Engineering the Biosynthesis of Cytochalasans for the Development of New Molecular Tools

细胞chalasans是一种重要的真菌天然产物，具有广泛的生物活性。这些化合物可以发展成为一类非常有用的分子工具，但它们很难从自然界或通过合成手段获得。然而，现在对它们的生物合成已经有了足够的了解，可以开始合理地设计它们。在这个项目中，我们的目标是在真菌中使用新的合成生物学方法来操纵生物合成基因，这些基因参与了许多细胞chalasans的生产。我们将从设计宿主生物开始，使我们能够进行更多的基因操作，包括联合敲除和异源表达实验。我们将尝试在链长和甲基化模式方面系统地重新设计细胞松弛素的聚酮部分。我们还将通过重建其生物合成蛋白质来改变氨基酸伙伴。我们将通过混合和匹配来自不同途径和真菌的剪裁基因来设计后期的剪裁步骤。最后，我们将结合突变合成方法和半合成方法来扩大有用的细胞查拉桑分子工具的范围，这些工具将被CytoLabs联盟的其他人用于探索细胞查拉桑在体内和体外对肌动蛋白的活性；并用于确定新的非肌动蛋白靶点。我们将向CytoLabs提供化合物，供其他人用于生物检测，并与合成化学家合作，对合成中间体进行生物转化，特别是在后期裁剪反应方面。我们将与德国科学家合作进行真菌基因组和转录组测序，并与法国科学家合作扩大固体发酵的规模。该项目将致力于真菌天然产物途径的合理操纵和开发有效的合成生物学工具，以制造有用的材料。这些将在CytoLabs联盟中发挥关键作用，在探索细胞生物学、生物间相互作用、植物病理学和体外酶学方面发挥重要作用。

Cytochalasans are important fungal natural products which have a very wide range of potent bioactivities. These compounds could be developed into a class of very useful molecular tools, but they are hard to access from nature or by synthetic means. However, enough is now known of their biosynthesis to start to be able to rationally engineer them. In this project we will aim to use new synthetic biology methods in fungi to manipulate biosynthetic genes involved in the production of a number of cytochalasans. We will start by engineering host organisms to allow us to do more genetic manipulations including combined knockout and heterologous expression experiments. We will attempt to systematically re-engineer the polyketide moiety of cytochalasins in terms of chain-length and methylation pattern. We will also change the amino acid partner by rebuilding its biosynthetic proteins. We will engineer late stage tailoring steps by mixing and matching tailoring genes from diverse pathways and fungi. Finally, we will combine mutasynthesis methods with semi-synthesis to expand the range of useful cytochalasan molecular tools which will be used by others in the CytoLabs consortium for exploring cytochalasan activity in vivo and in vitro vs actin; and for the determination of new non-actin targets. We will supply compounds to the CytoLabs compound collection for use by others in bio-assays, and also work with synthetic chemists for the biotransformation of synthetic intermediates, particularly in respect of late-stage tailoring reactions. We will collaborate with scientists in Germany for fungal genome and transcriptome sequencing, and with scientists in France for scale-up of solid fermentations. The project will work towards the rational manipulation of fungal natural products pathways and development effective synthetic biology tools for the manufacture of useful materials. These will play a key role in the CytoLabs consortium with important uses in exploring cell biology, inter-organism interactions, plant pathology and in vitro enzymology.