Coenzyme F₄₂₀ is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F₄₂₀-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F₄₂₀ levels are produced in microorganisms, F₄₂₀ availability is a serious bottleneck for research and application. Recent advances in our understanding of the F₄₂₀ biosynthesis enabled heterologous overproduction of F₄₂₀ in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F₄₂₀ biosynthesis in E. coli. However, it still led to the production of low amounts of F₄₂₀ and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module (“core”) addressed parts of the riboflavin biosynthesis pathway and F_O synthase for the conversion of GTP to the stable F₄₂₀ intermediate F_O. The enzymes of the second module (“decoration”) were chosen to turn F_O into F₄₂₀. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F₄₂₀-derived fluorescence. This approach yielded the highest titer of coenzyme F₄₂₀ produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F₄₂₀-dependent bioprocesses as well as applications of F₄₂₀-dependent enzymes in biocatalysis.

辅酶 F ₄₂₀参与生物过程，例如链霉菌的抗生素生物合成、结核分枝杆菌中的前药活化和古细菌中的产甲烷作用。F ₄₂₀依赖性酶作为有机化学中的生物催化剂也引起了人们的兴趣。然而，由于微生物中仅产生低水平的 F _{420 ，因此 F 420}的可用性是研究和应用的严重瓶颈。_{我们对 F 420}生物合成的理解的最新进展使大肠杆菌中F ₄₂₀的异源过量生产成为可能，但产量仍然适中。为了解决这个问题，我们合理地为 F 设计了一个合成操纵子_420在大肠杆菌中的生物合成。然而，它仍然导致生产少量的 F ₄₂₀和不需要的副产品。为了大大提高产量和纯度，选择了一种筛选方法来询问基于多样化启动子和核糖体结合位点的组合文库的基因表达空间。整个通路由双操纵子构建体编码。第一个模块（“核心”）涉及部分核黄素生物合成途径和 F _O合酶，用于将 GTP 转化为稳定的 F ₄₂₀中间体 F _O。选择第二个模块（“装饰”）的酶将 F _O转化为 F ₄₂₀. 最终构建体包括 T7 启动子强度和核糖体结合位点活性的变化，以改变参与途径的八个基因的表达比率。荧光激活细胞分选用于分离该文库的克隆，该文库显示出强 F ₄₂₀衍生荧光。这种方法产生了迄今为止在广泛使用的大肠杆菌中产生的最高滴度的辅酶 F _{420 。}在标准 LB 培养基中的生产提供了一种高效且简单的生产过程，这将有助于对未探索的 F ₄₂₀依赖性生物过程的基础研究以及 F ₄₂₀依赖性酶在生物催化中的应用。