Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440

Kevin J. McNaught; Eugene Kuatsjah; Michael Zahn; Érica T. Prates; Huiling Shao; Gayle J. Bentley; Andrew R. Pickford; Josephine N. Gruber; Kelley V. Hestmark; Daniel A. Jacobson; Brenton C. Poirier; Chen Ling; Myrsini San Marchi; William E. Michener; Carrie D. Nicora; Jacob N. Sanders; Caralyn J. Szostkiewicz; Dušan Veličković; Mowei Zhou; Nathalie Munoz; Young Mo Kim; Jon K. Magnuson; Kristin E. Burnum-Johnson; K. N. Houk; John E. McGeehan; Christopher W. Johnson; Gregg T. Beckham

doi:10.1016/j.ymben.2023.02.006

Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440

Kevin J. McNaught, Eugene Kuatsjah, Michael Zahn, Érica T. Prates, Huiling Shao, Gayle J. Bentley, Andrew R. Pickford, Josephine N. Gruber, Kelley V. Hestmark, Daniel A. Jacobson, Brenton C. Poirier, Chen Ling, Myrsini San Marchi, William E. Michener, Carrie D. Nicora, Jacob N. Sanders, Caralyn J. Szostkiewicz, Dušan Veličković, Mowei Zhou, Nathalie MunozYoung Mo Kim, Jon K. Magnuson, Kristin E. Burnum-Johnson, K. N. Houk, John E. McGeehan, Christopher W. Johnson, Gregg T. Beckham

Computational and Predictive Biology

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

Original language	English
Pages (from-to)	193-203
Number of pages	11
Journal	Metabolic Engineering
Volume	76
DOIs	https://doi.org/10.1016/j.ymben.2023.02.006
State	Published - Mar 2023

Funding

This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding was provided to KJM, GJB, JNG KVH, BCP, CL, WEM, CJS, CDN, DV, MZhou, NM, YK, JKM, KEB, CJW, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office (BETO) for the Agile BioFoundry. EK, ETP, DAJ, and GTB acknowledge funding from The Center for Bioenergy Innovation , a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . MZahn, ARP, and JEM acknowledge Research England for Expanding Excellence in England (E3) funding. This research was supported by the National Science Foundation ( CHE-1764328 ) to KNH. A portion of this research was performed on a project award (10.46936/reso.proj. 2020.51637/60000235) from the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830 . This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562 . This research used resources of the Oak Ridge Leadership Computing Facility, which is a DOE Office of Science User Facility supported under Contract DE-AC05-00OR22725 . This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding was provided to KJM, GJB, JNG KVH, BCP, CL, WEM, CJS, CDN, DV, MZhou, NM, YK, JKM, KEB, CJW, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office (BETO) for the Agile BioFoundry. EK, ETP, DAJ, and GTB acknowledge funding from The Center for Bioenergy Innovation, a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. MZahn, ARP, and JEM acknowledge Research England for Expanding Excellence in England (E3) funding. This research was supported by the National Science Foundation (CHE-1764328) to KNH. A portion of this research was performed on a project award (10.46936/reso.proj. 2020.51637/60000235) from the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562. This research used resources of the Oak Ridge Leadership Computing Facility, which is a DOE Office of Science User Facility supported under Contract DE-AC05-00OR22725. We thank the Diamond Light Source (Didcot, UK) for beamtime (proposal MX-23269) and the beamline staff at I03 for support. We thank Dr. Adam Guss for providing the temperature-sensitive plasmid (pGW26). The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

Keywords

Decarboxylase
Fatty acid biosynthesis
Hotdog fold
Pseudomonas putida

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McNaught, K. J., Kuatsjah, E., Zahn, M., Prates, É. T., Shao, H., Bentley, G. J., Pickford, A. R., Gruber, J. N., Hestmark, K. V., Jacobson, D. A., Poirier, B. C., Ling, C., San Marchi, M., Michener, W. E., Nicora, C. D., Sanders, J. N., Szostkiewicz, C. J., Veličković, D., Zhou, M., ... Beckham, G. T. (2023). Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440. Metabolic Engineering, 76, 193-203. https://doi.org/10.1016/j.ymben.2023.02.006

@article{b4448fabc8304fd2b9fc888012201928,

title = "Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440",

abstract = "Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.",

keywords = "Decarboxylase, Fatty acid biosynthesis, Hotdog fold, Pseudomonas putida",

author = "McNaught, {Kevin J.} and Eugene Kuatsjah and Michael Zahn and Prates, {{\'E}rica T.} and Huiling Shao and Bentley, {Gayle J.} and Pickford, {Andrew R.} and Gruber, {Josephine N.} and Hestmark, {Kelley V.} and Jacobson, {Daniel A.} and Poirier, {Brenton C.} and Chen Ling and {San Marchi}, Myrsini and Michener, {William E.} and Nicora, {Carrie D.} and Sanders, {Jacob N.} and Szostkiewicz, {Caralyn J.} and Du{\v s}an Veli{\v c}kovi{\'c} and Mowei Zhou and Nathalie Munoz and Kim, {Young Mo} and Magnuson, {Jon K.} and Burnum-Johnson, {Kristin E.} and Houk, {K. N.} and McGeehan, {John E.} and Johnson, {Christopher W.} and Beckham, {Gregg T.}",

note = "Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = mar,

doi = "10.1016/j.ymben.2023.02.006",

language = "English",

volume = "76",

pages = "193--203",

journal = "Metabolic Engineering",

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McNaught, KJ, Kuatsjah, E, Zahn, M, Prates, ÉT, Shao, H, Bentley, GJ, Pickford, AR, Gruber, JN, Hestmark, KV, Jacobson, DA, Poirier, BC, Ling, C, San Marchi, M, Michener, WE, Nicora, CD, Sanders, JN, Szostkiewicz, CJ, Veličković, D, Zhou, M, Munoz, N, Kim, YM, Magnuson, JK, Burnum-Johnson, KE, Houk, KN, McGeehan, JE, Johnson, CW & Beckham, GT 2023, 'Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440', Metabolic Engineering, vol. 76, pp. 193-203. https://doi.org/10.1016/j.ymben.2023.02.006

TY - JOUR

T1 - Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440

AU - McNaught, Kevin J.

AU - Kuatsjah, Eugene

AU - Zahn, Michael

AU - Prates, Érica T.

AU - Shao, Huiling

AU - Bentley, Gayle J.

AU - Pickford, Andrew R.

AU - Gruber, Josephine N.

AU - Hestmark, Kelley V.

AU - Jacobson, Daniel A.

AU - Poirier, Brenton C.

AU - Ling, Chen

AU - San Marchi, Myrsini

AU - Michener, William E.

AU - Nicora, Carrie D.

AU - Sanders, Jacob N.

AU - Szostkiewicz, Caralyn J.

AU - Veličković, Dušan

AU - Zhou, Mowei

AU - Munoz, Nathalie

AU - Kim, Young Mo

AU - Magnuson, Jon K.

AU - Burnum-Johnson, Kristin E.

AU - Houk, K. N.

AU - McGeehan, John E.

AU - Johnson, Christopher W.

AU - Beckham, Gregg T.

PY - 2023/3

Y1 - 2023/3

N2 - Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

AB - Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

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KW - Fatty acid biosynthesis

KW - Hotdog fold

KW - Pseudomonas putida

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DO - 10.1016/j.ymben.2023.02.006

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SN - 1096-7176

VL - 76

SP - 193

EP - 203

JO - Metabolic Engineering

JF - Metabolic Engineering

ER -

Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440

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Keywords

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