皮状丝孢酵母群体效应分子的发现及其对形态转换和油脂合成的调控机制研究

The dimorphism fungi Trichosporon cutaneum was an important candidate for microbial oil production for its high oil content and can hydrolyze lignocellulose. Our previous study showed that the quorum sensing occurs in the fermentation broth and quorum-sensing molecules (QSMs) produced by T. cutaneum are able to regulate the cell morphology and lipids synthesis. In present study, we applied the technology of EESI-MS, LC-MS and GC-MS to analyze and separate the QSMs based on the quorum sensing of T. cutaneum which elicited by increasing the inoculum size. The effect of QSMs on cell morphology and lipids synthesis was further investigated by injecting the additional QSMs. On the basis of above results, we applied the RNA-seq technique to compare the gene expression difference between the yeast and pseudohyphal forms of T. cutaneum cell under the quorum sensing condition. Additionally, the real-time quantitative PCR method was also used to check the differentially expressed gene so as to elucidate the molecular mechanism of morphologic transition and lipids synthesis of T. cutaneum which caused by quorum sensing. The results of this study were significant no only to discover the T. cutaneum morphologic transition pattern, but also enrich the theory of morphologic transition and improve the fermentation level of dimorphism fungi.

皮状丝孢酵母(Trichosporon cutaneum)为二型态真菌，其含油量高并可利用木质纤维素，是微生物油脂生产的重要候选菌种。我们在前期研究中，发现皮状丝孢酵母具有群体效应，同时能产生群体效应分子(QSMs)来调控其细胞形态和油脂合成。本项目以皮状丝孢酵母为对象，首先在诱导其产生群体效应的基础上利用电喷雾萃取电离-质谱、液质联用和气质联用等技术手段鉴定皮状丝孢酵母发酵液中的QSMs。然后通过外添法来确证QSMs对皮状丝孢酵母细胞形态转换与油脂合成的作用。最后利用转录组测序(RNA-seq)技术比较皮状丝孢酵母产生群体效应时的两种细胞形态间的基因表达差异，并利用实时荧光定量PCR技术进行验证，以揭示QSMs介导的皮状丝孢酵母形态转换及油脂合成的分子机制。该研究结果不仅能阐明皮状丝孢酵母的形态转换和油脂合成规律，而且对于丰富二型态真菌形态转换理论和提高二型态真菌的发酵水平具有重要意义。

皮状丝孢酵母（Trichosporon cutaneum）为二型态真菌，其含油量高并可利用木质纤维素，是微生物油脂生产的重要候选菌种。本项目以皮状丝孢酵母为对象，研究群体效应分子（QSMs）对皮状丝孢酵母细胞形态转换与油脂合成的作用及其分子机制。课题组首先验证了皮状丝孢酵母群体感应现象的存在，并利用HPLC和GC-MS等对潜在QSMs进行初步分离。在此基础上，分别将不同的群体效应分子（法尼醇、色醇和酪醇）添加到培养基中以研究其对细胞形态的影响。结果表明，法尼醇、色醇和酪醇均对皮状丝孢酵母的细胞形态转换有影响。其中法尼醇的最佳添加量为25μmol/L，与添加时间无关，但与氮源浓度关系密切。在低浓度氮源条件下，法尼醇的添加对细胞形态和油脂产量无显著差异，但当培养基中酵母提取物浓度达到3g/L时，法尼醇能明显改变皮状丝孢酵母的细胞形态和促进油脂的合成。特别是在5g/L的条件下，添加法尼醇能使胞内油脂产量提升65%。通过转录组分析表明，法尼醇可能主要是通过影响MAPK和cAMP-PKA信号途径中的与菌丝形成有关的基因表达而调控细胞形态的。此外，法尼醇还能够降低异柠檬酸脱氢酶的表达使胞内柠檬酸积累，裂解酶的作用下分解成乙酰-CoA和草酰乙酸，并能强化磷酸戊糖途径，从而有利于胞内脂肪酸的合成。该研究结果不仅揭示了QSMs介导的皮状丝孢酵母形态转换及油脂合成的分子机制，而且对于丰富二型态真菌形态转换理论和提高二型态真菌的发酵水平具有重要意义。