SummaryPeroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002(CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H 2 O 2 . This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA β-oxidation involving H 2 O 2 -producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.
Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO 2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.
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