Cordycepin, a nucleoside analog, is widely used in medicine and health products. However, the production of cordycepin from Cordyceps militaris faces the challenges of low productivity and high rate of greenhouse gas emissions. In this study, by optimizing the cordycepin biosynthesis pathway through promoter combination, Kozak sequence, and enzyme fusion, enhancing the methanol assimilation capacity in peroxisomes, adjusting the synthesis of NADPH and ATP, and combining the enhanced supply of adenosine and 3′-AMP, the cordycepin high-yield strain Pp29 was constructed, which produced 1551.44 mg/L cordycepin by shake-flask fermentation. In fed-batch fermentation, Pp29 achieved the highest yield (8.11 g/L, 67.64 mg/g DCW, and 1.35 g/L/d) to date in 10 L fermenter, and the CO2-eq emissions were 1.9–17.3 times lower than C. militaris and other yeast systems. This study provide basis for Pichia pastoris to be used as chassis cell for synthesizing cordycepin and other nucleoside analogs by methanol as carbon source.

中文翻译：

---

使用工程化的 Pichia pastoris 细胞工厂从甲醇中低碳和过量生产虫草素


虫草素是一种核苷类似物，广泛用于医药和保健品。然而，从蛹虫夏草生产冬虫夏草素面临着生产力低和温室气体排放率高的挑战。本研究通过启动子组合、Kozak 序列和酶融合优化虫草素生物合成途径，增强过氧化物酶体中甲醇的同化能力，调节 NADPH 和 ATP 的合成，结合腺苷和 3′-AMP 的增强供应，构建虫草素高产菌株 Pp29，通过摇瓶发酵生产 1551.44 mg/L 虫草素。在补料分批发酵中，Pp29 在 10 L 发酵罐中实现了迄今为止最高的产量（8.11 g/L、67.64 mg/g DCW 和 1.35 g/L/d），CO2-eq 排放量比 C. militaris 和其他酵母系统低 1.9-17.3 倍。本研究为毕赤酵母作为底盘细胞以甲醇为碳源合成虫草素和其他核苷类似物提供了基础。