We seek to develop a novel, direct, and generally applicable selection strategy for identifying enzymatic activity and specificity towards small-molecule, drug-like compounds. Both site-directed mutagenesis and combinatorial protein engineering (directed evolution) have successfully improved biocatalysts, however, full utilization of directed evolution is hampered by the lack of a general selection strategy to identify variants with improved properties. Our method comprises two steps: i) a nuclear receptor is engineered to activate transcription in response to a small-molecule reaction product, coupling the presence of the small molecule to survival of a microbe (i.e. genetic selection); ii) an enzyme is engineered through genetic selection to catalyze formation of the desired product. While applicable to any small molecule-enzyme pair, in this project we develop the method on the enzyme-catalyzed synthesis of beta-lactam antibiotics. This proposal seeks to improve ligand-activated growth (Aim 1), to develop enzyme-activated growth (Aim 2), to enhance enzyme substrate specificity to include beta-lactamase-resistant structures (Aim 3), and to synthesize biocatalytically a synthetic antibiotic currently not accessible through enzymatic synthesis (Aim 4). Our long-range goal is the application of this method to any drug-like target molecule, including non-lactam-based antibiotics and other drug classes.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Huntley, Clayton C
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Georgia Institute of Technology
Schools of Arts and Sciences
United States
Zip Code
Gryder, Berkley E; Rood, Michael K; Johnson, Kenyetta A et al. (2013) Histone deacetylase inhibitors equipped with estrogen receptor modulation activity. J Med Chem 56:5782-96
Blum, Janna K; Ricketts, M Daniel; Bommarius, Andreas S (2012) Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method. J Biotechnol 160:214-21
Shaffer, Hally Anne; Rood, Michael Kenneth; Kashlan, Badar et al. (2012) BAPJ69-4A: a yeast two-hybrid strain for both positive and negative genetic selection. J Microbiol Methods 91:22-9
Ousley, Amanda M; Castillo, Hilda S; Duraj-Thatte, Anna et al. (2011) A human vitamin D receptor mutant activated by cholecalciferol. J Steroid Biochem Mol Biol 125:202-10
Bommarius, Andreas S; Blum, Janna K; Abrahamson, Michael J (2011) Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst. Curr Opin Chem Biol 15:194-200
Rogers, Thomas A; Bommarius, Andreas S (2010) Utilizing Simple Biochemical Measurements to Predict Lifetime Output of Biocatalysts in Continuous Isothermal Processes. Chem Eng Sci 65:2118-2124
Blum, Janna K; Deaguero, Andria L; Perez, Carolina V et al. (2010) Ampicillin Synthesis Using a Two-Enzyme Cascade with Both ?-Amino Ester Hydrolase and Penicillin G Acylase. ChemCatChem 2:987-991
Blum, Janna K; Bommarius, Andreas S (2010) Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin. J Mol Catal B Enzym 67:21-28
Rogers, Thomas A; Daniel, Roy M; Bommarius, Andreas S (2009) Deactivation of TEM-1 ?-Lactamase Investigated by Isothermal Batch and Non-Isothermal Continuous Enzyme Membrane Reactor Methods. ChemCatChem 1:131-137
Polizzi, Karen M; Chaparro-Riggers, Javier F; Vazquez-Figueroa, Eduardo et al. (2006) Structure-guided consensus approach to create a more thermostable penicillin G acylase. Biotechnol J 1:531-6