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1.
World J Microbiol Biotechnol ; 36(1): 17, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31912247

RESUMO

In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.


Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos/análise , Microalgas/crescimento & desenvolvimento , Nitrogênio/metabolismo , Biomassa , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Regulação Enzimológica da Expressão Gênica , Metabolismo dos Lipídeos , Microalgas/genética , Microalgas/metabolismo , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Ácido alfa-Linoleico/metabolismo
2.
J Agric Food Chem ; 68(1): 402-408, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31809034

RESUMO

The functional ingredients of microalgal biomass are receiving substantial recognition as the global demands for health supplements produced from natural sources are on the rise. Paramylon, a conglomerate of ß-1,3-glucans, is one of the major valuable sources derived from Euglena gracilis having multiple applications, thus necessitating the development of an efficient quantification method. Here, we employed a DNA aptamer to quantify the amount of paramylon produced by E. gracilis. Paramylon-specific aptamers were isolated by the systematic evolution of ligands by exponential enrichment (SELEX) process. To evaluate the potential aptamers, the binding affinity between aptamer candidates and paramylon granules was confirmed by a confocal laser scanning microscope and the dissociation constants of the selected aptamers were determined by nonlinear regression analysis. The selected DNA aptamer was successfully used for the quantification of paramylon, and the results were compared to those obtained by the standard methods. The new approach was also used for quantification of paramylon from E. gracilis cells cultured to different cell stages and physiologies. It can be concluded that the aptamer-based protocol for the measurement of paramylon proposed in this study is highly accurate and comparatively less time-consuming.


Assuntos
Aptâmeros de Nucleotídeos/genética , DNA de Cadeia Simples/genética , Euglena gracilis/química , Glucanos/análise , Extratos Vegetais/análise , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Euglena gracilis/genética , Euglena gracilis/metabolismo , Glucanos/metabolismo , Microalgas/química , Microalgas/genética , Microalgas/metabolismo , Extratos Vegetais/metabolismo
3.
Pol J Microbiol ; 68(4): 527-539, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880896

RESUMO

Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.


Assuntos
Microalgas/isolamento & purificação , Clorófitas/classificação , Clorófitas/genética , Diatomáceas/classificação , Diatomáceas/genética , Diatomáceas/isolamento & purificação , Água Doce/análise , Ilhas , Microalgas/classificação , Microalgas/genética , Filogenia , Análise de Sequência de DNA
4.
Microb Cell Fact ; 18(1): 161, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547820

RESUMO

BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.


Assuntos
Dióxido de Carbono/metabolismo , Diatomáceas/crescimento & desenvolvimento , Diatomáceas/genética , Ácidos Graxos/metabolismo , Glucosefosfato Desidrogenase/genética , Dióxido de Carbono/análise , Diatomáceas/metabolismo , Engenharia Genética , Glucosefosfato Desidrogenase/metabolismo , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fotossíntese
5.
J Agric Food Chem ; 67(34): 9569-9578, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31385495

RESUMO

Acetyl-CoA synthetase (ACS) plays a key role in microalgal lipid biosynthesis and acetyl-CoA industrial production. In the present study, two ACSs were cloned and characterized from the oleaginous microalga Chromochloris zofingiensis. In vitro kinetic analysis showed that the Km values of CzACS1 and CzACS2 for potassium acetate were 0.99 and 0.81 mM, respectively. Moreover, CzACS1 and CzACS2 had outstanding catalytic efficiencies (kcat/Km), which were 70.67 and 79.98 s-1 mM-1, respectively, and these values were higher than that of other reported ACSs. CzACS1 and CzACS2 exhibited differential expression patterns at the transcriptional level under various conditions. Screening a recombinant library of 52 transcription factors (TFs) constructed in the present study via yeast one-hybrid assay pointed to seven TFs with potential involvement in the regulation of the two ACS genes. Expression correlation analysis implied that GATA20 was likely an important regulator of CzACS2 and that ERF9 could regulate two CzACSs simultaneously.


Assuntos
Acetato-CoA Ligase/metabolismo , Clorófitas/enzimologia , Regulação Enzimológica da Expressão Gênica , Microalgas/enzimologia , Acetato-CoA Ligase/química , Acetato-CoA Ligase/genética , Biocatálise , Clorófitas/química , Clorófitas/genética , Cinética , Metabolismo dos Lipídeos , Microalgas/química , Microalgas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
J Microbiol Biotechnol ; 29(9): 1453-1459, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31387339

RESUMO

Zeaxanthin is an important pigment in the photo-protection mechanism of microalgae. However, zeaxanthin epoxidase, an enzyme involved in the accumulation and conversion of zeaxanthin, has not been extensively studied in microalgae. In this work, we report the expression pattern of zeaxanthin epoxidase in Dunaliella tertiolecta (DtZEP) at different light and diverse salinity conditions. To confirm the responsiveness to light conditions, the ZEP expression pattern was investigated in photoperiodic (16 h of light and 8 h of dark) and continuous (24 h of light and 0 h of dark) light conditions. mRNA expression levels in photoperiodic conditions fluctuated along with the light/dark cycle, whereas those in continuous light remained unchanged. In varying salinity conditions, the highest mRNA and protein levels were detected in cells cultured in 1.5 M NaCl, and ZEP expression levels in cells shifted from 0.6 M NaCl to 1.5 M NaCl increased gradually. These results show that mRNA expression of DtZEP responds rapidly to the light/dark cycle or increased salinity, whereas changes in protein synthesis do not occur within a short period. Taken together, we show that DtZEP gene expression responds rapidly to light irradiation and hyperosmotic stress. In addition, ZEP expression patterns in light or salinity conditions are similar to those of higher plants, even though the habitat of D. tertiolecta is different.


Assuntos
Clorofíceas/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Microalgas/enzimologia , Oxirredutases/genética , Vias Biossintéticas/genética , Carotenoides/metabolismo , Clorofíceas/genética , Clorofíceas/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Microalgas/genética , Microalgas/metabolismo , Oxirredutases/metabolismo , Fotoperíodo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Salinidade , Cloreto de Sódio/farmacologia
7.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
8.
Mar Drugs ; 17(5)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035409

RESUMO

Short chain fatty acids (SCFAs) are valued as a functional material in cosmetics. Cyanobacteria can accumulate SCFAs under some conditions, the related mechanism is unclear. Two potential genes Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH) in Synechococcus sp. PCC 7942 have homology with fabB/F and fabH encoding ß-ketoacyl ACP synthases (I/II/III) in plants. Therefore, effects of culture time and cerulenin on SCFAs accumulation, expression levels and functions of these two potential genes were studied. The results showed Synechococcus sp. PCC 7942 accumulated high SCFAs (C12 + C14) in early growth stage (day 4) and at 7.5g/L cerulenin concentration, reaching to 2.44% and 2.84% of the total fatty acids respectively, where fabB/F expression was down-regulated. Fatty acid composition analysis showed C14 increased by 65.19% and 130% respectively, when fabB/F and fabH were antisense expressed. C14 increased by 10.79% (fab(B/F)-) and 6.47% (fabH-) under mutation conditions, while C8 increased by six times in fab(B/F)- mutant strain. These results suggested fabB/F is involved in fatty acid elongation (C <18) and the elongation of cis-16:1 to cis-18:1 fatty acid in Synechococcus sp. PCC 7942, while fabH was involved in elongation of fatty acid synthesis, which were further confirmed in complementary experiments of E. coli. The research could provide the scientific basis for the breeding of SCFA-rich microalgae species.


Assuntos
Ácidos Graxos Voláteis/biossíntese , Microalgas/metabolismo , Synechococcus/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cosméticos/química , Redes e Vias Metabólicas/genética , Microalgas/genética , Homologia de Sequência de Aminoácidos , Synechococcus/genética
9.
Mol Biotechnol ; 61(8): 541-561, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31140149

RESUMO

Targeted genome editing using RNA-guided endonucleases is an emerging tool in algal biotechnology. Recently, CRISPR-Cas systems have been widely used to manipulate the genome of some freshwater and marine microalgae. Among two different classes, and six distinct types of CRISPR systems, Cas9-driven type II system has been widely used in most of the studies for targeted knock-in, knock-out and knock-down of desired genes in algae. CRISPR technology has been demonstrated in microalgae including diatoms to manifest the function of the particular gene (s) and developing industrial traits, such as improving lipid content and biomass productivity. Instead of these, there are a lot of gears to be defined about improving efficiency and specificity of targeted genome engineering of microalgae using CRISPR-Cas system. Optimization of tools and methods of CRISPR technology can undoubtedly transform the research toward the industrial-scale production of commodity chemicals, food and biofuels using photosynthetic cell factories. This review has been focused on the efforts made so far to targeted genome engineering of microalgae, identified scopes about the hurdles related to construction and delivery of CRISPR-Cas components, algae transformation toolbox, and outlined the future prospect toward developing the CRISPR platform for high-throughput genome-editing of microalgae.


Assuntos
Biotecnologia , Sistemas CRISPR-Cas , Edição de Genes , Microalgas , Microalgas/genética , Microalgas/metabolismo , Fotobiorreatores
10.
Environ Pollut ; 250: 186-195, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30995572

RESUMO

The ultra-high Cd polluted environment is a special habitat in nature. Analysis of the biological adaptation and resistance mechanism of Auxenochlorella protothecoides UTEX234 to ultra-high Cd stress would offer some inspiring understanding on Cd detoxification mechanism and help discovering highly active bioremediation agents. In this study, integrated analyses of the transcriptome, multi-physiological and biochemical data and fatty acid profilings of UTEX2341 were performed for the first time. It was found that exogenous Ca ions could alleviate Cd stress. Manganese-dependent superoxide dismutase and peroxidase also participated in intracellular detoxification. And non-enzymatic antioxidants rather than one specific enzymatic antioxidant were suggested to be used as "core antioxidants", which witnessed better performance in Cd detoxification. In addition, Cd stress improved sixteen alkane value and biofuel yield and quality.


Assuntos
Cádmio/metabolismo , Clorófitas/fisiologia , Poluentes Ambientais/metabolismo , Lipídeos/biossíntese , Microalgas/fisiologia , Adaptação Fisiológica , Antioxidantes/metabolismo , Biodegradação Ambiental , Biocombustíveis , Cádmio/toxicidade , Clorófitas/genética , Clorófitas/metabolismo , Poluentes Ambientais/toxicidade , Microalgas/genética , Microalgas/metabolismo , Transcriptoma
11.
Lipids Health Dis ; 18(1): 56, 2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832728

RESUMO

BACKGROUND: ω-3 polyunsaturated fatty acids (PUFAs) are synthesized from α-Linolenic acid (ALA, C18:3ω3) and play important roles in anti-inflammatory and antioxidant responses in mammal cells. ALA is an essential fatty acid which cannot be produced within the human body and must be acquired through diet. The purpose of this study was to evaluate the potential of a novel microalgal strain (HDMA-20) as a source of ω-3 PUFAs including ALA and eicosatetraenoic acid (ETA, C20:4ω3). METHOD: Phylogenetic Neighbor-Joining analysis based on 18S ribosomal DNA sequence was used to identify the microalga strain HDMA-20. Autotrophic condition was chosen to cultivate HDMA-20 to reduce the cultivation cost. GC-MS was used to determine the fatty acid composition of HDMA-20 lipid. RESULTS: A microalgal strain (HDMA-20) from Lake Chengfeng (Daqing, Heilongjiang province, China) was found to accumulate high content of ω-3 PUFAs (63.4% of total lipid), with ALA and eicosatetraenoic acid (ETA, C20:4ω3) accounting for 35.4 and 9.6% of total lipid, respectively. Phylogenetic analysis based on 18S ribosomal DNA sequences suggested that the HDMA-20 belonged to genus Monoraphidium (Selenastraceae, Sphaeropleales) and its 18S rDNA sequence information turned out to be new molecular record of Monoraphidium species. The biomass productivity and lipid content of HDMA-20 were also investigated under autotrophic condition. The biomass productivity of HDMA-20 reached 36.3 mg L- 1 day- 1, and the lipid contents was 22.6% of dry weight. CONCLUSION: HDMA-20 not only represent an additional source of ALA, but also a totally new source of ETA. The high content of ω-3 PUFAs, especially ALA, of HDMA-20, makes it suitable as a source of nutrition supplements for human health. In addition, HDMA-20 exhibited good properties in growth and lipid accumulation, implying its potential for cost-effective ω-3 PUFAs production in future.


Assuntos
Ácidos Araquidônicos/isolamento & purificação , Clorofíceas/metabolismo , Suplementos Nutricionais/análise , Microalgas/metabolismo , Ácido alfa-Linoleico/isolamento & purificação , Ácidos Araquidônicos/biossíntese , Processos Autotróficos/fisiologia , Biomassa , China , Clorofíceas/classificação , Clorofíceas/genética , Clorofíceas/crescimento & desenvolvimento , Suplementos Nutricionais/provisão & distribução , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lagos , Metaboloma/fisiologia , Microalgas/classificação , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Filogenia , RNA Ribossômico 18S/genética , Ácido alfa-Linoleico/biossíntese
12.
Appl Microbiol Biotechnol ; 103(8): 3487-3499, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30899985

RESUMO

The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5' untranslated region. This region was identified as the full-length promoter and named PromA (- 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (- 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (- 2727 to - 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (- 2727 to - 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.


Assuntos
Proteínas de Algas/genética , Chlamydomonas reinhardtii/genética , Glicerolfosfato Desidrogenase/genética , Microalgas/genética , Regiões Promotoras Genéticas , Transgenes/genética , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/enzimologia , Clonagem Molecular , Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Microalgas/química , Microalgas/enzimologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Elementos de Resposta , Cloreto de Sódio/química , Cloreto de Sódio/farmacologia
13.
Appl Microbiol Biotechnol ; 103(8): 3239-3248, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30877356

RESUMO

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Microalgas/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica , Marcação de Genes , Engenharia Genética , RNA Guia/genética , Transformação Genética
14.
Mar Genomics ; 46: 8-15, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30852185

RESUMO

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes for the assembly of a Type 9 Secretion System (T9SS) and the associated gliding motility apparatus. In addition to these common features, pathogenic strains AD1 and BL5, encode genes for the production of flexirubin type pigments and a number of unique non-ribosomal peptide synthesis (NRPS) gene clusters, suggesting a role for these uncharacterised traits in virulence. This study provides valuable insight into the potential ecological role of Aquimarina in the marine environment and the complex factors driving pathogenesis and symbiosis in this genus.


Assuntos
Flavobacteriaceae/genética , Genômica , Interações Hospedeiro-Patógeno/genética , Microalgas/genética , Microalgas/microbiologia , Rodófitas/genética , Rodófitas/microbiologia , Flavobacteriaceae/patogenicidade , Simbiose
15.
Microb Cell Fact ; 18(1): 53, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30866936

RESUMO

BACKGROUND: Microalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesis RESULTS: NeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years. CONCLUSIONS: Our results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.


Assuntos
Aciltransferases/genética , Microalgas/genética , Microalgas/metabolismo , Organismos Geneticamente Modificados/genética , Triglicerídeos , Biocombustíveis , Plastídeos/genética , Triglicerídeos/biossíntese , Triglicerídeos/genética , Triglicerídeos/metabolismo
16.
Crit Rev Biotechnol ; 39(3): 422-435, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30744439

RESUMO

Microalgae are photosynthetic microorganisms with potential for biofuel production, CO2 mitigation and wastewater treatment; indeed they have the capacity to assimilate pollutants in wastewaters. Light supply and distribution among the microalgae culture is one of the major challenges of photo-bioreactor design, with many studies focusing on microalgae culture systems such as raceway ponds (RWP), widely used and cost-effective systems for algal biomass production. This review focuses on possible improvements of the RWP design in order to achieve optimal microalgal growth conditions and high biomass productivities, to minimize energy consumption and to lower the capital costs of the pond. The improvement strategy is based on three aspects: (1) hydrodynamic characteristics of the raceway pond, (2) evaluation of hydrodynamic and mass transfer capacities of the pond and (3) design of the RWP. Finally, a possible optimal design for the RWP is discussed in the context of wastewater treatment.


Assuntos
Microalgas/metabolismo , Águas Residuárias/química , Purificação da Água/métodos , Biocombustíveis , Biomassa , Reatores Biológicos , Humanos , Hidrodinâmica , Microalgas/química , Microalgas/genética , Fotossíntese/genética , Tanques/química , Poluentes Químicos da Água/química , Poluentes Químicos da Água/toxicidade
17.
Nat Commun ; 10(1): 853, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787273

RESUMO

Cytosolic lipid droplets are endoplasmic reticulum-derived organelles typically found in seeds as reservoirs for physiological energy and carbon to fuel germination. Here, we report synthetic biology approaches to co-produce high-value sesqui- or diterpenoids together with lipid droplets in plant leaves. The formation of cytosolic lipid droplets is enhanced in the transient Nicotiana benthamiana system through ectopic production of WRINKLED1, a key regulator of plastid fatty acid biosynthesis, and a microalgal lipid droplet surface protein. Engineering of the pathways providing the universal C5-building blocks for terpenoids and installation of terpenoid biosynthetic pathways through direction of the enzymes to native and non-native compartments boost the production of target terpenoids. We show that anchoring of distinct biosynthetic steps onto the surface of lipid droplets leads to efficient production of terpenoid scaffolds and functionalized terpenoids. The co-produced lipid droplets "trap" the terpenoids in the cells.


Assuntos
Materiais Biocompatíveis/metabolismo , Citosol/metabolismo , Gotículas Lipídicas/metabolismo , Folhas de Planta/metabolismo , Terpenos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Engenharia Metabólica/métodos , Microalgas/genética , Microalgas/metabolismo , Microscopia Confocal , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Biologia Sintética/métodos , Tabaco/genética , Tabaco/metabolismo
18.
Proc Natl Acad Sci U S A ; 116(9): 3385-3390, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808735

RESUMO

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.


Assuntos
Transferência de Energia/genética , Microalgas/metabolismo , Fotossíntese/genética , Zeaxantinas/química , Carotenoides/genética , Carotenoides/metabolismo , Clorofila/química , Clorofila/genética , Clorofila/metabolismo , Luz , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Microalgas/química , Microalgas/genética , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Xantofilas/química , Xantofilas/genética , Xantofilas/metabolismo , Zeaxantinas/genética , Zeaxantinas/metabolismo
19.
ISME J ; 13(8): 1899-1910, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30809012

RESUMO

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 132,173-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.


Assuntos
Clorofila/metabolismo , Eucariotos/metabolismo , Oxigênio/metabolismo , Cloroplastos/metabolismo , Ecossistema , Eucariotos/classificação , Eucariotos/genética , Microalgas/classificação , Microalgas/genética , Microalgas/metabolismo , Fotossíntese , Filogenia , Simbiose
20.
World J Microbiol Biotechnol ; 35(2): 35, 2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30712106

RESUMO

As major primary producers in marine environments, diatoms are considered a valuable feedstock of biologically active compounds for application in several biotechnological fields. Due to their metabolic plasticity, especially for light perception and use and in order to make microalgal production more environmentally sustainable, marine diatoms are considered good candidates for the large-scale cultivation. Among physical parameters, light plays a primary role. Even if sunlight is cost-effective, the employment of artificial light becomes a winning strategy if a high-value microalgal biomass is produced. Several researches on marine diatoms are designed to study the influence of different light regimens to increase biomass production enriched in biotechnologically high-value compounds (lipids, carotenoids, proteins, polysaccharides), or with emphasised photonic properties of the frustule.


Assuntos
Produtos Biológicos/metabolismo , Biotecnologia , Diatomáceas/metabolismo , Diatomáceas/efeitos da radiação , Microalgas/metabolismo , Microalgas/efeitos da radiação , Diatomáceas/genética , Diatomáceas/crescimento & desenvolvimento , Luz , Microalgas/genética , Microalgas/crescimento & desenvolvimento
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