Engineering ω-transaminase for efficient dihydroxyacetone transamination in serinol biosynthesis starting from methanol.

Serinol (2-amino-1,3-propanediol) is an important pharmaceutical intermediate, but conventional chemical or microbial routes are hampered by high energy demand, product toxicity, or complex regulation. Here, we report a modular cell-free enzyme cascade, termed the methanol-to-serinol pathway (MSP), that efficiently converts methanol-a low-cost C1 feedstock-into serinol with high carbon yield. The cascade comprises two modules: Module 1 employs an alcohol oxidase and an engineered formolase to generate dihydroxyacetone (DHA), while Module 2 uses a tailored ω-transaminase for direct one-step amination. To overcome the rate-limiting DHA amination, we applied an "ALF" scanning strategy and identified a triple-mutant Cv-ωTA (Y153F/Y168F/C418F) with 6.3-fold higher specific activity than the wild type. Fitness landscape analysis revealed strong non-additive interactions, highlighting the synergistic effect of these three mutations. Molecular dynamics simulations revealed structural changes underlying the activity boost. By incorporating a pyruvate-removal system to drive the equilibrium toward product formation, the integrated cascade achieved 43.86 mM (4 g/L) serinol from 150 mM methanol in 7 h, corresponding to 87.7 % carbon yield and a productivity of 0.57 g/L/h. This work establishes a carbon-efficient route for serinol biosynthesis and provides a generalizable strategy for sustainable C1 biomanufacturing.