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1.
Eur J Protistol ; 71: 125639, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31550629

RESUMO

Opalinids are a large group of anaerobic protists, mainly inhabiting the cloacae of amphibians (frogs and toads). The classification of this group has not been fully resolved, because of a lack of molecular information. Here, we give a redescription of Opalina triangulata Metcalf, 1923, collected from the rectum of the frog Fejervarya limnocharis, based on detailed morphological and molecular data. Our phylogenetic analyses confirmed the monophyly of Opalinata. Within it, Opalinea were monophyletic with O. triangulata and O. undulata as well as two Protoopalina species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea. Meanwhile, the ITS2 secondary structural similarities as well as G-C content revealed greater similitudes between Opalina species and P. lacertae than with Blastocystis hominis, which is in accordance with their position as sister clades in the SSU rDNA-based phylogenies.


Assuntos
Anuros/parasitologia , Filogenia , Estramenópilas/classificação , Animais , DNA Ribossômico/genética , Estramenópilas/citologia , Estramenópilas/genética
2.
J Agric Food Chem ; 67(34): 9683-9693, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31379160

RESUMO

Schizochytrium sp. A-2 is a heterotrophic marine fungus used for the commercial production of docosahexaenoic acid (DHA). However, the pattern of the distribution of DHA and how DHA is channeled into phospholipid (PL) and triacylglycerol (TAG) are unknown. In this study, we systematically analyzed the distribution of DHA in TAG and PL during the growth of the cell. The migration of DHA from PL to TAG was presumed during the fermentation cycle. DHA and docosapentaenoic acid were accumulated in both TAG and phosphatidylcholine (PC), whereas eicosapentaenoic acid was mainly deposited in PC. RNA seq revealed that malic enzyme may provide lipogenic NADPH. In addition, long-chain acyl-CoA synthase and acyl-CoA:lysophosphatidylcholine acyltransferase may participate in the accumulation of DHA in PL. No phosphatidylcholine:diacylglycerol cholinephosphotransferase was identified from the genome sequence. In contrast, phospholipid:diacylglycerol acyltransferase-mediated acyl-CoA-independent TAG synthesis pathway and phospholipase C may contribute to the channeling of DHA from PC to TAG.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Estramenópilas/genética , Estramenópilas/metabolismo , Transcriptoma , 1-Acilglicerofosfocolina O-Aciltransferase/genética , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Transporte Biológico , Fosfolipídeos/metabolismo , Estramenópilas/química , Triglicerídeos/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
3.
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
4.
Bioresour Technol ; 287: 121415, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31078814

RESUMO

Newly-isolated thraustochytrid strains from coastal waters of China were characterized as bioresource of squalene and the culture condition for the top producer was systematically optimized. Phylogenetic analysis revealed that eight squalene-producing isolates were closely related to genus Aurantiochytrium and one to genus Labyrinthula. The top producer, Aurantiochytrium sp. TWZ-97, produced squalene up to 188.6 mg/L at 28 °C in a 5-L bioreactor containing optimal medium (glucose: 40 g/L, monosodium glutamate: 3 g/L, yeast extract: 25 g/L, and NaCl: 6 g/L), which was 6-fold higher than that under unoptimized condition. Transcriptome analysis revealed for the first time the presence of seven key genes of mevalonate pathway for squalene biosynthesis in strain TWZ-97. Medium optimization yielded a 2.23-fold higher expression of the squalene synthase gene under optimal condition compared to unoptimized. This study provides a potential thraustochytrid strain TWZ-97 as bioresource of squalene and uncovers novel information about its squalene biosynthesis pathway for future strain improvement.


Assuntos
Vias Biossintéticas , Esqualeno/metabolismo , Estramenópilas/metabolismo , Reatores Biológicos , Filogenia , Estramenópilas/genética
5.
Protist ; 170(2): 209-232, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31100647

RESUMO

Strain HS-399 was isolated from a mangrove swamp in Biscayne Bay (Florida, USA) and selected for its capacity to accumulate lipids (84.0±1.0% DW), particularly docosahexaenoic acid (DHA; 22:6 n-3) (28.3±0.1% DW). Molecular phylogenetic analysis demonstrated that the new organism belonged to the genus Aurantiochytrium, and when the whole nuclear genome was blasted against the type species (and only described species), A. limacinum SR21, there was a 5.38% difference at the protein level. We described our new organism as Aurantiochytrium acetophilum sp. nov. (Thraustochytriaceae, Thraustochytriales) using light microscopy, electron microscopy, substrate assimilation, biochemical composition and nuclear genomic data. We found some characteristics of biotechnological relevance that were not previously described in this family. First, strain HS-399 of A. acetophilum was extremely tolerant to acetate toxicity, and it used this substrate as a sole carbon source. Second, we observed putative gametes that fused together to form a zygote. Zygote fate and the life stage with meiosis were not determined; however, we found several meiosis genes in the genome, further supporting the possibility of breeding for these industrially relevant organisms.


Assuntos
Genoma de Protozoário , Filogenia , Estramenópilas/classificação , Estramenópilas/genética , Reprodução , Especificidade da Espécie , Estramenópilas/fisiologia
6.
J Agric Food Chem ; 67(19): 5560-5570, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30901205

RESUMO

The phytohormone 6-benzylaminopurine (6-BAP) significantly improves lipid synthesis of oleaginous microorganisms with the great potential applied in lipid production. In the current study, the lipid and DHA productions in oleaginous Aurantiochytrium sp. were found to be improved by 48.7% and 55.3%, respectively, induced by 6-BAP treatments. Then, using high-throughput RNA-seq technology, the overall de novo assembly of the cDNA sequence data generated 53871 unigenes, and 15902 of these were annotated in at least one database. The comparative transcriptomic profiles of cells with and without 6-BAP treatments revealed that a total of 717 were differently expressed genes (DE), with 472 upregulated and 245 downregulated. Further annotation and categorization indicated that some DE genes were involved in pathways crucial to lipid and DHA productions, such as fatty acid synthesis, central carbon metabolism, transcriptional factor, signal transduction, and mevalonate pathway. A regulation mode of 6-BAP, in turn, perception and transduction of 6-BAP signal, transcription factor, expression regulations of the downstream genes, and metabolic changes, respectively, was put forward for the first time in the present study. This research illuminates the transcriptomic mechanism of phytohormone stimulation of lipid and DHA production in an oleaginous microorganism and provides the potential targets modified using genetic engineering for improving lipid and DHA productivity.


Assuntos
Compostos de Benzil/farmacologia , Ácidos Docosa-Hexaenoicos/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Reguladores de Crescimento de Planta/farmacologia , Purinas/farmacologia , Estramenópilas/efeitos dos fármacos , Estramenópilas/genética , Compostos de Benzil/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Reguladores de Crescimento de Planta/química , Purinas/química , Estramenópilas/metabolismo
7.
Genes (Basel) ; 10(3)2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909525

RESUMO

The complex evolution of chloroplasts in microalgae has resulted in highly diverse pigment profiles. Freshwater raphidophytes, for example, display a very different pigment composition to marine raphidophytes. To investigate potential differences in the evolutionary origin of chloroplasts in these two groups of raphidophytes, the plastid genomes of the freshwater species Gonyostomum semen and Vacuolaria virescens were sequenced. To exclusively sequence the organelle genomes, chloroplasts were manually isolated and amplified using single-cell whole-genome-amplification. Assembled and annotated chloroplast genes of the two species were phylogenetically compared to the marine raphidophyte Heterosigma akashiwo and other evolutionarily more diverse microalgae. These phylogenetic comparisons confirmed the high relatedness of all investigated raphidophyte species despite their large differences in pigment composition. Notable differences regarding the presence of light-independent protochlorophyllide oxidoreductase (LIPOR) genes among raphidophyte algae were also revealed in this study. The whole-genome amplification approach proved to be useful for isolation of chloroplast DNA from nuclear DNA. Although only approximately 50% of the genomes were covered, this was sufficient for a multiple gene phylogeny representing large parts of the chloroplast genes.


Assuntos
Cloroplastos/genética , Estramenópilas/genética , Sequenciamento Completo do Genoma/métodos , Genoma de Cloroplastos , Anotação de Sequência Molecular , Filogenia , Análise de Célula Única , Estramenópilas/classificação
8.
PLoS One ; 14(1): e0211164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30699157

RESUMO

Thraustochytrids possess docosahexaenoic acid (DHA, 22:6n-3) as acyl chain(s) of triacylglycerol (TG) and phosphatidylcholine (PC), some of which contain multiple DHAs. However, little is known about how these DHA-rich glycerolipids are produced in thraustochytrids. In this study, we identified PLAT2 in Aurantiochytrium limacinum F26-b as a glycerol-3-phosphate (G3P) acyltransferase (GPAT) by heterologous expression of the gene in budding yeast. Subsequently, we found that GPAT activity was reduced by disruption of the PLAT2 gene in A. limacinum, resulting in a decrease in DHA-containing lysophosphatidic acid (LPA 22:6). Conversely, overexpression of PLAT2 increased both GPAT activity and LPA 22:6. These results indicate that PLAT2 is a GPAT that transfers DHA to G3P in vivo as well as in vitro. Overexpression of the PLAT2 gene increased the production of a two DHA-containing diacylglycerol (DG 44:12), followed by an increase in the three DHA-containing TG (TG 66:18), two-DHA-containing TG (TG 60:12), and two DHA-containing PC (PC 44:12). However, overexpression of PLAT2 did not increase DHA-free DG (DG32:0), which was preferentially converted to three 16:0-containing TG (TG 48:0) but not two 16:0-containing PC (PC 32:0). Collectively, we revealed that DHA-rich glycerolipids are produced from a precursor, LPA 22:6, which is generated by incorporating DHA to G3P by PLAT2 in the A. limacinum.


Assuntos
Diglicerídeos/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Lisofosfolipídeos/metabolismo , Estramenópilas/enzimologia , Triglicerídeos/metabolismo , Diglicerídeos/genética , Ácidos Docosa-Hexaenoicos/genética , Lisofosfolipídeos/genética , Estramenópilas/genética , Triglicerídeos/genética
9.
Genome Biol Evol ; 11(2): 362-379, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629162

RESUMO

Eustigmatophytes, a class of stramenopile algae (ochrophytes), include not only the extensively studied biotechnologically important genus Nannochloropsis but also a rapidly expanding diversity of lineages with much less well characterized biology. Recent discoveries have led to exciting additions to our knowledge about eustigmatophytes. Some proved to harbor bacterial endosymbionts representing a novel genus, Candidatus Phycorickettsia, and an operon of unclear function (ebo) obtained by horizontal gene transfer from the endosymbiont lineage was found in the plastid genomes of still other eustigmatophytes. To shed more light on the latter event, as well as to generally improve our understanding of the eustigmatophyte evolutionary history, we sequenced plastid genomes of seven phylogenetically diverse representatives (including new isolates representing undescribed taxa). A phylogenomic analysis of plastid genome-encoded proteins resolved the phylogenetic relationships among the main eustigmatophyte lineages and provided a framework for the interpretation of plastid gene gains and losses in the group. The ebo operon gain was inferred to have probably occurred within the order Eustigmatales, after the divergence of the two basalmost lineages (a newly discovered hitherto undescribed strain and the Pseudellipsoidion group). When looking for nuclear genes potentially compensating for plastid gene losses, we noticed a gene for a plastid-targeted acyl carrier protein that was apparently acquired by horizontal gene transfer from Phycorickettsia. The presence of this gene in all eustigmatophytes studied, including representatives of both principal clades (Eustigmatales and Goniochloridales), is a genetic footprint indicating that the eustigmatophyte-Phycorickettsia partnership started no later than in the last eustigmatophyte common ancestor.


Assuntos
Evolução Biológica , Genomas de Plastídeos , Óperon , Rickettsiaceae/genética , Estramenópilas/genética , Sequência de Aminoácidos , Estramenópilas/microbiologia , Simbiose
10.
BMC Evol Biol ; 19(1): 20, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30634905

RESUMO

BACKGROUND: The Synurophyceae is one of most important photosynthetic stramenopile algal lineages in freshwater ecosystems. They are characterized by siliceous scales covering the cell or colony surface and possess plastids of red-algal secondary or tertiary endosymbiotic origin. Despite their ecological and evolutionary significance, the relationships amongst extant Synurophyceae are unclear, as is their relationship to most other stramenopiles. RESULTS: Here we report a comparative analysis of plastid genomes sequenced from five representative synurophycean algae. Most of these plastid genomes are highly conserved with respect to genome structure and coding capacity, with the exception of gene re-arrangements and partial duplications at the boundary of the inverted repeat and single-copy regions. Several lineage-specific gene loss/gain events and intron insertions were detected (e.g., cemA, dnaB, syfB, and trnL). CONCLUSIONS: Unexpectedly, the cemA gene of Synurophyceae shows a strong relationship with sequences from members of the green-algal lineage, suggesting the occurrence of a lateral gene transfer event. Using a molecular clock approach based on silica fossil record data, we infer the timing of genome re-arrangement and gene gain/loss events in the plastid genomes of Synurophyceae.


Assuntos
Variação Genética , Genomas de Plastídeos , Genômica , Sequências Repetidas Invertidas/genética , Estramenópilas/genética , Sequência de Bases , DNA Circular/genética , Evolução Molecular , Dosagem de Genes , Conformação de Ácido Nucleico , Filogenia , RNA de Transferência/química , RNA de Transferência/genética
11.
Mar Biotechnol (NY) ; 21(1): 99-110, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30456696

RESUMO

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.


Assuntos
Proteínas de Algas/genética , Meios de Cultura/farmacologia , Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Eicosapentaenoico/biossíntese , Proteoma/genética , Estramenópilas/efeitos dos fármacos , Proteínas de Algas/classificação , Proteínas de Algas/isolamento & purificação , Biomassa , Carotenoides/biossíntese , Carotenoides/isolamento & purificação , Meios de Cultura/química , Ácidos Docosa-Hexaenoicos/isolamento & purificação , Ácido Eicosapentaenoico/isolamento & purificação , Expressão Gênica , Ontologia Genética , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Maltose/metabolismo , Maltose/farmacologia , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/isolamento & purificação , RNA Ribossômico 18S/genética , Água do Mar/química , Análise de Sequência de DNA , Amido/metabolismo , Amido/farmacologia , Estramenópilas/genética , Estramenópilas/crescimento & desenvolvimento , Estramenópilas/metabolismo
12.
Protist ; 170(1): 38-51, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30576874

RESUMO

Heterosigma akashiwo is a raphidophyte known for forming ichthyotoxic blooms. In order to predict the potential impacts of rising CO2 on H. akashiwo it is necessary to understand the factors influencing growth rates over a range of CO2 concentrations. Here we examined the physiology and gene expression response of H. akashiwo to concentrations from 200 to 1000ppm CO2. Growth rate data were combined from this and previous studies and fit with a CO2 limitation-inhibition model that revealed an apparent growth optimum around 600-800ppm CO2. Physiological changes included a significant increase in C:N ratio at ∼800ppm CO2 and a significant decrease in hydrogen peroxide concentration at ∼1000ppm. Whole transcriptome sequencing of H. akashiwo revealed sharp distinctions in metabolic pathway gene expression between ∼600 and ∼800ppm CO2. Hierarchical clustering by co-expression identified groups of genes with significant correlations to CO2 and growth rate. Genes with significant differential expression with CO2 included carbon concentrating mechanism genes such as beta-carbonic anhydrases and a bicarbonate transporter, which may underpin shifts in physiology. Genes involved in cell motility were significantly changed by both elevated CO2 and growth rate, suggesting that future ocean conditions could modify swimming behavior in this species.


Assuntos
Dióxido de Carbono/análise , Expressão Gênica , Estramenópilas/fisiologia , Estramenópilas/genética
13.
Planta ; 249(3): 647-661, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30341489

RESUMO

MAIN CONCLUSION: Comparative genomic analysis of cytochromes P450 revealed high diversification and dynamic changes in stramenopiles, associated with transcriptional responsiveness to various environmental stimuli. Comparative genomic and molecular evolution approaches were used to characterize cytochromes P450 (P450) diversity in stramenopiles. Phylogenetic analysis pointed to a high diversity of P450 in stramenopiles and identified three major clans. The CYP51 and CYP97 clans were present in brown algae, diatoms and Nannochloropsis gaditana, whereas the CYP5014 clan mainly includes oomycetes. Gene gain and loss patterns revealed that six CYP families-CYP51, CYP97, CYP5160, CYP5021, CYP5022, and CYP5165-predated the split of brown algae and diatoms. After they diverged, diatoms gained more CYP families, especially in the cold-adapted species Fragilariopsis cylindrus, in which eight new CYP families were found. Selection analysis revealed that the expanded CYP51 family in the brown alga Cladosiphon okamuranus exhibited a more relaxed selection constraint compared with those of other brown algae and diatoms. Our RNA-seq data further evidenced that most of P450s in Saccharina japonica are highly expressed in large sporophytes, which could potentially promote the large kelp formation in this developmental stage. A survey of Ectocarpus siliculosus and diatom transcriptomes showed that many P450s are responsive to stress, nutrient limitation or light quality, suggesting pivotal roles in detoxification or metabolic processes under adverse environmental conditions. The information provided in this study will be helpful in designing functional experiments and interpreting P450 roles in this particular lineage.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Variação Genética/genética , Estramenópilas/genética , Genômica , Feófitas/enzimologia , Feófitas/genética , Filogenia , Alinhamento de Sequência , Estramenópilas/enzimologia , Transcriptoma
14.
Trends Biotechnol ; 37(4): 344-346, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30376959

RESUMO

Polyunsaturated fatty acids (PUFAs) are important for human health. They are traditionally extracted from animals and plants but can be alternatively derived from oleaginous microbes, and engineering microbial metabolism can improve PUFA accumulation. The next frontier is to engineer more efficient PUFA-producing microbes using systems and synthetic biology tools.


Assuntos
Ácidos Graxos Insaturados/biossíntese , Fungos/genética , Fungos/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Estramenópilas/genética , Estramenópilas/metabolismo , Biotecnologia/métodos , Ácidos Graxos Insaturados/isolamento & purificação , Humanos
15.
Microb Ecol ; 77(2): 394-405, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30083828

RESUMO

The heterotrophic labyrinthulomycete protists have long been known to play an important role in the nutrient cycling of coastal seawater. Yet, their spatiotemporal abundance and diversity in polluted coastal waters remain poorly discussed, due in part to the paucity of a rapid detection method. To this end, we developed a qPCR detection method based on a newly designed primer pair targeting their 18S rRNA gene. Using this method, we studied the population dynamics of labyrinthulomycete protists in nutrient-rich (Shenzhen Bay) and low-nutrient (Daya) coastal habitats along the Pearl River Delta. We found a significantly (P < 0.05) higher abundance of Labyrinthulomycetes in the Shenzhen bay (average 3455 gene copies mL-1) than that in Daya Bay (average 378 gene copies mL-1). Their abundance gradient positively correlated (P < 0.05) with the levels of inorganic nitrogen and phosphates. Further characterization of the molecular diversity of these protists in Shenzhen Bay using different primer sets revealed the presence of several genera besides a large number of unclassified OTUs. Regardless of the primer biases, our results show significant (P < 0.05) spatiotemporal changes in the molecular abundance and diversity of these heterotrophic protists. Overall, this study provides a rapid molecular detection tool for Labyrinthulomycetes and expands our current understanding of their dynamics controlled by physicochemical gradients in coastal waters.


Assuntos
Biodiversidade , Rios/parasitologia , Água do Mar/parasitologia , Estramenópilas/isolamento & purificação , Nitrogênio/análise , Nitrogênio/metabolismo , Fosfatos/análise , Fosfatos/metabolismo , Filogenia , RNA Ribossômico 18S/genética , Rios/química , Água do Mar/química , Estramenópilas/classificação , Estramenópilas/genética , Estramenópilas/metabolismo
16.
PLoS One ; 13(12): e0208853, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30533058

RESUMO

Aurantiochytrium limacinum has received attention because of its abundance of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). DHA is synthesized through the polyketide synthase (PKS) pathway in A. limacinum. The related enzymes of the PKS pathway are mainly expressed by three gene clusters, called pks1, pks2 and pks3. In this study, the full-length pks3 gene was obtained by polymerase chain reaction amplification and Genome Walking technology. Based on a domain analysis of the deduced amino acid sequence of the pks3 gene, 3-ketoacyl-ACP reductase (KR) and dehydratase (DH) enzyme domains were identified. Herein, A. limacinum OUC168 was engineered by gene knock-in of KR and DH using the 18S rDNA sequence as the homologous recombination site. Total fatty acid contents and the degree of unsaturation of total fatty acids increased after the kr or dh gene was knocked in. The cloning and functional study of the pks3 gene of A. limacinum establishes a foundation for revealing the DHA synthetic pathway. Gene knock-in of the enzyme domain associated with PKS synthesis has the potential to provide effective recombinant strains with higher DHA content for industrial applications.


Assuntos
Hidroliases/genética , Oxirredutases/genética , Estramenópilas/genética , Clonagem Molecular , Ácidos Docosa-Hexaenoicos/metabolismo
17.
Sci Rep ; 8(1): 15357, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337591

RESUMO

Picoeukaryotes play prominent roles in the biogeochemical cycles in marine ecosystems. However, their molecular diversity studies have been confined in marine surface waters or shallow coastal sediments. Here, we investigated the diversity and metabolic activity of picoeukaryotic communities at depths ranging from the surface to the abyssopelagic zone in the western Pacific Ocean above the north and south slopes of the Mariana Trench. This was achieved by amplifying and sequencing the V4 region of both 18S ribosomal DNA and cDNA using Illumina HiSeq sequencing. Our study revealed: (1) Four super-groups (i.e., Alveolata, Opisthokonta, Rhizaria and Stramenopiles) dominated the picoeukaryote assemblages through the water column, although they accounted for different proportions at DNA and cDNA levels. Our data expand the deep-sea assemblages from current bathypelagic to abyssopelagic zones. (2) Using the cDNA-DNA ratio as a proxy of relative metabolic activity, the highest activity for most subgroups was usually found in the mesopelagic zone; and (3) Population shift along the vertical scale was more prominent than that on the horizontal differences, which might be explained by the sharp physicochemical gradients along the water depths. Overall, our study provides a better understanding of the diversity and metabolic activity of picoeukaryotes in water columns of the deep ocean in response to varying environmental conditions.


Assuntos
Alveolados , Biodiversidade , Biota , Células Eucarióticas , Rhizaria , Estramenópilas , Alveolados/classificação , Alveolados/genética , Alveolados/crescimento & desenvolvimento , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/crescimento & desenvolvimento , Biota/fisiologia , Demografia , Ecossistema , Células Eucarióticas/classificação , Oceano Pacífico , Filogenia , RNA Ribossômico 18S/genética , Rhizaria/classificação , Rhizaria/genética , Rhizaria/crescimento & desenvolvimento , Água do Mar/química , Análise de Sequência de DNA , Estramenópilas/classificação , Estramenópilas/genética , Estramenópilas/crescimento & desenvolvimento
18.
PLoS One ; 13(8): e0201438, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30071073

RESUMO

Despite the advance of knowledge about the factors and potential mechanisms triggering the ichthyotoxicity in microalgae, these remain unclear or are controversial for several species (e.g. Heterosigma). Neither typical toxicity tests carried out with cell extracts nor direct exposure to harmful species were proved suitable to unravel the mechanism of harm. Ichthyotoxic species show a complex harmful effect on fish, which is mediated through various mechanisms depending on the species. In this work, we present a method to study sub-lethal effects triggered by reactive oxygen species of a population of harmful algae in vivo over a fish cell line. To that end, Transwell co-cultures in which causative and target species are separated by a 0.4 µm pore membrane were carried out. This allowed the evaluation of the effect of the released molecules by cells in a rapid and compact test. In our method, the harmful effect was sensed through the transcriptional activation of sub-lethal marker Hsp70b in the CHSE214 salmon cell line. The method was tested with the raphidophyte Heterosigma akashiwo and Dunaliella tertiolecta (as negative control). It was shown that superoxide intracellular content and its release are not linked in these species. The methodology allowed proving that reactive oxygen species produced by H. akashiwo are able to induce the transcriptional activation of sub-lethal marker Hsp70b. However, neither loss of viability nor apoptosis was observed in CHSE214 salmon cell line except when exposed to direct contact with the raphidophyte cells (or their extract). Consequently, ROS was not concluded to be the main cause of ichthyotoxicity in H. akashiwo.


Assuntos
Proteínas de Choque Térmico HSP70/biossíntese , Microalgas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Estramenópilas/crescimento & desenvolvimento , Ativação Transcricional , Animais , Linhagem Celular , Técnicas de Cocultura , Proteínas de Choque Térmico HSP70/genética , Microalgas/genética , Salmão , Estramenópilas/genética
19.
Mar Biotechnol (NY) ; 20(6): 792-802, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30136198

RESUMO

Schizochytrium sp. is a kind of marine microalgae with great potential as promising sustainable source of polyunsaturated fatty acids (PUFAs). Polyketide synthase-like (PKS synthase) is supposed to be one of the main ways to synthesize PUFAs in Schizochytrium sp. In order to study the exact relationship between PKS and PUFA biosynthesis, chain length factor (CLF) and dehydrogenase (DH) were cloned from the PKS gene cluster in Schizochytrium sp., then disrupted by homologous recombination. The results showed that DH- and CLF-disrupted strains had significant decreases (65.85 and 84.24%) in PUFA yield, while the saturated fatty acid (SFA) proportion in lipids was slightly increased. Meanwhile, the disruption of CLF decreased the C-22 PUFA proportion by 57.51% without effect on C-20 PUFA accumulation while DH-disrupted mutant decreased the production of each PUFA. Combined with analysis of protein prediction, it indicated that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis, especially for the final elongation from C-20 to C-22 PUFAs. Metabolomics analysis also suggested that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs, thus weakening glycolysis and tricarboxylic acid (TCA) cycle pathways. This study offers a broad new vision to research the mechanism of PUFA synthesis in Schizochytrium sp.


Assuntos
Metabolômica/métodos , Estramenópilas/química , Estramenópilas/genética , Ácidos Graxos Insaturados/metabolismo , Estramenópilas/metabolismo
20.
Proc Natl Acad Sci U S A ; 115(30): E7015-E7022, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987047

RESUMO

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.


Assuntos
Acil-CoA Oxidase , Sistemas CRISPR-Cas , Edição de Genes , Lipídeos , Característica Quantitativa Herdável , Estramenópilas , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos/biossíntese , Lipídeos/genética , Estramenópilas/genética , Estramenópilas/metabolismo
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