Exploring prpC Enzyme Activity in ΔgltA ΔprpC Escherichia coli K12

Undergraduate #67
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Leah Thomas - Milwaukee School of Engineering
Co-Author(s): Shu Pan and Jennifer L. Reed, University of Wisconsin-Madison, Madison, WI

Citrate synthase (gltA) carries out the first step in the citric acid cycle to produce citrate from oxaloacetate and acetyl-CoA. If gltA is not present within a cell, some amino acids cannot be made and ATP production is reduced, preventing growth in minimal media. Our computational algorithm predicted that gltA gene is essential for Escherichia coli cells to grow in glucose minimal media. Another enzyme, 2-methylcitrate synthase (prpC), carries out a similar reaction converting oxaloacetate and propionyl-CoA into 2-methylcitrate and has some activity with acetyl-CoA. The goal of our project was to engineer PrpC to better catalyze the citrate synthase reaction. First, a host strain was constructed, which was a ΔgltAΔprpC double knockout that was unable to grow in glucose minimal media. Then a plasmid library expressing prpC variants was created by error-prone PCR. The expression level of prpC gene was carefully controlled using a BIOFAB promoter and ribosomal binding site1 such that the amount of prpC produced was not enough to support maximal growth. The ΔgltAΔprpC host cells were electroporated with the library of plasmids and then plated on glucose minimal media plates. PrpC variants with “citrate synthase” activity rescued growth of ΔgltAΔprpC mutants on glucose minimal plates. From these experiments, the function of prpC could be altered from a 2-methylcitrate synthase to a citrate synthase. The experiments produced a variety of colonies from error-prone PCR, which resulted in colonies appearing at different rates. The different promoters and ribosomal binding sites were tested to find the lowest prpC expression levels to allow detection of the greatest increase in enzyme optimization. In the future, the growth rates of large colonies on the glucose minimal plates will be measured. The mutant that has the fastest growth rate will be sequenced to determine where the mutations occurred. The enzyme will be purified and tested for citrate synthase activity. Also, we could create more error-prone PCR libraries and perform more growth rate experiments to select for better prpC mutants.

1Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., … Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods, 354-360.

Funder Acknowledgement(s): We gratefully acknowledge the following funding for making this project possible: DMR0520527, DMR-0425880, NSF 1053712, and NSF EFRI-1240268.

Faculty Advisor: Jennifer L. Reed,