[P007] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P04-543] Inducible knockdown system enhanced extracellular lipid yields under high-light condition in the oleaginous diatom Fistulifera solaris

○Tomoki YAMANAKA¹, Kosuke Kataoka^1,2, Tsuyoshi Tanaka¹ (1. Tokyo University of Agriculture and Technology (Japan), 2. Comprehensive Research Organization, Waseda University (Japan))

Keywords:

Applied phycology,Bioengineering,Lipid metabolism,oleaginous diatom

The development of biofuels has gained attention as a strategy to reduce global CO₂ emissions, with microalgae emerging as a sustainable production platform. The oleaginous diatom Fistulifera solaris exhibits high lipid accumulation capacity, making it a strong candidate for biofuel production. However, high production costs remain a major obstacle to commercialization. One potential strategy to reduce costs is secretion-based production, in which free fatty acids (FFAs), major biofuel components, are released extracellularly and can be recovered without energy-intensive harvesting and extraction processes. We have previously generated a knockdown strain of a long-chain acyl-CoA synthetase (LACS) as an FFA-secreting strain, achieving approximately 67 mg L^-1 of extracellular FFA under normal light conditions (130 μmol photons m^-2 s^-1). However, this strain exhibited impaired growth and lower FFA productivity under high-light conditions (400 μmol photons m^-2 s^-1), likely due to lipotoxicity caused by excessive intracellular FFA accumulation. Since practical outdoor cultivation requires tolerance to strong light conditions, strategies to mitigate lipotoxicity are essential for efficient biofuel production. In this study, we established an inducible gene suppression system using the nitrate reductase (NR) promoter, whose activity responds to nitrogen sources in the culture medium. RNA interference mediated by antisense fragments under the control of the NR promoter enabled conditional knockdown of LACS expression. Gene suppression could be reversibly switched ON and OFF depending on the nitrogen source (NaNO₃ or NH₄Cl). In addition, effective suppression was achieved by nitrate spiking (NaNO₃ addition) without medium replacement. These induction methods exhibited comparable gene-silencing efficacy, indicating that the regulatory system functions independently of light intensity. To evaluate whether temporal control of LACS knockdown could alleviate lipotoxicity under high-light conditions, nitrate spiking was applied after cells reached the stationary phase. As a result, growth was restored, and extracellular FFA production exceeded 100 mg L^-1, surpassing that observed under all other tested conditions. This productivity approaches levels reported in cyanobacteria, where secretion-based oil production systems are relatively advanced. Our findings represent a notable advancement in FFA secretion systems in eukaryotic microalgae. These results demonstrate that combining NR promoter-mediated inducible control with an FFA secretion strategy effectively enhances productivity by mitigating lipotoxicity, even under high-light conditions. This study provides a practical framework for biofuel production using F. solaris.