Aims: 3-ketosteroid-Delta(1)-dehydrogenase (KSDD), a flavin adenine dinucleotide (FAD)-dependent enzyme involved in sterol metabolism, specifically catalyses the conversion of androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). However, the low KSDD activity and the toxic effects of hydrogen peroxide (H2O2) generated during the biotransformation of AD to ADD with FAD regeneration hinder its application on AD conversion. The aim of this work was to improve KSDD activity and eliminate the toxic effects of the generated H2O2 to enhance ADD production. Methods and Results: The ksdd gene obtained from Mycobacterium neoaurum JC-12 was codon-optimized to increase its expression level in Bacillus subtilis, and the KSDD activity reached 12.3 U mg(-1), which was sevenfold of that of codon-unoptimized gene. To improve AD conversion, catalase was co-expressed with KSDD in B. subtilis 168/pMA5-ksdd(opt)-katA to eliminate the toxic effects of H2O2 generated during AD conversion. Finally, under optimized bioconversion conditions, fed-batch strategy was carried out and the ADD yield improved to 8.76 g l(-1). Conclusions: This work demonstrates the potential to improve enzyme activity by codon-optimization and eliminate the toxic effects of H2O2 by co-expressing catalase. Significance and Impact of the Study: This study showed the highest ADD productivity ever reported and provides a promising strain for efficient ADD production in the pharmaceutical industry.