Multimodular Pathway Engineering of <i>Bacillus amyloliquefaciens</i> for Intensifying Iturin A Production

Iturin A is a potent lipopeptide with broad application potential. However, its microbial production remains constrained by limited precursor availability and metabolic inefficiencies. In this work, a modular metabolic engineering strategy was employed in <i>Bacillus amyloliquefaciens</i> to systematically enhance the biosynthetic capacity of multiple precursor pathways. Fatty acid synthesis was improved by deleting the transcriptional repressor <i>fapR</i>, overexpressing the key gene <i>fabG</i>, and disrupting <i>mmgA</i>, which is involved in acetyl-CoA metabolism. To increase the availability of proline and serine, their catabolic genes (<i>fadM</i> and <i>sdaAB</i>) were deleted, while the corresponding biosynthetic genes (<i>proBA</i> and <i>serC</i>) were overexpressed. Furthermore, the branched-chain amino acid pathway was reinforced by overexpressing the key gene <i>ilvD</i> and deleting the aminotransferase gene <i>gabT</i>. Nitrogen acquisition was also further enhanced by overexpressing the <i>opp</i> operon encoding an oligopeptide transporter. The resulting engineered strain, ITUz21, achieved an iturin A titer of 7.62 g/L and a productivity of 0.14 g/L/h in a 7.5 L bioreactor, representing the highest level reported to date. These findings demonstrate the effectiveness of coordinated precursor pathway engineering for improving microbial iturin A biosynthesis.