Cell-free systems for biosynthesis: towards a sustainable and economical approach

The advent of modern technologies has inspired biotechnologists to shift towards in vitro approaches that utilize cell-free environments for the generation of biochemicals and biomaterials. Despite several challenges, the development of cell-free systems (CFSs) is believed to open new venues in the production of biologically significant molecules and materials. Cell-free biosynthesis is helpful to overcome major discrepancies associated with conventional whole cell-based metabolic processes and offers several advantages, such as reduced structural and functional complexity, as well as the ability to bypass cell viability issues and use a controlled environment. Unfortunately, the implementation of these processes has been impeded by the economic constraints associated with the high-cost reagents, difficulties in the regeneration of energy-liberating molecules ( i.e. , cofactors), and the lack of scalable technologies. The development of efficient in situ cofactor regeneration strategies appears critical to the realization of economically feasible CFSs for industrial-scale production. This review addresses the main constraints associated with the development of CFSs, including the regeneration of cofactors by using various secondary energy sources (SESs) and methods. The development of advanced 'dual-energy systems' capable of regenerating cofactors from readily available energy sources is emphasized. It provides a comprehensive overview of the prospective development of a cost-effective cell-free system by discussing the cost of cofactors, enzymes, and raw materials, as well as discusses the efficacy and scalability of the process. Furthermore, potential directions for the future development of CFSs and their large-scale implementation are presented. This review provides new avenues into the development of sustainable and economically feasible CFSs useful for both novice readers and experts in the field. The advent of modern technologies has inspired biotechnologists to shift towards in vitro approaches that utilize cell-free environments for the generation of biochemicals and biomaterials.