title: Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, Northwestern University , Evanston, Illinois 60208, United States.|Robert H. Lurie Comprehensive Cancer Center, ∥Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Quentin M
authors.initials: QM
authors.lastname: Dudley
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, Northwestern University , Evanston, Illinois 60208, United States.|Robert H. Lurie Comprehensive Cancer Center, ∥Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Kim C
authors.initials: KC
authors.lastname: Anderson
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, Northwestern University , Evanston, Illinois 60208, United States.|Robert H. Lurie Comprehensive Cancer Center, ∥Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D053898:Biosynthetic Pathways; D001709:Biotechnology / Q000379:methods; D002474:Cell-Free System / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D045424:Complex Mixtures; D004798:Enzymes / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005612:Freeze Drying; D055786:Gene Knockout Techniques; D016680:Genome, Bacterial; D060847:Metabolic Engineering / Q000379:methods; D008798:Mevalonic Acid / Q000378:metabolism*; D058615:Synthetic Biology / Q000379:methods
chemical_list: D003067:Coenzymes; D045424:Complex Mixtures; D004798:Enzymes; D008798:Mevalonic Acid
keywords: Escherichia coli; cell-free metabolic engineering; cell-free synthetic biology; in vitro; metabolic pathway debugging; mevalonate