Abstract
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.
Highlights
Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology
Wagner and colleagues observed that the Walker strains suffered less from Sec translocon saturation, which causes the aggregation of endogenous proteins in the cytoplasm and results in perturbation of the membrane proteome[19]. They reported that three nucleotide changes in the –10 and + 1 regions of the lacUV5 promoter in the Walker strains are responsible for slowing transcription by T7 RNA polymerase (RNAP), improving membrane protein overexpression by reducing host toxicity[11]
To search for genetic factors that might be involved in the expanded capacity of strains C41(DE3) and C43(DE3) for the overexpression of membrane proteins, we sequenced the genomes of these two BL21(DE3) derivatives and examined genetic changes such as single nucleotide polymorphisms (SNPs), deletions, insertions or other polymorphisms (DIPs)
Summary
Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. Wagner and colleagues observed that the Walker strains suffered less from Sec translocon saturation, which causes the aggregation of endogenous proteins in the cytoplasm and results in perturbation of the membrane proteome[19] They reported that three nucleotide changes in the –10 and + 1 regions of the lacUV5 promoter in the Walker strains are responsible for slowing transcription by T7 RNAP, improving membrane protein overexpression by reducing host toxicity[11]. Based on these findings, they developed the Lemo(DE3) system, which allows T7 RNAP activity to be tuned using the T7 lysozyme, its natural inhibitor[11,20]. Alfasi and colleagues demonstrated that mutations at the regulatory region of the T7 RNAP gene accumulate in BL21(DE3) during production of recombinant proteins[21]
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