title: A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.
abstract: Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E-136, Evanston, IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Ashty S
authors.initials: AS
authors.lastname: Karim
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E-136, Evanston, IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA; Simpson Querrey Institute, Northwestern University, Chicago, IL 60611, USA. Electronic address: m-jewett@northwestern.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D053898:Biosynthetic Pathways / Q000502:physiology*; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010865:Pilot Projects
chemical_list: D009097:Multienzyme Complexes
keywords: Biosynthetic pathways; Cell-free metabolic engineering (CFME); Cell-free protein synthesis (CFPS); Design-build-test (DBT); Synthetic biology; n-butanol