Multistep metabolic engineering of Bacillus licheniformis to improve pulcherriminic acid production

Abstract

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 -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.\n\nImportancePulcherriminic acid is a cyclodipeptide that derived from cyclo(L-Leu-L-Leu), which has the same iron chelation group with hydroxamate siderophores. Pulcherriminic acid producing strains can inhibit the growths of various bacteria and plant pathogenic fungi. However, the expected pulcherriminic acid yield was not achieved. This study reports efficient microbial production of pulcherriminic acid in Bacillus licheniformis DWc9n* via multistep metabolic engineering strategies. The combination of these interventions resulted in the establishment of a pulcherriminic acid overproducing strain. In combination with bioprocess engineering efforts, pulcherriminic acid was produced at a final yield of 556 mg/L in a shake flask. This is the highest pulcherriminic acid yield ever reported so far using rationally engineered microbial cell factories.