Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally significant compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. We used transcriptomic analysis to identify glycosyltransferases that were upregulated in response to biosynthetic intermediates and produced plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produced a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases did not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

中文翻译：

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基因组工程本氏烟草中单萜吲哚生物碱生物合成的重建

单萜吲哚生物碱 (MIA) 是一类多样化的植物天然产物，其中包括许多具有药用价值的化合物。我们着手从本氏烟草的中枢代谢重建胡豆苷（所有 MIA 的关键中间体）的途径。该宿主的一个缺点是其丰富的背景代谢导致一些异源产生的分子的衍生化。我们使用转录组分析来鉴定响应生物合成中间体而上调的糖基转移酶，并产生在编码它们的基因中具有靶向突变的植物系。这些品系中早期 MIA 途径的表达产生了更有利的产品概况。Strictosidine 生物合成成功重组，通过 14 种酶的共表达获得最佳产量，其中一种来自荆芥（catmint）的主要乳胶蛋白样酶（MLPL）对于提高环烯醚萜通路的通量至关重要。内源性糖基转移酶的去除不会影响胡豆苷的产量，突出表明途径酶向稳定的生物合成中间体的代谢通量最大限度地减少了对宿主内源性代谢进行改造的需要。在植物中生产胡豆苷扩大了适用于生物合成的 MIA 产品的范围。强调途径酶到稳定的生物合成中间体的代谢通量最大限度地减少了对宿主内源代谢进行改造的需要。在植物中生产胡豆苷扩大了适用于生物合成的 MIA 产品的范围。强调途径酶到稳定的生物合成中间体的代谢通量最大限度地减少了对宿主内源代谢进行改造的需要。在植物中生产胡豆苷扩大了适用于生物合成的 MIA 产品的范围。