During the growth stages, the flux of acetyl-CoA used to synthesize malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC, EC 6.4.1.2), was 0.79 mmol gDW−1 h−1. In contrast during the ARA production stage, the corresponding flux was 1.43 mmol gDW−1 h−1 (81.0%). ACC catalyzes the first committal step of the fatty acid biosynthetic pathway, and should be overexpressed to maximize ARA production [39]. Glutamate has been shown to activate ACC, and adding glutamate to the culture medium led to an increase in total lipid (21.81%) and ARA yield (66.07%) [40]. As the M. alpina growth rate in batch culture ranged from 0.06 h−1 to 0.19 h-1 [1,7,8,41,42], the glutamate uptake rates used for the in silico analysis should be set between 0 and 1.8 mmol gDW−1 h−1. When the glutamate uptake rate was increased, ARA production increased from 0.128 mmol gDW−1 h−1 to 0.355 mmol gDW−1 h−1 (Additional file 5c).