A lysate proteome engineering strategy for enhancing cell-free metabolite production

David C. Garcia; Jaime Lorenzo N. Dinglasan; Him Shrestha; Paul E. Abraham; Robert L. Hettich; Mitchel J. Doktycz

doi:10.1016/j.mec.2021.e00162

A lysate proteome engineering strategy for enhancing cell-free metabolite production

David C. Garcia, Jaime Lorenzo N. Dinglasan, Him Shrestha, Paul E. Abraham, Robert L. Hettich, Mitchel J. Doktycz

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

14 Scopus citations

Abstract

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell's capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.

Original language	English
Article number	e00162
Journal	Metabolic Engineering Communications
Volume	12
DOIs	https://doi.org/10.1016/j.mec.2021.e00162
State	Published - Jun 2021

Funding

Special thanks to Ákos Nyerges for providing valuable advice regarding the use of the pORTMAGE system. This research was sponsored by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. D.C.G. is an NSF Graduate Fellow. This manuscript has been authored by UT-Battelle, LLC under Contract DE-AC05- 00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). Special thanks to Ákos Nyerges for providing valuable advice regarding the use of the pORTMAGE system. This research was sponsored by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area ( http://pmi.ornl.gov ). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. D.C.G. is an NSF Graduate Fellow. This manuscript has been authored by UT-Battelle, LLC under Contract DE-AC05- 00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).

Keywords

Cell lysates
Cell-free metabolic engineering
Genome engineering
In vitro biology
Proteomics

Access to Document

10.1016/j.mec.2021.e00162

Cite this

@article{801f3e49347b4a068eebadfde0618b32,

title = "A lysate proteome engineering strategy for enhancing cell-free metabolite production",

abstract = "Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell's capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.",

keywords = "Cell lysates, Cell-free metabolic engineering, Genome engineering, In vitro biology, Proteomics",

author = "Garcia, {David C.} and Dinglasan, {Jaime Lorenzo N.} and Him Shrestha and Abraham, {Paul E.} and Hettich, {Robert L.} and Doktycz, {Mitchel J.}",

note = "Publisher Copyright: {\textcopyright} 2021 The Authors",

year = "2021",

month = jun,

doi = "10.1016/j.mec.2021.e00162",

language = "English",

volume = "12",

journal = "Metabolic Engineering Communications",

issn = "2214-0301",

}

TY - JOUR

T1 - A lysate proteome engineering strategy for enhancing cell-free metabolite production

AU - Garcia, David C.

AU - Dinglasan, Jaime Lorenzo N.

AU - Shrestha, Him

AU - Abraham, Paul E.

AU - Hettich, Robert L.

AU - Doktycz, Mitchel J.

PY - 2021/6

Y1 - 2021/6

N2 - Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell's capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.

AB - Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell's capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.

KW - Cell lysates

KW - Cell-free metabolic engineering

KW - Genome engineering

KW - In vitro biology

KW - Proteomics

UR - http://www.scopus.com/inward/record.url?scp=85099945514&partnerID=8YFLogxK

U2 - 10.1016/j.mec.2021.e00162

DO - 10.1016/j.mec.2021.e00162

M3 - Article

AN - SCOPUS:85099945514

SN - 2214-0301

VL - 12

JO - Metabolic Engineering Communications

JF - Metabolic Engineering Communications

M1 - e00162

ER -

A lysate proteome engineering strategy for enhancing cell-free metabolite production

Abstract

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this