Abstract
Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.
Highlights
Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells
We hypothesized that the overexpression of translation machinery should benefit cell-free protein synthesis (CFPS) in two ways
The results show that the expression of translation machinery from a local genetic module results in a global proteome shift generally associated with a cellular state at high-growth rates[4,29]: upregulation of proteins involved in macromolecule synthesis
Summary
Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module We apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. The use of orthogonal genetic modules often faces the challenges of varying cellular context, such as growth rate, crosstalk, and noise[2] These challenges highlight the necessity to complement orthogonal genetic module design with a system-based approach that functions in conjunction with cell physiology[3]. Such systems-synthetic biology approaches have been applied in two major ways.
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