(620ce) Constructing a Biosynthetic Pathway to 3-Hydroxy-2-Pyrrolidinone
The specialty chemical 3-hydroxy-2-pyrrolidinone is the subject of biosynthetic pathway construction and metabolic engineering due to its favorable selling price ($2,600/kg) and because there are no known routes for biological production. In order to produce 3-hydroxy-2-pyrrolidinone biologically, a new biosynthetic pathway was proposed, and enzyme promiscuity is now being engineered so that enzymes of the pathway can accept and convert non-native substrates. This is a significant challenge in biosynthetic pathway synthesis that will ultimately allow the production of new chemicals and provide more efficient routes to existing chemical targets. We are approaching the problem of biosynthetic pathway design using simultaneous computational and experimental approaches. In particular, molecular docking and dynamics simulations offer understanding of how native and non-native substrates interact with active site residues of an enzyme. This approach is used to suggest enzyme engineering strategies that may be required for an enzyme to accept and convert a non-native substrate. In addition, a new enzyme activity screening technique has been developed using Raman spectroscopy that can probe the activity of engineered enzymes without enzyme purification or the development of specific colorimetric assays. Here, we also describe how these methods can be implemented in a semi-high-throughput assay to screen enzyme mutants for biosynthetic pathway construction.