3.2 Large amounts of required amino acids that derived acetyl-CoA and succinyl-CoA
The six amino acids are divided into three groups according to their specific intermediates in the tricarboxylic acid (TCA) cycle, as acetyl-CoA module (leucine, tryptophan, lysine), succinyl-CoA module (methionine), and α-ketoglutarate module (histidine, arginine) (Figure 1). As shown in Figure S2, 100 mg/L of leucine, tryptophan, and lysine could not support efficient cell growth, and hence the concentrations were further increased to 500 mg/L and 1,000 mg/L. As expected, to restore cell growth and FFA production, the required amounts of amino acids in acetyl-CoA module are extremely high, up to 1,000 mg/L (Figure 2). For strains deficient in synthesis of tryptophan and lysine, the cell growth and production capacity were basically similar to those of control strain with 500 mg/L of supplements. However, compared with control strain, only 70% of maximum biomass and FFA titers was obtained even in 1,000 mg/L of leucine (Figure 2A, D), demonstrating leucine metabolism may be closely related to acetyl-CoA for cell growth and product formation.
Similar to those in acetyl-CoA module, methionine in succinyl-CoA module also needs higher concentration to restore cell growth. While supplementing 500 mg/L and 1,000 mg/L of methionine to cultivate the auxotrophic strain, the growth and FFA were similar to the control strain, which was obviously improved than those in 100 mg/L (Figure 3). The degradation of methionine generates succinyl-CoA through a multi-step reaction involved in intermediates of methylmalonyl-coA and methylmalonyl-SCOA (Figure 1), whose absence may disturb central metabolism for cell growth and production.