Ultrahigh Throughput Microscale Mass Spectrometry for Pharmaceutical Prenylation Enzyme Engineering

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.Narrative Biocatalyst such as prenylation enzyme is a key element in the discovery, production, and pharmaceutical application of natural products; however, access to sufficiently functional biocatalysts often requires dedicated discovery and reengineering before they can be used. The goal of this project will be to develop a platform for high throughput engineering aromatic prenyltransferase, NphB, which has direct applications to produce novel pharmaceutical active compounds from the phenylpropanoids, cannabinoids, and aromatic chemical families. Furthermore, the platform developed during this project is highly extendable to other drug discovery or pharmaceutical discovery efforts.