Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

Cell-free expression(CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the constructionof a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms arenecessary to understand the principles behind life-like behavior.Here, we report the successful creation of a CFE system derived fromJCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, highribonuclease activity in Syn3A lysates impeded the establishment offunctional CFE systems. Now, we describe how an unusual cell lysismethod (nitrogen decompression) yielded Syn3A lysates with reducedribonuclease activity that supported in vitro expression.To improve the protein yields in the Syn3A CFE system, we optimizedthe Syn3A CFE reaction mixture using an active machine learning tool.The optimized reaction mixture improved the CFE 3.2-fold comparedto the preoptimized condition. This is the first report of a functionalCFE system derived from a minimal synthetic bacterium, enabling furtheradvances in bottom-up synthetic biology.

Automated summary

This article reports the successful creation of a CFE system derived from the minimal synthetic bacterium Syn3A, which is supported by an unusual cell lysis method that reduces ribonuclease activity. To improve protein yields, the researchers optimized the Syn3A CFE reaction mixture using an active machine learning tool. This is the first functional CFE system derived from a minimal synthetic bacterium, enabling further advancements in bottom-up synthetic biology.