Efficient Bioproduction of Indigo and Indirubin by Optimizing a Novel Terpenoid Cyclase XiaI in .

Blue indigo dye, an important natural colorant, is used for textiles and food additives worldwide, while another red isomer, indirubin, is the major active ingredient of a traditional Chinese medicine named "Danggui Longhui Wan" for treating various diseases including granulocytic leukemia, cancer, and Alzheimer's disease. In this work, we constructed a new and highly efficient indigoid production system by optimizing a novel terpenoid cyclase, XiaI, from the xiamycin biosynthetic pathway. Through introducing the flavin-reducing enzyme Fre, tryptophan-lysing and -importing enzymes TnaA and TnaB, and HO-degrading enzyme KatE and optimizing the fermentation parameters including temperature, the concentration of isopropyl-β-d-thiogalactopyranoside, and feeding of the l-tryptophan precursor, the final maximum productivity of indigoids by the recombinant strain BL21(DE3) (XiaI-Fre-TnaAB-KatE) was apparently improved to 101.9 mg/L, an approximately 60-fold improvement to that of the starting strain BL21(DE3) (XiaI) (1.7 mg/L). In addition, when the fermentation system was enlarged to 1 L in the flask (feeding with 5 mM tryptophan and 10 mM 2-hydroxyindole), the indigoid productivity further increased to 276.7 mg/L at 48 h, including an indigo productivity of 26.0 mg/L and an indirubin productivity of 250.7 mg/L, which has been the highest productivity of indirubin so far. This work provided a basis for the commercial production of bio-indigo and the clinical drug indirubin in the future.