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Background The development of affordable strategy to concurrently enhance microalgal biomass and biocomponents is warranted for commercial applications. Here, we investigated the efficacy of a cheap and natural polyphenol, tannic acid, on regulating metabolic and physiological properties of oleaginous microalga Phaeodactylum tricornutum to overproduce biocompounds. Result Tannic acid provision regulated the key metabolic pathways to enhance algal biomass and lipids in a dose-dependent manner without direct impact on photosynthesis. It reduced oxidative stress and reallocated carbon precursors towards lipogenesis. Lipidomic analyses showed that tannic acid provision unprecedentedly regulated the key lipogenic pathways, enhanced glyco- and neutral-lipids by 1.29- and 1.54-fold, respectively, whereas phospholipids were significantly altered. Tannic acid facilitated polyunsaturated fatty acid overproduction with a specific increment of EPA and DHA by 1.18- and 2.25-fold, respectively. Transcriptomic analysis demonstrated that tannic acid upregulated the expression of multiple genes involved in lipogenesis. Conclusion Here, we dissected the potential of tannic acid, a natural and cheaper polyphenol, on concurrently enhancing lipids and PUFAs without impairing physiological properties in P. tricornutum. The findings provide novel insights into the mechanistic roles of polyphenol as a potential chemical modulator.

The established human health benefits of carotenoids along with the contemporary consumption of natural carotenoids bring the necessity to sustainable production of carotenoids. Among, marine diatoms have emerged as the potential biological resources for carotenoid production; however, their relatively lower yield in native strains provides the impetus to genetically improve the diatoms to cope with the burgeoning demand. In this study, we genetically improved the diatom Phaeodactylum tricornutum by overexpressing key carotenogenic genes involved in methylerythritol phosphate (MEP) pathway. The genes with lower relative transcript level under optimum conditions such as CMK and CMS were selected and overexpressed in P. tricornutum individually. Both CMK and CMS overexpressing lines exhibited elevated growth and photosynthesis. The expression of key carotenogenic genes such as PSY, PDS, ZDS, CRT, and LCYB was significantly upregulated. Furthermore, total carotenoid content was significantly increased; particularly, fucoxanthin content was increased by 1.83- and 1.82-fold in engineered lines CMK and CMS, respectively. Together, the results identify the potential metabolic targets and also uncover the crucial role of MEP pathway in redirecting metabolic precursors towards carotenogenesis. KEY POINTS: • Low abundant genes CMS and CMK of MEP pathway were overexpressed in the diatom • Total carotenoid content was increased, particularly fucoxanthin • Critical metabolic nodes were uncovered to accelerate fucoxanthin biosynthesis.

The unicellular freshwater green alga

Background Despite the great potential of marine diatoms in biofuel sector, commercially viable biofuel production from native diatom strain is impractical. Targeted engineering of TAG pathway represents a promising approach; however, recruitment of potential candidate has been regarded as critical. Here, we identified a glycerol-3-phosphate acyltransferase 2 (GPAT2) isoform and overexpressed in Phaeodactylum tricornutum. Results GPAT2 overexpression did not impair growth and photosynthesis. GPAT2 overexpression reduced carbohydrates and protein content, however, lipid content were significantly increased. Specifically, TAG content was notably increased by 2.9-fold than phospho- and glyco-lipids. GPAT2 overexpression elicited the push-and-pull strategy by increasing the abundance of substrates for the subsequent metabolic enzymes, thereby increased the expression of LPAAT and DGAT. Besides, GPAT2-mediated lipid overproduction coordinated the expression of NADPH biosynthetic genes. GPAT2 altered the fatty acid profile in TAGs with C16:0 as the predominant fatty acid moieties. We further investigated the impact of GPAT2 on conferring abiotic stress, which exhibited enhanced tolerance to hyposaline (70%) and chilling (10 ºC) conditions via altered fatty acid saturation level. Conclusions Collectively, our results exemplified the critical role of GPAT2 in hyperaccumulating TAGs with altered fatty acid profile, which in turn uphold resistance to abiotic stress conditions.

Blooms of the dinoflagellate Prorocentrum donghaiense occur frequently along the East China coast and Yangtze River estuary and pose a potential threat to coastal ecosystems. However, information on the genetic diversity of P. donghaiense populations in the East China Sea remains limited despite its ecological significance. Here we developed nine microsatellite markers based on transcriptome sequencing and analyzed genetic diversity and structure of P. donghaiense population in the East China Sea. We found that the Nei’s genetic diversity index of P. donghaiense population varied from 0.019 to 0.551 with an average of 0.227, whereas Shannon’s index ranged from 0.055 to 0.968 with an average of 0.431. Moreover, four genetically distinct subpopulations were identified based on STRUCTURE analysis. These results clearly indicated that P. donghaiense population during the blooms had high genetic diversity in the East China Sea, which might form the basis for variability in phenotype of P. donghaiense population and empower its capability to adapt to changing environment. Collectively our findings uncovered the genetic distinctiveness in P. donghaiense populations between 2014 and 2016 and provide valuable insights into the genetic structure and diversity of P. donghaiense blooms.

BACKGROUND: Microalgal lipid overproduction has been achieved greatly by stress treatments, which drastically impede cellular biomass and overall productivity, hence providing the impetus to develop a viable strategy to uncouple lipid enhancement from stress associated growth impairment. RESULTS: We designed a sequential strategy in Nannochloropsis oceanica, to enhance cellular biomass and carbon sink by providing optimized bicarbonate source during the growth phase, followed by low‐temperature treatment to harness the increased cellular biomass and carbon precursors for lipid hyperaccumulation. Provision of bicarbonate (1.0 g/L) remarkably promoted cellular physiological properties, which in turn enhanced cellular biomass and photosynthetic carbon sink. Subsequent low‐temperature treatment (16 °C) significantly redirected the carbon flux towards lipogenesis and significantly altered fatty acid composition. The designed strategy resulted in significant enhancement of lipid productivity by 2.9‐fold than that of control cells and resulted in lipid accumulation up to 57% of dry cell weight without impeding cellular biomass. Interestingly, the sequential treatment altered fatty acid profile, particularly a 8.8‐fold increase in the content of eicosapentaenoic acid with health significance. CONCLUSION: Our study exemplifies the feasible strategy to potentiate microalgal biomass, and also to circumvent stress associated growth impairment and consequent algal lipid productivity. © 2019 Society of Chemical Industry

Provision of chemical modulators has emerged as an effective strategy to govern cell growth and development. Here, the impact of flavonoid quercetin on algal growth, lipid accumulation and transcriptional patterns was investigated in the green microalga Chlorella vulgaris. These results demonstrated that quercetin (15 μg/l) significantly enhanced the cellular biomass and photosynthetic efficiency, with up to 2.5-fold in the biomass in the stationary phase. Lipidomic analyses revealed that lipid content was increased by 1.8-fold. Furthermore, the functional mechanism of quercetin on the molecular level was dissected by transcriptomic analysis. Results revealed that quercetin upregulated the expression pattern of key genes involved in cellular signaling mechanisms such as phosphatidylinositol 4-kinase α, thus consequently enhanced cell growth. Altogether, the data present in this study demonstrate the dramatic role of quercetin on enhancing microalgal biomass and lipid accumulation by unprecedented regulation, of key metabolic nodes, for the first time and provide a novel insight into microalgal metabolism and regulation.

Tobacco wastewater is too difficult to decontaminate which poses a significant environmental problem due to the harmful and toxic components. Chlorella pyrenoidosa is a typical microalgal species with potential in removal of organic/inorganic pollutants and proves to be an ideal algal-based system for wastewater treatment. However, the strategy of tobacco related wastewater treatment using microalgae is in urgent need of development. In this study, C. pyrenoidosa was used to evaluate the removal efficiency of artificial tobacco wastewater. Under various solid-to-liquid (g/L) ratios, 1:1 ratio and acidic pH 5.0 were optimal for C. pyrenoidosa to grow with high performance of removal capacity to toxic pollutants (such as COD, NH3-N, nicotine, nitrosamines and heavy metals) with the alleviation of oxidative damage. Algal biomass could reach up to 540.24 mg/L. Furthermore, carbon flux of C. pyrenoidosa was reallocated from carbohydrate and protein biosynthesis to lipogenesis with a high lipid content of 268.60 mg/L at pH 5.0. Overall, this study demonstrates an efficient and sustainable strategy for tobacco wastewater treatment at acidic pH with the production of valuable microalgal products, which provides a promising biorefinery strategy for microalgal-based wastewater bioremediation.

Molybdenum disulfide nanoparticles (MoS2 NPs) hold tremendous properties in wide domain of applications. In this study, the impact of MoS2 NPs was investigated on algal physiological and metabolic properties and a two-stage strategy was acquired to enhance the commercial potential of Dunaliella salina. With 50 µg/L of MoS2 NPs exposure, cellular growth and biomass production were promoted by 1.47- and 1.33-fold than that in control, respectively. MoS2 NPs treated cells were subject to high light intensity for 7 days after 30 days of normal light cultivation, which showed that high light intensity gradually increased β-carotene content by 1.48-fold. Furthermore, analyses of primary metabolites showed that combinatorial approach significantly altered the biochemical composition of D. salina. Together, these findings demonstrated that MoS2 NPs at an optimum concentration combined with high light intensity could be a promising approach to concurrently enhance biomass and β-carotene production in microalgae.

Waste streams have emerged as potential feedstocks for biofuel production via microbial bioconversion. Metabolic engineering of the microalga Phaeodactylum tricornutum in its lipid biosynthetic pathways has been conducted with an aim to improve lipid production. However, there has been only limited achievement in satisfying biofuel demands by utilising extracellular organic carbons from low-cost waste streams. Herein, we present a successive staged cultivation mode, based on a previously engineered strain that co-overexpresses two key triacylglycerol biosynthesis genes. We first optimised microalgal biomass and lipid production by using food waste hydrolysate and crude glycerol as the cultivation media. Food waste hydrolysate (5% v/v) is a low-cost organic carbon source for enhanced microalgal biomass production, and the resulting lipid concentration was 1.08-fold higher with food-waste hydrolysate than that of the defined medium. Additionally, the resultant lipid concentration after using crude glycerol (100 mM) was 1.24-fold higher than that using the defined medium. Two carbon feeding modes (hybrid and sequential) were also performed to investigate the potential of engineered P. tricornutum with preliminary mechanistic analyses. The biodiesel properties of lipids produced in the hybrid mode were evaluated for potential application prospects. Collectively, this study demonstrates a waste stream utilisation strategy for efficient and sustainable microalgal biofuel production.

Though microalgae have been considered the potential resource for lipid production, native strains are unable to meet the industrial demand. Here, we aim to uncover the complex molecular relationship between algal growth and lipid accumulation. Transcriptomic analysis revealed the crucial role of plastidial fatty acid and triacylglycerol (TAG) biosynthetic machinery in lipid overproduction. The expression of key fatty acid biosynthetic genes such as acetyl-CoA carboxylase (ACCase), malonyl CoA‐acyl carrier protein transacylase (MCAT), 3-ketoacyl synthase (KAS), 3-ketoacyl-ACP reductase (KAR) increased during day 10-13 of cultivation, particularly plastidial TAG biosynthetic genes substantially increased. However, expression of genes involved in ER TAG biosynthesis increased only in the stationary phase, which implied the potential of plastidial TAG biosynthesis. This report provides a novel insight into the growth-phase dependent lipogenic orchestration, and also uncovers the signature genes and plastidial TAG biosynthesis that might be extrapolated for improving lipogenic traits of algae.

Additional file 1: Fig. S1. In silico analyses of GPAT2 sequence. (A) Protein structure of GPAT2. (B) Phylogeny analysis of GPAT2 from the deduced amino acid sequence of GPAT2 and GPAT of various organisms. Phylogenetic tree was constructed by MEGA using Neighbor joining method. Percentage of the replicate trees where the taxa clustered together in the bootstrap value (500 replicates) are provided in the branches. Arrow mark denotes the GPAT2 of P. tricornutum. Fig. S2. Analyses of photosynthetic parameters in transgenics. (A) Maximum quantum yield of photosystem II as indicated by Fv/Fm. (B) Determination of chlorophyll α content (pg/cell). (C) Non-photo chemical quenching (NPQ). (D) Electron transport rate (ETR) (µmol e-m-2s-1). Significant difference between WT and transgenics is indicated at the p

Antifreeze proteins (AFP) are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of LpAFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type (WT) plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content (RWC) and electrolyte leakage index (ELI) than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that LpAFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.

Microalgal metabolic engineering holds great promise for algal biofuels. However, identification of the key lipid metabolic target remains challenging due to its complex regulation. In this study, we advocated an alternative strategy that potentially rewired lipid metabolism by unprecedented mechanisms. PNPLA3, a human protein associated with non-alcoholic fatty liver disease (NAFLD), was firstly tested in microalgae for enhancing lipid accumulation. HsPNPLA3 was synthesized with a site mutation (I148M) and expressed in a model diatom Phaeodactylum tricornutum . Heterogeneous HsPNPLA3-I148M was successfully integrated, transcribed and expressed. Lipidomic analyses revealed that HsPNPLA3-I148M significantly elevated TAG content by 1.55-fold in algae, while algal growth and photosynthetic rate were not impaired. Fatty acid profile showed that content of C16:0, C18:1 and C20:4 was increased by 1.43-, 4.18- and 4.3-fold, respectively, which implied that HsPNPLA3-I148M might regulate the fatty acid substrate preference. Overall, the findings demonstrated that human PNPLA3 played a potential role in elevating TAG accumulation by regulating lipogenic enzymes and provide unprecedented insights into its functional significance.

Oleaginous microalgae have emerged as the promising feedstock for biofuels due to their various advantageous features over terrestrial plants. However, the microalgal fuel industry is still in its infancy due to the unavailability of robust algal strains with enhanced lipid content and biomass and economically feasible oil extraction strategies. Microalgal metabolic engineering shows the great potential to enhance lipid accumulation without impairing cell growth by targeted control of key metabolic nodes. Despite the recent advancements in synthetic biology, genetic improvement of microalgae without hindered cellular biomass remains an untapped tool for large-scale biofuel production. In this review, we therefore provide a comprehensive overview of various metabolic engineering strategies for lipid enhancement and the necessity of designing the appropriate strategy for scale-up processes. We have emphasized the need of streamlining the engineering strategies with the aid of recent advancements in DNA manipulation techniques to obviate the existing biological intricacies in lipogenesis. We outline the significance of recruiting potential engineering strategies such as transcription engineering, heterologous recombineering, and genome engineering to generate chassis algal strains successfully. Besides, this review highlights the designing of modular strategies by subjecting the engineered strains under sub-optimal conditions, mixotrophic cultivations and chemical treatment to overcome the existing bottlenecks in microalgal commercialization. This review also discusses the current large-scale algal cultivation units and the drawbacks. Overall, this review provides insights into employing the metabolic rewiring strategies to engineer the intricate lipogenic circuit in microalgae for enhancing biofuel potential.

Microalgae encounter a range of adverse conditions which restricts algal biomass, thereby the primary productivity is constrained. Death-specific proteins (DSP) play a crucial role in orchestrating cellular growth under adverse conditions. However, the functional role of DSPs in diatoms remains scant. Here, we identified a DSP (designated as PtDSP) in the model diatom Phaeodactylum tricornutum and elucidated its role in alleviating cellular growth and photosynthesis in a stress-dependent manner. Plastid-localized PtDSP upheld the cellular physiological properties under specific stress conditions. Overexpression of PtDSP potentiated the cells to withstand iron deprivation rather than nitrogen and phosphorus deprivation. Under iron deprivation, PtDSP orchestrated the expression of key genes involved in photosynthesis, thereby combated iron deprivation. This report epitomizes the role of DSP in governing diatom physiological properties in a stress-dependent manner and advances our understanding of the systematic response of diatoms to environmental changes.

Metabolic engineering has been hailed as a promising tool to impart oil productivity in fast-growing microalgae, thereby generating industry-suitable strains. However, complex regulatory networks in lipogenesis hampered the success to unify the abundance of metabolic carbon precursors and reducing equivalents towards lipogenesis. To obviate the biological bottlenecks, we attempted to rewire the pyruvate pathway, which could enhance the supply of NADPH by heterologously overexpressing a malic enzyme from the oleaginous diatom Phaeodactylum tricornutum (PtME) in fast-growing microalga Tetradesmus obliquus (previously known as Scenedesmus obliquus). PtME overexpression did not impair physiological properties and upheld lipogenesis by facilitating the adequate provision of CO2 and NADPH, respectively, through malate decarboxylation. The malate-pyruvate pathway played a role in elevating lipid content by 2.4-fold via coupling the carbon precursors and reducing equivalents. The malic enzyme activity increased by 9.9-fold, thereby elevated lipogenic NADPH. Together, these data exemplify the crucial role of PtME in pyruvate cycle for amalgamating the provision of metabolic carbon precursors and NADPH towards lipid accumulation and lay a strong foundation for metabolic engineering in algal industrialization.

Lipid droplets (LDs) are lipid monolayer-enclosed organelles comprising a lipid core and surface associated-proteins. However, the protein components and their regulatory functions in LDs have remained largely unknown in oleaginous diatoms. In this study, we identified a gene encoding lipid droplet (LD)-associated protein ( PtLDP1 ) in Phaeodactylum tricornutum and examined its function. The PtLDP1 showed homology to the diatom-oleosome-associated protein 1 (DOAP1) from Fistulifera . Overexpression of the PtLDP1 gene elevated lipid content, enlarged LD size and increased relative expression levels of key genes involved in triacylglycerol (TAG) and fatty acid biosynthesis. In contrast, knockdown of PtLDP1 by RNAi decreased lipid and TAG content, and subsequently reduced LD size. In addition, LDs were isolated from P. tricornutum cells and the proteome of LDs was identified by mass spectrometry. We found that PtLDP1 was a significant protein in the LD proteome. Importantly, labeling of enhanced yellow fluorescent protein (EYFP) confirmed that the PtLDP1 was localized to the LDs. Altogether, our data suggest that the PtLDP1 could be an important LD-associated protein contributing to regulation of TAG synthesis and lipogenesis. The findings will provide new targets for genetic improvement of oleaginous microalgae.

Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as promising alternatives to depleting fish oils. However, the overproduction of LC-PUFAs in microalgae has remained challenging. Here, we report a sequential metabolic engineering strategy that systematically overcomes the metabolic bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein transacylase, catalyzing the first committed step in type II fatty acid synthesis, and desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in Phaeodactylum tricornutum. Engineered microalgae hyper-accumulated LC-PUFAs, with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contents of up to 18.98 μg/mg and 9.15 μg/mg (dry weight), respectively. Importantly, eicosapentaenoic acid (EPA) was accumulated up to a highest record of 85.35 μg/mg by metabolic engineering. ARA and EPA were accumulated mainly in triacylglycerides, whereas DHA was found exclusively in phospholipids. Combinatorial expression of these critical enzymes led to the optimal increment of LC-PUFAs without unbalanced metabolic flux and demonstrated the practical feasibility of generating sustainable LC-PUFA production.

There is a huge interest in exploiting microalgae as an alternative bioenergy resource. Genetic improvement of microalgal strains with significant commercial potential is urgently demanded. Seipin has been shown to be associated with adipocyte differentiation and lipid droplet (LD) formation in mammals. However, very little is known about seipin in microalgae. Here, we identified a putative seipin in the oleaginous marine diatom Phaeodactylum tricornutum and developed a transgenic strain with overexpressed seipin. The transgenic strain possessed higher neutral lipid content and larger LDs than the wild type, and neutral lipid content was increased by 57%. Moreover, in this transgenic strain, the relative content of saturated fatty acids in total fatty acids was increased significantly, which improved lipid quality for the oil refinery. Our findings elucidated the significant potential role of seipin in regulating lipid accumulation and LD formation in microalgae for the first time.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

Burgeoning demand for long-chain polyunsaturated fatty acids (LC-PUFA) due to their established pharmacological and economic significances along with the declining sources of fish urge to explore the sustainable sources of LC-PUFA. Being the predominant LC-PUFA source for marine fishes at the base of the aquatic food web, microalgae has been hailed as a promising natural source for LC-PUFA. However, the potential of algal systems to overproduce LC-PUFA via metabolic engineering is warranted to meet the ever-increasing demand. In this study, we identified and overexpressed Δ6-desaturase, the key enzyme involved in fatty acid desaturation and exemplified its potential on elevating PUFA and lipid content in Nannochloropsis oceanica. Δ6-desaturase overexpression enhanced growth and photosynthetic efficiency. Transgenic cells exhibited a remarkable increase in EPA content and reached up to 652.35 mg/g DW, , the highest EPA production in transgenic N. oceanica by expressing a single key enzyme without impeding growthin a transgenic heterokontgreen alga. Total lipid content was significantly increased by 1.7-fold in the transgenic cell than WT. Together, these findings exemplify a potential candidate for LC-PUFA overproduction and also open a new avenue for sustainable production of microalgal PUFAs.

This study investigates the prospective of utilizing kitchen wastewater and food wastes, biofuels industry byproducts as alternative water and carbon sources. Kitchen wastewater did not impede cellular growth rate of the evolved Phaeodactylum strain E70, which indicates its potential as an alternative to freshwater resources. Among the organic wastes assessed, food waste hydrolysate significantly increased cell growth. Supplement of crude glycerol in cultivation medium enhances the total fatty acid content. Mixed food waste hydrolysate and crude glycerol remarkably increased both the cell density and total fatty acid content. Also, the supplement of butylated hydroxytoluene alleviated the oxidative stress induced by impurities in organic wastes and concomitantly increased microalgal total fatty acids and polyunsaturated fatty acids content. The experimental results reported in this study show that a waste-based biorefinery could lead to utilization of organic waste resources for the efficient production of value-added products.

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 μF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 μF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.