Abstract
Abstract
In this study, the methylotrophic yeast
Komagataella phaffii
GS115 was engineered to express the wild-type flavin-containing monooxygenase from
Methylophaga aminisulfidivorans
strain SK1 (MaFMO), enabling the biocatalytic conversion of externally supplied indole into the bisindole pigments indigo and indirubin. The effects of indole concentration, methanol concentration, induction time, agitation speed, and supplementation with L-cysteine or Tween 80 were individually evaluated. In the substrate concentration experiment, 3 mM indole resulted in the highest indigo titer (19.97 ± 0.68 mg/L) and an indirubin titer of 2.29 ± 0.19 mg/L. Increasing the indole concentration to 5 mM markedly decreased pigment formation, suggesting substrate-associated inhibition and/or indole-induced cellular stress. Methanol concentrations ranging from 0.5 to 2.0% (v/v) had relatively limited effects on pigment titers. In a separate time-course experiment, extending the induction period to 72 h increased the indigo and indirubin titers to 38.93 ± 3.77 and 2.70 ± 0.96 mg/L, respectively. Supplementation with L-cysteine altered the apparent product distribution, whereas supplementation with 7% (v/v) Tween 80 strongly inhibited pigment production. These results demonstrate the functional activity of MaFMO in
K. phaffii
and establish a proof-of-concept yeast platform for the biotransformation of indole into indigo and indirubin. Further optimization of substrate feeding, oxygen transfer, redox balance, and host metabolism will be necessary to improve pigment titer, productivity, and product selectivity.