With the gradual rise of enzyme engineering

it has played an essential role in synthetic biology

medicine

and biomanufacturing. However

due to the limitation of the cell membrane

the complexity of cellular metabolism

the difficulty of controlling the reaction environment

and the toxicity of some metabolic products in traditional

in vivo

enzyme engineering

it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds. Recently

cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies. This cell-free method has no limitation of the cell membrane and no need to maintain cell viability

and each biosynthetic pathway is highly flexible. This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize. This article aims to discuss the latest advances in cell-free enzyme engineering

assess the trend of this developing topical filed

and analyze its prospects.