ObjectiveTo strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).Resultsl-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 degrees C increased from 10 to 210min, 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 15min increased from 39 to 53 degrees 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 0.993M and 16,469, in contrast to 0.635M and 10,531 with wild-type l-TD, respectively.ConclusionsBy 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.