TY - JOUR
T1 - In vitro prototyping of limonene biosynthesis using cell-free protein synthesis
AU - Dudley, Quentin M.
AU - Karim, Ashty S.
AU - Nash, Connor J.
AU - Jewett, Michael C.
N1 - Funding Information:
We gratefully acknowledge the Department of Energy (BER grant: DE-SC0018249 ), the Joint Genome Institute Community Science Program (Project 503280 ), the David and Lucile Packard Foundation ( 2011–37152 ), and the Dreyfus Teacher-Scholar Program for funding and support. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 . QMD is funded, in part, by the Northwestern Molecular Biophysics Training Program supported by NIH via NIGMS ( 5T32 GM008382 ). We also thank Will Bothfeld for helpful discussions and Do Soon Kim for advice on developing supplemental figures.
Publisher Copyright:
© 2020 International Metabolic Engineering Society
PY - 2020/9
Y1 - 2020/9
N2 - 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. 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.
AB - 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. 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.
KW - Cell-free metabolic engineering
KW - Cell-free metabolic pathway prototyping
KW - Cell-free protein synthesis
KW - Limonene
KW - Synthetic biology
KW - iPROBE
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U2 - 10.1016/j.ymben.2020.05.006
DO - 10.1016/j.ymben.2020.05.006
M3 - Article
C2 - 32464283
AN - SCOPUS:85087727828
VL - 61
SP - 251
EP - 260
JO - Metabolic Engineering
JF - Metabolic Engineering
SN - 1096-7176
ER -