Featured Laboratory for Biosynthesis and Target Discovery of Active Components of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Binzhou Medical University, Yantai, China.
Appl Environ Microbiol. 2024 Oct 23;90(10):e0119124. doi: 10.1128/aem.01191-24. Epub 2024 Sep 16.
Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from , 5--jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and mA writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5--jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5--jinkoheremol synthase TPS18, resulting in the variant TPS18, with the most improved properties. Finally, the titer of 5--jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5--jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5--jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5--jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5--jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.
利用微生物底盘的合成生物学正在成为生产天然化学品的有力工具。在本研究中，我们构建了一个微生物平台，用于高水平生产具有强杀菌活性的倍半萜金合霍雷姆醇。首先，在工程酵母中优化了甲羟戊酸和甾醇生物合成途径，以增加前体法呢基焦磷酸的生物合成代谢通量。然后，在酵母中实施了转录因子 Hac1 和 mA 书写器 Ime4 为基础的代谢工程策略，以进一步提高 5--金合霍雷姆醇的产量。接下来，进行了蛋白质工程改造，以提高 5--金合霍雷姆醇合酶 TPS18 的催化活性和增强其稳定性，得到了具有最佳性能的变体 TPS18。最后，通过在摇瓶培养中优化碳源，将 5--金合霍雷姆醇的产量提高到 875.25mg/L。据我们所知，这是首次构建高效微生物细胞工厂，用于可持续生产这种抗真菌倍半萜的研究。
重要性：生物杀菌剂代表了防治作物真菌病害的一种新的可持续工具。然而，由于生物杀菌剂生产成本高，其使用并不像预期的那样普遍。利用微生物底盘的合成生物学正在成为生产天然化学品的有力工具。我们之前鉴定了一种有前途的倍半萜类生物杀菌剂，5--金合霍雷姆醇。在这里，我们构建了一个微生物平台，用于这种化学物质的高水平生产。首先采用萜烯生物合成途径的代谢工程来增加 5--金合霍雷姆醇生产的代谢通量。然而，关键酶 TPS18 的有限催化活性限制了 5--金合霍雷姆醇的进一步产量。通过使用蛋白质工程，我们提高了其催化效率，并结合调节因子的优化，实现了 5--金合霍雷姆醇的最高产量。我们的工作有助于更有效地大规模生产这种抗真菌倍半萜。
