The Enzyme-Bag Platform for Cytochrome P450 Biotransformation and High-Throughput Drug Metabolism Screening

A comprehensive and scalable understanding of human drug metabolism is essential for advancing pharmacology, toxicology, and precision medicine. Traditional in-vitro models, such as human liver microsomes and purified enzyme systems, are often constrained by high cost, technical complexity, and limited physiological relevance. To address these challenges, we present a streamlined and versatile biotransformation platform based on Schizosaccharomyces pombe, leveraging recombinant expression of human cytochrome P450 (CYP) enzymes integrated with a semi-intact whole-cell approach known as the enzyme-bag system. This protocol encompasses five core components: (1) microbial cultivation of recombinant yeast strains, (2) construction of a complete human CYPome gene library, (3) generation of enzyme bags through selective permeabilization, (4) execution of CYP-mediated biotransformations, and (5) high-throughput screening using hierarchical enzyme-bag cocktails. The resulting enzyme bags retain membrane-associated CYP activity while allowing direct substrate access, enabling robust, physiologically relevant enzymatic assays without the need for labor-intensive microsomal preparations or purified proteins. The platform supports rapid enzyme-bag preparation (~3 h) and efficient biotransformations (~4 h), compatible with diverse detection modalities, including luminescence-based assays and chromatographic or mass spectrometric techniques for metabolite profiling. It supports cofactor regeneration, is scalable, and enables functional analysis of all 57 human CYP enzymes-including orphan CYPs-within a unified yeast platform. Furthermore, this approach has been extended to phase II enzymes (UGTs, SULTs), insect P450s, and alternative hosts, such as Saccharomyces cerevisiae, broadening its applicability across diverse systems. Recent advancements in high-throughput screening using enzyme-bag cocktails enable rapid enzyme identification and substrate mapping, significantly enhancing throughput while preserving specificity. This platform offers a transformative tool for drug metabolism studies, enabling efficient characterization of enzyme activity, substrate specificity, and inhibitor profiling. By bridging physiological relevance with experimental simplicity, the enzyme-bag system sets a new standard for in vitro metabolic research, accelerating discovery in pharmacokinetics, toxicology, synthetic biology, and drug development.