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Investigating natural and novel PKS-NRPS genes

研究天然和新型 PKS-NRPS 基因

基本信息

批准号：
BB/E007791/1
负责人：
Colin Lazarus
金额：
$ 46.82万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE007791%2F1
关键词：
Investigating natural novel PKS NRPS

项目摘要

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.

许多生物体产生次生代谢物，这些代谢物是化合物，通常具有复杂的结构，对于产生它们的生物体的正常生长和发育来说不是必需的。聚酮化合物（PK）是一类由许多细菌和真菌产生的次级代谢产物。有些药代动力学是有毒的，必须避免，但其他药代具有有用的药理学特性，可用于人类和兽医医学以及农业。其中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。该项目旨在揭示丝状真菌产生 PK 的方式。许多编码多种酶的基因有助于指定特定的 PK 结构。这些酶中最重要的是聚酮合酶（PKS），它组装基本碳主链，并且该主链通过其他酶的活性进行修饰。该项目涉及一类特定的 PKS 蛋白，这些蛋白具有完整的非核糖体肽合酶 (NRPS) 模块，其功能是将特定氨基酸添加到 PK 主链上。这项工作的最终目标是能够操纵参与 PK 合成的基因，以产生具有所需生物特性的新型化学物质。为此，需要了解 PKS 和 PKS-NRPS 等复杂酶中的各种酶和功能域。这种理解可以通过单独查看系统各部分的活动以及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，可以简化从基因片段构建大基因以及将最终产物转移到质粒载体中以进行进一步分析。该系统还简化了含有不同来源 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，我们将能够从多个基因中发现 NRPS 模块的功能/其中一些基因仅存在于基因组序列中。我们将把 NRPS 模块附加到已删除其自身 NRPS 模块的 PKS 上，并查看将哪些氨基酸添加到 PK 主链中。使用相同的混合搭配方法，但规模更小，我们将剖析 NRPS 模块，以发现氨基酸特异性所在。我们希望能够从这种实验方法发展到仅通过检查 DNA 序列来预测氨基酸特异性。不具有剪裁酶的宿主生物体中的嵌合基因的活性将产生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 的基因簇中来生产其他新化合物。所有新型化合物都具有直接或经过合理修饰后用于生物干预的潜力。在另一系列实验中，我们将比较一对非常相似的 PKS-NRPS，以发现结构差异如何影响它们的功能。一对的区别仅在于是否存在短蛋白质片段，该片段可能赋予具有该片段的酶额外的活性。我们将寻找具有附加片段的基因和删除片段的相同基因的表达产物结构上的差异。已知另一对生产的产品仅在 PK 链长度上有所不同。通过比较我们预测的非常相似的基因，我们希望识别出负责“编程”酶经历的循环次数（决定链长度）的序列，并且我们将通过将基因从五酮化合物生产者转换为六酮化合物生产者来测试这一点。