1. Optimization of bacterial toluene monooxygenases

Bacterial toluene monooxygenases are key enzymes in the degradation of aromatic hydrocarbons. Due to their quite broad range of substrates these enzymes are often able to oxidize a variety of organic pollutants, including chloro-aliphatics such as TCE. The ability of monooxygenases to introduce an hydroxyl group on an aromatic ring can also be exploited for bioconversion purposes, that is to produce molecules of industrial interest through environmental-friendly, high-specificity processes.
We are using three different approaches to improve the activity of toluene monooxygenases: 1. construction of chimerical enzymatic complexes; 2. PCR random mutagenesis; 3. in vitro evolution by family DNA shuffling.

2. Bacterial degradation of chloro-aromatics

Chloro-aromatics are widespread environmental pollutants often quite recalcitrant to biodegradation. We isolated an Arthrobacter strain able to degrade chloro-benzoic acids and elucidated the catabolic pathway. The most interesting step is the dehalogenation of the substrates that leads to formation of the corresponding hydroxybenzoic acids. This ability can be exploited to design a new pathway for chlorotoluene degradation. Indeed, 4-chlorotoluene degradation has been achieved exploiting the combined activities of Arthrobacter and of a Pseudomonas strain, able to convert 4-chlorotoluene into 4-chlorobenzoic acid. We are cloning the dehalogenase-encoding Arthrobacter genes with the final aim to construct a Pseudomonas strain able to degrade the different isomers of chlorotoluene.

3. Regulation of catabolic operons in PseudomonasIn pseudomonas catabolic operons are subjected to both specific and global regulation. We have reconstructed the regulatory circuitry of the P. stutzeri OX1 toluene monooxygenase encoding operon and discovered a peculiar phenomenon, the effector-independent growth-phase dependent activation of transcription, never described before for other catabolic operons controlled by activator/promoter elements belonging to the same family of those isolated from P. stutzeri. The phenomenon is specifically triggered by the P. stutzeri activator protein following carbon starvation and we are investigating its genetic and physiological aspects. It The genetic dissection of the activator encoding gene is under way with the aim to identify the protein domain(s) specifically involved. We are also trying to identify the molecular signal that links carbon starvation to gratuitous transcription.