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ACS Synth Biol. 2017 May 19;6(5):875-883. doi: 10.1021/acssynbio.6b00366. Epub 2017 Feb 16.

An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in Escherichia coli.

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Department of Microbiology, Cornell University , Ithaca, New York 14853, United States.
School of Chemical and Biomolecular Engineering, Cornell University , Ithaca, New York 14853, United States.


The extracellular expression of recombinant proteins using laboratory strains of Escherichia coli is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (i.e., 1010-12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of E. coli single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanisms-enabling the exploration of protein secretion in pathogenesis as well as the creation of designer E. coli strains with greatly expanded secretomes-all without the need for expensive exogenous reagents, assay instruments, or robotic automation.


bacterial expression; genetic selection; microbial cell factory; protein translocation; recombinant protein; secretion pathway engineering

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