l-2-Aminobutyric acid (l-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. It can be produced by transaminase or dehydrogenase from alpha-ketobutyric acid, which can be synthesized enzymatically from the bulk amino acid, l-threonine. Deamination of l-threonine followed by a hydrogenation reaction gave almost the theoretical yield and was estimated to be more cost-effective than the established chemical process. l-Threonine deaminase from Escherichia coli, l-leucine dehydrogenase from Bacillus cereus, and formate dehydrogenase from Pseudomonas sp. were over-expressed in E. coli and used for one-pot production of l-ABA with formate as a co-substrate for NADH regeneration. 30 mol l-threonine were converted to 29.2 mol l-ABA at 97.3 % of theoretical yield and with productivity of 6.37 g l(-1) h(-1) at 50 l. This process offers a promising approach to fulfil industrial requirements for l-ABA.