聚酮合酶pks7和pks11对海洋草酸青霉合成Oxalicumone A及其环境适应性的调控机制研究

Fungal polyketide synthases (PKSs), whose recombinant or disrupted domains may form new compounds, not only regulate the biosynthesis of fungal polyketides, but also involve in regulation of fungal development and environmental adaptation. But roles of many fungal PKSs, such as PKSs in Penicillium oxalicum, are unknown, which limits the development and usage of new polyketides. Oxalicumone A (POA1), a novel dihydrothiophene-condensed cytotoxic chromone, was extracted from P. oxalicum SCSGAF 0023 isolated from gorgonian Muricella flexuosa in the South China Sea by ourselves and might perform as a lead compound for further study. However, its low yield is not enough for us to optimize the structure and carry out further pharmacological studies.Therefore, the subject focuses on the functional characterizations of two PKSs (pks7 and pks11) ,which take part in the biosynthesis of POA1 in P.oxalicum SCSGAF 0023. Recombinant strains in yeast and disrupted and over-expressed strains in P. oxalicum SCSGAF 0023 are obtained in order to analyze the differences of second metabolites, multi-phenotypic traits, transcriptional expression and the acitivies of the related enzymes. The differences demonstrate the molecular mechanisms of pks7 and pks11 in regulating the biosynthesis of POA1, fungal development and environmental adaptation in P. oxalicum SCSGAF 0023.The results of above studies may be helpful for the explanations of the biosynthesis of dihydrothiophene-condensed chromones, the improvement of POA1 production and the research on the correlation between secondary metabolites and environmental adaptation.

聚酮合酶PKSs参与聚酮化合物的合成和微生物生长发育及环境适应性的调控，其结构域改变可产生新化合物。但许多真菌（如草酸青霉）PKSs功能不明确，限制了新化合物的开发利用。Oxalicumone A (POA1)是本课题组从海洋草酸青霉SCSGAF 0023中分离的二氢噻吩并色酮骨架的新化合物，可作为候选的抗肿瘤先导化合物；但产量低而不利于后续结构优化及药理研究。本项目拟对POA1合成相关的pks7和pks11进行酵母异源表达和SCSGAF 0023单/双基因敲除及过表达，分析各菌次级代谢产物差异，明确合成POA1的中间体；比较敲除、过表达及原始菌的生长发育和胁迫环境适应性，测定相关基因转录及蛋白酶活，揭示2个PKSs对SCSGAF 0023生长发育与环境适应性的调控机制。该研究将为二氢噻吩并色酮骨架合成提供突破点，为提高POA1产量及研究次级代谢产物与生态环境适应性的相互关系提供理论依据。

聚酮合酶PKSs参与聚酮化合物的合成和微生物生长发育及环境适应性的调控，其结构域改变可产生新化合物。但许多真菌PKSs功能不明确，限制了新化合物的开发利用。Oxalicumone A (POA1)是本课题组从海洋草酸青霉SCSGAF0023中分离的二氢噻吩并色酮骨架的新化合物，可作为候选的抗肿瘤先导化合物；但产量低而不利于后续结构优化及药理研究。因此，本项目首先通过核糖体工程技术结合物理化学诱变筛选出3株POA1产量提高10%以上的突变株，并通过发酵条件优化实验发现C/N、氮源种类显著影响POA1产量，为提高低产量的活性产物提供技术支持及理论基础。其次，通过在草酸青霉SCSGAF0023中建立农杆菌介导真菌转化体系，根据同源重组原理获得分别位于细胞质、线粒体、细胞质膜上的3个聚酮合酶（pks3、pks7、pks11）的敲除菌株，比较敲除菌株与野生菌株在生长发育、环境适应性及POA1合成上的差异，发现草酸青霉SCSGAF0023聚酮合酶pks3、pks7对POA1合成、真菌生长发育及环境适应性无影响，而pks11正向调控真菌产孢及POA1合成。最后，以pks3为例，在球孢白僵菌中验证与草酸青霉SCSGAF0023同源的聚酮合酶功能。通过比较野生菌株、敲除菌株、回补菌株间的生长及次级代谢产物产量差异，发现与草酸青霉SCSGAF0023的pks3同源的球孢白僵菌pks正向调控真菌的萌发、产孢、耐热及耐紫外能力，负向调控真菌氧化胁迫、胞壁干扰、耐冷及合成linoleic acid能力。进一步qRT-PCR监测效应基因的转录表达情况发现球孢白僵菌pks敲除株通过抑制产孢相关转录因子flbA和疏水蛋白hyd1表达而抑制产孢，而通过提高过氧化氢酶和超氧化歧化酶、抑制几丁质酶的表达而提高真菌对氧化胁迫剂胞壁干扰的抗性。这些结果为提高POA1产量及研究聚酮合酶、次级代谢产物、生态环境适应性间的相互关系提供理论依据。