Construction of an Efficient Non-natural Enzyme System for Preparation of Testosterone in High Space-Time Yield

Testosterone (TS), an important hormone pharmaceutical and precursor for the synthesis of other steroids, is traditionally produced by a polluting and costly multistep chemical synthesis. Producing TS by a biological method is eco-friendly, while its application is still limited by its low substrate-loading, low conversion, long duration, and low time-space yield. Focusing on these problems, a new enzyme system was constructed in which the carbonyl reductase PmCR was employed to reduce 4-androstene-3,17-dione (4-AD) into TS for the first time coupled with the formate dehydrogenase BstFDH_m. Based on molecular dynamics (MD) simulation, the enzyme system was upgraded by rationally designing PmCR, and the variant L136S was screened with a 14-fold enhanced catalytic efficiency kcat/Km and the highest performance in 4-AD reduction. The testosterone production of the L136S-BstFDH_m system was further enhanced by condition optimization and competence to synthesize TS with the highest reported 4-AD loading (28.8 g/L), conversion (100%), and testosterone time-space yield (2.90 g/L/h) using a microbial method, highlighting its promising application in TS synthesis. The successful attempt in this study not only provides a promising biocatalyst and efficient approach for TS green synthesis but also opens the door to the application of carbonyl reductase in the green synthesis of diverse pharmaceutical sterols.