Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.

苯乙烯是一种重要的石油衍生分子，经过聚合可制成多功能塑料，包括一次性银器和泡沫包装材料。由于气候变化日益严重以及化石燃料供应有限，寻找更可持续的方法（例如生物合成）来生产苯乙烯至关重要。最近的代谢工程努力使大肠杆菌中的苯乙烯生物生产成为可能，但苯乙烯的毒性和挥发性限制了细胞中的生物合成。为了解决这些限制，我们开发了一个无细胞苯乙烯生物合成平台。无细胞系统提供了一个开放的反应环境，没有细胞活力的限制，这允许对反应条件和更大的碳通量进行精确控制，以形成产物而不是细胞生长。苯乙烯生产所需的两种生物合成酶是通过无细胞蛋白质合成产生的，并以规定的比例与补充的 L-苯丙氨酸和缓冲液混合。通过改变反应中的时间、温度、pH 值和酶浓度，这种方法将苯乙烯的无细胞滴度从 5.36 ± 0.63 mM 增加到 40.33 ± 1.03 mM，这是使用生物合成实现的最高量，无需工艺修改和产物去除策略.