Nannochloropsis as an Emerging Algal Chassis for Light-Driven Synthesis of Lipids and High-Value Products

In light of the escalating global energy crisis, microalgae have emerged as highly promising producers of biofuel and high-value products. Among these microalgae, Nannochloropsis has received significant attention due to its capacity to generate not only triacylglycerol (TAG) but also eicosapentaenoic acid (EPA) and valuable carotenoids. Recent advancements in genetic tools and the field of synthetic biology have revolutionized Nannochloropsis into a powerful biofactory. This comprehensive review provides an initial overview of the current state of cultivation and utilization of the Nannochloropsis genus. Subsequently, our review examines the metabolic pathways governing lipids and carotenoids, emphasizing strategies to enhance oil production and optimize carbon flux redirection toward target products. Additionally, we summarize the utilization of advanced genetic manipulation techniques in Nannochloropsis. Together, the insights presented in this review highlight the immense potential of Nannochloropsis as a valuable model for biofuels and synthetic biology. By effectively integrating genetic tools and metabolic engineering, the realization of this potential becomes increasingly feasible.


Introduction
Up until now, fossil-derived fuels have served as the main global energy sources.The ever-increasing energy demand, depleted reserves of fossil fuels, and environmental concerns associated with fossil fuel burning, however, have led to the exploration of alternative energy that is green, renewable, and sustainable.Biomass-derived oils have a similar structure to fossil fuels, are energy rich, and represent important sources for making biodiesel.Currently, plant oils contribute to the major feedstocks for biodiesel production.Biodiesel from plants, however, is a long way from meeting the existing demand and will cause the conflict of food versus fuel.Algae has gradually come to people's attention because of its advantages of fast growth, no competition with grain, and higher oil production potential than plants [1,2].However, it remains economically infeasible for algae to serve as a separate biofuel producer.Combining oil production with high-value co-products using synthetic biology is a solution.
Nannochloropsis, a genus of Eustigmatophyceae microalgae, holds immense promise due to its unique attributes, including rapid growth, a high lipid content, and the ability to thrive in diverse environmental conditions; these characteristics make it an ideal candidate for producing oils, omega-3 fatty acids, and a wide array of high-value compounds [3].Nannochloropsis, with its lipid content exceeding 30% of its dry weight, emerges as a highly promising feedstock for biodiesel production.In a study, the type 1 diacylglycerol acyltransferase (DGAT) gene derived from Arabidopsis thaliana (AtDGAT) was heterologously expressed in Nannochloropsis oceanica using electroporation.In comparison to the wildtype N. oceanica, the overexpression of AtDGAT resulted in a significant increase in C16 (C16:0 + C16:1) levels and a slight decrease in C18 (C18:0 + C18:1), providing novel insights into the genetic engineering of oleaginous microalgae [4].In addition, there have been many studies exploring Nannochloropsis as feed for Pacific white shrimp, Atlantic salmon, kuruma shrimp, and European sea bass [5,6].Adissin et al. [7] used algal powder to take the place of fish oil and finally improved shrimp growth.More interestingly, this strategy changed the fatty acid profile of kuruma shrimp, with a big increase in polyunsaturated fatty acids (PUFAs).Nannochloropsis can be used in food as well.Lafarga et al. [8] tried to add 2.0% Nannochloropsis to functional breads and performed an evaluation of their physicochemical properties, organoleptic properties, and nutritional properties.The new breads with a high amount of bioaccessible polyphenols showed a highly improved antioxidant capacity.Additionally, the non-living cells of N. oceanica can be effective adsorbents for treating textile wastewater containing azo dyes [9].The bioactive extracts of Nannochloropsis are interesting targets in the medical and cosmetic industries.Khattib et al. [10] isolated an active compound of lyso-diacylglyceryl-N,N,N-trimethyl-homoserine (lyso-DGTS) from Nannochloropsis, which can interact with high-density lipoprotein to improve its anti-atherosclerosis abilities.Moreover, violaxanthin has significant skin-protective activity against induced oxidative stress, such as UVB-mediated cellular damage [11].Due to the high abundance of violaxanthin (32% of total carotenoids), Nannochloropsis can be used as a functional ingredient in cosmetic formulations [11].It has been found that Nannochloropsis produces high levels of vitamin D 3 naturally, which is not common in other algae [12].
Synthetic biology, with its genetic and metabolic manipulative prowess, allows for the fine tuning of Nannochloropsis to optimize the production of lipids, proteins, carotenoids, and other high-value compounds [13,14].Synthetic biology provides the essential toolbox for reprogramming Nannochloropsis at the genetic level.It involves a spectrum of tools, such as the revolutionary CRISPR-Cas9 gene-editing system, custom-designed promoters, and metabolic pathway engineering [15,16].These tools empower researchers to tailor Nannochloropsis strains for trait improvements, including but not restricted to growth, oils, omega-3 PUFAs, and high-value carotenoids.
This review aims to provide a comprehensive overview of the current state of Nannochloropsis-based biorefining driven by synthetic biology [13].It will delve into the taxonomy and growth characteristics of Nannochloropsis, the fundamental principles of synthetic biology, the tools and techniques employed in genetic and metabolic engineering, and the various biorefining strategies being pursued.Furthermore, it will explore the challenges faced in scaling up Nannochloropsis-based biorefining processes, address environmental considerations and sustainability, and touch upon the regulatory and ethical aspects governing this rapidly advancing field.

Growth Physiology of Nannochloropsis
Nannochloropsis is a genus of unicellular and non-motile marine microalgae classified within the phylum Heterokontophyta, the class Eustigmatophyceae, and the family Eustigmataceae.This genus was initially defined by Hibberd and comprises six well-documented species: Nannochloropsis gaditana, Nannochloropsis granulata, N. oceanica, Nannochloropsis oculata, Nannochloropsis salina, and Nannochloropsis australis [17,18].Nannochloropsis is characterized by small, unicellular, non-motile, and spherical or ovoid cells.It is a single-celled organism, typically ranging from 2 to 8 µm in size, constituting an entire living entity within a solitary cell.This distinction sets it apart from multicellular organisms, which are formed through collaboration between multiple cells [3].The cells are encased within a unique cell-wall structure composed of complex polysaccharides, offering protection and stability.The inner layer of the cell wall in Nannochloropsis displays a porous structure with a delicate fibrous substructure and support structures linking this layer to the plasma membrane [19].In certain Nannochloropsis species, small amounts of additional sugars (such as rhamnose, mannose, ribose, xylose, fucose, and galactose) may be present [20].The ultrastructure of Nannochloropsis cells, including the organization of chloroplasts and other organelles, has been studied in great detail using electron microscopy techniques, shedding light on the intricacies of its cellular architecture [3].
Nannochloropsis exhibits rapid growth rates under optimal conditions, making it an attractive candidate for biotechnological purposes.Its growth physiology encompasses several key factors, including environmental requirements, nutrient uptake, photosynthesis, and metabolic pathways.The efficiency of photosynthesis in Nannochloropsis is influenced by the light intensity, quality, and photoperiod.

Environmental Factors
The cultivation of Nannochloropsis is highly dependent on various environmental factors.These factors play a crucial role in determining the growth rate, lipid content, and overall health of Nannochloropsis cultures.Understanding and managing these environmental conditions are essential for successful and efficient cultivation.Adequate light is essential for their growth.
As the light intensity increased from low to high levels, the growth rates of N. oceanica decreased in both autotrophic and mixotrophic cultures [21].Comparatively, under light intensities of 40, 80, 120, and 160 µmol m −2 s −1 , the biomass concentration and productivity of N. gaditana were highest at the light intensity of 120 µmol m −2 s −1 and a photoperiod comprising a 16 h light/8 h dark cycle [22].Similarly, Nannochloropsis sp.demonstrated optimal growth with the maximum cell concentration under the conditions of a light intensity of 100 µmol m −2 s −1 and a photoperiod comprising a 18 h light/6 h dark cycle [23].The light exposure significantly influences the production of compounds, particularly lipid synthesis, in Nannochloropsis.When subjected to light at an intensity of 500 µmol m −2 s −1 , N. oceanica cells exhibited a 45% reduction in total protein content and a 38% decrease in sugar content, accompanied by a substantial 44% increase in total lipid content and more than 2-fold increases in β-carotene, zeaxanthin, astaxanthin, and canthaxanthin.The analysis using isobaric tags for relative and absolute quantification (iTRAQ) revealed that in carbon metabolism, glycolysis, the Calvin cycle, and the tricarboxylic acid (TCA) cycle were activated to channel more acetyl-CoA towards lipid and carotenoid biosynthesis [24].The lower light intensity (30 and 50 µmol m −2 s −1 ), on the other hand, could promote the eicosapentaenoic acid (EPA) content in N. oculata to rise by 126% [25].A similar trend is observed in Nitzschia laevis.Under the influence of elongases, C16:0-acyl carrier protein (ACP) can convert into C18:0, followed by the formation of C18:1 under the action of ∆9 desaturase.Additionally, C18:0 has the potential to generate C18:2 through stearoyl-ACP desaturase and ∆12-fatty acid desaturase (FAD).The up-regulation of these pathways suggests that low light conditions promote the biosynthesis of C18:1 and C18:2, which is beneficial for EPA biosynthesis [26].Light wavelengths can influence the growth physiology of microalgal cells as well.It has been reported that low-light red light-emitting diodes (LEDs) could lead to a higher cell density and more fatty acids [27].Analyzing different light wavelengths from red, green, blue, and white LEDs, it was observed that under blue light, Nannochloropsis sp. in both phototrophic and mixotrophic cultures exhibited the highest growth rates, with the fatty acid content reaching 20.45% and 15.11%, respectively [28].The establishment of light-dark cycles contributes to the enhancement of cell photosynthetic efficiency; continuous light illumination, one the other hand, can result in energy loss and a decrease in cell density [28].A significant correlation between metabolic status and light absorption rate was observed by Matsui et al. [29].
Temperature is another significant element of Nannochloropsis growth.Study has shown that the optimal growth temperature for N. oceanica falls between 25 and 29 • C, while growth ceases completely at temperatures above 31 • C and below 9 • C [30].The suboptimal temperatures (<20 • C) were investigated, and the lowest growth rate but highest quantity of EPA occurred at 5 • C for N. salina [31].Similarly, low-temperature stress at 10 • C favored the accumulation of EPA in N. oculata, enabling the EPA content to reach 12.8 mg L −1 , which was 158% more than that obtained under 25 • C conditions [25].By contrast, high temperature promoted lipid accumulation, with N. gaditana reaching the highest lipid content at 30 • C within the examined temperature ranges [32].A sequential treatment approach was employed for N. oceanica, involving the initial addition of bicarbonate as the carbon source, followed by the application of low temperature to facilitate the redirection of carbon flux towards PUFA synthesis [33].A study utilized a dual strategy involving random mutagenesis and adaptive laboratory evolution to acquire thermotolerant strains of N. oculata.Metabolomics and lipidomics unveiled a reconfiguration of the central carbon metabolism and membrane lipid synthesis in these thermotolerant strains [34].
Furthermore, it is imperative to consider the impact of symbiotic bacteria and fungi on the Nannochloropsis culture process.Fulbright et al. [35], for instance, isolated Bacillus pumilus from a underperforming N. salina culture in a 200 L system, and demonstrated its inhibitory effect on cell growth.On the other hand, there are symbiotic organisms that can be beneficial to Nannochloropsis growth.Du et al. [36], for example, combined the marine alga N. oceanica with the oleaginous fungus Mortierella elongata, resulting in increased biomass and production of triacylglycerols (TAGs).

Nutritional Factors
To harness their substantial potential in enhancing biomass and lipid production, carbon sources are frequently employed as supplements during cultivation.These carbon sources can be categorized into inorganic carbon, such as carbon dioxide [37] and bicarbonate [38], as well as organic carbon, including acetate [39], glucose [40], and glycerol [41].Notably, a synergistic effect can be achieved by combining organic carbon and inorganic carbon.For instance, the addition of glycerol and carbon dioxide in the culture medium of N. oculata resulted in a significant increase in biomass and lipid production [42].Furthermore, the interaction of carbon and nitrogen is of paramount importance for algae growth.The presence of acetate can greatly reduce the toxicity of ammonium, allowing it to be used as a nitrogen source in N. oculata.With 1 mM of ammonium and 32 mM of acetate in the culture, biomass and lipid production could be increased 1.5-fold and 9.5-fold, respectively [43].It is worth noting that the presence of carbon promotes nitrogen utilization, and vice versa.When nitrate was introduced, glycerol consumption in N. salina was observed to shift from 60% to 79% [44].
Nitrogen and phosphorus comprise two of the most limiting factors in medium composition, and their deprivation often leads to lipid overproduction.Nitrogen availability is crucial for cell metabolism and the physiological and biochemical state in algae [45].Phosphorus, on the other hand, is a component of several essential molecules that play a vital role in lipid turnover and remodeling [46].The luxury uptake of phosphorus can act as a regulator of gene expression and an energy source [47].The simultaneous occurrence of phosphorus stress and nitrogen stress has garnered interest.When the N:P supply ratio is raised to at least 32:1, phosphorus usage can be halved without harming the biomass productivity [48].
The presence of certain metal ions at relatively low concentrations is crucial for regulating cellular activities and plays important roles in growth and oil metabolism; these ions include Fe 2+ .Increasing the concentration of Fe 2+ to 0.5 mmol L −1 results in a significant increase in PUFAs as a proportion of the total fatty acids in Nannochloropsis [49].The optimal salinity range for Nannochloropsis growth and EPA accumulation falls between 27% and 29% [50].Within a certain range, increasing the carbon dioxide concentration significantly boosts the EPA content in Nannochloropsis [51].
Moreover, combined factors have the potential to perform better than individual ones in lipid production.For example, the combination of salinity and light intensity could promote increased dry weight and lipid production in Nannochloropsis [52].With combined nitrogen deprivation and high light exposure, N. oculata showed enhanced TAG accumulation compared to either nitrogen deprivation or high light exposure alone [53].

TAG Biosynthesis
TAGs are the main storage lipids in eukaryotic cells and have been widely used for foods, light industrial products, and biofuels.In the biofuel sector, TAGs are harnessed as a source of fatty acids for the transesterification process, which converts them into biodiesel.This chemical transformation involves reacting TAGs with an alcohol, typically methanol or ethanol, in the presence of a catalyst, resulting in the production of biodiesel (fatty acid methyl or ethyl esters) and glycerol as a byproduct.The renewable and sustainable nature of TAGs, especially those derived from sources like microalgae, makes them highly desirable for biofuels [54].It is crucial to understand the pathways and key enzymes involved in TAG biosynthesis, as outlined in Figure 1.
Acetyl-CoA serves as the crucial carbon precursor for de novo fatty acid biosynthesis and can be derived from the conversion of pyruvate [55].This conversion is negatively regulated by the enzyme pyruvate dehydrogenase kinase (PDK).Attenuating PDK has the potential to enhance TAG accumulation without impairing cell growth for N. salina [56].Acetyl-CoA carboxylase (ACCase) is involved in the first step of de novo fatty acid biosynthesis, converting acetyl-CoA to malonyl CoA-ACP; N. oceanica is predicted to include multiple isoforms of ACCase [55].As a key enzyme in the type II fatty acid synthesis (FAS II) pathway, malonyl CoA-ACP transacylase (MCAT) has been found to contribute to the increased lipid production and cell growth in N. oceanica [57].The synthesized acyl-ACPs are either utilized in the chloroplast or released as free fatty acids, which are transported to the endoplasmic reticulum (ER) via transport proteins and long-chain acyl-CoA synthetase (LACS) [58].In Arabidopsis, the half-size ATP-binding cassette (ABC) transporter subfamily A (ABCA) located in the ER is responsible for transporting acyl-CoA to the ER [59].In Nannochloropsis, LACSs are believed to play a critical role in activating and facilitating the formation of acyl-CoA, which is essential for TAG synthesis, lipid elongation, and β-oxidation [60].Transporter proteins are also predicted to exist in Nannochloropsis but have yet to be functionally identified [55].
There are multiple pathways towards TAG assembly.The Kennedy pathway serves as the primary TAG assembly pathway, comprising four enzymes of glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), phosphatidic acid phosphatase (PAP), and DGAT [61].GPAT, the initial enzyme in the Kennedy pathway, catalyzes the transacylation of glycerol-3-phosphate (G3P) at the sn-1 position to lysophosphatidic acid (LPA).There are two GPAT isoforms in N. oceanica; the efforts to generate a disruption mutant of each GPAT have failed, indicating that both are essential for the alga [62].Moreover, the overexpression of the ER-located GPAT in N. oceanica can lead to an increase in non-polar lipids [63].In N. oceanica, four LPATs (LPAT1-LPAT4) have been identified, and they catalyze the transacylation of LPA at the sn-2 position to form phosphatidic acid (PA), with a preference for C16:0-CoA over C18:0-CoA as the acyl donor [62], similar to LPATs from the green alga Chlamydomonas reinhardtii [58].The dephosphorylation of PA at the sn-3 position yields diacylglycerol (DAG), the precursor of TAG, which is catalyzed by PAP.It has been reported in N. oceanica that four putative cytoplasmic PAP2 genes are stimulated by TAG-induction conditions, indicative of their roles in TAG biosynthesis [55].
The final step in TAG assembly involves the transacylation of DAG to TAG, catalyzed by DGAT.In N. oceanica, 13 putative homologs of DGAT have been identified, including 2 DGAT1 genes (DGAT1A-B) and 11 DGAT2 genes (DGAT2A-K, also known as DGTT1-11) [17].Among these, seven DGAT genes are upregulated under nitrogendepleted conditions, including DGAT1A and six DGAT2 genes [55].DGAT1A prefers C16 and C18 saturated/monounsaturated fatty acids as substrates, and its overexpression can result in a ~39% increase in the TAG content of N. oceanica under nitrogen deprivation [64].As for DGAT2 genes, they have been reported to show different preferences on the sub-strates of acyl-CoA and DAG, play roles in TAG synthesis, and have great potential in the manipulation of N. oceanica for enhancing TAG levels and/or modifying fatty acid compositions [65][66][67][68][69].
TAGs are the main storage lipids in eukaryotic cells and have been widely used for foods, light industrial products, and biofuels.In the biofuel sector, TAGs are harnessed as a source of fatty acids for the transesterification process, which converts them into biodiesel.This chemical transformation involves reacting TAGs with an alcohol, typically methanol or ethanol, in the presence of a catalyst, resulting in the production of biodiesel (fatty acid methyl or ethyl esters) and glycerol as a byproduct.The renewable and sustainable nature of TAGs, especially those derived from sources like microalgae, makes them highly desirable for biofuels [54].It is crucial to understand the pathways and key enzymes involved in TAG biosynthesis, as outlined in Figure 1.Another well-characterized pathway for TAG synthesis in microalgae is acylindependent and is mediated by phospholipid:diacylglycerol acyltransferase (PDAT).This enzyme transfers an acyl group from the sn-2 position of phospholipids (PLs) to form TAG [70].Similar to in C. reinhardtii, PDAT in N. oceanica functions parallel to the DGATmediated pathway for TAG biosynthesis and shows preferences for the acyl donors [71,72].It is worth mentioning that N. oceanica PDAT contributes more to the stress-associated TAG synthesis than to the basal TAG synthesis, seemingly distinct from C. reinhardtii PDAT [71].
It is generally accepted that EPA biosynthesis in Nannochloropsis occurs in the ER, where the key desaturases and elongases are predicted to be located.This is further confirmed via the ER subcellular localization experiment for ∆6-FAE, a key enzyme in EPA biosynthesis in N. oceanica [75].It should be noted that in Nannochloropsis, similar to in diatoms, EPA is mainly distributed in the chloroplast membrane lipids [69], raising the question that how EPA is channeled from the ER into chloroplasts.While there have been several studies dealing with this, it remains largely unknown [83,84].Additionally, EPA in chloroplast membrane lipids can be transferred to neutral lipids (NLs) like TAG during nitrogen deprivation, as demonstrated through 13 C isotopic labeling in the microalgae N. gaditana [85].The abundance of EPA in TAG, nevertheless, is relatively low for Nannochloropsis [69].Nannochloropsis also produces long-chain hydroxy fatty acids (LCHFAs), long-chain alkenols (LCAs), and long-chain alkyl diols (LCDs); some enzymes involved in the biosynthesis of these compounds have been identified, including polyketide synthases (PKSs) and 3-hydroxyacyl dehydratases (HADs) [86].

Carotenoid Biosynthesis
The Nannochloropsis chloroplast evolves from the secondary endosymbiosis and is surrounded by four membranes.Unlike green algae with chlorophylls a and b or diatoms with chlorophylls a and c, Nannochloropsis contains only chlorophyll a in its light-harvesting pigment-protein complexes (LHCs) [87].The proteomic analysis identified 17 LHC-type proteins among the 21 known in the genus Nannochloropsis, including LHCR-type proteins (related to red algae LHCI), LHCV proteins, and others [88].LHCV also refers to violaxanthin/vaucheriaxanthin chlorophyll protein (VCP), where the two central carotenoids are surrounded by five chlorophyll a molecules, preserving the LHC superfamily structure [89].The energy transfer efficiency of the VCP complex, from carotenoids to chlorophyll a, can reach up to 90% [90].The two carotenoids serve as the accessory pigments in light harvesting for Nannochloropsis [91].β-carotene is another prominent pigment in Nannochloropsis.
So far, the functional characterization of carotenogenic enzymes for N. oceanica remains limited.It has been reported that PDS overexpression has little effect on carotenoid profiles, while LCYB overexpression benefits the accumulation of carotenoids, particularly β-carotene; in this context, LCYB, rather than PDS, mediates a rate-limiting step for carotenoid biosynthesis in N. oceanica [92].There are two phylogenetically distant ZEP isoforms in N. oceanica, namely, ZEP1 and ZEP2.The functional roles of the two ZEPs have been revealed in a recent study, which supports the hypothesis that both ZEP1 and ZEP2, localized in the chloroplast, overlap in epoxidating zeaxanthin to violaxanthin for the light-dependent growth of N. oceanica [93].It is worth noting that ZEP1 appears to be more functional than NoZEP2 [93].N. oceanica harbors one VDE; deactivating this enzyme via insertional disruption can attenuate zeaxanthin accumulation substantially in response to high light and thus leads to growth impairment [94].Likely, zeaxanthin contributes to the induction of the non-photochemical quenching and scavenging of reactive oxygen species, thus playing an essential role in protecting the alga from excess light.While violaxanthin is essential for N. oceanica, neoxanthin and vaucheriaxanthin are not, as a disruption to VDL that leads to the abolishment of the two carotenoids is viable [95].As expected, the VDL-deficient mutant accumulates more violaxanthin [95].
Violaxanthin exhibits significant antioxidative capacity and anti-inflammatory activity against cancer cells [96].A previous study has demonstrated that extracts from Nannochloropsis, primarily composed of violaxanthin, can protect against UV-B-induced damage [11].In this context, the pursuit of violaxanthin enhancement in Nannochloropsis is an intriguing avenue of exploration.The overexpression of ZEPs can enable N. oceanica to synthesize more violaxanthin, yet the increase remains to be enlarged [93].Although N. oceanica synthesizes astaxanthin, a valuable ketocarotenoid with broad applications [97], the level is very low and generally detectable under stress conditions [24].The enzymes responsible for astaxanthin biosynthesis in the alga have not been identified.Given the relative abundance of β-carotene, the direct precursor of astaxanthin, introducing a strong β-carotenoid ketolase has the potential to allow N. oceanica for synthesizing astaxanthin in addition to EPA [98,99].Considering the function of ZEP1/2 [93], suppressing its expression may reallocate carotenoid flux towards astaxanthin synthesis in the engineered N. oceanica.The accumulated astaxanthin may, in turn, endow the alga with an improved production performance under high light, as is the case for C. reinhardtii [100].Moreover, allowing astaxanthin storage in the cytosolic lipid droplets, like in the astaxanthin-rich Haematococcus pluvialis [101], will be a promising strategy for a further increase in astaxanthin in the engineered N. oceanica.
Nannochloropsis can be employed as a potential host for the production of recombinant proteins.For example, N. oceanica has been genetically engineered to produce the surface protein VP2 of the infectious pancreatic necrosis virus, which causes high mortality rates in young salmonids [105].Nannochloropsis is also a promising candidate for the production of marine antimicrobial peptides.These peptides have garnered attention due to their natural ability to combat various pathogens and microbes in the marine environment.The oral administration of antimicrobial peptide-producing N. oculata can enable substantial improvements in the survival rate of medaka after infection with Vibrio parahaemolyticus [106].Moreover, the biosynthesis of a fish growth hormone has been achieved in N. oculata; this hormone can promote the growth of red tilapia larvae [107].Overall, genetic engineering and synthetic biology hold the key to utilizing Nannochloropsis to produce targeted products.

Cultivation Strategy for Nannochloropsis Production
To optimize Nannochloropsis production, a well-thought-out cultivation strategy is essential.Nitrogen deprivation is a well-established strategy for inducing lipid accumulation in Nannochloropsis.When nutrients are limited, the algal cells tend to divert energy and carbon toward lipid biosynthesis.Nitrogen deprivation can trigger TAG accumulation in N. gaditana, accompanied by a reduction in polar lipids and the partial translocation of EPA from polar lipids to TAG [108].A reduction in EPA content is also observed for N. gaditana responding to nitrogen deprivation, which is independent of light regimes and intensities [109].There is a considerable decrease in chlorophyll content upon nitrogen deprivation, accompanied by changes in carotenoids [54].
Multi-stage cultivation represents an effective strategy for maximizing lipid production from Nannochloropsis.In the initial stage, Nannochloropsis is cultivated under nutrientreplete conditions to grow fast and achieve high cell densities.Following the growth phase, the culture is subjected to stress-inducing conditions.Common stressors include nutrient limitations, such as nitrogen and phosphorus deprivation.Stress conditions divert the energy and metabolic resources of the algal cells away from growth to the synthesis of lipids.Such a two-stage cultivation approach can lead to more than a 2-fold increase in lipids for Nannochloropsis [110].Another effort using a two-stage continuous cultivation approach for N. gaditana, which involved four photobioreactors connected in series, resulted in a remarkable 7.7-fold increase in biomass and a 46% increase in lipid productivity [111].
A fed-batch culture is a sophisticated strategy for production improvements.In this approach, nutrients are supplied incrementally during the growth phase, allowing for higher cell densities and enhanced productivity.This strategy has been employed for the cultivation of various Nannochloropsis species, allowing a considerably higher EPA abundance (based on total fatty acids) and yield in N. oculata [112] and greater biomass production and EPA yield in N. oceanca [69].Implementing these cultivation strategies, alongside ongoing research and innovation, will play a pivotal role in realizing the full potential of Nannochloropsis for sustainable and economically viable production.

Genetic Resources
Several species of Nannochloropsis have been sequenced, with a nuclear genome size ~29 Mb nuclear genome that encodes a total of over 10,000 genes [17,[113][114][115][116][117].There are increasing number of transcriptomes regarding Nannochloropsis under different conditions [45,46,55,[118][119][120][121][122][123][124].These genome and transcriptome data help us to understand the biology of Nannochloropsis and its metabolic adaptability to respond to dynamic environmental changes, and, on the other hand, provide genetic resources for the genetic engineering of this organism.

Transformation Methods, Selectable Markers, and Reporters
Nannochloropsis species contain a rigid cell wall, which serves a barrier for the penetration of transgenes.The first successful nuclear transformation of Nannochloropsis was conducted in 2011 via electroporation with a very high voltage [125].Since then, the electroporation-mediated transformation method has been established for various Nannochloropsis species [115].Compared to linearized plasmids, the use of a PCR-amplified cassette can improve Nannochloropsis' transformation efficiency [126].The co-transformation of Nannochloropsis with two genes has also been achieved [126].With the assistance of secondary metabolites from myxobacteria that help weaken the algal cell wall, a 2.7-fold increase in transformation efficiency can be reached for N. salina [127].Other nuclear transformation methods besides electroporation have been developed as well, such as agrobacterium-mediated transformation for Nannochloropsis sp.[128] and particle bombardment for N. oceanica [129].Moreover, an electroporation-mediated chloroplast transformation method has been developed, opening up possibilities for plastid genome editing in Nannochloropsis [130].These diverse transformation techniques offer researchers tools to engineer Nannochloropsis for target applications.
Screening transformants of Nannochloropsis, regardless of the transformation methods, requires the assistance of proper selectable markers.The most widely used selectable marker is the bleomycin-resistant gene, which confers Nannochloropsis with resistance against zeocin [125].Additional feasible markers for Nannochloropsis transformation include hygromycin-B phosphotransferase, blasticidin-S deaminase, aminoglycoside 3 ′ -phosphotransferase, and nourseothricin acetyltransferase, which are resistant to hygromycin B, blasticidin, G418, and nourseothricin, respectively [15].These antibiotic resistance genes are not desirable, as they have their limitations and face regulatory hurdles, particularly for food uses, driving the development of endogenous genes as alternative selectable markers.One promising candidate is the mutated PDS gene, which has been employed in several algae to confer resistance to herbicides [131][132][133][134].While selectable markers facilitate the screening of transgene integration into the nuclear genome, reporters play a pivotal role in monitoring transgene expression at the protein level or subcellular localization when fused with transgenes.The workable reporters in Nannochloropsis include green fluorescent protein [135], sfCherry fluorescent protein [136], luciferases (Nlux, Flux) [74], and purple chromoprotein [137], among others.
To enable the efficient expression of transgenes in Nannochloropsis, regulatory elements are needed, for example, promoters, terminators, enhancers, and transit peptides.Generally, strong endogenous promoters are used for transgene expression in Nannochloropsis, including those from ubiquitin extension protein (UEP) [115,136], β-tubulin (βtub) [56,138], lipid droplet surface protein (LDSP) [17,74], elongation factor (EF) [66,74], violaxanthin/chlorophyll a-binding protein 2 (VCP2) [64], and ribosomal subunits (Ribi) [74].The frequently used terminators are those from LDSP, VCP1, and heat shock protein (HS) [15].To facilitate the transcriptional expression of transgenes, a leader-enhancing sequence may be placed upstream of the transgene [65].To guide the compartmentation of expressed proteins, transit peptides are needed, for example, the chloroplast transit peptides from glycine cleavage system protein L (GCSL) and VCP1 [135,139].There is an increasing need for the characterization of more regulatory elements (e.g., inducible promoters, dose-dependent promoters) to meet the needs of diverse uses in the genetic engineering of Nannochloropsis.

Genetic Tools
The implementation of Nannochloropsis engineering for trait manipulation requires sophisticated genetic tools.Overexpression represents a frequently employed approach for the engineering of Nannochloropsis, which typically involves a single gene of interest.To achieve the expression of multiple genes, these are assembled in separate vectors for Nannochloropsis transformation [69,74].This, however, requires the use of multiple antibiotic resistance markers.To reduce the use of antibiotic resistance markers, a highcapacity gene-stacking toolkit has recently been developed that takes advantage of the Gateway cloning technique and allows the easy assembly of multiple gene expression cassettes (with individual promoters and terminators) into a single vector for N. oceanica transformation [15].The transgenes can also be assembled in a single vector, fused by selfcleaving 2A peptides, and have proved workable for multigene expression in N. oceanica [74] and N. salina [140].This technique eliminates the use of multiple promoters and terminators and thus can reduce the size of vectors.Nevertheless, even with the optimization of 2A types, the cleavage rate of 2A-fused proteins is not high, reaching no more than 50% in N. oceanica [74].To enable the synthetic biology of Nannochloropsis, a more sophisticated and standardized toolkit is needed.A modular cloning toolkit has been developed for the green alga Chlamydomonas; the toolkit is based on the Golden Gate cloning method with standard syntax and comprises over 100 openly distributed genetic parts [141].This provides insights into the development of such a toolkit for Nannochloropsis.
Besides overexpression, suppression is a common employed strategy to understand gene function and can be achieved via knockdown or knockout.In Nannochloropsis, RNA interference (RNAi)-mediated gene knockdown has been proven feasible, with the knockdown efficiency reaching up to 80% [56,64,71,93,142,143].As for gene knockout, the homologous recombination (HR) approach has long been applied to Nannochloropsis, with a starting effort in inactivating the nitrate reductase gene [125].However, due to the low HR efficiency in Nannochloropsis, HR-mediated gene knockout has only been achieved by a limited number of studies [62,144].The CRISPR/Cas system represents an emerging technology in gene editing and has been widely applied to various organisms.Using the nitrate reductase gene as the target, the CRISPR/Cas9-mediated gene editing of Nannochloropsis was first developed in N. oceanica but with a very low efficiency of less than 1% [138].With the assistance of homologous DNA templates, the CRISPR/Cas9-mediated gene knockout efficiency in Nannochloropsis can be improved and varied depending on the target genes [145].To eliminate the restriction of selectable markers, the combination of CRISPR/Cas9 with an inducible Cre recombinase has been introduced into N. gaditana; this can enable the removal of marker genes and thus the potentially unlimited stacking of gene knockouts in the alga [146].As well as being integrated into the nuclear genome, the CRISPR/Cas9 in the plasmid with the presence of the CEN/ARS6 region from S. cerevisiae can be maintained as circular extrachromosal DNA in N. oceanica; once targeted mutations are generated, the episomal DNA can be removed in the absence of selection pressure, resulting in marker-free non-transgenic engineered strains [147].Furthermore, Cas ribonucleoprotein can be delivered into N. oceanica via electroporation for gene editing, thus eliminating the involvement of the plasmid; the editing efficiency using the Cas12a from Francisella novicida can reach up to 90% [148].With this Cas12a, an episomal plasmid-based CRISPR/Cas12a system has been developed, capable of performing multiplexed gene editing in N. oceanica [149].Moreover, CRISPR/Cas9-based transcription interference and activation systems have also been established for N. oceanica [149,150].

Conclusions
Nannochloropsis harbors advantageous features and has attracted increasing interest from academic and commercial stakeholders.It has emerged as a focal point in synthetic biology due to its versatile genetic characteristics and potential applications in biofuel production, bioremediation, and the synthesis of valuable compounds.Yet there are many things to do to increase the production potential of Nannochloropsis.Firstly, the cultivation strategies for Nannochloropsis need to be upgraded, with an emphasis on large-scale production for various applications.The development of next-generation photobioreactors and corresponding cultivation strategies will be instrumental in achieving commercial-scale biorefining.Secondly, the application of advanced omics technologies will further our understanding of Nannochloropsis' biology and metabolism, enabling more precise engineering for trait improvements.Thirdly, synthetic biology tools will continue to play a pivotal role in tailoring Nannochloropsis.This will depend on the development of versatile and robust toolkits.Learning from other algae such as C. reinhardtii and P. tricornutum would provide valuable insights and solutions for advancing the synthetic biology of Nannochloropsis.Nannochloropsis-based biorefineries hold great potential for the sustainable production of biofuels, high-value chemicals, and nutraceuticals.A key focus will be to optimize production processes to ensure economic viability and to meet the growing demand for renewable and environmentally friendly products.Navigating the potential applications of Nannochloropsis in synthetic biology is not without its set of challenges.One of the primary hurdles lies in achieving a meticulous level of control over the genetic modifications made to this microalgae genus.Ensuring the stability and predictability of the outcomes is essential for the successful integration of Nannochloropsis-based technologies into industrial processes.The precision required in genetic manipulation becomes increasingly critical as efforts are made to scale up production and consistently meet the demands of various industries.
In conclusion, Nannochloropsis represents a promising light-driven cell factory in the realm of synthetic biology.The inherent ability to synthesize abundant lipids and valuable pigments makes Nannochloropsis an ideal candidate for the sustainable production of biofuels and high-value compounds.Through the concerted efforts of researchers and the continued development of synthetic biology tools, we anticipate substantial advancements in strain engineering, large-scale production, and biorefining.With ongoing research and innovation, Nannochloropsis is set to contribute to shaping a more sustainable and greener future.