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

Dudley, Quentin M.; Karim, Ashty S.; Nash, Connor J.; Jewett, Michael C.

doi:10.1016/j.ymben.2020.05.006

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

Journal Article · Mon May 25 00:00:00 EDT 2020 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2020.05.006· OSTI ID:1660449

^[1];

^[2]; Nash, Connor J. ^[2];

^[2]

Northwestern Univ., Evanston, IL (United States); Earlham Institute, Norwich Research Park, Colney Lane, Norwich (United Kingdom)
Northwestern Univ., Evanston, IL (United States)

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.

View Accepted Manuscript (DOE)

View Accepted Manuscript (Publisher)

Cite

Export

Save

Research Organization:: Northwestern Univ., Evanston, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; National Institutes of Health (NIH)

Grant/Contract Number:: SC0018249; AC02-05CH11231; 503280

OSTI ID:: 1660449

Alternate ID(s):: OSTI ID: 1638279; OSTI ID: 2298993

Journal Information:: Metabolic Engineering, Vol. 61; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

References (61)

Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering Alonso-Gutierrez, Jorge; Kim, Eun-Mi; Batth, Tanveer S. Metabolic Engineering, Vol. 28 https://doi.org/10.1016/j.ymben.2014.11.011	journal	March 2015
Paracoccus zeaxanthinifaciens sp. nov., a zeaxanthin-producing bacterium Berry, Alan; Janssens, Danielle; Hümbelin, Markus International Journal of Systematic and Evolutionary Microbiology, Vol. 53, Issue 1 https://doi.org/10.1099/ijs.0.02368-0	journal	January 2003
Synthetic non-oxidative glycolysis enables complete carbon conservation Bogorad, Igor W.; Lin, Tzu-Shyang; Liao, James C. Nature, Vol. 502, Issue 7473 https://doi.org/10.1038/nature12575	journal	September 2013
Terpenoid biomaterials Bohlmann, Jörg; Keeling, Christopher I. The Plant Journal, Vol. 54, Issue 4 https://doi.org/10.1111/j.1365-313X.2008.03449.x	journal	May 2008
Cell-free protein synthesis: Applications come of age Carlson, Erik D.; Gan, Rui; Hodgman, C. Eric Biotechnology Advances, Vol. 30, Issue 5 https://doi.org/10.1016/j.biotechadv.2011.09.016	journal	September 2012
A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology Casini, Arturo; Chang, Fang-Yuan; Eluere, Raissa Journal of the American Chemical Society, Vol. 140, Issue 12 https://doi.org/10.1021/jacs.7b13292	journal	February 2018
Two-step pathway for isoprenoid synthesis Chatzivasileiou, Alkiviadis Orfefs; Ward, Valerie; Edgar, Steven McBride Proceedings of the National Academy of Sciences, Vol. 116, Issue 2 https://doi.org/10.1073/pnas.1812935116	journal	December 2018
De novo design of protein logic gates Chen, Zibo; Kibler, Ryan D.; Hunt, Andrew Science, Vol. 368, Issue 6486 https://doi.org/10.1126/science.aay2790	journal	April 2020
Orthogonal Engineering of Biosynthetic Pathway for Efficient Production of Limonene in Saccharomyces cerevisiae Cheng, Si; Liu, Xue; Jiang, Guozhen ACS Synthetic Biology, Vol. 8, Issue 5 https://doi.org/10.1021/acssynbio.9b00135	journal	April 2019
The isoprenoid alcohol pathway, a synthetic route for isoprenoid biosynthesis Clomburg, James M.; Qian, Shuai; Tan, Zaigao Proceedings of the National Academy of Sciences, Vol. 116, Issue 26 https://doi.org/10.1073/pnas.1821004116	journal	June 2019
A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli Des Soye, Benjamin J.; Gerbasi, Vincent R.; Thomas, Paul M. Cell Chemical Biology, Vol. 26, Issue 12 https://doi.org/10.1016/j.chembiol.2019.10.008	journal	December 2019
An in Vitro Biosynthesis of Sesquiterpenes Starting from Acetic Acid Dirkmann, Michael; Nowack, Julia; Schulz, Frank ChemBioChem, Vol. 19, Issue 20 https://doi.org/10.1002/cbic.201800128	journal	October 2018
Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis Dudley, Quentin M.; Anderson, Kim C.; Jewett, Michael C. ACS Synthetic Biology, Vol. 5, Issue 12 https://doi.org/10.1021/acssynbio.6b00154	journal	July 2016
Cell-free metabolic engineering: Biomanufacturing beyond the cell Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C. Biotechnology Journal, Vol. 10, Issue 1 https://doi.org/10.1002/biot.201400330	journal	October 2014
MUSCLE: multiple sequence alignment with high accuracy and high throughput Edgar, R. C. Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797 https://doi.org/10.1093/nar/gkh340	journal	March 2004
The Tomato Terpene Synthase Gene Family Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T. H. Plant Physiology, Vol. 157, Issue 2 https://doi.org/10.1104/pp.111.179648	journal	August 2011
Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M. Nucleic Acids Research, Vol. 41, Issue 14 https://doi.org/10.1093/nar/gkt489	journal	June 2013
Isoprenoid Drugs, Biofuels, and Chemicals—Artemisinin, Farnesene, and Beyond George, Kevin W.; Alonso-Gutierrez, Jorge; Keasling, Jay D. Biotechnology of Isoprenoids https://doi.org/10.1007/10_2014_288	book	January 2015
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
BioBits™ Explorer: A modular synthetic biology education kit Huang, Ally; Nguyen, Peter Q.; Stark, Jessica C. Science Advances, Vol. 4, Issue 8 https://doi.org/10.1126/sciadv.aat5105	journal	August 2018
Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate Ignea, Codruta; Raadam, Morten H.; Motawia, Mohammed S. Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-11290-x	journal	August 2019
Machine Learning of Designed Translational Control Allows Predictive Pathway Optimization in Escherichia coli Jervis, Adrian J.; Carbonell, Pablo; Vinaixa, Maria ACS Synthetic Biology, Vol. 8, Issue 1 https://doi.org/10.1021/acssynbio.8b00398	journal	December 2018
An integrated cell-free metabolic platform for protein production and synthetic biology Jewett, Michael C.; Calhoun, Kara A.; Voloshin, Alexei Molecular Systems Biology, Vol. 4, Issue 1, Article No. 220 https://doi.org/10.1038/msb.2008.57	journal	October 2008
Mimicking theEscherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis Jewett, Michael C.; Swartz, James R. Biotechnology and Bioengineering, Vol. 86, Issue 1 https://doi.org/10.1002/bit.20026	journal	January 2004
Biotechnological production of limonene in microorganisms Jongedijk, Esmer; Cankar, Katarina; Buchhaupt, Markus Applied Microbiology and Biotechnology, Vol. 100, Issue 7 https://doi.org/10.1007/s00253-016-7337-7	journal	February 2016
Controlling cell-free metabolism through physiochemical perturbations Karim, Ashty S.; Heggestad, Jacob T.; Crowe, Samantha A. Metabolic Engineering, Vol. 45 https://doi.org/10.1016/j.ymben.2017.11.005	journal	January 2018
A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery Karim, Ashty S.; Jewett, Michael C. Metabolic Engineering, Vol. 36 https://doi.org/10.1016/j.ymben.2016.03.002	journal	July 2016
Improving Solubility of Shewanella o neidensis MR-1 and Clostridium t hermocellum JW-20 Proteins Expressed into Esherichia c oli Kataeva, Irina; Chang, Jessie; Xu, Hao Journal of Proteome Research, Vol. 4, Issue 6 https://doi.org/10.1021/pr050108j	journal	December 2005
Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics Kelwick, Richard; Ricci, Luca; Chee, Soo Mei Synthetic Biology, Vol. 3, Issue 1 https://doi.org/10.1093/synbio/ysy016	journal	January 2018
A cell-free biosynthesis platform for modular construction of protein glycosylation pathways Kightlinger, Weston; Duncker, Katherine E.; Ramesh, Ashvita Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-12024-9	journal	November 2019
A synthetic biochemistry platform for cell free production of monoterpenes from glucose Korman, Tyler P.; Opgenorth, Paul H.; Bowie, James U. Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms15526	journal	May 2017
A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates : Production of Isoprene from Glycolysis Intermediates Korman, Tyler P.; Sahachartsiri, Bobby; Li, Dan Protein Science, Vol. 23, Issue 5 https://doi.org/10.1002/pro.2436	journal	March 2014
High-throughput preparation methods of crude extract for robust cell-free protein synthesis Kwon, Yong-Chan; Jewett, Michael C. Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep08663	journal	March 2015
Establishing a high yielding streptomyces -based cell-free protein synthesis system: Establishing a Streptomyces -Based CFPS System Li, Jian; Wang, He; Kwon, Yong-Chan Biotechnology and Bioengineering, Vol. 114, Issue 6 https://doi.org/10.1002/bit.26253	journal	February 2017
Improvement of isoprene production in Escherichia coli by rational optimization of RBSs and key enzymes screening Li, Meijie; Chen, Hailin; Liu, Changqing Microbial Cell Factories, Vol. 18, Issue 1 https://doi.org/10.1186/s12934-018-1051-3	journal	January 2019
Sequential Glycosylation of Proteins with Substrate-Specific N -Glycosyltransferases Lin, Liang; Kightlinger, Weston; Prabhu, Sunaina Kiran ACS Central Science, Vol. 6, Issue 2 https://doi.org/10.1021/acscentsci.9b00021	journal	February 2020
Quantitative analysis and engineering of fatty acid biosynthesis in E. coli Liu, Tiangang; Vora, Harmit; Khosla, Chaitan Metabolic Engineering, Vol. 12, Issue 4 https://doi.org/10.1016/j.ymben.2010.02.003	journal	July 2010
An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism Lund, Sean; Hall, Rachael; Williams, Gavin J. ACS Synthetic Biology, Vol. 8, Issue 2 https://doi.org/10.1021/acssynbio.8b00383	journal	January 2019
Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases Ma, Suzanne M.; Garcia, David E.; Redding-Johanson, Alyssa M. Metabolic Engineering, Vol. 13, Issue 5 https://doi.org/10.1016/j.ymben.2011.07.001	journal	September 2011
Harnessing evolutionary diversification of primary metabolism for plant synthetic biology Maeda, Hiroshi A. Journal of Biological Chemistry, Vol. 294, Issue 45 https://doi.org/10.1074/jbc.REV119.006132	journal	September 2019
Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors Meyer, Adam J.; Segall-Shapiro, Thomas H.; Glassey, Emerson Nature Chemical Biology, Vol. 15, Issue 2 https://doi.org/10.1038/s41589-018-0168-3	journal	November 2018
Metabolic Profiling of Escherichia coli -Based Cell-Free Expression Systems for Process Optimization Miguez, April M.; McNerney, Monica P.; Styczynski, Mark P. Industrial & Engineering Chemistry Research, Vol. 58, Issue 50 https://doi.org/10.1021/acs.iecr.9b03565	journal	September 2019
Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly Nowroozi, Farnaz F.; Baidoo, Edward E. K.; Ermakov, Simon Applied Microbiology and Biotechnology, Vol. 98, Issue 4 https://doi.org/10.1007/s00253-013-5361-4	journal	November 2013
High-level semi-synthetic production of the potent antimalarial artemisinin Paddon, C. J.; Westfall, P. J.; Pitera, D. J. Nature, Vol. 496, Issue 7446 https://doi.org/10.1038/nature12051	journal	April 2013
Identification and microbial production of a terpene-based advanced biofuel Peralta-Yahya, Pamela P.; Ouellet, Mario; Chan, Rossana Nature Communications, Vol. 2, Issue 1 https://doi.org/10.1038/ncomms1494	journal	September 2011
Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies Piserchio, Andrea; Ghose, Ranajeet; Cowburn, David Journal of Biomolecular NMR, Vol. 44, Issue 2 https://doi.org/10.1007/s10858-009-9318-y	journal	April 2009
Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei Primak, Yuliya A.; Du, Mai; Miller, Michael C. Applied and Environmental Microbiology, Vol. 77, Issue 21 https://doi.org/10.1128/AEM.05761-11	journal	September 2011
An Enzymatic Platform for the Synthesis of Isoprenoid Precursors Rodriguez, Sofia B.; Leyh, Thomas S. PLoS ONE, Vol. 9, Issue 8 https://doi.org/10.1371/journal.pone.0105594	journal	August 2014
BRENDA, the enzyme information system in 2011 Scheer, M.; Grote, A.; Chang, A. Nucleic Acids Research, Vol. 39, Issue Database https://doi.org/10.1093/nar/gkq1089	journal	November 2010
PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism Schmidt, Karin; Petersen, Jan; Munkert, Jennifer Phytochemistry, Vol. 156 https://doi.org/10.1016/j.phytochem.2018.08.012	journal	December 2018
In‐depth characterization of genome‐scale network reconstructions for the in vitro synthesis in cell‐free systems Schuh, Lisa Katharina; Weyler, Christian; Heinzle, Elmar Biotechnology and Bioengineering, Vol. 117, Issue 4 https://doi.org/10.1002/bit.27249	journal	January 2020
Cell-free gene expression: an expanded repertoire of applications Silverman, Adam D.; Karim, Ashty S.; Jewett, Michael C. Nature Reviews Genetics, Vol. 21, Issue 3 https://doi.org/10.1038/s41576-019-0186-3	journal	November 2019
Functional optimization of gene clusters by combinatorial design and assembly Smanski, Michael J.; Bhatia, Swapnil; Zhao, Dehua Nature Biotechnology, Vol. 32, Issue 12, p. 1241-1249 https://doi.org/10.1038/nbt.3063	journal	November 2014
BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts Stark, Jessica C.; Huang, Ally; Hsu, Karen J. ACS Synthetic Biology, Vol. 8, Issue 5 https://doi.org/10.1021/acssynbio.8b00381	journal	March 2019
BioBits™ Bright: A fluorescent synthetic biology education kit Stark, Jessica C.; Huang, Ally; Nguyen, Peter Q. Science Advances, Vol. 4, Issue 8 https://doi.org/10.1126/sciadv.aat5107	journal	August 2018
High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli Tsuruta, Hiroko; Paddon, Christopher J.; Eng, Diana PLoS ONE, Vol. 4, Issue 2 https://doi.org/10.1371/journal.pone.0004489	journal	February 2009
Cell free biosynthesis of isoprenoids from isopentenol Ward, Valerie C. A.; Chatzivasileiou, Alkiviadis Orfefs; Stephanopoulos, Gregory Biotechnology and Bioengineering, Vol. 116, Issue 12 https://doi.org/10.1002/bit.27146	journal	September 2019
Systematic Optimization of Limonene Production in Engineered Escherichia coli Wu, Jihua; Cheng, Si; Cao, Jiayu Journal of Agricultural and Food Chemistry, Vol. 67, Issue 25 https://doi.org/10.1021/acs.jafc.9b01427	journal	May 2019
Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production Yang, Liyang; Wang, Chonglong; Zhou, Jia Microbial Cell Factories, Vol. 15, Issue 1 https://doi.org/10.1186/s12934-016-0409-7	journal	January 2016
Fine‐tuning the (2 S )‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy Zhou, Shenghu; Lyu, Yunbin; Li, Huazhong Biotechnology and Bioengineering, Vol. 116, Issue 6 https://doi.org/10.1002/bit.26941	journal	February 2019
In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli : In Vitro Reconstitution of Mevalonate Pathway Zhu, Fayin; Zhong, Xiaofang; Hu, Mengzhu Biotechnology and Bioengineering, Vol. 111, Issue 7 https://doi.org/10.1002/bit.25198	journal	February 2014

Similar Records

Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Journal Article · Mon Jan 14 00:00:00 EST 2019 · Synthetic Biology · OSTI ID:1660449

Dudley, Quentin M.; Nash, Connor J.; Jewett, Michael C.

In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design

Journal Article · Mon Jun 15 00:00:00 EDT 2020 · Nature Chemical Biology · OSTI ID:1660449

Karim, Ashty S.; Dudley, Quentin M.; Juminaga, Alex; +17 more

Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002

Journal Article · Thu Jun 19 00:00:00 EDT 2014 · Frontiers in Bioengineering and Biotechnology, 2:Artcle No. 21 · OSTI ID:1660449

Davies, Fiona K.; Work, Victoria H.; Beliaev, Alex S.; +1 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
cell-free metabolic engineering
limonene
iPROBE
cell-free metabolic pathway prototyping
cell-free protein synthesis
synthetic biology

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

Citation Formats

References (61)

Similar Records

Related Subjects