Synthetic Biology Applications of a New Generation of Antibody Catalysts

长期以来，抗体作为催化剂一直是研究和讨论的主题，但迄今为止，将这一概念应用于有用的治疗或工业环境的初步尝试证明令人失望。与传统酶催化剂相比，显著降低的催化速率仍然是缺乏进展的关键原因，但最近的技术发展已经推出了新一代抗体格式及其在“印迹”酶催化位点[2]中的应用表明，这个话题现在值得重新审视。例如，VHH抗体格式能够在细胞内保持功能活性，并且可以在高浓度下表达，这突出了其在特定情况下克服任何内在低催化率的潜力。特别是，它们可能在催化工程微生物中有用的合成过程中发现用途，而用酶是不切实际或不可能做到这一点的。也有可能设想使用细胞内抗体催化剂文库来生成天然产物样化合物文库，用于针对药物相关靶标的抑制剂筛选。催化抗体在降解诸如阿尔茨海默病[3]等疾病中聚集或错误折叠的蛋白质方面也可能具有治疗效用。这项由bbsrc赞助的博士研究计划将利用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，并为催化抗体的未来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。使用非专有的UCB抗体噬菌体文库来鉴定SYNBIOCHEM特别感兴趣的反应中的催化抗体，并且它们已经具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驱动合成不同的单萜类支架从香叶二磷酸。在第二阶段的工作中，我们将从那些已知不能在大肠杆菌中功能性表达的酶中寻找酶的活性。孕酮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，被认为是硬核内酯生物合成的关键酶，因为它是导致5b配置中间体的途径的第一个立体特异性酶。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/部件的替代途径，因为在大肠杆菌等标准表达宿主中不可能表达天然酶。2. VHH催化抗体在微生物细胞内的表达，以测试它们是否可以用于合成生物学目的，以生产新的化学产品3。详细分析了使用催化抗体生成有用小分子文库的潜力，该文库基于化学上多样化的单萜类支架，用于生成针对治疗相关靶标（由UCB定义）的“击中”。我们设想在UCB 6个月的实习将对学生获得我们抗体库的使用和应用及其细胞内表达的知识至关重要。

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.