Simran Kundral

Biochemical and molecular characterization of Cytochrome P450 (CYPs) from extremophilic sources and their biotechnological applications

Cytochrome P450 enzymes (CYPs) are heme-containing monooxygenases, broadly distributed among living organisms, which play crucial roles in natural product biosynthesis e.g. steroids and the degradation of xenobiotics e.g. drug metabolism. P450s are considered amongst the most versatile biocatalysts in nature because of the wide variety of substrate structures they accept and the many types of reactions they catalyse. (Li et al., 2020). The genomes of extremophiles are reported to encode P450s. The ubiquity of the P450 and the range of transformations they catalyse suggests that they have evolved to encompass both structural stability and conformational flexibility. Prokaryotic marine extremophiles have been reported to utilize CYPs (CYP153 family) to catalyse the oxidization of medium-length alkanes (Di Donato et al., 2019). Additionally, a number of other potential halophilic P450 genes have been identified in halophilic archaea (Muller 2012). A number of thermophilic extremophiles have been reported to utilize CYPs as part of their metabolism and include: Thermobifida fusca (CYP154H1) (Shallmey et. al. 2001), Sulfolobus solfataricus (CYP119A1) (Wright et. al. 1996, Yano et. al. 2000), Sulfolobus tokodaii sp. strain 7 (CYP119A2) (Suzuki et. al. 2002), Thermus thermophilus (CYP175A) (Yano et. al. 2003), and Picrophilus torridus (CYP231A2) (Ho et. al. 2008). Once again, a number of other potential P450-encoding genes have been reported to be present in other thermophilic and psychrophilic organisms (Harris et. al. 2018). The diverse range of substrates metabolized by P450s and the broad range of reactions catalysed make these enzymes attractive candidates for industrially useful biocatalysis. P450s typically catalyse region and stereoselective oxidations of nonactivated CH bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, flavours and fragrances and as bioremediation agents (Li et al., 2020). Other chemical and biochemical applications of CYPs include protein, redox-partner, substrate, and electron source engineering in efforts to efficiently produce pharmaceuticals and other chemicals (Zou et. al. 2020). Significant efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. However, the harsh conditions of industrial applications (high temperature, low pH etc) impose limitations on the utility of CYPs due to their instability in such environments. Thus, there has been considerable interest and effort invested in finding or engineering extremophilic P450 systems (Harris et. al. 2018). The significant biotechnological impact of extremophiles and the uniqueness of the cytochrome P450 system make these organisms an attractive targets in which to investigate alternative cytochrome P450 redox systems. Taking into account the unquestionable importance of these systems in the metabolism of all organisms and the lack of knowledge about them in extremophiles, this work aims to provide provide a detailed structural, biochemical and phylogenetic characterization of extremeophilic CYPs and their diverse applications. The work will focus on screening cytochromes P450 in extremophiles with parallel computational analysis with known CYPs to establish their homology for eventual structural comparisons. Moreover, the work will be directed towards the over-expression of CYPs in suitable hosts to provide amounts sufficient for spectroscopic characterization and possible crystallisation. Further, their comparison with comparable mesophilic CYPs will be undertaken to understand the structural & functional adaptations to an extremophilic environment. Finally, potential functional applications will be explored in biotechnological, biochemical, pharmaceutical and environmental arenas. In summary, CYPs are remarkable enzymes with great potential as biocatalysts because of the wide range of substrates they accept and the variety of oxidative transformations they catalyse. Their biotechnological exploitation is somewhat limited due to their instability to the sometimes harsh conditions of industrial transformations. One way to overcome this limitation is by uitlilisation of CYPs already adapted to the harsh environments in which extremophiles live. Genome sequencing has revealed a number of potential CYPs in such organisms but very few have been characterized.