Cell-free systems: A synthetic biology tool for rapid prototyping in metabolic engineering.

Microbial cell factories provide sustainable alternatives to petroleum-based chemical production using cost-effective substrates. A deep understanding of their metabolism is essential to harness their potential along with continuous efforts to improve productivity and yield. However, the construction and evaluation of numerous genetic variants are time-consuming and labor-intensive. Cell-free systems (CFSs) serve as powerful platforms for rapid prototyping of genetic circuits, metabolic pathways, and enzyme functionality. They offer numerous advantages, including minimizing unwanted metabolic interference, precise control of reaction conditions, reduced labor, and shorter Design-Build-Test-Learn cycles. Additionally, the introduction of in vitro compartmentalization strategies in CFSs enables ultra-high-throughput screening in physically separated spaces, which significantly enhances prototyping efficiency. This review highlights the latest examples of using CFS to overcome prototyping limitations in living cells with a focus on rapid prototyping, particularly regarding gene regulation, enzymes, and multienzymatic reactions in bacteria. Finally, this review evaluates CFSs as a versatile prototyping platform and discusses its future applications, emphasizing its potential for producing high-value chemicals through microbial biosynthesis.