A dynamic, high affinity, modular scaffold toolkit for control of protein colocalization and intracellular metabolic flux
Date
2021
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Modern metabolic engineering uses platform organisms such as E. coli and S. cerevisiae to produce a variety of chemical compounds valuable to various industrial areas such as medical and food/flavor. Production of such compounds is usually achieved by introduction of non-native metabolic pathways. However, often the introduction of these non-native pathways can lead to metabolite imbalances that can cause slow growth, or low titers. Traditional approaches to optimize product yield involve pathway engineering or directed protein evolution. An alternative approach inspired by nature, scaffolds enzymes of interest to create metabolons that can increase pathway flux through substrate channeling. However, these synthetic scaffolds remain static and provide limited relief from metabolite imbalances. To address this issue, a dynamic, high affinity, modular scaffold toolkit that relies on RNA-RNA interactions for dis-/assembly and CRISPR/Cas6 proteins for enzyme localization on the RNA scaffold is proposed. ☐ The first step in demonstrating the functionality of the proposed scaffold was proving that the scaffold can assemble with high affinity and specificity in vitro and in vivo. Scaffold assembly was demonstrated both in vitro and in vivo only when all components of the scaffold were expressed concurrently. Furthermore, complementary RNA sequences were required to drive scaffold assembly. Scaffold stability was demonstrated via long term growth studies, and the physical tethering of the two proteins was shown via coimmunoprecipitation. Different scaffold hybridization lengths underscored the importance of protein orientation on the scaffold. The scaffold’s ability to assemble is contingent upon the expression of both the scaffold RNA and the two orthogonal Cas6-enzyme fusion proteins. ☐ The scaffold’s dynamics are perhaps its most novel characteristic. Conditional dynamic scaffold disassembly via toehold mediated strand displacement was demonstrated to occur only in the presence of short RNA triggers with appropriate sequences. A cycling system that can transition from assembled to disassembled and back to assembled state via a pair of trigger RNAs allowed for even finer control of protein colocalization in real time. Implementation of a riboswitch gated trigger sequence showed that conditional scaffold disassembly can be driven by metabolite sensing RNA constructs, which can allow the cell to self-regulate the scaffold state. Finally, a trigger-mediated scaffold assembly system drove scaffold assembly upon trigger expression. The development of wide array of scaffold architectures for dynamic dis-/assembly gives the user freedom of design when implementing the system for their desired metabolic engineering goals. ☐ The capability of the scaffold to control intracellular metabolic flux was demonstrated with the non-native indole-3-acetic acid pathway and one of the native malate production pathways. Scaffolding of the indole-3-acetic acid pathway enzymes showed increases in product titer upon scaffold assembly, which demonstrated the system’s capabilities when scaffolding consecutive enzymes. Scaffolding of the malate pathway enzymes demonstrated a novel way to control metabolic flux by scaffolding of two enzymes that both produce substrates that are combined downstream. The control of metabolism by the scaffold in multiple configurations showed the scaffold’s broad potential applications and modularity.
Description
Keywords
Colocalization, Dynamic, Metabolic engineering, Scaffold, Synthetic biology