期刊
ACS SYNTHETIC BIOLOGY
卷 7, 期 5, 页码 1291-1302出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.7b00400
关键词
extracellular expression; cell growth; membrane permeability; lipoprotein family; genome engineering; apparent molecular size
资金
- National Natural Science Foundation of China [81430082, 81703402]
- National Science and Technology Major Project [2018ZX09201001-003-002]
- China Postdoctoral Science Foundation [2017M611957]
- Fundamental Research Funds for the Central Universities [2015XPT02]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- Ministry of Education of China
- State Administration of Foreign Expert Affairs of China [111-2-07]
Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.
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