Enzyme Cascades and Synthetic Biology Routes to Non-Natural Alkaloids

几个世纪以来，植物和来自植物的药物化合物被用来治疗疼痛、感染、哮喘和癌症，其中许多植物天然产品被用于现代医学，例如：镇痛剂吗啡和可待因；抗疟疾的奎宁；治疗癌症的长春花碱和高三尖杉酯碱，以及治疗哮喘的麻黄碱。生物碱家族有14,000多种天然化合物，其中许多具有生物活性。最近，人们对利用微生物生产其中一些天然化合物产生了浓厚的兴趣，而不是将它们从世界上一些地区难以接近的植物中分离出来。实现这一目标的合成生物学方法被证明是非常有效的，最近的文章强调了从酵母中生产吗啡的可能性。在以前的工作中，我们研究了一些早期步骤和酶，这些步骤和酶使用氨基酸并向植物合成苄基异喹啉生物碱的途径中的关键中间体转化，该生物碱是一个超过2500种生物碱家族。我们现在的目标是使用这些酶，以及其他没有在植物途径中使用的酶，以及在生物催化应用中很少探索的新酶。这些酶将用于实验室细菌，以产生自然界中没有的一系列结构多样化的化合物。化合物将进行抗菌性能筛选。酶将被用于体外级联反应，以产生三个、四个、五个或更多个多个酶途径，并且通过在细菌中建立这些途径，我们将能够使用可再生资源以可重复的方式产生新的化合物。

Plants and medicinal compounds from plants have been used for centuries to treat pain, infections, asthma, cancer and many of these plant natural products are used in modern medicine, for example: the analgesics morphine and codeine; anti-malarial quinine for malaria; vinblastine and homoharringtonine for cancer treatment, and ephedrine for asthma. There are over 14,000 natural compounds of the alkaloid family and many possess biological activities. Recently, there has been intensive interest in producing some of these natural compounds using microbes, rather than isolating them from plants in parts of the world that are difficult to access. Synthetic biology approaches to achieve this are proving to be extremely powerful and recent articles have highlighted the possibility of producing morphine from yeast.In previous work we have investigated some of the early steps and enzymes that use amino acids and perform transformations towards a key intermediate in the pathways that plants use to synthesise the benzylisoquinoline alkaloids, a family of over 2500 alkaloids. We now aim to use these enzymes, together with other enzymes not used in the plant pathway, and new enzymes that have been little explored in biocatalytic applications. The enzymes will be used in a laboratory bacterium to generate a wide range of structurally diverse compounds that are not found in nature. Compounds will screened for antibacterial properties. Enzymes will be used in in vitro cascades to generate three, four, five, or more multiple enzyme pathways, and also by establishing the pathways in bacteria we will be able to generate new compounds in a reproducible way using a renewable resource.