Abstract:

Modern agriculture urgently demands for green biological pesticides. Polyoxins are a class of nucleoside antibiotics with a broad spectrum of biological activities. Polyoxins show remarkable potency towards diverse crop diseases due to their competitive inhibition of the chitin synthetase's activity during the building of fungal cell wall and insect crust. This study aimed to construct a high polyoxin B-producing strain, which is one of the most bioactive ingredients in polyoxin derivatives. First, a high polyoxin B-producing mutant strain Pol-12, showing a 1.2-fold higher yield of polyoxin B than the wild-type strain, was obtained from the random mutants generated by ultraviolet mutagenesis of the starting strain Streptomyces ansochromogenes that was isolated from soil and stored by this laboratory. Second, the polyoxin biosynthetic gene cluster pol was directly cloned into p15A vector by ExoCET direct cloning method; and the original promoter and the kasOp* strong promoter were respectively added upstream of the first gene of pol. Third, the resulting shuttle vectors were used to transform the Pol-12 strain by interspecies conjugation and the gene cluster pol was integrated into the chromosome by integrase phiC31, leading to the pol-duplicated strains S. ansochromogenes Pol-12::P_ori-pol (M1) and S. ansochromogenes Pol-12::P_kasOp*-pol (M2). Compared with Pol-12, the yield of polyoxin B was increased by 22 and 33 times in M1 and M2, respectively. These results indicate that UV mutagenesis together with genetic engineering breeding can be applied in construction of high polyoxin-producing strains. Increase of the copy number of biosynthetic gene cluster and strong promoter insertion are effective for titer-improvement of polyoxin B.

Key words: polyoxins, Streptomyces, UV mutagenesis, ExoCET direct cloning, genetic engineering breeding

摘要：

多氧霉素是一种抑制几丁质生物合成的广谱抗真菌类抗生素，对多种真菌引起的农作物病害具有显著的防治效果，且对人和动植物无害，是一种绿色安全的生物农药，目前仍然是全球应用最广泛的抗真菌农药之一。多氧霉素的主要作用机制在于竞争性抑制真菌细胞壁合成中几丁质合成酶的活性，因此对农作物真菌病害具有显著的防治效果。现代农业的发展对于绿色生物农药的需求日益增长，本研究的目的是构建多氧霉素关键活性成分——多氧霉素B的高产菌株。从一株自土壤环境中分离得到的金色链霉菌（Streptomyces ansochromogenes）出发，首先通过紫外诱变初步筛选多氧霉素B的高产突变菌株；然后利用ExoCET直接克隆技术对多氧霉素基因簇pol进行克隆，并在基因簇第1个基因上游分别添加原始启动子和kasOp*强启动子，通过整合酶phiC31将基因簇整合到突变株染色体上构建pol倍增菌株，HPLC-MS检测比较多氧霉素B的产量。通过紫外诱变育种和筛选获得了链霉菌突变株Pol-12菌株，其产量较野生型菌株提高了1.2倍。为进一步提高多氧霉素产量，利用ExoCET直接克隆技术将pol克隆至p15A载体，并通过接合转移转化Pol-12菌株获得pol倍增菌株S. ansochromogenes Pol-12：：P_ori-pol（M1）和S. ansochromogenes Pol-12：：P_kasOp*-pol（M2）。与受体菌Pol-12相比，菌株M1和M2多氧霉素B的产量分别提高了22倍和33倍。因此得出结论：紫外随机诱变育种联合基因工程定向育种可有效应用于多氧霉素高产菌株的构建，增加基因簇的拷贝数以及强启动子插入有效提高了多氧霉素B的产量。

关键词: 多氧霉素, 链霉菌, 紫外诱变, ExoCET直接克隆, 基因工程育种