Structure-guided engineering of benzaldehyde lyase for efficient synthesis of dihydroxyacetone and one-pot biosynthesis of 2-amino-1,3-propanediol†

2-Amino-1,3-propanediol (serinol) is ubiquitous in bioactive compounds and pharmaceutical agents. As a key precursor, dihydroxyacetone (DHA) plays an essential role in the biosynthesis of serinol. The carboligation of C1 compound formaldehyde (HCHO) catalyzed by formolase (FLS) is considered as a highly valuable approach to obtain DHA. Until now, only two FLSs have been discovered by engineering the benzaldehyde lyase (BAL) from Pseudomonas fluorescens biovar I and the benzoylformate decarboxylase (BFD) from Pseudomonas putida, but their low activity and chemoselectivity hinder their industrial application. In this study, a novel FLS with high catalytic efficiency (14.60 s⁻¹ M⁻¹) and excellent chemoselectivity (>99%) for the synthesis of DHA was discovered by engineering a BAL from Herbiconiux sp. SALV-R1. Meanwhile, molecular dynamics studies demonstrated that reducing the volume of the binding pocket significantly stabilized the cofactor in the enzyme, which in turn increased the activity of FLS. In addition, a series of de novo FLSs were successfully obtained by introducing six key mutations within the active pocket of wild-type BALs. Furthermore, a one-pot, two-step enzymatic process involving FLS-catalyzed carboligation of HCHO and subsequent transamination with transaminases was designed and implemented, providing a new and efficient method to produce serinol (91% isolated yield) from HCHO and thus contributing to the valorization of C1 compounds in a cheap and environment-friendly way.