TY - JOUR
T1 - A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery
AU - Karim, Ashty S.
AU - Jewett, Michael C.
N1 - Funding Information:
This work is funded by the DARPA Program ( D14PC00005/0001 ). Additional support was from the David and Lucile Packard Foundation and the Camille Dreyfus Teacher Scholar Award (to M.C.J.). A.S.K. is an NSF Graduate Fellow.
PY - 2016/7/1
Y1 - 2016/7/1
N2 - 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.
AB - 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.
KW - Biosynthetic pathways
KW - Cell-free metabolic engineering (CFME)
KW - Cell-free protein synthesis (CFPS)
KW - Design-build-test (DBT)
KW - N-butanol
KW - Synthetic biology
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U2 - 10.1016/j.ymben.2016.03.002
DO - 10.1016/j.ymben.2016.03.002
M3 - Article
C2 - 26996382
AN - SCOPUS:84962739598
VL - 36
SP - 116
EP - 126
JO - Metabolic Engineering
JF - Metabolic Engineering
SN - 1096-7176
ER -