In Vitro and in Vivo Biocatalysis Based on Oxidative Flavoenzymes: Feasibility Studies

This thesis comprises two research projects in biocatalysis: the first conducted at the University of Padua in the research group of Prof. Elisabetta Bergantino, and the second at the University of Groningen in the group of Prof. Marco Fraaije. The first project focused on the genetic modification of the cyanobacteria Synechocystis sp. PCC 6803 to develop a whole-cell platform for sustainable indigo production. The strain Syn_FMO, engineered to express the flavin-dependent monooxygenase (mFMO) from Methylophaga aminisulfidivorans and capable of converting indole to indigo, was further modified by inserting the PtUGT_stable gene from Polygonum tinctorium, which encodes a UDP-dependent glycosyltransferase. This enzyme is expected to enable the formation of indican, a soluble indigo derivative that facilitates recovery and downstream processing. The PtUGT_stable gene was successfully cloned into two recently developed integrative vectors: pN8Psll1626 and pN15Ptrc1O. The resulting constructs, pN8_PtUGT_Stable and pN15_PtUGT_Stable, were used to transform Syn_FMO, with the aim of obtaining a new strain co-expressing both mFMO and PtUGT_stable. Several rounds of subcloning at increasing antibiotic concentrations remain to be performed to verify successful gene insertion and to achieve homoplasmy. Successful transformation would represent a step forward in establishing a light-driven, sustainable biosynthetic platform for indigo production. The second project examines two flavoprotein oxidases, eugenol oxidase (EUGO) and 5-(hydroxymethyl)furfural oxidase (HMFO), through studies on solvent engineering and protein thermostabilization. Investigations in Deep Eutectic Solvents (DESs) confirmed that a mixture of glucose, fructose, and water (1:1:6), markedly enhanced enzyme thermal stability, resulting in up to a 2000-fold increase in HMFO’s half-life at 50 °C. However, this effect is largely attributable to the sugar components rather than the eutectic nature of the solvent. Additional DESs were tested as reaction media, revealing the importance of factors such as viscosity, miscibility, and oxygen solubility. In parallel, protein engineering was performed on HMFO to obtain more thermostable variants. Computationally predicted mutations were experimentally tested, identifying four single mutations that increased melting temperatures. However, combining these mutations did not produce additive effects, suggesting potential negative interactions between mutations. Finally, kinetic analyses of 8BxHMFO toward 5-(hydroxymethyl)furfural (HMF) were performed, allowing determination of both steady-state (kcat and KM) and pre-steady-state (kred and kox) kinetic parameters. Overall, this work provides valuable insights into the development of sustainable biocatalytic platforms and the enhancement of enzyme stability and performance, contributing to the advancement of practical applications of engineered enzymes in industrial bioprocesses.