Fantao Kong
email: kongfantaohit@163.com

Yasuyo Yamaoka

Takeshi Ohama

Youngsook Lee

Yonghua Li- Beisson

Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii

Keywords: Chlamydomonas reinhardtii, Genome Editing, Metabolic Engineering, Oil content, Transgene Expression, Triacylglycerol amiRNA, artificial microRNA, CRISPR, clustered regularly interspaced short fatty acid, FP, fluorescent protein, gRNA, guide RNA, N, nitrogen, RNAi, RNA interference, RNPs, ribonucleoproteins, S, sulfur, TAG, triacylglycerol, TALEN, Chlamydomonas reinhardtii, Genome Editing, Metabolic Engineering, Oil content, Transgene Expression, Triacylglycerol

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organismis is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-theart genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis, and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.

Introduction

Microalgae represent a polyphyletic group of photosynthetic eukaryotic microorganisms, and are sources of a number of value-added products, including proteins, pigments, carbohydrates, carotenoids, vitamins, anti-oxidants, and polyunsaturated very long chain fatty acids [START_REF] Chew | Microalgae biorefinery: High value products perspectives[END_REF]. Due to their high biomass productivity and diverse ecological habitats, microalgae have recently been considered an alternative sustainable resource for biofuels. However, the sustainable industrial production of oil, i.e., triacylglycerols (TAGs), from any microalgal species has yet to be realized, due to bottlenecks at both the biological and biotechnological levels. One of the most challenging issues is to increase oil productivity, because most microalgae accumulate large amounts of oil only when subjected to stress conditions (e.g., nutrient depletion), when cell growth is usually impaired [START_REF] Merchant | TAG, You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF]. Therefore, current research is intensively focused on deciphering the molecular mechanisms underlying lipid biosynthesis and the relationship between lipid biosynthesis and biomass growth [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances[END_REF], Kong et al. 2018a).

Among microalgal species, the unicellular eukaryotic green alga Chlamydomonas reinhardtii (hereafter, Chlamydomonas) is a well-established algal model, due to its haploid genotype, fast growth, and sequenced genome [START_REF] Merchant | The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions[END_REF]. Chlamydomonas is commonly used to study a number of biological processes, including photosynthesis and flagella-mediated motility. Over the past decade, spurred on by a renewed interest in microalgal oil, Chlamydomonas has emerged as a model organism for studies of lipid metabolism [START_REF] Du | Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae[END_REF]Benning, 2016, Li-Beisson et al. 2015).

Furthermore, the emergence of synthetic biology provides an opportunity to expedite the development of Chlamydomonas and other microalgal species as a useful industrial biotechnology platform. Synthetic biology, which is based on a combination of standardized biological components (e.g., promoters, terminators, and selection markers), predictive modeling, and iterative design and testing, was first used in bacterial systems and has since advanced to eukaryotic systems, including algae [START_REF] Crozet | Birth of a photosynthetic chassis: a MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii[END_REF]. A modular cloning toolkit, comprising 119 functionally validated genetic parts, was developed for Chlamydomonas based on Golden Gate cloning with standard syntax [START_REF] Crozet | Birth of a photosynthetic chassis: a MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii[END_REF].

This toolkit has established Chlamydomonas as the next chassis for sustainable synthetic biology.

Here, we present an overview of molecular approaches, such as metabolic engineering and gene disruption strategies, that enable the genetic manipulation of microalgae with the

Gene delivery tools and selection systems for Chlamydomonas

Transformation methods

Various techniques have been established to deliver DNA into the Chlamydomonas nucleus, including biolistics, electroporation, Agrobacterium-mediated transformation, or even simply vortexing with glass beads, silicon carbide whiskers, or 3-aminopropyl-functionalized magnesium (reviewed by [START_REF] Jinkerson | Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome[END_REF]. Biolistic transformation was the first method developed for this purpose and was mediated by microprojectiles, such as tungsten powder and gold particles, and, more recently, by size-controlled mesoporous TiO 2 nanoparticles, which are effective at lower density and are less prone to aggregation than are tungsten or gold particles [START_REF] Hou | Mesoporous TiO2 nanoparticles: A new material for biolistic bombardment[END_REF]. Biolistic methods can be used to transform nuclear, chloroplastic, and mitochondrial genomes. This method is efficient but expensive due to the requirement for specialized equipment. Consequently, cheaper and simpler methods (e.g., Agrobacterium-mediated transformation and electroporation) have been developed.

Agrobacterium-mediated transformation yields a slightly lower proportion of transgenesilenced transformants than does electroporation, but does not provide significant advantages over electroporation-mediated transformation in Chlamydomonas, due to its labor-intensive nature and large number of false positives [START_REF] Mini | Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study[END_REF]. Thus, electroporation is currently one of the most efficient methods for delivering DNA into the nuclei of cells [START_REF] Shimogawara | High-efficiency transformation of Chlamydomonas reinhardtii by electroporation[END_REF][START_REF] Yamano | Rapid transformation of Chlamydomonas reinhardtii without cell-wall removal[END_REF].

Selectable markers and reporter genes

Several antibiotic resistance genes have been developed for nuclear or chloroplast transformation in Chlamydomonas. Among these, aadA, encoding an aminoglycoside 3'adenylyltransferase (spectinomycin and streptomycin resistance), is widely used in both nuclear and chloroplast transformation [START_REF] Meslet-Cladière | Novel Shuttle Markers for Nuclear Transformation of the Green Alga Chlamydomonas reinhardtii[END_REF]. The GC-rich ble gene, conferring resistance to zeocin, is an effective selectable marker for efficient transgene expression when fused to the target gene [START_REF] Fuhrmann | A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii[END_REF][START_REF] Plucinak | Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii[END_REF], but the underlying mechanism is not well understood. It is possible that bleomycin resistance proteins (BLEs) function by sequestering zeocin in a one-to-one ratio, but not through enzymatic [START_REF] Rasala | Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii[END_REF]. However, the use of ble as a selectable maker is limited, because zeocin is light sensitive and may function as a mutagen when applied at high concentrations [START_REF] Lin | Verification of mutagen function of Zeocin in Nannochloropsis oceanica through transcriptome analysis[END_REF]. This prompted the development of other selectable markers, such as aphVIII and aph7″, which confer paromomycin and hygromycin-resistance, respectively. AphVIII is currently one of the most widely used selectable marker in Chlamydomonas, because it confers a stable drug-resistant phenotype [START_REF] Meslet-Cladière | Novel Shuttle Markers for Nuclear Transformation of the Green Alga Chlamydomonas reinhardtii[END_REF].

In addition to selectable markers, reporter genes have also become important tools in microalgal genetic engineering. For instance, to monitor gene expression and protein localization in vivo, various fluorescent proteins covering most fluorescent ranges (i.e., yellow YFP, blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange tdTomato, and red mCherry) have been adapted for optimal expression in Chlamydomonas [START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF][START_REF] Rasala | Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii[END_REF].

Forward genetics in Chlamydomonas

Mutagenesis

Mutagenesis mediated by mutagens (e.g, ethylmethanesulfonate) has been used to generate a library of Chlamydomonas mutants that was subjected to forward genetic screens for altered oil content [START_REF] Xie | Highthroughput fluorescence-activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants[END_REF][START_REF] Lee | Enhancement of lipid productivity by ethyl methane sulfonate-mediated random mutagenesis and proteomic analysis in Chlamydomonas reinhardtii[END_REF]). However, a major drawback of using these mutagens is that point mutations are randomly produced, making it challenging to identify the causative gene(s) for the phenotype.

Random insertional mutagenesis of Chlamydomonas using drug resistance cassettes, which randomly integrate exogenous DNA into the nuclear genome, has been used to generate tens of thousands of transformants in numerous forward genetics studies. Insertional mutants have been generated at large scale and screened for phenotypes related to lipid metabolism, motility, and photosynthesis [START_REF] Cagnon | Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii[END_REF], Cheng et al. 2017[START_REF] Dent | Large-scale insertional mutagenesis of Chlamydomonas supports phylogenomic functional prediction of photosynthetic genes and analysis of classical acetate-requiring mutants[END_REF]. An advantage of this approach is that insertional mutations can be mapped without the need for extensive genetic crosses.

Examples of medium-to-high-throughput screening developed for Chlamydomonas

Oil content can be estimated by staining with fluorescent dyes (e.g., Nile red and BODIPY) and can be combined flow cytometry analysis for high-throughput studies. This strategy has been used to screen for Chlamydomonas mutants with altered oil content [START_REF] Cagnon | Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii[END_REF], Cheng et al. 2017[START_REF] Tsai | The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas[END_REF]. Fluorescence-activated cell sorting of cells that have been stained with Nile red allows for the efficient screening of tens of thousands of Chlamydomonas mutants [START_REF] Terashima | A fluorescenceactivated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants[END_REF][START_REF] Xie | Highthroughput fluorescence-activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants[END_REF]. This method also allows for quantification of lipid content while preserving cell viability, as Nile red staining does not hinder growth, and requires only five weeks from mutagenesis to mutant isolation [START_REF] Terashima | A fluorescenceactivated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants[END_REF].

Another approach commonly used to study lipid metabolism is a screen for mutants with altered FA composition. Differences in FA composition can be analyzed using a gas chromatography-flame ionization detector (GC-FID) after lipids are converted to their fatty acid methyl esters. Direct transmethylation of cell pellets by acid-catalyzed transmethylation coupled with a robotized GC-FID was developed for high-throughput screening for Chlamydomonas mutants with altered FA composition [START_REF] Nguyen | The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids[END_REF]. Using this method, we previously isolated a mutant defective in the ω-3 FA desaturase (named crfad7) in a screen of 2,000 insertional mutants [START_REF] Nguyen | The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids[END_REF]. CrFAD7 is localized to plastids and contributes to the formation of all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid species [START_REF] Nguyen | The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids[END_REF].

Genetic mapping of insertional mutants

Various PCR-based techniques have been developed to identify insertion sites within the nuclear genome of Chlamydomonas, including Genome Walker, restriction enzyme sitedirected amplification-PCR (RESDA-PCR), thermal asymmetric interlaced-PCR, 3-rapid amplification of cDNA ends, SiteFinding-PCR, and hairpin-PCR (reviewed by [START_REF] Li | High-Throughput Genetics Strategies for Identifying New Components of Lipid Metabolism in the Green Alga Chlamydomonas reinhardtii[END_REF]. Among these methods, RESDA-PCR is the most commonly used and offers a high probability of identifying the insertion sites (González-Ballester et al. 2005, Kong andLi-Beisson 2018). However, this approach is labor-intensive and only identifies a handful of insertion sites at once, rendering it unsuitable for the simultaneous mapping of tens of thousands of Chlamydomonas insertional mutants. Additionally, the success of these PCRbased approaches is determined largely by the PCR product size, and requires intact PCR primer binding sites in the cassette, which are occasionally disturbed by cassette truncation, concatemerization after integration, and the presence of "junk DNA" from a distant locus (Li et al. 2016).

Prompted by these challenges, a method for mapping insertion cassettes in pools of [START_REF] Zhang | High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA[END_REF]. Approximately >100,000 insertions were successfully mapped using ChlaMmeSeq (CLiP:https://www.chlamylibrary.org/). In this method, a type II restriction enzyme site (MmeI) that facilitates subsequent high-throughput sequencing is included at both ends of the transformation cassette. However, in some instances, upon fragmentation of the cassette, the binding sites for sequencing primers are eliminated, making it difficult or impossible to use ChlaMmeSeq to map that insertion site.

Due to recent decreases in the cost of sequencing technologies, whole-genome re-sequencing has been used to identify point mutations and map target genes. Using this approach, we previously identified the site of genetic lesion for a Chlamydomonas mutant named constitutive-oil-accumulation 1 (coa1), which was found to harbor a >200 kb deletion [START_REF] Goold | Whole Genome Re-Sequencing Identifies a Quantitative Trait Locus Repressing Carbon Reserve Accumulation during Optimal Growth in Chlamydomonas reinhardtii[END_REF].

Reverse genetics in Chlamydomonas

A successful reverse genetic approach relies on the selection of sutiable target gene(s) for genetic manipulation. There are two major ways to identify and select target genes in Chlamydomonas. First, Chlamydomonas genes that are homologous to ones already known to increase seed oil content in terrestrial plants or in other organisms can be identified. This case requires selection of the genes and rigorous confirmation of the activity of the proteins encoded by the genes. However, many genes in the Chlamydomonas database are not well annotated or characterized. In general, the Chlamydomonas genome most closely resembles those of plants, followed by those of animals and fungi. This is particularly the case for genes involved in lipid metabolism, as de novo FA synthesis occurs in the chloroplast, an organelle common to plants and algae. However, the flagellar and basal body proteins tend to show greater similarity to those of animals [START_REF] Merchant | The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions[END_REF]). To select suitable target genes for reverse genetics, one can search for genes homologous to those already reported as being effective in model plant databases, such as Arabidopsis TAIR (https://www.arabidopsis.org).

Further steps in selecting a target gene may include constructing phylogenetic trees; aligning the amino acid sequences of the predicted proteins; performing a subcellular localization analysis, using PredAlgo [START_REF] Tardif | PredAlgo: A New Subcellular Localization Prediction Tool Dedicated to Green Algae[END_REF]; and verifying the expression profiles, using the Chlamy Network Portal (http://networks.systemsbiology.net/chlamy-portal/) or AlCOdb (http://alcodb.jp/coexpression/search). Second, candidate genes can be identified in comparative transcriptomic studies, gene co-expression analyses, or proteomics studies. Acyltransferases and lipid droplet-associated proteins are of particular interest to many scientists working in the field of lipid production.

Strategies to improve nuclear transgene expression

Codon optimization

Although foreign genes are easily integrated into the nuclear genome, transgene expression levels are notoriously poor in Chlamydomonas [START_REF] Kong | Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains[END_REF][START_REF] Lumbreras | Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron[END_REF][START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF]). The efficiency of gene expression partially depends on the nucleotide composition (particularly the GC content) of the inserted DNA fragment and on its consistency with the codon-usage of the host organism [START_REF] Barahimipour | Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii[END_REF][START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF]. Chlamydomonas coding sequences have an average GC content of 68% [START_REF] Merchant | The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions[END_REF]). An unfavorable GC content affects gene expression at the level of chromatin structure. Recently, codon usage was reported to be more critical than GC content for gene expression efficiency in Chlamydomonas [START_REF] Barahimipour | Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii[END_REF]. Codon optimization through gene re-synthesis improves transgene expression, even if Chlamydomonas contains a high GC content.

Choice of promoters

Both constitutive and inducible promoters have been developed for Chlamydomonas.

Established constitutive promoters present in Chlamydomonas include the Hsp70A, RbcS2, and PsaD promoters and the Hsp70A-RbcS2 chimeric promoter, and the inducible promoters include those present in the copper-(Cyc6) and alcohol-inducible (AlcR-P alcA ) promoter systems [START_REF] Fischer | The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii[END_REF][START_REF] Lee | Development of an alcohol-inducible gene expression system for recombinant protein expression in Chlamydomonas reinhardtii[END_REF][START_REF] Quinn | Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators[END_REF]. The AlcR-P alcA system is composed of a regulatory protein (AlcR) and a promoter controlling alcA expression (P alcA ), which has previously been used for inducible transgene expression in plants, and was also recently used in Chlamydomonas [START_REF] Lee | Development of an alcohol-inducible gene expression system for recombinant protein expression in Chlamydomonas reinhardtii[END_REF]). In addition to the aforementioned traditional promoters, [START_REF] Scranton | Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii[END_REF] generated a set of synthetic algal promoters (e.g., sap11) that contain a new DNA motif that is essential for promoter function and highly conserved in Chlamydomonas. The synthetic algal promoters were shown to drive higher expression of transgenes in Chlamydomonas than the strongest known endogenous promoter, the chimeric Hsp70A-RbcS2 promoter. Endogenous gene flanking sequences in Chlamydomonas have also been shown to enhance the expression level of a transgene. For example, a 1,425-bp upstream region of the endogenous gene ARG7 can drive the expression of the ARS2 reporter as strongly as the Hsp70A-RbcS2 promoter [START_REF] Specht | An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas[END_REF]. [START_REF] López-Paz | Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression[END_REF] recently reported that RPL23 flanking sequences (including promoters, terminators, and UTRs) resulted in stable expression of luciferase or sh-ble, at levels much higher than Hsp70A-RbcS2 or the PsaD promoter in Chlamydomonas.

Synthetic intron

Inclusion of a native intron in the cDNA of a transgene can improve expression level. For example, when the first intron of Chlamydomonas RbcS2 (RbcS2i1) is introduced into the coding region of ble, it dramatically increases the transformation frequency and the level of ble expression in a manner independent of orientation [START_REF] Lumbreras | Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron[END_REF]. Interestingly, [START_REF] Eichler-Stahlberg | Strategies to facilitate transgene expression in Chlamydomonas reinhardtii[END_REF] found that the addition of RbcS2i1 (e.g., one to three copies) resulted in increased expression levels of the luciferase gene in Chlamydomonas. They also showed that a particular number and genomic placement of the inserted RbcS2i1 had an overproportionally stimulating effect on expression.

Recently, it was reported that the repetitive dispersal of RbcS2i1 throughout the codon-optimized coding sequences (CDS) of transgenes enabled robust expression from the nuclear genome of Chlamydomonas. Using this strategy, the large sesquiterpene synthase gene AgBs as well as genes encoding two farnesyl pyrophosphate synthases, ERG20 and ispA, were efficiently expressed (Lauersen et al. 2016a, Wichmann et al. 2018). The optimal RbcS2i1 insertion site (...NG/GN...) in the codon-optimized transgene CDS for efficient splicing was also systematically determined [START_REF] Baier | Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii[END_REF]. Possible reasons for the "intron-mediated efficient transgene expression" phenomenon could either be that introns contain enhancers or that introns facilitate transcript maturation and export from the nucleus; however, the mechanism remains to be elucidated.

Cleavable fusion peptide system

Nuclear transgene expression levels can be improved by incorporating resistance to a selective agent. [START_REF] Rasala | Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide[END_REF] developed the ble2A system in Chlamydomonas, which enables efficient selection of transformants by transcriptional fusion of the heterologous gene to the ble selectable marker via self-cleavage of viral 2A peptides (20 amino acids). The effectiveness of this system was demonstrated by expressing various fluorescent proteins in Chlamydomonas [START_REF] Rasala | Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii[END_REF]. A significantly improved ble2A system, either using an extended 2A peptide (39 amino acids) or combining it with an improved ultraviolet-mutant strain (UVM4), was reported to drive the high-level expression of dicistronic nuclear genes [START_REF] Kong | Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains[END_REF][START_REF] Plucinak | Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii[END_REF]. However, the ability to use only one selectable marker limits the utility of the ble2A system.

Recently, [START_REF] Dong | A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii[END_REF] developed the IFT25P-IFT25-2A E system, which uses the Chlamydomonas endogenous intraflagellar transport 25 (IFT25) gene instead of ble. They showed that combining a more efficient endogenous gene that is expressed at high levels (e.g., IFT25) in-frame with the 2A peptide improves transgene expression levels [START_REF] Dong | A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii[END_REF].

A viral 2A peptide-based toolbox has also been used in Nannochloropsis salina [START_REF] Unkefer | Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts[END_REF]. A modified 2A peptide, P2A (60 amino acids), was found to be most efficient when placed in-frame with either the ble or aph7″ marker for bicistronic expression in Nannochloropsis oceanica [START_REF] Poliner | A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production[END_REF]. The viral 2A peptide system is a useful tool for transgene expression in Chlamydomonas; however, the self-cleavage efficiency of the 2A peptide varies based on the transgene used, and the mechanism by which the 2A system drives high expression of the transgene is unclear [START_REF] Rasala | Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii[END_REF][START_REF] Kong | Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains[END_REF].

Background strains

Mutants defective in gene silencing mechanisms are useful recipients for efficient transgene expression. Several Chlamydomonas mutant lines that show improved transgene expression levels have been generated (e.g., UVM4 and UVM11) [START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF]). These strains have been used as background strains for protein subcellular localization studies, as host strains for protein secretion, and for the production of terpene squalene (Kajikawa et al. 2015[START_REF] Lauersen | Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii[END_REF]). However, these UVM strains are still subjected to transgene silencing, probably because the gene silencing mechanisms have not been completely knocked out [START_REF] Kong | Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains[END_REF][START_REF] Kurniasih | UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems[END_REF]).

We previously found that the met1 mutant, which is deficient in one of the key enzymes mediating epigenetic gene silencing, showed significantly improved expression levels of nuclear transgenes, similar to UVM strains [START_REF] Kong | Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains[END_REF]. When the met1 strain was further mutated by UV irradiation, we obtained met1(UVM)-47A and B mutants, which enable robust expression of nuclear transgenes regardless of the genomic position of the insertion, due to disruption of DNA methylation-dependent and -independent silencing systems. Moreover, met1(UVM) mutants were more capable of robustly expressing native, fluorescent, or heterologous proteins than were other Chlamydomonas strains tested [START_REF] Kurniasih | UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems[END_REF].

Tools for targeted gene repression or disruption

In the case of essential genes, full disruption of the target gene results in cell lethality; therefore, RNAi-mediated gene repression is a more useful approach for assigning functions to these genes. RNAi approaches include expressing the antisense sequence or an inverted repeat of a gene targeted for knockdown [START_REF] Schroda | RNA silencing in Chlamydomonas: mechanisms and tools[END_REF]). The Maa7/X-IR (NE-537) plasmid tool and web-based RNAi design software (e.g., THE RNAi WEB, http://www.rnaiweb.com/) facilitate the use of RNAi technology to modulate transgene expression in Chlamydomonas [START_REF] Rohr | Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas[END_REF]). Despite their effectiveness, transgenes are often not stable in Chlamydomonas, and are subsequently silenced. For example, we and others have reported that RNAi silencing efficiency is reduced in Chlamydomonas after successive mitotic cell divisions [START_REF] Schroda | RNA silencing in Chlamydomonas: mechanisms and tools[END_REF][START_REF] Yamasaki | Unstable RNAi Effects Through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas reinhardtii[END_REF]. Unstable RNAi effects, including off-target effects, limit applications of RNAi in genetic engineering of Chlamydomonas. To overcome this obstacle, a novel artificial microRNA (amiRNA) system based on ligation of DNA oligonucleotides was developed, because microRNA is normally not associated with transcriptional silencing [START_REF] Molnar | Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii[END_REF]). To inactivate genes with higher accuracy and efficiency, CRISP (clustered regularly interspaced short palindromic repeats)-interference (CRISPi), which evolved from CRISPR/Cas9 (CRISPR associated nuclease 9) and uses the same type of guide RNA (gRNA), but nuclease-deficient Cas9 (Kao and Ng 2017), has recently been applied in Chlamydomonas.

Homologous recombination enables targeted gene integration, but under normal conditions, high-efficiency homologous recombination occurs only in a few algal species, such as Nannochloropsis sp. [START_REF] Kilian | High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp[END_REF]). Although homologous recombination has been shown to occur during nuclear transformation in Chlamydomonas [START_REF] Sodeinde | Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii[END_REF], the frequency of homologous recombination is very low compared to that of random insertion [START_REF] Zorin | Nuclear-gene targeting by using singlestranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii[END_REF].

Site-specific nuclease-mediated genome editing technologies, such as the ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), and CRISPR/Cas9 systems, are emerging as important alternative tools for gene knockout in microalgae. ZFN was initially used in a specially designed mutated Chlamydomonas strain, which is transformed by a non-functional aphVIII selectable marker interspaced with a short channelrhodopsin gene (COP3). The nonfunctional aphVIII was restored through ZFNmediated cleavage of COP3, when the cells were co-transformed with ZFNs and an aphVIII DNA template [START_REF] Sizova | Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases[END_REF]. Recently, the same group optimized ZFN editing protocols for several Chlamydomonas strains (i.e., CC3403 and CC503), making ZFN-based editing a reliable and predictable tool for homologous recombination [START_REF] Greiner | Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9[END_REF]).

However, ZFN methodology is somewhat laborious, as a specific ZFN protein must be engineered for each target and the targeting efficiency and specificity are poor.

The TALEN system is composed of a variable number of four different DNA recognition domains, each specifically recognizing one of the four nucleotides. Gene activation induced by artificially designed TALE (dTALE) has been studied in multiple organisms including Chlamydomonas. For example, dTALE was used to target the promoters of periplasmic arylsulfatase (ARS) genes in Chlamydomonas, resulting in their overexpression at both the mRNA and protein levels [START_REF] Gao | TALE activation of endogenous genes in Chlamydomonas reinhardtii[END_REF]. In another study, a putative inorganic carbon transporter (HLA3), proposed to be involved in inorganic carbon uptake, was overexpressed using dTALE in Chlamydomonas [START_REF] Gao | Expression activation and functional analysis of HLA3, a putative inorganic carbon transporter in Chlamydomonas reinhardtii[END_REF]. Furthermore, TALE in the form of TALEN was also used to induce the targeted knockout of a gene involved in a carbohydrate storage pathway (e.g., the gene encoding UDP-glucose pyrophosphorylase) in Phaeodactylum tricornutum, resulting in a strain producing higher levels of TAG [START_REF] Daboussi | Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology[END_REF]).

The CRISPR-Cas9 system, consisting of two key molecules, cas9 protein and gRNA, is a faster and more accurate genome-editing tool than ZFN and TALEN. Since its discovery in 2012, the CRISPR-Cas9 system has been widely applied to land plants [START_REF] Langner | CRISPR Crops: Plant Genome Editing Toward Disease Resistance[END_REF]. The first trial of the CRISPR-Cas9 system in microalgae was carried out in Chlamydomonas, but resulted in a low targeting efficiency, with no surviving transformants. This is probably due to the toxicity of constitutive Cas9 expression [START_REF] Jiang | Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii[END_REF].

Cytotoxicity was avoided by directly delivering Cas9 protein-gRNA ribonucleoproteins (RNPs) assembled in vitro into Chlamydomonas. However, the gene targeting efficiency (targeted colonies/preselected colonies) was still low, ranging from 0.17% to 1.4% [START_REF] Shin | CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii[END_REF]). To disrupt genes in a cost-effective, routine manner, [START_REF] Greiner | Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9[END_REF] optimized CRISPR/Cas9 protocols, improving the targeting efficiency (ranging from 5% to 15%). Using this modified method, the team disrupted eight photoreceptor genes in four different Chlamydomonas strains [START_REF] Greiner | Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9[END_REF]. Deliverng another pre-assembled RNP, Lachnospiraceae Cpf1 (an ortholog of Cas9), along with single-stranded DNA repair templates, resulted in efficient editing and replacement of the targeted DNA at frequencies of as high as 10% in Chlamydomonas. Moreover, this method allows for transgene-free generation of sequence-specific mutations and for epitope tagging of endogenous genes [START_REF] Ferenczi | Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and singlestranded DNA[END_REF]. Some examples of nuclease-directed genome editing in Chlamydomonas are provided in Table 1.

Plastidial genome engineering

The chloroplast genome of Chlamydomonas is a primary target in efforts to produce useful recombinant proteins, predominantly because it can be readily engineered via homologous recombination and lacks transgene silencing (Rasala and Mayfield 2011). Robust heterologous protein accumulation (≥ 5% total soluble protein) by Chlamydomonas chloroplasts was achieved by using the endogenous psbA promoter and the corresponding 5' UTR to drive expression of the target gene; however, the endogenous psbA gene had to be removed, rendering the cells non-photosynthetic (Minai et al. 2006, Rasala andMayfield 2011). To address this problem, the 16S rRNA-atpA UTR hybrid promoter and synthetic regulatory elements (i.e., UTRs) have been developed to efficiently drive transgene expression in chloroplasts without having to remove enodgenous psbA (Rasala et al. 2011, Specht and[START_REF] Specht | Synthetic oligonucleotide libraries reveal novel regulatory elements in Chlamydomonas chloroplast mRNAs[END_REF]. Cleavable fusion of the target gene to the C-terminal of rbcL also increased foreign protein accumulation. Using this approach, the luciferase activity was increased up to 5-fold in the wild type and 33-fold in the rbcL knock-out background [START_REF] Muto | Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast[END_REF].

Modification of the N-terminus of a transgene can also enhance foreign protein accumulation in algae. Recently, fusion of the downstream box (the first 15 codons following the start codon) of the TetC gene to the N-terminus of a transgene was shown to enhance foreign protein accumulation in Chlamydomonas chloroplasts. Its effect is probably related to translation or to other post-transcriptional processes [START_REF] Richter | A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts[END_REF]. Two intercistronic expression elements from psbN-psbH and tscA-chlN have recently been identified [START_REF] Macedo-Osorio | Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons[END_REF]) that enable the expression of foreign genes (i.e., aphA-6 and gfp) in Chlamydomonas chloroplasts in a synthetic operon-like manner. This approach could be useful for stacking genes for metabolic engineering or synthetic biology circuits in Chlamydomonas chloroplasts [START_REF] Macedo-Osorio | Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons[END_REF]. However, chloroplasts are deficient in post-translational modifications, such as glycosylation of proteins, which limits chloroplasts as a robust platform for protein production. In addition to being a suitable expression platform for efficient protein production, plastids can be used to produce lipids in Chlamydomonas. For example, plastid enigineering through overexpressing plastid-localized 2-lysophosphatidic acid acyltransferase 1 (CrLPAAT1) [START_REF] Yamaoka | Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content[END_REF]) can increase oil content in Chlamydomonas, as discussed below.

Examples of genetic approaches for increasing oil content in Chlamydomonas

Over the past decade, significant advances in metabolic engineering have been made to modify microalgal genomes to increase oil content. For instance, the genetic engineering tools detailed above have been used to modify lipid metabolism in Chlamydomonas through the following main approaches (Figure 1

Enhancing TAG biosynthesis

Glycerolipids are one of the most abundant lipid classes in microalgae. Their assembly starts with the stepwise acylation of glycerol 3-phosphate (G3P) to phosphatidic acid (PA) using fatty acyl donors. The 2-lysophosphatidic acid acyltransferase (LPAAT) catalyzes the acylation at the sn-2 position of 2-lysophosphatidic acid to produce PA, the first common precursor of membrane and storage lipids. We reported that Chlamydomonas encodes a plastid-targeted LPAAT (CrLPAAT1) and that overexpression of CrLPAAT1 in plastids led to a more than 20% increase in oil content under nitrogen (N)-deficient conditions [START_REF] Yamaoka | Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content[END_REF]. Likewise, transgenic Chlamydomonas strains overexpressing CrLPAAT2 showed greater accumulation of TAG than the parental strain [START_REF] Kim | Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas[END_REF]. By contrast, RNAimediated suppression of CrLPAAT2 reduced the TAG content of the RNAi lines under N deprivation [START_REF] Kim | Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas[END_REF]).

G3P dehydrogenase (GPDH) catalyzes the reversible redox conversion of dihydroxyacetone phosphate to G3P. Recently, the codon-optimized LPAAT gene from Brassica napus and GPDH1 from Saccharomyces cerevisiae were heterologously expressed in Chlamydomonas driven by the Hsp70A-RbcS2 chimeric promoter, respectively. The LPAAT and GPDH1 transgenetic lines showed a 44.5% and 67.5% increase in lipid contents, respectively, after triple heat-shock treatments [START_REF] Wang | Effect of overexpression of LPAAT and GPD1 on lipid synthesis and composition in green microalga Chlamydomonas reinhardtii[END_REF]). These results are consistent with those of an earlier transcriptome analysis in which GPDH and LPAAT were found to be significantly upregulated when high levels of lipids accumulated in Chlamydomonas [START_REF] Lv | Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation[END_REF].

Phospholipid:diacylglycerol acyltransferase (PDAT), an acyl-CoA-independent acyltransferase, catalyzes the transfer of an acyl group from the sn-2 position of a phospholipid to a diacylglycerol to make TAG. This PDAT pathway is considered to be another important of TAG biosynthesis from DAG, in addition to the acyl-CoAdependent pathway catalyzed by diacylglycerol acyltransferase (DGAT) [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]. Heterologous expression of Saccharomyces cerevisiae PDAT in Chlamydomonas increased the contents of total fatty acids (FAs) and TAG by 22% and 32%, respectively, under N-replete conditions [START_REF] Zhu | The synchronous TAG production with the growth by the expression of chloroplast transit peptide-fused ScPDAT in Chlamydomonas reinhardtii[END_REF].

The FAs used for TAG biosynthesis can also be derived from recycled membrane lipids (i.e., monogalactosyldiacylglycerol [MGDG] and digalactosyldiacylglycerol [DGDG]).

It was reported that PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION 1) from Chlamydomonas can hydrolyze MGDG to produce free FAs and lyso-MGDG in vitro, and the PGD1 knockout mutant had lower levels of TAG than the wild type following N deprivation (Li et al. 2012a). Furthermore, perturbing lipid trafficking or altering membrane lipid composition has been shown to result in TAG over-accumulation. For example, in tgd2 (trigalactosyldiacylglycerol) mutants, which are defective in lipid trafficking, excess levels of MGDG are produced that are postulated to contribute to additional acyl-groups for TAG synthesis [START_REF] Warakanont | Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue[END_REF]. Additionally, the Chlamydomonas fdx5 mutant, which has a defect in the ferredoxin5 gene, was shown to have changes in thylakoid membrane ultrastructure and lipid profiles in darkness (e.g., the ratio of MGDG to DGDG strongly declined), resulting in strains that hyper-accumulate TAG [START_REF] Yang | Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism[END_REF].

Increasing acetyl-CoA supply

Acetyl-CoA is an essential carbon precursor for de novo FA biosynthesis; therefore, increasing the acetyl-CoA supply by metabolic engineering could improve oil content.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme belonging to the family of decarboxylases in plants and microalgae. PEPC controls the carbon flux that enters the tricarboxylic acid (TCA) cycle, and plays an important role in carbon partitioning of substrates in competition with lipid synthesis [START_REF] Deng | Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii[END_REF][START_REF] Izui | Phosphoenolpyruvate carboxylase: a new era of structural biology[END_REF]). In Chlamydomonas, inhibiting PEPC1 activity by RNAi or CRISPRi-based techniques increased the oil content by 20%, probably by enhancing the flow of carbon to FA synthesis (Deng et al. 2014, Kao and[START_REF] Kao | CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii[END_REF]. Down-regulating both PEPC isoforms (CrPEPC1 and CrPEPC2) by amiRNA resulted in a 29% to 48% increase in total FA levels, respectively, which was suggested to occur due to the flux of carbon away from the TCA cycle, and the subsequent increase in the availability of acetyl-CoA for FA biosynthesis [START_REF] Wang | Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii[END_REF]. Consistent with this hypothesis, the expression levels of TAG biosynthesis-related genes, such as DGAT2 and phosphatidate phosphatase (PAP2), were increased in CrPEPC1 RNAi strains [START_REF] Deng | Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii[END_REF].

Manipulating transcription factors/regulators

Manipulating the expression of transcription factor (TF) genes usually affects multiple steps/genes of the same pathway, thereby upregulating the entire pathway rather than a specific enzymatic step. TF engineering is a promising strategy, because it can circumvent the secondary bottlenecks commonly observed when only a single gene is targeted [START_REF] Courchesne | Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches[END_REF]. TFs showing positive or negative correlations with lipid biosynthetic genes at the RNA expression level could be potential targets for engineering of oil content [START_REF] Hu | Genome-wide identification of transcription factors and transcription-factor binding sites in oleaginous microalgae Nannochloropsis[END_REF]. Dof-binding sequences are abundant in the promoter regions of many genes related to lipid biosynthesis [START_REF] Wang | Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean[END_REF], indicating that Dof genes might contribute to lipid biosynthesis by upregulating genes associated with FA biosynthesis. Indeed, a study in higher plants showed that overexpression of Dof-type TF genes (GmDof1 and GmDof14) from soybean (Glycine max) enhanced TAG accumulation in the seeds of transgenic Arabidopsis plants [START_REF] Wang | The soybean Dof-type transcription factor genes, GmDof4 and GmDof11, enhance lipid content in the seeds of transgenic Arabidopsis plants[END_REF]. A codon-optimized soybean DOF-type TF gene (GenBank ID: DQ857261.1) was subsequently heterologously expressed in Chlamydomonas, and the resulting transgenic lines had an up to 2-fold increase in lipid content [START_REF] Ibáñez-Salazar | Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii[END_REF]. [START_REF] Ngan | Lineagespecific chromatin signatures reveal a regulator of lipid metabolism in microalgae[END_REF] identified another TF named PSR1 (PHOSPHORUS STRESS RESPONSE 1), which triggers cytosolic lipid accumulation, as a pivotal switch in carbon storage metabolism in Chlamydomonas. Overexpression of PSR1 increased TAG accumulation 2-fold (on a dry weight basis) in cells grown under normal culture conditions without growth inhibition [START_REF] Ngan | Lineagespecific chromatin signatures reveal a regulator of lipid metabolism in microalgae[END_REF].

We recently isolated a Chlamydomonas mutant (starch degradation 1, std1) deficient in a dual-specificity tyrosinephosphorylation-regulated kinase (DYRK) [START_REF] Schulz-Raffelt | Hyper-accumulation of starch and oil in a Chlamydomonas mutant affected in a plantspecific DYRK kinase[END_REF]). The std1 mutant accumulates higher levels of oil than the wild type under photoautotrophic N starvation conditions. By contrast, another DYRK, named TAR1 (TAG ACCUMULATION REGULATOR 1), was found to positively regulate TAG accumulation under mixotrophic N and S deficiency, and the tar1-1 mutant accumulated lower levels of TAG than did the wild-type strain (Kajikawa et al. 2015b). The effect on oil content has not yet been evaluated for tar1 under photoautotrophic conditions. This plant-specific DYRK kinase (DYRKP), which has been proposed to act as a positive or negative regulator of sink capacity and thereby integrate nutrient and energy signals [START_REF] Schulz-Raffelt | Hyper-accumulation of starch and oil in a Chlamydomonas mutant affected in a plantspecific DYRK kinase[END_REF], represents a promising target for improving energy density in microalgae.

Additionally, VIP1, an inositol hexakisphosphate/diphosphoinositol phosphate kinase involved in phosphatidylinositol signaling, has recently been reported to show synergism with target of rapamycin kinase signaling, which governs carbon metabolism and lipid accumulation in Chlamydomonas. The null vip1-1 mutant exhibits increased levels of TAG and a higher content of lipid droplets in both N-replete and -deplete conditions [START_REF] Couso | Synergism between inositol polyphosphates and TOR kinase signaling in nutrient sensing, growth control and lipid metabolism in Chlamydomonas[END_REF]. A comprehensive understanding of the underlying mechanism by which TFs regulate lipid alteration and the discovery of TFs regulating algal lipid metabolism will facilitate targeted engineering strategies to improve lipid production in microalgae.

Blocking competing pathways

Disrupting competing metabolic pathways of lipid biosynthesis has been routinely used to enhance lipid productivity. In addition to TAG, starch is another primary carbon sink in green microalgae. Previous work showed that knocking-out starch synthesis could increase oil content [START_REF] Li | Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyperaccumulates triacylglycerol[END_REF][START_REF] Work | Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains[END_REF]; however, this does not always seem to be the case [START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF][START_REF] Vonlanthen | Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids[END_REF], and varied depending on the background strains and cultivation conditions. The relationship between starch synthesis and TAG accumulation merits further investigation.

Reducing lipid degradation

Cellular lipid content is the end result of an equilibrium between synthesis and degradation.

Therefore, lipid hydrolytic enzymes (i.e., lipases) are also potential targets for engineering efforts to increase TAG content. In Chlamydomonas, the lipase CrLIP1 was found to be a DAG lipase with broad substrate specificity. Silencing of CrLIP1 resulted in an increase in oil content following N replenishment upon N starvation due to the arrest of TAG hydrolysis (Li et al. 2012). TAG degradation is also directly linked to the cell cycle in Chlamydomonas. The insertional null mutant cht7 (compromised hydrolysis of triacylglycerols 7) is severely impaired in TAG degradation, and the TAG level in this mutant remains 10-fold higher than that of wild-type cells upon N resupply after N starvation, due to a defect in exiting quiescence [START_REF] Tsai | The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas[END_REF].

Recently, we reported that functional FA -oxidation is involved in lipid homeostasis during nutrient stress in Chlamydomonas. In the insertional knockout mutants for ACX2 (ACYL-COA OXIDASE 2), a key enzyme in peroxisomal FA -oxidation, oil remobilization is severely blocked (by 60-80%) upon N resupply after N starvation and oil content is increased by 20% during N starvation [START_REF] Kong | Chlamydomonas carries out fatty acid β-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF]. In Chlamydomonas, with the exception of isocitrate lyase (ICL), all other enzymes of the glyoxylate cycle are localized in peroxisomes (Lauersen et al. 2016b). The null icl mutant overaccumulated total FAs when cultivated mixotrophically, presumably due to a reduction in the -oxidation of FAs [START_REF] Plancke | Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth[END_REF]. Overall, reduced lipid turnover may enhance the net oil content; therefore, identifying and engineering genes involved in lipid degradation is a promising avenue toward increasing oil content in microalgae.

Modulating redox state

The FA synthase complex is activated by NADPH [START_REF] Rawsthorne | Carbon flux and fatty acid synthesis in plants[END_REF], and a positive link between the level of NADPH and FA content has been established in microalgae [START_REF] Osada | Enhanced NADPH production in the pentose phosphate pathway accelerates lipid accumulation in the oleaginous diatom Fistulifera solaris[END_REF][START_REF] Xue | Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation[END_REF]. Malic enzyme (ME) catalyzes the conversion of malate to pyruvate and simultaneously reduces NADP + to NADPH. The total lipid content and the neutral lipid content were increased 2.5-fold and 3.2-fold via transgenic overexpression of PtME in Phaeodactylum tricornutum and Chlorella pyrenoidosa, respectively [START_REF] Xue | Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation[END_REF][START_REF] Xue | The pivotal role of malic enzyme in enhancing oil accumulation in green microalga Chlorella pyrenoidosa[END_REF].

Recently, the enzyme glucose-6-phosphate dehydrogenase (G6PD), which is involved in the formation of NADPH in the pentose phosphate pathway, was reported to contribute to lipid synthesis in the oleaginous diatoms Phaeodactylum tricornutum and Fistulifera solaris.

Overexpression of G6PD elevated lipid productivity 2.7-fold in Phaeodactylum tricornutum during the stationary phase, and 1.5-fold in Fistulifera under N depletion [START_REF] Osada | Enhanced NADPH production in the pentose phosphate pathway accelerates lipid accumulation in the oleaginous diatom Fistulifera solaris[END_REF][START_REF] Xue | Glucose-6phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply[END_REF].

Malate dehydrogenase (MDH) catalyzes the reversible conversion of NAD(P)H to NAD(P) + via the reduction of oxaloacetate to malate [START_REF] Mettler | Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle[END_REF]. Recently, we isolated and characterized a Chlamydomonas mutant deficient in the peroxisomal NAD +dependent MDH2. In its absence, mdh2 mutant cells are compromised in TAG breakdown and showed an increased capacity to make NAD(P)H, resulting in a >50% increase in storage lipids and a 200% increase in starch during photoautotrophic N deprivation (Kong et al. 2018b). Enhancing the NADPH supply using metabolic engineering techniques seems to be a promising strategy for boosting lipid accumulation in microalgae.

Conclusion and prospectives

Here we reviewed the essential techniques for improving the oil content of Chlamydomonas, from transformation methodologies to genome engineering strategies. The state-of-the-art CRISPR-Cas9 system shows great potential for precise genetic engineering, and has been successfully used in Chlamydomonas, diatoms, and Nannochloropsis. The continued progress of synthetic biology in Chlamydomonas will facilitate the development of the microalgal biotechnology sector.

We also described cases in which genetic tools were succesfully used to increase oil content in Chlamydomonas. It is apparent that microalgae that accumulate oils have acquired not only divergent enzymes that produce these FAs, but also a large number of enzymes (e.g., acyltransferases) that can effectively channel these FAs into oil. Understanding and manipulating the regulatory processes of lipid metabolism may turn out to be even more effective than manipulating specific enzymes. Thus, boosting oil accumulation in microalgae may require a combination of strategies involving both genetic engineering tools and synthetic biology approaches employing many genes. A breakthrough in this technology is hopefully not far off.

F

  metabolic pathways and elucidating physiological mechanisms. We provide examples of successful efforts to genetically manipulate Chlamydomonas to increase oil content, and discuss future challenges and directions in microalgal synthetic biology.

  of Chlamydomonas mutants, named Mme1-based insertion site sequencing (ChlaMmeSeq), was developed

  ): (1) Enhancing TAG biosynthesis; (2) Increasing acetyl-CoA supply; (3) Manipulating transcription factors/regulators; (4) Blocking competing pathways; (5) Reducing lipid degradation; and (6) Modulating redox state. Some examples of engineering attempts are described below.

Figure 1 .

 1 Figure 1. The main approaches of genetic engineering of Chlamydomonas for altered oil content. 160x79mm (300 x 300 DPI)

21

  Kong, and French Agence Nationale pour la Recherche (ANR-MUsCA) awarded to Y. Li-

	Beisson.
	F F F F F F F F F F
	o r o r o r o r o r o r o r o r o r o r o r
	P P P P P P P P P P P
	e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r
	R e v R e v R e v R e v R e v R e v R e v R e v R e v R e v R e v
	i e w i e w i e w i e w i e w i e w i e w i e w i e w i e w i e w

Table 1 . Summary of current uses of nuclease-directed genome editing in Chlamydomonas. Editing method Chlamydomonas species Targeted gene/Marker gene Transformation method Comment Reference

 1 PHOT, photoreceptor phototropin; PHT7, a putative phosphate transporter; TALEN, transcription activator-like effector nuclease; UVR8, a homomultimeric UV-B photoreceptor; VGCC, voltage-gated calcium channel; ZEP, zeaxanthin epoxidase, ZNF, zinc finger nuclease."---" indicates selectable marker gene was not used.

	ZNF	CC4350	COP3/aphVIII	Glass beads	The first application of	Sizova et
					ZNF to knockout gene in	al. (2012)
					Chlamydomonas
		CC3403, CC503	COP3/ARG7, aphVIII	Electroporation	ZNF mediated-targeting	Greiner et
					of photoreceptor genes	al. (2017)
	TALEN	CC849	ARS1, ARS2/ble	Electroporation	The first success case of	Gao et al.
					TALEN mediated-gene	(2014)
					expression activation in
					Chlamydomonas
		CC849	HLA3/ble	Electroporation	TALE mediated gene	Gao et al.
					activation	(2015)
	CRISPR/Cas9	CC503	FKB12/---	Electroporation	First trail of	Jiang et
					CRISPR/Cas9 system in	al. (2014)
					Chlamydomonas, but
					lack of surviving
					transformants
		CC124	MAA7, CpSRP43,	Electroporation	Succeeded in obtaining	Shin et al.
			ChlM /aph7″		stable Chlamydomonas	(2016)
					transformants by
					delivering preassembled
					Cas9-gRNA RNPs
		CC4349	ZEP, CpFTSY/---	Electroporation	Generation of two-gene	Baek et al.
					knockout mutant by	(2016)
					delivering Cas9-gRNA
					RNPs
		CC3403	COP1/2, COP5,	Electroporation	Targetting photoreceptor	Greiner et
			PHOT, PSY1/aphVIII		genes in	al. (2017)
					Chlamydomonas via
					CRISPR/Cas9
		CC3403, CC125,	aCRY, COP1/2,	Electroporation	Targetting photoreceptor	Greiner et
		SAG73.72	COP5, MAT3, PHOT,		genes by delivering	al. (2017)
			POLQ, KU80, UVR8,		exogenously supplied
			VGCC/ARG7, aph7″,		recombinant Cas9-gRNA
			aphVIII		RNPs
		CC1883	FKB12, CpFTSY,	Electroporation	The first CRISPR/Cpf1	Ferenczi
			CpSRP43, PHT7/---		RNPs-mediated efficient	et al.
					genome editing in	(2017)
					Chlamydomonas
		CC4349	LTD/---	Electroporation	Photosynthesis gene	Jeong et

32 Abbreviations: aCRY, animal-like cryptochrome; aph7″, hygromycin B resistance gene; aphVIII, aminoglycoside 3-phosphotransferase (paromomycin resistance gene); ALS, acetolactate synthase; ARG, argininosuccinate lyase; ARS, arylsulfatase; ble, bleomycin resistance gene; CRISPR/Cas9, clustered regularly interspersed palindromic repeats associated protein 9; COP, light-gated proton channel rhodopsin; CpSRP, chloroplast signal recognition particle; ChlM, Mg-protoporphyrin IX S-adenosyl methionine O-methyl transferase; CpFTSY, signal recognition particle receptor; FKB12, peptidyl-prolyl cis-trans isomerase; HLA3 (also known as MRP1), a putative inorganic carbon transporter; KU80, ATP-dependent DNA helicase 2 subunit 2; LTD, light-harvesting chlorophyll-and carotenoid-binding proteins translocation defect gene; MAT3, retinoblastoma; MAA7, beta subunit of tryptophan synthase; PSY1, PHYTOENE SYNTHASE 1 ; PLA2, phospholipase A2; POLQ, DNA polymerase theta; 33 Legends

to figure Figure 1. The main approaches of genetic engineering of Chlamydomonas for altered oil content.

  CrLPAAT1 and 2, Chlamydomonas lysophosphatidic acid acyltransferase 1 and 2; CrLIP1, Chlamydomonas lipase 1; cracx2, Chlamydomonas acyl-CoA oxidase 2; DOFs, Dof-type transcription factor genes; ER, endoplasmic reticulum; FA, fatty acid; fdx5, ferredoxin5; icl, isocitrate lyase; lcs2, long-chain acyl-CoA synthetase; LD, lipid droplet; mldp, major lipid droplet protein; mdh2, malate dehydrogenase 2; nrr1, nitrogen-responsive regulator 1; OE,

	Abbreviations: amiRNAi, artificial microRNA interference; CRISPRi, clustered regularly
	interspaced short palindromic repeats interference; cht7, compromised hydrolysis of
	triacylglycerols 7; CrPEPC1/2, Chlamydomonas phosphoenolpyruvate carboxylase1/2;

over-expressing strains; PSR1, phosphorus stress response 1; pgd1, plastid galactoglycerolipid degradation 1; pdat1, phospholipid:diacylglycerol acyltransferase 1; RNAi, RNA interference; std1, starch degradation 1; sta6 and 7; starch-less mutant 6 and 7; TAG, triacylglycerol; tgd2, trigalactosyldiacylglycerol 2; tar1, triacylglycerol accumulation regulator 1; vip1, a predicted diphosphoinositol pentakisphosphate kinase.

Plant & Cell Physiology

Acknowledgements

This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2015M3A6A2065746) and a grant from the BioGreen 21 Program (No. PJ013412), Rural Development Administration, Republic of Korea awarded to Y. Lee, and the Korea Research Fellowship Program funded by the Ministry of Science, ICT and Future Planning through the National Research Foundation of Korea (2016H1D3A1909463) to Y. Yamaoka. The work was also supported by the Start-up Fund of Introduction of Talent Program awarded to F.

Disclosures

The authors have no conflicts of interest to declare.