MICROBIAL PRODUCTION OF INDIRUBIN DERIVATIVES USING A CYTOCHROME P450 ENZYME FROM ISATIS TINCTORIA L.

Author

Sandipty Kayastha, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2023

Degree Name

Doctor of Philosophy

Department

Plant Biology

First Advisor

Anterola, Aldwin

Abstract

CYP71B102 is a cytochrome P450 enzyme in woad (Isatis tinctoria L.), which catalyzes the hydroxylation of indole, leading to the formation of the anticancer compound indirubin. The I. tinctoria gene CYP71B102 and Arabidopsis thaliana P450 reductase AtR2 were heterologously expressed together in E. coli (LJSK023 strain), which then produced a pink-colored water-soluble extract hypothesized to contain indirubin derivatives having anticancer and antimicrobial activities. Shake flask experiments with the LJSK023 strain in LB media supplemented with 2 mM L-tryptophan enabled the production of 254 ± 49.0 mg/L of water-soluble extract at the yield of 0.62 mg compounds/g L-tryptophan. Both CYP71B102 and AtR2 were codon optimized and expressed in E. coli (LJSK236 strain), which enabled the production of 144 ± 24.0 mg/L of water-soluble extract at the yield of 0.35 mg compounds/g L-tryptophan. The inhibitory activities of the LJSK023 and LJSK236 extracts against prokaryotic and eukaryotic organisms were tested by performing spot and 96-well plate assays. The results revealed that both extracts inhibited the growth of Saccharomyces cerevisiae, Salmonella typhimurium, Candida albicans, and Cryptococcus neoformans. In addition, a trypan blue staining assay was carried out to determine the viability of HeLa cells treated with or without the LJSK023 extract. The data showed that LJSK023 extract (8.12 ng/μL) inhibited the proliferation of HeLa cells over time. Then the chemical structures of the compounds present in the extracts were investigated by spectroscopic analysis using UV-Visible, Raman, and tandem mass spectrometry (LC-MS/MS), which altogether suggested the presence of indirubin-like compounds. In particular, LC-MS/MS suggested the presence of three potential structures for the novel indirubin derivatives in the extract. Finally, a protocol was developed using His GraviTrap column to purify the codon optimized chimeric protein (CYP71B102 linked to AtR2). Therefore, this study demonstrated the potential for microbial production of high-value-added novel water-soluble indirubin derivatives exhibiting potent antimicrobial and anticancer effects. In the future, these preliminary findings can be leveraged to scale up the microbial production of novel water-soluble indirubin derivatives in engineered E. coli strains. Furthermore, the molecular mechanism of these novel indirubin derivatives can be explored to assess their potential as lead compounds for the development of new therapeutic agents followed by in-depth biochemical characterization of the novel CYP71B102 enzyme.