Strain Selection Of Bacitracin High-yield Bacillus Licheniformis

Bacitracin, comprised of 12 amino acid residues, is a kind of polypeptide antibiotics. It can inhibit most of Gram-positive bacteria and some Gram-negative bacteria. The main producing strain are Bacillus licheniformis and Bacillus subtilis. Currently, bacitracin is widely used in feed antibiotics. In this study, the strain, Bacillus licheniformis DW2, was used as the starting strain, induced by physical and chemical methods to screen high-yield bacitracin mutant. And the theory of molecular biology and genetic engineering was used to construct a engineering bacteria, in which the gene of vgb can express. At the same time, a microplate detection based on the antibacterial activity of bacitracin was established. The major findings are as follows:(1) Screening for the mutant strain produicng the high yield bacitracin:659 strains were obtained by UV and DES composite mutation. The bacitracin production of these strains were prescreened by agar diffusion plate.4 mutants were selected. The highest yield of mutant bacitracin was only increased by 1.4% compared with that of B. licheniformis DW2. Therefore, The result showed that physical and chemical treatment may be not an effective way to obtain a high-yielded bacitracin strain.(2) pHY300-vgb and pHY300, which could expression in Bacillus sp., were constructed:They were imported in Bacillus licheniformis DW2 by electroporation, and obtained Bacillus licheniformis (pHY300-vgb)and Bacillus licheniformis (pHY300), respectively. The transformation was vertified to be successful. VHb expression activity in B. licheniformis (pHY300-vgb) was detected by the CO-difference spectra analysis. Shake flask experiments showed that, when the liquid volume was 150 mL in 500 mL flask, thought B. licheniformis (pHY300) and B. licheniformis (pHY300-vgb) fermentations’ inhibition zone were both smaller than DW2, the zone of B. licheniformis (pHY300-vgb) was 15.46% bigger than that of B. licheniformis (pHY300). The results in 3 L bioreactor were:In spite of that, the biomass and fermentation titers of B. licheniformis DW2 were both higher than that of B. licheniformis (pHY300) and B. licheniformis (pHY300-vgb), but the unit production of the B. licheniformis (pHY300-vgb) strain was increased by 9.47%, compared with B. licheniformis DW2, and compared with B. licheniformis (pHY300), biomass and fermentation titers of were incresssed by 23.31% and 42.55%, respectively. It was confirmed that, in some condition, VHb protein expression could improve the bacitracin production and cell growth.(3) To establish a microplate method for quantitative bacitracin initially. The study show that using initial OD600=0.2,37℃,800 r/min, a incubation period of 6 h, in the bacitracin concentration from 0.01 U/mL to 0.04 U/mL, this method had a good repeatability and stability. The aim was to set up a rapid and high-through analysis method to detect bacitracin titer.