Overexpression of acetyl-CoA synthetase (ACS) enhances the biosynthesis of neutral lipids and starch in the green microalga Chlamydomonas reinhardtii

Genetic engineering can be the solution to achieve the economically feasible production of microalgal based biofuels and other bulk materials. A good number of microalgal species can grow mixotrophically using acetate as carbon source. Moreover, experimental evidence suggests that the biosynthesis of acetyl-CoA could be a limiting step in the complex multifactor-dependent biosynthesis of acylglycerides and point to acetyl-CoA synthetase (ACS) as a key enzyme in the process. In order to test this hypothesis we have engineered the model chlorophyte Chlamydomonas reinhardtii to overexpress the endogenous chloroplastic acetyl-CoA synthetase, ACS2. Expression of the ACS2 encoding gene under the control of the strong constitutive RBCS2 promoter in nitrogen-replete cultures resulted in a 2-fold increase in starch content and 60% higher acyl-CoA pool compared to the parental line. Under nitrogen deprivation, the Cr-acs2 transformant shows 6-fold higher levels of ACS2 transcript and a 2.4-fold higher accumulation of triacylglycerol (TAG) than the untransformed control. Analysis of lipid species and fatty acid profiles in the Cr-acs2 transformant revealed a higher content than the parental strain in the major glycolipids and suggests that the enhanced synthesis of triacylglycerol in the transformant is not achieved at the expense of membrane lipids, but is due to an increase in the carbon flux towards the synthesis of acetyl-CoA in the chloroplast. These data demonstrate the potential of engineering the chloroplastic ACS to increase the carbon flux towards the synthesis of fatty acids as an alternative strategy to enhance the biosynthesis of lipids in microalgae.