Engineering of an L-threonine aldolase from Firmicutes bacterium for the stereoselective synthesis of Droxidopa
Adol condensation mediated by L-threonine aldolase (LTA) is a promising route to synthesize β-hydroxy-α-amino acids, such as the anti-Parkinson's disease drug L- threo -3,4-dihydroxyphenylserine (L- threo -DOPS; droxidopa). However, the biocatalytic approach generally suffered from poor stereoselectivity at the C β chiral center. Herein, several LTAs from diverse microorganisms were examined for the stereoselective synthesis of L- threo -DOPS, and an LTA from Firmicutes bacterium ( Fb LTA) was identified with relatively high stereoselectivity. A semi-rational design was then employed to further enhance the stereoselectivity, resulting in the identification of beneficial mutations and the generation of beneficial mutants with markedly elevated stereoselectivity (up to 80 % de) over the wild-type (23 % de). Molecular dynamics (MD) simulation revealed that the significant improvement of stereoselectivity could be attributed to the structural reconstitution of substrate tunnels. Finally, the best mutant was applied to a scaled-up reaction, affording 1.3 g/L yield of L- threo -DOPS with 80 % de. This study provides new insights into the stereoselectivity control of LTAs and potential engineered LTAs for the manufacture of L- threo -DOPS.
危险品化学品经营许可证（带存储）