Modifying The Ornithine And The Central Metabolic Pathway To Promote The Bacitracin Production In Bacillus Licheniformis

Bacitracin is a cyclic dodecyl peptide antibiotic containing 11 amino acids and12 amino acid residues in a non-ribosomal manner.It is an effective bacteriocide against Gram-positive bacteria and some Gram-negative bacteria.Bacitracin is widely used as an antibacterial feed additive for livestock since it is not easily absorbed by the intestine and easily excreted.Many previous studies have demonstrated that the availability of bacitracin precursor amino acids is one of the core factors in the production of bacitracin.As one of the prerequisite amino acids for the synthesis of bacitracin,L-ornithine plays an important role in the synthesis of bacitracin.The precursor amino acids of several other synthetic bacitracin including L-ornithine are derived from the TCA cycle.Therefore,this study mainly focuses on the improvement bacitracin synthesis by enhancing Bacillus licheniformis DW2L-ornithine anabolism and precisely regulating important genes of the central metabolism.This study mainly strengthens the biosynthesis of L-ornithine from two aspects.Firstly,the rate-limiting enzyme of L-ornithine biosynthesis acetylglutamate kinase encoded by arg B,arginase encoded by roc F,NAD~+kinase encoded by ppn K1 and ppn K2 were overexpressed.The results show that the over-expression of ppn K1 is most effective for the biosynthesis of L-ornithine and bacitracin.The bacitracin production and intracellular ornithine content of the engineering strain DW2/p HY-ppn K1 increased by 31.9%and 25.9%compared with the control strain,respectively.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 pro B and pro J to prevent proline biosynthesis and the disruption of the gene encoding the arginine repressor Arg R 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%and11.9%.Finally,several most effective efforts were combined to construct the optimal strain DW2?pro B?pro J?arg R::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 TCA cycle serves as the central metabolism,and its intermediate substances are precursors for the synthesis of many amino acids and it provides energy for the synthesis and transport of amino acids.Pyruvate carboxylase catalyzes pyruvate to produce oxaloacetate.And isocitrate dehydrogenase is one of the main enzymes regulated the TCA cycle.Therefore,in the central metabolic pathway,pyruvate carboxylase and isocitrate dehydrogenase in B.licheniformis DW2 were upregulated via promoter replacement.The promoters of the above two genes were replaced by5'-UTR with different strengths to achieve different degrees of gene expression,and the bacitracin titer was improved accordingly.Compared with the control strain,the bacitracin production of the engineered strains DW2-Prepeat3-pyc A and DW2-Prepeat7-icd increased by 11.5%and 12.8%,respectively.Therefore,it is speculated that isocitrate dehydrogenase and pyruvate carboxylase increase the supply of bacitracin synthetic precursor amino acids by affecting the strength of the TCA cycle,and ultimately increase the production of bacitracin.