Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction

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.

[1]  S H White,et al.  Hydrophobic interactions of peptides with membrane interfaces. , 1998, Biochimica et biophysica acta.

[2]  T. Stevens,et al.  Do more complex organisms have a greater proportion of membrane proteins in their genomes? , 2000, Proteins.

[3]  H. Mori,et al.  Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[4]  K. V. van Wijk,et al.  Consequences of Membrane Protein Overexpression in Escherichia coli*S , 2007, Molecular & Cellular Proteomics.

[5]  S. Emr,et al.  D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport , 1984, Journal of bacteriology.

[6]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[7]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Richard E. Lenski,et al.  Mechanisms Causing Rapid and Parallel Losses of Ribose Catabolism in Evolving Populations of Escherichia coli B , 2001, Journal of bacteriology.

[9]  Haeyoung Jeong,et al.  An Optimized Strategy for Genome Assembly of Sanger/pyrosequencing Hybrid Data using Available Software , 2008 .

[10]  J. Gowrishankar,et al.  IS186 Insertion at a Hot Spot in thelon Promoter as a Basis for Lon Protease Deficiency ofEscherichia coli B: Identification of a Consensus Target Sequence for IS186 Transposition , 2001, Journal of bacteriology.

[11]  L. Vuillard,et al.  The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). , 2004, Protein expression and purification.

[12]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[13]  R. H. Fillingame,et al.  Structure of the Membrane Domain of Subunit b of the Escherichia coli F0F1 ATP Synthase* , 1999, The Journal of Biological Chemistry.

[14]  F. Neidhardt,et al.  lon gene product of Escherichia coli is a heat-shock protein , 1984, Journal of bacteriology.

[15]  C Cruz,et al.  Genetic studies of the lac repressor. XIV. Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as "spacers" which do not require a specific sequence. , 1994, Journal of molecular biology.

[16]  A. Flower,et al.  Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase , 2005, Microbial cell factories.

[17]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[18]  G. Chang,et al.  Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer , 1996, Science.

[19]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[20]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[21]  M. Lewis,et al.  Structural analysis of lac repressor bound to allosteric effectors. , 2007, Journal of molecular biology.

[22]  R. Hall,et al.  Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins. , 2011, Journal of biotechnology.

[23]  K. Lundstrom,et al.  Structural genomics and drug discovery , 2007, Journal of cellular and molecular medicine.

[24]  K. Matthews,et al.  Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins. , 1991, The Journal of biological chemistry.

[25]  G. Larsson,et al.  Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli , 2011, Microbial cell factories.

[26]  B. Miroux,et al.  A general approach for heterologous membrane protein expression in Escherichia coli: the uncoupling protein, UCP1, as an example. , 2003, Methods in molecular biology.

[27]  K. Jung,et al.  First Insights into the Unexplored Two-Component System YehU/YehT in Escherichia coli , 2012, Journal of bacteriology.

[28]  D. Drew,et al.  Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). , 2012, Journal of molecular biology.

[29]  K. Altendorf,et al.  Structure and function of the F(o) complex of the ATP synthase from Escherichia coli. , 2000, The Journal of experimental biology.

[30]  Jeffrey E. Barrick,et al.  Genome evolution and adaptation in a long-term experiment with Escherichia coli , 2009, Nature.

[31]  Kui Hyeon Kang,et al.  Rapid and efficient construction of markerless deletions in the Escherichia coli genome , 2008, Nucleic acids research.

[32]  A. Robinson.  Production of membrane proteins : strategies for expression and isolation , 2011 .

[33]  J. Walker,et al.  Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. , 1996, Journal of molecular biology.

[34]  H. Bernstein,et al.  Physiological Basis for Conservation of the Signal Recognition Particle Targeting Pathway in Escherichia coli , 2001, Journal of bacteriology.

[35]  S W Lin,et al.  Vectors for selective expression of cloned DNAs by T7 RNA polymerase. , 1987, Gene.

[36]  P. Green,et al.  Consed: a graphical tool for sequence finishing. , 1998, Genome research.

[37]  Improving protein folding efficiency by directed evolution using the GFP folding reporter. , 2003, Methods in molecular biology.

[38]  G. Georgiou,et al.  Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli , 2010, Applied and Environmental Microbiology.

[39]  Samuel Wagner,et al.  Tuning Escherichia coli for membrane protein overexpression , 2008, Proceedings of the National Academy of Sciences.

[40]  B. Nannenga,et al.  Reprogramming chaperone pathways to improve membrane protein expression in Escherichia coli , 2011, Protein science : a publication of the Protein Society.

[41]  P. Genevaux,et al.  De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields. , 2015, Cell reports.

[42]  J. Gowrishankar,et al.  IS 186 Insertion at a Hot Spot in the lon Promoter as a Basis for Lon Protease Deficiency of Escherichia coli B: Identification of a Consensus Target Sequence for IS 186 Transposition , 2001 .

[43]  G. Georgiou,et al.  Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor. , 2008, Biotechnology and bioengineering.

[44]  Inge Jonassen,et al.  Systematic exploration of error sources in pyrosequencing flowgram data , 2011, Bioinform..

[45]  R. Lenski,et al.  Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). , 2009, Journal of molecular biology.

[46]  J. Bowie,et al.  Genetic selection system for improving recombinant membrane protein expression in E. coli , 2009, Protein science : a publication of the Protein Society.

[47]  D. Drew,et al.  Optimization of membrane protein overexpression and purification using GFP fusions , 2006, Nature Methods.