The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l⁻¹. We expect iPROBE to accelerate design–build–test cycles for industrial biotechnology.

由于转化特性，经过验证的遗传部分数量减少以及缺乏高通量的工作流程，因此与工业相关的非模式生物的生物合成途径的设计和优化面临挑战。在这里，我们描述了体外原型制作和快速优化生物合成酶（iPROBE）来加速该过程的平台。在iPROBE中，细胞裂解液通过无细胞蛋白质合成富集了生物合成酶，然后以混合搭配的方式组装了代谢途径，以评估途径的性能。我们通过筛选54种不同的无细胞途径生产3-羟基丁酸酯并使用数据驱动设计优化跨205个排列的六步丁醇途径来证明iPROBE。观察到很强的相关性（r = 0.79），然后我们扩大了最高性能的途径，将梭状芽孢杆菌的体内3-HB产量提高了20倍，达到14.63±0.48 g l ^-1。我们希望iPROBE能够加快工业生物技术的设计，建造，测试周期。