Microbial Synthesis of Therapeutic Bile Acids for Alzheimer's Disease

摘要 这一STTR阶段第二阶段的提案旨在设计和放大微生物体内的合成代谢途径 东道主生产UDCA和相关化合物。此外，由工程合成的UDCA生产的UDCA 代谢途径将被用来合成衍生物，用于在我们的阿尔茨海默病细胞和动物身上进行测试 模特们。拟议的工作具有很高的智力价值，原因如下：(I)UDCA生物合成 不会出现在任何可规模化种植以满足全球需求的有机体中，(Ii)UDCA需要多个 生物合成途径(动物胆酸由肠道微生物群转化为UDCA)将不得不 在单个单元中重组，以及(Iii)这一工程工作的预期规模和复杂性(组合 来自四个生物体的十几个基因)处于可行的边缘。技术障碍包括 发现了将麦角甾醇转化为胆固醇的新酶(已知存在于咸虾中)， 在酵母中实现15个重组基因产物的正确定位和酶活性，并平衡 每个基因的表达水平，以支持酵母中UDCA的高效生产。合并后的 Metselex/明尼苏达大学研究小组将利用他们的多基因途径平台克服这些障碍 重构和高通量DNA组装和分析方法。使用这些工具/方法的经验 在设计类似的合成代谢途径方面的成功使这个团队有资格进行这些研究和 将使他们能够实现他们的第二阶段目标。

ABSTRACT This Phase STTR Phase II proposal aims to engineer and scale-up a synthetic metabolic pathway in a microbial host to produce UDCA and related compounds. Additionally, the UDCA produced from the engineered synthetic metabolic pathway will be used to synthesize derivatives for testing in our Alzheimer’s Disease cell and animal models. The proposed work has high intellectual merit for the following reasons: (i) UDCA biosynthesis does not occur in any organism whose cultivation can be scaled to meet global demand, (ii) UDCA requires a multi- organism biosynthetic pathway (animal cholic acids converted to UDCA by the gut microbiome) that will have to be reconstituted in a single cell, and (iii) the anticipated scale and complexity of this engineering effort (combining more than a dozen genes from four organisms) are on the edge of what is feasible. Technical hurdles involve the discovery of new enzymes that convert ergosterol to cholesterol (a process known to exist in brine shrimp), achieving proper localization and enzymatic activity for fifteen recombinant gene products in yeast, and balancing the expression levels of each gene to support high-titer production of UDCA in yeast. The combined Metselex/University of Minnesota team will overcome these hurdles using their platform for multi-gene pathway refactoring and high-throughput DNA assembly and analytical methods. Experience with these tools/approaches and success in engineering similar synthetic metabolic pathways qualifies this team conduct these studies and will enable them to accomplish their Phase II goals.