Recombinant Production of a Novel Cytochrome P450 Enzyme from Antarctica by Metagenomics


Creative Commons License

Keleş I., Mumcu H., Güneş Develioğlu Y., Çelik Balcı N., Karacan I., Gül Karagüler N., ...More

Presentation, pp.3-4, 2023

  • Publication Type: Other Publication / Presentation
  • Publication Date: 2023
  • Page Numbers: pp.3-4
  • Istanbul Technical University Affiliated: Yes

Abstract

The cytochromes P450 are a heme-bound enzyme superfamily involved in several important pathways in living organisms such as fatty acid, steroid, secondary metabolite metabolisms and xenobiotic detoxification. They catalyze more than twenty different chemical reactions including hydroxylation, oxidation, alkylation, epoxidation, and C-C bond cleavage. P450s are naturally expressed in a broad range of living organisms from prokaryotes to eukaryotes and even non-living entities such as viruses. These versatile and highly abundant enzymes have great potential to be used in various industrial fields. However, low stability and low activity restrict the usage of P450s under extreme industrial conditions. Extremozymes which are natively resistant to harsh environmental conditions, might overcome these limitations and enhance the potential of P450s. To date, the number of well-characterized extremophilic P450s do not meet the demand of the market. Thus, the discovery of novel extremophilic P450s and their biochemical and biophysical characterization is crucial for their large-scale production and application. Metagenomics is a powerful tool for novel enzyme discovery from environmental samples thanks to the recent improvements in next-generation sequencing technologies and bioinformatics tools. This study aims at the cloning, heterologous expression, and purification of a novel P450 enzyme from the metagenomics data of soil samples from King George Island, Antarctica. Firstly, the environmental DNA from soil samples was extracted, total DNA was sequenced, and metagenome assembly was done. After library generation, full-length sequences were obtained, and P450s were determined by using NCBI and CYPED databanks. The selected P450 sequence was synthesized and cloned into a pET28a (+) expression vector with specific restriction sites. The vector containing the gene of interest was transformed into an Escherichia coli C43 host cell. The overexpressed P450 enzyme was purified with immobilized metal affinity chromatography (IMAC). In the near future, the biochemical and biophysical characterization of this overexpressed P450 enzyme will be completed.