Ultrahigh Throughput Microscale Mass Spectrometry for Pharmaceutical Prenylation Enzyme Engineering

项目摘要 生物催化剂可以是合成天然产物类药物的关键工具，也可以作为强大的成分 药剂师的药物改装试剂盒。例如，芳香族戊烯基转移酶NphB在 常见的戊烯基化化合物或衍生主链的合成包括大麻素，α酸，β酸， 苯丙烷类化合物、挥发油类和水杨素类化合物。同样酶还具有作为化合物的通用工具的用途 功能化。我们的合作伙伴BioMediCan已经开发了一个基于这种酶的全细胞生物催化平台 生产天然和非天然稀有大麻素。然而，尽管酶的所有吸引人的品质和用途- 基于或全细胞生物催化，NphB以及许多其他酶都没有进化出具有这种特征的 工业或制药目标所需的。这些限制的出现是因为传统的 当前超高通量微流控芯片的分析方法和窄检测能力 技巧。为开发新型高效平台和改进NphB生物催化剂，流体发现公司将 具有高通量样品处理能力的微流控生物催化样品制备系统 和低成本，并使其能够与基质辅助激光解吸/电离(MALDI)质量无缝耦合 光谱灵敏度高，适用范围广。这种被称为μMALDI-TOF的方法将不仅允许流体 Discovery和BioMedian将迈出成为新化合物和酶的主要供应商的第一步 工具，而且还通过高度可扩展和通用的 放映平台。我们的第一阶段目标是建立和展示这个平台在工程上的能力 NphB在全细胞生物催化中的应用。在第二阶段，我们将继续通过NphB工程进行工作，以改善和 扩展现有全细胞生物催化剂平台，启动签约服务，及早获取合成生物学 公司。最终，我们计划利用我们产品的通用性在新一代 用于绿色化学药物生产或作为分子多样化工具的生物催化剂 复合改性和优化。

Project Summary Biocatalyst can be key tools in the synthesis of natural product-based pharmaceuticals and as powerful components of the pharmaceutical chemist’s drug modification kit. For example, the aromatic prenyltransferase NphB has utility in the synthesis of common prenylated compounds or derived backbones include cannabinoids, alpha acids, beta acids, phenylpropanoids, naphterpins, and marinones. The same enzyme also has utility as a general tool for compound functionalization. Our partner, BioMediCan has developed a whole cell biocatalysis platform based on this enzyme for production of natural and nonnatural rare cannabinoids. However, for all the attractive qualities and uses of enzyme- based or whole-cell biocatalysis, NphB as well as many other enzymes have not evolved with the characteristics required for industrial or pharmaceutical goals. These limitations arise because of the limitations of conventional analytical methods and the narrow detection capabilities of the current suite of ultrahigh-throughput microfluidic techniques. To develop novel high efficient platform and to improve the NphB biocatalyst, Fluid Discovery will engineer a microfluidic based biocatalysis sample preparation system with capacities of high throughput sample processing and low cost and enable its seamless coupling with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for high sensitivity and general applicability. This method, called μMALDI-TOF, will not only allow Fluid Discovery and BioMediCan to take the first steps towards becoming a key provider of novel compounds and enzymatic tools, but also provide a quantum leap in biocatalyst discovery and engineering through a highly scalable and universal screening platform. Our Phase I goal is to establish and demonstrate the capacity of this platform on engineering NphB in whole cell biocatalysis. In Phase II, we will continue our work via engineering NphB for improving and expanding the current whole-cell biocatalyst platform and initiate contracted service to early access synthetic biology companies. Ultimately, we plan to leverage the generality of our product for applications in the generation of biocatalysts for either green chemistry drug production or as tools for the molecular diversification that underlies lead compound modification and optimization.