TY - JOUR
T1 - Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis
AU - Dudley, Quentin M.
AU - Anderson, Kim C.
AU - Jewett, Michael C.
N1 - Funding Information:
We gratefully acknowledge the National Science Foundation (MCB-0943393), the Office of Naval Research (N00014-11-1- 0363), the Army Research Office (W911NF-11-1-0445), the NSF Materials Network Grant (DMR - 1108350), the David and Lucile Packard Foundation (2011-37152), ARPA-E (DEAR0000435),the Camille Dreyfus Teacher Scholar Award, and the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust for support. QMD is funded, in part, by the Northwestern Molecular Biophysics Training Program supported by NIH via NIGMS (5T32 GM008382). We would also like to thank Jay Keasling43 for graciously sharing plasmids pGW322, pGW350, and pGW360.
PY - 2016/12/16
Y1 - 2016/12/16
N2 - Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L-1 (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L-1·hr-1. We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD+ and demonstrates the ability to rapidly prototype and debug enzymatic pathways in vitro for compelling metabolic engineering and synthetic biology applications.
AB - Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L-1 (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L-1·hr-1. We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD+ and demonstrates the ability to rapidly prototype and debug enzymatic pathways in vitro for compelling metabolic engineering and synthetic biology applications.
KW - Escherichia coli
KW - cell-free metabolic engineering
KW - cell-free synthetic biology
KW - in vitro
KW - metabolic pathway debugging
KW - mevalonate
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U2 - 10.1021/acssynbio.6b00154
DO - 10.1021/acssynbio.6b00154
M3 - Article
C2 - 27476989
AN - SCOPUS:85006489426
VL - 5
SP - 1578
EP - 1588
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
SN - 2161-5063
IS - 12
ER -