Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein
Abstract
Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. In this work, we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered “SufFeScient” derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.
- Authors:
-
- Marquette Univ., Milwaukee, WI (United States)
- Univ. of Wisconsin, Madison, WI (United States)
- Marquette Univ., Milwaukee, WI (United States); Saint Louis Univ. School of Medicine, MO (United States)
- Univ. of Illinois at Urbana-Champaign, IL (United States)
- Publication Date:
- Research Org.:
- Marquette Univ., Milwaukee, WI (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1593945
- Grant/Contract Number:
- SC0017866; R01-GM115894
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Bacteriology
- Additional Journal Information:
- Journal Volume: 202; Journal Issue: 3; Journal ID: ISSN 0021-9193
- Publisher:
- American Society for Microbiology
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; electron transfer; Fe-S protein overproduction; E. coli B; iron-sulfur biogenesis; Suf pathway; Fe-S cluster biogenesis; Fe-S protein overexpression; Suf
Citation Formats
Corless, Elliot I., Mettert, Erin L., Kiley, Patricia J., Antony, Edwin, and Metcalf, William W. Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein. United States: N. p., 2019.
Web. doi:10.1128/JB.00496-19.
Corless, Elliot I., Mettert, Erin L., Kiley, Patricia J., Antony, Edwin, & Metcalf, William W. Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein. United States. doi:https://doi.org/10.1128/JB.00496-19
Corless, Elliot I., Mettert, Erin L., Kiley, Patricia J., Antony, Edwin, and Metcalf, William W. Mon .
"Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein". United States. doi:https://doi.org/10.1128/JB.00496-19. https://www.osti.gov/servlets/purl/1593945.
@article{osti_1593945,
title = {Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein},
author = {Corless, Elliot I. and Mettert, Erin L. and Kiley, Patricia J. and Antony, Edwin and Metcalf, William W.},
abstractNote = {Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. In this work, we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered “SufFeScient” derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.},
doi = {10.1128/JB.00496-19},
journal = {Journal of Bacteriology},
number = 3,
volume = 202,
place = {United States},
year = {2019},
month = {11}
}
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Works referenced in this record:
Iron/sulfur proteins biogenesis in prokaryotes: Formation, regulation and diversity
journal, March 2013
- Roche, Béatrice; Aussel, Laurent; Ezraty, Benjamin
- Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1827, Issue 3
Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex
journal, January 2013
- Moser, J.; Lange, C.; Krausze, J.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 6
Sequence-Specific Binding to a Subset of IscR-Regulated Promoters Does Not Require IscR Fe–S Cluster Ligation
journal, March 2009
- Nesbit, A. D.; Giel, J. L.; Rose, J. C.
- Journal of Molecular Biology, Vol. 387, Issue 1
Building Fe–S proteins: bacterial strategies
journal, June 2010
- Py, Béatrice; Barras, Frédéric
- Nature Reviews Microbiology, Vol. 8, Issue 6
Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria
journal, June 2015
- Wayne Outten, F.
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1853, Issue 6
Crystal Structure of the Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase Catalytic Complex (ChlN/ChlB) 2
journal, June 2010
- Bröcker, Markus J.; Schomburg, Sebastian; Heinz, Dirk W.
- Journal of Biological Chemistry, Vol. 285, Issue 35
Reconstitution of Light-independent Protochlorophyllide Reductase from Purified Bchl and BchN-BchB Subunits
journal, May 2000
- Fujita, Yuichi; Bauer, Carl E.
- Journal of Biological Chemistry, Vol. 275, Issue 31
Coordinate Regulation of the Suf and Isc Fe-S Cluster Biogenesis Pathways by IscR Is Essential for Viability of Escherichia coli
journal, September 2014
- Mettert, E. L.; Kiley, P. J.
- Journal of Bacteriology, Vol. 196, Issue 24
Iron-sulfur clusters: ever-expanding roles
journal, April 2006
- Fontecave, Marc
- Nature Chemical Biology, Vol. 2, Issue 4
Tracing Ancestors and Relatives of Escherichia coli B, and the Derivation of B Strains REL606 and BL21(DE3)
journal, December 2009
- Daegelen, Patrick; Studier, F. William; Lenski, Richard E.
- Journal of Molecular Biology, Vol. 394, Issue 4
Understanding the Differences between Genome Sequences of Escherichia coli B Strains REL606 and BL21(DE3) and Comparison of the E. coli B and K-12 Genomes
journal, December 2009
- Studier, F. William; Daegelen, Patrick; Lenski, Richard E.
- Journal of Molecular Biology, Vol. 394, Issue 4
Multi-omics Quantification of Species Variation of Escherichia coli Links Molecular Features with Strain Phenotypes
journal, September 2016
- Monk, Jonathan M.; Koza, Anna; Campodonico, Miguel A.
- Cell Systems, Vol. 3, Issue 3
Genomic and transcriptomic landscape of Escherichia coli BL21(DE3)
journal, March 2017
- Kim, Sinyeon; Jeong, Haeyoung; Kim, Eun-Youn
- Nucleic Acids Research, Vol. 45, Issue 9
Iron–sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding
journal, December 2017
- Pérard, J.; Ollagnier de Choudens, Sandrine
- JBIC Journal of Biological Inorganic Chemistry, Vol. 23, Issue 4
Dark-operative protochlorophyllide oxidoreductase generates substrate radicals by an iron-sulphur cluster in bacteriochlorophyll biosynthesis
journal, June 2014
- Nomata, Jiro; Kondo, Toru; Mizoguchi, Tadashi
- Scientific Reports, Vol. 4, Issue 1
Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron-sulfur clusters
journal, December 2015
- Tanaka, Naoyuki; Kanazawa, Miaki; Tonosaki, Keitaro
- Molecular Microbiology, Vol. 99, Issue 5
The Impact of O2 on the Fe–S Cluster Biogenesis Requirements of Escherichia coli FNR
journal, December 2008
- Mettert, Erin L.; Outten, F. Wayne; Wanta, Brendan
- Journal of Molecular Biology, Vol. 384, Issue 4
IspH Protein of Escherichia c oli : Studies on Iron−Sulfur Cluster Implementation and Catalysis
journal, October 2004
- Gräwert, Tobias; Kaiser, Johannes; Zepeck, Ferdinand
- Journal of the American Chemical Society, Vol. 126, Issue 40
Life without Fe-S clusters: Life without Fe-S clusters
journal, December 2015
- Rocha, Agostinho G.; Dancis, Andrew
- Molecular Microbiology, Vol. 99, Issue 5
Interchangeability and Distinct Properties of Bacterial Fe-S Cluster Assembly Systems: Functional Replacement of the isc and suf Operons in Escherichia coli with the nifSU-Like Operon from Helicobacter pylori
journal, August 2004
- Tokumoto, U.
- Journal of Biochemistry, Vol. 136, Issue 2
Metabolic Deficiences Revealed in the Biotechnologically Important Model Bacterium Escherichia coli BL21(DE3)
journal, August 2011
- Pinske, Constanze; Bönn, Markus; Krüger, Sara
- PLoS ONE, Vol. 6, Issue 8, Article No. e22830
The role of Fe–S proteins in sensing and regulation in bacteria
journal, April 2003
- Kiley, Patricia J.; Beinert, Helmut
- Current Opinion in Microbiology, Vol. 6, Issue 2
A Third Bacterial System for the Assembly of Iron-Sulfur Clusters with Homologs in Archaea and Plastids
journal, June 2002
- Takahashi, Yasuhiro; Tokumoto, Umechiyo
- Journal of Biological Chemistry, Vol. 277, Issue 32
Iron-sulfur proteins: ancient structures, still full of surprises
journal, February 2000
- Beinert, H.
- JBIC Journal of Biological Inorganic Chemistry, Vol. 5, Issue 1
Nitrogenase Fe protein-like Fe-S cluster is conserved in L-protein (BchL) of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus
journal, October 2006
- Nomata, Jiro; Kitashima, Masaharu; Inoue, Kazuhito
- FEBS Letters, Vol. 580, Issue 26
Interplay between Oxygen and Fe–S Cluster Biogenesis: Insights from the Suf Pathway
journal, September 2014
- Boyd, Eric S.; Thomas, Khaleh M.; Dai, Yuyuan
- Biochemistry, Vol. 53, Issue 37
Metabolic network reconstruction and phenome analysis of the industrial microbe, Escherichia coli BL21(DE3)
journal, September 2018
- Kim, Hanseol; Kim, Sinyeon; Yoon, Sung Ho
- PLOS ONE, Vol. 13, Issue 9
A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli: suf operon role during iron starvation
journal, April 2004
- Outten, F. Wayne; Djaman, Ouliana; Storz, Gisela
- Molecular Microbiology, Vol. 52, Issue 3
Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)
journal, December 2009
- Jeong, Haeyoung; Barbe, Valérie; Lee, Choong Hoon
- Journal of Molecular Biology, Vol. 394, Issue 4
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
journal, May 2000
- Datsenko, K. A.; Wanner, B. L.
- Proceedings of the National Academy of Sciences, Vol. 97, Issue 12, p. 6640-6645
Effect of iron–sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase
journal, April 2003
- Kriek, Marco; Peters, Liv; Takahashi, Yasahiro
- Protein Expression and Purification, Vol. 28, Issue 2
How Is Fe-S Cluster Formation Regulated?
journal, October 2015
- Mettert, Erin L.; Kiley, Patricia J.
- Annual Review of Microbiology, Vol. 69, Issue 1
Hyperproduction of Recombinant Ferredoxins in Escherichia coli by Coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 Gene Cluster
journal, July 1999
- Nakamura, M.; Saeki, K.; Takahashi, Y.
- Journal of Biochemistry, Vol. 126, Issue 1
Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus
journal, June 2005
- Nomata, Jiro; Swem, Lee R.; Bauer, Carl E.
- Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1708, Issue 2
NB-protein (BchN-BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe-S clusters
journal, March 2008
- Nomata, Jiro; Ogawa, Takuro; Kitashima, Masaharu
- FEBS Letters, Vol. 582, Issue 9
Iron-Sulfur (Fe/S) Protein Biogenesis: Phylogenomic and Genetic Studies of A-Type Carriers
journal, May 2009
- Vinella, Daniel; Brochier-Armanet, Céline; Loiseau, Laurent
- PLoS Genetics, Vol. 5, Issue 5
Assembly of Fe/S proteins in bacterial systems
journal, June 2015
- Blanc, B.; Gerez, C.; Ollagnier de Choudens, S.
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1853, Issue 6
X-ray crystal structure of the light-independent protochlorophyllide reductase
journal, May 2010
- Muraki, Norifumi; Nomata, Jiro; Ebata, Kozue
- Nature, Vol. 465, Issue 7294