Investigating natural and novel PKS-NRPS genes

许多生物体产生次级代谢物，这些次级代谢物是通常具有复杂结构的化合物，这些化合物对于产生它们的生物体的正常生长和发育是不必要的。聚酮化合物是一类由细菌和真菌产生的次生代谢产物。一些PK是有毒的，必须避免使用，但其他PK具有有用的药理学特性，用于人类和兽医学以及农业。这些包括用于对抗感染的各种抗生素，以及减少胆固醇合成的他汀类药物。本项目旨在阐明丝状真菌制造PK的方式。许多基因，编码许多酶，有助于指定一个特定的PK结构。这些酶中最重要的是聚酮合酶（PKS），其组装碱性碳骨架，并且该骨架被其他酶的活性修饰。该项目涉及一类特殊的PKS蛋白，这些蛋白具有完整的非核糖体肽合酶（NRPS）模块，其功能是将特定的氨基酸添加到PK骨架。这项工作的最终目标是能够操纵参与PK合成的基因，以产生具有理想生物学特性的新型化学品。要做到这一点，需要了解各种酶和复杂酶如PKS和PKS-NRPS内的功能结构域。这种理解可以通过孤立地观察系统各部分的活动以及通过混合和匹配组件来查看结果来获得。由于PKS-NRPS是非常大的多功能蛋白质，因此很难操纵编码它们的基因。为了准备这个项目，我们开发了一个系统，该系统简化了从基因片段构建大基因和将最终产物转移到质粒载体中的过程，在质粒载体中需要进行进一步的分析。该系统还简化了含有来自不同来源的DNA的嵌合基因的构建（和转移）。这将使我们能够调查PKS-NRPS活动的各个方面。首先，我们将能够从几个基因中发现NRPS模块的功能，其中一些基因只存在于基因组序列中。我们将把NRPS模块连接到已经去除了自己的NRPS模块的PKS上，看看有什么氨基酸被添加到PK骨架上。使用相同的混合匹配方法，但在更精细的尺度上，我们将解剖NRPS模块，以发现氨基酸特异性所在的位置。我们希望能够从这种实验方法进展到仅通过仔细检查DNA序列来预测氨基酸特异性。嵌合基因在不具有剪裁酶的宿主生物体中的活性将产生在自然界中通常不会遇到的新产物。我们将通过将嵌合基因直接放入分离原始PKS-NRPS的基因簇中来产生额外的新化合物。所有新的化合物都具有直接或经合理修饰后用于生物干预的潜力。在另一系列实验中，我们将比较非常相似的PKS-NRPS对，以发现结构差异如何影响它们的功能。其中一对的差异仅在于是否存在一个短的蛋白质片段，该片段可能赋予具有该片段的酶额外的活性。我们将寻找具有额外片段的基因和去除片段的相同基因表达产物的结构差异。已知另一对产生的产物仅在PK链的长度上不同。通过比较我们预测的非常相似的基因，我们希望确定负责“编程”酶所经历的循环数（决定链长）的序列，我们将通过将基因从pentaketide生产者转换为hexaketide生产者来测试这一点。

Many organisms produce secondary metabolites, which are compounds, often with complicated structures, that are not necessary for the normal growth and development of the organism that produces them. Polyketides (PKs) are a class of secondary metabolites that are produced by many bacteria and fungi. Some PKs are toxic and must be avoided, but others have useful pharmacological properties and are used in human and veterinary medicine and agriculture. These include various antibiotics used in combating infection, and the statins, which reduce cholesterol synthesis. This project seeks to shed light on the way in which PKs are made by filamentous fungi. Many genes, encoding many enzymes, contribute to specifying a particular PK structure. Foremost among these enzymes is the polyketide synthase (PKS), which assembles a basic carbon backbone, and this backbone is modified by the activities of other enzymes. This project concerns a particular class of PKS proteins, ones that have an integral non-ribosomal peptide synthase (NRPS) module, whose function it is to add a specific amino acid to the PK backbone. An ultimate objective of the work is to be able to manipulate the genes involved in PK synthesis in order to generate novel chemicals with desirable biological properties. To do this requires understanding of the various enzymes and functional domains within complex enzymes like PKSs and PKS-NRPSs. Such understanding can be obtained by looking at the activities of parts of the system in isolation and by mixing and matching components to see what results. Because PKS-NRPSs are very large multifunctional proteins it is very difficult to manipulate the genes that encode them. In preparation for this project we have developed a system that simplifies both the construction of large genes from gene fragments and the transfer of the final product into the plasmid vectors in which they are required for further analysis. The system also simplifies the construction (and transfer) of chimaeric genes containing DNA from different sources. This will enable us to investigate various aspects of the activities of PKS-NRPSs. First, we will be able to discover the function of NRPS modules from several genes / some of which are only known to exist from genome sequences. We will attach NRPS modules to a PKS that has had its own NRPS module removed and see what amino acid gets added to the PK backbone. Using the same mix-and-match approach, but on a finer scale, we will then dissect the NRPS modules to discover where the amino-acid specificity resides. We would hope to be able to progress from this experimental approach towards making predictions of amino-acid specificity from scrutiny of DNA sequences alone. Activity of the chimaeric genes in a host organism that does not have the tailoring enzymes will produce novel products that are not usually encountered in nature. We will produce additional novel compounds by putting the chimaeric genes directly into the gene cluster from which the original PKS-NRPS was isolated. All novel compounds have the potential for use in biological interventions directly or after rational modification. In another series of experiments we will compare pairs of very similar PKS-NRPSs to discover how differences in structure affect their function. One pair differs only in the presence or absence of a short protein segment that may confer an extra activity on the enzyme that has it. We will look for differences in structure of the products of expressing the gene that has the additional segment and the same gene with segment removed. Another pair is known to produce products that differ only in the length of the PK chain. By comparing what we predict to be very similar genes we expect to identify sequences responsible for 'programming' the number of cycles that the enzyme goes through (which determines chain length), and we will test this by converting the gene from a pentaketide producer to a hexaketide producer.