Research ArticleGENETICS

Transcriptional regulation of microalgae for concurrent lipid overproduction and secretion

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Science Advances  30 Jan 2019:
Vol. 5, no. 1, eaau3795
DOI: 10.1126/sciadv.aau3795
  • Fig. 1 Physiological and biochemical analyses of NobZIP1-engineered cells.

    (A) Schematic map of the NobZIP1 overexpression cassette. The NobZIP1-coding region was cloned under the control of an Hsp20 promoter and a TfcpA terminator. An omega leader motif was inserted in between the promoter and NobZIP1 gene to enhance the translation. (B) Schematic map of the hpNobZIP1 expression cassette to silence NobZIP1. (C) Maximum quantum yield of photosystem II (PSII) as measured by Fv/Fm. (D) Growth curve analysis. Relative fluorescence intensity of extracellular lipids (E) per 106 cell and (F) per milliliter, as determined by Nile red fluorescence analysis. Relative fluorescence intensity of intracellular lipids as determined by Nile red fluorescence analysis (G) per cell and (H) fluorescence intensity ×106 per milliliter. Total extracellular (percentage of total cell dry weight) (I) and intracellular (J) lipid content (percentage of total cell dry weight). (K) Total carbohydrate content, as determined by the phenol-sulfuric acid method. (L) Total protein content, as measured by bicinchoninic acid (BCA) assay. Error bars represent mean values ± SD for three separate experiments. Significant difference is indicated at P < 0.05 (*) or P < 0.01 (**) level.

  • Fig. 2 Ultrastructural analyses of NobZIP1-engineered cells.

    WT cells were encapsulated by rigid cell wall (left), and the enlarged image of cell wall was given in the box; NobZIP1-overexpressing cells exhibited loosen cell wall and highly enriched in oil bodies (middle). Enlarged image of cell wall was given in the box and the alteration in the cell wall structure was indicated by arrow marks. CW, cell wall; Chl, chloroplast; Ob, oil bodies. Scale bars, 500 nm.

  • Fig. 3 ChIP-qPCR analysis.

    ChIP analysis of (A) ACBP, (B) KAS, (C) LC-FACS, (D) LPAAT, (E) CPS, and (F) UGDH. Each value represents mean ± SD (n = 3). qPCR was carried out to elucidate the regulatory role of NobZIP1 in regulating transcription of predicted key target genes. (G) ACBP, (H) KAS, (I) LC-FACS, (J) LPAAT, (K) CPS, and (L) UGDH. β-Actin was used as an internal reference gene. Significant difference is indicated at P < 0.05 (*) or P < 0.01 (**) level. Each value represents mean ± SD (n = 3).

  • Fig. 4 Physiological and biochemical analyses of UGDH-overexpressing and UGDH-silencing cells.

    (A) Schematic map of the siUGDH expression cassette to silence NobZIP1. (B) Schematic representation of the UGDH overexpression cassette. The UGDH-coding region was under the control of an Hsp20 promoter and a TfcpA terminator. An omega leader motif was inserted in between the promoter and the UGDH gene to enhance the translation. (C) Maximum photochemical efficiency of PSII, as measured by Fv/Fm. (D) Growth curve analysis. Relative fluorescence intensity of extracellular lipids (E) per 106 cell and (F) per milliliter, as determined by Nile red fluorescence analysis. Relative fluorescence intensity of intracellular lipids, as determined by Nile red fluorescence analysis (G) per cell and (H) fluorescence intensity ×106 per milliliter. Total extracellular (percentage of total cell dry weight) (I) and intracellular (percentage of total cell dry weight) (J) lipid content (%). (K) Total carbohydrate content as determined by the phenol-sulfuric acid method. (L) Total protein content as determined by the BCA method. Error bars represent mean values ± SD for three separate experiments. Significant difference is indicated at P < 0.05 (*) or P < 0.01 (**) level.

  • Fig. 5 Ultrastructural analyses of UGDH-engineered cells by TEM.

    WT cells were encapsulated by rigid cell wall (left), and the enlarged image of cell wall was given in the box; UGDH-silencing cells exhibited thin cell wall and were observed to have large oil bodies than that of WT (right). Enlarged view of cell wall was given in the box, and the alteration in the cell wall structure was indicated by arrow marks. Scale bars, 1 μm.

  • Fig. 6 Schematic representation of the mechanistic role of NobZIP1 in N. oceanica.

    Expression of NobZIP1 up-regulates the expression of KAS, ACBP, LC-FACS, LPAAT (red) and down-regulates the expression of UGDH (green). The targets of NobZIP1 were represented by blue arrow marks, as revealed by ChIP analysis. The role of UGDH on cell wall formation and carbon flux direction toward lipogenesis was revealed by genetic engineering of UGDH in transgenic cells, which substantiate the NobZIP1-mediated concurrent lipid overproduction and secretion in transgenic cells. NobZIP that remarkably up- and down-regulated the target genes are indicated by red and green lines, respectively. TAG, triacylglyceride; ER, endoplasmic reticulum.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau3795/DC1

    Fig. S1. Sequence analysis and characterization of NobZIP1 engineered cells.

    Fig. S2. Laser scanning confocal microscopic analysis of Nile red–stained NobZIP1-engineered cells.

    Fig. S3. Sequence analysis and characterization of UGDH overexpressing and silencing cells.

    Fig. S4. Confocal microscopic analysis of Nile red–stained UGDH-engineered and CPS-silencing cells.

    Table S1. List of oligonucleotide primers and their sequences used in this study.

    Movie S1. Laser scanning confocal fluorescence of Nile red–stained cells identifying the lipid droplets mobilization across the cell wall and revealing the lipid secretion phenomenon across the weakened cell wall in overexpressing cells NobIP1-1 (movie S1), whereas no such mechanism was observed in WT (movie S2).

    Movie S2. Laser scanning confocal fluorescence of Nile red–stained cells identifying the lipid droplets mobilization across the cell wall and revealing the lipid secretion phenomenon across the weakened cell wall in overexpressing cells NobIP1-1 (movie S1), whereas no such mechanism was observed in WT (movie S2).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Sequence analysis and characterization of NobZIP1 engineered cells.
    • Fig. S2. Laser scanning confocal microscopic analysis of Nile red–stained NobZIP1-engineered cells.
    • Fig. S3. Sequence analysis and characterization of UGDH overexpressing and silencing cells.
    • Fig. S4. Confocal microscopic analysis of Nile red–stained UGDH-engineered and CPS-silencing cells.
    • Table S1. List of oligonucleotide primers and their sequences used in this study.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Laser scanning confocal fluorescence of Nile red–stained cells identifying the lipid droplets mobilization across the cell wall and revealing the lipid secretion phenomenon across the weakened cell wall in overexpressing cells NobIP1-1 (movie S1), whereas no such mechanism was observed in WT (movie S2).
    • Movie S2 (.avi format). Laser scanning confocal fluorescence of Nile red–stained cells identifying the lipid droplets mobilization across the cell wall and revealing the lipid secretion phenomenon across the weakened cell wall in overexpressing cells NobIP1-1 (movie S1), whereas no such mechanism was observed in WT (movie S2).

    Files in this Data Supplement:

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