Identification and Engineering of an l -Threonine Aldolase from Agrobacterium pusense for Efficient l

Abstract

l-Serine is a high-value amino acid, but enzymatic production is limited by insufficient catalytic activity and limited formaldehyde tolerance. A metagenomic library derived from glufosinate-exposed soil was mined to identify an l-threonine aldolase from Agrobacterium pusense (ApLTA). The substrate tunnel and active pocket were engineered through tunnel analysis, structural comparison, consensus design, and residue plasticity-guided polarity scanning. The resulting quadruple mutant D88M/T202S/A57S/F234M (Mut4) exhibited 48.5-fold higher catalytic efficiency than wild-type ApLTA. At 1 M formaldehyde loading, Mut4 increased l-serine concentration from 23.0 to 87.3 g L–1, with a space–time yield of 21.8 g L–1 h–1, the highest reported for LTA-catalyzed l-serine synthesis. Mut4 also showed enhanced activity toward multiple aromatic aldehydes. Molecular dynamics simulations further revealed the molecular basis for its enhanced catalytic efficiency. This study provides the first demonstration of highly efficient l-serine synthesis using an engineered LTA and offers a new strategy for enzymatic l-serine production.