High-Efficiency Biocatalytic Production of Androsta-1,4-diene-3,17-dione via a Dual-Enzyme Coupling System in Escherichia coli

Androsta-1,4-diene-3,17-dione (ADD) is a key intermediate in the synthesis of various steroidal pharmaceuticals, but the biocatalytic conversion of androst-4-ene-3,17-dione (AD) to ADD is limited by hydrogen peroxide (H₂O₂), a byproduct that inhibits the enzyme 3-ketosteroid-∆1-dehydrogenase (KstD). This study aimed to improve the bioconversion efficiency by introducing catalase (katA) to decompose H₂O₂, thereby alleviating its toxic effects on the enzyme. The kstD₂ gene from Mycobacterium neoaurum DSM 1381 was mutated using error-prone PCR to generate the KstD₂^ep variant, which was then coupled with the katA gene in E. coli strains. The engineered strain E. coli BL21-pET28a-KstD₂^ep-L-katA exhibited the highest catalytic efficiency under optimized conditions, achieving a 98.6% conversion of 40 g/L AD to ADD in 14 h (optimized conditions: 40 °C, pH 8.0, 40 g/L wet cell concentration, and 1:1 cosolvent HP-β-CD with AD). Fermentation in a 5L fermenter further increased the conversion to 98.2%, using 80 g/L AD in repeated batch feeding, significantly improving the conversion efficiency compared to shake flask conditions. These results suggest that the coupled KstD₂^ep and catalase system, along with optimized fermentation parameters, could provide an efficient and scalable biocatalytic process for the industrial production of ADD and related steroidal compounds.