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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: A novel d-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a d-allulose 3-epimerase family enzyme based on its high specificity for d-allulose. It usually converts both d-fructose and d-tagatose to respectively d-allulose and d-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. Results: SaDAE showed a high activity on d-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s−1 mM−1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward d-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. Conclusions: We redesigned SaDAE for thermostability and biocatalytic production of d-allulose. The research will aid the development of industrial biocatalysts for d-allulose. [ABSTRACT FROM AUTHOR]