Masters Thesis

Engineering DgcZ at the inhibition site and GTP binding site for enhanced c-di-GMP production

Bacterial second messenger, c-di-GMP, regulates important functions such as biofilm formation, motility, and virulence. C-di-GMP also has an immunostimulatory property, so can be used as a vaccine adjuvant. In this project, DgcZ (a diguanylate cyclase) was engineered to create hyperactive mutants in c-di-GMP production. For this, two protein sites were targeted: substrate (GTP) binding site and product (c-di-GMP) inhibition site. At the GTP binding site, three residues (L134, D182 and R204) were tested through codon randomization and in vivo c-di-GMP production assay, but none of the positions provided high activity DgcZ mutants superior to wild type DgcZ. In contrast, the engineering at the product inhibition site was fruitful as superior mutants could be made at both R197 and E200 positions. So far, R197T, E200A and E200G were identified to be better than wild type DgcZ in in vivo c-di-GMP production based on both Clp toxicity-masking assay and c-di-GMP toxicity assay. These mutants were toxic to the host cell when highly produced. The toxicity problem was circumvented by tagging one of the mutants (E200A) with PelB, a signal sequence to the periplasmic space, at the N- terminus and the fused PelB-E200A was not toxic to the cells. In the future, the resultant PelB-E200A needs to be tested thoroughly through protein expression, purification and in vitro c-di-GMP production assay for mass production of c-di-GMP.