Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

中文翻译：

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通过酵母基因组的系统重构和反义转录调控工具包实现天麻素的高滴度生产


天麻素是一种酚苷，是天麻的重要成分，天麻以其镇静、催眠、抗惊厥和神经保护活性而闻名。在微生物宿主中对植物天然产物进行工程异源生产是植物提取的一种安全、经济高效且可扩展的替代方案。在这里，我们介绍了一种工程化的 Yarrowia lipolytica 酵母的构建，该酵母实现了天麻素的高滴度生产。我们通过增强莽草酸通路的通量和优化葡萄糖基转移系统，系统地重构了酵母基因组。我们在酵母基因组上引入了五十多种基因修饰，包括酶筛选、限速步骤缓解、启动子选择、基因组整合位点优化、竞争途径下调和消除天麻素降解。同时，我们开发了一种铜诱导的反义转录调控 （CATR） 工具。开发的 CATR 工具包通过在 Y. lipolytica 中添加铜实现了紫罗兰素合成的动态抑制和激活。该策略进一步用于动态调节丙酮酸激酶节点，以有效地将糖酵解通量重定向到莽草酸通路，同时保持细胞以适当的速率生长。综上所述，这些努力在 5 L 生物反应器中产生 9477.1 mg/L 的天麻素和 13.4 g/L 的天麻素，葡萄糖产量为 0.149 g/g，突出了微生物发酵中大规模和可持续生产天麻素的潜力。