2024 AIChE Annual Meeting
Biosynthesis and Incorporation of Aromatic Non-Standard Amino Acids from Readily Accessible Precursors
The genetic code consists of twenty amino acids, however, with today’s knowledge of biology, chemistry, and engineering, scientists are able to create and incorporate nonstandard amino acids, or nsAAs, into proteins for specialized purposes for pharmaceutical and industrial applications. Synthetically produced nsAAs are often not enantiomerically pure and may be costly. When supplementing nsAAs to cells, often high concentrations are required in order to achieve the desired incorporation. Coupling nsAA biosynthesis with an orthogonal translation system (OTS) all within a single cell allows for a cheaper and stereoselective path for nsAA production and incorporation. So far, the production of aromatic nsAAs with a β-hydroxy group has been established using L-threonine transaldolase enzymes from diverse aldehyde substrates and L-threonine co-substrate. Unfortunately, the β-hydroxy group prevents previously engineered OTSs from incorporating the nsAA into proteins to our knowledge. We have designed an enzyme cascade which can remove the β-hydroxy group to form an aromatic nsAA ready for incorporation. Through in vitro and in vivo testing in E. coli, the pathway enzymes were characterized and conditions optimized for production and incorporation of nsAAs into protein using genetic code expansion techniques with an engineered aminoacyl-tRNA synthetase and tRNA pair and a green fluorescent protein (GFP) reporter. The biosynthetic pathway enzymes show broad specificity on aromatic compounds and can further extend to include a carboxylic acid reductase in order to initialize from carboxylic acids, which may be less expensive, less toxic, or more soluble than the aldehyde analogs. Using high-performance liquid chromatography (HPLC), we can quantify the yield of the nsAA biosynthesis. HPLC yields for a variety of nsAAs in the in vitro cascade are above 95% with enantioselectivity at greater than 99% e.e.. In a live cell context for coupled biosynthesis and incorporation, the GFP reporter can be purified and mass spectrometry used to quantify the accuracy of the desired incorporation of nsAA. This enzymatic pathway may open a world of new chemistries which can be incorporated into proteins for various functionalities.