雄甾-4-烯-3,17-二酮11α羟化突变株Metarhizium anisopliae M28-203的代谢调控

Metabolic regulation of androst-4-ene-3,17-dione 11α-hydroxylation mutant strain Metarhizium anisopliae M28-203

目的从营养代谢调控和转化条件控制两个方面入手提高雄甾-4-烯-3,17-二酮（androst-4-ene-3,17-dione,AD）11α羟化突变株Metarhizium anisopliaeM28-203转化效率。方法通过正交试验确定发酵培养基配方,对影响转化的一系列因素如：pH、发酵时间、通气量、底物投料量及溶解性、细胞通透性进行控制,考察以上因素对羟化反应的影响,确定最佳转化条件。结果该突变株培养基最佳配方为葡萄糖30 g/L、玉米浆20 g/L、蚕蛹粉5 g/L、硫酸铵2.5 g/L、磷酸氢二钾1 g/L、硫酸镁0.5 g/L和硫酸亚铁0.02 g/L。在pH 6.0~6.5、菌龄48~60 h2、50 mL摇瓶装液量为30和40 mL、转化时间72 h时能取得最佳转化效果。在底物投料量为2 mg/mL时,采用4%无水乙醇溶解底物或加入0.75 mg/mL Tween 80均有利于羟化反应,在以上优化条件下转化率分别达到62.5%和66.8%。结论突变株Metarhizium anisopliaeM28-203能有效地在AD上引入11α羟基,为工业生产依普利酮及其他甾体药物提供关键中间体。

Objective To improve the transformation efficiency of androst-4-ene-3,17-dione（AD） 11α-hydroxylation mutant strain Metarhizium anisopliae M28-203 through regulations of nutrition metabolism and biotransformation conditions. Methods The optimal fermentation culture was determined by orthogonal test.The effects of pH,fermentation time,air flow,substrate addition method and substrate concentration on hydroxylation were investigated and the optimal transformation condition was constructed. Results The optimal fermentation culture contained glucose 30 g/L,corn steep 20 g/L,silkworm chrysalis powder 5 g/L,（NH4）2SO4 2.5 g/L,K2HPO4 1 g/L,MgSO4 0.5 g/L and FeSO4 0.02 g/L.The optimal transformation condition was pH 6.0-6.5,48-60 h mycelium age,30 and 40 mL medium volume in 250 mL shake flask and 72 hour transformation time.Both 4% ethanol and 0.75 mg/mL Tween 80 were helpful for substrate solubility,and then improved transformation efficiency.While conversion rate arrived at 62.5% and 66.8% respectively under the optimal culture conditions when substrate concentration was 2 mg/mL. Conclusions The mutant strain Metarhizium anisopliae M28-203 can introduce 11α-hydroxyl to its precursor AD effectively,which provides the key intermediate for the production of Eplerenone and other steroid drugs.