Metabolic plasticity of the starchless mutant of Chlorella sorokiniana and mechanisms underlying its enhanced lipid production revealed by comparative metabolomics analysis

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The unicellular green alga Chlorella sorokiniana is of great industrial interest for biofuels production due to its rapid growth, high lipid content, and capabilities in tolerating broad environment and cultivation conditions. C. sorokiniana can grow under both heterotrophic and photoautotrophic conditions, which offers opportunities to produce lipids from this species/strain by employing coupled heterotrophic and phototrophic cultivation mode. In this study, a starchless mutant SLM2 was successfully obtained by high-throughput screening of the chemical mutagenesis pool of a C. sorokiniana strain that has the potential to achieve high cell density under heterotrophic conditions, aiming to enhance its lipid content and productivity. Unexpectedly, enhanced lipid production was only observed in photoautrophically-grown SLM2 cells subjected to stress conditions (i.e. high-light and nutrient limitation). When the heterotrophically-grown cells were subjected to the same stress conditions that can trigger lipid accumulation, less differences in lipid productivities were observed between wild type (WT) and SLM2. To understand metabolic basis underlying such a discrepancy, comparative time-course metabolomics analysis was performed for SLM2 and WT from different cultivation conditions. The results highlighted up-regulated metabolic flux related to the increased lipid biosynthesis in the phototrophically-grown starchless mutant cells, which included enhanced oxidative pentose phosphate pathway in a mode favoring NADPH production, and the augmented glycolysis pathway providing precursors for both fatty acids and glycerol backbone synthesis. In SLM2, the classical TCA cycle is down-regulated, while its bypass the gamma-aminobutyric acid (GABA) shunt is triggered, which is speculated to contribute to enhanced lipid accumulation in the starchless mutant as well. In addition to revealing the metabolic plasticity possessed by starchless mutant, it is suggested that photoautotrophic cultivation mode is more suitable than coupled heterotrophic and photoautotrophic cultivation mode for production of lipids by using the starchless mutant SLM2 obtained in this study.