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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. [ABSTRACT FROM AUTHOR]

Background: 3‐ketosteroid‐Δ1‐dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2‐desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3‐ketosteroid‐Δ1‐dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3). Results: KsdD4 exhibited optimal activity at pH 7.0 and 35°C. KsdD4 had the highest activity toward 4‐androstene‐3,17‐dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure‐based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate‐binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side‐chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed‐batch bioconversion using resting cells resulted in efficient production of 2.66 g L−1 androst‐1,4‐diene‐3,17‐dione (ADD). Conclusion: Biochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed‐batch strategy improved the bioconversion of steroids. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]