Engineering Yeast for De Novo Synthesis of the Insect Repellent Nepetalactone,ACS Synthetic Biology

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Engineering Yeast for De Novo Synthesis of the Insect Repellent Nepetalactone
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-11-08 , DOI: 10.1021/acssynbio.1c00420
Meghan E Davies _{1,

2} , Daniel Tsyplenkov _{1,

2} , Vincent J J Martin _{1,

2}

Affiliation

While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe for the first time de novo nepetalactone synthesis in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introducing eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, and copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity and increasing geraniol-8-hydroxylase gene copy number improved 8-hydroxygeraniol titers. The accumulation of an unwanted metabolite implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologues were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in 3.10 mg/L/OD₆₀₀ of nepetalactone from simple sugar in microtiter plates. This optimized pathway will benefit the development of high yielding strains for the scale up production of nepetalactone.

中文翻译：

从头合成驱虫剂荆芥内酯的工程酵母

虽然猫薄荷中的活性成分荆芥内酯是一种有效的驱虫剂，但其在植物体内的低积累限制了其作为替代驱虫剂的商业可行性。在这里，我们首次描述了酿酒酵母中荆芥内酯的从头合成，实现可持续和可扩展的生产。荆芥内酯的生产需要引入八个外源基因，包括细胞色素 P450 geraniol-8-hydroxylase，这是异源途径的瓶颈。geraniol-8-hydroxylase 和细胞色素 P450 还原酶变体的组合评估以及拷贝数变化被用来克服这一瓶颈。我们发现几种还原酶提高了羟基化活性，增加 geraniol-8-hydroxylase 基因拷贝数提高了 8-hydroxygeraniol 滴度。不需要的代谢物的积累意味着碳通过该途径的效率低下。使用先前显示使用单萜中间体作为底物的天然酵母老黄色酶，删除了两个同源物。这些缺失增加了 8-羟基香叶醇的产量，产生 3.10 mg/L/OD微量滴定板中来自单糖的_{600荆芥内酯。}这种优化的途径将有利于开发用于扩大荆芥内酯生产的高产菌株。

更新日期：2021-11-19

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