Increased lipid production by heterologous expression of AtWRI1 transcription factor in Nannochloropsis salina

Abstract

Background: Genetic engineering of microalgae is necessary to produce economically feasible strains for biofuel production. Current efforts are focused on the manipulation of individual metabolic genes, but the outcomes are not sufficiently stable and/or efficient for large-scale production of biofuels and other materials. Transcription factors (TFs) are emerging as good alternatives for engineering of microalgae, not only to increase production of biomaterials but to enhance stress tolerance. Here, we investigated an AP2 type TF Wrinkled1 in Arabidopsis (AtWRI1) known as a key regulator of lipid biosynthesis in plants, and applied it to industrial microalgae, Nannochloropsis salina.

Results: We expressed AtWRI1 TF heterologously in N. salina, named NsAtWRI1, in an effort to re-enact its key regulatory function of lipid accumulation. Stable integration AtWRI1 was confirmed by RESDA PCR, and its expression was confirmed by Western blotting using the FLAG tag. Characterizations of transformants revealed that the neutral and total lipid contents were greater in NsAtWRI1 transformants than in WT under both normal and stress conditions from day 8. Especially, total lipid contents were 36.5 and 44.7% higher in NsAtWRI1 2-3 than in WT under normal and osmotic stress condition, respectively. FAME contents of NsAtWRI1 2-3 were also increased compared to WT. As a result, FAME yield of NsAtWRI1 2-3 was increased to 768 mg/L/day, which was 64% higher than that of WT under the normal condition. We identified candidates of AtWRI1-regulated genes by searching for the presence of the AW-box in promoter regions, among which lipid metabolic genes were further analyzed by qRT-PCR. Overall, qRT-PCR results on day 1 indicated that AtWRI1 down-regulated TAGL and DAGK, and up-regulated PPDK, LPL, LPGAT1, and PDH, resulting in enhanced lipid production in NsAtWRI1 transformants from early growth phase.

Conclusion: AtWRI1 TF regulated several genes involved in lipid synthesis in N. salina, resulting in enhancement of neutral lipid and FAME production. These findings suggest that heterologous expression of AtWRI1 TF can be utilized for efficient biofuel production in industrial microalgae.

Keywords: Biofuels; Microalgae; Nannochloropsis salina; TF engineering; Transcription factor; Wrinkled1.

Fig. 1

The pNsAtWRI1 vector and identification of the plasmid in the transformants. a Schematic map of the pNsAtWRI1 plasmid. b Detection of plasmids in NsAtWRI1 and WT. Genomic sequences of AtWRI1 (1.3 kb) and 18S rDNA (380 bp) were PCR amplified and the products were electrophoresed on agarose gels. c Western blotting of FLAG-tagged NsAtWRI1. The expected size of FLAG-tagged AtWRI1 was 49.4 kD, but running around 55 kD. AtpB (the CF₁ ß subunit of ATP synthase of expected size 72.6 kD) was used as a loading control. d Relative intensity of AtWRI1-FLAG protein, which was calculated as the ratio of AtWRI1-FLAG vs AtpB. WT wild type, N normal conditions, NL nitrogen limitation, O osmotic stress

Fig. 2

Growth of the NsAtWRI1 transformants under various culture conditions. Growth curve based on cell density under normal (a), N limitation (b), and osmotic stress conditions (c). The data points represent the average of samples and error bars indicate standard deviation (n = 4)

Fig. 3

Total lipid analyses of NsAtWRI1 transformants under various culture conditions. Total lipid contents on day 8 (a) and day 12 (b). Total lipid yield on day 8 (c) and day 12 (d). The data points represent the average of samples and error bars indicate standard deviation (n = 4). Significant differences against WT for the same condition, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

FAME quantitation of NsAtWRI1 transformants under various culture conditions. FAME contents on day 8 (a) and day 12 (b). FAME yield on day 8 (c) and day 12 (d). The data points represent the average of samples and error bars indicate standard deviation (n = 4). Significant differences against WT for the same condition, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Expression profiles of AtWRI1-regulated candidate genes involved in lipid synthesis in NsAtWRI1 2–3. mRNA was obtained from cells which were incubated for 1 day under normal, N limitation and osmotic stress conditions. The expression levels of these genes were determined by qRT-PCR, normalized by that of actin. The data points represent the average of samples and error bars indicate standard deviation (n = 3). Significant differences against WT for the same condition, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). TAGL triacylglycerol lipase, DAGK diacylglycerol kinase, LPL lysophospholipase, LPGAT1 lysophosphatidylglycerol acyltransferase 1, DGAT diacylglycerol acyltransferase family protein, PPDK pyruvate phosphate dikinase, PDH dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase mitochondrial-like