Increased productivity of L-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of L-threonine deaminase.

OBJECTIVE: To strengthen NADH regeneration in the biosynthesis of L-2-aminobutyric acid (L-ABA). RESULTS: L-Threonine deaminase (L-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type L-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable L-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for L-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of L-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type L-TD, respectively. CONCLUSIONS: By using the engineered L-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of L-ABA and total turnover number of coenzyme.