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.

The marine diatom, Phaeodactylum tricornutum, has become a model for studying lipid metabolism and its triacylglycerol (TAG) synthesis pathway makes it an ideal target for metabolic engineering to improve lipid productivity. However, the genetic background and metabolic networks of fatty acid biosynthesis in diatoms are not well understood. Glycerol-3-phosphate acyltransferase (GPAT) is the critical enzyme that catalyzes the first step of TAG formation. So far, characterization of GPAT in marine microalgae has not been reported, especially at the level of comprehensive sequence-structure and functional analysis. A GPAT was cloned from P. tricornutum and overexpressed in P. tricornutum. Volumes of oil bodies were produced and the neutral lipid content was increased by twofold determined by Nile red fluorescence staining. Fatty acid composition was analyzed by GC–MS, which showed significantly higher proportion of unsaturated fatty acids compared to wild type. These results suggested that the identified GPAT could upregulate TAG biosynthesis in P. tricornutum. Moreover, this study offers insight into the lipid metabolism of diatoms and supports the role of microalgal strains for biofuels production.

The fast growing photosynthetic microalgae have been widely used in aquaculture, food, health, and biofuels. Recent findings in the diatom has proposed a pivotal role of NADP-malic enzyme in generation of NADPH as an important supply of reducing power for fatty acid biosynthesis. To test the lipogenic malic enzyme for fatty acid synthesis in green algae, here the malic enzyme gene PtME from the oleaginous diatom Phaeodactylum tricornutum was expressed in a representative green microalga Chlorella pyrenoidosa. The engineered C. pyrenoidosa strain showed higher enzymatic activity of malic enzyme which subsequently promoted fatty acid synthesis. The neutral lipid content was significantly increased by up to 3.2-fold than wild type determined by Nile red staining, and total lipid content reached 40.9 % (dry cell weight). The engineered strain exhibited further lipid accumulation subjected to nitrogen deprivation condition. Upon nitrogen deprivation, engineered microalgae accumulated total lipid up to 58.7 % (dry cell weight), a 4.6-fold increase over the wild type cells under normal culture condition. At cellular level, increased volume and number of oil bodies were observed in the engineered microalgal cells. These findings suggested that malic enzyme is a pivotal regulator in lipid accumulation in green microalga C. pyrenoidosa, and presenting a breakthrough of generating ideal algal strains for algal nutrition and biofuels.

Microalgal metabolic engineering holds great promise for the overproduction of a wide range of commercial bioproducts. It demands simultaneous manipulation of multiple metabolic nodes. However, high-efficiency promoters have been lacking. Here we report a strong constitutive promoter Pt211 in expressing multiple target genes in oleaginous microalga Phaeodactylum tricornutum. Pt211 was revealed to contain significant cis-acting elements. GUS reporter and principal genes glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase 2 (DGAT2) involved in triacylglycerol biosynthesis were tested under driven of Pt211 in P. tricornutum. GUS staining and qPCR analysis showed strong GUS expression. DGAT2 and GPAT linked with a designed 2A sequence exhibited higher transcript abundances than WT, while algal growth and photosynthesis were not impaired. The total lipid content increased notably by 2.6-fold compared to WT and reached up to 57.5% (dry cell weight). Overall, our findings report a strong promoter and a strategy for coordinated manipulation of complex metabolic pathways.

Okadaic acid (OA) is a marine biotoxin associated with diarrhetic shellfish poisoning (DSP), posing some threat to human beings. The oral toxicity of OA is complex, and the mechanism of toxicity is not clear. The interaction between OA and gut microbiota may provide a reasonable explanation for the complex toxicity of OA. Due to the complex environment in vivo, an in vitro study may be better for the interactions between OA and gut microbiome. Here, we conducted an in vitro fermentation experiment of gut bacteria in the presence of 0–1000 nM OA. The remolding ability of OA on bacterial composition was investigated by 16S rDNA sequencing, and differential metabolites in fermentation system with different concentration of OA was detected by LC-MS/MS. We found that OA inhibited some specific bacterial genera but promoted others. In addition, eight possible metabolites of OA, including dinophysistoxin-2 (DTX-2), were detected in the fermentation system. The abundance of Faecalitalea was strongly correlated with the possible metabolites of OA, suggesting that Faecalitalea may be involved in the metabolism of OA in vitro. Our findings confirmed the direct interaction between OA and gut bacteria, which helps to reveal the metabolic process of OA and provide valuable evidence for elucidating the complex toxicity of OA.