Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate
Abstract
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.
- Authors:
-
[1];
[3];
[3];
[3];
[1]
- 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)
- Publication Date:
- Research Org.:
- Michigan State Univ., East Lansing, MI (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- 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)
- OSTI Identifier:
- 1902049
- Alternate Identifier(s):
- OSTI ID: 1896683
- Grant/Contract Number:
- FG02-91ER20021; AC02-05CH11231; MCB 1733552
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Proceedings of the National Academy of Sciences of the United States of America
- Additional Journal Information:
- Journal Volume: 119; Journal Issue: 8; Journal ID: ISSN 0027-8424
- Publisher:
- National Academy of Sciences
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; bacterial microcompartment; formate assimilation; synthetic biology; metabolic engineering
Citation Formats
Kirst, Henning, Ferlez, Bryan H., Lindner, Steffen N., Cotton, Charles R., Bar-Even, Arren, and Kerfeld, Cheryl A. Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate. United States: N. p., 2022.
Web. doi:10.1073/pnas.2116871119.
Kirst, Henning, Ferlez, Bryan H., Lindner, Steffen N., Cotton, Charles R., Bar-Even, Arren, & Kerfeld, Cheryl A. Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate. United States. https://doi.org/10.1073/pnas.2116871119
Kirst, Henning, Ferlez, Bryan H., Lindner, Steffen N., Cotton, Charles R., Bar-Even, Arren, and Kerfeld, Cheryl A. Tue .
"Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate". United States. https://doi.org/10.1073/pnas.2116871119. https://www.osti.gov/servlets/purl/1902049.
@article{osti_1902049,
title = {Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate},
author = {Kirst, Henning and Ferlez, Bryan H. and Lindner, Steffen N. and Cotton, Charles R. and Bar-Even, Arren and Kerfeld, Cheryl A.},
abstractNote = {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.},
doi = {10.1073/pnas.2116871119},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 8,
volume = 119,
place = {United States},
year = {Tue Feb 22 00:00:00 EST 2022},
month = {Tue Feb 22 00:00:00 EST 2022}
}
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Fig. 1: HO-shell architectures and design of the synthetic formate and acetate utilizing BMC. (A) The HO-shell is composed of three different types of building blocks: BMC-H (blue, hexamers), BMC-T (green, trimers/pseudohexamers), and BMC-P (yellow, pentamers) proteins. In the HO-shell are three BMC-T proteins, single-layer T1, and double-layer T2 andmore »
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