Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate

Kirst, Henning; Ferlez, Bryan H.; Lindner, Steffen N.; Cotton, Charles R.; Bar-Even, Arren; Kerfeld, Cheryl A.

doi:10.1073/pnas.2116871119

Title: Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate

Journal Article · 22 February 2022 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.2116871119 · OSTI ID:1902049

^[1]; Ferlez, Bryan H. ^[2];

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Michigan State Univ., East Lansing, MI (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Michigan State Univ., East Lansing, MI (United States)
Max Planck Inst. of Molecular Plant Physiology, Potsdam-Golm (Germany)

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don’t compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a “wiffleball” architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.

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Research Organization:: Michigan State Univ., East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Basic Energy Sciences (BES). Materials Sciences & Engineering Division; National Science Foundation (NSF)

Grant/Contract Number:: FG02-91ER20021; AC02-05CH11231; MCB 1733552

OSTI ID:: 1902049

Alternate ID(s):: OSTI ID: 1896683

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 119, Issue 8; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

References (53)

Biofuels: Environment, technology and food security Escobar, José C.; Lora, Electo S.; Venturini, Osvaldo J. Renewable and Sustainable Energy Reviews, Vol. 13, Issue 6-7 https://doi.org/10.1016/j.rser.2008.08.014	journal	August 2009
Production of first and second generation biofuels: A comprehensive review Naik, S. N.; Goud, Vaibhav V.; Rout, Prasant K. Renewable and Sustainable Energy Reviews, Vol. 14, Issue 2 https://doi.org/10.1016/j.rser.2009.10.003	journal	February 2010
The formate bio-economy Yishai, Oren; Lindner, Steffen N.; Gonzalez de la Cruz, Jorge Current Opinion in Chemical Biology, Vol. 35 https://doi.org/10.1016/j.cbpa.2016.07.005	journal	December 2016
Photocatalytic CO2 reduction with high turnover frequency and selectivity of formic acid formation using Ru(II) multinuclear complexes Tamaki, Y.; Morimoto, T.; Koike, K. Proceedings of the National Academy of Sciences, Vol. 109, Issue 39 https://doi.org/10.1073/pnas.1118336109	journal	August 2012
Formate Assimilation: The Metabolic Architecture of Natural and Synthetic Pathways Bar-Even, Arren Biochemistry, Vol. 55, Issue 28 https://doi.org/10.1021/acs.biochem.6b00495	journal	July 2016
Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes Bar-Even, Arren; Noor, Elad; Flamholz, Avi Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1827, Issue 8-9 https://doi.org/10.1016/j.bbabio.2012.10.013	journal	August 2013
Engineered Assimilation of Exogenous and Endogenous Formate in Escherichia coli Yishai, Oren; Goldbach, Leander; Tenenboim, Hezi ACS Synthetic Biology, Vol. 6, Issue 9 https://doi.org/10.1021/acssynbio.7b00086	journal	June 2017
Pyruvate Formate-Lyase Enables Efficient Growth of Escherichia coli on Acetate and Formate Zelcbuch, Lior; Lindner, Steffen N.; Zegman, Yonatan Biochemistry, Vol. 55, Issue 17 https://doi.org/10.1021/acs.biochem.6b00184	journal	April 2016
Eukaryotic Pyruvate Formate Lyase and Its Activating Enzyme Were Acquired Laterally from a Firmicute Stairs, C. W.; Roger, A. J.; Hampl, V. Molecular Biology and Evolution, Vol. 28, Issue 7 https://doi.org/10.1093/molbev/msr032	journal	February 2011
Bacterial microcompartments: catalysis-enhancing metabolic modules for next generation metabolic and biomedical engineering Kirst, Henning; Kerfeld, Cheryl A. BMC Biology, Vol. 17, Issue 1 https://doi.org/10.1186/s12915-019-0691-z	journal	October 2019
Bacterial Microcompartments Kerfeld, Cheryl A.; Heinhorst, Sabine; Cannon, Gordon C. Annual Review of Microbiology, Vol. 64, Issue 1 https://doi.org/10.1146/annurev.micro.112408.134211	journal	October 2010
A catalog of the diversity and ubiquity of bacterial microcompartments Sutter, Markus; Melnicki, Matthew R.; Schulz, Frederik Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-24126-4	journal	June 2021
Clues to the function of bacterial microcompartments from ancillary genes Kirst, Henning; Kerfeld, Cheryl A. Biochemical Society Transactions, Vol. 49, Issue 3 https://doi.org/10.1042/BST20200632	journal	June 2021
A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method Axen, Seth D.; Erbilgin, Onur; Kerfeld, Cheryl A. PLoS Computational Biology, Vol. 10, Issue 10 https://doi.org/10.1371/journal.pcbi.1003898	journal	October 2014
Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments Zarzycki, Jan; Erbilgin, Onur; Kerfeld, Cheryl A. Applied and Environmental Microbiology, Vol. 81, Issue 24 https://doi.org/10.1128/AEM.02587-15	journal	September 2015
Glycyl Radical Enzyme-Associated Microcompartments: Redox-Replete Bacterial Organelles Ferlez, Bryan; Sutter, Markus; Kerfeld, Cheryl A. mBio, Vol. 10, Issue 1 https://doi.org/10.1128/mBio.02327-18	journal	January 2019
In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/srep42757	journal	February 2017
Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme Craciun, S.; Balskus, E. P. Proceedings of the National Academy of Sciences, Vol. 109, Issue 52 https://doi.org/10.1073/pnas.1215689109	journal	November 2012
Engineered bacterial microcompartments: apps for programming metabolism Kerfeld, Cheryl A.; Sutter, Markus Current Opinion in Biotechnology, Vol. 65 https://doi.org/10.1016/j.copbio.2020.05.001	journal	October 2020
Protein Structures Forming the Shell of Primitive Bacterial Organelles Kerfeld, C. A. Science, Vol. 309, Issue 5736 https://doi.org/10.1126/science.1113397	journal	August 2005
Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport Klein, Michael G.; Zwart, Peter; Bagby, Sarah C. Journal of Molecular Biology, Vol. 392, Issue 2 https://doi.org/10.1016/j.jmb.2009.03.056	journal	September 2009
Atomic-Level Models of the Bacterial Carboxysome Shell Tanaka, S.; Kerfeld, C. A.; Sawaya, M. R. Science, Vol. 319, Issue 5866 https://doi.org/10.1126/science.1151458	journal	February 2008
Assembly principles and structure of a 6.5-MDa bacterial microcompartment shell Sutter, Markus; Greber, Basil; Aussignargues, Clement Science, Vol. 356, Issue 6344 https://doi.org/10.1126/science.aan3289	journal	June 2017
Programmed loading and rapid purification of engineered bacterial microcompartment shells Hagen, Andrew; Sutter, Markus; Sloan, Nancy Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05162-z	journal	July 2018
Structural Characterization of a Synthetic Tandem-Domain Bacterial Microcompartment Shell Protein Capable of Forming Icosahedral Shell Assemblies Sutter, Markus; McGuire, Sean; Ferlez, Bryan ACS Synthetic Biology, Vol. 8, Issue 4 https://doi.org/10.1021/acssynbio.9b00011	journal	March 2019
Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function Lassila, Jonathan K.; Bernstein, Susan L.; Kinney, James N. Journal of Molecular Biology, Vol. 426, Issue 11 https://doi.org/10.1016/j.jmb.2014.02.025	journal	May 2014
De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments Lee, Matthew J.; Mantell, Judith; Brown, Ian R. Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05922-x	journal	August 2018
A designed bacterial microcompartment shell with tunable composition and precision cargo loading Ferlez, Bryan; Sutter, Markus; Kerfeld, Cheryl A. Metabolic Engineering, Vol. 54 https://doi.org/10.1016/j.ymben.2019.04.011	journal	July 2019
In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures Hagen, Andrew R.; Plegaria, Jefferson S.; Sloan, Nancy Nano Letters, Vol. 18, Issue 11 https://doi.org/10.1021/acs.nanolett.8b02991	journal	October 2018
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin Zakeri, B.; Fierer, J. O.; Celik, E. Proceedings of the National Academy of Sciences, Vol. 109, Issue 12 https://doi.org/10.1073/pnas.1115485109	journal	February 2012
Programmable polyproteams built using twin peptide superglues Veggiani, Gianluca; Nakamura, Tomohiko; Brenner, Michael D. Proceedings of the National Academy of Sciences, Vol. 113, Issue 5 https://doi.org/10.1073/pnas.1519214113	journal	January 2016
Synthetic scaffolds for pathway enhancement Siu, Ka-Hei; Chen, Rebecca P.; Sun, Qing Current Opinion in Biotechnology, Vol. 36 https://doi.org/10.1016/j.copbio.2015.08.009	journal	December 2015
Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation: Escherichia coli phosphotransacetylase Campos-Bermudez, Valeria Alina; Bologna, Federico Pablo; Andreo, Carlos Santiago FEBS Journal, Vol. 277, Issue 8 https://doi.org/10.1111/j.1742-4658.2010.07617.x	journal	March 2010
Characterization of Escherichia coli EutD: a phosphotransacetylase of the ethanolamine operon Bologna, Federico P.; Campos-Bermudez, Valeria A.; Saavedra, Damián D. The Journal of Microbiology, Vol. 48, Issue 5, p. 629-636 https://doi.org/10.1007/s12275-010-0091-0	journal	October 2010
Streamlined Construction of the Cyanobacterial CO ₂ -Fixing Organelle via Protein Domain Fusions for Use in Plant Synthetic Biology Gonzalez-Esquer, C. Raul; Shubitowski, Tyler B.; Kerfeld, Cheryl A. The Plant Cell, Vol. 27, Issue 9 https://doi.org/10.1105/tpc.15.00329	journal	August 2015
The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypopthosphite Unkrig, Volker; Neugebauer, Franz A.; Knappe, Joachim European Journal of Biochemistry, Vol. 184, Issue 3 https://doi.org/10.1111/j.1432-1033.1989.tb15072.x	journal	October 1989
Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor Lawrence, Andrew D.; Frank, Stefanie; Newnham, Sarah ACS Synthetic Biology, Vol. 3, Issue 7 https://doi.org/10.1021/sb4001118	journal	November 2013
Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli Liang, Mingzhi; Frank, Stefanie; Lünsdorf, Heinrich Biotechnology Journal, Vol. 12, Issue 3 https://doi.org/10.1002/biot.201600415	journal	February 2017
Elucidating Essential Role of Conserved Carboxysomal Protein CcmN Reveals Common Feature of Bacterial Microcompartment Assembly Kinney, James N.; Salmeen, Annette; Cai, Fei Journal of Biological Chemistry, Vol. 287, Issue 21 https://doi.org/10.1074/jbc.M112.355305	journal	March 2012
Short N-terminal sequences package proteins into bacterial microcompartments Fan, C.; Cheng, S.; Liu, Y. Proceedings of the National Academy of Sciences, Vol. 107, Issue 16 https://doi.org/10.1073/pnas.0913199107	journal	March 2010
Engineered Protein Nano-Compartments for Targeted Enzyme Localization Choudhary, Swati; Quin, Maureen B.; Sanders, Mark A. PLoS ONE, Vol. 7, Issue 3 https://doi.org/10.1371/journal.pone.0033342	journal	March 2012
De novo design of signal sequences to localize cargo to the 1,2-propanediol utilization microcompartment : Design of Signal Sequences Jakobson, Christopher M.; Slininger Lee, Marilyn F.; Tullman-Ercek, Danielle Protein Science, Vol. 26, Issue 5 https://doi.org/10.1002/pro.3144	journal	April 2017
The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle Erbilgin, Onur; Sutter, Markus; Kerfeld, Cheryl A. PLOS Biology, Vol. 14, Issue 3 https://doi.org/10.1371/journal.pbio.1002399	journal	March 2016
Bacterial microcompartments Kerfeld, Cheryl A.; Aussignargues, Clement; Zarzycki, Jan Nature Reviews Microbiology, Vol. 16, Issue 5 https://doi.org/10.1038/nrmicro.2018.10	journal	March 2018
Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: Isolation and structural properties Conradt, Harald; Hohmann-Berger, Marita; Hohmann, Hans-Peter Archives of Biochemistry and Biophysics, Vol. 228, Issue 1 https://doi.org/10.1016/0003-9861(84)90054-7	journal	January 1984
X-ray Structure of Pyruvate Formate-Lyase in Complex with Pyruvate and CoA Becker, Andreas; Kabsch, Wolfgang Journal of Biological Chemistry, Vol. 277, Issue 42 https://doi.org/10.1074/jbc.M205821200	journal	October 2002
Equilibrium Constant for Conversion of Pyruvate to Acetyl Phosphate and Formate Tanaka, Nobumasa; Johnson, Marvin J. Journal of Bacteriology, Vol. 108, Issue 3 https://doi.org/10.1128/jb.108.3.1107-1111.1971	journal	December 1971
Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein Andorfer, Mary C.; Backman, Lindsey R. F.; Li, Phoebe L. Journal of Biological Chemistry, Vol. 297, Issue 6 https://doi.org/10.1016/j.jbc.2021.101423	journal	December 2021
Pyruvate Formate-Lyase of Escherichia coli: the Acetyl-Enzyme Intermediate Knappe, Joachim; Blaschkowski, Hans P.; Grobner, Peter European Journal of Biochemistry, Vol. 50, Issue 1 https://doi.org/10.1111/j.1432-1033.1974.tb03894.x	journal	December 1974
Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production Li, Tianpei; Jiang, Qiuyao; Huang, Jiafeng Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-19280-0	journal	October 2020
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
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels Shevchenko, Andrej; Wilm, Matthias; Vorm, Ole Analytical Chemistry, Vol. 68, Issue 5 https://doi.org/10.1021/ac950914h	journal	January 1996