Salmon Melissa, Laurendon Caroline, Vardakou Maria, Cheema Jitender, Defernez Marianne, Green Sol, Faraldos Juan A, O'Maille Paul E
John Innes Centre, Department of Metabolic Biology, Norwich Research Park, Norwich NR4 7UH, UK.
John Innes Centre, Computational and Systems Biology, Norwich Research Park, Norwich NR4 7UH, UK.
Nat Commun. 2015 Feb 3;6:6143. doi: 10.1038/ncomms7143.
The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-β-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of ~27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism.
萜类环化作用的出现对于化学多样性的进化扩展至关重要，但仍未得到充分探索。在此，我们报告首次发现了一个控制青蒿萜类环化作用起始的上位性残基网络。我们从分别产生环状或线性萜类的一对萜类合酶——紫穗槐-4,11-二烯合酶（ADS）和（E）-β-法尼烯合酶（BFS）入手。通过将来自环状产物的天然氨基酸替换与线性产物进行组合育种，构建了一个约27,000种酶的文库。我们发现一个显性突变足以激活环化作用，并且与另外两个残基共同构成了一个强烈上位性相互作用的网络，该网络可激活、抑制或重新激活环化作用。值得注意的是，这个由等效残基组成的上位性网络也控制着来自日本柚子的BFS同源物中的环化作用。对突变轨迹的适应度景观分析为特殊代谢的一个主要时期提供了定量见解。
