Rational Design of Agmatinase SpeB and Spermidine Synthase SpeE Enhances Spermidine Production in Bacillus amyloliquefaciens.

Spermidine, a natural polyamine, is commonly used as a dietary supplement. Microbial synthesis of spermidine is a sustainable and effective alternative to traditional extraction methods but is constrained by low yields. In this study, enzyme engineering strategies were applied to increase the spermidine yield in Bacillus amyloliquefaciens. Alanine scanning mutagenesis of agmatinase SpeB increased spermidine yield by 78.3%. Enzyme kinetic analysis and molecular dynamics (MD) simulations further revealed that the increase in yield was attributed to the enhanced substrate affinity of SpeB. Additionally, molecular docking, negative feedback regulation, and substrate channel engineering were used to engineer spermidine synthase SpeE1, resulting in a 178.2% increase in spermidine yield. Through fed-batch fermentation in shake flasks, the coexpression-engineered strain PM1/SpeE1E159P/S157L/Q32N-SpeBH151A/Y144A exhibited a 5.6-fold increase in spermidine yield, reaching up to 851.15 mg/L, compared to the control strain PM1/SpeE-SpeB. This work provides an effective enzyme engineering approach for the high-level microbial synthesis of spermidine.