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 a platform for de novo nepetalactone production in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introduction of eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, which represented the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, as well as copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity, with a higher geraniol-8-hydroxylase ratio further increasing 8-hydroxygeraniol titers. Another roadblock was the accumulation of an unwanted metabolite that implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologs were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in a final de novo accumulation of 3.10 mg/L/OD₆₀₀ of nepetalactone from simple sugar in microtiter plates. Our pathway optimization will aid in the development of high yielding monoterpene S. cerevisiae strains.

中文翻译：

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用于从头合成驱虫剂 - 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，但其植物积累量低限制了其作为替代驱虫剂的商业可行性。在这里，我们描述了在酿酒酵母中从头生产荆芥内酯的平台，实现可持续和可扩展的生产。荆芥内酯生产需要引入八种外源基因，包括细胞色素 P450 香叶醇-8-羟化酶，这是异源途径的瓶颈。使用香叶醇-8-羟化酶和细胞色素 P450 还原酶变体以及拷贝数变异的组合评估来克服这一瓶颈。我们发现几种还原酶提高了羟基化活性，更高的香叶醇-8-羟化酶比率进一步增加了 8-羟基香叶醇的滴度。另一个障碍是不需要的代谢物的积累，这意味着碳通过该途径的通道效率低下。对于先前显示使用单萜中间体作为底物的天然酵母老黄色酶，两个同源物都被删除。在微量滴定板中从单糖中从头积累 3.10 mg/L/OD ₆₀₀荆芥内酯。我们的通路优化将有助于开发高产单萜酿酒酵母菌株。