Boosting Iturin A Production in Bacillus velezensis by Multimodule Engineering, Operon Restructuring, and Pickering Emulsion Fermentation

Abstract Iturin A is a valuable natural cyclic lipopeptide with broad application potential in multiple fields such as food preservation and agricultural production. However, its industrial production remains limited due to low fermentation yield. In this study, iturin A biosynthesis in Bacillus velezensis was enhanced by modifying the genes involved in fatty acid precursors, amino acid precursors, regulators, and transporters, such as accAD, serC, and comA. Additionally, insertion of a terminator–promoter regulatory element into the ituA–ituB intergenic region prevented upstream polar effects and reinitiated downstream transcription, which increased the transcript abundance and stability of the ituB–ituC region to further improve iturin A yield. Optimization of the N-terminal-coding sequence of ituD in the engineered strain ZD-F23 further elevated iturin A yield to 3.85 g/L in shake flasks, representing a 7.95-fold increase over the wild strain ZD122 (0.43 g/L). Finally, iturin A yield was increased to 4.59 g/L in shake flasks by coupling microbial fermentation with Pickering emulsion techniques in an optimized oil-in-water system. This value represented the highest iturin A titer among previous B. velezensis strains. In summary, this study establishes a multilevel engineering strategy integrating transcriptional reprogramming with emulsion-based fermentation, demonstrating the potential for scalable biomanufacturing of iturin A.