Prof. Kristala L Jones Prather

Professor Prather is interested in combining the traditions of metabolic engineering with the practices of biocatalysis to construct novel metabolic pathways for the production of non-natural compounds (small molecules) in microbial hosts. Non-natural products may be naturally-occurring in other organisms and only produced in the target organism following transfer of the metabolic pathway from the source to target (e.g., carotenoid production in E. coli), or the products themselves may be novel, i.e., structurally distinct from those found in nature. The goal is to create biosynthetic pathways for novel production formation by combining a series of enzymatic reactions that are not known to exist in sequence in nature for the formation of a designated product. To do so, the aim is to build upon principles employed in the field of biocatalysis, that is, the use of enzymes or whole cells for single-reaction conversions of an externally-supplied substrate. While a biocatalytic transformation is typically employed as an alternative to one step of an organic chemical synthesis route; it is usually done in the context of the full synthesis, which relies on the principles of retrosynthesis. As such, biocatalysis is product-focused (though the starting substrate is usually pre-determined from the overall synthesis scheme), and the products formed are usually non-natural, produced from non-natural substrates. The choice of enzymes is based only on consideration of the required chemical conversion. Using a "retrobiosynthetic" approach (i.e., focusing on the desired product) and based on our knowledge of natural biochemical reactions, we wish to propose pathways consisting of a series of enzyme-catalyzed steps that are likely to result in formation of the target product. The available starting reactants consist of cellular metabolites or commonly used nutrients (e.g., glucose) such that the ultimate pathways can be linked to basic metabolism. The final configuration of these production systems is envisioned as custom-designed "microbial factories" for organic chemical production.

Prather is also interested in the optimal manner in which these pathways should be constructed with respect to gene dosage levels and the choice of starting substrates. The most common approach for the introduction of new genes in a host cell involves the use of readily available, high-copy-number plasmids. Yet the use of such plasmids is well-known to include the additional burden of maintaining the plasmid DNA and expressing its encoded genes, a burden that may be reflected in a re-organization of the host cell's basic metabolism. Therefore, the optimal cell for production purposes may require that recombinant gene copy numbers be limited to balance enzyme levels and activity against gene levels. Conversely, if metabolites involved in basic host maintenance and propagation are required substrates for the final product of interest, the gene levels required to effectively divert such substrates towards alternative pathways may be quite high. Interest is in characterizing "metabolically engineered" cells in order to define the recombinant pathway characteristics that necessarily require higher gene dosages for adequate expression and those that benefit from lower gene dosages for maximum productivity. The knowledge gained can guide transfer and optimization of naturally-occurring metabolic pathways into more tractable hosts such as E. coli as well as provide useful insights into which substrates are more easily diverted from their intended pathways towards product formation (i.e., result in high productivity from a low gene dosage) and would likely be better suited for use as starting reagents in our novel, retrobiosynthetic pathways.