Kato Yuichi, Fujihara Yusuke, Vavricka Christopher J, Chang Jo-Shu, Hasunuma Tomohisa, Kondo Akihiko
1Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501 Japan.
2Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501 Japan.
Biotechnol Biofuels. 2019 Feb 21;12:39. doi: 10.1186/s13068-019-1380-4. eCollection 2019.
Light/dark cycling is an inevitable outdoor culture condition for microalgal biofuel production; however, the influence of this cycling on cellular lipid production has not been clearly established. The general aim of this study was to determine the influence of light/dark cycling on microalgal biomass production and lipid accumulation. To achieve this goal, specific causative mechanisms were investigated using a metabolomics approach. Laboratory scale photoautotrophic cultivations of the oleaginous green microalga sp. JSC4 were performed under continuous light (LL) and light/dark (LD) conditions.
Lipid accumulation and carbohydrate degradation were delayed under the LD condition compared with that under the LL condition. Metabolomic analysis revealed accumulation of phosphoenolpyruvate and decrease of glycerol 3-phosphate under the LD condition, suggesting that the imbalance of these metabolites is a source of delayed lipid accumulation. When accounting for light dosage, biomass yield under the LD condition was significantly higher than that under the LL condition. Dynamic metabolic profiling showed higher levels of lipid/carbohydrate anabolism (including production of 3-phosphoglycerate, fructose 6-phosphate, glucose 6-phosphate, phosphoenolpyruvate and acetyl-CoA) from CO under the LD condition, indicating higher CO fixation than that of the LL condition.
Photoperiods define lipid accumulation and biomass production, and light/dark cycling was determined as a critical obstacle for lipid production in JSC4. Conversions of phosphoenolpyruvate to pyruvate and 3-phosphoglycerate to glycerol 3-phosphate are the candidate rate-limiting steps responsible for delayed lipid accumulation. The accumulation of substrates including ribulose 5-phosphate could be explained by the close relationship of increased biomass yield with enhanced CO fixation. The present study investigated the influence of light/dark cycling on lipid production by direct comparison with continuous illumination for the first time, and revealed underlying metabolic mechanisms and candidate metabolic rate-limiting steps during light/dark cycling. These findings suggest promising targets to metabolically engineer improved lipid production.
光/暗循环是微藻生物燃料生产中不可避免的室外培养条件；然而，这种循环对细胞脂质生产的影响尚未明确。本研究的总体目标是确定光/暗循环对微藻生物量生产和脂质积累的影响。为实现这一目标，采用代谢组学方法研究了具体的致病机制。在连续光照（LL）和光/暗（LD）条件下，对产油绿微藻JSC4进行了实验室规模的光合自养培养。
与LL条件相比，LD条件下脂质积累和碳水化合物降解延迟。代谢组学分析显示，LD条件下磷酸烯醇丙酮酸积累，3-磷酸甘油减少，表明这些代谢物的失衡是脂质积累延迟的一个原因。在考虑光照剂量时，LD条件下的生物量产量显著高于LL条件。动态代谢谱显示，LD条件下来自CO的脂质/碳水化合物合成代谢水平较高（包括3-磷酸甘油酸、6-磷酸果糖、6-磷酸葡萄糖、磷酸烯醇丙酮酸和乙酰辅酶A的产生），表明CO固定率高于LL条件。
光周期决定脂质积累和生物量生产，光/暗循环被确定为JSC4中脂质生产的关键障碍。磷酸烯醇丙酮酸向丙酮酸的转化以及3-磷酸甘油酸向3-磷酸甘油的转化是导致脂质积累延迟的候选限速步骤。包括5-磷酸核酮糖在内的底物积累可以通过生物量产量增加与CO固定增强之间的密切关系来解释。本研究首次通过与连续光照直接比较，研究了光/暗循环对脂质生产的影响，并揭示了光/暗循环过程中的潜在代谢机制和候选代谢限速步骤。这些发现为代谢工程改善脂质生产提供了有前景的靶点。
