Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼ 5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation.

中文翻译：

---

酿酒酵母中植物异育亨宾和棒南青生物碱的工程生物合成

单萜吲哚生物碱（MIA）是一类天然产物，由数千种结构独特的生物活性化合物组成，具有显着的治疗价值。由于与本地植物物种分离和这些结构复杂分子的有机合成相关的困难，非常需要使用工程宿主微生物生产 MIA。在这项工作中，我们报告了完全整合的酿酒酵母菌株的工程设计，该菌株允许从头获得胡豆素，这是数千种浓度为 30-40 mg/L 的 MIA 的通用前体。优化工作基于先前报道的酵母菌株，该酵母菌株被设计为通过线粒体中甲羟戊酸途径的区室化来产生高滴度的单萜前体香叶醇。我们的方法包括使用 CRISPR-dCas9 干扰来识别对单萜滴度产生负面影响的线粒体二磷酸转运蛋白，然后进行基因失活；增加细胞呼吸和线粒体生物发生的转录调节因子的过度表达。菌株构建包括在各种组成型和诱导型启动子的作用下将编码 MIA 生物合成酶和辅助酶的基因战略性整合到基因组中。在成功从头生产胡胡豆素后，通过引入额外的下游酶，在宿主中重构了属于异育亨宾和棒南花碱家族的复杂生物碱。我们证明蛇纹石/阿尔斯通碱对可以以~ 5 mg/L 的滴度生产，而帽柱木碱的前体冠花碱可以以〜1 mg/L 的滴度生产。喂食卤化色胺会导致两个科中生物碱类似物的生物合成。总的来说，我们的酵母菌株代表了一个很好的起点，可以进一步设计该途径中的生物合成瓶颈，并通过微生物发酵获得更多的 MIA 和类似物。