Improvement of NADPH-dependent P450-mediated biotransformation of 7α,15α-diOH-DHEA from DHEA by a dual cosubstrate-coupled system

• Low intracellular NADPH/NADP was demonstrated to be a limiting factor during DHEA biotransformation.
• Hydroxylation yield from DHEA was increased significantly by a dual cosubstrate-coupled system.
• High substrate concentration (15 g/L) and high yield were obtained by a fed-batch transformation model.

Hydroxylation of DHEA to 7α,15α-diOH-DHEA was catalyzed by NADPH-dependent cytochrome P450 monooxygenase from Colletotrichum lini. By adding coenzyme precursor nicotinic acid, the NADPH/NADP ratio was significantly increased, and the 7α,15α-diOH-DHEA molar conversion was enhanced from 37.37% to 50.85%. To improve the availability of intracellular NADPH, a dual cosubstrate-coupled system consisting of nicotinic acid and glucose was investigated in C. lini. Using 20 mM nicotinic acid and 15 g/L glucose as cosubstrate for NADPH regeneration, the 7α,15α-diOH-DHEA molar conversion was dramatically increased by 74.58%. The conversion course was simultaneously shortened by 30 h. Moreover, a fed-batch transformation model was established to diminish DHEA toxicity to C. lini and further increase DHEA concentration. The maximum concentration of DHEA was elevated to 15 g/L using a three-batch transformation in a coenzyme regeneration system, and 7α,15α-diOH-DHEA production of 11.21 g/L could be achieved after 60 h of biotransformation. These results demonstrated that this strategy was promising for steroids hydroxylation.

Figure optionsDownload as PowerPoint slide