Enzymology and engineering of the biosynthesis of polyether antibiotics

该项目旨在联合收割机剑桥两个已建立的研究小组的努力，以解决化学和生物化学中一个重大的悬而未决的问题：在自然界中生产某种称为聚醚的天然抗生素的方法。聚醚是一种抗生素，其临床使用受到其毒性以及合成或化学修饰的困难的限制，但最近发现（例如）对抗药性疟疾寄生虫非常有效，这是一个主要的全球健康威胁。因此，人们对开发新的生物学方法来合成这些分子的文库以作为潜在的低毒性改进药物的起点进行测试非常感兴趣。我们已经知道，从细菌细胞内的简单构件构建如此复杂的小分子需要多个步骤，每个步骤都由一种酶催化。其中一些被物理地拴在一起形成大量的多酶复合物，这是迄今为止发现的最复杂的生物催化剂，但所有这些都被精心安排以提供平稳的级联或反应链，这样就不会浪费任何东西，通常会产生单一的最终产品。这项研究的机会出现，因为我们之一（PFL）最近（在BBSRC的支持下）首次成功地克隆和测序了四个控制四种不同聚醚生物合成的巨大基因簇，而不是一个;他的研究小组已经开发出遗传学方法来利用一种工业细菌菌株，这种菌株过度生产一种叫做莫能菌素的聚醚，因此产物以比野生型高1000倍的水平积累，使得甚至少量产物的分析成为可能。了解聚醚生物合成的分子细节将需要不同的实验方法，包括细菌细胞内基因的遗传操作，以查看改变的蓝图以何种方式指导改变的最终产物的合成;并尝试使用纯化的酶和人工底物在试管中重建部分催化作用。我们的目标不仅是提供一个新的认识，如何个别酶的性质是调制时，组合成这样一个集成的系统，而且要学习如何改变基因的规则，或联合收割机的基因从两个或多个聚醚途径，故意转移生物合成，使新的目标天然产物。我们的联合项目旨在加速这一令人兴奋的技术的进一步发展，其在制药行业的广泛潜在利益。

This project seeks to combine the efforts of two established research teams in Cambridge to solve a major outstanding problem in chemistry and biochemistry: the way in which a certain large group of natural antibiotics called polyethers are produced in Nature. Polyethers are antibiotics, whose clinical use has been restricted by their toxicity and by the difficulty of synthesising them or modifying them chemically, but which have been recently discovered (for example) to be highly effective against drug-resistant malarial parasites, a major global health threat. There is therefore great interest in developing new biological ways of synthesising libraries of such molecules to test as the starting point for potentially improved drugs of lower toxicity. We already know that to build up such complex small molecules from the simple building blocks inside bacterial cells requires multiple steps, each one catalysed by an enzyme. Some of these are physically tethered together into massive multienzyme complexes, the most complex biological catalysts so far discovered, but all are orchestrated to provide a smooth cascade or chain of reactions so that nothing is wasted and typically a single end-product is made. The opportunity for this research arises because one of us (PFL) has recently (with BBSRC support) succeeded for the first time in cloning and sequencing not one but four giant gene clusters governing the biosynthesis of four different polyethers; and his group has developed genetic methods to harness an industrial bacterial strain which overproduces one of these polyethers called monensin, so that products accumulate at levels up to 1000-fold higher than from the wild type, making the analysis of even minor products possible. Understanding polyether biosynthesis in molecular detail will require diverse experimental approaches including genetic manipulation of the genes inside the bacterial cells to see in what way the altered blueprint directs the synthesis of an altered end-product; and attempts to reconstitute portions of the catalysis in the test tube using purified enzymes and artificial substrates. We aim not only to provide a new appreciation of how the properties of individual enzymes are modulated when combined into such an integrated system, but also to learn the rules of how to alter the genes, or combine the genes from two or more polyether pathways, so as deliberately to divert the biosynthesis to make new target natural products. Our joint project aims to accelerate the further development of this exciting technology, with its broad potential benefits in the pharmaceutical industry.