Researcher(s)
- Abigail Spangler, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware
Abstract
The genetic code consists of twenty amino acids, however with today’s knowledge of biology, chemistry, and engineering, over two hundred nonstandard amino acids (nsAAs) have been produced and incorporated into peptides/proteins for specialized purposes for pharmaceutical and industrial applications. This project focuses on the production of nsAAs from a β-hydroxy nsAA which is the product of threonine aldolase (TA) or threonine transaldolase (TTA) enzymes, both of which demonstrate broad substrate specificity. However, in order to incorporate the broad range of β-hydroxy nsAA products into proteins, we must first make the nsAA analog which is compatible with the orthogonal incorporation machinery. We demonstrated removal of the β-hydroxy group using the enzymes phenylserine dehydratase from Ralstonia pickettii (RpPSDH) and the aminotransferase enzyme native to Escherichia coli, TyrB, which are used to form a keto acid intermediate and then undergoes transamination to form the amino acid. We characterized this enzyme cascade both in vitro and in vivo in various E. coli strains to better understand the ideal conditions for the maximum production yield. Enzymatic activity can be tracked by analyzing the presence of the substrates and products. Both high-performance liquid chromatography (HPLC) and the addition of ferric chloride solution read on a plate reader at 640 nm can track the substrate production and depletion over time. Because β-hydroxy nsAAs are not readily commercially available and custom synthesis is cost prohibitive, we further demonstrate these enzymes, coupled with TTA enzymes, can produce nonstandard amino acids in vitro starting from the inexpensive aldehyde precursor. The next step will ultimately be targeting the formation of nsAAs in vivo for industrial and biomedical applications.