Improved DNA/protein delivery in microalgae – A simple and reliable method for the prediction of optimal electroporation settings

Muñoz, Camilo F.; Jaeger, Lenny de; Sturme, Mark H. J.; Lip, Ka Y.F.; Olijslager, Justus W.J.; Springer, Jan; Wolbert, E.J.H.; Martens, Dirk E.; Eggink, Gerrit; Weusthuis, Ruud A.; Wijffels, René H.

doi:10.1016/j.algal.2018.06.021

Cited by 40 publications

(13 citation statements)

References 30 publications

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“…N. oleoabundans cells were harvested and washed in electroporation buffer as described by Muñoz et al (2018). The transformation mixture containing 2 µg · ml -1 of linearized plasmid and 25 µg · ml -1 of boiled salmon sperm DNA (D1626, Sigma) were incubated on ice for 15 min.…”

Section: Methodsmentioning

confidence: 99%

“…Antibiotic resistant colonies obtained on plate were transferred to new plates containing 75 µg · ml -1 of hygromycin B. Identification of positive transformants was performed by PCR amplification of the hygromycin resistance gene ( aph7 ) with primers used by Munoz et al (2018). DNA extraction and colony PCR were performed by using Phire Plant Direct PCR Master Mix (ThermoFisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

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Effect of Single and Combined Expression of Lysophosphatidic Acid Acyltransferase, Glycerol-3-Phosphate Acyltransferase, and Diacylglycerol Acyltransferase on Lipid Accumulation and Composition in Neochloris oleoabundans

et al. 2019

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Microalgal lipids are promising feedstocks for food and biofuels. Since lipid production by microalgae is not yet economically feasible, genetic engineering is becoming a promising strategy to achieve higher lipid accumulation and productivities. Enzymes involved in the Kennedy pathway such as glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT) catalyze key steps in the formation of triacylglycerol, which is the main constituent of lipids in N. oleoabundans. The overexpression of these enzymes in the targeted strain has a great potential to further increase their triacylglycerol content. We overexpressed single and multiple encoding genes for LPAT, GPAT, and DGAT from Acutodesmus obliquus in N. oleoabundans. Strains overexpressing single genes produced up to 52% and 45% g · gDW-1, which corresponds to 1.3- and 1.4-fold increase in total fatty acids and triacylglycerols, respectively. The orchestrated expression of the three genes resulted in 49% and 39% g · gDW-1, which is 1.2-folds increase in total fatty acids and triacylglycerols. Single expression of LPAT, GPAT, and DGAT genes resulted in higher lipid productivities during starvation without a significant effect on growth and photosynthetic activity during replete conditions. On the other hand, the simultaneous expression of LPAT, GPAT, and DGAT genes resulted in 52% lower growth rate, 14% lower photosynthetic activity and 4-folds increase in cell diameter. Moreover, the multigene expressing line showed a decrease in carbohydrates and protein content and an increase in pigments during nitrogen starved condition. The single and multiple expression of heterologous genes LPAT, GPAT, and DGAT showed to significantly enhanced the lipid accumulation in N. oleoabundans. Single gene expression resulted in higher lipid production and productivities without having a significant impact in the physiological status of the strains. This approach shows the potential for the generation of microalgal strains with higher economical potential for the production of lipids.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effect of Single and Combined Expression of Lysophosphatidic Acid Acyltransferase, Glycerol-3-Phosphate Acyltransferase, and Diacylglycerol Acyltransferase on Lipid Accumulation and Composition in Neochloris oleoabundans

et al. 2019

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“…The T. suecica cells will be placed in 6-well plates and 250 V will be applied for electroporation [31]. This technique will increase the permeability of cell membranes by placing cells in an electric field [36]. The cells will be transfected with 2.0 µg Cas-9 RNP and 0.4 µg of the linear HSP90 HA plasmid [37].…”

Section: Methodsmentioning

confidence: 99%

Genetic Modification of the HSP90 Gene Using CRISPR-Cas9 to Enhance Thermotolerance in T. Suecica

Soni

Chopra

et al. 2020

URNCST Journal

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Phytoplankton are marine microorganisms that play a key role in the production of oxygen and serve as the foundation of the marine food chain. Over the past century, the population of phytoplankton has declined significantly with the onset of climate change. Although phytoplankton have the capacity to adapt to rising ocean temperatures, rapid environmental changes, including increased top-down control and thermal stratification, reduce populations before adaptations are incorporated into the genome. To enhance survival rates, thermotolerance in common algal strains can be enhanced through increased expression of the conserved Heat Shock Protein 90 (HSP90). Trials will be conducted on the common algal species, Tetraselmis suecica (T. suecica), for its considerable size, photosynthetic rate, and nutrient-rich properties. Thermotolerance will be augmented by splicing the HSP90 gene into the T. suecica metallothionein (Mt) promoter using CRISPR-Cas9. A period of incubation in a copper sulphate solution ensures Mt promoter stimulation, thereby increasing HSP90 expression. The efficacy of the proposed methods will be measured by comparing HSP90 protein production between transgenic and wild-type T. suecica cultures. The genomic incorporation of the modified HSP90 gene enables future populations to exhibit thermotolerance in the presence of heavy metals in the ocean beyond its basal level of expression. By accelerating the adaptation of thermotolerance, the overall fitness of T. suecica can be increased to re-establish its population under warmer oceanic conditions. By applying similar methods to other phytoplankton, the repopulation of various species can increase biodiversity and global net primary productivity.

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“…One of the most often used methods for the genetic transformation of microalgae is electroporation (Muñoz et al, 2018) that requires the incoming DNA to pass through the cell wall (pellicle) and cell membrane. Electroporation has been successfully applied for a range of microalgae including Chlamydomonas reinhardtii, Chlorella vulgaris, Neochloris oleoabundans , and Acutodesmus obliquus (Muñoz et al, 2018; Nelson & Lefebvre, 1995; Shimogawara, Fujiwara, Grossman, & Usuda, 1998). Electroporation of E. gracilis was for the first time reported by Khatiwada, Kautto, Sunna, Sun, and Nevalainen (2019), however, despite optimization of the voltage strength, concentration of the transforming DNA and using linear DNA to drive integration into the genome, stable transformants were not obtained.…”

Section: Molecular Characteristics and Tools Of E Gracilismentioning

confidence: 99%

Section: Molecular Characteristics and Tools Of E Gracilismentioning

confidence: 99%

Molecular tools and applications of Euglena gracilis: From biorefineries to bioremediation

Khatiwada

Sunna

Nevalainen

2020

Biotech & Bioengineering

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Euglena gracilis is a promising source of commercially important metabolites such as vitamins, wax esters, paramylon, and amino acids. However, the molecular tools available to create improved Euglena strains are limited compared to other microorganisms that are currently exploited in the biotechnology industry. The complex How to cite this article: Khatiwada B, Sunna A, Nevalainen H. Molecular tools and applications of Euglena gracilis: From biorefineries to bioremediation. Biotechnology and Bioengineering.

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Improved DNA/protein delivery in microalgae – A simple and reliable method for the prediction of optimal electroporation settings

Cited by 40 publications

References 30 publications

Effect of Single and Combined Expression of Lysophosphatidic Acid Acyltransferase, Glycerol-3-Phosphate Acyltransferase, and Diacylglycerol Acyltransferase on Lipid Accumulation and Composition in Neochloris oleoabundans

Effect of Single and Combined Expression of Lysophosphatidic Acid Acyltransferase, Glycerol-3-Phosphate Acyltransferase, and Diacylglycerol Acyltransferase on Lipid Accumulation and Composition in Neochloris oleoabundans

Genetic Modification of the HSP90 Gene Using CRISPR-Cas9 to Enhance Thermotolerance in T. Suecica

Molecular tools and applications of Euglena gracilis: From biorefineries to bioremediation

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