Genome engineering via gene editing technologies in microalgae.

CRISPR-Cas has revolutionized genetic modification with its comparative simplicity and accuracy, and it can be used even at the genomic level. Microalgae are excellent feedstocks for biofuels and nutraceuticals because they contain high levels of fatty acids, carotenoids, and other metabolites; however, genome engineering for microalgae is not yet as developed as for other model organisms. Microalgal engineering at the genetic and metabolic levels is relatively well established, and a few genomic resources are available.

Aureochromes maintain polyunsaturated fatty acid content in Nannochloropsis oceanica.

Nannochloropsis oceanica, like other stramenopile microalgae, is rich in long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA). We observed that fatty acid desaturases (FADs) involved in LC-PUFA biosynthesis were among the strongest blue light-induced genes in N. oceanica CCMP1779.

Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS).

Manipulating fatty-acid profile at unit chain-length resolution in the model industrial oleaginous microalgae Nannochloropsis.

The chain length (CL) of fatty acids (FAs) is pivotal to oil property, yet to what extent it can be customized in industrial oleaginous microalgae is unknown. In Nannochloropsis oceanica, to modulate long-chain FAs (LCFAs), we first discovered a fungi/bacteria-originated polyketide synthase (PKS) system which involves a cytoplasmic acyl-ACP thioesterase (NoTE1). NoTE1 hydrolyzes C16:0-, C16:1- and C18:1-ACP in vitro and thus intercepts the specific acyl-ACPs elongated by PKS for polyunsaturated FA biosynthesis, resulting in elevation of C16/C18 monounsaturated FAs when overproduced and increase of C20 when knocked out.

Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages.

Industrial microalgae are promising photosynthetic cell factories, yet tools for large-scale targeted genome engineering are limited. Here for the model industrial oleaginous microalga Nannochloropsis oceanica, we established a method to precisely and serially delete large genome fragments of ~100 kb from its 30.01 Mb nuclear genome.

The NanDeSyn database for Nannochloropsis systems and synthetic biology.

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.

Genetic Impairment of Cellulose Biosynthesis Increases Cell Wall Fragility and Improves Lipid Extractability from Oleaginous Alga .

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene () expression would lead to protein accumulation without the accumulation of storage C polymers in .

Development and characterization of a mutant with simultaneously enhanced growth and lipid production.

Background: The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of to improve their growth and lipid contents have been published.

Heterologous synthesis of chlorophyll in enhances growth and lipid production by increasing photosynthetic efficiency.

Background: Chlorophylls play important roles in photosynthesis, and thus are critical for growth and related metabolic pathways in photosynthetic organisms. They are particularly important in microalgae, emerging as the next generation feedstock for biomass and biofuels. are industrial microalgae for these purposes, but are peculiar in that they lack accessory chlorophylls.

Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor.

Microalgae are promising feedstocks for sustainable and eco-friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix-loop-helix transcription factor (NsbHLH2).

Isolation, phenotypic characterization and genome wide analysis of a strain naturally modified under laboratory conditions: towards enhanced microalgal biomass and lipid production for biofuels.

Background: Microalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing.

Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris.

Photosynthesis of microalgae enables conversion of light energy into chemical energy to produce biomass and biomaterials. However, the efficiency of this process must be enhanced, and truncation of light-harvesting complex (LHC) has been suggested to improve photosynthetic efficiency. We reported an EMS-induced mutant (E5) showing partially reduced LHC in Chlorella vulgaris.

Current status and perspectives of genome editing technology for microalgae.

Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases.

Increased lipid production by heterologous expression of AtWRI1 transcription factor in .

Background: Genetic engineering of microalgae is necessary to produce economically feasible strains for biofuel production. Current efforts are focused on the manipulation of individual metabolic genes, but the outcomes are not sufficiently stable and/or efficient for large-scale production of biofuels and other materials. Transcription factors (TFs) are emerging as good alternatives for engineering of microalgae, not only to increase production of biomaterials but to enhance stress tolerance.

Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina.

Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel-based fuels and chemicals. Genetic engineering of microalgae is prerequisite to maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials.

Transcriptional Regulation of Cellulose Biosynthesis during the Early Phase of Nitrogen Deprivation in Nannochloropsis salina.

Microalgal photosynthesis provides energy and carbon-containing precursors for the biosynthesis of storage carbohydrates such as starch, chrysolaminarin, lipids, and cell wall components. Under mild nitrogen deficiency (N-), some Nannochloropsis species accumulate lipid by augmenting cytosolic fatty acid biosynthesis with a temporary increase in laminarin. Accordingly, biosynthesis of the cellulose-rich cell wall should change in response to N- stress because this biosynthetic pathway begins with utilisation of the hexose phosphate pool supplied from photosynthesis.

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9.

Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina.

Background: Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes.

Heterologous overexpression of sfCherry fluorescent protein in .

Oleaginous microalgae of the genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Sh under the control of TUB and UEP promoters, cloned from , was used to confer resistance to Zeocin antibiotics, resulting in 5.

Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana.

Triiodide resin (TR) was used to generate axenic cultures of microalgae by employing the antibacterial capability of triiodide. A Nannochloropsis gaditana culture contaminated with bacteria was passed through a column filled with TR using the gravity flow. Based on analyses of flow cytometry and vital staining using a fluorescent dye SYTOX Green, three cycles of TR treatments remarkably reduced the number of viable bacteria but had little effects on the microalgae.

Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7.

Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III-secreted effector HopU1 is a mono-ADP-ribosyltransferase required for full virulence on Arabidopsis thaliana. HopU1 targets several RNA-binding proteins including GRP7, whose role in immunity is still unclear.

Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity.

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.

The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas.

Changes in chromatin organization are emerging as key regulators in nearly every aspect of DNA-templated metabolism in eukaryotes. Histones undergo many, largely reversible, posttranslational modifications that affect chromatin structure. Some modifications, such as trimethylation of histone H3 on Lys 4 (H3K4me3), correlate with transcriptional activation, whereas others, such as methylation of histone H3 on Lys 27 (H3K27me), are associated with silent chromatin.

A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity.

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure.

Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas.

Histone Lys methylation plays an important role in determining chromatin states and is mostly catalyzed by SET domain-containing proteins. The outcome, transcriptional repression or activation, depends on the methylated histone residue, the degree of methylation, and the chromatin context. Dimethylation or trimethylation of histone H3 Lys 4 (H3K4me2 or H3K4me3) has been correlated with transcriptionally competent/active genes.