20.109(F10) Novel Antimicrobial Synthesis or Engineering Neurons for Epilepsy Treatment
Polyketide and Non-Ribosomal Polypeptide Synthases
Combinatorial Antibotic Biosynthesis by the Modular Rearrangement of Nonribosomal Peptide Synthases
Ever since Fleming discovered Penicillin, it has been known that Fungi produce antibiotics that give them competitive advantage.However, synthesis of antimicrobials de novo has been challenging. Fungi possess enormous enzymatic complexes known as Polyketide and Non-Ribosomal Polypeptide Synthases to produce antimicrobials such as Erythromycin and Vancomycin. These synthases are constituted of catalytic modules that catalyze a specific reaction leading up to the production of the final product.Luckily, the domains of Polyketide and Non-Ribosomal Polypeptide Synthases are linearly and compactly arranged in Fungi genome. Therefore, not only is it feasible to predict the final product of a synthase based on the genomic arrangement of its modules, it's also possible to modify them in an attempt to modify the final product. Hence, it is possible to synthesis novel antibiotics in vivo or in vitro by rearranging the domains for these synthases. In fact, it is possible to create a library of polyketides and polypeptides that can serve as a pool for selection of a hit.
Challenges and Questions:
- Selection of system: in vitro or in vivo?
- Stabilization of purified synthases in vitro.
- Dearth of sequenced synthases is a limitation.
- Possible to reconstitute a synthase with modules from other types of synthases?
- What kind of high-throughput assays are available for selection of a hit?
- Can novel antibiotics possible overcome drug resistance?
- If in-vivo, what type of model cell can process all the necessary post-translational modifications?
- How is the specificity of loading modules affected?
- How would we deal with iterative modules?
- Possible means of modifying modules: PCR Site Directed Mutagenesis
- Mass Spectroscopy to Determine Identity of Modules
- Radioactive labeled substrates
Engineering Neurons for Epilepsy Treatment
Using halorhodopsins, scientists have discovered a way to regulate neuron activity in transgenic neurons.Neurons initiate a variety of processes ranging from motor responses and perception,to higher order cognition, based on their "activity"( i.e. successful firing of an action potential or inhibition of action potential) and spatial arrangement. Halorhodopsins which are naturally regulated by light and engineered neurons infected with halorhodospins coupled to membrane transport proteins in neurons allow scientists to initiate or inhibit actions potentials in a neuron.Epilepsy is a neurological disorder cause by hyperactivity of neurons. If we can develop a method to selectively infect neuron cell types that are responsible for the erratic firing of action potentials in epileptic patients we can develop a more efficient means to stop sudden epileptic seizures through light. However there are numerous challenges to overcome such as conferring selective infection of neuronal population,determining the duration of transgenes in vivo neuron populations,etc..
<"A Genetic Method for Selective and Quickly Reversible Silencing of Mammalian Neurons">Lechner and Lein</ref>
<"Harnessing the Power of the Genome in the Search for New Antibiotics">Rosamond and Allsop</ref>