Cell-free expression of RuBisCO for ATP production in the synthetic cells.

Recent advances in bottom-up synthetic biology have made it possible to reconstitute cellular systems from non-living components, yielding artificial cells with potential applications in industry, medicine and basic research. Although a variety of cellular functions and components have been reconstituted in previous studies, sustained biological energy production remains a challenge. ATP synthesis via ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), a central enzyme in biological CO2 fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested. In this study, we test RuBisCO expression and its activity-mediated ATP synthesis in a reconstituted Escherichia coli-based cell-free translation system. We then construct a system in which ATP is synthesized by RuBisCO activity in giant vesicles and used as energy for translation reactions. These results represent an advance toward independent energy production in artificial cells. Graphical Abstract.

Progresses in Cell-Free In Vitro Evolution.

Biopolymers, such as proteins and RNA, are integral components of living organisms and have evolved through a process of repeated mutation and selection. The technique of "cell-free in vitro evolution" is a powerful experimental approach for developing biopolymers with desired functions and structural properties. Since Spiegelman's pioneering work over 50 years ago, biopolymers with a wide range of functions have been developed using in vitro evolution in cell-free systems. The use of cell-free systems offers several advantages, including the ability to synthesize a wider range of proteins without the limitations imposed by cytotoxicity, and the capacity for higher throughput and larger library sizes than cell-based evolutionary experiments. In this chapter, we provide a comprehensive overview of the progress made in the field of cell-free in vitro evolution by categorizing evolution into directed and undirected. The biopolymers produced by these methods are valuable assets in medicine and industry, and as a means of exploring the potential of biopolymers.

In Vitro Transcription/Translation-Coupled DNA Replication through Partial Regeneration of 20 Aminoacyl-tRNA Synthetases.

The in vitro reconstruction of life-like self-reproducing systems is a major challenge in in vitro synthetic biology. Self-reproduction requires regeneration of all molecules involved in DNA replication, transcription, and translation. This study demonstrated the continuous DNA replication and partial regeneration of major translation factors, 20 aminoacyl-tRNA synthetases (aaRS), in a reconstituted transcription/translation system (PURE system) for the first time. First, we replicated each DNA that encodes one of the 20 aaRSs through aaRS expression from the DNA by serial transfer experiments. Thereafter, we successively increased the number of aaRS genes and achieved simultaneous, continuous replication of DNA that encodes all 20 aaRSs, which comprised approximately half the number of protein factors in the PURE system, except for ribosomes, by employing dialyzed reaction and sequence optimization. This study provides a step-by-step methodology for continuous DNA replication with an increasing number of self-regenerative genes toward self-reproducing artificial systems.