期刊
METABOLIC ENGINEERING
卷 52, 期 -, 页码 153-167出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2018.12.001
关键词
Site-specific recombination; Multi-copy integration; Actinomycetes; Natural product; Biosynthetic gene cluster (BGC)
资金
- National Natural Science Foundation of China [31430004, 31630003, 31570072, 31770088]
- National Postdoctoral Program for Innovative Talents [BX201700265]
- China Postdoctoral Science Foundation [2017M621545]
- Science and Technology Commission of Shanghai Municipality [18ZR1446700, 16490712100]
- National Mega-project for Innovative Drugs [2018ZX09711001-006-012]
- Derivative Bank of Chinese Biological Resources, CAS [ZSYS-016]
Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of multiple integrases-multiple attB sites. This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据