Optimizing Heterologous Expression of CYP124 in Escherichia coli using Box-Behnken Design

Gunawan, Catherine Marta; Nurfadila, Masyarrah Halida; Utami, Ratna Annisa; Pramastya, Hegar

doi:10.30491/jabr.2024.470675.1766

Optimizing Heterologous Expression of CYP124 in Escherichia coli using Box-Behnken Design

Document Type : Original Article

Authors

Catherine Marta Gunawan ¹

Masyarrah Halida Nurfadila ¹

Ratna Annisa Utami ²

Hegar Pramastya ¹

¹ Laboratory of Combinatorial Biosynthesis, Department of Pharmaceutical Biology, School of Pharmacy, Institut Teknologi Bandung, Jalan Ganesha No. 10 Bandung, West Java 40132, Indonesia

² Laboratory of Pharmaceutical Biotechnology, Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Jalan Ganesha No. 10 Bandung, West Java 40132, Indonesia

10.30491/jabr.2024.470675.1766

Abstract

Introduction: CYP124 is a cytochrome P450 enzyme from Mycobacterium tuberculosis capable of catalyzing ω-hydroxylation of methyl-branched lipids, isoprenoid alcohols, sterols, cholesterol analogs, and vitamin D3. It is also suspected to be involved in the bacterium's resistance to macrophages. Due to its capabilities, CYP124 has potential as a drug target and biocatalyst. Therefore, there is a need to produce sufficient quantities of CYP124 for biochemical assays, protein engineering, and drug development. The study optimized CYP124 production in Escherichia coli DH5α using Response Surface Methodology (RSM) with a Box-Behnken Design (BBD).
Materials and Methods: FeCl3, δ-aminolevulinic acid (5-ALA), and IPTG concentrations were optimized using BBD. A total of 15 experimental runs with various factor combinations were performed, and a three-dimensional response surface was generated to analyze the effects and identify the optimal concentrations of each factor, with R/Z value and CYP124 yield as responses.
Results: The optimal supplement combination for CYP124 production included 0.10 mM FeCl₃, 1 mM 5-ALA, and 0.2427 mM IPTG. The model predicted the highest CYP124 yield to be 0.041 mg/L. Purified CYP124 bound to farnesol, its substrate, with a dissociation constant of 1.00 ± 0.14 µM.
Conclusions: The findings underscore the significance of ALA supplementation in regulating CYP450 expression and emphasize the need to optimize FeCl3 and IPTG concentrations as cofactors and inducers, respectively.

Keywords

Cytochrome P450

Medium Supplements

Response Surface Methodology

Mycobacterium tuberculosis