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.