Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant Coleus forskohlii. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast Saccharomyces cerevisiae, and upon co-expression of the three CytB5s - identified in C. forskohlii transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, CfCytB5A, was used. Co-expression of CfCytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.

中文翻译：

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并非所有细胞色素 b5 都是一样的：特异性 CytB5 如何促进酿酒酵母中的毛喉素生物合成


细胞色素 B5 或 CytB5 是小的血红素结合蛋白，在所有生命界中无处不在，主要作为参与氧化反应的酶的电子供体。它们通常作为细胞色素 P450 （CYP） 的氧化还原伴侣发挥作用，细胞色素 P450 （CYP） 是参与多种生化过程的酶超家族。在植物中，CYPs 催化植物特化代谢物生物合成中的关键反应，其活性取决于电子供体，通常来自细胞色素 P450 氧化还原酶 （CPR 或 POR）。在真核微粒体 CYP 中，CytB5 经常参与电子转移过程，尽管它们的确切作用仍未得到充分研究，尤其是在植物系统中。在这项研究中，我们评估了 CytB5s 在酵母中植物特化代谢物异源生物技术生产中的作用。为此，我们以毛喉素的生物合成为案例研究 - 一种完全由植物毛喉鞘蕊花产生的生物活性二萜类化合物。毛喉素的完整生物合成途径是已知的，包括三种 CYP 酶。我们在酵母酿酒酵母中重建了整个毛喉素通路，并且在三种 CytB5 共表达时 - 在 C. forskohlii 转录组中鉴定 - 仅当使用特异性 CytB5 时，才注意到与 CYP 相关的瓶颈步骤的缓解，CfCytB5A，被使用。CfCytB5A 在酵母中的共表达与毛喉素通路工程相结合，在生物反应器中产生滴度为 1.81 g/L 的毛喉素。我们的研究结果表明，CytB5 不仅在植物特化代谢中发挥重要作用，而且它们可以与特定的 CYP 精确相互作用，这表明 CytB5 的特性远未被了解。 此外，我们的工作强调了当 CytB5 的生物合成途径涉及 CYP 酶时，CytB5s 如何作为植物代谢物可持续微生物生产中不可或缺的组成部分。