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

Huifang Yin; Hongping Chen; Meng Yan; Zhikun Li; Rongdi Yang; Yanjiao Li; Yanfang Wang; Jianyi Guan; Huili Mao; Yan Wang; Yuyang Zhang

doi:10.1021/acsomega.1c02679

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

ACS Omega. 2021 Jul 27;6(31):20569-20576. doi: 10.1021/acsomega.1c02679. eCollection 2021 Aug 10.

Authors

Huifang Yin^{1

2}, Hongping Chen¹, Meng Yan¹, Zhikun Li¹, Rongdi Yang¹, Yanjiao Li¹, Yanfang Wang¹, Jianyi Guan^{1

2}, Huili Mao¹, Yan Wang^{1

2}, Yuyang Zhang^{1

2}

Affiliations

¹ School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, China.
² Synthetic Biology Engineering Lab of Henan Province, Xinxiang, 453003 Henan, China.

Abstract

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 H₂O₂-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 Escherichia coli 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 E. coli 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.