Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster

LIU Ruxin; DU Lei; XU Xiaoqing; DING Jinpeng; ZHANG Wei; LI Shengying

doi:10.12211/2096-8280.2020-032

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Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster

Invited Review | 更新时间：2025-03-20

- Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster
- Synthetic Biology Journal Vol. 1, Issue 5, Pages: 609-620(2020)
- 作者机构：
  
  1.山东大学微生物技术研究院，山东青岛 266237
  2.山东省合成生物学重点实验室，中国科学院青岛生物能源与过程研究所，山东青岛 266101
  3.乳山韩威生物科技有限公司，山东乳山 264502
- 作者简介：
  
  lishengying@sdu.edu.cn
- 基金信息：
- DOI：10.12211/2096-8280.2020-032
  CLC： Q 939.9
- Received：22 March 2020，
  
  Revised：2020-04-08，
  
  Published：31 October 2020
- 稿件说明：
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刘如欣, 杜磊, 徐晓庆, 丁金鹏, 张伟, 李盛英. 基于紫外诱变与生物合成基因簇倍增的多氧霉素高产菌株构建[J]. 合成生物学, 2020, 1(5): 609-620

LIU Ruxin, DU Lei, XU Xiaoqing, DING Jinpeng, ZHANG Wei, LI Shengying. Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster[J]. Synthetic Biology Journal, 2020, 1(5): 609-620
刘如欣, 杜磊, 徐晓庆, 丁金鹏, 张伟, 李盛英. 基于紫外诱变与生物合成基因簇倍增的多氧霉素高产菌株构建[J]. 合成生物学, 2020, 1(5): 609-620 DOI： 10.12211/2096-8280.2020-032.

LIU Ruxin, DU Lei, XU Xiaoqing, DING Jinpeng, ZHANG Wei, LI Shengying. Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster[J]. Synthetic Biology Journal, 2020, 1(5): 609-620 DOI： 10.12211/2096-8280.2020-032.

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

phi

C31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。

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 t

he 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

phi

C31

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.

关键词

Keywords

references

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通讯作者：作者简介:李盛英（1978—），男，博士，教授。研究方向为合成生物学，微生物学及生物化学。

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