•A gene designated as PtLDP1 was markedly induced during N deprivation.•Knockdown of PtLDP1 led to reduced sizes of LDs and TAG accumulation.•Overexpression of PtLDP1 led to increased sizes of LDs and TAG accumulation.•The PtLDP1 was a major protein in LD proteome.•The PtLDP1 was localized to LDs.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.

•G6PD promotes fatty acid biosynthesis by supplying NADPH in the unicellular microalga.•G6PD is localized to the chloroplast.•G6PD overexpression stimulates PPP pathway and subsequent fatty acid synthesis.•G6PD overexpression rewires the primary metabolism towards lipid accumulation.Oleaginous microalgae have great prospects in the fields of feed, nutrition, biofuel, etc. However, biomass and lipid productivity in microalgae remain a major economic and technological bottleneck. Here we present a novel regulatory target, glucose-6-phosphate dehydrogenase (G6PD) from the pentose phosphate pathway (PPP), in boosting microalgal lipid accumulation. G6PD, involved in the formation of NADPH demanded in fatty acid biosynthesis as reducing power, was characterized in oleaginous microalga Phaeodactylum tricornutum. In G6PD overexpressing microalgae, transcript abundance of G6PD increased by 4.4-fold, and G6PD enzyme activity increased by more than 3.1-fold with enhanced NADPH production. Consequently, the lipid content increased by 2.7-fold and reached up to 55.7% of dry weight, while cell growth was not apparently affected. The fatty acid composition exhibited significant changes, including a remarkable increase in monounsaturated fatty acids C16:1 and C18:1 concomitant with a decrease in polyunsaturated fatty acids C20:5 and C22:6. G6PD was localized to the chloroplast and its overexpression stimulated an increase in the number and size of oil bodies. Proteomic and metabolomic analyzes revealed that G6PD play a key role in regulating pentose phosphate pathway and subsequently upregulating NADPH consuming pathways such as fatty acid synthesis, thus eventually leading to lipid accumulation. Our findings show the critical role of G6PD in microalgal lipid accumulation by enhancing NADPH supply and demonstrate that G6PD is a promising target for metabolic engineering.

Microalgae have emerged as a potential feedstock for biofuels and bioactive components. However, lack of microalgal strains with promising triacylglycerol (TAG) content and desirable fatty acid composition have hindered its commercial feasibility. Attempts on lipid overproduction by metabolic engineering remain largely challenging in microalgae.In this study, a microalgal 1-acyl-sn-glycerol-3-phosphate acyltransferase designated AGPAT1 was identified in the model diatom Phaeodactylum tricornutum. AGPAT1 contained four conserved acyltransferase motifs I–IV. Subcellular localization prediction and thereafter immuno-electron microscopy revealed the localization of AGPAT1 to plastid membranes. AGPAT1 overexpression significantly altered the primary metabolism, with increased total lipid content but decreased content of total carbohydrates and soluble proteins. Intriguingly, AGPAT1 overexpression coordinated the expression of other key genes such as DGAT2 and GPAT involved in TAG synthesis, and consequently increased TAG content by 1.81-fold with a significant increase in polyunsaturated fatty acids, particularly EPA and DHA. Moreover, besides increased lipid droplets in the cytosol, ultrastructural observation showed a number of TAG-rich plastoglobuli formed in plastids.The results suggested that AGPAT1 overexpression could elevate TAG biosynthesis and, moreover, revealed the occurrence of plastidial TAG synthesis in the diatom. Overall, our data provide a new insight into microalgal lipid metabolism and candidate target for metabolic engineering.

Microalgae are important primary producers in the marine ecosystem and excellent sources of lipids and other bioactive compounds. The marine diatom Phaeodactylum tricornutum accumulates eicosapentaenoic acid (EPA, 20:5n-3) as its major component of fatty acids. To improve the EPA production, delta 5 desaturase, which plays a role in EPA biosynthetic pathway, was characterized in P. tricornutum. An annotated delta 5 desaturase PtD5b gene was cloned and overexpressed in P. tricornutum. The transgene was integrated into the genome demonstrated by Southern blot, and the overexpression of PtD5b was verified by qPCR and Western blot analysis. Fatty acid composition exhibited a significant increase in the unsaturated fatty acids. Monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) showed an increase of 75% and 64%, respectively. In particular, EPA showed an increase of 58% in engineered microalgae. Meanwhile, neutral lipid content showed an increase up to 65% in engineered microalgae. More importantly, engineered cells showed a similar growth rate with the wild type, thus keeping high biomass productivity. This work provides an effective way to improve the production of microalgal value-added bioproducts by metabolic engineering.

The marine diatom Phaeodactylum tricornutum is attracting considerable interest as a candidate for biofuel production due to its fast growth and high lipid content. Nitrogen deficiency can increase the lipid content in certain microalgae species, including P. tricornutum. However, the molecular basis of such changes remains unclear without analyzing metabolism at the proteomic level. We attempted to systematically analyze protein expression level changes of P. tricornutum upon N deprivation. We observed translational level changes that could overall redirect the metabolic network from carbon flux towards lipid accumulation. N deprivation led to an increase in the expression of genes involved in nitrogen assimilation and fatty acid biosynthesis and a concomitant decrease in photosynthesis and lipid catabolism enzymes. These molecular level changes are consistent with the observed physiological changes, e.g., in photosynthesis rate and saturated lipid content. Our results provide information at the proteomic level of the key enzymes involved in carbon flux towards lipid accumulation in P. tricornutum and suggest candidates for genetic manipulation in microalgae breeding for biodiesel production.

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.

Plants are proven effective bioreactors for the production of heterologous proteins including those desired by the biopharmaceutical industry. However, the potential of plants as bioreactors is limited by the availability of characterized plant promoters that can drive target gene expression in relatively distant plant species. Seeds are ideal for protein storage because seed proteins can be kept stably for several months. Hence, a strong promoter that can direct the expression and accumulation of target proteins within seeds represents a powerful tool in plant biotechnology. Toward this end, an effort was made to identify such a promoter from Vigna radiata (mung bean) to drive expression in dicot seeds. A 784-bp 5′-flanking sequence of the gene encoding the 8S globulin α′ subunit (8SGα′) of the V. radiata seed storage protein was isolated by genome walking. When the 5′-flanking region was analyzed with bioinformatics tools, numerous putative cis-elements were identified. The Green Fluorescent Protein (GFP) regulated by this promoter was observed to be transiently expressed in protoplasts derived from V. radiata cotyledons. Finally, transgenic Arabidopsis plants expressing the β-glucuronidase (GUS) reporter gene driven from the 8S globulin α′ promoter showed strong GUS expression in transgenic embryos in both histochemical and quantitative GUS assays, confirming high expression within seeds. Therefore, the V. radiata 8S α′ promoter has shown potential in directing expression in seeds for bioreactor applications.

The succession of dominant phytoplankton species plays an important role in harmful algal blooms. However, the molecular mechanism of algal succession remains largely unclear, including the most commonly occurring diatom/dinoflagellate succession. Here we investigated the responses of the diatom Phaeodactylum tricornutum during mixed culture with the potentially toxic dinoflagellate Alexandrium tamarense. The growth of P. tricornutum was significantly inhibited within 24 h in mixed culture. Organelles such as the chloroplasts and mitochondria of P. tricornutum were severely damaged. Transcriptional responses in P. tricornutum were revealed by RNA-seq. Genes involved in glycolysis, TCA cycle, β-oxidation, carbon fixation and oxidation phosphorylation were downregulated, indicating the inhibition of energy metabolism. Several genes associated with check points and cell cycle were also downregulated, suggesting the suppression of DNA replication and cell division. Taking into account the upregulation of genes involved in endocytosis and transporter ABCB1, P. tricornutum could perceive certain allelochemicals released from A. tamarense, which played a role in their interaction. Additionally, a family of leucine-rich repeat receptor-like kinases was upregulated, suggesting that flagellin-sensitive mediated cell-cell interactions are responsible for toxic effect other than allelopathy. Here we showed a molecular overview of interactions between diatom and dinoflagellate, thereby provide molecular insight into the unraveled mechanisms on the effects of toxic species on non-toxic species.

•Few Vigna seed promoter sequences have been reported.•A new 8SGα promoter that conferred seed-specific expression in Arabidopsis was identified.•The 8SGα promoter strength was observed to be higher than the conventional 35S CaMV promoter in seeds.•This 8SGα promoter provides an additional choice for the expression of multiple proteins in seeds.Vigna radiata (mung bean) is an important crop plant and is a major protein source in developing countries. Mung bean 8S globulins constitute nearly 90% of total seed storage protein and consist of three subunits designated as 8SGα, 8SGα′ and 8SGβ. The 5′-flanking sequences of 8SGα′ has been reported to confer high expression in transgenic Arabidopsis seeds. In this study, a 472-bp 5′-flanking sequence of 8SGα was identified by genome walking. Computational analysis subsequently revealed the presence of numerous putative seed-specific cis-elements within. The 8SGα promoter was then fused to the gene encoding β-glucuronidase (GUS) to create a reporter construct for Arabidopsis thaliana transformation. The spatial and temporal expression of 8SGα∷GUS, as investigated using GUS histochemical assays, showed GUS expression exclusively in transgenic Arabidopsis seeds. Quantitative GUS assays revealed that the 8SGα promoter showed 2- to 4-fold higher activity than the Cauliflower Mosaic Virus (CaMV) 35S promoter. This study has identified a seed-specific promoter of high promoter strength, which is potentially useful for directing foreign protein expression in seed bioreactors.