冠突散囊菌莽草酸合成途径功能研究

The shikimate pathway is an important pathway to synthesize shikimic acid and its derivatives with important medicinal value. In bacteria, reactions two to six of the shikimate pathway are controled by five separate genes, but in fungi, a Arom fusion gene serves the same purpose. The genome analysis of Eurotium cristatum shows the organism including two shikimate pathways, the first pathway using Erythrose 4-phosphate and Phosphoenolpyruvate as the substrate is regulated by Arom fusion gene, the second pathway using quinate as the substrate is regulated by QuiB、aroD and aroE, which is never reported in fungi. In this study, Arom, QuiB, aroD and aroE will be deleted using gene knockout. The correlation of two pathways is revealed by the analysis of shikimate acid yield of mutants and wild type strain; the regulation mechanism of two pathways is also showed by transcriptome analysis. The study will discover a new shikimate biosynthesis pathway and its regulation mechanism, and also get some potential strains with the shikimate acid production, these results are of great theoretic value and practical significance.

莽草酸途径是合成具有重要药用价值的莽草酸及其衍生物的关键途径。在真菌中莽草酸途径的第二到第六步由Arom融合基因调控，而细菌的每步反应由单一基因调控。本研究对冠突散囊菌基因组分析发现它既有以赤藓糖-4-磷酸和磷酸烯醇式丙酮酸为底物催化莽草酸形成的融合基因（AROM）途径，又有以奎宁酸为底物，QuiB、aroD和aroE三个基因分步催化形成莽草酸的途径，后者在真菌中没有报道。本研究采用基因敲除技术获得Arom、QuiB、aroD和aroE基因的突变株，通过突变株和野生型菌株的莽草酸产量分析，揭示两条途径对莽草酸合成的影响及相互关系；通过突变株及野生型菌株的转录组分析，揭示冠突散囊菌莽草酸途径关键基因表达对莽草酸合成的调控机制。研究结果可发现真菌中新的莽草酸合成途径及其调控机制，还可获得具有生产莽草酸潜力的菌株，具有理论价值和实践意义。

莽草酸途径是合成具有重要药用价值的莽草酸及其衍生物的关键途径。冠突散囊菌的基因组分析显示它具有以赤藓糖-4-磷酸和磷酸烯醇式丙酮酸为底物形成莽草酸的aroF、aroM和aroC基因，也有以奎宁酸为底物，生产莽草酸的QutB、aroD和aroE基因。本项目为拓展生产莽草酸的微生物资源，对冠突散囊菌进行了转录组和代谢组联合分析，共获得差异表达基因4636个，11类代谢物，它们分布在79条KEGG路径。项目对莽草酸途径部分基因及其酶活进行了检测，结果显示了冠突散囊菌的莽草酸途径相关基因和酶的运行特性。最后，我们开展了莽草酸途径相关基因aroF、aroM、aroC、QutB和aroQ的功能研究，其中aroF、aroM、aroC基因均成功构建了敲除载体，但未获得敲除株。QutB获得了敲除株，aroQ获得了敲除株和超表达菌株，并以野生型为对照、对突变株和超表达的代谢物进行了检测，检测结果表明QutB的敲除有利于绿原酸等功能有机酸的形成。aroQ的结果还在检测中。总之，本项目解析了冠突散囊菌有性发育早期的代谢物组成，对形成莽草酸的相关基因开展了功能研究，这为认识真菌莽草酸途径和冠突散囊菌的保健功能提供了基础。