Multistep metabolic engineering of Bacillus licheniformis to improve pulcherriminic acid production

Pulcherriminic acid, a cyclodipeptide, possesses the excellent antibacterial activities by chelating iron ions from environment, however, the low yield has hindered its application. In this study, high-level production of pulcherriminic acid was achieved by the multistep metabolic engineering of Bacillus licheniformis DWc9n*. Firstly, leucine (Leu) supply was increased by overexpressing the genes ilvBHC-leuABCD and ilvD involved in the Leu synthetic pathway and deleting the gene bkdAB encoding a branched-chain α-keto acid dehydrogenase. The intracellular Leu content and pulcherriminic acid yield of strain W2 reached 147.4 mg/g DCW and 189.9 mg/L respectively, which corresponded to a 226.7% and 49.1% higher than those of DWc9n*. Secondly, the strain W3 was obtained by overexpressing leucyl-tRNA synthase LeuS in W2, which lead to a 190% improvement compared to DWc9n*, pulcherriminic acid yield increase to 367.7 mg/L. Thirdly, overexpression of the cytochrome synthase gene cluster yvmC-cypX further increased the yield of pulcherriminic acid to 507.4 mg/L. Finally, the secretion capability was improved by overexpressing the pulcherriminic acid transporter gene yvmA. This resulted in the 556.1 mg/L pulcherriminic acid was produced in W4/pHY-yvmA, increased by 340% as compared with DWc9n*, and it is the highest pulcherriminic acid production currently reported. Taken together, this study provided an efficient strategy for enhancing pulcherriminic acid production and might also be applied for high-level production of other cyclodipeptides.