Nannochloropsis oceanica CCAP 849/10 was purchased from NCMA (National Center for Marine Algae and Microbiota) and cultivated in the filter-sterilized f/2 medium at 22 ± 1°C with an irradiance of 200 μmol photons m^–2s^–1 under 12/12 h light/dark photoperiod (Li et al., 2016).

Total RNA was prepared from N. oceanica using RNA/DNA/Protein isolation kit (Omega, United States) and subsequently, retro-transcribed into cDNA using the Prime Script^TM RT reagent kit (Takara, China). The full-length coding region of Δ6-desaturase (referred to as NoD6) was amplified using the primers NOD6-F and NOD6-R (Table 1) and the amplicon was purified using EZNA gel extraction kit (Omega, United States). The resultant NoD6 amplicon was cloned into pNa03 under the control of fcpC promoter and fcpA terminator using ClonExpress II One Step kit (Vazyme, China) according to the manufacturer’s specification (Figure 1A). Besides, an omega motif leader sequence was cloned between promoter and the transgene to facilitate its translation. The recombinant expression vector was linearized and electroporated into N. oceanica by using a Bio-Rad Gene Pulser Xcell electroporator following the protocol described by Li et al. (2016). Thereafter, the electroporated cells were harvested and preliminarily screened on the solid medium containing zeocin (5 μg/ml, Invitrogen) and the screened cells were cultivated in the selection liquid f/2 medium as described elsewhere (Li et al., 2016).

The putative transgenic strain was evaluated by single colony genomic PCR using the primers 89A and 91R that amplify the flanking region of the introduced NoD6 and the vector backbone as described previously (Li et al., 2016). Relative transcript level of NoD6 was determined by qPCR using a SYBR Green SuperMix for qPCR (Invitrogen, United States) on ABI Prism 7500 Sequence Detection System (Applied Biosystems, United States) as reported previously (Li et al., 2016). Briefly, total RNA was prepared from transgenics and WT using RNAiso Plus (Takara, Japan) and used as the template for the synthesis of first strand cDNA using the PrimeScript RT Reagent Kit (Takara, Japan) as per the provider’s instruction. The qPCR was performed in 96-well plates (20 μl reaction volume) as the manufacturer’s specifications (Applied Biosystems, United States). The threshold cycle value (Ct) for each reaction was determined and the relative mRNA abundance was determined after normalization to tublin gene.

The growth rate of transgenic and WT cells was determined by direct cell count method (Balamurugan et al., 2017) and estimating the specific growth rate (Sandnes et al., 2005). Photosynthetic efficiency of the cells was determined by measuring the maximum quantum yield of photosystem II (Fv/Fm) (Li et al., 2016).

Relative neutral lipid content of transgenic cell was measured by qualitative Nile-red (Sigma, United States) fluorometric analysis as described previously (Li et al., 2016). Lipid content of transgenic and WT cells was further quantitatively determined by gravimetric method. Total lipids were extracted from the freeze-dried microalgal cells as described by Bligh and Dyer, 1959 and the extracted lipid content was determined gravimetrically (Li et al., 2016). Nile-red stained cells were observed under confocal microscope LSM 510 Meta (Zeiss, Germany) as described previously (Li et al., 2016).

Total lipids were fractionated into phospholipid by solid-phase extraction (SPE) through gravity on a disposable pre-packed silica cartridges (500 mg, 6 cc Sep-Pak, Waters) conditioned with chloroform (5 ml). Thereafter, the extracted lipid sample (1 ml) was loaded on the chloroform-conditioned column and the PL was eluted into the pre-weighed glass vials with 10 ml of methanol. The fractionated lipids were transmethylated and the fatty acid composition of the extracted lipids was determined by using gas chromatography-mass spectrometry (GC-MS) as described elsewhere (Li et al., 2016).

All experiments were carried out in biological triplicate. The data were represented as mean ± SD (n = 3). The data obtained were subjected to one−way analysis of variance (ANOVA) followed by Tukey’s test using GraphPad Prism 7.0 (GraphPad, San Diego, CA, United States).

BLAST analysis using P. tricornutum Δ6-desaturase against N. oceanica genome, identified Δ6-desaturase coding region (Accession number KY214451.1) with high sequence similarity (E-value 4e-175). Pfam and SMART tools (Letunic and Bork, 2018; El-gebali et al., 2019) predicted the presence of four transmembrane domains and cytochrome b5 domain (31–101 aa), which could facilitate the acquisition of electrons from NADH cytochrome b5 reductase (Meesapyodsuk and Qiu, 2012; Figure 1B). Phylogenetic analysis by MEGA showed that NoD6 exhibited high homology with Δ6-desaturase of N. oculata and both were clustered into the same clade, while distinctly separated from others (Figure 1C). To elucidate its role in LC-PUFA biosynthesis, we cloned NoD6 in pNa03 expression vector and overexpressed in N. oceanica.

The putative transgenic algal cells were screened for zeocin resistance and cultivated on the selection medium supplemented with zeocin for at least four successive subcultures and thereafter, evaluated by molecular approaches. Genomic PCR was performed to identify the integration of transgene in the host, which resulted in the presence of 1.5-kb amplicon in transgenic cells, which is in accordance with the expected amplicon range, but no such band was detected in WT (Figure 1D). qPCR analysis showed that the relative transcript level of NoD6 was significantly increased by 2.6-fold in transgenic cells than WT (Figure 1E), however, enzymatic studies are needed to confirm the protein expression in transgenic cells. These results showed that NoD6 was successfully introduced and transcribed in the transgenic cells.

Concurrent enhancement of biomass and desired products is considered a critical parameter for commercial applications (Li et al., 2019). Hence, we examined the impact of NoD6 overexpression on cellular physiological parameters. Interestingly, NoD6 overexpression increased growth rate in transgenic cells compared to WT and it is worth mentioning that growth was significantly higher during stationary phase (Figure 2A), which is consistent with previous reports. Previous study has shown that expression of Δ12- and Δ5-desaturase enhanced growth in N. oceanica (Poliner et al., 2018). Similarly, expression of Δ5-elongase from Ostreococcus tauri enhanced growth in P. tricornutum during stationary phase. Thereafter, we examined the photosynthetic efficiency by determining Fv/Fm, which implied that NoD6 overexpression increased Fv/Fm during exponential phase, meanwhile Fv/Fm was reduced in transgenic cells during the stationary phase (Figure 2B). Besides, the specific growth rate was also found to be increased in transgenic cells (Figure 2C). Being the major membrane lipids of chloroplasts, PUFAs play a crucial role in maintaining plastidial membrane integrity and photosynthesis (Valentine and Valentine, 2004). Particularly, the potential of Nannochloropsis sp. to hyperaccumulate EPA in cellular membrane and thylakoids has been reported (Kaye et al., 2015). The abundance of PUFAs in plastids could enhance the rate of electron transport, thereby enhance photosynthetic efficiency (Chen et al., 2013). Consistently, our results showed that NoD6 overexpression enhanced photosynthetic efficiency, which enables the transgenic cells to harness the photosynthetic carbon sink toward enhanced growth and metabolite production during stationary phase which is in good agreement with previous studies (Thawechai et al., 2016).

As fatty acids are the key precursors for lipogenesis, fatty acid hyperaccumulation could enhance lipid production (Peng et al., 2014). To assess the effect of NoD6 overexpression on lipid accumulation and to elucidate the lipogenic potential of NoD6, we determined lipid content by fluorometric and gravimetric analyses. Nile-red analysis showed that lipid fluorescence was gradually increased, particularly, lipid fluorescence was significantly higher in transgenic cells than WT at 13th day (Figure 2D). Congruently, the gravimetric analysis showed that TAG was significantly increased and reached up to 50 mg/L in transgenic cells (Figure 2E). Laser scanning confocal microscopic analysis of the Nile-red stained cells corroborated the fluorometric and gravimetric lipid analysis, which showed that volume of lipid droplets was increased in transgenic cells than WT (Figure 2G). Interestingly, total lipid productivity was remarkably increased in transgenic cells and reached up to 21 mg/gDCW/day (Figure 2F). Delta-6 fatty acid desaturase overexpression significantly elevated total lipid in P. tricornutum (Zhu et al., 2017). Elongase overexpression increased TAG in Thalassiosira pseudonana (Cook and Hildebrand, 2015). Simultaneous expression of MCAT and Δ5-desaturase increased total lipid content by 2.61-fold in P. tricornutum (Wang et al., 2017). Congruently, individual overexpression of Δ5-desaturase elevated neutral lipid up to 65% in transgenic P. tricornutum (Peng et al., 2014). Effective consumption of fatty acid moieties for the generation of PUFA by NoD6 could result in the activation of upstream fatty acid biogenesis and provide adequate fatty acid precursors for lipogenesis, thereby overproducing lipids in the transgenic cell which is consistent with the previous reports (Chen et al., 2017).

Given the established health benefits of LC-PUFAs and their burgeoning demand, there exists a pressing need to develop a potential engineering strategy to overproduce algal LC-PUFAs (Martins et al., 2013). Consequently, tremendous efforts have been devoted to enhance algal PUFA content through various strategies (Yang et al., 2013; Hamilton et al., 2016). Stress treatments enhanced LC-PUFA content but impeded biomass and overall productivity, thereby obstructing the algal commercial potential (Mühlroth et al., 2013). On the other hand, microalgal genetic engineering yields mixed success owing to intricacies in LC-PUFA metabolism despite its potential (Peng et al., 2014). Hence, it is crucial to identify the key node to exploit its full potential (Li et al., 2019). To investigate the impact of NoD6 on LC-PUFA biosynthesis, we determined the fatty acid composition of total lipids and phospholipids during 10th day of cultivation, which showed that NoD6 overexpression significantly altered fatty acid composition (Figures 3D,E). Interestingly, fatty acids such as palmitic acid (PA), ARA and DHA were significantly increased. Total saturated and monounsaturated fatty acids were significantly reduced in transgenic cells, particularly, C16:1 was found to be significantly reduced. The reduced C16:1 content was is in accordance with the enhanced desaturation of oleic acid and subsequent desaturation of LA to GLA (Kaye et al., 2015). It is worth noting that LC-PUFAs such as ARA, EPA and DHA were increased by 2.04-, 1.53- and 2.32-fold, respectively in NoD6 overexpressing cells (Figures 3A–C), which accounts to 11.58, 62.35, and 5.80 μg/mg, respectively. Among the three hyperaccumulated LC-PUFAs, EPA was found to be predominant which constitutes up to 62.35 mg/g DCW at the 10th day of cultivation, however, EPA content was found to be decreased thereafter (Figure 3B). Meanwhile, DHA content was significantly higher in transgenic cells during 10th and 13th day, however, DHA content during the 13th day was found to be remarkably higher than that of the 10th day (Figure 3C).

Given the crucial role of Δ6-desaturase in catalyzing the first committed step of generating EPA from ALA, it emerged as the potential metabolic target for LC-PUFA overproduction (Zhu et al., 2017). Various reports demonstrated the exploitation of elongases, desaturases, fatty acid synthase or combinatorial multiple gene expression to hyperaccumulate algal LC-PUFAs (Martins et al., 2013; Gong et al., 2014), however, promising strategies and molecular candidates are yet to be characterized (Domergue et al., 2002). Zhu et al. (2017) reported the overexpression of Δ6-desaturase enhanced EPA up to 38.101 mg/g DW in P. tricornutum (Zhu et al., 2017). Expression of multiple desaturase genes increased EPA by 25% in Nannochloropsis oceanica (Poliner et al., 2018). Similarly, overexpression of endogenous elongases overproduced EPA and DHA, by 1.4- and 4.5-fold, respectively in transgenic T. pseudonana (Cook and Hildebrand, 2015). In contrast, our study showed the potential of NoD6 gene on remarkably increasing EPA and DHA which reached the highest record up to 62.35 and 5.80 mg/g of DCW, respectively in transgenic N. oceanica. The increased EPA content enhanced physiological properties of the transgenic cells which is reflected in the enhanced photosynthetic efficiency and subsequent primary metabolite content (Thawechai et al., 2016). Taken together, these findings demonstrated the unprecedented regulatory role of NoD6 on enhancing LC-PUFAs, providing the balanced carbon flux in the form of acyl-CoA moieties. Previous studies have reported the abundance of PUFAs in the acyl-CoA pool could govern the adequate provision of carbon precursors for acylation into neutral lipids (Hamilton et al., 2014). Congruently, increased PUFA content has been reported to elicit the upstream metabolic pathways of fatty acid biosynthesis and enhance the availability of carbon metabolic precursors (Peng et al., 2014), which in turn adequately provide the carbon precursors for growth and lipogenesis.