Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov., isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Central Andes in Chile.

Two novel Micromonospora strains, STR1-7T and STR1S-6T, were isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Atacama Desert, Chile. Chemotaxonomic, cultural and phenotypic features confirmed that the isolates belonged to the genus Micromonospora. They grew from 20 to 37 °C, from pH7 to 8 and in the presence of up to 3 %, w/v NaCl. The isolates formed distinct branches in Micromonospora gene trees based on 16S rRNA gene sequences and on a multi-locus sequence analysis of conserved house-keeping genes. A phylogenomic tree generated from the draft genomes of the isolates and their closest phylogenetic neighbours showed that isolate STR1-7T is most closely related to Micromonospora orduensis S2509T, and isolate STR1S-6 T forms a distinct branch that is most closely related to 12 validly named Micromonospora species, including Micromonospora saelicesensis the earliest proposed member of the group. The isolates were separated from one another and from their closest phylogenomic neighbours using a combination of chemotaxonomic, genomic and phenotypic features, and by low average nucleotide index and digital DNA-DNA hybridization values. Consequently, it is proposed that isolates STR1-7T and STR1S-6T be recognized as representing new species in the genus Micromonospora, namely as Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov.; the type strains are STR1-7T (=CECT 9665T=LMG 30768T) and STR1S-6T (=CECT 9666T=LMG 30770T), respectively. Genome mining showed that the isolates have the capacity to produce novel specialized metabolites, notably antibiotics and compounds that promote plant growth, as well as a broad-range of stress-related genes that provide an insight into how they cope with harsh abiotic conditions that prevail in high-altitude Atacama Desert soils.

Downsizing Class II Lasso Peptides: Genome Mining-Guided Isolation of Huascopeptin Containing the First Gly1-Asp7 Macrocycle.

A new lasso peptide, huascopeptin, was isolated following genome-mined discovery of a new biosynthetic gene cluster in extremotolerant Streptomyces huasconensis HST28T from Salar de Huasco, Atacama Desert, Chile. Compound 1 is a 13-residue class II lasso peptide containing a novel Gly1-Asp7 macrolactam ring, a three-residue loop, and a three-residue tail, making it the smallest lasso peptide isolated to date. The lasso structure was confirmed using NOE restraint-based molecular dynamics simulations.

Atacama Database: a platform of the microbiome of the Atacama Desert.

The Atacama Desert is one of the oldest and driest places on Earth. In the last decade, microbial richness and diversity has been acknowledged as an important biological resource of this region. Owing to the value of the microbial diversity apparent in potential biotechnology applications and conservation purposes, it is necessary to catalogue these microbial communities to promote research activities and help to preserve the wide range of ecological niches of the Atacama region. A prototype Atacama Database has been designed and it provides a description of the rich microbial diversity of the Atacama Desert, and helps to visualise available literature resources. Data has been collected, curated, and organised into several categories to generate a single record for each organism in the database that covers classification, isolation metadata, morphology, physiology, genome and metabolism information. The current version of Atacama Database contains 2302 microorganisms and includes cultured and uncultured organisms retrieved from different environments within the desert between 1984 and 2016. These organisms are distributed in bacterial, archaeal or eukaryotic domains, along with those that are unclassified taxonomically. The initial prototype of the Atacama Database includes a basic search and taxonomic and advanced search tools to allow identification and comparison of microbial populations, and space distribution within this biome. A geolocation search was implemented to visualise the microbial diversity of the ecological niches defined by sectors and extract general information of the sampling sites. This effort will aid understanding of the microbial ecology of the desert, microbial population dynamics, seasonal behaviour, impact of climate change over time, and reveal further biotechnological applications of these microorganisms. The Atacama Database is freely available at: https://www.atacamadb.cl.

Micromonospora acroterricola sp. nov., a novel actinobacterium isolated from a high altitude Atacama Desert soil.

A Micromonospora strain, designated 5R2A7T, isolated from a high altitude Atacama Desert soil was examined by using a polyphasic approach. Strain 5R2A7T was found to have morphological, chemotaxonomic and cultural characteristics typical of members of the genus Micromonospora. The cell wall contains meso- and hydroxy-diaminopimelic acid, the major whole-cell sugars are glucose, ribose and xylose, the predominant menaquinones MK-10(H4), MK-10(H6), MK-10(H8) and MK-9(H6), the major polar lipids diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown glycolipid, and the predominant cellular fatty acids iso-C16 : 0, iso-C15 : 0 and 10-methyl C17 : 0. The digital genomic DNA G+C content is 72.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain 5R2A7T was closely related to Micromonospora coriariae DSM 44875T (99.8 %) and Micromonospora cremea CR30T (99.7 %), and was separated readily from the latter, its closest phylogenetic neighbour, based on gyrB and multilocus sequence data, by low average nucleotide identity (92.59 %) and in silico DNA-DNA relatedness (51.7 %) values calculated from draft genome assemblies and by a range of chemotaxonomic and phenotypic properties. Consequently, strain 5R2A7T is considered to represent a novel species of Micromonospora for which the name Micromonospora acroterricola sp. nov. is proposed. The type strain is 5R2A7T (=LMG 30755T=CECT 9656T).

Use of genome-scale models to get new insights into the marine actinomycete genus Salinispora.

BACKGROUND: There is little published regarding metabolism of Salinispora species. In continuation with efforts performed towards this goal, this study is focused on new insights into the metabolism of the three-identified species of Salinispora using constraints-based modeling. At present, only one manually curated genome-scale metabolic model (GSM) for Salinispora tropica strain CNB-440T has been built despite the role of Salinispora strains in drug discovery. RESULTS: Here, we updated, and expanded the scope of the model of Salinispora tropica CNB-440T, and GSMs were constructed for two sequenced type strains covering the three-identified species. We also constructed a Salinispora core model that contains the genes shared by 93 sequenced strains and a few non-conserved genes associated with essential reactions. The models predicted no auxotrophies for essential amino acids, which was corroborated experimentally using a defined minimal medium (DMM). Experimental observations suggest possible sulfur accumulation. The Core metabolic content shows that the biosynthesis of specialised metabolites is the less conserved subsystem. Sets of reactions were analyzed to explore the differences between the reconstructions. Unique reactions associated to each GSM were mainly due to genome sequence data except for the ST-CNB440 reconstruction. In this case, additional reactions were added from experimental evidence. This reveals that by reaction content the ST-CNB440 model is different from the other species models. The differences identified in reaction content between models gave rise to different functional predictions of essential nutrient usage by each species in DMM. Furthermore, models were used to evaluate in silico single gene knockouts under DMM and complex medium. Cluster analysis of these results shows that ST-CNB440, and SP-CNR114 models are more similar when considering predicted essential genes. CONCLUSIONS: Models were built for each of the three currently identified Salinispora species, and a core model representing the conserved metabolic capabilities of Salinispora was constructed. Models will allow in silico metabolism studies of Salinispora strains, and help researchers to guide and increase the production of specialised metabolites. Also, models can be used as templates to build GSMs models of closely related organisms with high biotechnology potential.

Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the Antarctic bacteria Pseudoalteromonas sp. 2-3.

α-Amylase is an endo-acting enzyme which catalyzes random hydrolysis of starch. These enzymes are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. The use of active enzymes at low temperatures has a high potential because these enzymes would avoid the demand for heating during the process thereby reducing costs. In this work, the gene of α-amylase from Pseudoalteromonas sp. 2-3 (Antarctic bacteria) has been sequenced and expressed in Escherichia coli BL21(DE3). The ORF of the α-amylase gene cloned into pET22b(+) is 1824 bp long and codes for a protein of 607 amino acid residues including a His6-tag. The mature protein has a calculated molecular mass of 68.8 kDa. Recombinant α-amylase was purified with Ni-NTA affinity chromatography. The purified enzyme is active on potato starch with a Km of 6.94 mg/ml and Vmax of 0.27 mg/ml*min. The pH optimum is 8.0 and the optimal temperature is 20 °C. This enzyme was strongly activated by Ca2+; results consistent with other α-amylases. To the best of our knowledge, this enzyme has the lowest temperature optimum so far reported for α-amylases.

Geodermatophilus chilensis sp. nov., from soil of the Yungay core-region of the Atacama Desert, Chile.

A polyphasic study was undertaken to establish the taxonomic status of three representative Geodermatophilus strains isolated from an extreme hyper-arid Atacama Desert soil. The strains, isolates B12T, B20 and B25, were found to have chemotaxonomic and morphological properties characteristic of the genus Geodermatophilus. The isolates shared a broad range of chemotaxonomic, cultural and physiological features, formed a well-supported branch in the Geodermatophilus 16S rRNA gene tree in which they were most closely associated with the type strain of Geodermatophilus obscurus. They were distinguished from the latter by BOX-PCR fingerprint patterns and by chemotaxonomic and other phenotypic properties. Average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between the whole genome sequences of isolate B12T and G. obscurus DSM 43160T were 89.28%, 87.27% and 37.4%, respectively, metrics consistent with its classification as a separate species. On the basis of these data, it is proposed that the isolates be assigned to the genus Geodermatophilus as Geodermatophilus chilensis sp. nov. with isolate B12T (CECT 9483T=NCIMB 15089T) as the type strain. Analysis of the whole genome sequence of G. chilensis B12T with 5341 open reading frames and a genome size of 5.5Mb highlighted genes and gene clusters that encode for properties relevant to its adaptation to extreme environmental conditions prevalent in extreme hyper-arid Atacama Desert soils.

Introducing the Atacama Desert.

This brief introduction is intended to orientate the reader with respect to the principal environmental and historical features of the Atacama Desert, the oldest and continuously driest non-polar temperate desert on Earth. Exploration of its microbiology is relatively recent but both fundamental and applied research activities have grown dramatically in recent years reflecting the substantial interest in its microbial diversity, ecology, biogeochemistry, natural product potential and Mars-analogue properties of this unique and invigorating environment.

Relevance of Local Flexibility Near the Active Site for Enzymatic Catalysis: Biochemical Characterization and Engineering of Cellulase Cel5A From Bacillus agaradherans.

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations.

Amycolatopsis vastitatis sp. nov., an isolate from a high altitude subsurface soil on Cerro Chajnantor, northern Chile.

The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H4) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5T (= NCIMB 14970T = NRRL B-65279T).

Streptomyces asenjonii sp. nov., isolated from hyper-arid Atacama Desert soils and emended description of Streptomyces viridosporus Pridham et al. 1958.

A polyphasic study was undertaken to establish the taxonomic status of Streptomyces strains isolated from hyper-arid Atacama Desert soils. Analysis of the 16S rRNA gene sequences of the isolates showed that they formed a well-defined lineage that was loosely associated with the type strains of several Streptomyces species. Multi-locus sequence analysis based on five housekeeping gene alleles showed that the strains form a homogeneous taxon that is closely related to the type strains of Streptomyces ghanaensis and Streptomyces viridosporus. Representative isolates were shown to have chemotaxonomic and morphological properties consistent with their classification in the genus Streptomyces. The isolates have many phenotypic features in common, some of which distinguish them from S. ghanaensis NRRL B-12104T, their near phylogenetic neighbour. On the basis of these genotypic and phenotypic data it is proposed that the isolates be recognised as a new species within the genus Streptomyces, named Streptomyces asenjonii sp. nov. The type strain of the species is KNN35.1bT (NCIMB 15082T = NRRL B-65050T). Some of the isolates, including the type strain, showed antibacterial activity in standard plug assays. In addition, MLSA, average nucleotide identity and phenotypic data show that the type strains of S. ghanaensis and S. viridosporus belong to the same species. Consequently, it is proposed that the former be recognised as a heterotypic synonym of the latter and an emended description is given for S. viridosporus.

Acidithiobacillus ferrooxidans's comprehensive model driven analysis of the electron transfer metabolism and synthetic strain design for biomining applications.

Acidithiobacillus ferrooxidans is a gram-negative chemolithoautotrophic γ-proteobacterium. It typically grows at an external pH of 2 using the oxidation of ferrous ions by oxygen, producing ferric ions and water, while fixing carbon dioxide from the environment. A. ferrooxidans is of great interest for biomining and environmental applications, as it can process mineral ores and alleviate the negative environmental consequences derived from the mining processes. In this study, the first genome-scale metabolic reconstruction of A. ferrooxidans ATCC 23270 was generated (iMC507). A total of 587 metabolic and transport/exchange reactions, 507 genes and 573 metabolites organized in over 42 subsystems were incorporated into the model. Based on a new genetic algorithm approach, that integrates flux balance analysis, chemiosmotic theory, and physiological data, the proton translocation stoichiometry for a number of enzymes and maintenance parameters under aerobic chemolithoautotrophic conditions using three different electron donors were estimated. Furthermore, a detailed electron transfer and carbon flux distributions during chemolithoautotrophic growth using ferrous ion, tetrathionate and thiosulfate were determined and reported. Finally, 134 growth-coupled designs were calculated that enables Extracellular Polysaccharide production. iMC507 serves as a knowledgebase for summarizing and categorizing the information currently available for A. ferrooxidans and enables the understanding and engineering of Acidithiobacillus and similar species from a comprehensive model-driven perspective for biomining applications.

Chaxapeptin, a Lasso Peptide from Extremotolerant Streptomyces leeuwenhoekii Strain C58 from the Hyperarid Atacama Desert.

Lasso peptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that possess a unique "lariat knot" structural motif. Genome mining-targeted discovery of new natural products from microbes obtained from extreme environments has led to the identification of a gene cluster directing the biosynthesis of a new lasso peptide, designated as chaxapeptin 1, in the genome of Streptomyces leeuwenhoekii strain C58 isolated from the Atacama Desert. Subsequently, 1 was isolated and characterized using high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance methods. The lasso nature of 1 was confirmed by calculating its nuclear Overhauser effect restraint-based solution structure. Chaxapeptin 1 displayed a significant inhibitory activity in a cell invasion assay with human lung cancer cell line A549.

Production of cell-penetrating peptides in Escherichia coli using an intein-mediated system.

Cell-penetrating peptides are molecules with the ability to cross membranes and enter cells. Attention has been put on these peptides as a tool for drug delivery research, as they are able to serve as delivery vectors for large molecules. Intracellular delivery of bioactive peptides is a very promising research area for clinical applications, since peptides are able to simulate protein regions and thus modulate key intracellular protein-protein interactions. Therefore, evaluation of different strategies for production of these peptides is necessary. In this work, an intein-mediated system was used to evaluate Escherichia coli recombinant production of p53pAnt and PNC27 anticancer cell-penetrating peptides. It was demonstrated that the pTXB1 and the pTYB11 vector systems are suitable for production of this kind of peptides. The production process involves a low-temperature induction process and an efficient on-column intein-mediated cleavage, which allowed an effective peptide recovery using a single chromatographic step.

Generation of an atlas for commodity chemical production in Escherichia coli and a novel pathway prediction algorithm, GEM-Path.

The production of 75% of the current drug molecules and 35% of all chemicals could be achieved through bioprocessing (Arundel and Sawaya, 2009). To accelerate the transition from a petroleum-based chemical industry to a sustainable bio-based industry, systems metabolic engineering has emerged to computationally design metabolic pathways for chemical production. Although algorithms able to provide specific metabolic interventions and heterologous production pathways are available, a systematic analysis for all possible production routes to commodity chemicals in Escherichia coli is lacking. Furthermore, a pathway prediction algorithm that combines direct integration of genome-scale models at each step of the search to reduce the search space does not exist. Previous work (Feist et al., 2010) performed a model-driven evaluation of the growth-coupled production potential for E. coli to produce multiple native compounds from different feedstocks. In this study, we extended this analysis for non-native compounds by using an integrated approach through heterologous pathway integration and growth-coupled metabolite production design. In addition to integration with genome-scale model integration, the GEM-Path algorithm developed in this work also contains a novel approach to address reaction promiscuity. In total, 245 unique synthetic pathways for 20 large volume compounds were predicted. Host metabolism with these synthetic pathways was then analyzed for feasible growth-coupled production and designs could be identified for 1271 of the 6615 conditions evaluated. This study characterizes the potential for E. coli to produce commodity chemicals, and outlines a generic strain design workflow to design production strains.

Design and implementation of a high yield production system for recombinant expression of peptides.

BACKGROUND: Making peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. A non common but interesting research area is related to peptides with intracellular targets, which opens up new possibilities, allowing the modulation of processes occurring within the cell or interference with signaling pathways. However, if the bioactive sequence requires fusion to a carrier peptide to allow access into the cell, the resulting peptide could be such a length that traditional production could be difficult. The goal of the present study was the development of a flexible recombinant expression and purification system for peptides, as a contribution to the discovery and development of these potentially new drugs. RESULTS: In this work, a high throughput recombinant expression and purification system for production of cell penetrating peptides in Escherichia coli has been designed and implemented. The system designed produces target peptides in an insoluble form by fusion to a hexahistidine tagged ketosteroid isomerase which is then separated by a highly efficient thrombin cleavage reaction procedure. The expression system was tested on the anticancer peptides p53pAnt and PNC27. These peptides comprise the C-terminal region and the N-terminal region of the protein p53, respectively, fused by its carboxyl terminal extreme to the cell penetrating peptide Penetratin. High yields of purified recombinant fused peptides were obtained in both cases; nevertheless, thrombin cleavage reaction was successful only for p53pAnt peptide release. The features of the system, together with the procedure developed, allow achievement of high production yields of over 30 mg of highly pure p53pAnt peptide per g of dry cell mass. It is proposed that the system could be used for production of other peptides at a similar yield. CONCLUSIONS: This study provides a system suitable for recombinant production of peptides for scientific research, including biological assays.

Cold adaptation, ca2+ dependency and autolytic stability are related features in a highly active cold-adapted trypsin resistant to autoproteolysis engineered for biotechnological applications.

Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca(2+) dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10(°) C and at least 50% more active than pig trypsin up to 50(°) C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca(2+) concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca(2+) dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.

Streptomyces bullii sp. nov., isolated from a hyper-arid Atacama Desert soil.

A Streptomyces strain isolated from a hyper-arid Atacama Desert soil was characterised using a polyphasic taxonomic approach. The strain, designated C2(T), had chemical and morphological properties typical of the genus Streptomyces. The isolate formed a branch in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces chromofuscus and was also loosely related to Streptomyces fragilis NRRL 2424(T). DNA:DNA relatedness values between the isolate and its two phylogenetic neighbours showed that it formed a distinct genomic species. The strain was readily distinguished from these organisms using a combination of morphological and phenotypic data. Based on the genotypic and phenotypic results, isolate C2(T) represents a novel species in the genus Streptomyces, for which the name Streptomyces bullii sp. nov. is proposed. The type strain is C2(T) (=CGMCC 4.7019(T) = KACC 15426(T)).

Elution relationships to model affinity chromatography using a general rate model.

Different mathematical models with different degrees of complexity have been proposed to model affinity chromatography. In this work, in particular, a general rate model has been studied that considers axial dispersion, external film mass transfer, intraparticle diffusion, and kinetic effects investigating the influence in the simulations of two different relationships between the properties of the mobile phase and the affinity of different proteins to the ligand bound to the matrix. Two systems were used: Blue Sepharose and Protein A. With Blue Sepharose, an increasing linear salt gradient was used, and with Protein A, a decreasing semi-linear pH gradient. The kinetic parameters obtained in each of the two elution (adsorption/desorption) relationships studied (a power law type and an exponential type) led to very good agreements between experimental and simulated elution curves of mixtures of proteins finding that for more symmetrical peaks, the preferred elution relationship should be the exponential one, in contrast to the more asymmetrical peaks which shapes are better simulated by the power law relationship.