Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis. However, to create industrially competitive strains to efficiently convert lignocellulose to high-value chemicals, current challenges must be addressed. Redox constraints, allosteric regulation, and transport-related limitations are important bottlenecks limiting the commercial production of renewable chemicals from lignocellulose. Advances in metabolic engineering techniques have enabled researchers to engineer microbial strains that overcome some of these challenges but new approaches that facilitate the commercial viability of lignocellulose valorization are needed. Biological systems are complex with a plethora of regulatory systems that must be carefully modulated to efficiently produce and excrete the desired metabolites. In this work, I explore metabolic engineering strategies to address some of the biological constraints limiting bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective lignocellulose bioconversion.

This study was conducted to understand the reactivity of APE1 in repairing abasic sites associated with clustered DNA damages and to determine if the efficiency of APE1 enzyme is affected by the type of bases (purines or pyrimidines) neighboring the AP site. DNA damages are always occurring in living cells and if left uncorrected can lead to various problems such as diseases and even cell death. Cells are able to recognize and correct these DNA damages to prevent further damages to the genome, and the Base Excision Repair (BER) pathway is one of the mechanisms used in repairing DNA damages. A former student in the Levitus Lab, Elana Maria Shepherd Stennett, henceforth referred to as Elana worked on this project. She observed that the activity of the APE1 enzyme increased some when the base opposing the abasic site was changed from thymine (T) to adenine (A) while no difference was observed when the surrounding bases were changed. Thus, this experiment was conducted to further study the results she obtained and to possibly validate her findings. The AP sites used in this study are natural abasic sites created by UDG glycosylase enzyme from a double stranded uracil-containing DNA samples ordered from IDT technologies. Each reaction was carried out at physiological temperature (37degrees Celsius) and analyzed using polyacrylamide gel electrophoresis.