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1.
Biotechnol Bioeng ; 121(6): 1986-2001, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38500406

RESUMEN

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.


Asunto(s)
Modelos Biológicos , Estramenopilos , Estramenopilos/metabolismo , Estramenopilos/genética , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Redes y Vías Metabólicas/genética , Aminoácidos/metabolismo , Biomasa , Metabolismo de los Lípidos , Nutrientes/metabolismo , Adenosina Trifosfato/metabolismo
2.
Int J Syst Evol Microbiol ; 73(12)2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38059605

RESUMEN

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.


Asunto(s)
Fabaceae , Micromonospora , Técnicas de Tipificación Bacteriana , Ácidos Grasos/química , Análisis de Secuencia de ADN , Chile , Filogenia , Rizosfera , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Composición de Base
3.
Mar Drugs ; 21(11)2023 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-37999410

RESUMEN

Coenzyme Q (CoQ; ubiquinone) is an essential component of the respiratory chain. It is also a potent antioxidant that prevents oxidative damage to DNA, biological membranes, and lipoproteins. CoQ comprises a six-carbon ring with polar substituents that interact with electron acceptors and donors, and a hydrophobic polyisoprenoid chain that allows for its localization in cellular membranes. Human CoQ has 10 isoprenoid units (CoQ10) within the polyisoprenoid chain. Few microorganisms produce CoQ10. This work shows that Thraustochytrium sp. RT2316-16 produces CoQ10 and CoQ9. The CoQ10 content in RT2316-16 depended strongly on the composition of the growth medium and the age of the culture, whereas the CoQ9 content was less variable probably because it served a different function in the cell. Adding p-hydroxybenzoic acid to the culture media positively influenced the CoQ10 content of the cell. The absence of some B vitamins and p-aminobenzoic acid in the culture medium negatively affected the growth of RT2316-16, but reduced the decline in CoQ10 that otherwise occurred during growth. The highest content of CoQ9 and CoQ10 in the biomass were 855 µg g-1 and 10 mg g-1, respectively. The results presented here suggest that the thraustochytrid RT2316-16 can be a potential vehicle for producing CoQ10. Metabolic signals that trigger the synthesis of CoQ10 in RT2316-16 need to be determined for optimizing culture conditions.


Asunto(s)
Antioxidantes , Ubiquinona , Humanos , Antioxidantes/metabolismo , Membranas Mitocondriales/metabolismo , Estrés Oxidativo , Membrana Celular/metabolismo
4.
J Transl Med ; 20(1): 373, 2022 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-35982500

RESUMEN

BACKGROUND: Recently, extensive cancer genomic studies have revealed mutational and clinical data of large cohorts of cancer patients. For example, the Pan-Lung Cancer 2016 dataset (part of The Cancer Genome Atlas project), summarises the mutational and clinical profiles of different subtypes of Lung Cancer (LC). Mutational and clinical signatures have been used independently for tumour typification and prediction of metastasis in LC patients. Is it then possible to achieve better typifications and predictions when combining both data streams? METHODS: In a cohort of 1144 Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LSCC) patients, we studied the number of missense mutations (hereafter, the Total Mutational Load TML) and distribution of clinical variables, for different classes of patients. Using the TML and different sets of clinical variables (tumour stage, age, sex, smoking status, and packs of cigarettes smoked per year), we built Random Forest classification models that calculate the likelihood of developing metastasis. RESULTS: We found that LC patients different in age, smoking status, and tumour type had significantly different mean TMLs. Although TML was an informative feature, its effect was secondary to the "tumour stage" feature. However, its contribution to the classification is not redundant with the latter; models trained using both TML and tumour stage performed better than models trained using only one of these variables. We found that models trained in the entire dataset (i.e., without using dimensionality reduction techniques) and without resampling achieved the highest performance, with an F1 score of 0.64 (95%CrI [0.62, 0.66]). CONCLUSIONS: Clinical variables and TML should be considered together when assessing the likelihood of LC patients progressing to metastatic states, as the information these encode is not redundant. Altogether, we provide new evidence of the need for comprehensive diagnostic tools for metastasis.


Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación/genética
5.
Mar Drugs ; 20(7)2022 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-35877709

RESUMEN

The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.


Asunto(s)
Técnicas de Cultivo Celular por Lotes , Estramenopilos , Ácido Ascórbico , Biomasa , Cantaxantina , Carotenoides , Glucosa , Glicerol , Fosfolípidos , beta Caroteno
6.
Mar Drugs ; 21(1)2022 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-36662188

RESUMEN

The psychrophilic marine microorganism Thraustochytrium sp. RT2316-16 can produce carotenoids as well as lipids containing the omega-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid and docosahexaenoic acid. This work reports on the effects of the composition of the culture medium, including certain amino acids, on growth and lipid synthesis by RT2316-16. Compared with the culture on glutamate, the use of lysine, alanine, or serine, increased the content of the omega-3 PUFA in total lipids. In the media that contained yeast extract, glutamate, and glucose, lipid accumulation occurred when organic ammonium was exhausted earlier than glucose. In contrast, lipid mobilization was promoted if glucose was exhausted while organic ammonium (supplied by yeast extract and glutamate) remained in the medium. The total content of carotenoids in the lipid-free biomass decreased during the first 12 to 24 h of culture, simultaneously with a decrease in the total lipid content of the biomass. The experimental data suggested a possible interrelationship between the metabolism of carotenoids and lipids. A high content of omega-3 PUFA in the total lipids could be obtained by growing the thraustochytrid in a medium with a low glucose concentration (6 g L-1) and a high concentration of organic nitrogen (yeast extract 12 g L-1; glutamate 1.06 g L-1), after glucose was exhausted. These observations may guide the development of a strategy to enhance omega-3 PUFA in the biomass.


Asunto(s)
Ácidos Grasos Omega-3 , Estramenopilos , Nitrógeno/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Estramenopilos/metabolismo , Carotenoides/metabolismo , Glucosa/metabolismo , Glutamatos/metabolismo , Ácidos Grasos/metabolismo
7.
Molecules ; 27(21)2022 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-36364011

RESUMEN

Bacteria belonging to the phylum Actinobacteria are a very good source of antibiotics, and indeed dominate the current clinical antibiotic space. This paper reports Mutactimycin AP, a new compound belonging to an anthracycline-type family of antibiotics, isolated from a Saccharothrix sp. This actinobacterial strain was isolated from the rhizosphere of lupine plants growing in the extreme hyper-arid Atacama Desert. Structural characterization was carried out using electrospray ionization-mass spectrometry (ESI-MS) and NMR spectroscopy in combination with molecular modelling. The compound was tested against the ESKAPE pathogens, where it showed activity against MRSA and five strains associated with bovine mastitis, where it showed activity against Enterococcus pseudoavium and Staphylycoccus Aureus subsp. Aureus.


Asunto(s)
Actinobacteria , Actinomycetales , Bovinos , Animales , Femenino , Actinobacteria/química , Microbiología del Suelo , Bacterias , Antibacterianos/farmacología , Clima Desértico
8.
Annu Rev Microbiol ; 70: 215-34, 2016 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-27607552

RESUMEN

The Atacama Desert of northern Chile is the oldest and most arid nonpolar environment on Earth. It is a coastal desert covering approximately 180,000 km(2), and together with the greater Atacama region it comprises a dramatically wide range of ecological niches. Long known and exploited for its mineral resources, the Atacama Desert harbors a rich microbial diversity that has only recently been discovered; the great majority of it has not yet been recovered in culture or even taxonomically identified. This review traces the progress of microbiology research in the Atacama and dispels the popular view that this region is virtually devoid of life. We examine reasons for such research activity and demonstrate that microbial life is the latest recognized and least explored resource in this inspiring biome.


Asunto(s)
Bacterias/aislamiento & purificación , Biodiversidad , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Chile , Clima Desértico , Ecosistema , Ambiente
9.
Mar Drugs ; 19(7)2021 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-34356811

RESUMEN

Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and carotenoids are needed as human dietary supplements and are essential components in commercial feeds for the production of aquacultured seafood. Microorganisms such as thraustochytrids are potential natural sources of these compounds. This research reports on the lipid and carotenoid production capacity of thraustochytrids that were isolated from coastal waters of Antarctica. Of the 22 isolates, 21 produced lipids containing EPA+DHA, and the amount of these fatty acids exceeded 20% of the total fatty acids in 12 isolates. Ten isolates were shown to produce carotenoids (27.4-63.9 µg/g dry biomass). The isolate RT2316-16, identified as Thraustochytrium sp., was the best producer of biomass (7.2 g/L in five days) rich in carotenoids (63.9 µg/g) and, therefore, became the focus of this investigation. The main carotenoids in RT2316-16 were ß-carotene and canthaxanthin. The content of EPA+DHA in the total lipids (34 ± 3% w/w in dry biomass) depended on the stage of growth of RT2316-16. Lipid and carotenoid content of the biomass and its concentration could be enhanced by modifying the composition of the culture medium. The estimated genome size of RT2316-16 was 44 Mb. Of the 5656 genes predicted from the genome, 4559 were annotated. These included genes of most of the enzymes in the elongation and desaturation pathway of synthesis of ω-3 polyunsaturated fatty acids. Carotenoid precursors in RT2316-16 were synthesized through the mevalonate pathway. A ß-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.


Asunto(s)
Carotenoides/química , Ácidos Grasos Omega-3/química , Estramenopilos , Animales , Regiones Antárticas , Organismos Acuáticos
10.
Biotechnol Bioeng ; 117(10): 3006-3017, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32557613

RESUMEN

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.


Asunto(s)
Organismos Acuáticos/metabolismo , Biotecnología/métodos , Medios de Cultivo/química , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Fermentación , Estramenopilos/metabolismo , Regiones Antárticas , Organismos Acuáticos/crecimiento & desarrollo , Organismos Acuáticos/aislamiento & purificación , Biomasa , Frío , Ácidos Docosahexaenoicos/análisis , Ácido Eicosapentaenoico/análisis , Estramenopilos/crecimiento & desarrollo , Estramenopilos/aislamiento & purificación
11.
J Org Chem ; 85(3): 1661-1667, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-31931565

RESUMEN

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.


Asunto(s)
Péptidos , Streptomyces , Familia de Multigenes , Streptomyces/genética
12.
Antonie Van Leeuwenhoek ; 113(2): 185-195, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31535335

RESUMEN

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.


Asunto(s)
Bases de Datos Factuales , Archaea/genética , Archaea/fisiología , Bacterias/genética , Biotecnología , Clima Desértico , Microbiota/fisiología , Microbiología del Suelo
13.
Mar Drugs ; 18(11)2020 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-33217919

RESUMEN

Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid's response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.


Asunto(s)
Organismos Acuáticos/genética , Eucariontes/genética , Regulación de la Expresión Génica , Metabolismo de los Lípidos/genética , Temperatura , Transcriptoma , Regiones Antárticas , Organismos Acuáticos/crecimiento & desarrollo , Organismos Acuáticos/metabolismo , delta-5 Desaturasa de Ácido Graso , Eucariontes/crecimiento & desarrollo , Eucariontes/metabolismo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/biosíntesis
14.
Plant J ; 96(5): 1076-1088, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30168220

RESUMEN

A metabolic network model of the green microalga Chlamydomonas reinhardtii was used to characterize photoautotrophic and heterotrophic (i.e. growth on stored compounds) growth under light and dark, respectively. The metabolic network comprised 2514 reactions distributed among nine intracellular compartments and the extracellular space. The metabolic network included all the key biochemical pathways for synthesis and metabolism of starch and triacylglycerols (TAGs). Under light and nitrogen limitation, the model simulated the accumulation of the energy-rich compounds (TAGs and starch) in the cell. In the dark, the model could simulate cell growth and maintenance on stored compounds. The model-predicted consumption rates of storage compounds (starch or TAGs) to enable growth in the dark, were found to be greater than the rates of synthesis under light. This implied utilization of the storage compounds for cell maintenance in the dark. Under constant illumination, the simulations of cell growth and intracellular starch content agreed closely with independent experimental data. In other simulations, compared with the case without photorespiration, light uptake rate increased 1.04-fold when the ratio of the rates of oxygenation and carboxylation (Rubisco) was 0.1. Although extensive experimental work exists on culture and physiology of microalgae, it does not allow quantitative predictions of the influence of dark metabolism on the productivity of metabolites to be made. This limitation is overcome using the present model. A metabolic network model of Chlamydomonas reinhardtii is shown to simulate growth and synthesis of energy-rich compounds (triacylglycerols and starch) under light. The same model also simulates dark growth and maintenance through consumption of the stored energy-rich compounds.


Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Respiración de la Célula , Chlamydomonas reinhardtii/crecimiento & desarrollo , Oscuridad , Luz , Redes y Vías Metabólicas , Modelos Biológicos , Fotosíntesis , Almidón/metabolismo , Triglicéridos/metabolismo
15.
Appl Environ Microbiol ; 85(23)2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31562169

RESUMEN

Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.


Asunto(s)
Proteínas Bacterianas/genética , Expresión Génica , Familia de Multigenes , Péptidos/genética , Streptomyces/genética , Proteínas Bacterianas/metabolismo , Péptidos/metabolismo , Streptomyces/metabolismo
16.
Protein Expr Purif ; 155: 78-85, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30496815

RESUMEN

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.


Asunto(s)
Pseudoalteromonas/enzimología , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Regiones Antárticas , Clonación Molecular , Frío , Activación Enzimática , Estabilidad de Enzimas , Filogenia , Pseudoalteromonas/química , Pseudoalteromonas/genética , Pseudoalteromonas/metabolismo , Almidón/metabolismo , alfa-Amilasas/química , alfa-Amilasas/genética
17.
Antonie Van Leeuwenhoek ; 112(12): 1863-1874, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31407134

RESUMEN

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes.


Asunto(s)
Actinobacteria/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Actinobacteria/clasificación , Actinobacteria/genética , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Microbiología del Suelo , América del Sur
18.
Biotechnol Bioeng ; 115(7): 1815-1828, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29578590

RESUMEN

The first genome scale model (GSM) for Streptomyces leeuwenhoekii C34 was developed to study the biosynthesis pathways of specialized metabolites and to find metabolic engineering targets for enhancing their production. The model, iVR1007, consists of 1,722 reactions, 1,463 metabolites, and 1,007 genes, it includes the biosynthesis pathways of chaxamycins, chaxalactins, desferrioxamines, ectoine, and other specialized metabolites. iVR1007 was validated using experimental information of growth on 166 different sources of carbon, nitrogen and phosphorous, showing an 83.7% accuracy. The model was used to predict metabolic engineering targets for enhancing the biosynthesis of chaxamycins and chaxalactins. Gene knockouts, such as sle03600 (L-homoserine O-acetyltransferase), and sle39090 (trehalose-phosphate synthase), that enhance the production of the specialized metabolites by increasing the pool of precursors were identified. Using the algorithm of flux scanning based on enforced objective flux (FSEOF) implemented in python, 35 and 25 over-expression targets for increasing the production of chaxamycin A and chaxalactin A, respectively, that were not directly associated with their biosynthesis routes were identified. Nineteen over-expression targets that were common to the two specialized metabolites studied, like the over-expression of the acetyl carboxylase complex (sle47660 (accA) and any of the following genes: sle44630 (accA_1) or sle39830 (accA_2) or sle27560 (bccA) or sle59710) were identified. The predicted knockouts and over-expression targets will be used to perform metabolic engineering of S. leeuwenhoekii C34 and obtain overproducer strains.


Asunto(s)
Genoma Bacteriano , Redes y Vías Metabólicas/genética , Modelos Biológicos , Streptomyces/genética , Biología de Sistemas/métodos , Antibacterianos/biosíntesis , Ingeniería Metabólica/métodos , Metabolismo Secundario
19.
Int J Syst Evol Microbiol ; 68(9): 2712-2721, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29969090

RESUMEN

A polyphasic study was undertaken to establish the taxonomic status of a Blastococcus strain isolated from an extreme hyper-arid Atacama Desert soil. The isolate, strain P6T, was found to have chemotaxonomic and morphological properties consistent with its classification in the genus Blastococcus. It was shown to form a well-supported branch in the Blastococcus 16S rRNA gene tree together with the type strains of Blastococcus capsensis and Blastococcus saxobsidens and was distinguished from the latter, its close phylogenetic neighbour, by a broad range of phenotypic properties. The draft genome sequence of isolate P6T showed 84.6 % average nucleotide identity, 83.0 % average amino acid identity and a digital DNA-DNA hybridisation value of 27.8 % in comparison with the genome sequence of B. saxobsidens DSM 44509T, values consistent with its assignment to a separate species. Based on these data it is proposed that isolate P6T (NCIMB 15090T=NRRL B-65468T) be assigned to the genus Blastococcus as Blastococcus atacamensis sp. nov. Analysis of the whole genome sequence of B. atacamensis P6T, with 3778 open reading frames and a genome size of 3.9 Mb showed the presence of genes and gene clusters that encode for properties that reflect its adaptation to the extreme environmental conditions that prevail in Atacama Desert soils.


Asunto(s)
Actinomycetales/clasificación , Clima Desértico , Filogenia , Microbiología del Suelo , Actinomycetales/genética , Actinomycetales/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , Chile , ADN Bacteriano/genética , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN
20.
Antonie Van Leeuwenhoek ; 111(8): 1479-1491, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29396707

RESUMEN

Metabolic modelling is a useful tool that enables the rational design of metabolic engineering experiments and the study of the unique capabilities of biotechnologically important microorganisms. The extreme abiotic conditions of the Atacama Desert have selected microbial diversity with exceptional characteristics that can be applied in the mining industry for bioleaching processes and for production of specialised metabolites with antimicrobial, antifungal, antiviral, antitumoral, among other activities. In this review we summarise the scientific data available of the use of metabolic modelling and flux analysis to improve the performance of Atacama Desert microorganisms in biotechnological applications.


Asunto(s)
Bacterias/metabolismo , Biotecnología , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas , Modelos Biológicos , Microbiología del Suelo , Bacterias/clasificación , Chile , Clima Desértico , Genoma Bacteriano/genética , Metabolómica
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