Not all cytochrome b5s are created equal: how a specific CytB5 boosts forskolin biosynthesis in Saccharomyces cerevisiae

酿酒酵母 福斯科林 生物合成 生物化学 生物 酵母 受体
作者
Victor Forman,Dan Luo,Sotirios C. Kampranis,Dan Stærk,Birger Lindberg Møller,Irini Pateraki
出处
期刊:Metabolic Engineering [Elsevier BV]
标识
DOI:10.1016/j.ymben.2024.10.008
摘要

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.
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