Heavy-ion Irradiating And Screening Of Pleuromutilin High-yielding Strains

Pleuromutilin is terpenoid antibiotic produced by Clitopilus fungi, and it combines the 50 S ribosome subunit to perform its antibiotic activity through fit-induced mechanism in order to inhibit bacterial protein synthesis. Pleuromutilin derivatives Tiamulin and valnemulin have an efficient antibiotic activity towards staphylococcus, streptococcus and klebsiella,and is applied to treat pig diseases like swine dysentery and endemic pneumonia. Pleuromutilin antibiotics have the dvantages in high bioavailability, low toxicity and less residues. So far pleuromutilin industrial production in China hits the bottleneck of low pleuromutilin producing strain titer, long fermentation period and the unideal fermentation conditions, therefore, screening of pleuromutilin producing strains has a considerable economic benefits.Protoplast Mutagenesis is an important mean of industrial breeding which protoplasts are treated by physical or chemical mutagens, and after their regeneration, the mutant strains are screened and bred. Protoplast preparation is the prerequisite for the successful application of protoplast mutagenesis and other protoplast technology, and protoplast yield and regeneration rate is affected by many conditions. Through the singular factor experiments and the orthogonal experiment, this study measured effects of factors including ages of hypha, enzymes and their concentrations, temperature and osmotic stabilizers on the Clitopilus pinsitus protoplast preparation. According to the results, the optimal preparation condition is: 5d of hypha age, 0.4mol/L mannitol as the osmotic stabilizer, 1.5% snailase and 1.5% cellulase, 25℃, hydrolyzing 1.5h. The preparation yielding is up to 5.9×107 protoplast/mL and the regeneration rate is 1.20‰.Heavy ion beam which has the characters of the high LET value, the sharp Bragg peak of dose distribution, and the higher relative biological effect, leads to a large number of stable mutants. Clitopilus pinsitus protoplasts were irradiated by ¹²C⁶⁺ ion beam at varied absorbed doses for mutagenesis, and high-yielding pleuromutilin mutant strains were screened using high-throughput method and solid fermentation. The results showed that the optimum ¹²C⁶⁺ ion beam absorbed dose is 1.5 Gy, and under this condition the mutation rate is 43.62%.High-throughput screening is a novel application in the field of microbial pharmaceutical technology, which is to select microbial strains basing on their characteristics. The technic accelerates and facilitates the screening of microbial secondary metabolites, and pushes forward the development of innovative drugs in China and microbial. Due to the better sensitive of Staphylococcus aureus towards pleuromutilin, the experiments established a high-throughput screening of agar plug method. The experimental results show that pleuromutinlin yields and antibacterial zone diameters have a significant positive correlation, and the Pearson correlation coefficient was 0.956（P < 0.01）, which means through the observation of the sizes of the diameters of inhibition zone, pleuromutilin high yield strains can be screened. By agar column prescreening and 96-plate solid fermentation, the mutant strains, C.pin15I5 E and C.pin15II6 B, were selected. The yields of the strain increased by 16.17% and 15.47%, respectively, compared with that of the original strain.The resreach used the Logistic equation and the Luedeking-Piret equation to eastbilsh the fermentation kinetic model of pleuromutilin high-yielding mutant strains including cell growth, product formation and substrate consumption, in order to better understand the mechanmism between cell growth and pleuromutilin formation for improving the fermentation product index.The research suggests that applying heavy ion irradiation to protoplast is a possible method to breed mutant strains.