Abstract L‐Homophenylalanine (L‐HPA) is a vital building block for the synthesis of numerous chiral drugs. However, the high cost of starting materials limits the industrial production of L‐HPA. In this study, an enzymatic‐spontaneous chemical cascade route for L‐HPA production was designed based on retrosynthetic analysis. This route, using simple benzaldehyde and pyruvate as starting materials, is extremely cost‐effective. The enzymes were screened and further assembled in E.?coli, and TipheDH was identified as the rate‐limiting enzyme. Therefore, TipheDH was engineered to improve its specific activity (by 82?%) and expression level (by 254?%), thus generating the best strain (W14). W14 exhibited the optimum enzyme activity ratio (1.7?:?1.1?:?1?:?1.8) and demonstrated production of 100.9?g?L?1 of L‐HPA (with 94?% conversion, >99?% ee) in a 5‐L reactor. This route effectively exploits the power of cascades and offers insight into avenues for synthesizing other valuable chemicals from inexpensive building blocks.
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