Viability of engineered biocatalysts in biotransformation

This project aims to exploit engineered biofilms as biocatalysts in the biotransformations of enantiomerically pure compounds for fine chemical and pharmaceutical industry. It aims for conditions to be designed which would improve reactions and formation of the engineered biofilms. Tsoligkas et al. (2012) has previously engineered a biofilm to act as a biocatalyst using tryptophan synthase, TrpBA produced from plasmid pSTB7 to catalyse the biotransformation of haloindoles to L-halotryptophans. To build on this work, biofilm formation and how the cells react to the biotransformation were investigated through flow cytometry and analysis of colony forming units (CFU). For biofilms to be formed from Escherichia coli (E. Coli) K-12, it was found that the plasmid pT7-csgD had to be present or the strain required an ompR234 point mutation to allow production of curli for extracellular polymeric substances to form a biofilm. This demonstrates the importance of CsgD as a regulator for formation, as without an increase in cellular concentration E. coli cells failed to attach to glass surfaces. From planktonic data it is apparent that carrying out the biotransformation with 5-chloroindole has a toxic effect on metabolically active E. coli PHL644 pSTB7. The source of this toxicity is not clear, it may be due to the products of the reaction, the chloroindole being metabolised or incorporated into the cellular proteins. Efflux data indicates that cells are incubated with fluoroindole have decreased efflux, an advantage for biotransformation