RESUMO
Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.
Assuntos
Organismos Aquáticos , Processos Fototróficos , Bombas de Próton , Rodopsinas Microbianas , Organismos Aquáticos/metabolismo , Organismos Aquáticos/efeitos da radiação , Bactérias/metabolismo , Bactérias/efeitos da radiação , Carotenoides/metabolismo , Cor , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Processos Heterotróficos/efeitos da radiação , Luz , Oceanos e Mares , Processos Fototróficos/efeitos da radiação , Bombas de Próton/metabolismo , Bombas de Próton/efeitos da radiação , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/efeitos da radiação , Zeaxantinas/metabolismo , Zeaxantinas/efeitos da radiação , Luteína/metabolismo , Luteína/efeitos da radiação , Metagenoma , LagosRESUMO
Carotenoids are a large group of health-promoting compounds used in many industrial sectors, such as foods, feeds, pharmaceuticals, cosmetics, nutraceuticals, and colorants. Considering the global population growth and environmental challenges, it is essential to find new sustainable sources of carotenoids beyond those obtained from agriculture. This review focuses on the potential use of marine archaea, bacteria, algae, and yeast as biological factories of carotenoids. A wide variety of carotenoids, including novel ones, were identified in these organisms. The role of carotenoids in marine organisms and their potential health-promoting actions have also been discussed. Marine organisms have a great capacity to synthesize a wide variety of carotenoids, which can be obtained in a renewable manner without depleting natural resources. Thus, it is concluded that they represent a key sustainable source of carotenoids that could help Europe achieve its Green Deal and Recovery Plan. Additionally, the lack of standards, clinical studies, and toxicity analysis reduces the use of marine organisms as sources of traditional and novel carotenoids. Therefore, further research on the processing of marine organisms, the biosynthetic pathways, extraction procedures, and examination of their content is needed to increase carotenoid productivity, document their safety, and decrease costs for their industrial implementation.
Assuntos
Microalgas , Alga Marinha , Carotenoides/farmacologia , Carotenoides/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Microalgas/metabolismo , Archaea , Organismos Aquáticos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/metabolismo , Bactérias , LevedurasRESUMO
Halophilic archaea represent a promising natural source of carotenoids. However, little information is available about these archaeal metabolites and their biological effects. In the present work, carotenoids of strains Haloferax sp. ME16, Halogeometricum sp. ME3 and Haloarcula sp. BT9, isolated from Algerian salt lakes, were produced, extracted and identified by high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Analytical results revealed a variation in the composition depending on the strain with a predominance of bacterioruberin. The evaluation of antioxidant capacity using ABTS [(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays showed that these extracts have a strong antioxidant potential, in particular those of Haloferax sp. ME16 which displayed antioxidant power significantly higher than that of ascorbic acid used as standard. Antibacterial activity of carotenoid extracts against four human-pathogenic strains and four fish-pathogenic strains was evaluated by agar disk diffusion method. The results showed a good antibacterial activity. These findings suggest that the C50 carotenoids from the studied strains offer promising prospects for biotechnological applications.
Assuntos
Carotenoides , Lagos , Animais , Antioxidantes/análise , Antioxidantes/farmacologia , Carotenoides/análise , Cromatografia Líquida de Alta Pressão , Humanos , Extratos VegetaisRESUMO
INTRODUCTION: The only curative treatment for severe aplastic anemia in children is an allogeneic stem cell transplant; however, few patients have a matched related or unrelated donor. Haploidentical stem cell transplantation (haplo-SCT) using bone marrow (BM) and peripheral blood stem cells (PBSC) has been recently described as effective and safe. In this study, we retrospectively report the outcome of twelve pediatric patients who underwent haplo-SCT using only PBSC. METHODS: The conditioning regimen consisted on rabbit anti-thymocyte globulin (r-ATG) 2.5 mg/kg/d on days -7, -6,-5, and -4, and cyclophosphamide (Cy) 50 mg/kg/d on days -3 and -2. We used Cy 50 mg/kg/d on days +3 and +4, tacrolimus and mycophenolic acid as graft versus host disease (GVHD) prophylaxis. RESULTS: The median follow-up was 1,099 days (45-1258 days). The overall survival rate up-to-date is 83.3%. In 10 of the 12 patients, a sustained graft was achieved. None of the patients had acute or chronic GVHD. CONCLUSIONS: Haplo-SCT could be established as a first-line treatment when there is no matched related or unrelated donor. According to this short sample and previous reports, PBSC are a feasible option effectively used as the sole source of stem cells. Additionally, post-transplant cyclophosphamide remains a good strategy for GVHD prevention.
Assuntos
Anemia Aplástica/terapia , Antígenos CD34 , Transplante de Células-Tronco Hematopoéticas , Transplante Haploidêntico , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , México , Estudos Retrospectivos , Condicionamento Pré-TransplanteRESUMO
Cadmium is one of the most common heavy metals in contaminated aquatic environments and one of the most toxic contaminants for phytoplankton. Nevertheless, there are not enough studies focused on the effect of this metal in algae. Through a proteomic approach, this work shows how Cd can alter the growth, cell morphology and metabolism of the microalga Chlorella sorokiniana. Using the sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), we concluded that exposure of Chlorella sorokiniana to 250 µM Cd2+ for 40 h caused downregulation of different metabolic pathways, such as photosynthesis, oxidative phosphorylation, glycolysis, TCA cycle and ribosomal proteins biosynthesis. However, photorespiration, antioxidant enzymes, gluconeogenesis, starch catabolism, and biosynthesis of glutamate, cysteine, glycine and serine were upregulated, under the same conditions. Finally, exposure to Cd also led to changes in the metabolism of carotenoids and lipids. In addition, the high tolerance of Chlorella sorokiniana to Cd points to this microalga as a potential microorganism to be used in bioremediation processes.
Assuntos
Cádmio/toxicidade , Chlorella/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Proteoma/metabolismo , Poluentes Químicos da Água/toxicidade , Antioxidantes/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Carotenoides/metabolismo , Chlorella/metabolismo , Espectrometria de Massas , Metais Pesados/metabolismo , Microalgas/metabolismo , Fotossíntese/efeitos dos fármacos , ProteômicaRESUMO
Antimicrobial peptides (AMPs) form part of the innate immune response, which is of vital importance in fish, especially in eggs and early larval stages. Compared to antibiotics, AMPs show action against a wider spectrum of pathogens, including viruses, fungi and parasites, are more friendly to the environment, and do not seem to generate resistance in bacteria. Thus, we have tested in vitro the potential use of several synthetic peptides as antimicrobial agents in aquaculture: frog Caerin1.1, European sea bass Dicentracin (Dic) and NK-lysin peptides (NKLPs) and sole NKLP27. Our results demonstrate that the highest bactericidal activity against both human and fish pathogens was obtained with Caerin1.1 followed by sea bass Dic and NKLPs, having the sea bass NKLP20.2 none to negligible activity. Interestingly, Aeromonas salmonicida was refractory to all the fish peptides tested. Regarding the antiviral activity, synthetic peptides were able to inhibit the viral infection of nodavirus (NNV), viral septicaemia haemorrhagic virus (VHSV), infectious pancreatic necrosis virus (IPNV) and spring viremia carp virus (SVCV), which are some of the most devastating virus for aquaculture. However, their effectiveness was highly dependent on the type of virus. Strikingly, IPNV resulted the most resistant virus since Caeerin1.1 and sea bass NKLP20.2 were unable to reduce its titre and the other peptides tested only reduced it to values in the 43-78% range. These data demonstrate that synthetic peptides have great antibacterial and antiviral in vitro activity against important fish pathogens and point to their use as potential therapeutic agents in aquaculture.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Proteínas de Peixes/farmacologia , Animais , Anuros , Bass , Linguados , Proteolipídeos/farmacologiaRESUMO
A set of 110 extremely halophilic archaeal strains were isolated from seven distinct saline habitats located in different regions of Algeria. The physicochemical characterization of the samples showed that these habitats were thalassohaline. The carotenoid production from isolated strains varied from 0.1 to 3.68 µg/ml. Based on their physiological characteristics and pigment production, 43 strains were selected and identified by means of phenotypic tests and 16S ribosomal RNA gene sequencing. Phylogenetic analysis indicated that the isolates corresponded to the class Halobacteria and were closely related to genera Halorubrum, Haloarcula, Haloferax, Natrinema, Halogeometricum, Haloterrigena, and Halopiger. Carotenoids of the highest producer, strain Halorubrum sp. BS2 were identified using high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Bacterioruberin and bisanhydrobacterioruberin were the predominant carotenoids. The scavenging activity of these carotenoids reached 99% at a concentration of 18 µg/ml, which was much higher than that of ascorbic acid used as a reference compound. These carotenoids also exhibited significant antibacterial activities against four human-pathogenic strains and four fish-pathogenic strains. Variations in salinity, agitation rate, temperature, and light intensity were found to influence growth and carotenoid production of Halorubrum sp. BS2. Our results suggest that halophilic archaea represent a potential source for carotenoids, which are characterized by high antioxidant and antibacterial activities.
Assuntos
Antibacterianos/metabolismo , Antioxidantes/metabolismo , Carotenoides/metabolismo , Halorubrum/classificação , Halorubrum/metabolismo , Argélia , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Bioprospecção , Carotenoides/farmacologia , DNA Arqueal/genética , Halorubrum/isolamento & purificação , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , SalinidadeRESUMO
Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.
Assuntos
Microalgas/genética , Plasmídeos/genética , Transgenes/genética , Antibacterianos/farmacologia , Marcadores Genéticos/genética , Canamicina Quinase/genética , Paromomicina/farmacologia , Regiões Promotoras Genéticas/genética , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Transformação Genética/genéticaRESUMO
The stereocontrolled synthesis of naturally occurring products containing a 5,5-spiroketal molecular structure represents a major synthetic problem. Moreover, in a previous work, the stereocontrolled synthesis of cephalosporolide E (ceph E), which presumably was obtained from its epimer congener (ceph F) through an acid-mediated equilibration process, was reported. Consequently, we performed a theoretical investigation to provide relevant information regarding the title question, and it was found that the higher thermodynamic stability of ceph E, relative to ceph F, is caused by an n â π* interaction between a lone electron pair of the oxygen atom of the spiroketal ring (nO) and the antibonding orbital of the carbonyl group (π*C=O). Although similar stereoelectronic interactions have been disclosed in other molecular structures, its presence in ceph E, and very likely in other related naturally occurring products, represents a novel nonanomeric stabilizing effect that should be introduced into the chemical literature.
RESUMO
The solar salterns located in the Odiel marshlands, in southwest Spain, are an excellent example of a hypersaline environment inhabited by microbial populations specialized in thriving under conditions of high salinity, which remains poorly explored. Traditional culture-dependent taxonomic studies have usually under-estimated the biodiversity in saline environments due to the difficulties that many of these species have to grow at laboratory conditions. Here we compare two molecular methods to profile the microbial population present in the Odiel saltern hypersaline water ponds (33% salinity). On the one hand, the construction and characterization of two clone PCR amplified-16S rRNA libraries, and on the other, a high throughput 16S rRNA sequencing approach based on the Illumina MiSeq platform. The results reveal that both methods are comparable for the estimation of major genera, although massive sequencing provides more information about the less abundant ones. The obtained data indicate that Salinibacter ruber is the most abundant genus, followed by the archaea genera, Halorubrum and Haloquadratum. However, more than 100 additional species can be detected by Next Generation Sequencing (NGS). In addition, a preliminary study to test the biotechnological applications of this microbial population, based on its ability to produce and excrete haloenzymes, is shown.
Assuntos
Bacteroidetes/genética , Halobacteriales/genética , Salinidade , Microbiologia da Água , Bacteroidetes/isolamento & purificação , Biodiversidade , Biotecnologia/métodos , DNA Bacteriano/isolamento & purificação , Variação Genética , Halobacteriales/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Espanha , Áreas AlagadasRESUMO
Carotenoids are a wide group of lipophylic isoprenoids synthesized by all photosynthetic organisms and also by some non-photosynthetic bacteria and fungi. Animals, which cannot synthesize carotenoids de novo, must include them in their diet to fulfil essential provitamin, antioxidant, or colouring requirements. Carotenoids are indispensable in light harvesting and energy transfer during photosynthesis and in the protection of the photosynthetic apparatus against photooxidative damage. In this review, we outline the factors inducing carotenoid accumulation in microalgae, the knowledge acquired on the metabolic pathways responsible for their biosynthesis, and the recent achievements in the genetic engineering of this pathway. Despite the considerable progress achieved in understanding and engineering algal carotenogenesis, many aspects remain to be elucidated. The increasing number of sequenced microalgal genomes and the data generated by high-throughput technologies will enable a better understanding of carotenoid biosynthesis in microalgae. Moreover, the growing number of industrial microalgal species genetically modified will allow the production of novel strains with enhanced carotenoid contents.
Assuntos
Carotenoides/metabolismo , Microalgas/metabolismo , Microalgas/genéticaRESUMO
Persistent HGF/Met signaling drives tumor growth and dissemination. Proteoglycans within the tumor microenvironment might control HGF availability and signaling by affecting its accessibility to Met (HGF receptor), likely defining whether acute or sustained HGF/Met signaling cues take place. Given that betaglycan (BG, also known as type III TGFß receptor or TGFBR3), a multi-faceted proteoglycan TGFß co-receptor, can be found within the tumor microenvironment, we addressed its hypothetical role in oncogenic HGF signaling. We found that HGF/Met promotes lung cancer and endothelial cells migration via PI3K and mTOR. This effect was enhanced by recombinant soluble betaglycan (solBG) via a mechanism attributable to its glycosaminoglycan chains, as a mutant without them did not modulate HGF effects. Moreover, soluble betaglycan extended the effect of HGF-induced phosphorylation of Met, Akt, and Erk, and membrane recruitment of the RhoGEF P-Rex1. Data-mining analysis of lung cancer patient datasets revealed a significant correlation between high MET receptor, HGF, and PREX1 expression and reduced patient survival. Soluble betaglycan showed biochemical interaction with HGF and, together, they increased tumor growth in immunocompetent mice. In conclusion, the oncogenic properties of the HGF/Met pathway are enhanced and sustained by GAG-containing soluble betaglycan.
RESUMO
Nordic microalgae are a group of photosynthetic organisms acclimated to growth at low temperature and in varying light conditions; the subarctic climate offers bright days with moderate temperatures during summer and cold and dark winter months. The robustness to these natural stress conditions makes the species interesting for large-scale cultivation in harsh environments and for the production of high-value compounds. The aim of this study was to explore the ability of nineteen species of Nordic microalgae to produce different bioactive compounds, such as carotenoids or polyphenols. The results showed that some of these strains are able to produce high amounts of carotenoids (over 12 mg·g-1 dry weight) and phenolic compounds (over 20 mg GAE·g-1 dry weight). Based on these profiles, six species were selected for cultivation under high light and cold stress (500 µmol·m-2·s-1 and 10 ËC). The strains Chlorococcum sp. (MC1) and Scenedesmus sp. (B2-2) exhibited similar values of biomass productivity under standard or stress conditions, but produced higher concentrations of carotenoids (an increase of 40% and 25%, respectively), phenolic compounds (an increase of 40% and 30%, respectively), and showed higher antioxidant capacity (an increase of 15% and 20%, respectively) during stress. The results highlight the ability of these Nordic microalgae as outstanding producers of bioactive compounds, justifying their cultivation at large scale in Nordic environments.
Assuntos
Antioxidantes , Microalgas , Carotenoides , Polifenóis , Fenóis , BiomassaRESUMO
In recent years, there has been an increasing concern related to the contamination of aqueous ecosystems by heavy metals, highlighting the need to improve the current techniques for remediation. This work intends to address the problem of removing heavy metals from waterbodies by combining two complementary methodologies: adsorption to a copolymer synthesized by inverse vulcanization of sulfur and vegetable oils and phytoremediation by the microalga Chlorella sorokiniana to enhance the metal adsorption. After studying the tolerance and growth of Chlorella sorokiniana in the presence of the copolymer, the adsorption of highly concentrated Cd2+ (50 mg L-1) by the copolymer and microalgae on their own and the combined immobilized system (AlgaPol) was compared. Additionally, adsorption studies have been performed on mixtures of the heavy metals Cd2+ and Cu2+ at a concentration of 8 mg L-1 each. AlgaPol biofilm is able to remove these metals from the growth medium by more than 90%. The excellent metal adsorption capacity of this biofilm can be kinetically described by a pseudo-second-order model.
Assuntos
Chlorella , Metais Pesados , Microalgas , Águas Residuárias , Cádmio , Biodegradação Ambiental , Ecossistema , Metais Pesados/análise , AdsorçãoRESUMO
The production of reactive oxygen species (ROS) plays an important role in the progression of many inflammatory diseases. The search for antioxidants with the ability for scavenging free radicals from the body cells that reduce oxidative damage is essential to prevent and treat these pathologies. Haloarchaea are extremely halophilic microorganisms that inhabit hypersaline environments, such as saltworks or salt lakes, where they have to tolerate high salinity, and elevated ultraviolet (UV) and infrared radiations. To cope with these extreme conditions, haloarchaea have developed singular mechanisms to maintain an osmotic balance with the medium, and are endowed with unique compounds, not found in other species, with bioactive properties that have not been fully explored. This study aims to assess the potential of haloarchaea as a new source of natural antioxidant and anti-inflammatory agents. A carotenoid-producing haloarchaea was isolated from Odiel Saltworks (OS) and identified on the basis of its 16S rRNA coding gene sequence as a new strain belonging to the genus Haloarcula. The Haloarcula sp. OS acetone extract (HAE) obtained from the biomass contained bacterioruberin and mainly C18 fatty acids, and showed potent antioxidant capacity using ABTS assay. This study further demonstrates, for the first time, that pretreatment with HAE of lipopolysaccharide (LPS)-stimulated macrophages results in a reduction in ROS production, a decrease in the pro-inflammatory cytokines TNF-α and IL-6 levels, and up-regulation of the factor Nrf2 and its target gene heme oxygenase-1 (HO-1), supporting the potential of the HAE as a therapeutic agent in the treatment of oxidative stress-related inflammatory diseases.
RESUMO
The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from D-mannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid's conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.
Assuntos
Células Eucarióticas , Polímeros , Humanos , Polímeros/química , Manitol , Transfecção , Plasmídeos/genética , DNA/química , Engenharia Genética , Vetores Genéticos/genéticaRESUMO
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene ß-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and ß-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.
Assuntos
Chlorella/genética , Liases Intramoleculares/genética , Microalgas/genética , Nitrogênio/farmacologia , Cantaxantina/biossíntese , Carotenoides/biossíntese , Chlorella/efeitos dos fármacos , Chlorella/enzimologia , Escherichia coli/genética , Luz , Luteína/biossíntese , Microalgas/efeitos dos fármacos , RNA Mensageiro/genética , Estresse Fisiológico/genética , Transcrição Gênica/genética , Xantofilas/biossíntese , Zeaxantinas , beta Caroteno/biossínteseRESUMO
The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.
Assuntos
Chlamydomonas reinhardtii/genética , Canamicina Quinase/genética , Paromomicina/farmacologia , Streptomyces/genética , Antibacterianos/farmacologia , Chlamydomonas reinhardtii/efeitos dos fármacos , DNA Bacteriano/genética , Regulação Enzimológica da Expressão Gênica , Marcadores Genéticos , Microalgas/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , TransgenesRESUMO
Terpenoids are a diverse class of naturally occurring compounds consisting of more than 50,000 structurally different molecules and are found in all living organisms. Many terpenoid compounds, in particular those isolated from plants, have applications in various commercial sectors including medicine, agriculture and cosmetics. However, these high value terpenoids are produced in relatively small quantities in their natural hosts and their chemical synthesis for large scale production is costly and complicated. Therefore, there is much focus on producing these compounds in novel biological hosts using metabolic engineering technologies. As a photosynthetic system, the unicellular green alga C. reinhardtii is of particular interest as the most well-studied model alga with well-established molecular tools for genetic manipulation. However, the direct manipulation of terpenoid biosynthetic pathways in C. reinhardtii necessitates a thorough understanding of the basic terpenoid metabolism. To gain a better understanding of the methylerythritol phosphate (MEP) pathway that leads to terpenoid biosynthesis in the chloroplast of C. reinhardtii, hence this study has investigated the effect of over-expressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) on plastidic downstream terpenoids. We produced marker-free chloroplast transformants of C. reinhardtii lines that express an additional cyanobacterial gene for DXS. The analysis of terpenoid content for the transgenic line demonstrates that overexpressing DXS resulted in a two-fold decrease in the chlorophyll levels while carotenoid levels showed variable changes: zeaxanthin and antherxanthin levels increased several-fold, lutein levels dropped to approximately half, but ß-carotene and violaxanthin did not show a significant change.
RESUMO
Replacement of fossil fuels has to be accompanied by the incorporation of bio-based procedures for the production of fine chemicals. With this aim, the microalga Chlamydomonas reinhardtii was selected for its ability to accumulate starch, an environmentally-friendly alternative source of chemical building blocks, such as 5'-hydroxymethylfurfural or levulinic acid. The content of appreciated lipophilic coproducts was assessed in the selected microalga cultured at different nutritional conditions; and the parameters for the acidic hydrolysis of the algal biomass, obtained after pigments extraction, were optimized using a Central Composite Design. Response Surface Methodology predicted that the optimal hydrolysis conditions were elevated temperature, high DMSO % and short hydrolysis time for glucose. LA was favored at long times and high acid % and 5'-HMF at lower acid % and high DMSO %. Chlamydomonas can therefore be used as a sustainable feedstock for the simultaneous production of high-added value lipophilic compounds and platform chemicals.