Building synthetic enzyme fusions for chiral molecule production.

旋光性胺作为生物活性化合物在制药和化学工业中发挥着重要作用。由于传统的化学方法复杂且昂贵，生物催化途径是可取的。它们的生物生产途径之一是将醇转化为胺。目前，还没有已知的酶可以将醇直接转化为胺，但是通过醇氧化和随后的还原性胺化或转氨化两步转化是可能的。在酶级联中，反应工程是必要的，以确保流向产物的通量，并避免通常是活性中间体的积累。相比之下，天然存在的多功能酶已经进化到将活性底物保持在酶复合物内，直到进行关键转化。在体内，多功能酶被认为通过增加反应速率和化学稳定性或防止活性中间体的毒性而赋予选择性优势。该项目的目的是表征在恶臭假单胞菌KT2440（一种具有多种氨基酸和脂肪酸代谢途径的非致病性土壤细菌）中转氨酶和脱氢酶蛋白质PP_2782之间的自然融合，以利用其生物合成能力进行新型酶融合工程。利用这种自然融合作为指导模板，并能够合成具有挑战性的化学生产的新型生物活性化合物，设计和测试了许多由特征良好的酶组成的合成融合物。

Strategic Research Priority: World Class Underpinning ResearchOptically active amines play an important role as bioactive compounds in pharmaceutical and chemical industries. Because classical chemical approaches are complex and expensive, biocatalytic routes are desirable. One route for their bio-production involves conversion of alcohols to amines. Currently, there are no known enzymes that can convert alcohols directly to amines, however a two-step transformation is possible via alcohol oxidation and a subsequent reductive amination or transamination.In enzyme cascades, reaction engineering is necessary to ensure flux towards the products, and to avoid accumulation of, often, reactive intermediates. In contrast, naturally occurring multifunctional enzymes have evolved to keep reactive substrates within enzyme complexes until key transformations have been carried out. In vivo, multifunctional enzymes are thought to confer selective advantage via increased reaction rates and chemical stability or prevention of toxicity from reactive intermediates.The aim of this project was to characterise a natural fusion between a transaminase and a dehydrogenase, protein PP_2782, in Pseudomonas putida KT2440 (a non-pathogenic soil bacterium that has a diverse range of amino and fatty acid metabolic pathways), in order to exploit its biosynthetic abilities for novel enzyme fusion engineering. Using this natural fusion as a guide template, and to enable the synthesis of novel bioactive compounds that are challenging to produce chemically, a number of synthetic fusions comprising well-characterised enzymes was engineered and tested.