Dynamism of Metabolic Carbon Flow of Starch and Lipids in Chlamydomonas debaryana

Abstract

Microalgae have the potential to recycle CO₂ as starch and triacylglycerol (TAG), which provide alternative source of biofuel and high added-value chemicals. Starch accumulates in the chloroplast, whereas TAG accumulates in the cytoplasmic lipid droplets (LD). Preferential accumulation of starch or TAG may be achieved by switching intracellular metabolic carbon flow, but our knowledge on this control remains limited. Are these two products mutually exclusive? Or, does starch act as a precursor to TAG synthesis, or vice versa? To answer these questions, we analyzed carbon flow in starch and lipids using a stable isotope ¹³C in Chlamydomonas debaryana NIES-2212, which accumulates, without nutrient limitation, starch in the exponential growth phase and TAG in the stationary phase. Pulse labeling experiments as well as pulse labeling and chase experiments were conducted, and then, gas chromatography-mass spectrometry (GC-MS) analysis was performed on starch-derived glucose and lipid-bound fatty acids. We exploited the previously developed method of isotopomer analysis to estimate the proportion of various pools with different isotopic abundance. Starch turned over rapidly to provide carbon for the synthesis of fatty acids in the exponential phase cells. Most fatty acids showed rapid and slow components of metabolism, whereas oleic acid decayed according to a single exponential curve. Highly labeled population of fatty acids that accumulated during the initial labeling decreased rapidly, and replaced by low abundance population during the chase time, indicating that highly labeled fatty acids were degraded and the resulting carbons were re-used in the re-synthesis with about 9-fold unlabeled, newly fixed carbons. Elongation of C16-C18 acids in vivo was indicated by partially labeled C18 acids. The accumulation of TAG in the stationary growth phase was accounted for by both de novo synthesis and remodeling of membrane lipids. These results suggest that de novo synthesis of starch and TAG was rapid and transient, and also almost independent to each other, but there is a pool of starch quickly turning over for the synthesis of fatty acids. Fatty acids were also subject to re-synthesis. Evidence was also provided for remodeling of lipids, namely, re-use of acyl groups in polar lipids for TAG synthesis.

Keywords: biofuel production; green alga; isotopomer analysis; lipid biosynthesis; metabolic analysis; stable isotope.