Characterization of acetate catabolism in Chlamydomonas reinhardtii reveals distinct roles for ACS1 and ACK2 in regulating cell growth and carbon storage

Acetate metabolism drives mixotrophic and heterotrophic growth in some microalgae. Acetyl-CoA synthetase (ACS) and acetate kinase (ACK) are often considered the main enzymes involved in acetate catabolism in microalgae; however, their contributions to metabolic flux and carbon allocation are not fully understood. In this study, the functions of cytosolic ACS1 and mitochondrial ACK2 were characterized using two knockout mutants of the model microalga Chlamydomonas reinhardtii. The acs1 mutant exhibited a growth-oriented phenotype, characterized by 29.8% faster cell growth at 96 h and up to a 15.5% higher acetate depletion rate, yet showed a 38.3% lower triacylglycerol (TAG) content at 48 h under heterotrophic conditions. By contrast, the ack2 mutant exhibited an altered carbon-allocation phenotype under heterotrophic conditions. Despite an up to 32.4% lower respiratory oxygen consumption rate and a 27.7% reduction in cell density, ack2 exhibited a 39.3% higher biomass concentration and a 90.4% greater dry weight per cell than the wild type at 96 h. Biochemical analysis revealed that ack2 accumulated 23.3% more carbohydrate than the wild type at 120 h under heterotrophic conditions, whereas its TAG level remained comparable to that of the wild type. These findings suggest that, under heterotrophic conditions, the loss of cytosolic ACS1 facilitates cell growth and division at the expense of TAG biosynthesis, whereas the loss of mitochondrial ACK2 regulates growth by affecting carbon flux toward biomass and carbohydrate accumulation. This work provides insight into acetate catabolism in C. reinhardtii and suggests targets for engineering microalgae for production of biomass and bioproducts.