Multistep metabolic engineering of Bacillus licheniformis to improve pulcherriminic acid production

The cyclodipeptide pulcherriminic acid produced by Bacillus licheniformis is derived from cyclo(L-Leu-L-Leu) and possesses excellent antibacterial activities. In this study, we achieved high-level production of pulcherriminic acid via multistep metabolic engineering of B. licheniformis DWc9n*. Firstly, we increased leucine (Leu) supply by overexpressing the ilvBHC-leuABCD operon and ilvD involved in Leu biosynthesis to obtain strain W1, and the engineered strain W2 was further attained by deletion of gene bkdAB encoding branched-chain α-keto acid dehydrogenase in W1. As a result, intracellular Leu content and pulcherriminic acid yield of W2 strain reached 147.4 mg/g DCW (dry cell weight) and 189.9 mg/L, which were 227.6% and 48.9% higher than those of DWc9n*, respectively. Secondly, the strain W3 was constructed through overexpressing leucyl-tRNA synthase gene leuS in W2, and it produced 367.7 mg/L pulcherriminic acid. Thirdly, the original promoter of pulcherriminic acid synthetase cluster yvmC-cypX in W3 was replaced with a proven strong promoter PbacA to attain the strain W4, and its pulcherriminic acid yield was increased to 507.4 mg/L. Finally, pulcherriminic acid secretion was strengthened via overexpressing the transporter gene yvmA in W4, and W4/pHY-yvmA obtained 556.1 mg/L pulcherriminic acid yield, increased by 337.8% compared to DWc9n*, which is currently the highest pulcherriminic acid yield to the best of our knowledge. Taken together, we provided an efficient strategy for enhancing pulcherriminic acid production, which could apply to high-level production of other cyclodipeptides. Importance Pulcherriminic acid is a cyclodipeptide derived from cyclo(L-Leu-L-Leu), which shares same iron chelation group with hydroxamate sidephores. Generally, pulcherriminic acid-producing strains could be perfect candidate for antibacterial and anti-plant pathogenic fungi agent. This study attained a promising pulcherriminic acid producing strain W4/pHY-yvmA via a multistep metabolic modification. The engineered strain W4/pHY-yvmA could be able to achieve 556.1 mg/L pulcherriminic acid production, which is the highest yield so far to the best of our knowledge.