建立土曲霉高效基因打靶平台的研究

Aspergillus terreus is a kind of important filamentous fungus, which is mainly used in the industrial production of itcaonic acid - one of the most significant bio-based platform chemicals.Currently, itaconic acid yield is primarily improved through traditional mutagenesis (UV , radiation and chemical), but no big breakthrough has been made. Studies towards functional genomics and genetic engineering of A. terreus have become very essential with determination of its complete genome sequence. However，the low efficiency of homologous recombination and few suitable selection markers for A. terreus have hampered the application of gene targeting technology.Therefore, it is of great significance to develop a highly efficient gene-targeting platform for constructing a superior A.terreus strain with higher itaconic acid producing ability.. In this study, we attempt to improve theefficiency of homologous recombination by disrupting the ligD gene which is involved in the final step of nohomologousend joining in order to make genetargeting technologyapplicable, efficient, and easyin A.terreus; To overcome the limited availability of selection markers in A.terreus, we plan to develop a transformation system based on the bidirectional selection marker pyrG gene,establish a modified Cre/loxP recombination system for marker recycling, andwill create the conditions for the genetic manipulation of multi-gene and multi-site integration. This efficient gene-targeting system will be constructed inA.terreuswith higher itaconic acid yield, which will not onlylay the foundation for studying its physiological characteristicsby functional genomics，but also provide guidance for the mechanisms of itaconic acid accumulation and further genetic engineering of A. terreus.

土曲霉是一种重要的丝状真菌，能工业化生产重要的平台化合物-衣康酸。目前主要通过传统诱变方式提高衣康酸生产能力,长时间来都未能取得重大突破。随着基因组序列的公布，土曲霉功能基因组学和基因工程改造研究显得尤为重要。但是由于土曲霉同源重组效率低下、筛选标签有限，严重限制了基因打靶技术的应用。因此，开发一个高效的基因打靶平台对于构建优质高产衣康酸菌株具有重要意义。. 本项目拟通过敲除控制非同源重组的ligD基因来提高土曲霉的同源重组效率，使基因打靶变得高效可行；建立基于双向筛选标签pyrG的转化体系，然后通过改良的Cre-loxP重组系统切除细胞内pyrG标签，实现筛选标签的循环利用，为多基因、多位点的遗传操作创造条件。在衣康酸高产土曲霉中建立这个高效的基因打靶平台，不仅可以为功能基因组学研究土曲霉的生理特性打下基础，还可以为解析衣康酸积累机制和进一步基因工程改造提供指导。

土曲霉是一种具有重要经济价值的丝状真菌，已经被应用于衣康酸和洛伐他汀的工业生产。在功能基因组分析和菌株改造研究过程中，一系列的遗传改造是不可避免的。然而，系统性的土曲霉遗传改造受到同源重组效率低下和筛选标签不足的严重限制。为了解决该问题，我们分别在产衣康酸和洛伐他汀的工业生产土曲霉菌株中构建了一个高效的土曲霉基因打靶平台。首先，通过敲除ku80基因，将同源重组效率显著提高到95%。随后，通过敲除pyrG基因，构建了一个基于尿嘧啶营养缺陷型的双向筛选遗传转化系统，为筛选标签切除奠定基础。最后，通过直接向原生质体中引入商业化的Cre重组酶的方式，应用Cre/loxP位点特异性重组系统在体内高效地切除pyrGAn筛选标签。这将为在土曲霉中进行连续的多基因、多位点遗传改造提高一个高效的操作平台。.这一高效的基因打靶平台促进了本课题组在土曲霉产衣康酸或他汀药物方面的研究。 首先，利用该平台鉴定了高产衣康酸土曲霉菌株CIAT01的衣康酸降解途径，为进一步菌株改良奠定基础。另外，利用该平台对产洛伐他汀土曲霉菌株HZ01进行代谢工程改造，构建了能直接发酵生产辛伐他汀前体物monacolin J的土曲霉细胞工厂，大大简化了降血脂药物辛伐他汀的生产工艺。由此可见，对于进一步研究土曲霉中高附加值化合物的生物合成机制和菌株改造，本项目构建的高效遗传操作平台是一个重要的前期基础。

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