Construction of a dual-enzyme cascade system for the directed biosynthesis of indigotin and indirubin from indican...

Indigotin and indirubin are bisindole alkaloids derived from plants such as Strobilanthes cusia (Nees) Ktze, with significant industrial application value. Currently, their primary preparation methods rely on plant extraction, which not only involves complex processes but also results in low yields. Although chemical synthesis is efficient, it necessitates the use of large quantities of organic reagents, contributing to environmental pollution. Additionally, both catalysts and by-products can have toxic effects on the environment and human health. With the rise of green production concepts, biocatalysis technologies that align with sustainable development principles offer new pathways for the biosynthesis of indigotin and indirubin. This study improved the flavin-dependent monooxygenase MeFMO, derived from Methylophaga sp. SK1, resulting in a mutant strain MeFMO^Q318L/W319F with a 2.1-fold increase in catalytic activity. By integrating the engineered MeFMO^Q318L/W319F with the β-glucosidase Asbg1 into a one-pot cascade system, a clean production platform was established, enabling efficient conversion of indigotin or indirubin from indican fermentation broth while eliminating intermediate purification steps. Additionally, a tryptophan-based bioconversion process was developed by integrating a heterologous indican biosynthetic module, an NADPH regeneration system, and a subsequent enzymatic oxidation-hydrolysis cascade. To address interference from fermentation broth impurities, a three-step pretreatment strategy was established to obtain a purified and concentrated indican solution. Following process optimization, the indigotin yield reached 413 mg/L with a molar conversion rate of 99.1 %, while indirubin production reached 795 mg/L with a molar conversion rate of 80.0 %.