Improving membrane protein expression and function using genomic edits

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; Herbert, Robin A.; Mukhopadhyay, Aindrila

doi:10.1038/s41598-017-12901-7

Title: Improving membrane protein expression and function using genomic edits

Journal Article · Thu Oct 12 00:00:00 EDT 2017 · Scientific Reports

DOI:https://doi.org/10.1038/s41598-017-12901-7· OSTI ID:1419447

Jensen, Heather M. ^[1]; Eng, Thomas ^[1]; Chubukov, Victor ^[1]; Herbert, Robin A. ^[1]; Mukhopadhyay, Aindrila ^[1]

Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression of five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1419447

Journal Information:: Scientific Reports, Vol. 7, Issue 1; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 19 works

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References (45)

Experimentally based topology models for E. coli inner membrane proteins Rapp, Mikaela; Drew, David; Daley, Daniel O. Protein Science, Vol. 13, Issue 4 https://doi.org/10.1110/ps.03553804	journal	April 2004
Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli Goldbeck, Cheryl P.; Jensen, Heather M.; TerAvest, Michaela A. ACS Synthetic Biology, Vol. 2, Issue 3 https://doi.org/10.1021/sb300119v	journal	August 2012
Enhancing Tolerance to Short-Chain Alcohols by Engineering the Escherichia coli AcrB Efflux Pump to Secrete the Non-native Substrate n-Butanol Fisher, Michael A.; Boyarskiy, Sergey; Yamada, Masaki R. ACS Synthetic Biology, Vol. 3, Issue 1, p. 30-40 https://doi.org/10.1021/sb400065q	journal	September 2013
Reprogramming chaperone pathways to improve membrane protein expression in Escherichia coli Nannenga, Brent L.; Baneyx, François Protein Science, Vol. 20, Issue 8 https://doi.org/10.1002/pro.669	journal	July 2011
Expanded microbial genome coverage and improved protein family annotation in the COG database Galperin, Michael Y.; Makarova, Kira S.; Wolf, Yuri I. Nucleic Acids Research, Vol. 43, Issue D1 https://doi.org/10.1093/nar/gku1223	journal	November 2014
Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria Anderson, J. Christopher; Clarke, Elizabeth J.; Arkin, Adam P. Journal of Molecular Biology, Vol. 355, Issue 4 https://doi.org/10.1016/j.jmb.2005.10.076	journal	January 2006
Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep19512	journal	January 2016
Engineering microbial biofuel tolerance and export using efflux pumps Dunlop, Mary J.; Dossani, Zain Y.; Szmidt, Heather L. Molecular Systems Biology, Vol. 7, Issue 1 https://doi.org/10.1038/msb.2011.21	journal	January 2011
Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome Papanastasiou, Malvina; Orfanoudaki, Georgia; Kountourakis, Nikos PROTEOMICS, Vol. 16, Issue 1 https://doi.org/10.1002/pmic.201500304	journal	November 2015
Tuning Escherichia coli for membrane protein overexpression Wagner, S.; Klepsch, M. M.; Schlegel, S. Proceedings of the National Academy of Sciences, Vol. 105, Issue 38 https://doi.org/10.1073/pnas.0804090105	journal	September 2008
Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels Miroux, Bruno; Walker, John E. Journal of Molecular Biology, Vol. 260, Issue 3 https://doi.org/10.1006/jmbi.1996.0399	journal	July 1996
Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues vonHeijne, Gunnar Nature, Vol. 341, Issue 6241 https://doi.org/10.1038/341456a0	journal	October 1989
Global Topology Analysis of the Escherichia coli Inner Membrane Proteome Daley, D. O. Science, Vol. 308, Issue 5726 https://doi.org/10.1126/science.1109730	journal	May 2005
Structural basis of Sec-independent membrane protein insertion by YidC Kumazaki, Kaoru; Chiba, Shinobu; Takemoto, Mizuki Nature, Vol. 509, Issue 7501 https://doi.org/10.1038/nature13167	journal	April 2014
Overlapping functions of components of a bacterial Sec-independent protein export pathway Sargent, F. The EMBO Journal, Vol. 17, Issue 13 https://doi.org/10.1093/emboj/17.13.3640	journal	July 1998
Truncation Analysis of TatA and TatB Defines the Minimal Functional Units Required for Protein Translocation Lee, P. A.; Buchanan, G.; Stanley, N. R. Journal of Bacteriology, Vol. 184, Issue 21 https://doi.org/10.1128/JB.184.21.5871-5879.2002	journal	November 2002
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki Molecular Systems Biology, Vol. 2, Article No. 2006.0008 https://doi.org/10.1038/msb4100050	journal	February 2006
Circos: An information aesthetic for comparative genomics Krzywinski, M.; Schein, J.; Birol, I. Genome Research, Vol. 19, Issue 9 https://doi.org/10.1101/gr.092759.109	journal	June 2009
j5 DNA Assembly Design Automation Software Hillson, Nathan J.; Rosengarten, Rafael D.; Keasling, Jay D. ACS Synthetic Biology, Vol. 1, Issue 1, p. 14-21 https://doi.org/10.1021/sb2000116	journal	December 2011
CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli Jensen, Heather M.; TerAvest, Michaela A.; Kokish, Mark G. ACS Synthetic Biology, Vol. 5, Issue 7 https://doi.org/10.1021/acssynbio.5b00279	journal	April 2016
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 Skretas, Georgios; Georgiou, George Biotechnology and Bioengineering, Vol. 102, Issue 2 https://doi.org/10.1002/bit.22097	journal	February 2009
The Mtr Pathway of Shewanella oneidensis MR-1 Couples Substrate Utilization to Current Production in Escherichia coli TerAvest, Michaela A.; Zajdel, Tom J.; Ajo-Franklin, Caroline M. ChemElectroChem, Vol. 1, Issue 11 https://doi.org/10.1002/celc.201402194	journal	August 2014
Predicting transmembrane protein topology with a hidden markov model: application to complete genomes11Edited by F. Cohen Krogh, Anders; Larsson, Björn; von Heijne, Gunnar Journal of Molecular Biology, Vol. 305, Issue 3 https://doi.org/10.1006/jmbi.2000.4315	journal	January 2001
Spatiotemporal control of cell signalling using a light-switchable protein interaction Levskaya, Anselm; Weiner, Orion D.; Lim, Wendell A. Nature, Vol. 461, Issue 7266 https://doi.org/10.1038/nature08446	journal	September 2009
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in <em>Escherichia coli</em> Bird, Louise E.; Rada, Heather; Verma, Anil Journal of Visualized Experiments, Issue 95 https://doi.org/10.3791/52357	journal	January 2015
Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction Kwon, Soon-Kyeong; Kim, Seong Keun; Lee, Dae-Hee Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep16076	journal	November 2015
BglBricks: A flexible standard for biological part assembly Anderson, J. Christopher; Dueber, John E.; Leguia, Mariana Journal of Biological Engineering, Vol. 4, Issue 1 https://doi.org/10.1186/1754-1611-4-1	journal	January 2010
Tolerance engineering in bacteria for the production of advanced biofuels and chemicals Mukhopadhyay, Aindrila Trends in Microbiology, Vol. 23, Issue 8 https://doi.org/10.1016/j.tim.2015.04.008	journal	August 2015
arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon. Wall, D.; Delaney, J. M.; Fayet, O. Journal of Bacteriology, Vol. 174, Issue 20 https://doi.org/10.1128/jb.174.20.6554-6562.1992	journal	October 1992
Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol Boyarskiy, Sergey; Davis López, Stephanie; Kong, Niwen Metabolic Engineering, Vol. 33 https://doi.org/10.1016/j.ymben.2015.11.005	journal	January 2016
De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields Schlegel, Susan; Genevaux, Pierre; de Gier, Jan-Willem Cell Reports, Vol. 10, Issue 10 https://doi.org/10.1016/j.celrep.2015.02.029	journal	March 2015
Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J. mBio, Vol. 6, Issue 3, Article No. e00306-15 https://doi.org/10.1128/mBio.00306-15	journal	May 2015
Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli Bäcklund, Emma; Ignatushchenko, Marina; Larsson, Gen Microbial Cell Factories, Vol. 10, Issue 1 https://doi.org/10.1186/1475-2859-10-35	journal	January 2011
The Sec translocase du Plessis, David J. F.; Nouwen, Nico; Driessen, Arnold J. M. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1808, Issue 3 https://doi.org/10.1016/j.bbamem.2010.08.016	journal	March 2011
Consequences of Membrane Protein Overexpression in Escherichia coli Wagner, Samuel; Baars, Louise; Ytterberg, A. Jimmy Molecular & Cellular Proteomics, Vol. 6, Issue 9 https://doi.org/10.1074/mcp.M600431-MCP200	journal	April 2007
Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3) Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/srep45089	journal	March 2017
Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production Gialama, Dimitra; Kostelidou, Kalliopi; Michou, Myrsini ACS Synthetic Biology, Vol. 6, Issue 2 https://doi.org/10.1021/acssynbio.6b00174	journal	November 2016
Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli Skretas, G.; Georgiou, G. Applied and Environmental Microbiology, Vol. 76, Issue 17 https://doi.org/10.1128/AEM.00963-10	journal	July 2010
Genetic selection system for improving recombinant membrane protein expression in E. coli Massey-Gendel, Elizabeth; Zhao, Anni; Boulting, Gabriella Protein Science, Vol. 18, Issue 2 https://doi.org/10.1002/pro.39	journal	February 2009
Improving Microbial Biogasoline Production in Escherichia coli Using Tolerance Engineering Foo, Jee Loon; Jensen, Heather M.; Dahl, Robert H. mBio, Vol. 5, Issue 6 https://doi.org/10.1128/mBio.01932-14	journal	November 2014
Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors Unden, G.; Bongaerts, J. Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1320, Issue 3 https://doi.org/10.1016/s0005-2728(97)00034-0	journal	July 1997
Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle de Gier, Jan-Willem L.; Mansournia, Parvaneh; Valent, Quido A. FEBS Letters, Vol. 399, Issue 3 https://doi.org/10.1016/s0014-5793(96)01354-3	journal	December 1996
Physiological Response to Membrane Protein Overexpression in E. coli Gubellini, Francesca; Verdon, Grégory; Karpowich, Nathan K. Molecular & Cellular Proteomics, Vol. 10, Issue 10 https://doi.org/10.1074/mcp.m111.007930	journal	June 2011
Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis Zweers, J. C.; Wiegert, T.; van Dijl, J. M. Applied and Environmental Microbiology, Vol. 75, Issue 23 https://doi.org/10.1128/aem.01560-09	journal	October 2009
Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3) Thomas, Baumgarten,; Susan, Schlegel,; Samuel, Wagner, ETH Zurich https://doi.org/10.3929/ethz-b-000130066	text	January 2017

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