Systematic metabolic pathway modification to boost L-ornithine supply for bacitracin production in Bacillus...

Bacitracin is a cyclic dodecyl peptide antibiotic that is an effective bacteriocide against Gram-positive and some Gram-negative bacteria. Bacitracin has been widely used as an antibacterial feed additive for livestock since it is not absorbed easily by the intestine and is easily excreted. Precursor availability has been proven to be one of the core factors for bacitracin production by many previous studies. In this study, we focused on enhancing the supply of the precursor amino acid L-ornithine to enhance bacitracin production by Bacillus licheniformis DW2 through systematic metabolic pathway modification. Several genes encoding rate-limiting enzymes for L-ornithine biosynthesis were episomally overexpressed, including argB, rocF, ppnk1, and ppnk2. The results showed that the overexpression of ppnK1 was the most effective for both L-ornithine and bacitracin biosynthesis. Secondly, the competitive branch pathways for L-ornithine biosynthesis were blocked, and the repressor was also deleted to boost L-ornithine biosynthesis. The results suggested that the deletion of genes proB and proJ to prevent proline biosynthesis and the disruption of the gene encoding the arginine repressor ArgR could enhance the intracellular concentration of L-ornithine by 49% and 2.1 times respectively, and the bacitracin production also increased accordingly by 6.6% and 11.9% respectively. Finally, several most effective efforts were combined to construct the optimal strain DW2ΔproBΔproJΔargR::ppnk1. In the optimal strain, the NADPH availability was improved and the expression levels of several essential genes for L-ornithine biosynthesis were upregulated, resulting in the enhancement of both L-ornithine and bacitracin production by 71.4% and 16.5% respectively. The final bacitracin production titer was 950 U/mL, which reached the level for industrial production.