“The research for this work has received funding from the European Union (EU) project ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Programme  Research and Innovation actions H2020-LEIT BIO-2014-1”

Meet Michele Tavanti – Researcher at the University of Manchester 

MicheleTavantiMichele Tavanti has recently joined the research group of Prof. Nicholas J. Turner, based at the Manchester Institute of Biotechnology as a first-year chemistry PhD student, currently working on developing CYP450 monooxygenases.

Michele received a Bachelor’s degree in Biotechnology from the University of Perugia in 2013 and then he earned a Master’s Degree in Industrial Biotechnology from the University of Padua in 2015, with a thesis about the mutational analysis of GraFDH, a formate dehydrogenase with double cofactor specificity.

Cytochrome P450 enzymes (CYPs) are heme-containing monooxygenases able to catalyse the activation of molecular oxygen, enabling the insertion of a single atom of oxygen into an organic substrate, which would be extremely challenging from a synthetic chemical standpoint. Auxiliary redox proteins shuttles electron equivalents from NAD(P)H allowing the reaction to proceed (Fig.1).

                       

MTFig1CytochromesP450sFig. 1: Cytochromes P450s are heme-enzymes capable of catalysing the monooxygenation of a substrate assisted by redox partners.

 

These exceptional biocatalysts are found in all the kingdoms of life, and among them, human CYPs have attracted the interest as they play an important role in drug metabolism and hence have a significant role in the effectiveness of pharmaceutical activity of drugs.

In particular, CYPs play a major role in drug metabolism: they are highly involved in the conversion of drugs to water-soluble metabolite ready for excretion. Large-scale synthesis of these metabolites is crucial during the process of drug development, since pharmacokinetics, metabolite toxicity and interactions have to be thoroughly evaluated. To this end, heterologous expressed CYPs could be wonderful candidate for target molecules synthesis. Moreover, given the vast range of reaction catalysed and substrate specificities, these enzymes could be also used in other sectors such as flavour and fragrances or food industries, for instance.

However, many inherent limitations of these enzymes have led to successful applications of P450s only on a small scale. Thus, many research groups have focused on engineering and improving these enzymes in robustbiocatalysts. For example, both rational protein design and directed evolution have been applied with the aim of improving the catalytic efficiency, substrate scope, stability and solvent tolerance.

The availability of human-like CYPs would be excellent biocatalytic tools given their broad substrate specificity. Thus, prokaryotic CYPs have been engineered towards “human like” activities. Moreover, thanks to the genomic data explosion, various self-sufficient bacterial CYPs have been discovered in which the redox partners are fused to the heme-domain of the enzyme, generating efficient single-unit biocatalysts.

The CYP P450-RhF from Rhodococcus sp. NCIMB 9784 is one of the first examples of such a system and, within the ROBOX consortium, the University of Manchester will focus on creating libraries of this enzyme to convert pharmaceutically interesting molecules and generating new chimeric self-sufficient enzymes based on P450-RhF architecture. Expression of promising candidates will be improved in order to move towards large-scale applications