Genomic Iterative Replacements of Large Synthetic DNA Fragments in Corynebacterium glutamicum

谷氨酸棒杆菌 合成生物学 基因组 生物 代谢工程 遗传学 全基因组测序 比较基因组学 基因组学 质粒 计算生物学 细菌人工染色体 DNA 基因组工程 基因 基因组编辑
作者
Yanrui Ye,Minmin Zhong,Zhanhua Zhang,Tai Chen,Yue Shen,Zhanglin Lin,Yun Wang
出处
期刊:ACS Synthetic Biology [American Chemical Society]
卷期号:11 (4): 1588-1599 被引量:7
标识
DOI:10.1021/acssynbio.1c00644
摘要

Synthetic genomics will advance our understanding of life and allow us to rebuild the genomes of industrial microorganisms for enhancing performances. Corynebacterium glutamicum, a Gram-positive bacterium, is an important industrial workhorse. However, its genome synthesis is impeded by the low efficiencies in DNA delivery and in genomic recombination/replacement. In the present study, we describe a genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants are selected by the alternative use of kanR and speR selectable markers. As a proof of concept, we systematically redesigned and replaced a 54.3 kb wild-type sequence of C. glutamicumATCC13032 with its 55.1 kb synthetic counterpart with several novel features, including decoupled genes, the standard PCRTags, and 20 loxPsym sites, which was for the first time incorporated into a bacterial genome. The resulting strain semi-synCG-A1 had a phenotype and fitness similar to the wild-type strain under various stress conditions. The stability of the synthetic genome region faithfully maintained over 100 generations of nonselective growth. Genomic deletions, inversions, and translocations occurred in the synthetic genome region upon induction of synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE), revealing potential genetic flexibility for C. glutamicum. This strategy can be used for the synthesis of a larger region of the genome and facilitate the endeavors for metabolic engineering and synthetic biology of C. glutamicum.
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