Insight into cordycepin biosynthesis of <i>Cordyceps militaris</i>: Comparison between a liquid surface culture and a submerged culture through transcriptomic analysis

Cordyceps militaris produces cordycepin, which is known to be a bioactive compound. Currently, cordycepin hyperproduction of C. militaris was carried out in a liquid surface culture because of its low productivity in a submerged culture, however the reason was not known. In this study, 4.92 g/L of cordycepin was produced at the 15th day of C. militaris NBRC 103752 liquid surface culture, but only 1 mg/L was produced in the submerged culture. RNA-Seq was used to clarify the gene expression profiles of the cordycepin biosynthetic pathways of the submerged culture and the liquid surface culture. From this analysis, 1036 genes were shown to be upregulated and 557 genes were downregulated in the liquid surface culture compared with the submerged culture. Specifically, adenylosuccinate synthetase and phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthase in purine nucleotide metabolism were significantly upregulated in the liquid surface culture. Thick mycelia formation in the liquid surface culture was found to induce the expression of hypoxia-related genes (GABA shunt, glutamate synthetase precursor, and succinate-semialdehyde dehydrogenase). Cytochrome P450 oxidoreductases containing heme were also found to be significantly enriched, suggesting that a hypoxic condition might be created in the liquid surface culture. These results suggest that hypoxic conditions are more suitable for cordycepin production in the liquid surface culture compared with the submerged culture. Our analysis paves the way for unraveling the cordycepin biosynthesis pathway and for improving cordycepin production in C. militaris.蛹虫草（Cordyceps militaris）可合成虫草素（cordycepin），该物质是一类已被广泛研究的生物活性化合物。目前，由于蛹虫草在深层发酵培养中的虫草素产量极低，其虫草素的高产制备多采用表面液体培养策略，但该培养方式提升产量的具体机制尚未明确。本研究以蛹虫草NBRC 103752菌株为研究材料，发现其表面液体培养体系在培养至第15天时可积累4.92 g/L虫草素，而深层发酵培养体系仅能产生1 mg/L虫草素。为阐明深层发酵与表面液体培养下虫草素生物合成通路的基因表达差异，本研究采用RNA测序（RNA-Seq）技术对两种培养体系的转录组进行分析。结果显示，相较于深层发酵培养，表面液体培养体系中有1036个基因显著上调，557个基因显著下调。具体而言，嘌呤核苷酸代谢通路中的腺苷酸琥珀酸合成酶与磷酸核糖氨基咪唑琥珀基甲酰胺（SAICAR）合成酶在表面液体培养体系中显著上调。研究发现，表面液体培养中形成的致密菌丝体可诱导缺氧相关基因的表达，包括γ-氨基丁酸旁路（GABA shunt）、谷氨酸合成酶前体以及琥珀酸半醛脱氢酶。此外，携带血红素辅基的细胞色素P450氧化还原酶也呈现显著富集特征，这提示表面液体培养环境中可能存在缺氧微环境。上述结果表明，相较于深层发酵培养，缺氧条件更有利于表面液体培养体系中的虫草素合成。本研究的转录组分析为解析虫草素生物合成通路以及优化蛹虫草的虫草素产量提供了重要的理论依据与研究方向。