Toward low-cost biomanufacturing through in vitro synthetic biology: bottom-up design

While most in vitro synthetic biology projects are usually used for the purposes of basic science research or the formation of high-value products, cell-free synthetic pathway biotransformation (SyPaB), which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, would be used for low-cost biomanufacturing. In this article, we present bottom-up design principles for SyPaB from basic building blocks (enzymes and/or immobilized enzymes) to basic modules, such as NAD(P)H regeneration, NAD(P)H consumption, ATP regeneration, and extra ATP removal. A combination of thermostable enzymes (called thermoenzymes) with immobilization on solid supports, especially nano-materials and/or electrodes, would greatly prolong enzyme lifetime, enhance mass transfer, and facilitate product/biocatalyst separation. With developments in stable building blocks and modules (called biocatalytic modules), SyPaB has the potential to become a low-cost biomanufacturing platform for biofuels production and even biological CO2 fixation.