Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments  

作　　者：Jiming Zhuo Binbin Ma Jingjing Xu Weihong Hu Jihui Zhang Huarong Tan Yuqing Tian 

机构地区：[1]State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China [2]University of Chinese Academy of Sciences, Beijing 100049, China [3]School of Life Sciences, Yunnan University, Kunming 650091, China

出　　处：《Science China(Life Sciences)》2017年第9期968-979,共12页中国科学（生命科学英文版）

基　　金：supported by grants from the Ministry of Science and Technology of China(2013CB734001 and 2015CB150600);the National Natural Science Foundation of China(31370097 and 31571281)

摘　　要：Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.Genetic modification of large DNA fragments（gene clusters） is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction（PCR） targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties（HPHT） in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid（CPOAA）from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.

关 键 词：large DNA fragment PCR targeting Gibson assembly gene cluster hybrid antibiotic 

分 类 号：Q78[生物学—分子生物学]