Presentation Information
[P04-555]Targeted knockout of LHCF impacts on oil production in the marine oleaginous diatom Fistulifera solaris
○Ryoma Oishi1, Satoshi Murata1, Ryota Kumakubo1, Haru Fukuda1, Sawa Suzuki1, Yoshiaki Maeda1, Kosuke Kataoka1,2, Tsuyoshi Tanaka1 (1. Graduate School of Engineering, Tokyo University of Agriculture and Technology (Japan), 2. Comprehensive Research Organization, Waseda University (Japan))
Keywords:
Diatom,Light-harvesting complex,Photosynthetic efficiency
[Purpose]
Microalgae are promising hosts for sustainable biofuel production, however biomass and lipid productivity often decline in mass cultivation because dense cultures create heterogeneous light environments, causing photoinhibition in surface cells and light limitation in deeper layers. Fistulifera solaris is an oleaginous diatom with high lipid productivity and rapid growth, and both CRISPR-Cas9 editing and outdoor cultivation have been demonstrated. This study evaluated whether lhcf disruption could reduce antenna size and improve growth, biomass, and oil production under high-light conditions simulating outdoor cultivation.
[Method]
Based on RNA-seq profiles, the most highly expressed gene among 27 predicted lhcf genes in F. solaris, was selected as the target. A CRISPR-Cas9 plasmid was constructed for gene knockout, and cells were transformed by biolistic bombardment. Targeted mutations were confirmed by PCR amplification and Sanger sequencing. Growth and physiological characteristics were evaluated during 7-day cultivation in f/2 and 2f medium at 25ºC under low light (130 µmol photons/m2/s) / high light (400 µmol photons/m2/s) conditions. Biomass and oil production, pigment contents, and photosynthetic performance were assessed by spectrophotometry/HPLC, and PAM chlorophyll fluorescence.
[Results]
CRISPR-Cas9-mediated disruption of targetgene was achieved, and multiple independent mutant lines carrying frameshift mutations (1-bp, 11-bp, or 46-bp deletions) were obtained. All lhcf mutants showed reduced chlorophyll and fucoxanthin contents vs wild type, indicating reduced antenna size. Under low light conditions (130 µmol photons/m2/s, f/2 medium), some mutants showed reduced growth. This growth reduction under low light suggests that knockout of LHCF reduced light-harvesting capacity and limited efficient photon capture. In contrast, under high light and nutrient-rich conditions (400 µmol photons/m2/s, 2f medium), growth of the mutants was comparable to or higher than that of the wild type, and one mutant line exhibited increased late-phase biomass and oil production. In addition, Fv/Fm and Fv'/Fm' values were higher in the mutants under high light, suggesting mitigation of PSⅡ photodamage.
[Conclusion]
In this study, we demonstrated CRISPR-Cas9-based editing of lhcf target gene in the oleaginous diatom F. solaris. Our results indicate that antenna-size optimization enhances light-use efficiency and oil production, providing a foundation of molecular breeding of diatoms for biofuel production.
Microalgae are promising hosts for sustainable biofuel production, however biomass and lipid productivity often decline in mass cultivation because dense cultures create heterogeneous light environments, causing photoinhibition in surface cells and light limitation in deeper layers. Fistulifera solaris is an oleaginous diatom with high lipid productivity and rapid growth, and both CRISPR-Cas9 editing and outdoor cultivation have been demonstrated. This study evaluated whether lhcf disruption could reduce antenna size and improve growth, biomass, and oil production under high-light conditions simulating outdoor cultivation.
[Method]
Based on RNA-seq profiles, the most highly expressed gene among 27 predicted lhcf genes in F. solaris, was selected as the target. A CRISPR-Cas9 plasmid was constructed for gene knockout, and cells were transformed by biolistic bombardment. Targeted mutations were confirmed by PCR amplification and Sanger sequencing. Growth and physiological characteristics were evaluated during 7-day cultivation in f/2 and 2f medium at 25ºC under low light (130 µmol photons/m2/s) / high light (400 µmol photons/m2/s) conditions. Biomass and oil production, pigment contents, and photosynthetic performance were assessed by spectrophotometry/HPLC, and PAM chlorophyll fluorescence.
[Results]
CRISPR-Cas9-mediated disruption of targetgene was achieved, and multiple independent mutant lines carrying frameshift mutations (1-bp, 11-bp, or 46-bp deletions) were obtained. All lhcf mutants showed reduced chlorophyll and fucoxanthin contents vs wild type, indicating reduced antenna size. Under low light conditions (130 µmol photons/m2/s, f/2 medium), some mutants showed reduced growth. This growth reduction under low light suggests that knockout of LHCF reduced light-harvesting capacity and limited efficient photon capture. In contrast, under high light and nutrient-rich conditions (400 µmol photons/m2/s, 2f medium), growth of the mutants was comparable to or higher than that of the wild type, and one mutant line exhibited increased late-phase biomass and oil production. In addition, Fv/Fm and Fv'/Fm' values were higher in the mutants under high light, suggesting mitigation of PSⅡ photodamage.
[Conclusion]
In this study, we demonstrated CRISPR-Cas9-based editing of lhcf target gene in the oleaginous diatom F. solaris. Our results indicate that antenna-size optimization enhances light-use efficiency and oil production, providing a foundation of molecular breeding of diatoms for biofuel production.
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