Enhancing L-asparagine Production Through In Vivo ATP Regeneration System Utilizing Glucose Metabolism of Escherichia coli.

L-asparaginase synthetase, an ATP-dependent enzyme, necessitates ATP for its catalytic activity. However, the integration of L-asparaginase synthetase into industrial processes is curtailed by the prohibitive cost of ATP. To address this limitation, this study explores the construction of an efficient ATP regeneration system using the glucose metabolism of Escherichia coli, synergistically coupled with L-asparaginase synthetase catalysis. The optimal conditions for L-asparagine yield were determined in shake flasks. A total of 2.7 g/L was the highest yield achieved under specific parameters, including 0.1 mol/L of substrate, 0.2 mol/L glucose, 0.01 mol/L MgCl₂ at pH 7.5, a temperature of 37 °C, and agitation at 300 r/min over 12 h. The process was then scaled to a 3-L fermenter, optimizing the addition rates of the substrate and magnesium chloride, and employing a constant glucose feed of 10 g/L/h. The scale-up process led to a significant enhancement in the production of L-asparagine. The yield of L-asparagine was increased to 38.49 g/L after 20 h of conversion, and the molar conversion rate reached 29.16%. This strategy has proven to be effective in improving the efficiency of L-asparagine production. When compared to in vitro ATP regeneration methods, this in vivo approach showcased superior efficiency and reduced costs. These findings furnish pivotal insights that may propel the enzymatic synthesis of L-asparagine toward viable industrial application.