Reconstruction of Efficient Flavin Adenine Dinucleotide (FAD) Regeneration Cycle to Enhance C1,2‐Dehydrogenation of Steroids in <i>Saccharomyces cerevisiae</i>

Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous enzymes involved in critical physiological activities and industrial reactions. A notable example is 3-ketosteroid Δ1-dehydrogenase (KSTD), which catalyzes the C1,2-dehydrogenation of steroids, a pivotal step in steroid biotransformation. This reaction enhances the biological activity and value of steroids, making them essential precursors for pharmaceutically significant compounds. However, microbial conversion of steroids encounters challenges such as insufficient supply of FAD cofactor. To address this issue, we firstly heterologously expressed the kstd gene to achieve C1,2-dehydrogenation in Saccharomyces cerevisiae. We then developed approaches to enhance FAD supply and regeneration: the FAD biosynthesis pathway from Bacillus subtilis was integrated into S. cerevisiae to enhance FAD supply, and the formate dehydrogenase and NADH oxidase were incorporated to enhance FAD regeneration. Thus, the resulting recombinant S. cerevisiae strain SC-BEFNK enabled a high conversion efficiency of the substrate 4-androstenedione (AD) to 1,4-diene-3,17-dione (ADD), producing 0.95 g/L ADD from 1 g/L AD in 60 h, with a molar conversion ratio of 96.1%. Upon optimization of the fermentation conditions, this strain completely converted 5 g/L of AD in 48 h, achieving a molar conversion ratio of 98.1%. This yeast-based system was further shown to be applicable to the dehydrogenation reaction of a number of different steroids and held great potential for other FAD-dependent enzymatic processes.