Research ArticleBIOENGINEERING

Intracellular spectral recompositioning of light enhances algal photosynthetic efficiency

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Science Advances  01 Sep 2017:
Vol. 3, no. 9, e1603096
DOI: 10.1126/sciadv.1603096
  • Fig. 1 The ISR strategy for the cultivation of engineered P. tricornutum cells.

    (A) Optimization of light spectrum, transfer, and utilization in the eGFP transformant cells. GFPs absorb a portion of wasted blue light and re-emits it as green, which can be captured by LHCs/FCPs and used in PSII. FCPs, fucoxanthin chlorophyll proteins; ATP, adenosine 5′-triphosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate. hv, where h is the Planck’s constant and ν is the frequency of photon, represents the photons. Dashed lines in color represent transmission of light. (B) Schematic mechanisms for improving photosynthesis and phenotype characteristics in the eGFP transformants through nucleus transformation. OD600, optical density at 600 nm.

  • Fig. 2 Cultivation of P. tricornutum cells under combined red and blue LED illumination.

    (A) LED illumination applied in the experiments consisted of 25% blue lights and 75% red lights with an overall intensity of 400 μmol photons m−2 s−1. (B) Fluorescent microscopy images of P. tricornutum cells stained with 2.0 μM BODIPY 505/515; stained lipid vesicles are seen as green fluorescence bodies in the image. (C) Comparisons of photosynthetic efficiencies in P. tricornutum cultures. The values were averaged from three independent experiments. Error bars indicate SEM. *P < 0.05, statistically significant difference between the WT and stained cells (with BODIPY).

  • Fig. 3 Engineered diatoms for enhanced growth performance and energy conversion efficiency through the ISR strategy.

    (A) Fluorescent microscopy images of the nitrate-inducible eGFP transformants with construct pPha-NR/eGFP. (B) Shifting of absorption spectra in the eGFP transformants. (C) Emission spectra of the WT and eGFP transformants ranging from 485 to 600 nm. Excitation wavelength, 455 nm. (D) Quantum yields of PSII in WT and transformants using flat-panel PBRs. Values were averaged from three independent experiments. *P < 0.05, statistically significant difference between the WT and the transformants. (E) Photosynthetic energy conversion efficiencies in the WT and transformants using flat-panel PBRs. Values were averaged from three independent experiments. Error bars indicate SEM. *P < 0.05, statistically significant difference between the WT and the transformants.

  • Fig. 4 Comparison of photophysiology between WT and eGFP transformant strains.

    (A) The theoretical photosynthetic efficiency and maximal NPQ level. (B) NPQ induction under a light intensity simulating the high-light condition in flat-panel PBRs. LL and HL represent low-light (50 μmol photons m−2 s−1) and high-light (200 μmol photons m−2 s−1) conditions, respectively. Values were averaged from three independent experiments. Error bars indicate SEM.

  • Fig. 5 Global analysis and identification of DEGs.

    (A) Genome-scale analysis of all identified genes in the eGFP transformants and WT under high-light condition. The Venn diagram indicates the numbers of shared and unique genes in the WT and eGFP transformants. The heat map compares the different profiles of gene expression levels between the WT and the eGFP transformants. (B) Numbers of up-regulated and down-regulated genes are shown in the eGFP transformants in comparison with the WT. (C) Genome-scale analysis of all identified genes between high- and low-light conditions in the WT strain. The Venn diagram indicates the numbers of shared and unique genes in high- and low-light conditions. The heat map shows the different profiles of gene expression levels between high- and low-light conditions. (D) The numbers of up-regulated and down-regulated genes are shown under high-light conditions in comparison with low-light conditions using the WT. For (A) and (C), values in the color bar indicate log10 (FPKM) values. For (B) and (D), DEGs are shown with red dots, and non-DEGs are shown with black dots in the volcano plot; the horizontal line indicates the fold changes, and the vertical line indicates the significance threshold (FDR < 0.05).

  • Fig. 6 Gene set enrichment analysis.

    (A) Identification of up-regulated genes highlighting the photosynthesis process in the eGFP transformants in comparison with the WT under high-intensity light condition. GO terms are represented as nodes in the graph, the color gradient (yellow to orange) represents the statistical term enrichment significance (P < 0.05), white (no color) indicates no significant difference, and node sizes indicate the relative numbers of genes that represent the GO term. (B) Identified DEGs involved in stress response in the WT strain between high- and low-intensity light conditions. The log2 (FPKM) values are shown for the WT under both high- and low-light conditions and the eGFP transformants under high-light condition.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1603096/DC1

    fig. S1. Effects of the dye ATTO 465 on the shifting of absorption spectrum in P. tricornutum cell suspensions.

    fig. S2. Effects of the dye BODIPY 505/515 on the shifting of absorption spectrum in P. tricornutum cell suspensions.

    fig. S3. Evaluation of the biocompatibility of fluorescence dyes after staining for 30 min.

    fig. S4. Photostability of fluorescent BODIPY 505/515 staining on diatom cells within 24 hours.

    fig. S5. Fluorescent microscopy images of the WT and eGFP transformants of P. tricornutum cells.

    fig. S6. Selection of positive eGFP transformants.

    fig. S7. Comparison of growth performance between the WT and the chloroplast-localized eGFP transformants over 24 hours using flat-panel PBRs.

    fig. S8. The enhancement of green fluorescence in the eGFP transformants.

    fig. S9. The regain of green fluorescence in the nitrate-inducible eGFP transformants upon presence of 5 mM nitrate (KNO3).

    fig. S10. Comparison of growth performance between the WT and the nitrate-inducible eGFP transformants over 24 hours using flat-panel PBRs.

    fig. S11. The light supply setup of OPSs.

    fig. S12. Cultivation of diatoms in OPSs.

    fig. S13. GSEA of down-regulated DEGs in the eGFP transformants.

    fig. S14. Analysis of LHCX proteins by Western blotting in cells under high-intensity light condition.

    fig. S15. Heat map of expression profile of DEGs based on RNA-seq data in relation to photosynthesis.

    fig. S16. GSEA of up-regulated genes between low- and high-intensity light conditions through BiNGO.

    fig. S17. GSEA of down-regulated genes between low- and high-intensity light conditions through BiNGO.

    fig. S18. A schematic process for generation of genetically modified P. tricornutum strains.

    table S1. Carbon fraction analysis in dry biomass.

    table S2. Comparison of detected level of LHCX proteins between the WT and the eGFP transformants.

    data set S1. Shared genes between the WT and the eGFP transformants under high-intensity light condition.

    data set S2. Genes uniquely present in the eGFP transformants under high-intensity light condition.

    data set S3. GSEA of up-regulated genes in the eGFP transformants under high-intensity light condition.

    data set S4. GSEA of down-regulated genes in the eGFP transformants under high-intensity light condition.

    data set S5. List of DEGs in the WT under high-light intensity condition compared to that under low-intensity light condition.

    data set S6. Key identified genes involved in photosynthesis.

    data set S7. List of identified genes in relation to photosynthesis, as shown in fig. S14.

    data set S8. DEGs involved in stress response in the WT based on high- and low-intensity light conditions.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Effects of the dye ATTO 465 on the shifting of absorption spectrum in P. tricornutum cell suspensions.
    • fig. S2. Effects of the dye BODIPY 505/515 on the shifting of absorption spectrum in P. tricornutum cell suspensions.
    • fig. S3. Evaluation of the biocompatibility of fluorescence dyes after staining for 30 min.
    • fig. S4. Photostability of fluorescent BODIPY 505/515staining on diatom cells within 24 hours.
    • fig. S5. Fluorescent microscopy images of the WT and eGFP transformants of P. tricornutum cells.
    • fig. S6. Selection of positive eGFP transformants.
    • fig. S7. Comparison of growth performance between the WT and the chloroplast-localized eGFP transformants over 24 hours using flat-panel PBRs.
    • fig. S8. The enhancement of green fluorescence in the eGFP transformants.
    • fig. S9. The regain of green fluorescence in the nitrate-inducible eGFP transformants upon presence of 5 mM nitrate (KNO3).
    • fig. S10. Comparison of growth performance between the WT and the nitrateinducible eGFP transformants over 24 hours using flat-panel PBRs.
    • fig. S11. The light supply setup of OPSs.
    • fig. S12. Cultivation of diatoms in OPSs.
    • fig. S13. GSEA of down-regulated DEGs in the eGFP transformants.
    • fig. S14. Analysis of LHCX proteins by Western blotting in cells under high-intensity light condition.
    • fig. S15. Heat map of expression profile of DEGs based on RNA-seq data in relation to photosynthesis.
    • fig. S16. GSEA of up-regulated genes between low- and high-intensity light conditions through BiNGO.
    • fig. S17. GSEA of down-regulated genes between low- and high-intensity light conditions through BiNGO.
    • fig. S18. A schematic process for generation of genetically modified P. tricornutum strains.
    • table S1. Carbon fraction analysis in dry biomass.
    • table S2. Comparison of detected level of LHCX proteins between the WT and the eGFP transformants.

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    Other Supplementary Material for this manuscript includes the following:

    • data set S1 (Microsoft Excel format). Shared genes between the WT and the eGFP transformants under high-intensity light condition.
    • data set S2 (Microsoft Excel format). Genes uniquely present in the eGFP transformants under high-intensity light condition.
    • data set S3 (Microsoft Excel format). GSEA of up-regulated genes in the eGFP transformants under high-intensity light condition.
    • data set S4 (Microsoft Excel format). GSEA of down-regulated genes in the eGFP transformants under high-intensity light condition.
    • data set S5 (Microsoft Excel format). List of DEGs in the WT under high-intensity light condition compared to that under low-intensity light condition.
    • data set S6 (Microsoft Excel format). Key identified genes involved in photosynthesis.
    • data set S7 (Microsoft Excel format). List of identified genes in relation to photosynthesis, as shown in fig. S14.
    • data set S8 (Microsoft Excel format). DEGs involved in stress response in the WT based on high- and low-intensity light conditions.

    Download Data Sets S1 to S8

    Files in this Data Supplement:

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