Diatom milking? A review and new approaches

The rise of human populations and the growth of cities contribute to the depletion of natural resources, increase their cost, and create potential climatic changes. To overcome difficulties in supplying populations and reducing the resource cost, a search for alternative pharmaceutical, nanotechnology, and energy sources has begun. Among the alternative sources, microalgae are the most promising because they use carbon dioxide (CO2) to produce biomass and/or valuable compounds. Once produced, the biomass is ordinarily harvested and processed (downstream program). Drying, grinding, and extraction steps are destructive to the microalgal biomass that then needs to be renewed. The extraction and purification processes generate organic wastes and require substantial energy inputs. Altogether, it is urgent to develop alternative downstream processes. Among the possibilities, milking invokes the concept that the extraction should not kill the algal cells. Therefore, it does not require growing the algae anew. In this review, we discuss research on milking of diatoms. The main themes are (a) development of alternative methods to extract and harvest high added value compounds; (b) design of photobioreactors; (c) biodiversity and (d) stress physiology, illustrated with original results dealing with oleaginous diatoms

Comparison of two Phaeodactylum tricornutum ecotypes under nitrogen starvation and resupply reveals distinct lipid accumulation strategies but a common degradation process

IntroductionPhaeodactylum tricornutum is a model species frequently used to study lipid metabolism in diatoms. When exposed to a nutrient limitation or starvation, diatoms are known to accumulate neutral lipids in cytoplasmic lipid droplets (LDs). Those lipids are produced partly de novo and partly from the recycle of plastid membrane lipids. Under a nitrogen resupply, the accumulated lipids are catabolized, a phenomenon about which only a few data are available. Various strains of P. tricornutum have been isolated around the world that may differ in lipid accumulation patterns.MethodsTo get further information on this topic, two genetically distant ecotypes of P. tricornutum (Pt1 and Pt4) have been cultivated under nitrogen deprivation during 11 days followed by a resupply period of 3 days. The importance of cytoplasmic LDs relative to the plastid was assessed by a combination of confocal laser scanning microscopy and cell volume estimation using bright field microscopy pictures.Results and discussionWe observed that in addition to a basal population of small LDs (0.005 μm3 to 0.7 μm3) present in both strains all along the experiment, Pt4 cells immediately produced two large LDs (up to 12 μm3 after 11 days) while Pt1 cells progressively produced a higher number of smaller LDs (up to 7 μm3 after 11 days). In this work we showed that, in addition to intracellular available space, lipid accumulation may be limited by the pre-starvation size of the plastid as a source of membrane lipids to be recycled. After resupplying nitrogen and for both ecotypes, a fragmentation of the largest LDs was observed as well as a possible migration of LDs to the vacuoles that would suggest an autophagic degradation. Altogether, our results deepen the understanding of LDs dynamics and open research avenues for a better knowledge of lipid degradation in diatoms

Bioinformatics-Based Screening Approach for the Identification and Characterization of Lipolytic Enzymes from the Marine Diatom <i>Phaeodactylum tricornutum</i>

Oleaginous diatoms accumulate lipids of biotechnological interest when exposed to nutrient stress conditions such as nitrogen starvation. While accumulation mechanisms are well-known and have been engineered to improve lipid production, degradation mechanisms remain poorly investigated in diatoms. Identifying lipid-degrading enzymes is the initial step to understanding the catabolic processes. In this study, an in silico screening of the genome of Phaeodactylum tricornutum led to the identification of 57 putative triacylglycerol lipases (EC 3.1.1.3) grouped in 4 families. Further analysis revealed the presence of conserved domains and catalytic residues of lipases. Physico-chemical characteristics and subcellular localization predictions highlighted that a majority of these putative proteins are hydrophilic and cytosolic, suggesting they could be recruited to lipid droplets directly from the cytosol. Among the 57 identified putative proteins, three lipases were identified as possibly involved in lipophagy due to a potential vacuolar localization. The expression of the mRNA corresponding to the 57 proteins was then searched in 3 transcriptomic datasets obtained under nitrogen starvation. Nine genes were highly regulated and were considered as encoding enzymes with a probable important function in lipid catabolism. A tertiary structure prediction of these nine candidates yielded eight functional 3D models. Among those, two downregulated enzymes, Phatr3_J54974 and Phatr3_EG00720, were highlighted as good targets for future functional genomics and purification studies to investigate their role in lipid degradation

Lipase-catalyzed production of lysophospholipids

Lysophospholipids, such as lysophosphatidic acid or lysophosphatidylcholine, are important bioactive lipids, involved in various normal and pathological cellular processes. They also have industrial and pharmaceutical uses such as emulsifiers or components of drug delivery systems. Lipases, which natural substrates are long chain triacylglycerols, are important biocatalysts for organic synthesis mainly due to their broad substrate specificity and their ability to display high catalytic activity in organic media. This paper describes the various lipase-catalyzed reactions implemented for the production of lysophospholipids. They include hydrolysis or alcoholysis of phospholipids and acylation of the glycerophosphoryl moiety. Special emphasis is made on our work dealing with the production of lysophospholipids rich in dososahexaenoic acid, an important dietary polyunsaturated fatty acid via the hydrolysis of phospholipids extracted from the microalga Isochrysis galbana

Lipase-catalyzed production of lysophospholipids

Lysophospholipids, such as lysophosphatidic acid or lysophosphatidylcholine, are important bioactive lipids, involved in various normal and pathological cellular processes. They also have industrial and pharmaceutical uses such as emulsifiers or components of drug delivery systems. Lipases, which natural substrates are long chain triacylglycerols, are important biocatalysts for organic synthesis mainly due to their broad substrate specificity and their ability to display high catalytic activity in organic media. This paper describes the various lipase-catalyzed reactions implemented for the production of lysophospholipids. They include hydrolysis or alcoholysis of phospholipids and acylation of the glycerophosphoryl moiety. Special emphasis is made on our work dealing with the production of lysophospholipids rich in dososahexaenoic acid, an important dietary polyunsaturated fatty acid via the hydrolysis of phospholipids extracted from the microalga Isochrysis galbana

Lipids and lipolytic enzymes of the microalga Isochrysis galbana

Marine microalgae are now well-known for their ability to produce omega-3 long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Among these microalgae, Isochrysis galbana has received increasing interest especially because of its high DHA content and its common use in hatchery to feed fish larvae and clams. Moreover, lipolysis occurring from the biomass harvest stage suggests that I. galbana may contain lipolytic enzymes with potential interesting selectivities. For these reasons, the potential of this microalga for the production of valuable lipids and lipolytic enzymes was investigated. Lipid analysis revealed that DHA is mainly located at the sn-2 position of the phospholipids. Thus, I. galbana was considered as an interesting starting material for the lipase catalyzed production of 1-lyso-2-DHA-phospholipids which are considered as convenient vehicles for the conveyance of DHA to the brain. Lipids from I. galbana can also be used for the enzyme-catalyzed production of structured phospholipids containing one DHA and one medium chain fatty acid in order to combine interesting therapeutic and biological benefits. Starting from total RNA extract from I. galbana, coding sequences of putative lipolytic enzymes were obtained by RACE and Nested PCR. The heterologous expression of a sequence designated IgTeCe was implemented. An expression plasmid was constructed by ligating the coding sequence to a plasmid vector and then cloned and expressed in E. coli. Results showed the effective functionality of plasmid construction for the production of a recombinant protein with the expected molecular mass. Moreover, local alignment using BLASTP and biochemical evidences support the hypothesis that the expressed protein is a thioesterase

Lipides et enzymes lipolytiques de la microalgue Isochrysis galbana

International audienceMarine microalgae are now well-known for their ability to produce omega-3 long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Among these microalgae, Isochrysis galbana has received increasing interest especially because of its high DHA content and its common use in hatchery to feed fish larvae and clams. Moreover, lipolysis occurring from the biomass harvest stage suggests that I. galbana may contain lipolytic enzymes with potential interesting selectivities. For these reasons, the potential of this microalga for the production of valuable lipids and lipolytic enzymes was investigated. Lipid analysis revealed that DHA is mainly located at the sn-2 position of the phospholipids. Thus, I. galbana was considered as an interesting starting material for the lipase catalyzed production of 1-lyso-2-DHA-phospholipids which are considered as convenient vehicles for the conveyance of DHA to the brain. Lipids from I. galbana can also be used for the enzyme-catalyzed production of structured phospholipids containing one DHA and one medium chain fatty acid in order to combine interesting therapeutic and biological benefits. Starting from total RNA extract from I. galbana, coding sequences of putative lipolytic enzymes were obtained by RACE and Nested PCR. The heterologous expression of a sequence designated IgTeCe was implemented. An expression plasmid was constructed by ligating the coding sequence to a plasmid vector and then cloned and expressed in E. coli. Results showed the effective functionality of plasmid construction for the production of a recombinant protein with the expected molecular mass. Moreover, local alignment using BLASTP and biochemical evidences support the hypothesis that the expressed protein is a thioesterase. Keywords: microalgae / v-3 polyunsaturated fatty acids / phospholipids / lipolytic enzymes Résumé-Lipides et enzymes lipolytiques de la microalgue Isochrysis galbana. Les microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série v-3 comme l'acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérêt croissant du fait d'un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D'autre part, la lipolyse souvent observée dès l'étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu'I. galbana pourrait contenir des enzymes lipolytiques intéressantes en termes de sélectivité. Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d'intérêt et d'enzymes lipolytiques a été étudié. L'analyse des lipides d'I. galbana a tout d'abord révélé que le DHA était majoritairement greffé sur la position sn-2 des phospholipides. Dans ce contexte, les phospholipides d'I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2-DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. À partir des lipides d'I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants. À partir des ARN totaux d'I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L'expression hétérologue d'une séquence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que laLes microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série ω-3 comme l’acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérèt croissant du fait d’un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D’autre part, la lipolyse souvent observée dès l’étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu’I. galbana pourrait contenir des enzymes lipolytiques intéressantes en terme de sélectivité.Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d’intérêt et d’enzymes lipolytiques a été étudié.L’analyse des lipides d’I. galbana a tout d’abord revélé que le DHA était majoritairement greffé sur la position sn-2 des phopholipides. Dans ce contexte, les phospholipids d’I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2–DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. A partir des lipides d’I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants.A partir des ARN totaux d’I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L’expression hétérologue d’une sequence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que la construction plasmidique permettait bien d’obtenir une protéine recombinante avec la masse moléculaire attendue. D’autre part, l’outil d’alignement local de séquences, BLASTP, ainsi que des données biochimiques ont permis de confirmer l’hypothèse que la protéine obtenue était une thioestérase

Optimization of protein electroextraction from microalgae by a flow process

International audienceClassical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24 h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size

Diatom Milking: A Review and New Approaches

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Correction: Vinayak, V., et al. Diatom Milking: A Review and New Approaches. Marine Drugs 2015, 13, 2629–2665

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