ⢠Hydroxyectoine production in H. salifodinae correlates with the salinity of medium ⢠Transcriptomic analysis reveals the limiting factors for hydroxyectoine production ⢠The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.
Subject(s)
Amino Acids, Diamino , Fermentation , Halomonas , Metabolic Engineering , Halomonas/genetics , Halomonas/metabolism , Metabolic Engineering/methods , Amino Acids, Diamino/biosynthesis , Amino Acids, Diamino/metabolism , Amino Acids, Diamino/genetics , Citric Acid Cycle/genetics , Gene Expression Profiling , Sodium Chloride/metabolism , Salinity , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Ketoglutaric Acids/metabolismABSTRACT
Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.
Subject(s)
Amino Acids, Diamino , Corynebacterium glutamicum , Escherichia coli , Fermentation , Halomonas , Metabolic Engineering , Amino Acids, Diamino/biosynthesis , Amino Acids, Diamino/metabolism , Amino Acids, Diamino/genetics , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Metabolic Engineering/methods , Halomonas/genetics , Halomonas/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Lysine/metabolism , Lysine/biosynthesis , Promoter Regions, Genetic , Operon/genetics , Aspartate Kinase/genetics , Aspartate Kinase/metabolism , Amino Acid Transport Systems, BasicABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Halomonas , Halomonas/genetics , Amino Acids, Diamino/genetics , Anti-Bacterial Agents , BiopolymersABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Escherichia coli , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering/methods , Amino Acids, Diamino/genetics , Amino Acids, Diamino/metabolism , FermentationABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Escherichia coli , Escherichia coli/genetics , Escherichia coli/metabolism , Amino Acids, Diamino/genetics , Amino Acids, Diamino/metabolism , Fermentation , Biosynthetic Pathways , Metabolic Engineering/methodsABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with an average age-of-onset of â¼60 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.
Subject(s)
Amino Acids, Diamino , Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Animals , Mice , Amyotrophic Lateral Sclerosis/chemically induced , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Neurodegenerative Diseases/complications , Motor Neurons/pathology , Phenotype , Amino Acids, Diamino/toxicity , Amino Acids, Diamino/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, AnimalABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Carbon Dioxide , Hydrogen , Bacteria , Amino Acids, Diamino/chemistry , Amino Acids, Diamino/geneticsABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Bacterial Proteins , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Amino Acids, Diamino/genetics , Amino Acids, Diamino/metabolism , Multigene Family , Membrane Transport Proteins/genetics , Archaea/genetics , Bacteria/geneticsABSTRACT
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.
Subject(s)
Amino Acid Transport Systems, Neutral , Amino Acids, Diamino , Calculi , Cystinuria , Mice , Animals , Cystinuria/genetics , Cystinuria/metabolism , Amino Acid Transport Systems, Basic/genetics , Cystine/metabolism , Mutation , Disease Models, Animal , Amino Acids, Diamino/genetics , Amino Acid Transport Systems, Neutral/geneticsABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Sodium Chloride , Amino Acids, Diamino/genetics , Amino Acids, Diamino/metabolism , RNA, Ribosomal, 16S/genetics , Sodium Chloride/metabolismABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Lathyrus , Abscisic Acid/metabolism , Amino Acids, Diamino/analysis , Amino Acids, Diamino/genetics , Amino Acids, Diamino/metabolism , Gene Expression , Lathyrus/chemistry , Lathyrus/genetics , Lathyrus/metabolismABSTRACT
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.
Subject(s)
Amino Acids, Diamino , Rhodospirillaceae , Seawater , Amino Acids, Diamino/genetics , Escherichia coli/genetics , Genes, Bacterial/genetics , Multigene Family , Phylogeny , RNA, Ribosomal, 16S/genetics , Rhodospirillaceae/classification , Rhodospirillaceae/genetics , Seawater/microbiology , Species SpecificityABSTRACT
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 (98.6â%), 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).
Subject(s)
Streptomyces/isolation & purification , Amino Acids, Diamino/genetics , Bacterial Typing Techniques/methods , Base Composition/genetics , DNA, Bacterial/genetics , Diaminopimelic Acid , Fatty Acids/genetics , Forests , Galactose/genetics , Glucose/genetics , Mannose/genetics , Nucleic Acid Hybridization/genetics , Phospholipids/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Ribose/genetics , Soil , Soil Microbiology , Streptomyces/genetics , Thailand , WetlandsABSTRACT
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.
Subject(s)
Amino Acids/chemistry , Genetic Code , Protein Biosynthesis , Proteins/chemistry , Synthetic Biology , Amino Acids/genetics , Amino Acids, Diamino/chemistry , Amino Acids, Diamino/genetics , Codon/genetics , Escherichia coli/genetics , Protein Processing, Post-Translational , Proteomics , Ribosomes/geneticsABSTRACT
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 100â¯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.
Subject(s)
Antioxidants/metabolism , Brevibacterium/physiology , Oryza/microbiology , Oryza/physiology , Plant Development/physiology , Proton-Translocating ATPases/metabolism , Salt-Tolerant Plants/microbiology , Salt-Tolerant Plants/physiology , Amino Acids, Diamino/genetics , Biomass , Brevibacterium/drug effects , Brevibacterium/enzymology , Carbon-Carbon Lyases , Catalase/metabolism , Genes, Bacterial/genetics , Genotype , Hydrogen Peroxide/metabolism , Lipid Peroxidation , Oryza/drug effects , Oryza/enzymology , Oxidative Stress/physiology , Plant Development/drug effects , Plant Leaves/enzymology , Plant Roots/microbiology , Plant Roots/physiology , Reactive Oxygen Species , Salinity , Sodium Chloride/pharmacology , Soil , Stress, Physiological/drug effects , Stress, Physiological/physiologyABSTRACT
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.
Subject(s)
Atriplex/microbiology , Bacillaceae/classification , Bacillaceae/genetics , Biodiversity , Osmoregulation/genetics , Salsola/microbiology , Salt-Tolerant Plants/microbiology , Amino Acids, Diamino/genetics , Bacillaceae/metabolism , DNA, Bacterial/genetics , Pakistan , Phylogeny , Soil MicrobiologyABSTRACT
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.
Subject(s)
Amino Acids, Diamino/biosynthesis , Escherichia coli/metabolism , Multigene Family/genetics , Osmosis , Pseudomonas stutzeri/metabolism , Amino Acids, Diamino/genetics , Escherichia coli/genetics , Microorganisms, Genetically-Modified/genetics , Microorganisms, Genetically-Modified/metabolism , Promoter Regions, Genetic/genetics , Pseudomonas stutzeri/genetics , SalinityABSTRACT
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.
Subject(s)
Amino Acids, Diamino/metabolism , Osmotic Pressure/physiology , Sodium Chloride/pharmacology , Virgibacillus/metabolism , Amino Acids, Diamino/genetics , Gene Expression Regulation, Bacterial , Salinity , Salt Tolerance/genetics , Salt Tolerance/physiology , Stress, Physiological/physiologyABSTRACT
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.