L-malic acid is an important organic acid produced during biological metabolism. In microorganisms, its production is related to multiple metabolic pathways, not only in the tricarboxylic acid cycle and its branch glyoxylate cycle, but also as a product of CO2 fixation reaction. Since L-malic acid only participates in the circulation in ordinary organisms and will not accumulate in large amounts, otherwise it will cause blockage of metabolic flow. When L-malic acid is produced by direct fermentation of glucose, Malic acid accumulation is only possible if there are other ways of supplementing the tetracarbonate. There are two ways to supplement tetracarbonate, glyoxylate cycle and pyruvate carboxylation branch, therefore, there may be three ways to produce L-malic acid.
The first way is to supplement tetracarbonate without other ways. Glucose is first acid hydrolyzed by sugar to synthesize pyruvate, and then through the tricarboxylic acid cycle to synthesize L-malic acid. The reaction formula of this way is:
In this way, 1 molecule of glucose can only generate 1 molecule of L-malic acid and release 2 molecules of CO2, the theoretical conversion rate of this way is only 74%.
The second way is to supplement tetracarbonate through the glyoxylate cycle. Glucose first synthesizes pyruvate through the glycolysis, and then synthesizes malic acid through the tricarboxylic acid cycle and the glyoxylate cycle. The reaction formula of this way is:
In this way, 3 molecules of glucose can generate 4 molecules of L-malic acid, and release 2 molecules of CO2, so the theoretical conversion rate of this way is 99%.
The third way is that glucose first generate phosphoenolpyruvate through the glycolysis, and then CO2 fixation reaction is used to supplement the tetracarbonate. Under the catalysis of phosphoenolpyruvate carboxytransferase (EC18.104.22.168) or phosphoenolpyruvate carboxykinase, phosphoenolpyruvate combines with exogenous CO2 to synthesize oxaloacetate, and then oxaloacetate is reduced to malate. The reaction formula of this way is:
In this way, the CO2 of pyruvate carboxylation comes from the CaCO3 added in the fermentation broth. Therefore, 1 molecule of glucose can generate 2 molecules of malic acid (calcium), and the theoretical conversion rate is 148.8%, and this way does not require oxygen.
Undoubtedly, increasing the ratio of pyruvate carboxylation can improve the utilization rate of carbon source, that is, can improve the yield of malic acid.