Indirubin Production Research Articles

Functional characterization of a novel flavin reductase from a deep-sea sediment metagenomic library and its application for indirubin production.

Microbial synthesis is a desirable approach to produce indirubin but suffers from low synthetic efficiency. Insufficient supply of reduced flavins is one major factor limiting synthetic efficiency. To address this, a novel flavin reductase, MoxB, was discovered through screening of the metagenomic library. MoxB showed a strong preference for NADH over NADPH as the electron source for FMN/FAD reduction and exhibited the highest activity at pH 8.0 and 30°C. It displayed remarkable thermostability by maintaining 80% of full activity after incubation at 60°C for 1 h. Furthermore, MoxB showed great organic solvent tolerance and its activity could be significantly increased by bivalent metal ions. In addition, heterologous expression of the moxB gene in the indirubin-producing E. coli significantly improved indirubin production up to 15.12-fold. This discovery expands the understanding of flavin reductases and provides a promising catalytic tool for microbial indirubin production.IMPORTANCEMuch effort has been exerted to produce indirubin using engineered Escherichia coli, but high-level production has not been achieved so far. Insufficient supply of reduced flavins is one key factor limiting the catalytic efficiency. However, the flavin reductases involved in indirubin biosynthesis have not been hitherto reported. Discovery of the novel flavin reductase MoxB provides a useful tool for enhancing indirubin production by E. coli. Overexpression of MoxB in indirubin-producing E. coli increased indirubin production by 15.12-fold in comparison to the control strain. Our results document the function of flavin reductase that reduces flavins during indirubin biosynthesis and provide an important foundation for using the flavin reductases to improve indirubin production by engineered microorganisms.

Silver and salicylic acid-chitosan nanoparticles alter indole alkaloid production and gene expression in root and shoot cultures of Isatis tinctoria and Isatis ermenekensis

Isatis spp. are well-known for their industrial significance due to natural sources of indigotin and indirubin, important indole alkaloids, used in the dye and pharmaceutical industries. In this study, silver nanoparticles (AgNP) and salicylic acid-chitosan nanoparticles (SA-CNP) were synthesized and applied to enhance the production of indigotin and indirubin in shoot and root cultures of Isatis tinctoria and Isatis ermenekensis. Different doses of AgNP and SA-CNP were administered to three-week-old shoot and root cultures, and the effects were assessed at 12, 24, and 48 h. The harvested samples were analyzed to quantify indigotin and indirubin levels. Furthermore, the expression levels of It-TSA and CYP79B2 genes, known to be involved in indole alkaloid biosynthesis, were determined. In I. tinctoria roots, the highest levels of indigotin and indirubin were observed after applying 150 mg L−1 of SA-CNP for 48 h while in I. ermenekensis shoots, indigotin and indirubin reached the maximum levels with the application of 8 mg L−1 AgNP for 48 h. NP application had no remarkable effects on the accumulation of indigotin and indirubin in I. tinctoria shoots and I. ermenekensis roots compared to controls. Additionally, shoot cultures demonstrated superior indirubin production, which significantly increased with AgNP applications. The gene expression analysis also exhibited significant correlations with the changes in indigotin and indirubin levels. The findings of this study lay the groundwork for enhancing in vitro production of indigotin and indirubin in Isatis species through NP applications, and for developing high-capacity production strategies by determining optimal dosages in scale-up studies.

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

Green production of indigo and indirubin by an engineered Baeyer–Villiger monooxygenase

Indigoids are well-known dye molecules of widespread industrial applications in textiles as well as more recently in pharmaceuticals. Historically they have been predominantly produced using chemical reactions that generate undesired by-products. The use of flavin-containing monooxygenases as biocatalysts is an environmentally-friendly alternative to the oxidation of indole leading to the production of indigoids such as indigo and indirubin. In this work, one such enzyme, an active site mutant of the Baeyer–Villiger monooxygenase from Acinetobacter radioresistens (Ar-BVMO), has been used for the transformation of indole into indigo or indirubin. Under optimal conditions, E. coli whole-cells expressing this mutant are capable of producing up to 154 mg/L of the blue indigo dye. Supplementation with 5 mM cysteine switches the production to the more medically relevant red dye, indirubin, where 138 mg/L can be obtained. In silico docking experiments performed with the 3D molecular model of this BVMO demonstrates that the enlarged active site of the mutant enzyme not only facilitates indole access but also its productive binding. Taken together, this work represents one of the first characterizations of a green process driven by a BVMO as a biocatalyst for the production of indigoids, specially the medically relevant indirubin.

Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin

Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π–π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.Graphical

Integrated process for the production of indigo and indirubin in an anaerobic environment at laboratory and pilot level

The production of indigo commonly requires an oxidation step in which indoxyl is oxidized to indigo. There are no investigations into the effect of oxygen transfer on indigo production. Herein, increased yield and purity of indigo were found with increased reaction volume, indicating a negative effect of aeration on indigo production in alkali conditions. Therefore, the condensation of indoxyl in the presence and absence of oxygen was assessed. The results showed consumption of indoxyl in aerobic conditions to be faster than in anaerobic conditions, while the utilization rate of indoxyl into indigo and indirubin was lower. In order to improve indoxyl utilization, the production of indigo/indirubin in an anaerobic environment was evaluated in a nitrogen gas environment during the indoxyl condensation step. The yield and purity of indigo were 4.5 mg/g and 43.5%, respectively, which were 1.1 and 1.5 times higher than those of the traditional process. Moreover, the alkaline condition under the nitrogen atmosphere provided a high production of indirubin. The cost of raw materials was found to be the most important factor for determining the cost of indigo production and could be reduced by improved indican utilization. Based on these findings, an anaerobic indigo/indirubin integrated production process was proposed for a pilot scale adaptation.

Effect of Silver Nanoparticles on Production of Indole Alkaloids in Isatis constricta

Silver nanoparticles (AgNPs) are widely used in many applications of biotechnology, including medicine and agriculture. They are released to the nature as waste materials, which can cause physiological and biochemical effects on plants. Indigo, indirubin and tryptanthrin are valuable indole alkaloid compounds in Isatis constricta due to both medicinal effects and dye properties. This research was conducted to determine the effects of different concentrations (0, 0.25, 0.5, 1, 1.5 and 2 mg L−1) of AgNPs on the production of indigo, indirubin and tryptanthrin compounds in leaves of in vitro grown shoots of I. constricta Davis. Indigo production was 1.15-fold of control (869 ± 8.33 µg g−1) in the leaves of shoots regenerated in Murashige and Skoog supplemented with 2 mg L−1 of AgNPs (1003 ± 11.42 µg g−1) on 5 days post-treatment. Tryptanthrin production showed an increase in all applications of AgNPs, but the highest increase was observed at a concentration of 2 mg L−1 (4.59 ± 0.046 µg g−1) and this increase was 1.71-fold of control (2.68 ± 0.031 µg g−1) on 5 days post-treatment. The production of indigo and tryptanthrin decreased on 10 and 15 days post-treatment with AgNPs. The contents of indirubin decreased during day 5-10-15 and at all concentrations of AgNPs compared to the control.

A Biomimetic, One-Step Transformation of Simple Indolic Compounds to Malassezia-Related Alkaloids with High AhR Potency and Efficacy.

Malassezia furfur isolates from diseased skin preferentially biosynthesize compounds which are among the most active known aryl-hydrocarbon receptor (AhR) inducers, such as indirubin, tryptanthrin, indolo[3,2-b]carbazole, and 6-formylindolo[3,2-b]carbazole. In our effort to study their production from Malassezia spp., we investigated the role of indole-3-carbaldehyde (I3A), the most abundant metabolite of Malassezia when grown on tryptophan agar, as a possible starting material for the biosynthesis of the alkaloids. Treatment of I3A with H2O2 and use of catalysts like diphenyldiselenide resulted in the simultaneous one-step transformation of I3A to indirubin and tryptanthrin in good yields. The same reaction was first applied on simple indole and then on substituted indoles and indole-3-carbaldehydes, leading to a series of mono- and bisubstituted indirubins and tryptanthrins bearing halogens, alkyl, or carbomethoxy groups. Afterward, they were evaluated for their AhR agonist activity in recombinant human and mouse hepatoma cell lines containing a stably transfected AhR-response luciferase reporter gene. Among them, 3,9-dibromotryptanthrin was found to be equipotent to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an AhR agonist, and 3-bromotryptanthrin was 10-times more potent than TCDD in the human HG2L7.5c1 cell line. In contrast, 3,9-dibromotryptanthrin and 3-bromotryptanthrin were ∼4000 and >10,000 times less potent than TCDD in the mouse H1L7.5c3 cell line, respectively, demonstrating that they are species-specific AhR agonists. Involvement of the AhR in the action of 3-bromotryptanthrin was confirmed by the ability of the AhR antagonists CH223191 and SR1 to inhibit 3-bromotryptanthrin-dependent reporter gene induction in human HG2L7.5c1 cells. In conclusion, I3A can be the starting material used by Malassezia for the production of both indirubin and tryptanthrin through an oxidation mechanism, and modification of these compounds can produce some highly potent, efficacious and species-selective AhR agonists.

Effects of Methyl Jasmonate and Putrescine on Tryptanthrin and Indirubin Production in in vitro Cultures of Isatis demiriziana Mısırdalı

Tryptantrin and indirubin are pharmacologically active compounds used in treatment of disseases such as cancer and Alzheimer's. In this study, we investigated the influences of different concentrations of methyl jasmonate (MeJa) and putrescine (Put) on tryptanthrin and indirubin production in leaf explants and development of Isatis demiriziana Mısırdalı grown in vitro. In all media treated with methyl jasmonate, tryptanthrin production in leaves of plantlets showed an increase. The highest increase in tryptanthrin production was observed in solid Murashige-Skoog (MS) medium containing 1.0 mM MeJa (154.026 ± 0.11 µg g-1), about 2.85-fold higher than the control (untreated plantlets) (40.017 ± 0.031 µg g-1). Production of tryptanthrin decreased about 2.56-fold in the leaves of plantlets treated with Put, when compared to control. The highest indirubin production was obtained in the leaves of plantlets grown in the MS medium containing 0.1 mM MeJa (11.274 ± 0.035 µg g-1) but treatments with Put didn’t show any positive affect on the indirubin production. Analysis of tryptanthrin and indirubin were performed using high performance liquid chromatography (HPLC).

Protein Engineering of Flavin-containing Monooxygenase from Corynebacterium glutamicum for Improved Production of Indigo and Indirubin

Protein Engineering of Flavin-containing Monooxygenase from Corynebacterium glutamicum for Improved Production of Indigo and Indirubin

Metabolic engineering of Escherichia coli for the production of indirubin from glucose

Indirubin is an indole alkaloid that can be used to treat various diseases including granulocytic leukemia, cancer, and Alzheimer’s disease. Microbial production of indirubin has so far been achieved by supplementation of rather expensive substrates such as indole or tryptophan. Here, we report the development of metabolically engineered Escherichia coli strain capable of producing indirubin directly from glucose. First, the Methylophaga aminisulfidivorans flavin-containing monooxygenase (FMO) and E. coli tryptophanase (TnaA) were introduced into E. coli in order to complete the biosynthetic pathway from tryptophan to indirubin. Further engineering was performed through rational strategies including disruption of the regulatory repressor gene trpR and removal of feedback inhibitions on AroG and TrpE. Then, combinatorial approach was employed by systematically screening eight genes involved in the common aromatic amino acid pathway. Moreover, availability of the aromatic precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate, was enhanced by inactivating the pykF (pyruvate kinase I) and pykA (pyruvate kinase II) genes, and by overexpressing the tktA gene (encoding transketolase), respectively. Fed-batch fermentation of the final engineered strain led to production of 0.056 g/L of indirubin directly from glucose. The metabolic engineering and synthetic biology strategies reported here thus allows microbial fermentative production of indirubin from glucose.

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C–C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Characterization of a flavin-containing monooxygenase from Corynebacterium glutamicum and its application to production of indigo and indirubin.

To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments. The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu(2+) and recovered by adding 1-10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l(-1) and 103 mg indirubin l(-1) from 2.5 g L-tryptophan l(-1). The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.

Production of Indirubin from Tryptophan by Recombinant Escherichia coli Containing Naphthalene Dioxygenase Genes from Comamonas sp. MQ

Indirubin, a red isomer of indigo, can be used for the treatment of various chronic diseases. However, the microbial production of indirubin did not receive much attention probably due to its low yield compared with indigo. In this study, the recombinant Escherichia coli containing the naphthalene dioxygenase (NDO) genes from Comamonas sp. MQ was used to produce indirubin from tryptophan. To enhance the production of indirubin, the induction conditions for NDO expression were optimized. The optimal induction conditions were carried out with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 30 °C when cells were grown to OD600 ≈ 1.20. Subsequently, the effects of medium composition on indirubin production were investigated by response surface methodology, and 9.37 ± 1.01 mg/l indirubin was produced from 3.28 g/l tryptophan. Meanwhile, the indirubin production was further improved by adding 2-oxindole and isatin to the tryptophan medium after induction. About 57.98 ± 2.62 mg/l indirubin was obtained by the addition of 500 mg/l 2-oxindole after 1-h induction, which was approximately 6.2-fold to that without additional 2-oxindole. The present study provided a possible way to improve the production of indirubin and should lay the foundation for the application of microbial indirubin production.

Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation

In our previous study, a batch fermentation of recombinant Escherichia coli DH5α cells harboring the fmo gene from Methylophaga aminisulfidivorans MPT produced indirubin (5.0mg/L) and indigo (920mg/L) in a 5L fermenter containing tryptophan medium (2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl). In this study, it was found that indirubin production greatly increased when 0.36g/L cysteine was added to the tryptophan medium, although cysteine inhibited the growth of the recombinant E. coli harboring the fmo gene. However, the addition of cysteine did not inhibit the expression level and activity of FMO in the cell. Indigo was synthesized by the dimerization of two 3-hydroxyindole molecules under the non-enzymatic reaction. Cysteine influenced the regioselectivity of FMO and enhanced the synthesis of 2-hydroxyindole instead of 3-hydroxyindole, which might function to increase indirubin production. The optimal culture conditions for indirubin production in tryptophan medium were determined from the response surface methodology analysis: 2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl, 0.36g/L (3mM) cysteine, pH 8.0 at 35°C. Under these conditions, the recombinant E. coli cells were capable of producing 223.6mg/L of indirubin from 2g/L of tryptophan. The intracellular accumulation of the indirubin crystals might stress the cell, which may be a main reason for the poor growth of the recombinant E. coli pBlue 1.7.

Easy transformation of indoles to substituted tryptanthrins with powerful AHR activity

Malassezia furfur isolates from diseased skin preferentially biosynthesize compounds which are among the most active known Aryl-hydrocarbon Receptor (AhR) inducers, such as indirubin and tryptanthrin. In our effort to study their production from Malassezia spp., we investigated the role of indole-3-carboxaldehyde (I3A), the most abundant metabolite of Malassezia when grown on tryptophan agar, as a possible starting material for the biosynthesis of both alkaloids. Treatment of I3A with H2O2 and use of diphenyldiselenide as a catalyst resulted in the simultaneous one-step transformation of I3A to indirubin and tryptanthrin in good yields. The same reaction was first applied on simple indole and then on substituted indoles and indole-3-carboxaldehydes, leading to a series of mono- and bi-substituted indirubins and tryptanthrins bearing halogens, alkyl or carbomethoxy groups. Afterwards, they were evaluated for their activity on AhR in four different cell lines stably transfected with a luciferase reporter gene. Among them, 3-bromotryptanthrin interestingly showed 10 times higher activity than dioxin in the human HepG2 7.5 cell line at 6h. In conclusion, I3A could be the starting material used by Malassezia for the production of both indirubin and tryptanthrin through an oxidating mechanism. Additionally, some of the prepared tryptanthrins showed significantly increased activity and selectivity against the human cell line.

Production of Indirubin under Acidic Conditions and the Application to Dyeing with Fresh Leaves of Indigo Plants

Production of Indirubin under Acidic Conditions and the Application to Dyeing with Fresh Leaves of Indigo Plants

Conditions for the Production of Indirubin in the Process of the Dyeing with Fresh Leaves of Indigo Plants

Conditions for the Production of Indirubin in the Process of the Dyeing with Fresh Leaves of Indigo Plants

Abstracts

Directed evolution of cytochrome P450 enzymes represents an attractive means of generating novel catalysts for specialized applications. Xenobiotic-metabolizing P450s are particularly well suited to this approach due to their inherent wide substrate specificity. In the present study, a novel method for DNA shuffling was developed using an initial restriction enzyme digestion step, followed by elimination of long parental sequences by size-selective filtration. P450 2C forms were subjected to a single round of shuffling then coexpressed with reductase in E. coli. A sample (54 clones) of the resultant library was assessed for sequence diversity, hemo- and apoprotein expression, and activity towards the substrate indole. All mutants showed a different RFLP pattern compared to all parents, suggesting that the library was free from contamination by parental forms. Haemoprotein expression was detectable in 45/54 (83%) of the mutants sampled. Indigo production was less than or comparable to the activities of one or more of the parental P450s, but three mutants showed indirubin production in excess of that seen with any parental form, representing a gain of function. In conclusion, a method is presented for the effective shuffling of P450 sequences to generate diverse libraries of mutant P450s containing a high proportion of correctly folded hemoprotein, and minimal contamination with parental forms.

Improved indirubin production in a two-phase suspension culture of Polygonum tinctorium using dimethylpolysiloxane

Dimethylpolysiloxane (DMPS), used as a second phase in a suspension culture of Polygonum tinctorium, increased indirubin production by up to 259% and 16% in shake flask and air-lift bioreactors, respectively. In the shake flask culture, indirubin production increased by up to 248 mg/l using 90% v/v DMPS (volume of DMPS/volume of medium). In an air-lift bioreactor culture grown at a perfusion rate of 0.05 day−1 the indirubin concentration reached a maximum of 130 mg/l using 50% (v/v) DMPS. For the same DMPS content, the lower indirubin production in an air-lift bioreactor was probably due to the mass transfer limitation of indirubin between the two phases. © Rapid Science Ltd. 1998