Synthetic Biology Applications of a New Generation of Antibody Catalysts

抗体作为催化剂的使用长期以来一直是研究和讨论的主题，但在有用的治疗或工业环境中使用这一概念的初步尝试迄今证明令人失望[1]。与传统的酶催化剂相比，显著降低的催化速率仍然是缺乏进展的关键原因，但最近推出了新一代抗体形式的技术发展，并将它们用于“印迹”酶催化部位[2]，这表明这个话题现在值得重新讨论。例如，VHH抗体形式能够维持细胞内的功能活性，并可以在高浓度下表达，这突显了它在特定环境下克服任何固有的低催化速率的潜力。特别是，当用酶来催化有用的合成过程是不切实际或不可能的时候，它们可能会在工程微生物中被用来催化有用的合成过程。还可以设想使用细胞内抗体催化剂库来生成天然产物类化合物的库，用于针对与药物相关的靶标进行抑制剂筛选。催化抗体还可能在降解阿尔茨海默病等疾病中聚集或错误折叠的蛋白质方面具有治疗作用[3]。这项由BBSRC赞助的博士研究提案将利用SYNBIOCHEM(曼彻斯特生物技术研究所)的合成生物学专业知识和UCB的抗体格式专业知识和文库来研究这一新的研究领域，并为催化抗体未来的合成生物学应用奠定基础。这项工作将分为三个主要研究领域：1.使用非专利的UCB抗体噬菌体文库来识别SYNBIOCHEM特别感兴趣的反应中的催化抗体，以及它们已经拥有可用于印迹过程的蛋白质和晶体结构。早期的目标将集中在构象催化上，以推动从香叶基二磷酸合成不同的单萜类支架。在这项工作的第二阶段，我们将针对已知不能在大肠杆菌中表达的酶的酶活性。黄体酮5B-还原酶就是一个很好的例子，它属于短链脱氢酶/还原酶(SDR)超家族，是腰果内酯生物合成的关键酶，因为它是导致5B构型中间体的第一个立体专一性酶。这两类酶都是代谢工程的主要目标。重要的是，第二阶段的研究将建立基于抗体技术的催化模块/部件的替代路线，对于这种技术，天然酶不可能在标准表达宿主(如大肠杆菌)中表达。2.VHH催化抗体在微生物中的细胞内表达，以测试它们是否可以用于合成生物学目的来生产新的化工产品。详细分析使用催化抗体生成有用的小分子文库的可能性，这些文库基于化学上不同的单萜类支架，用于生成针对治疗相关靶点(由UCB定义)的“命中”。我们预计在UCB放置6个月将是学生获得使用和应用我们的抗体库及其细胞内表达的知识所必需的。

The use of antibodies as catalysts has long been the topic of research and discussion but initial attempts to utilise the concept in useful therapeutic or industrial settings has so far proved disappointing [1]. Significantly reduced catalytic rates in comparison to traditional enzyme catalysts remains the key reason for this lack of progress but recent technological developments that have launched a new generation of antibody formats and their use in "imprinting" enzyme catalytic sites [2] suggest that this topic is now worth revisiting. The VHH antibody format, for example, is capable of maintaining functional activity within a cell and can be expressed at high concentrations, which highlights its potential for overcoming any intrinsic low catalytic rate under specific circumstances. In particular, they may find use in catalysing useful synthetic process within engineered microorganisms when it is impractical or impossible to do this with an enzyme. It is also possible to envisage the use of libraries of intracellular antibody catalysts to generate libraries of natural product-like chemical compounds for use in inhibitor screening against pharmaceutically-relevant targets. Catalytic antibodies may also find therapeutic utility in degrading aggregated or misfolded protein in diseases such as Alzheimer's Disease [3].This BBSRC-sponsored PhD study proposal will utilise the synthetic biology expertise at SYNBIOCHEM (Manchester Institute of Biotechnology) and the antibody format expertise and libraries at UCB to investigate this novel area of research and lay the groundwork for the future synthetic biology uses of catalytic antibodies. The work will be divided into three main areas of research:1. The use of non-proprietary UCB antibody phage libraries to identify catalytic antibodies in reactions of particular interest to SYNBIOCHEM and where they already possess protein and crystal structures that could be used in the imprinting process. Early targets will be centred on conformational catalysis in driving the synthesis of different monoterpenoid scaffolds from geranyl diphosphate. In the second phase of the work we will target enzyme activities from enzymes that are known not to express functionally in E. coli. A good example is progesterone 5B-reductase, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, and has been considered a key enzyme in cardenolide biosynthesis since it is the first stereospecific enzyme of the pathway leading to 5B-configured intermediates. Both these enzyme classes are major targets for metabolic engineering. Importantly, the second phase study will establish alternative routes to catalytic modules/parts based on antibody technology, for which expression of the natural enzyme is not possible in standard expression hosts such as E. coli. 2. The intracellular expression in microorganisms of VHH catalytic antibodies to test if they can be used for synthetic biology purposes in the production of novel chemical products3. A detailed analysis of the potential to use catalytic antibodies to generate libraries of useful small molecules based on the chemically diverse monoterpenoid scaffold for use in generating "hits" against a therapeutically relevant target (to be defined by UCB).We envisage that a 6 month placement at UCB will be essential for the student to gain knowledge in the use and application of our antibody libraries and their intracellular expression.