Systematic engineering of branch chain amino acid supply modules for the enhanced production of bacitracin from Bacillus licheniformis

Bacitracin is a broad-spectrum cyclic peptide antibiotic mainly produced by Bacillus, precursor amino acid supply served as the critical role during its synthesis. In this study, we systematically engineered branch-chain amino acid (BCAA) supply modules for bacitracin production. Firstly, we demonstrated that Ile and Leu acted as limiting precursors for bacitracin synthesis, and that BCAA synthetic pathways were strengthened via simultaneous overexpression of, feedback-resistance acetolactate synthase IlvBN(fbr), 2-isopropylmalate synthetase LeuA(fbr) and BCAA aminotransferase YbgE. Using this approach, bacitracin yield from strain DW-BCAA2 was 892.54 U/mL, an increase of 18.32% compared with that DW2 (754.32 U/mL). Secondly, the BCAA permeases, YvbW and BraB, which have higher affinities for Leu and Ile transportation, respectively, were both identified as BCAA importers, with their overexpression improving intracellular BCAA accumulations and bacitracin yields. Finally, the leucine-responsive family regulator, lrpC was deleted to generate the final strain DW-BCAA6, with intracellular concentrations of Ile, Leu and Val increased by 2.26-, 1.90- and 0.72-fold, respectively. The bacitracin yield from DW-BCAA6 was 1029.83 U/mL, an increase of 36.52%, and is the highest bacitracin yield reported. Equally, concentrations of other byproducts including acetic acid, acetoin and 2,3-butanediol were all reduced. Taken together, we devised an efficient strategy for the enhanced production of bacitracin, and a promising B. licheniformis DW-BCAA6 strain was constructed for industrial production of bacitracin.