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

Catucci, Gianluca; Turella, Simone; Cheropkina, Hanna; Angelis, M. De; Gilardi, Gianfranco; Sadeghi, Sheila J.

doi:10.1016/j.bcab.2022.102458

Cited by 10 publications

(13 citation statements)

References 51 publications

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“…This is 3-fold higher than the production obtained by E. coli whole-cell biocatalysis using a flavin monooxygenase from Methylophaga aminisulfidivorans for indigo [ 35 ]. For indirubin, the production upon supplementation with 200 µM L-tryptophan corresponds to 133.0 mg/L of cell culture, which is similar to the value recently reported for whole-cell biocatalysis using E. coli overexpressing an active site variant (R292A) of a BVMO from Acinetobacter radioresistens (138 mg/L) [ 36 ].…”

Section: Resultssupporting

confidence: 83%

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

Núñez-Navarro

Muñoz

Castillo

et al. 2022

IJMS

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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.

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Section: Resultssupporting

confidence: 83%

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

Núñez-Navarro

Muñoz

Castillo

et al. 2022

IJMS

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“…The data fitted to the Hill equation (Figure 6) and the values of the kinetic parameters ( k cat and K m ) are summarized in Table 1. The catalytic efficiency ( k cat / K m ) of indole hydroxylation by the F87G/T268V/F77I/E140D mutant reached 2849 mM −1 ⋅ min −1 , which were 712‐fold higher than that of R292A Ar‐BVMO [32] and 475‐fold higher than that of CYP102G4 [46] . And the catalytic efficiency ( k cat / K m ) was almost 2‐ to 15‐fold higher than those of the reported P450 enzymes, UPO and FMO [28,31,71,72] .…”

Section: Resultsmentioning

confidence: 93%

“…Many oxidizing enzymes have been examined for the synthesis of indigo through the hydroxylation and subsequent autooxidation of indole, a readily available chemical and common material (Figure 1A). [25][26][27][28][29][30][31][32] Among the enzymes, cytochrome P450 monooxygenase (P450 or CYP), a well-known and versatile bio-oxidation catalyst, has attracted special interest due to its catalytic capability for the hydroxylation of inert CÀ H bonds. [33][34][35][36][37][38][39][40][41] Several engineered P450s, including CYP2C19, [42] CYP2E1, [43] CYP2A6, [44,45] CYP102G4, [46,47] and P450BM3, [48] have supported the potential for the synthesis of indigoid dyes by rational design or directed evolution.…”

Section: Introductionmentioning

confidence: 99%

“…Although the synthesis of indigo by microorganisms has been widely reported in recent years, [21–24] enzymatic indigo synthesis is thought to be a more suitable biocatalytic strategy because it bypasses the challenges of product isolation and long reaction times encountered during the microbial synthesis of indigo. Many oxidizing enzymes have been examined for the synthesis of indigo through the hydroxylation and subsequent autooxidation of indole, a readily available chemical and common material (Figure 1A) [25–32] . Among the enzymes, cytochrome P450 monooxygenase (P450 or CYP), a well‐known and versatile bio‐oxidation catalyst, has attracted special interest due to its catalytic capability for the hydroxylation of inert C−H bonds [33–41] .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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Herein, 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.

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“…273 In addition, indigoids, such as the textile dye indigo and the pharmaceutical indirubin, were produced by indole oxidation using a mutant of a BVMO from A. radioresistens. 274 E. coli cells over-expressing the recombinant human flavin-containing monooxygenase 3 were used for the bioconversions of clomiphene and dasatinib drugs, GSK5182 (a candidate against type 2 diabetes mellitus), and the enzymatic inhibitor tozasertib to their corresponding N-oxide metabolites, with high yields and regio-selectivities. 275 Flavin-containing monooxygenase 1 has been used for nicotine oxidation.…”

Section: Bioconversion With Oxygenase Enzymesmentioning

confidence: 99%

Oxidoreductase enzymes: Characteristics, applications, and challenges as a biocatalyst

Cárdenas‐Moreno,

González‐Bacerio,

García Arellano

et al. 2023

Biotech and App Biochem

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Oxidoreductases are enzymes with distinctive characteristics that favor their use in different areas, such as agriculture, environmental management, medicine, and analytical chemistry. Among these enzymes, oxidases, dehydrogenases, peroxidases, and oxygenases are very interesting. Because their substrate diversity, they can be used in different biocatalytic processes by homogeneous and heterogeneous catalysis. Immobilization of these enzymes has favored their use in the solution of different biotechnological problems, with a notable increase in the study and optimization of this technology in the last years. In this review, the main structural and catalytical features of oxidoreductases, their substrate specificity, immobilization, and usage in biocatalytic processes, such as bioconversion, bioremediation, and biosensors obtainment, are presented.

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

Cited by 10 publications

References 51 publications

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

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

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

Oxidoreductase enzymes: Characteristics, applications, and challenges as a biocatalyst

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