Engineering artificial biosynthetic pathway enables simultaneous production and in-situ bio-dyeing of indigoids for textiles

Domain: chemistry
Published: 2025-12-26
Editorial outcome: major_revision

Abstract
The textile dyeing industry has shaped modern aesthetics through vibrant coloration technologies. Indigoid dyes embody this potential but are hindered by the hazardous chemo-synthesis and water-intensive chemo-dyeing processes. While biosynthesis and bio-dyeing efforts offer alternatives, disjointed synthesis-dyeing steps lead to unsustainable water and energy demands, and complex biosynthetic pathways cause low atom economy and inefficiency. Here, we report a one-pot one-step approach that unifies biosynthesis and bio-dyeing of indigoids to drastically reduce water usage in the dyeing process. By combining alcohol dehydrogenase with flavin-containing monooxygenase, we construct an artificial redox-neutral cascade biosynthetic pathway to produce bluish-violet indigoid dyes, which significantly enhances atom economy and achieves high production efficiency. Various natural and synthetic fabrics are dyed in-situ with the biosynthetic indigoids utilizing recombinant E. coli whole cells. Crucially, this system dyes synthetic nylon fibers with the historic purple hue.

Introduction
The textile dyeing industry has undergone rapid growth driven by advancements in synthetic chemistry, enriching human society with vibrant colors1. Bluish-violet indigoid dyes, long-renowned natural colorants, exemplify this legacy2,3. Indigo, one of the prominent indigoid dyes, which its blue hue can be traced back to the Indus Valley Civilization (~3000 BC) and was extracted from Indigofera tinctoria for dyeing cotton and linen fabrics4. Similarly, Tyrian purple, a rare 6,6′-dibromoindigo (6,6′-DBI)-based dye extracted from sea snails, symbolized the noble status and power of royalty within the Roman Empire5. At the end of the 19th century, BASF achieved the industrial chemical synthesis of indigo. The popularization of synthetic indigo and its fading effect on denim have spawned a global fashion trend, creating an early $100 billion/year market6. However, the widespread adoption of synthetic indigoid dyes has precipitated serious threats to human health and caused significant environmental burdens7. One is the toxic, carcinogenic, and hazardous chemical synthesis process, in which hydrogen cyanide, strong bases, and sodamide was employed8 (Fig. 1a). The other is the highly polluting and water-intensive dyeing process7 (Fig. 1a). In industry, large amounts of sodium dithionite need to be repeatedly added to reduce water-insoluble indigoid to its soluble leuco form, which enables the dyeing of fabrics with indigoid dyes9. This results in large quantities of corrosive sulfur-containing wastewater. It is estimated that a single medium-sized indigo dyeing factory can produce ~1000 tons of wastewater per day.

To mitigate the persistent environmental pollution and health hazard, recent biotechnological advances have pioneered eco-friendly alternatives, such as biosynthesis of indigoid dyes10,11 and enzymatic bio-dyeing systems8. A landmark example achieving sustainable and eco-friendly synthesis and dyeing of indigoid is the use of glucosyltransferase (UGT) and β-glucosidase (BGL)-dependent biochemical protection and deprotection strategies for indigo synthesis and dyeing8 (Fig. 1b). In engineered E. coli cells, fed tryptophan (Trp) was first converted to indoxyl, an unstable intermediate, by tryptophanase (TnaA) and flavin-containing monooxygenase (FMO). Indoxyl was then stabilized via UGT-mediated glycosylation into indican and released by BGL for cotton dyeing. This method eliminated the use of sodium dithionite, presenting a sustainable alternative to the conventional chemical dyeing approach. Despite its promise, this method separates biosynthesis and dyeing, still consuming substantial water and energy due to BGL usage, which also suffers from limited enzymatic robustness and high production cost12. Furthermore, whether this system is applicable to other indigoid dyes (e.g., Tyrian purple analogs) still remains to be explored. The development of a simple, efficient, and universal biological strategy that integrates indigoids synthesis and dyeing to eliminate water usage during dyeing process is therefore of great interest.

Other research focused on using recombinant microbes or enzymes for biosynthesis of indigoid dyes, which made considerable efforts to create sustainable synthetic approaches13. However, the widely reported three-step biosynthetic pathway (Fig. 1c), which is composed of two-component halogenase (HL)-catalyzed selective halogenation of tryptophan (omitted for indigo biosynthesis), tryptophanase (TnaA)-mediated decomposition of substituted tryptophan, and oxygenase (OX)-driven hydroxylation of substituted indoles, still face critical limitations: (1) low atom economy due to the C-C bond cleavage reactions catalyzed by TnaA, which could generate byproducts including pyruvate and ammonia; (2) requires at least five enzymes (including two NAD(P)H regeneration enzymes) for the synthesis of substituted indigos complicating catalytic balance regulation and imposing metabolic burden on host cells; (3) inevitable pot replacement or temperature regulation that control expression of the tnaA gene for enhancing the selectivity of the pathway. Hence, the development of an easy-operation, atom economical, and efficient one-pot one-step strategy for biosynthesizing indigoids that eliminates process control steps is highly attractive.

Here, we demonstrate a one-pot one-step approach for simultaneous biosynthesis and bio-dyeing of indigoids, eliminating water-intensive dyeing processes (Fig. 1d). Our strategy integrates a highly atom-economical artificial biosynthetic pathway coupling NAD+-dependent alcohol dehydrogenase (ADH)-catalyzed aminoalcohol oxidation with NAD(P)H-dependent flavin-containing monooxygenase (FMO)-catalyzed indole oxygenation. To eliminate exogenous cofactor dependence, we engineer a cofactor self-recycling redox-neutral cascade via a classic enzymatic hydrogen-borrowing mechanism. In recombinant E. coli whole cells co-expression of the ADH and FMO genes, indigoids (leuco form) including indigo and 6,6′-dibromoindigo (6,6′-DBI) are synthesized and directly fixed onto fabrics, enabling in-situ integration of biosynthesis and dyeing. This strategy can dye various natural and synthetic fibers, particularly when applying 6,6′-DBI to nylon fibers, achieving its inherent distinctive purple hue10.

Results
Design artificial biosynthesis pathway for 6-bromoindole
To design a proof-of-concept artificial biosynthetic pathway for indigoid dyes, retrosynthesis analysis was initially applied. Tyrian purple (primarily composed of 6,6′-DBI) was selected as the representative product due to its noble color and high commercial value. As shown in Fig. 2a, the 6,6′-DBI synthesis pathway was divided into two steps: the first involves the synthesis of 6-bromoindole (6-Br-indole), and the second involves the synthesis of 6,6′-DBI from 6-Br-indole. For the synthesis of 6-Br-indole, two routes including intermolecular (route 1) and intramolecular (route 2) oxidative condensation reactions were proposed inspired by classical metal-catalyzed hydrogen-borrowing (HB) reactions14 (Fig. 2a). In route 1, we suspected that alcohol dehydrogenase could catalyze the oxidation of diol substrate15, the resulting glyoxal could couple with substrate aniline forming 5-membered ring based on the aldimine condensation and C-C coupling. However, this condensation reaction usually needs high reaction temperature16. Thus, route 1 was excluded.

Route 2 begins from 2-amino-4-bromo-phenylethanol (1a), which can be synthesized from 4-bromo-2-nitrotoluene (Supplementary Fig. 1). Based on our previous study on the cascade catalysis of alcohol dehydrogenase and halogenase for the synthesis of halogenated indoles17, we hypothesized that it could undergo alcohol dehydrogenase-catalyzed oxidation followed by spontaneous intramolecular aldimine condensation and dehydration to generate 6-Br-indole. We selected twelve alcohol dehydrogenases belonging to short-18–21, medium-22–27, and long-chain28,29 families to verify the oxidation of the substrate and performed their phylogenetic analysis (Fig. 2b). Employing NAD+ or NADP+ as cofactors, the specific activity and conversion of each enzyme were tested in vitro (Fig. 2c and Supplementary Fig. 2). The results showed that PpADH, YsADH, and HLADH exhibited better catalytic efficiency compared with other tested ADHs. The high enzyme activity (10.5 U mg−1) and conversion rate (45%) were achieved in the case of that using HLADH and NAD+ as co-substrates for catalyzing. To further confirm the formation of 6-Br-indole, GC-MS analysis was conducted (Supplementary Fig. 3). These spectra indicated that 6-Br-indole was generated during the oxidation of aminoalcohol. However, the conversion of this reaction was limited to 45% (Fig. 2c). To gain deeper mechanistic insights (Fig. 2d and Supplementary Fig. 4), QM/MM and DFT calculations were employed to elucidate the catalytic mechanism. The results showed that DFT calculations yield a lower energy barrier from IM to 1c (16.5 kcal mol−1) compared to the barrier from IM to 1c’ obtained by QM/MM calculations (31.4 kcal mol−1) (Fig. 2e and Supplementary Figs. 5, 6), which indicates that 1a is first oxidized by HLADH, followed by spontaneous intramolecular aldimine condensation and dehydration to generate 6-Br-indole. The oxidation of 1a is apparently the limited step.

A conventional NADH oxidase (NOX)-coupled NAD+ regeneration system was then employed (Fig. 2f). The yield of 6-Br-indole increased to 96% in 8 h which is 2.1-fold higher compared with that of adding excess NAD+ (Supplementary Fig. 7). Residual substrate (4%) was attributed to partial amino group protonation at pH 7.0, hindering aldehyde-amine condensation30. Further Increasing the reaction pH to 9.0, >99% yield of 6-Br-indole was achieved in 4 h (Supplementary Fig. 8). The turnover frequency (TOF) reached 62,400 h−1, representing a 2-fold increase over the value at pH 7.0. This result motivated us to expand the substrate scope of this system. 2-amino-phenylethanol and its different substituent analogues (2a-21a) were tested (Fig. 2g). The results demonstrated that HLADH exhibited broad substrate scope toward substituted 2-amino-phenylethanols (-F, -Cl, -Br, -Me, -OMe) at various positions (4-, 5-, 6-, 7-), achieving yields ranging from 36% to 99% for corresponding substituted indole products (2d-21d). HLADH displayed high catalytic efficiency for substrates bearing substituents at the 5-, 6-, and 7-positions, with product yields reaching 76–99% under optimized reaction conditions. Moreover, the synthesis of indole (13d) achieved an excellent yield (>99%), consistent with our previous research31.

Construction and optimization of the HLADH-mFMO cascade
Next, we evaluated the biosynthesis of 6,6′-DBI from 6-Br-indole by using the NAD(P)H-dependent flavin-containing monooxygenases (FMOs) which were widely used for indigoid synthesis. Three FMOs including Methylophaga sp. strain SK1 FMO (mFMO)32, Corynebacterium glutamicum FMO (cFMO)33, Nitrincola lacisaponensis FMO (NiFMO)34 were selected for catalyzing 6-Br-indole to 6,6′-DBI. Using NADH or NADPH as cofactors, the specific activity and conversion of each enzyme towards 6-Br-indole was tested (Fig. 3a and Supplementary Fig. 9). The result showed that mFMO exhibited the higher activity among these enzymes either using NADH or NADPH, but cFMO had no activity towards 6-Br-indole. We found that NiFMO showed similar activity when adding NADH or NADPH. The result of kinetic analysis of NiFMO also revealed that the catalytic activity with NADH is comparable to that of NADPH (Table 1). This observation contradicted the findings of Loncar et al.34, who reported NiFMO showed no activity towards NADH. Subsequently, glucose dehydrogenase (GDH)-catalyzed cofactor regeneration system was employed to further enhance the catalytic efficiency (Fig. 3a). As expected, the yield of 6,6′-DBI showed significant improvements compared with that adding excess amount of NADH or NADPH except for cFMO. The yields of NADP+-involved reactions tested in this study were all higher than those of NAD+-involved, suggesting that NADP+ may be a preferable cofactor for this specific biotransformation under the experimental conditions. mFMO was selected for the following experiment due to its higher yield (40.8%) among these FMOs when using NADP+ as cofactor.

Recently, enzymatic borrowing hydrogen via NAD(P)H self-regeneration has emerged as an efficient redox-neutral strategy for synthesizing value-added chemicals35–37. Inspired by this approach, we explored the potential of integrating ADH with FMO for 6,6′-DBI synthesis. Based on the above studies, NAD+ is the optimal cofactor for HLADH, whereas NADPH is preferred by FMO. Accordingly, we assessed the performance of NADP+ and NAD+ in the cascade system. The results indicated that the yields of 6,6′-DBI reached 15.2% and 42.1%, respectively (Fig. 3b). Kinetic analysis showed that the kcat/KM values of mFMO for NADH and NADPH were 3.4 and 9.2 mM−1 s−1, respectively. In contrast, the values for HLADH with NAD+ and NADP+ were only 0.8 and 0.004 mM−1 s−1, respectively (Table 1 and Supplementary Fig. 10). These kinetic data clearly demonstrate that HLADH exhibits very low catalytic activity toward NADP+. Hence, NAD+ is the more suitable cofactor for this cascade system. After further optimization of pH and temperature, the yield of 6,6′-DBI increased to 48.7% (Supplementary Figs. 11, 12). The ratios of HLADH and mFMO were further optimized. A ratio of 1:2 was found to most effectively alleviate the inhibition caused by the accumulation of intermediate 1d, resulting in the 6,6′-DBI yield of 60.1% (Fig. 3b and Supplementary Fig. 13).

Expansion of substrate scope
Having established the HLADH-mFMO-catalyzed 6,6′-DBI cascade synthesis reaction with NAD+ as a hydrogen-transfer mediator, we next applied the procedure for the synthesis of a variety of indigoids 2f-21f by using aminoalcohols (2a-21a) as substrates to evaluate the versatility of this HLADH-mFMO cascade catalytic system (Fig. 4, Method A). Directly using indoles as the substrates catalyzed by mFMO was also conducted for comparison (Fig. 4, Method B). In the dual-enzyme cascade reaction, substituent-free aminoalcohol 13a afforded 92% indigo yield. All 19 aminoalcohol derivatives were converted, with yields ranging from 2% to 83% for the indigoids. It was observed that not only substrates 2a-12a with electron-withdrawing groups (EWGs: -F, -Cl, -Br), but also substrates 14a-21a which contain electron-donating groups (EDGs: -Me, -OMe) exhibited enhanced compatibility under the optimized conditions. As shown in Fig. 4, the yields of HLADH-mFMO system are generally higher than those catalyzed by single mFMO, thus demonstrating the benefits of the dual-enzyme cascade catalytic system.

Substituent electronic effects exhibited pronounced structure-activity relationships in both enzymatic systems. The yield of mFMO-mediated C3-hydroxylation correlated with the electronegativity of EWGs (-F>-Cl>-Br), with stronger electron-withdrawing groups demonstrating enhanced reaction efficiency and higher product yields. Positional dependence was observed: C5/C6/C7-EWG derivatives achieved optimal yields (35–83%), while C4-substituted analogues suffered obvious yield inhibition (<30%). Conversely, EDGs (-Me/-OMe) universally suppressed activity, with 4,4′- and 5,5′-EDGs resulting in extreme yield reductions (2–14%), while 6,6′-/7,7′-EDG derivatives demonstrated comparatively higher yields (13–33%).

Finally, we used a red-green-blue (RGB) color model to quantitatively analyze the enzyme-synthesized dyes. The RGB values were acquired using Photoshop’s Color Picker38. The color and absorption maximum of indigo dyes depend on the type and position of substituents on the benzene ring (Supplementary Fig. 14). The combination of indigo derivatives with diverse substituents via the dual-enzyme cascade resulted in a broad spectrum of colors. Overall, our constructed catalytic route of dual enzyme cascade greatly enhanced the yield of substituted indigos, enriched the substrate scope, and characterized the color profiles of indigoids.

Production of 6,6′-DBI in E. coli
After demonstrating the in vitro redox-neutral cascade reaction with HLADH and mFMO, recombinant E. coli whole cells were engineered to convert 1a to 6,6′-DBI, which were more economical and stable than free enzymes (Fig. 5a). A single tailored recombinant whole-cell biocatalyst containing all required enzymes is preferred for cascade biocatalysis to avoid multi-strain cultivation, enzyme purification, and large amounts of external cofactor addition39. To balance cascade enzyme expression in a single recombinant E. coli strain, 12 variants coexpressing HLADH and mFMO were constructed (Fig. 5b), and their yields were evaluated for 6,6′-DBI production (Fig. 5c). The gene of TnaA was knocked out to avoid the generation of endogenic indigo40. SDS-PAGE analysis revealed that all of the enzymes were produced in E. coli cells (Supplementary Fig. 15). All combinations were examined for the biotransformation of 2.5 mM 1a. With all the recombinant E. coli cells density at OD600 = 20, 6,6′-DBI was obtained in 0.2%−55.1% yield (Fig. 5c). M1 was selected for further study due to its capability to achieve the yield (55.1%) of 6,6′-DBI (Fig. 5c). The growth and conversion efficiency of M1 were optimized (Supplementary Fig. 16a, b). The conversion (64.7%) was achieved after 12 h of cultivation at 18°C with 0.1 mM IPTG. Furthermore, after optimizing the pH and temperature conditions in the whole-cell catalytic process, a maximum yield of 72.1% was achieved after 12 h at pH 8.0 and 30°C (Fig. 5d and Supplementary Fig. 16c, d). Finally, a 6,6′-DBI titer of 367.5 mg L−1 was achieved in 200 mL shake-flask whole-cell fermentation (Fig. 5e). The space-time yield of 6,6′-DBI (15.31 mg L−1 h−1) achieved in this study demonstrate the effectiveness of the one-pot one-step biosynthetic strategy for 6,6′-DBI production in E. coli10,11,41 (Supplementary Table 1). The route also exhibited better environmental performance, with improved atom economy and a reduced E-factor (Supplementary Table 2).

In-situ bio-dyeing strategy for 6,6′-DBI
The synthesis and dyeing processes collectively constitute the complete cycle of textile dyeing42. To further demonstrate the practical applicability of this system, we develop an innovative strategy combining indigoid biosynthesis with in-situ bio-dyeing strategy (Bio-in-situ strategy), where fabrics were directly added in the indigoid biosynthesis development buffer, enabling effective fiber coloration through spontaneous utilization of leuco intermediates generated during biosynthesis (Fig. 6a).

To assess the feasibility, the recombinant whole cell that generating 6,6′-DBI was directly employed for dyeing diverse fabric substrates (Fig. 6a). When the substrate 1a was added to the whole-cell reaction system, multi-fabric strips are immediately introduced into the reaction solution. Dyeing efficiencies through K/S (absorption coefficient, K, and scattering coefficient, S, a value used to determine the depth of color of a dyed fabric) value43 measurement at 580 nm revealed optimal dyeing was achieved at 24 h (Supplementary Table 3). Among all tested fabric strips, natural wool and synthetic nylon exhibited satisfactory coloration efficacy under the catalytic conditions. For dyeing synthetic nylon, Bio-in-situ strategy reproduced the authentic “regal purple” of Tyrian purple, whereas the chemical dyeing strategy (Bio-Chem strategy) resulted in a bluish coloration (Fig. 6b). Moreover, we analyzed brightness indicator (L*), red to green indicator (a*), yellow to blue indicator (b*) and color difference (ΔE*ab) (Supplementary Table 4). Statistical analysis revealed high color difference between nylon substrates colored via Bio-Chem and Bio-in-situ strategies. Specifically, nylon substrates colored via Bio-in-situ strategy exhibited significantly higher a* values (indicating a redder hue), while those processed through chemical dyeing showed markedly lower b* values (suggesting enhanced blueness).

We wondered if this color distinction was due to a change in the particle size of 6,6′-DBI dyed on nylon. Previous study has demonstrated that pigment particle size critically influences chromatic performance on fabrics44. To validate this hypothesis, we performed SEM analysis on dyed nylon fabrics from both methods to compare particle size distributions. SEM analysis revealed significant differences in pigment particle morphology between the two dyeing approaches (Fig. 6c). The dyed nylon by Bio-in-situ strategy exhibited smaller 6,6′-DBI particles. In contrast, samples dyed by Bio-Chem strategy showed substantially larger particles. This structural divergence directly correlates with the observed chromatic differences. The smaller particles enhance light absorption at longer wavelengths (590 nm red shoulder), consistent with the characteristic Tyrian purple hue44. Conversely, the larger particles promote stronger scattering at shorter wavelengths, amplifying the 560 nm blue shoulder44. This contrast not only confirms the integrative advantage of combining synthesis and coloration in a single process through the Bio-in-situ strategy but also highlights its ability to achieve historically accurate chromatic reproduction on synthetic textiles, particularly for recreating the prestigious Tyrian purple aesthetics on nylon fabrics.

To further investigate the universality of Bio-in-situ method, we systematically evaluated six structurally diverse indigoid dyes (Supplementary Fig. 17). Experimental results demonstrated uniformly achieved robust dyeing outcomes. Building upon our prior work on scale-up of Tyrian purple biosynthesis, this approach enables feasible implementation of Bio-in-situ method with minimal infrastructure investment for producing commercially viable dyed materials. To demonstrate practical applicability, we strategically integrated the technique with embroidery technology for custom pattern development. Nylon via in-situ coloration was precision-stitched onto blank textile substrates using a computerized embroidery system, fabricating a proof-of-concept fabric sample featuring purple “NJTECH” letters (Fig. 6d). This can demonstrate the possible forms and aesthetics of in-situ dyeing.

Methods
Chemicals and materials
LB medium was prepared from 5 g L−1 yeast extract, 10 g L−1 peptone and 10 g L−1 NaCl. Indole 13d and substituted indole 1d-13d, 14d-21d were purchased from Aladdin. 2-aminophenethanol 13a and indigo 13f were purchased from Sigma-Aldrich. Oligomer synthesis and sequencing were purchased and done through Generay Biotech (Chuzhou, China). Enzymes involved in restriction reaction, ligation and PCR were purchased from Vazyme (Nanjing, China). Multifabric strips were purchased from SDC Corporation, UK. 1a-13a, 14a-21a were synthesized from substituted 2-nitrotoluene by two step chemical method (Supplementary Fig. 2) and they were identified by 1H and 13C nuclear magnetic resonance (NMR). Characterization of substrates and products were shown in Supplementary Note 1.

Plasmids and strains construction
Primers, plasmids and strains in this study are listed in Supplementary Table 5 and Supplementary Data 1, 2, respectively. The genes for 12 alcohol dehydrogenases, 3 flavin-containing monooxygenases, SmNOX and BmGDH were synthesized and cloned into the NdeI-XhoI site of pET-28a (+) after codon optimization by Generay Biotech (Chuzhou, China) (Supplementary Table 6). Plasmids used in this study were constructed in the following protocol. The fragments for target gene and vectors (pRSFDuet-1, pETDuet-1 and pACYCDuet-1(Novagen)) were amplified by PCR using primers to clone the target gene into the HindIII and XhoI sites of the vector, respectively. The gene fragments, the vector fragments, ddH2O and 2 × MultiF Seamless Assembly Mix from TransGen Biotech (Beijing, China) were added into 1.5 mL tube and incubated at 50°C for 10 min. Then, the mixture was transformed into 100 μL competent cells using heat shock and plated on LB agar with appropriate antibiotics. The results were confirmed by sequencing. Strains were based on E. coli BL21 (DE3). The tnaA gene was knocked out of the genome to give the engineered strain E. coli BL21 (DE3) ΔtnaA. Briefly, the sequences of sgRNA for tnaA was obtained from NCBI and ligated to the plasmid pTargetF (Addgene), giving the modified pTargetF. The upstream and downstream homologous arms of target genes were amplified and merged, resulting in deletion fragments. The plasmid pEcCas (Addgene) was first transformed into E. coli BL21 (DE3) by chemical transformation and then used for the preparation of the electroporated competent cells. Next, the corresponding deletion fragment and the modified pTargetF were cotransformed into the above competent cells and then plated on LB agar with appropriate antibiotics. The correct single colony was selected and confirmed by sequencing. The plasmids pTargetF and pEcCas were further removed by the corresponding culture conditions with appropriate antibiotics and sugar, finally giving the engineered strain E. coli BL21 (DE3) ΔtnaA. The plasmids for protein expression were transformed into E. coli BL21 (DE3), while the plasmids for whole-cell biotransformation were transformed into the engineered strain E. coli BL21 (DE3) ΔtnaA, giving the engineered strains M1-M12.

Expression of genes and purification of recombinant proteins
A single colony of recombinant E. coli BL21 (DE3) containing ADHs, FMOs, SmNOX and BmGDH was inoculated into LB medium supplemented with appropriate antibiotics and cultured overnight at 37°C. Then, 20 mL of the seed cultures were inoculated into 400 mL of LB media and grown at 37°C until the cell density reached an optical density (OD600) of roughly 0.6–0.8. Protein expression was induced with 0.2 mM β-D-1-thiogalactopyranoside (IPTG) at 25°C for 20 h when the optical density (OD600) of engineered strain reached 0.6–0.8. Next, the cells were collected by centrifugation at 3,500g for 10 min and washed with KPi buffer pH 7.0. The above pellets were resuspended with lysis buffer (50 mM KPi buffer pH 7.0, 300 mM NaCl, 10 mM imidazole, and 5% (w/v) glycerol) at a concentration of 0.1 g mL−1. The resuspended solution was disrupted by ultrasonication in ice-chilled water for 15 min (3 s turn on, 5 s turn off). The soluble fractions were collected by centrifugation of the cell lysate at 13,000g for 30 min at 4°C. The above supernatant was collected and used for target protein purification by NI-NTA agarose column. The collected protein was desalted to remove imidazole through the desalting column (50 mM KPi buffer pH 7.0 and 150 mM NaCl). After the desalting process, the purified protein was loaded into 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 90 V for 15 min and 130 V for 60 min to verify the production of the recombinant proteins. Protein concentrations were measured using the Bradford assay. Finally, the purified protein was stocked at −80°C for further study.

Enzymatic activity assay
To determine the catalytic activity of ADHs towards 1a (FMOs towards 1d), enzyme activity was determined by spectrophotometric assay at room temperature measuring the reduction of NAD(P)+ (oxidation of NAD(P)H) at 340 nm (ɛ = 6.22 mM−1 cm−1). The reaction mixture (3 mL) was composed of 5 μM the purified ADHs, 1 mM 1a and 0.5 mM NAD(P)+ (1 mM 1d and 0.5 mM NAD(P)H for FMOs activity assay). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the reduction (or oxidation) of 1 μmol NAD(P)(H) per min under the assay conditions.

Kinetic assay of HLADH and mFMO
The kinetic parameters (Vmax, KM, kcat) were determined by assessing the initial reaction rates at different NAD(P)(H) concentrations at 30°C in 50 mM KPi buffer, pH 7.0. The experimental procedure and method used for sample analysis were similar to the catalytic activity assay. The Michaelis-Menten equation was used to obtain the kinetic parameters.

Optimum conditions for the dehydrogenation reaction step catalyzed by HLADH
To determine the optimal conditions for the dehydrogenation of 1a catalyzed by HLADH, the conversion of 1a to 1d was utilized. The reactions were performed in 50 mM KPi pH 7.0 and 2.5 mM 1a, 0.5 mM NAD+ and 0.2 mg mL−1 purified HLADH and SmNOX were added at the beginning. KPi buffer (pH 5.5−8.0, 50 mM), Tris-HCl buffer (pH 8.0−9.0, 50 mM) and CHES-NaOH (pH 9.0−10.0, 50 mM) were used to determine the optimum pH. The optimal reaction temperature was determined by a range of temperatures from 20°C to 45°C with an interval of 5°C.

In vitro reactions
To determine the feasibility of HLADH-mFMO cascade (in vitro), the one-pot one-step strategy was applied in vitro. In vitro reactions of HLADH-mFMO cascade were performed at a final volume of 1 mL in 2 mL Eppendorf tubes. For the synthesis of 6,6′-DBI from 1a, the reactions were performed in 50 mM KPi pH 7.0. 2.5 mM 1a, 0.5 mM NAD+ and 0.4 mg mL−1 purified HLADH and mFMO were added to initiate the reactions. The reactions were stopped by incubating at 95°C for 5 min, and the precipitated protein was removed by centrifugation before analysis via high-performance liquid chromatography (HPLC) (Supplementary Table 7).

In vivo whole-cell biocatalysis
The cells used for the whole-cell reactions were harvested and washed by KPi buffer. For the twelve strains (M1-M12) containing different combinations of plasmids in the HLADH-mFMO cascade, the reactions were conducted in a 50 mL falcon tube with a final volume of 5 mL. Fresh cells were resuspended in the buffer containing 50 mM KPi pH 8.0, reaching a final OD600 of 20. 2.5 mM substrate 1a and 0.5 mM NAD+ were added to the above system to initiate the reaction at 30°C and 250 rpm for 24 h. After 24 h, the samples were prepared by centrifugation for 5 min at 20,000g before analysis via ultraviolet–visible light (UV–vis) spectroscopy.

HPLC analysis of reaction mixture
For quantification of 1a and 1d, 200 μL of the sample from reaction mixture was collected in Eppendorf tube and centrifuged at 13,000g for 10 min. The supernatant was filtered through 0.22 μm filters for HPLC analysis. Phenol was used as an internal standard for quantification, and ten-point standard curves were generated according to the concentrations of 1a and 1d ranging from 0.01 to 2.5 mM. HPLC analysis was conducted as follows: HPLC-DAD, ZORBAX Eclipse Plus C18, MeCN:H2O = 1:1 for 30 min, flow rate: 0.4 mL min−1, injection volume: 10 µL. Analytes were detected by using a diode array detector (DAD) at a wavelength of 254 nm.

Quantitative analysis of 6,6′-DBI and indigoids
For extraction and quantification of 6,6′-DBI and indigoids, 400 μL of the reaction mixtures were centrifuged at 13,000g, 10 min, 4°C and the supernatant was removed. The pellet was suspended in an 800 μL of dimethylsulfoxide (DMSO) followed by vigorous vortexing. The mixtures were centrifuged at 13,000g, 10 min and the supernatants were diluted to half concentration with water. Absorbance of the samples was measured at the absorption maxima by ultraviolet–visible light (UV–vis) spectroscopy. Absorbances were converted into the concentrations of the 6,6′-DBI and indigoids from 0.005 to 0.25 mM yielding ten-point standard curves, where 6,6′-DBI and indigoids were dissolved in DMSO:H2O = 50%:50% (v/v).

NMR analysis of 6,6′-DBI and indigoids
NMR spectra were recorded on a Bruker Ascend 400 MHz NMR spectrometer at 400 MHz (1H NMR) (Supplementary Figs. 18–57). Chemical shifts are reported in parts per million (ppm). Tetramethylsilane (TMS) was used as the reference substance for 1H and 13C chemical shifts.

Scale-up preparation of 6,6′-DBI
Similar to whole-cell biocatalysis section, 6,6′-DBI was scaled up to a 200 mL solution. After the reaction, the samples were prepared by centrifugation for 5 min at 20,000g to collect the precipitates and subsequently washed by 50 mM KPi pH 7.0 (3 × 50 mL). The cleaned precipitate was lyophilized overnight to yield 6,6′-DBI crude extracts for further study.

Chemical dyeing procedure
The 6,6′-DBI cell dye was prepared by using the cells (OD600 = 20 in 200 mL of reaction mixture) collected from the reaction of the strain M1 in the consecutive one-pot one-step reaction system at a 1 L glass flask. After the reaction, the cells were collected and washed with water several times and freeze-dried. Dyeing solutions (distilled water 50 mL; tetrahydrofuran 7.5 mL, NaOH 0.5 g, Na2S2O4 0.5 g and chemical 6,6′-DBI 0.15 g or the produced cellular 6,6′-DBI dye) were prepared by heating with stirring on the 80°C hot plate for 15 min. After the heating, NH4Cl 0.5 g was added to the dyeing solution and subsequent heating was repeated at 80°C for 15 min. The fabrics (multifabric strips) were soaked into the solution and dyed using an automatic dyeing machine at 60°C for 15 min. After the dyeing procedure, the fabrics were rinsed with 1% acetic acid solution and were dried under room temperature. Further repeated steps of washing with water and drying at room temperature were proceeded three times.

In-situ bio-dyeing strategy
The strain M1 was grown up to OD600 = 0.6–0.8 for IPTG induction in 400 mL of LB medium (containing 50 mg L−1 kanamycin) in a 1 L glass flask at 37°C and 200 rpm, using 5 mL of seed culture of the same LB medium overnight grown under the same condition. After IPTG was added to a final concentration of 0.5 mM, the culture was incubated at 18°C and 200 rpm for an additional 20 h. The collected cells were pelleted by centrifugation (4,000g, 20 min, 4°C) and washed with KPi solution for the following whole-cell reaction. The pelleted M1 cells were resuspended in 50 mM KPi, pH 8.0, and they were diluted down to OD600 = 20. The whole-cell reaction was proceeded by adding 2.5 mM 1a and 0.5 mM NAD+ at a final volume of 200 mL and the fabrics (multifabric strips) were soaked into the solution at the beginning of the whole-cell reaction. The dyeing process was conducted in a shaking incubator at 30°C and 200 rpm for 24 h. After the dyeing procedure, the fabrics were washed with water and drying at room temperature proceeded three times.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General: Na et al. report the construction of an artificial redox-neutral enzymatic cascade to synthesize indigoids with high atom economy and production efficiency. Employing engineered E. coli whole cells they achieved the biosynthesis and in situ dyeing of indigoids on various natural and synthetic fabrics. The work is well described and explained, and contains well-thought-out figures. The English can be improved and the supplementary figures need better explanation.\nComments:\n24 whole cells\n65 that integrates\n67 recombinant microbes or enzymes\n69 which is composed of\n72 indoles, still face…..\n74 requires\n76 complicating….and imposing….\n81 demonstrate\n112 aniline forming…\n115 (1a0. Based on…\n125 and NAD+ as co-substrates\n127 spectra\n137 proposed\n144 2.1 fold higher….\n179 activity with NADH\nFig. 3C Why is Vmax with mFMO with NADPH lower than with NADH, while the kcat with NADPH is higher than with\nNADH?\n386 oxidation\n389 was applied to……, thereby developing…..\n392 for the biosynthesis of….\n421 various indoles or tryptophans\n425 to cellular toxicity\n426 was recently developed\n427 challenges of whole-cell biocatalysis\n428 oxidative stress and oxygen limitations? (see John Woodley papers)\n430 presents\n431 delivers\n432 retired?\nSupplementary Information\nTable S2 carrying the…..gene\nTable S3 carrying the…..gene\nFig. S1 0.2 mg/ml HLADH? (amount of reaction volume not indicated)\nFig. S5a temperature?\nFig. S5b pH? 0.2 mg/ml HLADH?\nFig. S6 similar comments as for Fig. S5\nFig. S7 0.2 mg/ml FMOs?\nFig. S8 enzyme concentrations?\nFig. S9 similar comments as for Fig. S5\nFig. S10a indicate substrate and incubation conditions\nFig. S11 indicate substrate and incubation conditions\nThe kcat values for the monooxygenases are very high. Are they in line with literature data?\nFig. S12 The spectra are difficult to read. Please use better discriminating colors.\nFig. S13 Give reference to Table S3 for composition M1-M12 strains", "reviewer_2": "Na et al. developed a biosynthetic pathway for converting 2-amino-4-bromo-phenylethanol to Tyrian purple, a brominated\nindigo, for dyeing nylon directly in the fermenter. A detailed retrobiosynthetic prediction and analysis was performed to\ndetermine an optimal series of bioconversions. They constructed this enzyme-catalyzed pathway and screened several\northologs of enzymes for the optimized activity and redox balancing, ultimately employing a NADP+-consuming ADH and a\nNADP+ regenerating FMO oxygenation reaction. While the titers and staining results are only at modest efficiencies, the\napproach is interesting.\nSome outstanding questions remain. What is the efficiency of the indigo staining? The smaller crystals than the chemically-\nstained control is concerning. Does this indicate that there is a considerably lower efficiency of leucoindigo oxidation in the\nfibers with this novel route compared to the chemical route? Similarly, the significantly more red coloration with the novel\nroute than the chemical staining is concerning. The rationale is that this color difference is because the crystals are smaller\nwith the novel method. However, are there side products produced that are more red, such as or similar to indirubin?\nMore minor issues:\nP7. 138. Confused about the explanation of the spontaneous reactions for the production of desired intermediate 1d rather\nthan off-pathway product 1c’. While the TS4 barrier is a bit higher (66.98 kcal/mol), than TS2 (44.03 kcal/mol), the energetic\nstate of the intermediate product is higher (-6.56 kcal/mol for 1c’) compared to that for 1c (+1.86 kcal/mol). More discussion\non this is needed. How much 1c’ byproduct is observed?\nP9. 192. Typo “HLADA” should be HLADH.\nP16. 332. “Fig. 6a and 6b meant to be Fig. 6c and 6d?\nTiters are incorrectly referred to as “yields” throughout the manuscript.", "reviewer_3": "The authors report a two-enzyme “borrowing-hydrogen” cascade (horse-liver alcohol dehydrogenase, HLADH, plus an\nFMN-dependent mono-oxygenase, mFMO) that converts 2-amino-phenylethanols into a range of indigoid dyes. By\nexpressing the pair in E. coli and immersing fabric directly in the fermentation broth, they couple biosynthesis with in-situ\ndyeing and claim record atom-economy, titers and successful coloring of nylon with a historically accurate Tyrian-purple\nhue. The topic is timely for sustainable dye manufacturing, but several novelty, mechanistic, and logic issues must be\nresolved before the work reaches the standard of Nature Communications. The work, at least in its current form, appears\nmore suitable for a niche biocatalysis journal\n1. While the topic of greener pigment production is timely, the present study does not move the field forward in a convincing\nway. The key reaction can already be reached more directly from indole or bromo-indole; the substrate-color relationships\nare well charted; and in-situ dyeing was proven several years ago.\n1.1 Reaction design – Starting from indole (or its halogenated analogues) already delivers higher titres with simpler\nbiocatalytic setups. The manuscript offers no cost, yield, or operational advantage for beginning with amino-alcohols.\n1.2 Substrate scope – Previous papers have mapped color shifts across indole derivatives; the new data largely reproduce\nthose trends.\n1.3 In-situ dyeing – Hsu et al. (Nat. Chem. Biol., 2018) demonstrated in-fibre trapping of labile intermediates and showed\nsuperior color depth. The current study does not surpass that benchmark.\n2. A gas-phase or implicit-solvent DFT calculation cannot validate an HLADH-catalyzed oxidation–cyclization pathway. The\nauthors should either run a QM/MM model based on the HLADH crystal structure and back it up with kinetic data (e.g.,\nturnover of the putative imine), or tone down the mechanistic speculation and remove the DFT section.\n3. There are also some logical issues:\n•Lines 189-209 – The NADH vs. NADPH preference could be easily settled experimentally and summerized in one\nsentence; the present narrative over-complicates the issue.\n•Lines 210-225 – Because the final application uses whole cells, optimising the in-vitro enzyme ratio adds little value.\n• Lines 119-123 – State how many enzymes were screened, spell each one out in full at first mention, and note clearly that\nthe screen was in vitro. “Better” is sufficient; “superior” is overstated.\n• Line 185 – “Yields of NADP⁺-involved reactions” is misleading; it reflects only the particular enzymes tested, not all\npossible NADP-dependent options. Other enzymes catalyze the same reaction may have better yields.\n• Line 192 – “HLADA” → “HLADH.”\n• Lines 309-319 – This historical context belongs in the Introduction.\nThe manuscript would benefit from a thorough language edit to tighten phrasing and remove redundancies.", "reviewer_4": "Review of NCOMMS-25-51643-T\nThis paper (NCOMMS-25-51643-T) “Engineered artificial biosynthesis pathway enables simultaneous production and in-situ\nbio-dyeing of indigoids for textiles” examined a new novel route of the synthesis of indigoids compounds, for example Tyrian\npurple, using alcohol dehydrogenase and flavin monooxygenase with redox-neutral cascade, i.e. cofactor(NAD+) balance.\nAlthough there are many defects and mistakes on the writing this manuscript, the rationales of this study are simple, creative\nand straightforward. This reviewer thinks that it is advisable to publish this paper with major revisions to improve the quality\nof this paper.\nMajor comments:\n1. In fact, the approach the authors proposing for the synthesis of indigoids in this manuscript is chemo-enzymatic(or cell)\nsynthesis, not genuine enzymatic(or biological) synthesis. Although the authors did not supply trypthophan(Trp) for the\nsynthesis of indigoids such as tyrian purple(6,6’-DBI), they are now using 2-amino-4-bromo-phenylethanol as the initial\nsubstrate for the reaction, so that the aminoalcohol should be synthesized from either 4-bromo-alanine or o-nitrotoluene,\nwhich should undergo either nitration or bromination(i.e. halogenation), respectively, and in addition, their conversions of the\nreaction are not 100%, so that several steps of product purification and usage of solvents are required to be used as the\nsubstrate. Such substrate supply should be correctly evaluated with balanced viewpoint. The authors did not mention\nanything about these points, for example synthetic yields, cost of chemicals, solvent toxicity/wastes, byproducts, and etc.,\nand its pros and cons for environmental and economical viewpoints should be provided in detail, and revised.\n2. In addition, the authors did not mention about the toxicity of 2-amino-4-bromo-phenylethanol or its substrate inhibition\nagainst the ADH reaction at above 2.5mM of the halogenated aminoalcohol concentration. The reviewer thinks that using 2-\namino-4-bromo-phenylethanol as the reaction substrate is in quite different situation compared to that using Trp, which is\ncurrently commercially produced in E. coli with higher concentrations and large quantity is supplied from glucose in the\nseveral companies. Then, such strains can be easily coupled to the strains developed from previous findings(see ref. 10,\n11), which used three-step enzyme reactions, i.e. tryptophase, halogenase and FMO(or indole oxygenase). In result, other\nspecific advantages of using ADH and FMO over tryptophanase/halogenase and FMO should be considered.\n3. To this reviewer, the reaction of alcohol dehydrogenase reaction employed in this paper can smartly overcome the low\nreaction rate of using halogenase. However, the beauty of using halogenase is very environment friendly halogenation\nreaction in water solvent system with halogen salts. Therefore, for the above revision, the pros and cons between chemical\nhalogenation and biological halogenation should be fairly compared and discussed precisely.\n4. The authors claim that this study is the realization of the first one pot one step synthesis(line 293-294), but the reviewer\ncannot agree with this phrase, since several references(see ref.10, 11, and below reference*) already demonstrated the\nconcept of one pot synthesis. It should be revised. In biological system, one cell system and two cell system are all can be\nexecuted in one pot system, if you sacrifice the production rate and/or conversion yield to a certain extent.\n*Reference: Namgung, S., Park, H.A., Kim, J., Lee, P.-G., Kim, B.-G., Yang, Y.-H., Choi, K.-Y., 2019. Ecofriendly one-pot\nbiosynthesis of indigo derivative dyes using CYP102G4 and PrnA halogenase. Dyes Pigments 162, 80–88.\n5. The authors elaborated and compared in situ Bio-dyeing process and Bio-Chem dyeing process in Fig. 6, and in addition,\nthe authors specified all the RGB values of the studied compounds in Fig. 4. This reviewer is very uneasy with this part of\nthe manuscript, since RGB value is not that accurate to compare precisely the dyeing state and colors, and the values will\nbe dynamically changed depending upon the coloring dye concentrations, dyeing time and the numbers of washing steps\nsubjected to the fabric. In result, the textile dying field experts are more concerned about the durability of the same color of\ndyes and economy of the process. The major defects of bio-colors for textile-dying is instability to the light, pH, oxidation etc.\nOne of the major reasons of using leuco-form of indigo dyes with Na2S2O4 in chemical dying process is not only the\nsolubility of the indigo dyes but also the durability issue.\nWhen the indigoid dyes exist as leuco-form, they are highly solubilized and diffused into the fabric core center, and the dyes\nare adsorbed internally and externally, and impregnated into the fabric, whereas in In situ Bio-dyeing the dyes are adsorbed\nto the surface of the fabric rather than impregnated. In result, this reviewer is afraid that when the fabrics are subjected to\nrepeated washing steps, the original colors of the dye in the fabric would not be maintained even within a couple of washing\ncycles. Although the authors claim that the result of In situ Bio-dyeing is quite comparable to that of Bio-Chem dyeing, the\nreviewer thinks that without such special dyeing know-how and after-treatment process, it is dangerous to mention or\ncompare the two different dyeing process and color difference. Since this manuscript is more focused on a novel\nconsecutive enzyme reactions and high production synthesis of various indigoids compounds, it is more advisable to\ndramatically reduce this part of the manuscript, elaborating the comparison of dying process and color changes and\ndifferences between the two processes.\nMinor comments:\n1. There are many typos and wrong expressions in the manuscripts, such as line 200 (analyze?), line 214( the ratio of A to B\nis ?), line 363(were?), line 429(are entail solved?), line 432(retired subsidiary products?), line 443(traditional), etc."}, "author_response": "Thank you very much for thorough evaluation of our manuscript and give us a chance to\nrespond to the Reviewers. Wehave considered your and Reviewer's comments carefully and\nmadecorrespondingchangesintherevision.Thechangesandresponsearepresentedbelow.\nThechangesarealsohighlightedinyellowbackgroundinthefile\"RevisedManuscript\".\nResponsetoreviewers:\nReviewer#1(RemarkstotheAuthor):\nNa et al. report the construction of an artificial redox-neutral enzymatic cascadeto synthesize\nindigoids with high atom economy and production efficiency. Employing engineered E. coli\nwhole cells they achieved the biosynthesis and in situ dyeing of indigoids on various natural\nandsyntheticfabrics.Theworkiswelldescribedandexplained, andcontainswell-thought-out\nfigures.TheEnglishcanbeimprovedandthesupplementaryfiguresneedbetterexplanation.\nA. Thank you for your positive assessment of our work and your constructive suggestions. In\nresponseto your comments, wehaveperformedacomprehensive revision of themanuscript.\nThe language has been polished to enhance clarity and fluency, with all modifications clearly\nhighlighted in yellow for your convenience. Furthermore, we have also revised the\nSupplementary Information to include more detailed captions and explanations for all\nsupplementaryfigures,makingthemcomprehensiveandeasytointerpret.\nComments:\nQ1: The manuscript contains some grammatical and spelling errors, including that line 24\nshould be \"whole cells\", line 65 should be \"that integrates\", line 67 should be \"recombinant\nmicrobes or enzymes\", line 69 should be \"which is composed of\", line 72 should be \"indoles,\nstillface…..\",line74shouldbe\"requires\",line76shouldbe\"complicating….andimposing….\",\nline 81 should be \"demonstrate\", line 88 should be \"FMO. Indigoids\", line 112 should be\n\"aniline forming…\", line 115 should be \"(1a). Based on…\", line 121 should be \"Employing\nNAD+….\", line 125 should be \"and NAD+ as co-substrates\", line 127 should be \"spectra\", line\n137 should be \"proposed\", line 144 should be \"2.1 fold higher….\", line 179 should be \"activity\nwith NADH\", line 342 should be \"We wondered if this color distinction….\", line 386 should be\n\"oxidation\", line 389 should be \"was applied to……, thereby developing…..\", line 392 should\nbe \"for the biosynthesis of….\", line 421 should be \"various indoles or tryptophans\", line 425\nshouldbe\"tocellulartoxicity\",line426shouldbe\"wasrecentlydeveloped\",line427shouldbe\n\"challenges of whole-cell biocatalysis\", line 430 should be \"presents\", and line 431 should be\n\"delivers\".\nA1: Thank you for your thorough review and for identifying these grammatical and spelling\nerrors throughout the manuscript. We have carefully reviewed all the points you highlighted\nandcorrectedeachof theseerrorsin therevisedmanuscript.Additionally,wehaveperformed\na full professional language edit to ensure the text meets the high standards expected for\npublication.\nQ2:Inline85,FMOorBVMO?\nA2: We thank the reviewer for raising this important point regarding the distinction between\nFMOs and BVMOs. We acknowledge that the original description of \"flavin monooxygenase\"\non line 85 of the manuscript was imprecise. Flavin-containing monooxygenases (FMOs)\nbelong to subgroup B2 of group B FPMOs, and Baeyer-Villiger monooxygenases (BVMOs)\nbelongtosubgroupB1(Biotechnol.Adv.,2021,51,107712).\nWe have revised the manuscript by consistently adopting the term \"flavin-containing\nmonooxygenase\" to accurately reflect the specific enzyme class used in this study. These\nchangeshavebeenimplementedonlines20,57,85,and169andarehighlightedinyellowfor\neasyreference.\nQ3:Inline253,canthisdifferenceinpositionaldependencewithEDGsbeexplained?\nA3: We thank the reviewer for raising this insightful question. To elucidate the mechanistic\nbasis underlying the positional dependence of electron-donating groups (EDGs), we\nperformed molecular docking and interaction analysis of mFMO with 4-methoxyindole\n(4-OMe-YD)and6-methoxyindole(6-OMe-YD),respectively(seefigurebelow).\nThe docking results reveal that in the 6-OMe-YD–FMO complex, the 6-OMe-YD molecule\nadopts a nearly parallel conformation relative to FAD, engaging in strong π–π stacking\ninteractions. Additionally, residue N73 forms a hydrogen bond with the methoxy group of the\nsubstrate. These interactions collectively stabilize the binding pose of 6-OMe-YD, facilitating\nthecatalyticreaction. Incontrast, 4-OMe-YD isrotatedbyapproximately180°relativetoFAD.\nAlthough N73 also forms a hydrogen bond with the methoxy group, W319 engages in a\nhydrogen bond with the nitrogen atom of the substrate, and Q318 participates in an amide–π\nstacking interaction. These alternative interactions result in a greater distance between\n4-OMe-YD and FAD, as well as a relatively less stable binding conformation within the active\nsite, ultimately leading to its lower reactivity compared to 6-OMe-YD. This provides a\nmechanisticexplanationforthepositionaldependenceofEDGs.\nFig.MoleculardockingresultsofthemFMOproteinstructurewith4-methoxyindoleand\n6-methoxyindole, respectively. a Docking result of the mFMO protein structure with\n6-methoxyindole.bAnalysisoftheinteractionforcesbetweenmFMOaminoacidresiduesand\n6-methoxyindole. c Docking result of the mFMO protein structure with 4-methoxyindole. d\nAnalysisoftheinteractionforcesbetweenmFMOaminoacidresiduesand4-methoxyindole.\nQ4: In Fig. 3c, why is V with mFMO with NADPH lower than with NADH, while the k with\nmax cat\nNADPHishigherthanwithNADH?\nA4: Thank you for pointing out this critical discrepancy in our kinetic data analysis. Upon\nreview,weidentifiedamiscalculationintheprocessingthat ledtoincorrect kineticparameters\ninFig.3c.\nWehavenowreassessedthekineticmeasurementsandmadecorrespondingcorrectionsto\nFig. 3c and Supplementary Figure 11 (shown below). The revised figures and kinetic\nparameters not only align reasonably with the observed reaction rates but also remain\ncoherentwiththecofactorpreference.\nTheupdatedfiguresareprovidedbelow:\nFig.3ScreeningofFMOandconstructionofHLADH-mFMOcascadesystem.\nSupplementary Figure 11. The kinetic plots of HLADH towards NAD+ and NADP+ and\nmFMOandNiFMOtowardsNADHandNADPH.\nQ5:Inline428,oxidativestressandoxygenlimitations?(seeJohnWoodleypapers)\nA5: Thank you for pointing out this important aspect and for the relevant reference to John\nWoodley’s work. We agree that oxidative stress and oxygen transfer limitations can\nsignificantly affect whole-cell biocatalytic performance, especially in oxygen-dependent\nreactionslikeFMOcatalysis.\nIn the revised manuscript, we have added a discussion on the impact of balancing oxygen\ntransport and oxidative stress on the catalytic activity of the FMO enzyme in lines 406-410,\nwithacitationtoWoodley’srelevantpublications(ref.54,55).\nThecontentfromlines406-410intherevisedmanuscriptispresentedbelow:\n“The enzyme FMO is widely used to catalyze the oxidization of indole to indigoid, while\noxygen stress is always accompanied by the oxidation process54,55. The central challenge for\nfuture research lies in integrating enzyme engineering and process engineering strategies to\npreciselybalancetheoxygentransferratewithoxidativestress.”\nQ6:Inline429,whatdoyoumeanwithentailsolved?limited?\nA6: Thank you for pointing out that the wording was not sufficiently clear. The phrase \"are\nentail solved by\" in the original line 429 was intended to convey that the limitations of the\ncurrent tryptophan biosynthesis pathway are effectively addressed by the HLADH-mFMO\ncascadesystem.Duringtherevisionofthemanuscript,thisphrasinghasbeenreplacedwitha\nmore direct explanation of the advantages of the HLADH-mFMO cascade system, as\npresentedinlines399-402.\nQ7:Inline432,retired?\nA7:Thankyouforcatchingthisincorrectwordchoice.Thesentencehasbeenrevisedandthe\nmodificationishighlightedinyellowonlines402-404foreasyreference.\nThecontentfromlines402-404intherevisedmanuscriptispresentedbelow:\n“Pham et al reported an E.coli fermentation approach for synthesis of indigo starting from\nglucose, with titers reaching 235 mg/L and 965 mg/L at flask and 3L bioreactor scales,\nrespectively.”\nQ8: The Supplementary Information contains several grammatical inaccuracies and issues\nwith unclear descriptions. Specifically,inTable S2 andTable S3, it should be clearly indicated\nthat the plasmids are “carrying the … gene”. Additionally, the figure captions for Fig. S1 and\nFig. S5-S11 lack clarity in describing key parameters such as enzyme concentration, pH,\ntemperature, and reaction conditions. These should be explicitly stated to ensure\nreproducibility.\nA8: Thank you for your detailed feedback on the Supplementary Information. We have\ncarefully revised Table S2 and Table S3 as suggested, now clearly stating “carrying the …\ngene”. Additionally, we have enriched the figure captions for Fig. S3 and Fig. S5-S11 with\nexplicit details regarding enzyme concentration, pH, temperature, and reaction conditions to\nimproveclarity.Allmodificationshavebeenhighlightedinyellowforyourconvenience.\nQ9: In Fig. S11, the k values for the monooxygenases are very high. Are they in line with\ncat\nliteraturedata?\nA9: Thank you for raising this important question regarding the kinetic parameters in\nSupplementary Figure 11. An error in unit conversion led to the inaccuracy in the reported\nkinetic parameters, where the “10-3” was missing from the k value. This has been corrected\ncat\nintherevisedversion(asshownbelow).\nWehaverepeatedallkineticmeasurementsunderconsistentconditionstoensureaccuracy.\nThe corrected values now align well with literature reports for related FMOs, which typically\nrange between 0.1-6.1 s-1 for various substrates such as indole, trimethylamine, and\nN,N-dimethylaniline (PNAS, 2008, 105, 6572–6577; Appl Microbiol Biotechnol, 2019, 103,\n1755–1764;Int.J.Mol.Sci.,2019,20,6148; ChemBioChem,2025,e202401073).\nTheupdatedfiguresareprovidedbelow:\nSupplementary Figure 11. The kinetic plots of HLADH towards NAD+ and NADP+ and\nmFMOandNiFMOtowardsNADHandNADPH.\nQ10:InFig.S12,thespectraaredifficulttoread. Pleaseusebetterdiscriminatingcolors.\nA10:Thankyouforyoursuggestion.Wehaverevisedthefigurebyreplottingthespectrausing\nfourmoredistinctandeasilydistinguishablecolorstoimprovereadability.\nTheupdatedfigureisprovidedbelow:\nSupplementaryFig.14.The(a-e)absorptionspectraand(f)maximumabsorptionofthe\nindigoidsinDMFsolution.\nQ11:InFig.S13,givereferencetoTableS3forcompositionM1-M12strains.\nA11:Thank you foryoursuggestion. WehaverevisedthecaptionofSupplementaryFigure 13\nto explicitly reference Supplementary Table 3, where the composition of strains M1-M12 is\ndetailed.\nTheupdatedcaptionnowreads:\n“Supplementary Figure 15. SDS-PAGE analysis of M1-M12. Yellow arrow: mFMO; Red\narrow:HLADH.ThecompositionofM1-M12strainscanbefoundinSupplementaryTable3.”\nReviewer#2(RemarkstotheAuthor):\nNa et al. developed a biosynthetic pathway for converting 2-amino-4-bromo-phenylethanol to\nTyrian purple, a brominated indigo, for dyeing nylon directly in the fermenter. A detailed\nretrobiosynthetic prediction and analysis was performed to determine an optimal series of\nbioconversions. They constructed this enzyme-catalyzed pathway and screened several\northologs of enzymes for the optimized activity and redox balancing, ultimately employing a\nNADP+-consuming ADH and a NADP+ regenerating FMO oxygenation reaction. While the\ntitersandstainingresultsareonlyatmodestefficiencies,theapproachisinteresting.\nA.Wearegratefulforyourthoughtfulcommentsandsuggestions.Wehaveaddressedeachof\nthem in detail and have incorporated the corresponding revisions into the manuscript. The\nmodificationshavebeenhighlightedinyellowtofacilitatethereviewprocess.\nComments:\nQ1:Whatistheefficiencyoftheindigostaining?\nA1:Thankyouforyourquestionregardingstainingefficiency.Ourresultsdemonstratethatthe\ncolor strength (K/S values) of the Bio-in-situ strategy is comparable to that of Bio-Chem\nstrategy at equal dye concentrations (Supplementary Table 7). We acknowledge that the\ncurrentefficiencyoftheBio-in-situstrategy,reflectedintheextendedprocessingtime(12hvs.\n0.5 h), presents a significant limitation. Therefore, with the aim of enhancing the overall\ncoloring efficiency, our ongoing research are focused on exploring strategies to increase the\n6,6’-DBIsynthesisrateandimproveitstransferefficiencytothesubstrate.\nQ2: The smaller crystals than the chemically-stained control is concerning. Does this indicate\nthatthereisaconsiderablylowerefficiencyofleucoindigooxidationinthefiberswiththisnovel\nroutecomparedtothechemicalroute?\nA2: Weappreciatethereviewer’sinsightful questionregardingtheparticlesize of the6,6’-DBI\ncrystals.WewouldliketoclarifythatthesmallerparticlesizeobservedintheBio-in-situdyeing\nstrategy does not indicate a lower oxidation efficiency of leuco indigo. Rather, it is a key\ncharacteristic of our novel dyeing methodology that contributes to its superior color\nperformance.\nAs supported by previous literature (Dyes Pigments, 2015, 117, 37-48) and our data in Fig.\n6c, the color manifestation of indigoid dyes is correlated with particle size. The smaller\nparticlesformedinourbio-in-situprocessenhancelightabsorptionintheredregion(~590nm),\nyielding the authentic ‘regal purple’hue characteristic of Tyrian Purple. Conversely, the larger\nparticles resulting from chemical dyeing scatter more blue light (~560 nm), leading to a less\ndesirablebluishtint.\nThe successful development of the deep purple color on fabrics across different dyeing\ndurations(Fig. 6b)providesdirectevidenceofefficientoxidation.Thedistinctionliesnotinthe\noxidation efficiency, but in the resultant particle size and its subsequent impact on color\npresentation.\nQ3: Similarly, the significantly more red coloration with the novel route than the chemical\nstaining is concerning. The rationale is that this color difference is because the crystals are\nsmaller with the novel method. However,are there side products produced that are morered,\nsuchasorsimilartoindirubin?\nA3: Thank you for raising this important question regarding the color difference observed with\nourbiologicalstainingmethod.Weagreethatthereddishhuewarrantscarefulexamination.\nNMR analysis of the product did not detect significant side products such as indirubin or\nother red derivatives in the stained samples (Supplementary Figure 38). The observed color\nshift is primarily attributed to differences in crystal size and distribution within the fibers, not\nchemical impurities, as supported by previous literature (Dyes Pigments, 2015, 117, 37-48)\nandcorroboratedinourexperiments(Fig.6c).Thesmallercrystalsizeresultingfromslowerin\nsitu oxidation in our system scatters light differently compared to larger crystals formed\nthroughrapidchemicaloxidation,leadingtoaperceptibleshifttowardareddertone.\nTheupdatedfiguresareprovidedbelow:\nFig.6Chemicalandinsitumethodsfordyeingfabricswith6,6’-DBI.\nQ4: On page 7, line 79, confused about the explanation of the spontaneous reactions for the\nproduction of desired intermediate 1d rather than off-pathway product 1c’. While the TS4\nbarrier is a bit higher (66.98 kcal/mol), than TS2 (44.03 kcal/mol), the energetic state of the\nintermediateproductishigher(-6.56kcal/molfor1c’)comparedtothatfor1c(+1.86kcal/mol).\nMorediscussiononthisisneeded.Howmuch1c’byproductisobserved?\nA4: We sincerely appreciate your insightful comments regarding the energy profile and the\nformationpathwayofbyproduct1c’.Wehavesupplementedourstudywithmoleculardocking,\nQM/MM,andDFTcalculationstoevaluatetheselectivityofthereactionpathways(Fig.2eand\nSupplementaryFig.5,6).Thecomputationalresultsrevealedthat,underenzymaticconditions,\nthe energy barrier for the IM → 1c’ pathway is relatively high, reaching 31.4 kcal/mol, and is\nthus kinetically unfavorable. In contrast, in the enzyme-free liquid phase, the rate-determining\nstepof the IM → 1c → 1d pathway exhibits a barrier of only 16.5 kcal/mol, indicating that this\nprocess is more favorable. This explains why the reaction preferentially proceeds via 1c to\nformthetargetintermediate1d.\nExperimentally, HPLC analysis of the reaction mixture showed no detectable signal\ncorresponding to the 1c’ standard (see figure below). In addition, we have included a\ndiscussiononthispointintherevisedmanuscript(lines129–133).\nThecontentfromlines129-133intherevisedmanuscriptispresentedbelow:\n“Togain deeper mechanistic insights (Fig. 2dand Supplementary Fig. 4), QM/MM and DFT\ncalculations were employed to elucidate the catalytic mechanism. The results show that DFT\ncalculationsyieldalowerenergybarrierfromIMto1c(16.5kcalmol-1)comparedtothebarrier\nfrom IM to 1c’ obtained by QM/MM calculations (31.4 kcal mol-1) (Fig. 2e and Supplementary\nFig.5,6)”\nFig.2eDFT-calculatedreactionsofIM→1c→1dandIM→1c’werecarriedout.\nFig.HPLCplotforthereactionsolutionand1c’standard.\nQ5:Onpage9,line192,typo“HLADA”shouldbeHLADH.\nA5:Thankyouforbringingthiserrortoourattention.Wehavecorrected“HLADA”to“HLADH”\nandhavethoroughlyreviewedthemanuscripttoconfirmconsistency.\nQ6:Onpage16,line332,“Fig.6aand6b”meanttobeFig.6cand6d?\nA6: Thank you for raising this point. In the revised manuscript, the relevant content has been\nremoved.TheK/SvaluesobtainedfromtheBio-in-situstrategyandtheBio-Chemstrategyare\nconsolidated into a single table for ease of comparison (Supplementary Table 7). This table\nclearly presents the differences in color depth across various fabric types under the two\nstrategies.\nQ7:Titersareincorrectlyreferredtoas“yields”throughoutthemanuscript.\nA7: Thank you for highlighting this important terminological imprecision. We have carefully\nrevised the entire manuscript to use “titer” when referring to concentration-based\nmeasurements(e.g.line266).\nThecontentfromtheline266intherevisedmanuscriptispresentedbelow:\n“Finally, a 6,6’-DBI titer of 367.5 mg/L was achieved in 200 mL shake-flask whole-cell\nfermentation(Fig.5e).”\nReviewer#3(RemarkstotheAuthor):\nThe authors report a two-enzyme “borrowing-hydrogen” cascade (horse-liver alcohol\ndehydrogenase, HLADH, plus an FMN-dependent mono-oxygenase, mFMO) that converts\n2-amino-phenylethanols into a range of indigoid dyes. By expressing the pair in E. coli and\nimmersingfabricdirectlyinthefermentationbroth,theycouplebiosynthesiswithin-situdyeing\nand claim record atom-economy, titers and successful coloring of nylon with a historically\naccurateTyrian-purple hue. The topic is timely for sustainable dye manufacturing, but several\nnovelty,mechanistic, andlogic issuesmustbe resolvedbeforetheworkreaches thestandard\nof Nature Communications. The work, at least in its current form, appears moresuitable for a\nnichebiocatalysisjournal.\nA. Thank you for taking the time to review our manuscript and providing such thorough\nfeedback. We highly value your acknowledgment of the timeliness of our work and fully\nunderstand the issues you raised regarding the need for further strengthening in terms of\nnovelty, mechanism, and logical coherence. In the revised version, we will focus on clarifying\nthe innovative aspects of the \"borrowing-hydrogen\" cascade mechanism and more precisely\ndefinetheclaimedinnovationsandapplicablescopeofourstudy,especiallybyprovidingmore\nsolid data and logical support regarding its connection to practical sustainable dyeing\napplications.\nComments:\nQ1: While the topic of greener pigment production is timely,the present study does not move\nthe field forward in a convincing way. The key reaction can already be reached more directly\nfrom indole or bromo-indole; the substrate-color relationships are well charted; and in-situ\ndyeingwasprovenseveralyearsago.\nA1:Thank you foryourthoughtfulcomments.Weappreciate theopportunitytoclarifyhow our\nworkprovidesameaningfulcontribution.\nWhileindoleandbromo-indoleareestablishedsubstrates,theypresentchallengesincluding\nsignificant toxicity, poor solubility, and limited scalability at high concentrations (Biotechnol.\nAdv.,2024, 73,108374).Oursystemaddressestheselimitationsbyemployingaminoalcohols\nasabenignprecursor.Theredox-neutralcascade,facilitatedbyADHandFMO,maintainslow\nindole intermediate levels and operates without sacrificial reagents or external enzymes for\ncofactorregeneration.\nFurthermore, although certain structure-color relationships are already known (Dyes\nPigments, 2020, 181, 108570), our system provides the first unified enzymatic platform\ncapable of synthesizing and applying diverse indigo dyes directly within the dyeing medium.\nOur substrate scope study not only offers a consistent qualitative analysis of the\nstructure-color relationships for 20 indigoids, but also extends the application of mFMO to the\nsynthesisofF-,Me-,andOMe-substitutedindigoids.\nCritically, our Bio-in-situ dyeing approach diverges fundamentally from earlier strategies: it\navoids the use of toxic Na S O (Fig. 1a, Nat. Chem. Biol., 2021, 17, 104-112) and costly\n2 2 4\nmulti-enzyme systems relying on glycosyltransferase/glucosidase (Fig. 1b, Nat. Chem. Biol.,\n2018,14,256-261).\nWehopewehavesuccessfullyclarifiedtheseconceptualandmethodologicaladvances.\nQ2: Reaction design – Starting from indole (or its halogenated analogues) already delivers\nhigher titers with simpler biocatalytic setups. The manuscript offers no cost, yield, or\noperationaladvantageforbeginningwithamino-alcohols.\nA2: We appreciate your valuable perspective. In response, we have conducted an in-depth\ndiscussionregardingthecost,yield,andoperationalconvenienceofthereactionsystem.\nComparative data indicate that our biosynthetic system achieved the highest reported titer\nandyieldof6,6’-DBItodate(SupplementaryTable6).\nFurthermore, we performed two sets of comparative experiments using 6-bromoindole as\nthe substrate in the presence of a monooxygenase: one with stoichiometric cofactors and the\nother supplemented with a glucose dehydrogenase-based cofactor regeneration system. The\nresults revealed that the yield of 6,6’-DBI was lower than that achieved with the\nHLADH-mFMOcascadesystem(Fig.3a).\nTo better illustrate the practical application and economic value of our strategy, we have\nsupplemented the discussion with a comparative analysis of substrate cost and overall\nefficiency(asshownbelow).\nAlthough indoleand its halogenatedderivatives arecommonlyusedstarting materials, their\ncytotoxicity and low aqueous solubility severely limit reaction concentration and scalability\n(Biotechnol.Adv.,2024,73,108374).Incontrast,ourrouteoffersdistinctadvantages:\n(1) Reaction rates are carefully regulated to minimize the accumulation of toxic indole\nintermediates;\n(2) The redox-neutral cascade mechanism enables cofactor self-recycling without requiring\nadditionalenzymesorstoichiometriccofactors;\n(3)Avarietyofindigoidscanbebiosynthesizedinaone-potreaction.\nSubstrate(pricefromSigma-Aldrich) Product Yield References\n2-amino-phenylethanol($4.2/g) Indigo 92% Thisstudy\nIndole($3.7/g) Indigo 80% ACSCatal.9,9539-9544\nL-Tryptophan($6.3/g) Indigo 85% Enzym.Microb.Technol.142,109692\nQ3: Substrate scope – Previous papers have mapped color shifts across indole derivatives;\nthenewdatalargelyreproducethosetrends.\nA3: Thank you for your valuable comments. We acknowledge that the color variation trends\nobservedforcertainsubstitutedindigoids(e.g.,Br-andCl-substitutedindigoids)areconsistent\nwith previously reported patterns in the literature (Dyes Pigments, 2020, 181, 108570). It is\nworth noting that the color changes for several substrates (2f, 4f, 5f, 6f, 10f, 16f, 21f) have\nbeen reported for the first time. Moreover, the enzymatic synthesis of F-, Me-, and\nOMe-substitutedindigoidsusingmFMOhasnotbeenpreviouslydocumented.\nOur study provides a unified qualitative analysis of the structure-color relationship across\nthese 20 mFMO-generated indigoids. It also highlights the broad applicability and high\nefficiency of the HLADH-mFMO cascade reaction under mild and sustainable reaction\nconditions.\nWe have revised the discussion (lines 380-386) to more clearly acknowledge established\ntrendswhileemphasizingtheenzymaticandprocess-levelinnovationsofourwork.\nThecontentfromthelines380-386intherevisedmanuscriptispresentedbelow:\n“Lee et al. reported the biosynthesis of Br- and Cl-substituted indigoids10, while we have\nexpanded the substrate scope of mFMO to include F-, Me-, and OMe-substituted indigoids.\nThis demonstrates the broad applicability of our newly developed redox-neutral enzymatic\ncascade for producing a wide range of indigoid dyes under mild and sustainable conditions.\nFurthermore,aunifiedqualitativeanalysisofthecolor–structurerelationship(RGBvaluesand\nUV–Vis absorption spectra) for 20 distinct mFMO-catalyzed indigoids provides guidance for\nsubsequentresearch.”\nQ4:In-situdyeing–Hsuetal.(Nat.Chem.Biol.,2018)demonstratedin-fibretrappingoflabile\nintermediates and showed superior color depth. The current study does not surpass that\nbenchmark.\nA4: Thank you for raising this comparison. We fully appreciate the groundbreaking work by\nHsu et al. (Nat. Chem. Biol., 2018), which cleverly utilized glycosyltransferases to stabilize\nreactive intermediates and achieved impressive dyeing performance. Our study offers a\ndistinct strategy: we focus on simultaneous biosynthesis and dyeing in one pot using whole\ncells, without requiring prior synthesis or stabilization of intermediates. The color depth is\ncomparableto that of traditional chemical staining methods, and our approach offerspractical\nadvantagessuchaseaseof operation,minimalreagentconsumption, andsuperiorscalability.\nOur study expands the toolbox for sustainable textile dyeing by introducing an efficient and\nintegrablebio-dyeingalternative.\nQ5: A gas-phase or implicit-solvent DFT calculation cannot validate an HLADH-catalyzed\noxidation–cyclization pathway. The authors should either run a QM/MM model based on the\nHLADH crystal structure and back it up with kinetic data (e.g., turnover of the putative imine),\nortonedownthemechanisticspeculationandremovetheDFTsection.\nA5: We sincerely appreciate your insightful and rigorous feedback. Your valuable comments\nregardingtheDFTcalculationswereparticularlyinspiring.Inresponsetothepointsraised,we\nhave conducted molecular docking, QM/MM, and additional DFT calculations to validate the\nreliabilityoftheproposedreactionpathway.\nMolecular docking revealed that the intermediate IM is unlikely to adopt a reactive\nconformation within the enzyme active site (Supplementary Fig. 5). QM/MM calculations\nfurther indicated a high energy barrier (31.4 kcal mol-1) for the enzymatic conversion of IM to\n1c’, suggesting this pathway is energetically unfavorable (Supplementary Fig. 6). In contrast,\nDFT calculations demonstrated a significantly lower barrier (16.5 kcal mol-1) for the\nnon-enzymaticconversionofIMinsolution,supportingthefeasibilityofthealternativepathway\nleadingto1d(Fig.2e).\nWehaveincorporatedthelatestfindingsfrommoleculardocking, QM/MM,andDFTstudies\ninto the revised manuscript and have refined the discussion regarding the mechanistic\nspeculationinlines129-133.\nThecontentfromlines129-133intherevisedmanuscriptispresentedbelow:\n“Togaindeepermechanisticinsights(Fig.2dandSupplementaryFig.4),QM/MM,andDFT\ncalculations were employed to elucidate the catalytic mechanism. The results show that DFT\ncalculationsyieldalowerenergybarrierfromIMto1c(16.5kcalmol-1)comparedtothebarrier\nfrom IM to 1c’ obtained by QM/MM calculations (31.4 kcal mol-1) (Fig. 2e and Supplementary\nFig.5,6)”\nTheupdatedfiguresareprovidedbelow:\nFig.2eDFT-calculatedreactionsofIM→1c→1dwerecarriedout.\nSupplementary Fig. 5. Optimal docked conformation of the HLADH-IM substrate\ncomplex. The crystal structure of HLADH (PDB: 1ADB) was used as the receptor. To better\nmimic the physiological environment, the original ligand CND was manually replaced with the\ncoenzymeNAD+.Moleculardockingoftheintermediate(IM)intotheactivesiteofHLADHwas\nperformedusingAutoDock.\nSupplementary Fig. 6. QM/MM calculation results for the IM→1c' reaction process. a\nPotential energy profile of the first dehydrogenation elementary step along the reaction\npathway.Thecalculatedenergybarrieris31.4kcalmol-1.Thissubstantialbarriersuggeststhat\nthe catalytic reaction is kinetically unfavorable. Furthermore, the step is endothermic,\nindicating it is also thermodynamically uphill. b Potential energy profile of the second\ndehydrogenationelementarystep.Thisstepfeaturesarelativelylowenergybarrier.Combined\nwith the results above, the first step is identified as the rate-determining step of the overall\nreaction.\nQ6:Lines189-209–TheNADH vs. NADPHpreferencecouldbeeasilysettledexperimentally\nandsummerizedinonesentence;thepresent narrativeover-complicatestheissue.\nA6: Thank you for this suggestion. Weagree that the cofactor preference could be presented\nmore concisely. We have revised this section to summarize the key experimental findings\nmore directly, stating clearly that NAD+ was identified as the optimal cofactor for the overall\ncascade based on kinetic and activity comparisons (lines 191-193). This change streamlines\nthenarrativewhilepreservingthescientificrationale.Weappreciateyourhelpfulfeedback.\nThecontentfromlines191-193intherevisedmanuscriptispresentedbelow:\n“Accordingly, we assessed the performance of NADP+ and NAD+ in the cascade system.\nThe results indicated that the yields of 6,6’-DBI reached 15.2% and 42.1%, respectively (Fig.\n3b).”\nQ7: Lines 210-225 – Because the final application uses whole cells, optimising the in-vitro\nenzymeratioaddslittlevalue.\nA7: Thank you for raising this important point. We agree that optimization under in vitro\nconditions does not directly translate to whole-cell applications. Therefore, we have\ndownplayed the emphasis on the experimental results of in vitro enzyme ratio optimization in\nthe manuscript. The content has been significantly reduced, with only one sentence retained\nfordescriptionintherevisedmanuscript(lines198-201).\nThecontentfromlines198-201intherevisedmanuscriptispresentedbelow:\n“The ratios of HLADH and mFMO were further optimized.Aratio of 1:2 was found to most\neffectively alleviate the inhibition caused by the accumulation of intermediate 1d, resulting in\nthehighest6,6’-DBIyieldof60.1%(Fig.3bandSupplementaryFig.12).”\nQ8:Lines119-123–Statehowmanyenzymeswerescreened,spelleachoneoutinfullatfirst\nmention, and note clearly that the screen was in vitro. “Better” is sufficient; “superior” is\noverstated.\nA8:Thankyouforyourconstructivesuggestions.Wehaverevisedthemanuscriptaccordingly.\n(1)Online119,wenowexplicitlystatethattenalcoholdehydrogenaseswerescreened.\n(2) The full names and sources of all ten ADHs are provided in the caption of Fig. 2b, as\nrecommended.\n(3)Thedescriptiononline122hasbeenrevisedtoclarifythatthescreeningwasperformed\ninvitro.\n(4)Theterm“superior”online123hasbeenreplacedwiththemoreappropriate“better”.\nQ9:Line185–“YieldsofNADP+-involvedreactions”ismisleading;itreflectsonlytheparticular\nenzymestested,notallpossibleNADP-dependentoptions.Otherenzymescatalyzethesame\nreactionmayhavebetteryields.\nA9: Thank you for raising this important point. We agree that the original wording could be\nmisinterpreted as a general claim about all NADP+-dependent enzymes rather than a specific\nobservationfromtheFMOstestedinourstudy.\nThecontentfromlines182-185intherevisedmanuscriptispresentedbelow:\n“The yields of NADP+-involved reactions tested in this study were all higher than those of\nNAD+-involved, suggesting that NADP+ may be a preferable cofactor for this specific\nbiotransformationundertheexperimentalconditions.”\nQ10:Line192–“HLADA”→“HLADH.”\nA10: We are grateful for the reviewer pointing out the typo. The correction from \"HLADA\" to\n\"HLADH\" has been made, and we have carefully checked all abbreviations in the manuscript\nforconsistency.\nQ11:Lines309-319–ThishistoricalcontextbelongsintheIntroduction.\nA11: We thank the reviewer for this constructive suggestion. We agree that the historical\ncontextofTyrianpurpleismoreappropriatelysituatedintheIntroduction.Accordingly,wehave\nsignificantly condensed and refocused the original paragraph (lines 303-309), removing the\nhistorical details and retaining only abrief conceptual link to highlight the innovative aspect of\nourinsitudyeingstrategy.Intheintroductionsection(lines45-50),thehistoricalbackgroundof\ndyeingisfullypresented.\nQ12: The manuscript would benefit from a thorough language edit to tighten phrasing and\nremoveredundancies.\nA12: Thank you for your valuable suggestion. We fully agree with your perspective and have\nconducted a thorough language edit of the entire manuscript to eliminate redundancies and\nimprovetheaccuracyandconcisenessoftheexpression.\nReviewer#4(RemarkstotheAuthor):\nThis paper (NCOMMS-25-51643-T) “Engineered artificial biosynthesis pathway enables\nsimultaneousproduction andin-situ bio-dyeingof indigoids fortextiles”examined anew novel\nroute of the synthesis of indigoids compounds, for example Tyrian purple, using alcohol\ndehydrogenase and flavin monooxygenase with redox-neutral cascade, i.e. cofactor(NAD+)\nbalance. Although there are many defects and mistakes on the writing this manuscript, the\nrationales of this study are simple, creative and straightforward. This reviewer thinks that it is\nadvisabletopublishthispaperwithmajorrevisionstoimprovethequalityofthispaper.\nA.Thankyouforyourpositiveassessmentofourwork’snoveltyandrationale, aswellasyour\nconstructive suggestions for revision. We fully acknowledge the language and presentational\nshortcomings in the current version of the manuscript. In response, we have undertaken\nprofessional language editing of the entire text to enhance clarity, precision, and readability,\nandhavethoroughlyaddressedalltheissuesraisedinthereviewcomments.\nComments:\nQ1:Infact,theapproachtheauthorsproposingforthesynthesisofindigoidsinthismanuscript\nis chemo-enzymatic (or cell) synthesis, not genuine enzymatic(or biological) synthesis.\nAlthough the authors did not supply trypthophan(Trp) for the synthesis of indigoids such as\ntyrian purple(6,6’-DBI), they are now using 2-amino-4-bromo-phenylethanol as the initial\nsubstrate for the reaction, so that the aminoalcohol should be synthesized from either\n4-bromo-alanine or o-nitrotoluene, which should undergo either nitration or bromination(i.e.\nhalogenation),respectively,andinaddition, theirconversionsof thereactionarenot 100%,so\nthat severalsteps ofproduct purificationandusage of solvents arerequiredtobeusedas the\nsubstrate. Such substrate supply should be correctly evaluated with balanced viewpoint. The\nauthors did not mention anything about these points, for example synthetic yields, cost of\nchemicals, solvent toxicity/wastes, byproducts, and etc., and its pros and cons for\nenvironmentalandeconomicalviewpointsshouldbeprovidedindetail,andrevised.\nA1: We sincerely thank the reviewer for their valuable comments and insightful suggestions.\nWefully agree that a comprehensive evaluation of the chemo-enzymatic route—including the\nsynthesis pathway, environmental footprint, and economic feasibility—is essential to properly\ncontextualizethevalueofourwork.Inresponsetothecomments,wehavenowprovided:(1)a\ndetailed account of the synthetic route and yield for 2-amino-4-bromophenylethanol\n(Supplementary Figure 1); and (2) a systematic comparison of three different routes—starting\nfrom nitrotoluene, aminoalcohol, and tryptophan, respectively—in terms of space-time yield,\nchemicalcost,atomefficiency,andE-factor(SupplementaryTable9).Therelevantsectionsof\nthemanuscripthavebeenupdatedaccordingly.\nOur analysis shows that although the substrate requires chemical synthesis, the overall\nprocessstilldemonstratesconsiderableadvantages,includingareducednumberofenzymatic\nsteps, improved atom economy, avoidance of cytotoxic intermediates, superior E-factor\nperformance,andoperationalsimplicityundermildaqueousconditions.\nThenewlyaddedsupplementarytableisprovidedbelow:\nSupplementaryTable9.Thecomparisonofsynthesisof6,6’-DBIbydifferentstrategies.\nSubstrate Space-timeyield Atomefficiency\nEntry E-factorb ref.\n(pricefromSigma-Aldrich) (mgproductL-1d-1) (%)a\nNat.Chem.Biol.,\n2021,17,104-112\n2-amino-4-bromo-phenylethanol\n2 367.4 84.6 55.7 Thiswork\n(Noresultsfound)\n4-bromo-1-methyl-2-nitrobenzene\n3 326.5 73.9 97.1 Thiswork\n($4.7/g)\naTheatomefficiency(%)wascalculatedaccordingtothefollowingformula(Chem.Rev.,2018,\n118,801-838;ChemSusChem,2022,15,e202200529):\nbThe E factor was determined as weight of the waste per kg of product according to the\nreported literature (Green Chem., 2010, 12, 81-86; Angew. Chem. Int. Ed., 2018, 57,\n12308-12312).\nQ2: In addition, the authors did not mention about the toxicity of\n2-amino-4-bromo-phenylethanol or its substrate inhibition against theADH reaction at above\n2.5mM of the halogenated aminoalcohol concentration. The reviewer thinks that using\n2-amino-4-bromo-phenylethanol as the reaction substrate is in quite different situation\ncompared to that using Trp, which is currently commercially produced in E. coli with higher\nconcentrations and large quantity is supplied from glucose in the several companies. Then,\nsuchstrainscanbeeasilycoupledtothestrainsdevelopedfrompreviousfindings(seeref.10,\n11),whichusedthree-stepenzymereactions,i.e.tryptophase,halogenaseandFMO(orindole\noxygenase). In result, other specific advantages of using ADH and FMO over\ntryptophanase/halogenaseandFMOshouldbeconsidered.\nA2: Thank you for raising these critical points regarding substrate toxicity, inhibition, and the\ncomparativeadvantagesofourpathway.WehaveevaluatedthecatalyticefficiencyoftheADH\nreactionat higher substrateconcentrations and elucidatedthe distinct advantages of theADH\nandFMOcascade.\nThe experimental results demonstrated that using the coupled system of HLADH and\nsmNOX for cofactor regeneration achieved complete conversion within 12 h at a 5 mM\nsubstrate concentration, affording 6-bromoindole in >99% yield. Even at 10 mM, full\nconversionwasalsoachievedwithin24hwithsimilarlyhighyield(>99%).\nComparedtothetryptophanase/halogenaseandFMOcascade,theADHandFMOcascade\nsystemoffersdistinctadvantages:\n(1)Itachieveshigheryieldandtiter;\n(2)Itmaintainscytotoxicintermediates(e.g.,indoleorhalogenatedindoles)atlowerlevels;\n(3) It requires fewer enzymatic steps and operates as a redox-neutral system, thereby\nminimizingcofactordemand;\n(4)Iteliminatestheneedforspatiotemporalseparationinadditionalreactionprocesses;\n(5)ItexhibitssuperioratomeconomyandalowerE-factor.\nQ3: To this reviewer, the reaction of alcohol dehydrogenase reaction employed in this paper\ncansmartlyovercomethelowreactionrateofusinghalogenase.However,thebeautyofusing\nhalogenase is very environment friendly halogenation reaction in water solvent system with\nhalogen salts. Therefore, for the above revision, the pros and cons between chemical\nhalogenationandbiologicalhalogenationshouldbefairlycomparedanddiscussedprecisely.\nA3: Thank you for your thoughtful comments. In the revised manuscript, we have added a\ndiscussion in lines 394-402 to provide a more balanced and precise comparison between\nchemicalhalogenationandbiohalogenation.\nThecontentfromlines394-402intherevisedmanuscriptispresentedbelow:\n“Biocatalytichalogenationoffershighselectivity,mildreactionconditions,andenvironmental\nfriendliness. However, its large-scale application is hindered by issues such as low reaction\nrates and narrow substrate scopedifficulties. In contrast, chemical halogenation is\nwell-established, efficient, and cost-effective, yet suffers from poor selectivity, harsh reaction\nconditions, and generation of hazardous waste. HLADH-mFMO cascade strategy, employing\nhalogenated amino alcohol precursors as substrates, effectively circumvents the low reaction\nrates associated with halogenase enzymes. The higher atomic economy and lower E-factor,\ndemonstratingsuperiorsustainabilityandeconomicfeasibility(SupplementaryTable8).”\nQ4: The authors claim that this study is the realization of the first one-pot one-step\nsynthesis(line 293-294), but the reviewer cannot agree with this phrase, since several\nreferences(seeref.10,11,andbelowreference*)alreadydemonstratedtheconceptofonepot\nsynthesis. It should be revised. In biological system, one cell system and two cell system are\nall can be executed in one pot system, if you sacrifice the production rate and/or conversion\nyieldtoacertainextent. *Reference: Namgung, S.,Park,H.A., Kim,J.,Lee, P.-G.,Kim,B.-G.,\nYang,Y.-H.,Choi,K.-Y.,2019.Ecofriendlyone-potbiosynthesisofindigoderivativedyesusing\nCYP102G4andPrnAhalogenase. DyesPigments162,80–88.\nA4: Thank you for this important correction. We agree that the phrase \"the first one-pot\none-step synthesis\" was an overstatement. We have removed the claim of \"first\" and revised\nthestatement(lines267-269).\nThecontentfromlines267-269intherevisedmanuscriptispresentedbelow:\n“The highest space-time yield of 6,6’-DBI (15.31 mg L-1 h-1) achieved in this study\ndemonstrates the effectiveness of the one-pot one-step biosynthetic strategy for 6,6’-DBI\nproductioninE.coli10,11,41(SupplementaryTable6)”\nQ5: The authors elaborated and compared in situ Bio-dyeing process and Bio-Chem dyeing\nprocess in Fig. 6, and in addition, the authors specified all the RGB values of the studied\ncompoundsinFig. 4.This reviewer isveryuneasywiththispart of themanuscript, sinceRGB\nvalueisnotthataccuratetocomparepreciselythedyeingstateandcolors,andthevalueswill\nbedynamicallychangeddependinguponthecoloringdyeconcentrations,dyeingtimeandthe\nnumbers of washing steps subjected to the fabric. In result, the textile dying field experts are\nmore concerned about the durability of the same color of dyes and economy of the process.\nThemajordefectsofbio-colorsfortextile-dyingisinstabilitytothelight,pH,oxidationetc.One\nof the major reasons of using leuco-form of indigo dyes with Na S O in chemical dying\n2 2 4\nprocessisnotonlythesolubilityoftheindigodyesbutalsothedurabilityissue.\nA5:Thankyouforyourvaluablefeedbackregardingthecoloranalysisandpracticalrelevance\nof our dyeing study. We fully agree that RGB values are not a reliable or standard metric in\ntextilescience.\nWe would like to clarify that the RGB values provided in Fig. 4 were not intended to\nquantitatively characterize the dyeing results of the in situ Bio-dyeing and Bio-Chem dyeing\nprocesses compared in Fig. 6. Instead, they were used for a qualitative comparison of the\ncolors exhibited by 20 different indigoid compounds at the same concentration, in order to\nassess the applicability of the substrate spectrum.This approach offers a unified standard for\ncomparingvariousindigoids,assimilarlyreportedinrelevantliterature(Syst.Biotechnol.,2023,\n8,452-461).\nTo properly evaluate the color and dyeing performance, the textiles were subjected to\nmultiple washing and drying cycles. The resulting color depth and chromatic properties were\nthenverifiedusingK/SvaluesandCIELAB(L*,a*,b*)measurements(Nat.Chem.Biol.,2021,\n17, 104-112). Furthermore, the main advantage of the proposed in situ dyeing process lies in\nits integration of dye synthesis and dyeing into a single step. This eliminates the need for\ncomplex product separation and avoids the environmental issues associated with using\nNa₂S₂O₄forreducinginsolubleindigo—acommonpracticeinconventionaldyeing.\nQ6: When the indigoid dyes exist as leuco-form, they are highly solubilized and diffused into\nthe fabric core center, and the dyes are adsorbed internally and externally, and impregnated\nintothefabric,whereasinInsituBio-dyeingthedyesareadsorbedtothesurfaceofthefabric\nratherthanimpregnated.Inresult,thisreviewerisafraidthatwhenthefabricsaresubjectedto\nrepeated washing steps, the original colors of the dye in the fabric would not be maintained\neven within a couple of washing cycles. Although the authors claim that the result of In situ\nBio-dyeing is quite comparable to that of Bio-Chem dyeing, the reviewer thinks that without\nsuch special dyeing know-how and after-treatment process, it is dangerous to mention or\ncompare the two different dyeing process and color difference. Since this manuscript is more\nfocused on a novel consecutive enzyme reactions and high production synthesis of various\nindigoids compounds, it is more advisable to dramatically reduce this part of the manuscript,\nelaborating the comparison of dying process and color changes and differences between the\ntwoprocesses.\nA6: Thank you for your important and insightful feedback regarding the practical limitations of\nour in situ bio-dyeing approach. Based on your suggestions, we have revised the manuscript\nto reduce direct comparisons between different dyeing processes, focusing instead on the\npotential of the Bio-in-situ strategy as a mild and environmentally friendly biosynthesis-dyeing\nprocess.\nWewouldliketoclarifythat intheBio-in-situdyeingprocess,thetextiles arefully immersed\nin the whole-cell reaction system, where the dye is uniformly dispersed in the solution while\ndye formation and the dyeing process occur simultaneously. Additionally, all dyed fabrics\nundergomultiplewashinganddryingcyclesbeforetesting(lines583-584).Theparticlesizeof\nthe pigment is the direct cause of the differences in red and blue tones in the fabrics (Dyes\nPigments,2015,117,37-48).\nQ7: There are many typos and wrong expressions in the manuscripts, such as line 200\n(analyze?), line214( theratio ofAtoBis ?), line363(were?), line429(are entailsolved?), line\n432(retiredsubsidiaryproducts?),line443(traditional),etc.\nA7: Thank you for your meticulous review and for identifying these errors in expression and\nterminology. We have carefully corrected all the instances in the revised manuscript you\nhighlighted:\n(1) Line200:“analyze”→“analysis”\n(2) Line214:astheentirepassagehasbeenrevised,theissuehasbeenresolved.\n(3) Line363:“were”→“was”\n(4) Line429:“areentailsolved”→“areeffectivelytackled”\n(5) Line 432: rephrased to “with titers reaching 235 mg/L and 965 mg/L at flask and 3L\nbioreactorscales,respectively”\n(6) Line443:“traditional”→“traditionally”\nAllcorrectionshavebeenhighlightedinyellowintherevisedmanuscriptforeasyreference.\nWehavealsoperformedafulllanguagechecktoensureconsistencyandaccuracythroughout\nthetext.\nOnce again, thank you very much for your time and your consideration of this paper.Should\nthere be any questions or suggestions, please feel free to contact me. We look forward to\nhearingfromyou.\nWithbestregards,\nYourssincerely\nChenjieZhu,Ph.D,Professor\nNationalEngineeringTechniqueResearchCenterforBiotechnology,\nCollegeofBiotechnologyandPharmaceuticalEngineering,NanjingTechUniversity\n30SPuzhuRd,Nanjing211816,Jiangsu,China\nEmail:zhucj@njtech.edu.cn."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript has much improved after addressing the comments of the reviewers.\nMinor issues that need attention are listed below:\n60 dithionite\n62 suffers\n67 biosynthesis of\n77 expression of the tnaA gene\n88 co-expression of the ADH and mFMO genes\n131 showed\n133 indicates\n194 3.4 and 9.2 mM-1 s-1\n195 0.8 and 0.004 mM<-1<s<-1sup>/sup>sup> \n199 to alleviate\n214 for comparison\n258 successfully produced\n318 at shorter wavelengths\n348 that couples\n493 to verify the production of the recombinant proteins\n500 of enzyme activity\n501 per min", "reviewer_2": "Thank you for your responses to my questions. I think your manuscript is considerably improved from the responses to each\nof the reviewers' comments.", "reviewer_3": "The authors have substantially strengthened the paper. They now (i) articulate practical advantages of the amino-\nalcohol/HLADH–FMO route versus indole/halo-indole starts, including toxicity/solubility and process considerations; (ii)\nclarify what is genuinely new in the substrate scope (extension to F-, Me- and OMe-substituted indigoids and a unified\nqualitative color–structure analysis); (iii) replace the earlier protein-free DFT with a more credible package of docking,\nQM/MM, and corrected kinetics. They also cleaned up several logical detours and fixed the language and figure issues\ncalled out previously.\nReviewer 4’s points 1–3 (trade-offs vs Trp/halogenase routes, substrate toxicity/inhibition, and fair comparison of chemical\nvs bio-halogenation) are now addressed with a balanced process-level analysis (space-time yield, atom efficiency, E-factor),\nnew conversion data at higher substrate loadings, and a clearer statement of the operational pros/cons of the\nADH–FMO cascade. Reviewer 4’s point 5 (use of RGB) is handled by clarifying that RGB was used only for qualitative substrate-\nspectrum comparison, while dye performance is evaluated with standard K/S and CIELAB metrics; Typographical and\nphrasing issues flagged by R4 have been corrected. Overall, Reviewer 4’s concerns are satisfactorily met in the revision.\nIn light of the revisions and accompanying data/analyses, I’m satisfied that the manuscript now fits the scope and standard of\nNature Communications. I support publication in its current form.", "reviewer_4": "Reviewer 4’spoints 1–3(trade-offsvsTrp/halogenase routes, substratetoxicity/inhibition, and\nfair comparison of chemical vs bio-halogenation) are now addressed with a balanced\nprocess-level analysis (space-time yield, atom efficiency, E-factor), new conversion data at\nhigher substrate loadings, and a clearer statement of the operational pros/cons of the\nADH–FMOcascade. Reviewer4’spoint5(useofRGB)is handledbyclarifyingthat RGBwas\nused only for qualitative substrate-spectrum comparison, while dye performance is evaluated\nwith standard K/S and CIELAB metrics; Typographical and phrasing issues flagged by R4\nhavebeencorrected.Overall,Reviewer4’sconcernsaresatisfactorilymetintherevision.\nInlightoftherevisionsandaccompanyingdata/analyses,I’msatisfiedthatthemanuscriptnow\nfitsthescopeandstandardofNatureCommunications.Isupportpublicationinitscurrentform."}, "author_response": "Wesincerely appreciate your positive evaluation and recognition of our manuscript. We have\ncarefully considered all comments from you and the reviewers and have prepared a\npoint-by-pointresponsetoeach,asdetailedbelow.\nResponsetoreviewers:\nReviewer#1(RemarkstotheAuthor):\nThemanuscripthasmuchimprovedafteraddressingthecommentsofthereviewers.\nA.Thankyouverymuchforyourpositivefeedbackandforacknowledgingtherevisions.\nComments:\nQ1: The manuscript contains some grammatical and spelling errors, including that line 60\nshould be \"dithionite\", line 62 should be \"suffers\", line 67 should be \"biosynthesis of\", line 77\nshould be \"expression of the tnaA gene\", line 88 should be \"co-expression of the ADH and\nmFMO genes\", line 131 should be \"showed\", line 133 should be \"indicates\", line 178 should\nbe\"Loncaretal.\",line194shouldbe\"3.4and9.2mM-1s-1\",line195shouldbe\"0.8and0.004\nmM-1s-1\",line199shouldbe\"toalleviate\",line124shouldbe\"forcomparison\",line257should\nbe \"SDS-PAGE analysis\", line 258 should be \"successfully produced\", line 318 should be \"at\nshorter wavelengths\", line 348 should be \"that couples\", line 476 should be \"Expression of\ngenes and\", line493 shouldbe \"toverifythe productionof therecombinant proteins\", line500\nshouldbe\"of enzymeactivity\",line501shouldbe\"permin\".\nA1: Thank you for your careful reading and for identifying these specific grammatical and\nspelling errors throughout our manuscript. We have carefully reviewed the entire manuscript\nandhavecorrectedalltheerrorsyouhelpfullypointedout.\nReviewer#2(RemarkstotheAuthor):\nThank you for your responses to my questions. I think your manuscript is considerably\nimprovedfromtheresponsestoeachofthereviewers'comments.\nA. Thank you for your positive feedback. We sincerely appreciate the time and expertise you\nhave dedicated to reviewing our work and providing constructive suggestions, which have\ngreatlystrengthenedthepaper.\nReviewer#3(RemarkstotheAuthor):\nThe authors have substantially strengthened the paper. They now (i) articulate practical\nadvantages of the amino-alcohol/HLADH–FMO route versus indole/halo-indole starts,\nincluding toxicity/solubility and process considerations; (ii) clarify what is genuinely new in the\nsubstrate scope (extension to F-, Me- and OMe-substituted indigoids and a unified qualitative\ncolor–structure analysis); (iii) replace the earlier protein-free DFT with a more credible\npackage of docking, QM/MM, and corrected kinetics. They also cleaned up several logical\ndetoursandfixedthelanguageandfigureissuescalledoutpreviously.\nA. Thank you for your thorough and thoughtful assessment of our revised manuscript. Your\nconstructive feedback throughout the review process has been invaluable in shaping the final\nversionofthiswork.\nReviewer#4(RemarkstotheAuthor):\nReviewer 4’spoints 1–3(trade-offsvsTrp/halogenase routes, substratetoxicity/inhibition, and\nfair comparison of chemical vs bio-halogenation) are now addressed with a balanced\nprocess-level analysis (space-time yield, atom efficiency, E-factor), new conversion data at\nhigher substrate loadings, and a clearer statement of the operational pros/cons of the\nADH–FMOcascade. Reviewer4’spoint5(useofRGB)is handledbyclarifyingthat RGBwas\nused only for qualitative substrate-spectrum comparison, while dye performance is evaluated\nwith standard K/S and CIELAB metrics; Typographical and phrasing issues flagged by R4\nhavebeencorrected.Overall,Reviewer4’sconcernsaresatisfactorilymetintherevision.\nInlightoftherevisionsandaccompanyingdata/analyses,I’msatisfiedthatthemanuscriptnow\nfitsthescopeandstandardofNatureCommunications.Isupportpublicationinitscurrentform.\nA. Thank you for your positive feedback. We are truly delighted and honored to have your\nsupportforthepublicationofthemanuscriptinitscurrentform.\nOnceagain, thank you very muchfor your precious timeand recognition of this manuscript. If\ntherebeanyquestionsorsuggestions,pleasefeelfreetocontactme.\nWithbestregards,\nYourssincerely\nChenjieZhu,Ph.D,Professor\nNationalEngineeringTechniqueResearchCenterforBiotechnology,\nCollegeofBiotechnologyandPharmaceuticalEngineering,NanjingTechUniversity\n30SPuzhuRd,Nanjing211816,Jiangsu,China\nEmail:zhucj@njtech.edu.cn."}]

Decision letter