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

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.

SUBMITTER: Yin H

PROVIDER: S-EPMC8359145 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. RESULTS: Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any yield published in scientific literature so far. CONCLUSIONS: This study provides for the first time a comprehensive and detailed in silico comparison of the most prominent heterologous hosts E. coli and S. cerevisiae as terpenoid factories giving an overview on several promising metabolic engineering strategies paving the way for an enhanced terpenoid yield.

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

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