Cell-free expression (CFE) systems are fundamental to reconstituting
metabolic pathwaysin vitrotoward the construction
of a synthetic cell. Although anEscherichia coli-based CFE system is well-established, simpler model organisms are
necessary to understand the principles behind life-like behavior.
Here, we report the successful creation of a CFE system derived from
JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high
ribonuclease activity in Syn3A lysates impeded the establishment of
functional CFE systems. Now, we describe how an unusual cell lysis
method (nitrogen decompression) yielded Syn3A lysates with reduced
ribonuclease activity that supportedin vitroexpression.
To improve the protein yields in the Syn3A CFE system, we optimized
the Syn3A CFE reaction mixture using an active machine learning tool.
The optimized reaction mixture improved the CFE 3.2-fold compared
to the preoptimized condition. This is the first report of a functional
CFE system derived from a minimal synthetic bacterium, enabling further
advances in bottom-up synthetic biology.