Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. To accomplish successful overexpression of membrane proteins in a workhorse organism such as E. coli, defined and rational optimization strategies based on an understanding of the genetic background of the toxicity-escape mechanism are desirable. To this end, we sequenced the genomes of E. coli C41(DE3) and its derivative C43(DE3), which were developed for membrane protein production. Comparative analysis of their genomes with those of their ancestral strain E. coli BL21(DE3) revealed various genetic changes in both strains. A series of E. coli variants that are able to tolerate transformation with or overexpression of membrane proteins were generated by in vitro evolution. Targeted sequencing of the evolved strains revealed the mutational hotspots among the acquired genetic changes. By these combinatorial approaches, we found non-synonymous changes in the lac repressor gene of the lac operon as well as nucleotide substitutions in the lacUV5 promoter of the DE3 region, by which the toxic effect to the host caused by overexpression of membrane proteins could be relieved. A mutation in lacI was demonstrated to be crucial for conferring tolerance to membrane protein overexpression.
|Publication status||Published - 2015|
Bibliographical noteFunding Information:
We thank John E. Walker for providing the pMW7(OGCP) and pMW7(Ecb) plasmids and the E. coli C41(DE3) and C43(DE3) strains, Sun Chang Kim for pREDI, Da Eun Jeong for helping with the flow cytometry experiments, and Eui-Jeon Woo for structural visualization of the LacI protein. This work was supported by the Global Frontier Intelligent Synthetic Biology Center (NRF-2012M3A6A8053632), the National Research Foundation of Korea (NRF-2011-0017670), and the 21C Frontier Microbial Genomics and Applications Center.
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