RESUMO
The eukaryotic green microalga Tetraselmis suecica is commonly used for aquaculture purposes because of its high stress tolerance and ease of culture in a wide spectrum of environments; they are therefore suitable candidates for biotechnology applications. To date, no data are available regarding chloroplast transformation vectors based on specific endogenous promoters and homologous targeting regions. We report on the identification of Tetraselmis suecica genes encoding the ribulose bisphosphate carboxylase/oxygenase large subunit protein, the photosystem II D1 protein and the ATP synthase CF1-beta subunit protein together with their untranslated regions (5'UTR, 3'UTR). The full-length ORFs of the putative genes with their regulatory sequences were obtained. We were also able to identify the downstream 3' end of the large subunit ribosomal RNA gene (23S) along with the 5S RNA end-to-end with the psbA gene on the complementary strand. The intergenic region between these genes appears to be a good target site for the integration of target proteins. Moreover, we identified a back-to-back promoter region among the rbcL and atpB genes. To assess the bidirectionality activities of both promoters, a dual reporter vector was constructed for Tetraselmis suecica transformation containing the cat and TurboGFP genes driven by the 5'rbcL/5'atpB divergent promoter. The vector included the 23S-5S and psbA nucleotide sequences as flanking regions. These flanking regions provided suitable insertion sites within the chloroplast genome for cassette integration via homologous recombination. Simultaneous expression of the chloramphenicol-resistant conferring gene and the gene coding for TurboGFP driven by 5'rbcL/5'atpB showed a potent natural bidirectional promoter as a reliable genetic tool.
Assuntos
Clorofíceas , Cloroplastos , Plasmídeos , Regiões Promotoras Genéticas , Ribulose-Bifosfato Carboxilase/genéticaRESUMO
Haematococcus pluvialis is the richest biological source of astaxanthin under unfavorable growing conditions. Many reports have discussed the optimal astaxanthin extraction methods. Free-astaxanthin could be still hindered by microalgae extracts composition or by prolonged extraction times. In this study we evaluated the effect of enzymolysis and saponification deesterification processes of astaxanthin and its carotenoid precursors under high irradiance and nitrogen deprivation stress time conditions. Results showed that cholesterol esterase facilitated astaxanthin deesterification (975.65⯵g mg-1 DW) while saponification positively affected zeaxanthin (1038.68⯵g mg-1 DW).
RESUMO
Microalgae are an economically important source of biomolecules and metabolites that can be exploited as feed, nutraceuticals and, potentially, as biofuels, among other biotechnological applications. Microalgae biotechnology involves both culture and metabolic pathways manipulation to obtain high-value products, such as omega-3 fatty acids and carotenoids. However, the introduction of genes and/or foreign regulatory sequences has caused public concern about the effect of genetically modified microalgae to achieve greater secondary metabolite accumulations. To placate these worries, we have focused on two cutting-edge concepts, cisgenesis and intragenesis in order to sustainably produce commercially relevant metabolites. This review provides updated background on current and future uses for microalgae molecular farming. We also discuss the development of genetic tools used in terrestrial plants to obtain genetically modified microalgae free of foreign DNA by means of (i) site-specific mutations, (ii) excision of selectable markers, (iii) zinc-finger nuclease and transcription activator-like effectors, and (iv) CRISPR/Cas9 systems. It is currently important to consider scientific debate not only from a technological standpoint but also in terms of conceptual, socioeconomic, ethical, and legal aspects.
Assuntos
Produtos Biológicos/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Metabolismo , Microalgas/genética , Microalgas/metabolismoRESUMO
Carotenoids are a class of isoprenoids synthesized by all photosynthetic organisms as well as by some non-photosynthetic bacteria and fungi with broad applications in food, feed and cosmetics, and also in the nutraceutical and pharmaceutical industries. Microalgae represent an important source of high-value products, which include carotenoids, among others. Carotenoids play key roles in light harvesting and energy transfer during photosynthesis and in the protection of the photosynthetic apparatus against photooxidative damage. Carotenoids are generally divided into carotenes and xanthophyls, but accumulation in microalgae can also be classified as primary (essential for survival) and secondary (by exposure to specific stimuli).In this chapter, we outline the high value carotenoids produced by commercially important microalgae, their production pathways, the improved production rates that can be achieved by genetic engineering as well as their biotechnological applications.
Assuntos
Vias Biossintéticas/genética , Carotenoides/genética , Microalgas/genética , Fotossíntese/genética , Biotecnologia , Carotenoides/biossíntese , Transferência de Energia/genética , Engenharia Genética , Luz , Microalgas/metabolismoRESUMO
The great phylogenetic diversity of microalgae is corresponded by a wide arrange of interesting and useful metabolites. Nonetheless metabolic engineering in microalgae has been limited, since specific transformation tools must be developed for each species for either the nuclear or chloroplast genomes. Microalgae as production platforms for metabolites offer several advantages over plants and other microorganisms, like the ability of GMO containment and reduced costs in culture media, respectively. Currently, microalgae have proved particularly well suited for the commercial production of omega-3 fatty acids and carotenoids. Therefore most metabolic engineering strategies have been developed for these metabolites. Microalgal biofuels have also drawn great attention recently, resulting in efforts for improving the production of hydrogen and photosynthates, particularly triacylglycerides. Metabolic pathways of microalgae have also been manipulated in order to improve photosynthetic growth under specific conditions and for achieving trophic conversion. Although these pathways are not strictly related to secondary metabolites, the synthetic biology approaches could potentially be translated to this field and will also be discussed.
RESUMO
Piscirickettsia salmonis is the bacterium that causes Piscirickettsiosis, a systemic disease of salmonid fish responsible for significant economic losses within the aquaculture industry worldwide. The growth of the bacterium for vaccine formulation has been traditionally accomplished by infecting eukaryotic cell lines, a process that involves high production costs and is time-consuming. Recent research has demonstrated that it is possible to culture pure P. salmonis in a blood containing (cell-free) medium. In the present work we demonstrate the growth of P. salmonis in a liquid medium free from blood and serum components, thus establishing a novel and simplified bacteriological medium. Additionally, the new media reported provides improved growth conditions for P. salmonis, where biomass concentrations of approximately 800 mg cell dry weight L(-1) were obtained, about eight times higher than those reported for the blood containing medium. A 2- level full factorial design was employed to evaluate the significance of the main medium components on cell growth and an optimal temperature range of 23-27°C was determined for the microorganism to grow in the novel liquid media. Therefore, these results represent a breakthrough regarding P. salmonis research in order to optimize pure P. salmonis growth in liquid blood and serum free medium.
Assuntos
Doenças dos Peixes/microbiologia , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/veterinária , Animais , Linhagem Celular , Meios de Cultura Livres de Soro , Técnicas de Cultura , Concentração de Íons de Hidrogênio , Piscirickettsia/patogenicidade , Infecções por Piscirickettsiaceae/microbiologia , Salmonidae/microbiologia , TemperaturaRESUMO
Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE) are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Peixes/microbiologia , Piscirickettsia/genética , Piscirickettsia/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Lisossomos/microbiologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Fagossomos/metabolismo , Fagossomos/microbiologia , Filogenia , Piscirickettsia/classificação , Piscirickettsia/fisiologia , Homologia de Sequência do Ácido NucleicoRESUMO
Piscirickettsia salmonis is a bacterial fish pathogen seriously threatening the sustainability of the Chilean salmon industry. The biology and life cycle of this bacterium is not completely understood and there are no reports explaining how it survives or persists in marine environments. This work provides descriptive data of P. salmonis behavior when it is exposed to stress conditions, producing large cell aggregates closely resembling typical biofilm structures. In order to track this putative biofilm, we used indirect fluorescence and scanning electron microscopy. Complex masses were observed over time; the bacteria appear to be embedded within a matrix which disappears when it is exposed to cellulase, suggesting a polysaccharide nature typical of biofilm formation. Two lectins (ConA and WGA) were used to characterize the matrix. Both lectins showed a strong reaction with the structure, validating the exopolysaccharide nature of the matrix. Recently, several studies have demonstrated a correlation between toxin/anti-toxin system expression at initial stages of biofilm formation. In this report, QRT-PCR analysis was used with the P. salmonis toxin/anti-toxin mazEF operon, showing induction of these genes at early stages of biofilm formation, suggesting that said formation may be an adaptive strategy for survival and persistence under stress conditions in marine environments.
Assuntos
Biofilmes/crescimento & desenvolvimento , Piscirickettsia/fisiologia , Estresse Fisiológico , Toxinas Bacterianas/biossíntese , Celulase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Lectinas/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Dados de Sequência Molecular , Piscirickettsia/crescimento & desenvolvimento , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
This is the first report of a functional toxin-antitoxin (TA) locus in Piscirickettsia salmonis. The P. salmonis TA operon (ps-Tox-Antox) is an autonomous genetic unit containing two genes, a regulatory promoter site and an overlapping putative operator region. The ORFs consist of a toxic ps-Tox gene (P. salmonis toxin) and its upstream partner ps-Antox (P. salmonis antitoxin). The regulatory promoter site contains two inverted repeat motifs between the -10 and -35 regions, which may represent an overlapping operator site, known to mediate transcriptional auto-repression in most TA complexes. The Ps-Tox protein contains a PIN domain, normally found in prokaryote TA operons, especially those of the VapBC and ChpK families. The expression in Escherichia coli of the ps-Tox gene results in growth inhibition of the bacterial host confirming its toxicity, which is neutralized by coexpression of the ps-Antox gene. Additionally, ps-Tox is an endoribonuclease whose activity is inhibited by the antitoxin. The bioinformatic modelling of the two putative novel proteins from P. salmonis matches with their predicted functional activity and confirms that the active site of the Ps-Tox PIN domain is conserved.
Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Genoma Bacteriano , Piscirickettsia/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Fases de Leitura Aberta , Óperon , Piscirickettsia/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
Piscirickettsia salmonis is a novel, aggressive, facultative Gram-negative bacterium that drastically affects salmon production at different latitudes, with particular impact in southern Chile. Initially, P. salmonis was described as a Rickettsia-like, obligate, intracellular Alphaproteobacteria, but it was reclassified recently as a facultative intracellular Gammaproteobacteria. This designation has prompted the independent growth of the bacterium to a pure state for detailed study of its biology, genetics and epidemiology, properties that are still relatively poorly characterized. The preliminary sequence analysis of a 992-bp fragment of pure P. salmonis DNA allowed us to characterize a novel and complete 863-bp insertion sequence in the bacterial genome (named ISPsa2), which has a novel 16/16bp perfectly inverted terminal repeat flanking a 726-bp ORF that encodes a putative transposase (Tnp-Psa). The coding sequence of the enzyme shares similarities to that described in some Bacillus species and particularly to those of the IS6 family. ISPsa2 carries its own promoter with standard -10 and -35 sequences, suggesting an interesting potential for plasticity in this pathogenic bacterium. Additionally, the presence of ISPsa2 was confirmed from three isolates of P. salmonis collected from different epizootics in Chile in 2010.