Metabolic Engineering of Main Transcription Factors in Carbon, Nitrogen, and Phosphorus Metabolisms for Enhanced Production of Bacitracin in Bacillus licheniformis

Primary metabolism plays a key role in the synthesis of secondary metabolite. In this study, the main transcription factors in carbon, nitrogen, and phosphorus metabolisms (CcpA, CcpC, CcpN, CodY, TnrA, GlnR, and PhoP) were engineered to improve bacitracin yield in Bacillus licheniformis DW2, an industrial strain for bacitracin production. First, our results demonstrated that deletions of ccpC and ccpN improved ATP and NADPH supplies, and the bacitracin yields were respectively increased by 14.02% and 16.06% compared with that of DW2, while it was decreased significantly in ccpA deficient strain DW2 Delta ccpA. Second, excessive branched chain amino acids (BCAAs) were accumulated in codY, tnrA, and glnR deletion strains DW2 Delta codY, DW2 Delta tnrA, and DW2 Delta glnR, which resulted in the nitrogen catabolite repressions and reductions of bacitracin yields. Moreover, overexpression of these regulators improved intracellular BCAA supplies, and further enhanced bacitracin yields by 14.17%, 12.98%, and 16.20%, respectively. Furthermore, our results confirmed that phosphate addition reduced bacitracin synthesis capability, and bacitracin yield was improved by 15.71% in gene phop deletion strain. On the contrary, overexpression of PhoP led to a 19.40% decrease of bacitracin yield. Finally, a combinatorial engineering of these above metabolic manipulations was applied, and bacitracin yield produced by the final strain DW2-CNCTGP (Simultaneously deleting ccpC, ccpN, phop and overexpressing glnR, codY, and tnrA in DW2) reached 1014.38 U/mL, increased by 35.72% compared to DW2, and this yield was the highest bacitracin yield currently reported. Taken together, this study implied that metabolic engineering of carbon, nitrogen, and phosphorus metabolism regulators is an efficient strategy to enhance bacitracin production, and provided a promising B. licheniformis strain for industrial production of bacitracin.