Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering Chaoyong Huang 1,2
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Liwei Guo 1
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Jingge Wang 1
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Ning Wang 1
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Yi-Xin Huo 1,2
Received: 26 March 2020 / Revised: 20 July 2020 / Accepted: 5 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering. Key points • This article reports an efficient genome engineering tool for E. coli. • The tool is advantageous for the manipulations of long DNA fragments. • The tool has been successfully applied for genome simplification. • The tool has been successfully applied for metabolic engineering. Keywords Genome engineering . Long fragment editing . Genetic stability . Genome simplification . Metabolic engineering
Introduction As a class of versatile living systems, bacteria are useful in many fields of synthetic biology. In bacteria, genetic information contained on the single-copy genome determines the
Chaoyong Huang and Liwei Guo contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10819-1) contains supplementary material, which is available to authorized users. * Yi-Xin Huo [email protected] 1
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
2
SIP-UCLA Institute for Technology Advancement, Suzhou 215123, China
characteristics of a specific strain. To understand bacterial characteristics and utilize them to explore the world and serve human life, researchers frequently conduct genome engineering to reprogram the genetic information of bacteria. Through DNA editing, researchers can add desired exogenous genetic information to
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