Multi-strategy engineering of transaminase enables a one-pot synergistic biocatalytic cascade to a key florfenicol chiral intermediate

Abstract

(1R,2R)-2-Amino-1-(4-(methylsulfonyl)phenyl)propane-1,3-diol [(1R,2R)-3] is a key chiral intermediate for the antibiotic florfenicol. Its asymmetric synthesis remains challenging due to the difficulty in constructing two contiguous stereocenters with high selectivity. Herein, we report a green and sustainable one-pot enzymatic cascade for the efficient synthesis of (1R,2R)-3 from inexpensive and readily available L/D-serine and 4-(methylsulfonyl)benzaldehyde (4-Mb). The cascade starts with the L-amino acid oxidase (AncLAAO)-mediated oxidation of L-serine to produce β-hydroxypyruvate (1). The subsequent pyruvate decarboxylase (SwPDC)-catalyzed decarboxylative condensation led to the asymmetric assembly of 1 and 4-Mb to yield (R)-1,3-dihydroxy-1-(4-(methylsulfonyl)phenyl)propan-2-one [(R)-2], and an engineered transaminase (ATA117) aminates (R)-2 using D-serine as the amine donor to produce the target product (1R,2R)-3. This design inherently enables a one-pot cascade for assess (1R,2R)-3 and prevents the accumulation of inhibitory pyruvate, embodying the principles of atom economy and waste prevention. The rate-limiting transaminase ATA117 was engineered through a multi-strategy approach. The resulting optimal variant, ATA117-Mu9 demonstrated a 39-fold increase in catalytic efficiency (kcat/Km) and a 4.65-fold improvement in (1R,2R)-3 yield (27.9 mM) over the wild-type enzyme while maintaining a high diastereomeric excess (de) of 96-98% throughout the engineering process. For scalable application, a whole-cell biocatalyst BL21(01) co-expressing ancLAAO, SwPDC and ATA117-M9 was constructed. In a 100 mL scale-up reaction, 50 mM of 4-Mb was completely converted to 47.9 mM of (1R,2R)-3 within 3 h, achieving a space-time yield of 3.91 g/L/h, the highest reported for this compound. In summary, this work establishes a simple, efficient, and environmentally benign route that eliminates heavy metals, minimizes waste, and uses benign reaction media, providing a promising green alternative for the industrial synthesis of florfenicol intermediates.