Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids

Threonine deaminase (TD, EC 4.3.1.19) mediates α,β-elimination (deamination), which has untapped potential in the synthesis of unnatural α-keto acids. However, the narrow substrate scope of wild-type TD limits its application. This issue could be overcome by engineering the substrate tunnel of the enzyme. Here, Corynebacterium glutamicum TD ( Cg TD) was used as a model, and its substrate tunnel was identified as a gating element. On the basis of the gating characteristics and constitution of the substrate access tunnel of Cg TD, an “open-gate” strategy was developed to improve its substrate scope toward bulky substrates. The best variant obtained following this approach, Cg TD Mu7, exhibited 90.6-fold greater catalytic efficiency ( k cat / K M ) toward a bulky substrate such as phenylserine. The improvement was due to more efficient substrate coupling, deprotonation, and imine hydrolysis. Cg TD Mu7 was used for efficient production of both natural and unnatural α-keto acids, including 2-oxobutyric acid (83.4 g L –1, 99% conversion), phenylpyruvic acid (72.5 g L –1, 95% conversion), 2-oxovaleric acid (21.1 g L –1, 91% conversion), and 2-oxo-4-phenylbutyric acid (29.9 g L –1, 84% conversion). The proposed strategy provides a theoretical basis for the synthesis of α-keto acids and is also effective for engineering other similar enzymes.