Chemical synthesis is the most commonly used method of producing industrially relevant molecules, yet this practice is often accompanied by various environmental hazards. Biological synthesis, on the other hand, does not produce any toxic byproducts, nor does it require expensive starting materials. In the long run, it is a better manufacturing solution in many respects.
One strategy for biological synthesis is to adapt pre-existing systems already in nature. For instance, E. coli produces proteinaceous components in its biofilm called curli fibers. These fibers self-polymerize outside of the cell, forming a macroscopic agglomeration of material when isolated in sufficient bulk. The Joshi lab has already demonstrated how various functional peptide domains can be added to the self-polymerizing units of curli, so the polymers can form the basis of a variety of functional materials.
Our project focuses on optimizing curli production on two fronts as a first step to developing the curli system into a robust platform for producing materials at industrially relevant yields. First, we alter ribosome binding site strengths associated with proteins involved in the curli pathway to optimize the stoichiometric ratios of these molecules in the cell. The alterations are informed by a model of curli production and export that determines the optimum ratio of pathway components to maximize the production of our desired product, extracellular curli fibers, and minimize the aggregation of unwanted byproducts within the cell. This ensures that each cell becomes a more efficient curli export machine. The second component of the project aims to maximize cell densities within culture media through the development of a bioreactor that maintains higher dissolved oxygen concentrations than standard shaker flasks. Eliminating the limiting factor of insufficient oxygenation allows bacterial cells to produce more curli per unit of feedstock. This aspect of the project aims to optimize the conditions for protein-producing cell cultures. Our work along these two lines will inform the development of the curli system as a feasible biosynthetic platform for producing scalable and programmable materials.
Curli fibers are the main proteinaceous component of E. coli biofilms, which are aggregates of microorganisms embedded in a self-produced matrix of various polymeric substances. Biofilms are often associated with pathogenicity, but they also provide opportunities for development as platforms for biological synthesis of functional materials. The secretion and polymerization pathway of curli fibers is well-documented, and is depicted here in this diagram:
Goyal, Parveen, et al. (2014)
There are a number of chaperone and membrane proteins involved in this pathway:
|csgA||Major subunit that aggregates and self-polymerizes to form curli fibers|
|csgB||Interacts with csgF to initiate nucleation of csgA fibers|
|csgC||Chaperone protein that interacts with csgA and csgB and prevents them from polymerizing inside the cell|
|csgE||Periplasmic protein that interacts with csgG at the outer membrane|
|csgF||Periplasmic protein that interacts with csgG at the outer membrane|
|csgG||Outer membrane lipoprotein that is required for stability and secretion of csgA and csgB|
We are particularly interested in curli fibers because they are extremely robust and can withstand exposure to extreme conditions like boiling in detergents or incubation in solvents. Thus, they present great potential for use in harsh environments.
Additionally, the Joshi lab has demonstrated that it is possible to fuse functional proteins to the csgA subunit and have both csgA retain its secretion and polymerization properties while having the fused protein maintain its primary function. For instance, the Joshi Lab successfully showed GFP fused to csgA retains its fluorescent properties, and enzymes can be immobilized on csgA to form catalytic biofilms. Proteins fused to csgA will not only be secreted using E. coli ’s natural secretion machinery, but it will also be embedded within a physical material that can be handled and applied in a variety of ways.
Nguyen, Peter Q., et al. (2014)
- Goyal, Parveen, et al. "Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG." Nature 516.7530 (2014): 250-253.
- Nguyen, Peter Q., et al. "Programmable biofilm-based materials from engineered curli nanofibres." Nature communications 5 (2014).