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Title: In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Abstract

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. Finally, we anticipatemore » that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.« less

Authors:
ORCiD logo [1]; ORCiD logo [2];  [2]; ORCiD logo [2]
  1. Northwestern Univ., Evanston, IL (United States); Earlham Institute, Norwich Research Park, Colney Lane, Norwich (United Kingdom)
  2. Northwestern Univ., Evanston, IL (United States)
Publication Date:
Research Org.:
Northwestern Univ., Evanston, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; National Institutes of Health (NIH)
OSTI Identifier:
1660449
Alternate Identifier(s):
OSTI ID: 1638279
Grant/Contract Number:  
SC0018249; AC02-05CH11231; 503280
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 61; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
cell-free metabolic engineering; limonene; iPROBE; cell-free metabolic pathway prototyping; cell-free protein synthesis; synthetic biology

Citation Formats

Dudley, Quentin M., Karim, Ashty S., Nash, Connor J., and Jewett, Michael C. In vitro prototyping of limonene biosynthesis using cell-free protein synthesis. United States: N. p., 2020. Web. doi:10.1016/j.ymben.2020.05.006.
Dudley, Quentin M., Karim, Ashty S., Nash, Connor J., & Jewett, Michael C. In vitro prototyping of limonene biosynthesis using cell-free protein synthesis. United States. doi:10.1016/j.ymben.2020.05.006.
Dudley, Quentin M., Karim, Ashty S., Nash, Connor J., and Jewett, Michael C. Mon . "In vitro prototyping of limonene biosynthesis using cell-free protein synthesis". United States. doi:10.1016/j.ymben.2020.05.006.
@article{osti_1660449,
title = {In vitro prototyping of limonene biosynthesis using cell-free protein synthesis},
author = {Dudley, Quentin M. and Karim, Ashty S. and Nash, Connor J. and Jewett, Michael C.},
abstractNote = {Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. Finally, we anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.},
doi = {10.1016/j.ymben.2020.05.006},
journal = {Metabolic Engineering},
issn = {1096-7176},
number = ,
volume = 61,
place = {United States},
year = {2020},
month = {5}
}

Journal Article:
Free Publicly Available Full Text
This content will become publicly available on May 25, 2021
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