Resistant Mutants Screening Of Pleuromutilin High-producing Clitopilus Prunulus

Pleuromutilins could inhibit the growth of a variety of Gram-positive and mycoplasmas strongly, and has better activity than other biotics to Gram-negative and some drug-resistants. bacterial strains. Pleuromutilins bind to the bacterial 50S ribosomal subunit, hindering correct positioning of the tRNAs for peptide transfer and consequently halting bacterial protein synthesis,and do not affect protein formation in eukaryotes and mammals. Pleuromutilin derivatives, taimulin and valnemulin have a long history in veterinary medicine, retapamulin is the first antibiotic of this class to be developed for use in human therapeutics. Good antibacterial activity and unique mechanism of antibacterial show that pleuromutilins is increasingly economically important.Pleuromutilin derivatives are usually chemical synthesized or biological transformation by pleuromutilin, so industrial production of pleuromutilin is very important. Pleuromutilin was first discovered in Pleurotus mutilis and pleurotus passeckerianus by Kavanagh et al, and is a tricyclic diterpene secondary metabolite. Pleuromutilin producers have been reported belong to Basidiomycetes Agaricales. Microbe as the core of fermentation, strain improvement is very important for the industrial production of pleuromutilin. Because of unclear genetic background and little knowledge about biosynthetic gene, mutation breeding technology combined with resistant screening is more suitable for strain improvement of Basidiomycetes.In this paper, the breeding of pleuromutilin producing strain used Clitopilus prunulus-04 as original strain, and screened by four resistant drugs:antibiotics nystatin and terbinafine phenol, surfactant sodium dodecyl benzene sulfonate (SDBS) and dodecyl trimethyl ammonium chloride (DTAC). Nystatin and terbinafine phenol both are antifungal antibiotics, can inhibit the biosynthesis of ergosterol, which is accompanied by changes in membrane permeability; surfactant inhibit microbial growth by changing the membrane permeability. So choose these drugs test as a screening model.These drugs have shown antibacterial activity on Clitopilus prunulus-04, the minimum inhibitory are:nystatin 6μg/ml, terbinafine phenol 120μg/ml, DTAC35μg/ml, SDBS70μg/ml.. Clitopilus prunulus-04 was treated with NTG concentration 0.4 mg/ml,25℃,210r/min oscillation 30 min, which get 83.30 percent of death rate. A large number of pleuromutilin high-producing strains were obtained from the original basidiomycetes strain Pleurotus mutilis after nitrosoguanidine mutagenesis and following continuous subculture in the broth with nystatin, terbinafine, SDBS, and DTAC respectively. Pleuromutilin yield of pn163,a nystatin resistant mutant with genetic stability, was increased by 38.50%.Moreover, the ergosterol and extracellular pleuromutilin produced by drug-resistants were detected by HPLC.Mutants with antibiotic resistance which antibiotics targeted on ergosterol biosynthesis pathway, got a higher forward mutation rate. The extracellular pleuromutilin produced by drug resistant mutants more than that of otiginal strain. And ergosterol of the antibiotic resistance mutants were decreased obviously. Biosynthesis of pleuromutilin associated with the cell membrane permeability.