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 (H 2 O 2 ), a byproduct that

inhibits the enzyme 3-ketosteroid-Δ1-dehydrogenase (KstD). This study aimed to improve the bioconversion effi ciency by

introducing catalase (katA) to decompose H 2 O 2 , thereby alleviating its toxic eff ects on the enzyme. The kstD 2 gene from

Mycobacterium neoaurum DSM 1381 was mutated using error-prone PCR to generate the KstD 2

ep variant, which was then

coupled with the katA gene in E. coli strains. The engineered strain E. coli BL21-pET28a-KstD 2

ep -L-katA exhibited the

highest catalytic effi ciency 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, signifi cantly improving

the conversion effi ciency compared to shake fl ask conditions. These results suggest that the coupled KstD 2

ep and catalase

system, along with optimized fermentation parameters, could provide an effi cient and scalable biocatalytic process for the

industrial production of ADD and related steroidal compounds.