Improvement Of Pristinamycin-producing Strain By Genome Shuffling

Pristinamycin, produced by Streptomyces pristinaespiralis, is a member of streptogramin family and is particularly active against gram-positive cocci, methicillin- and erythromycin A-resistant strains. Its modificatory compounds— dalfopristin/quinupristin have the same antibacterical activity as pristinamycin but with greater hydrosolubility. It has also the postantibiotic effect and is hard to cause antibiotic resistance.This research is aimed to improve the pristinamycin-producing strain by genome shuffling, and the results are as follows.First, the conditions for protoplast preparation, regeneration and fusion were optimized. The results showed that using 2 mg/ml lysozyme at 30℃ for 90 minutes protoplasts were obtained from mycelia of S. pristinaespiralis 11-2 which had cultured in a medium containing 0.5% glycin. The frequency of protoplast regeneration is 5.1%. When S. pristinaespiralis ZP-7, the regenerative strain of S. pristinaespiralis 11-2, was used as the start strain to prepare protoplast, the frequency of protoplast regeneration can reach 25.2% which is much higher than that of S. pristinaespiralis 11-2. When S. pristinaespiralis ZP-7 protoplasts were fused by 40% PEG6000, the frequency of fusion can reach 10.3‰.Secondly, 5. pristinaespiralis ZP-7 protoplast was mutated by UV in order to obtain a population of slightly improved phenotype called pop-UV. Then the pop-UV protoplasts were fused recursively for four rounds, and the comparative experiment was performed by protoplast preparation and regeneration without PEG-induced fusion. The results show that the population of F4, which was obtained after four rounds of protoplast fusion, remarkably improved the resistance to pristinamycin. They can grow on agar medium containing 100 μg/ml pristinamycin, while the original strain S. pristinaespiralis ZP-7's resistance to pristinamycin is about 20 μ g/ml. The compared P4's resistance to pristinamycin is about 35 Vμg/ml, only havingslightly more improvement than the starting strain. It is indicated that the improvement of F4 phenotypes is caused by recursive protoplast fusion (genome shuffling). Finally five shuffled strains-F4-5, F4-6, F4-10, F4-17 and F4-29 were obtained and their outoput reached 1.523g/L, 1.401g/L, 1.401g/L, 1.591g/L and 1.562g/L, respectively, which are 70.5%, 56.9%, 56.9%, 78.2% and 74.9% more than that of the start strain 5. pristinaespiralis ZP-7, whose output is 0.893g/L. fermentation with the shuffled strain 5. pristinaespiralis F4-17 was performed in 3 liter fermentor, the yield of pristinamycin reached 2.747g/L? which is increased by around 2 times compared with that of the start strain S. pristinaespiralis 2S-1.It is showed that genome shuffling within a population of bacteria can efficiently generate combinatorial libraries of new strains with mutation and recombination. When applied to a population phenotypically selected bacteria, many of these new strains show marked improvement in the selected phenotype.