Engineering nanoscale protein containers

The proposed work describes the characterization and modification of the protein-based propanediol utilization (Pdu) bacterial microcompartment for use as an artificial ‘organelle’ in bacteria or nanoscale container in extracellular environments. Applications for such protein-based containers include the production specialty chemicals, drug delivery, and nanomaterial fabrication. Despite much work on several native microcompartment systems in recent years, challenges remain that currently prevent their widespread use in these applications. Notably, the rules governing the assembly of the structures within the cell are not well understood, and to date there are no reports of compartment assembly outside of the cellular context. Moreover, little is known with respect to cargo encapsulation, especially in terms of loading and stoichiometry for more than one target cargo simultaneously. The PI will be assisted by a graduate student in carrying out the proposed research, which will: Objective 1: Determine the range of shell protein expression levels that allow for proper microcompartment assembly in vivo (years 1 and 2) Objective 2: Identify requirements for microcompartment shell formation in vitro (years 1-3) Objective 3: Control enzyme loading to the microcompartment lumen (years 1-3) Synthetic biology is a growing field with great potential for the development of microbes or even artificial cells to produce chemicals and materials on demand from a variety of energy and nutrient sources. Several advances led to the commercialization of a malaria drug precursor and a commodity chemical used to make fibers, but there are limits to the available methods used to push forward the field, particularly with respect to the complexity of cellular reactions and the need for segregation of some reactions. The work proposed here will enable the design of in vivo and in vitro protein containers that assemble to encapsulate cargo from engineered metabolic pathways to toxic materials, and could eventually be applied to uses such as drug delivery and biosensing. Moreover, this work aims to identify some of the design rules for multimeric protein structure assembly – essentially, to establish how the presence and concentration of each biological building blocks affects assembly into stable three-dimensional polyhedral structures. The methods developed will aid future studies of the function of the protein shells of bacterial microcompartments and may extend to virus-like particles. Together, these studies will enhance our understanding of natural systems involved in the microbiome while establishing guidelines for the design and loading of these structures for a variety of potentially transformative applications.