Synthesis Of Indigo Research Articles

Construction of Escherichia coli cell factory for efficient synthesis of indigo.

Indigo is widely used in dyes, medicines and semiconductors materials due to its excellent dyeing efficiency, antibacterial, antiviral, anticancer, anti-corrosion, and thermostability properties. Here, a biosynthetic pathway for indigo was designed, integrating two enzymes (EcTnaA, MaFMO) into a higher L-tryptophan-producing the strain Escherichia coli TRP. However, the lower catalytic activity of MaFMO was a bottleneck for increasing indigo titers. To overcome this limitation, the enzyme activity of MaFMO was enhanced through mechanism-guided rational design. The optimal mutant obtained in this study, MaFMOD197E, whose kcat/Km was 1.34 times that of the wild type, and its specific activity was 2.36 times that of the wild type. In addition, the expression levels of EcTnaA and MaFMOD197E were regulated by optimizing the promoters and increasing the copy number to generate the strain E. coli IND-13. Finally, in the optimal fermentation conditions (220 rpm, 0.05% Tween-80), the strain E. coli IND-13 achieved the indigo titer of 568.52 mg/L in a 5-L bioreactor, with the yield and productivity of 2.62 mg/g and 12.96 mg/L/h (the highest to date), respectively. The results presented here can lay a foundation for further construction of cell factories for indigo and its derivatives with industrial application potential.

Deep eutectic solvent-oriented integration of eco-friendly and efficient indican extraction and enzyme-based indigo preparation

Indigo, one of the oldest dyes, finds widespread usage across multiple industries, encompassing food, medicine, printing, dyeing, as well as cosmetics. Indican is recognized as a precursor substance crucial for the synthesis of indigo, however, the current indican extraction and indigo preparation are largely limited due to the low efficiency of indican extraction, by-product production, as well as serious environmental pollution. To address the serial of issues, we developed deep eutectic solvent (DESs)-oriented integration of the efficient extraction of indican and the green preparation of indigo from Strobilanthes cusia (Nees) Kuntze. DESs were rationally designed and screened to make them able to destroy cell walls to facilitate the precipitation of the desired substance for specific indican extraction. And then optimized to obtain the final extraction rate of 22.8 mg/g, which present the highest so far. More importantly, without additional treatment for DES-containing indican extractive, the β-glucosidase Asbg1 was able to convert indican to indigo at a molar conversion rate of 74.6%. By fully harnessing plant resources, the study introduced an innovative process that bypassed extreme acid-base conditions, ensuring rapidness and minimal pollution. This approach offered a promising avenue for the preparation of indigo, paving the way for sustainable production methods.

The Synthetic Approaches for Preparation of Indigo and Applications in Denim Industry.

This paper describes indigo chemistry and its brief scientific history beginning with the first characterization and chemical synthesis of indigo via various precursors such as isatin, cinnamic acid, 2-nitrobenzaldehyde, anthranilic acid, N-phenylglycine, aniline, and indole. Furthermore, alternative methods such as eco-friendly microbial synthesis of indigo using a variety of enzymes are reported: Cytochrome P450 monooxygenases, flavin-containing monooxygenases, and unspecific peroxygenases. Subsequently, the application of indigo in the denim industry, reduction methods (chemical, electrochemical, enzymatic and catalytic) and dyeing methods (all parameters in dyeing, ring dye) are discussed. In addition, the main reason for the use of indigo in the denim industry is briefly explained.

Construction of Biocatalysts Using the P450 Scaffold for the Synthesis of Indigo from Indole.

With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity with kcat of 1402 min-1 and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity (kcat/Km = 1388 mM-1 min-1) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo.

Evolving a P450BM3 Peroxygenase for the Production of Indigoid Dyes from Indoles

AbstractHerein, we describe an environmentally benign enzymatic approach for the preparation of indigoid from indole derivatives. A series of beneficial P450BM3 mutants were obtained using a stepwise approach involving site‐directed, random, and combinatory hot‐site mutations. Using H2O2 as the terminal oxidant and N‐(ω‐imidazolyl)‐hexanoyl‐L‐phenylalanine as a co‐catalyst, the quadruple‐mutant F87G/T268V/F77I/E140D efficiently catalyzed the 3‐hydroxylation of indole and subsequent autooxidation to indigo with higher efficiency than the flavin‐containing monooxygenase, PTDH‐mFMO, which was the best oxidizing enzyme for indigo synthesis reported to date. Following this procedure, 12 indigoid compounds were prepared using indole derivatives with various substituents as starting materials with moderate to high isolated yields (31.3–79.5 %). The catalytic efficiencies (kcat/Km) of the beneficial mutants for typical indole substrates ranged from 182–2849 mM−1 ⋅ min−1, almost 2‐ to 712‐fold higher than those of the reported P450 enzymes. This study provides a new enzymatic approach for the biosynthesis of indigoid dyes from indole derivatives.

Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3

AbstractNatural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4‐phenyl‐2‐butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active‐site variants of P450 BM3 using orthogonal high‐throughput workflows to identify the most streamlined route to all indigo‐forming variants. Among the three known and 13 new indigo‐forming variants, eight hydroxylated 4‐phenyl‐2‐butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3.

C&EN talks with Tammy Hsu, maker of ‘greener’ blue jeans

In the early 1880s, chemist Adolf von Baeyer devised a scheme that would revolutionize the fashion industry. For thousands of years prior, people had dyed fabric blue using powders painstakingly extracted from the leaves of indigo plants (Indigofera tinctoria). But thanks to Baeyer’s Nobel Prize–winning chemical synthesis of the indigo molecule, factories around the globe now churn out some 70,000 metric tons of the iconic dye each year—much of it to color the global wardrobe staple denim. Yet the $66 billion industry is not without its faults: large-scale indigo synthesis uses a lot of energy and water. It also requires the use of toxic chemicals like formaldehyde. In recent years, a number of suppliers have tried to make textile dyeing more sustainable and simultaneously replace problematic chemicals. One of the latest companies to pursue this goal is Tinctorium, a San Francisco Bay Area start-up that launched earlier this year. It

Expression of styAB is regulated by a two-component system during indigo biosynthesis in Pseudomonas putida

A two-component regulatory system involving StyS and StyR is involved in the regulation of indigo synthesis in Pseudomonas sp. However, the function of the styR gene in indigo synthesis and the detailed mechanisms through which StyR enhances the expression of the styAB operon are unclear. Accordingly, in this study, we constructed a styR/styS gene knockout mutant strain. By comparing the differences in indigo yields between the wild-type and mutant strains, we found that the styR gene mutant strain had no indigo synthesis ability, whereas the yield in the wild-type strain was 5.4 mg/L. Thus, these findings indicate that the styR gene plays a key role in the regulation of indigo synthesis. The interactions among StyS, StyR, and the styAB promoter were verified by electrophoresis mobility shift assays. The results showed that StyR interacts with the styAB promoter by binding to the palindrome in the styAB promoter. Moreover, the kinase function of StyS regulated StyR by transphosphorylating StyR during indigo biosynthesis in P. putida B3. Taken together, these findings provide important insights into the establishment of environmentally friendly indigo synthesis methods using P. putida.

Recent Advances in Several Organic Reaction Mechanisms

This Review is a brief account of our theoretical contributions in seven research communications in the field of reaction mechanisms. Some mechanisms were corrected as in the case of the Baeyer-Drewsen indigo synthesis. When two very different reaction mechanisms had been proposed, as in the Clemmensen Reduction, a unified theory was provided. In other cases there were no reaction mechanisms at all, as in the Baeyer-Emmerling synthesis of indigo and in the Froehde Reaction for opioids. This deficit has been solved. The reaction that controls fructosazone regiochemistry has been described, and an internal process in a mixed osazone formation has been explained. All the proposals are based on well known reactivities and we provide complete and coherent reaction series with commented steps.

Deciphering the bacterial composition in the rhizosphere of Baphicacanthus cusia (NeeS) Bremek

Rhizobacteria is an important ingredient for growth and health of medicinal herbs, and synthesis of pharmacological effective substances from it. In this study, we investigated the community structure and composition of rhizobacteria in Baphicacanthus cusia (NeeS) Bremek via 16S rRNA amplicon sequencing. We obtained an average of 3,371 and 3,730 OTUs for bulk soil and rhizosphere soil samples respectively. Beta diversity analysis suggested that the bacterial community in the rhizosphere was distinctive from that in the bulk soil, which indicates that B.cusia can specifically recruit microbes from bulk soil and host in the rhizosphere. Burkholderia was significantly enriched in the rhizosphere. Burkholderia is a potentially beneficial bacteria that has been reported to play a major role in the synthesis of indigo, which was a major effective substances in B. cusia. In addition, we found that Bacilli were depleted in the rhizosphere, which are useful for biocontrol of soil-borne diseases, and this may explain the continuous cropping obstacles in B. cusia. Our results revealed the structure and composition of bacterial diversity in B. cusia rhizosphere, and provided clues for improving the medicinal value of B. cusia in the future.

Self-Assembled Multimeric-Enzyme Nanoreactor for Robust and Efficient Biocatalysis.

The construction of artificial multienzyme nanodevices with desired spatial arrangements have shown great promise for improving the overall performance of targeted enzyme cascades. However, it is a challenge to rationally design and construct multiple oligomeric enzyme assemblies that can be used as stable and reusable catalysts. Herein, we report a novel approach to rapidly achieve ultrastable multimeric enzyme nanoclusters (MENCs) based on enzymes property of oligomerization and the SpyTag/SpyCatcher system of orthogonally reactive split peptides. The SpyCatcher peptide and its binding partner SpyTag were fused to a dimeric cytochrome P450 monooxygenase mutant (P450BM3m) and a tetrameric glucose dehydrogenase (GDH), respectively. The fusion proteins self-assembled into the MENCs, forming a covalently coupled supramolecular multienzyme nanodevices that facilitated NADPH regeneration and converted indole into a pigment indigo. We investigated the morphology of the MENCs and found these multimeric enzymes assembled into two-dimensional layerlike nanoscale architecture, ranging from a few to several hundred square microns in size. Importantly, enzymatic analysis revealed that the MENCs not only increased the initial rate by more than three times for the indigo synthesis, but also achieved significant improvements on stability and reusability compared to unassembled enzyme mixtures. This work demonstrates a versatile and efficient strategy to construct stable and multifunctional biocatalysts with potential applications in metabolic engineering and synthetic biology.

Complete Genome Sequence of Indigo-Producing Bacterium Celeribacter sp. Strain TSPH2.

ABSTRACTCeleribacter sp. strain TSPH2, a novel producer of indigo, was isolated from oil-contaminated sediment. We present here its genome sequence consisting of one circular chromosome (4 Mb) and one plasmid (0.15 Mb), with an overall G+C content of 60.9%. This strain contains oxygenase genes involved in indigo synthesis, such as flavin-containing monooxygenase.

Biosynthesis of indigo in Escherichia coli expressing self-sufficient CYP102A from Streptomyces cattleya

Cytochrome P450 monooxygenases (CYP) are a superfamily of heme-thiolate proteins which catalyze the incorporation of oxygen atoms into substrates. Here, a self-sufficient CYP102A from Streptomyces cattleya (CYP102A_scat) was cloned, produced recombinantly in Escherichia coli strain BL21 (DE3), and the characteristic features were investigated. However, unlike other self-sufficient CYP102A enzymes that have been reported, CYP102A_scat was found to be able to catalyze intracellular hydroxylation of indole molecules with 3-C specific regioselectivity. Consequently, E. coli strains producing CYP102A_scat could synthesize approximately 1.0 g/L of indigo in LB media. Optimization of indigo synthesis was investigated through additional feeding of indole precursors such as glucose, l-tryptophan, and indole. Indigo production reached up to 3.8 ± 0.1 g/L by adding 20 μM of extracellular indole and 0.2 mM of l-tryptophan to the LB media. To our knowledge, this is a record and the highest yield achieved so far.

Biotransformation of Indigo Pigment by Indigenously Isolated Pseudomonas sp. HAV-1 and Assessment of Its Antioxidant Property.

Chemical synthesis of indigo poses harsh environmental hazards and adverse human health effects. This necessitates an environment-friendly and producer-friendly approach for indigo production. The present study was thus significant as it reports an indigenously isolated potential indigo pigment producing culture identified as Pseudomonas sp. HAV-1 with noteworthy antioxidant property. The bioindigo pigment was characterized using various analytical techniques. The pigment production was enhanced from 412 μg mL−1 to 700 μg mL−1 by optimizing the growth parameters. Furthermore, the antioxidant property of indigo pigment is hitherto unexplored. This property can significantly append to its therapeutic potential. The bioindigo pigment produced by Pseudomonas sp. HAV-1 depicted 2.2 μM ascorbic acid equivalent antioxidant property. More to the point, the present work addresses a footstep towards green production of indigo.

Indigo dye production by enzymatic mimicking based on an iron(III)porphyrin

A novel indigo synthesis is based on a simple and cost-effective model system of the enzymes involved in the natural and biocatalytic productions. The method considers the oxidation of indole by hydrogen peroxide, being catalyzed by an iron(III)porphyrin in ethanol, as solvent, and no other additives. The yields of indigo and of the other oxidized indolinoid derivatives were found to be dependent on the metalloporphyrin system used and on the control of the oxidation conditions. Significant reductions of the environmental impact relatively to the present industrial production and of the costs relatively to the biocatalytic methodologies were obtained. The enhanced indigo production in the presence of the iron(III)porphyrin-ethanol catalytic system relatively to the manganese(III)porphyrin-acetonitrile system can be rationalized by the formation of different active species in the two systems.

Recent Advances in Microbial Synthesis of Indigo

Indigo is one of the organic pigments which is widely used in printing,dyeing and pharmaceutical industries.Microbial synthesis of indigo has recently attracted extensive attention worldwide.The research processes and development trends are reviewed in the present paper.Biosynthesis of indigo could be divided into three periods: Biosynthesis by wild microbes,whole-cell catalysis by engineering bacteria and biotransformation regulated by metabolic engineering.Most aromatic-degrading microbes and their relevant enzymes possess the ability to convert indole to indigo.New technologies such as directed evolution,metagenome and two phase reaction system could facilitate in-depth investigations of the enzyme resources,and they will play a crucial role in the indigo biosynthesis research.Meanwhile,hydroxyl-indoles and indigo derivatives produced in the process are promising pharmaceutical and chemical precursors with great research interests.However,the transformation interactions between intermediates and by-products are still unclear.Besides,low indigo yield and efficiency with high cost have hampered practical production.Therefore,it is essential to combine the molecular biology and metabolic engineering technologies to investigate the mechanisms and industrial application of indigo biosynthesis in the future.

ChemInform Abstract: Development of a Practical One‐Pot Synthesis of Indigo from Indole.

ChemInformVolume 42, Issue 20 Organic Dyes ChemInform Abstract: Development of a Practical One-Pot Synthesis of Indigo from Indole. Yoshihiro Yamamoto, Yoshihiro Yamamoto Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this authorYoshihisa Inoue, Yoshihisa Inoue Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this authorUsaji Takaki, Usaji Takaki Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this authorHiroharu Suzuki, Hiroharu Suzuki Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this author Yoshihiro Yamamoto, Yoshihiro Yamamoto Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this authorYoshihisa Inoue, Yoshihisa Inoue Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this authorUsaji Takaki, Usaji Takaki Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this authorHiroharu Suzuki, Hiroharu Suzuki Catal. Sci. Lab., Mitsui Chem., Inc., Sodegaura, Chiba 299-02, JapanSearch for more papers by this author First published: 21 April 2011 https://doi.org/10.1002/chin.201120178Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume42, Issue20May 17, 2011 RelatedInformation

Development of a Practical One-Pot Synthesis of Indigo from Indole

Abstract A novel and highly practical one-pot synthesis of indigo from indole via 3-position selective oxidation and dimerization of the indole framework was developed. Using 0.1 mol % of molybdenum complex and 2.2 equivalents of cumene hydroperoxide in tert-butyl alcohol, the reaction was complete in 7 h and pure indigo was obtained in 81% yield as a deep-blue solid just by filtration. The described one-step method renders the pure indigo readily available on a large scale using only inexpensive raw materials.

Organic synthesis "on water".

Organic synthesis "on water".

A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1–FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12 h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.