高产截短侧耳素担子菌的抗性突变株筛选

Resistant Mutant Screening of Pleuromutilin High-producing Clitopilus prunulus

截短侧耳素是由担子菌产生的一种三环二萜类化合物,通过抑制肽基转移酶的活性而使细菌蛋白质合成受阻。以截短侧耳素产生菌Clitopilus prunulus-04为出发菌株,经亚硝基胍诱变后分别进行制霉菌素、特比萘酚、十二烷基苯磺酸钠和十二烷基三甲基氯化铵抗性突变子筛选,获得了大量的产量提高的突变子。制霉菌素和特比萘酚抗性突变子的正突变率较高,其中,一株制霉菌素抗性突变子pn163的截短侧耳素产量比出发菌株提高了38.50%,且遗传稳定性较好。制霉菌素和特比萘酚抗性突变子在高产截短侧耳素的同时,也降低了麦角甾醇生物合成量,推测产量提高与细胞膜通透性有关。

Pleuromutilin is one of the tricyclic structural diterpenoid antibiotics, and is currently being used as an intermediate for semi-synthetic antibiotics such as veterinary antibiotics tiamulin, valnemulin, and humanused antibiotic retapamulin. These compounds inhibit bacterial protein synthesis by specifically targeting the large subunit （50S） of the bacterial ribosome and exhibit excellent in vitro and in vivo antibacterial activity against gram-positive pathogens . Recently, a series of novel water-soluble pleuromutilin derivatives bearing a purine ring have been developed. These developing pleuromutilin derivatives stimulate the need of pleuromutilin from Clitopilus prunulus. But the strain improvement about Clitopilus prunulus has not been reported. A large number of pleuromutilin high-producing strains were obtained from the original basidiomycetes strain Clitopilus prunulus after nitrosoguanidine mutagenesis and following continuous subculture in the broth with nystatin, terbinafine, sodium dodecyl benzene sulfonate, and lauryl trimethyl ammonium chloride respectively. Mutants with antibiotic resistance which antibiotics targeted on ergosterol biosynthesis pathway, got a higher forward mutation rate. The pleuronmtilin yield of mutant strain pn163, a nystatin resistant mutant with genetic stability, was increased by 38.50% while ergosterol was decreased to 29. 29% , which was supposed to correlate with cell membrane permeability.