牲畜链霉菌（Streptomyces　cattleya）thienamycin合成阻断变株的筛选、阻断部位的确定及变株原生质体的研究

Screening,block position determination and study on protoplast of thienamycin block mutant from Streptomyces cattleya

以ｔｈｉｅｎａｍｙｃｉｎ（ＴＮＭ）产生菌Ｓ．ｃａｔｔｌｅｙａ为出发菌株，利用终浓度为１．５ｍｇ／ｍｌ强诱变剂亚硝基胍在ｐＨ９．０、３７℃条件下对其孢子悬液处理３０ｍｉｎ，得到稳定的ＴＮＭ生物合成阻断变株Ｙ＿３。纸层析后进行茚三酮显迹和生物显迹表明Ｙ＿３变株积累一种无活性中间产物，其迁移率不同于ＴＮＭ。建立了Ｙ＿３变株中间产物的分离纯化方法。酸水解和氨基酸组分分析表明Ｙ＿３中间产物为谷氨酸和丙氨酸组成的二肽，可能是形成碳青霉烯双环母核的底物，提示丙氨酸可能也是ＴＮＭ的前体，Ｙ＿３变株阻断在形成碳青霉烯双环母核的环化步骤。Ｙ＿３变株具有较高的原生质体形成率和再生率。原生质体在４０℃进行处理可以在不影响再生率的前题下提高ＤＮＡ转化率。通过以上研究建立了以Ｙ＿３变株为宿主的ＴＮＭ环化酶基因克隆受体系统。

A stable mutant Y3 blocked in the thienamycin biosynthetic pathway was obtained from thienamycin producing strain S.cattleya by 1.5mg/ml NTG treatment at pH 9.0,37℃for 30min. The intermediate accumulated by Y3 was identified as a bipeptide consisting of glutamic acid and alanine by means of paper chromatography and amino acid composition analysis.We presume that this bipeptide may serve as precursor of the bicyclic ring system of thienamycin and alanine is another possible precursor of thienamycin. Protoplast formation rate was up to 90%when Y3 mycelitum was treated by 2.0mg/ml lysozyme at 37℃ for 90min and protoplast regeneration rate was 8.0%in SGGP medium containing 10.3%sucrose,The transformation rate was increased about 10 times using Koch-Light PEG1000 as mediater and by treating protoplast at 40℃ for 10min before transformation.Therefore,we set up the thienamycin cyclase gene cloning system from S.cattleya using Y3 block mutant as a host.