Highly efficient conversion of androstenedione into dehydroepiandrosterone through the construction of molecular machines

Abstract

Dehydroepiandrosterone (DHEA), a pivotal steroid hormone precursor, holds significant clinical and industrial value for its role in hormone synthesis. Traditional chemical and chemo-enzymatic production methods face challenges such as complex processes, low yields, and environmental concerns. This study presents a green, all-enzymatic route for the synthesis of DHEA from 4-androstene-3,17-dione (4-AD) using engineered molecular machines. By leveraging SpyCatcher-SpyTag and cohesin-dockerin interactions, we constructed dual- and triple-enzyme complexes to spatially organize 3β-ketosteroid isomerase, carbonyl reductase, and formate dehydrogenase. The dual-enzyme system achieved an 84% conversion rate for 10 g/L 4-AD, while the triple-enzyme complex further enhanced conversion to 90% (10 g/L) and 98% (2.5 g/L). This strategy overcomes the instability of the intermediate 5-androstene-3,17-dione (5-AD) through enzyme proximity, and eliminate chemical reactions. This work establishes a sustainable, highly efficient biocatalytic synthesis of DHEA, offering a novel strategy for challenging steroidal transformations and advancing green pharmaceutical manufacturing.

Keywords

3β-ketosteroid isomerase / 3β-ketosteroid dehydrogenase / Dockerin-cohesin / SpyCatcher-SpyTag / 4-androstenedione / Dehydroepiandrosterone

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Funding

National Key R and D Program of China(2019YFA0905300)

Innovative Research Group Project of the National Natural Science Foundation of China(32471543)

Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-CXRC-052)

RIGHTS & PERMISSIONS

Jiangnan University