Biosynthesis of pyomelanin from methanol with engineered Komagataella phaffii and its characterizations

Domain: physics
Published: 2026-03-17
Editorial outcome: major_revision

Abstract
Pyomelanin has been extensively applied in various fields. However, the yield of pyomelanin isolated from natural producers is low. Engineering microbial biosynthesis is considered a sustainable and economically feasible alternative. We utilize engineered Komagataella phaffii to increase the yield of homogentisic acid by 66-fold by balancing three different biosynthetic modules and further synthesizing pyomelanin by oxidative polymerization. During this process, we establish a pyomelanin colour screening system and apply this system to perform directed evolution of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase and semirational design modification of hydroxyphenylpyruvate dioxygenase. We report the difference of pyomelanin between oxidative polymerization under alkaline conditions and that under laccase treatment. After high-density fermentation in a 5-L fermenter for 204 h, strain Pyo29 achieve the highest titer (70.5 ± 0.7 g/L). Finally, we demonstrate the potential application value of pyomelanin. This study provides a feasible solution for high-yield pyomelanin biosynthesis with substantial industrial production.

Introduction
Pyomelanin, a crucial type of melanin, refers to a group of phenolic polymers and is a brown‒black pigment formed through the oxidation and polymerization of homogentisic acid (HGA), which is derived from L-tyrosine [1]. Pyomelanin exhibits diverse biological activities, including resistance to ultraviolet (UV) light and oxidative stress, antibacterial and anti-inflammatory effects, and roles in electron transfer and energy applications [2–7]. Despite these properties, pyomelanin has been less extensively studied than eumelanin, another major melanin group. With recent advances in metabolic engineering and synthetic biology, biosynthesis offers a promising, sustainable, and cost-effective approach for pyomelanin production [8–10].

Over the past decade, pyomelanin biosynthesis has depended mainly on the exogenous addition of L-tyrosine, which has greatly increased production costs. Furthermore, the absence of comprehensive metabolic regulation has hindered efforts to achieve high pyomelanin yields [11–13]. To date, the only report on de novo pyomelanin synthesis achieved production at the gram scale [14]. However, this study did not implement complete modular metabolic engineering of the biosynthetic pathway from carbon sources to pyomelanin, nor did it apply protein engineering strategies to address the low catalytic efficiency of key enzymes in the pathway. As a result, the production potential of pyomelanin remains underdeveloped.

The methylotrophic yeast Komagataella phaffii is classified as a “generally recognized as safe” (GRAS) organism by the U.S. Food and Drug Administration (FDA) [15,16]. Many studies have demonstrated its potential as an effective host for the production of L-tyrosine-derived compounds, including resveratrol, naringenin, and norcoclaurine [17,18]. Compared with bacteria, K. phaffii has advantages in terms of efficient and high-level expression of heterologous enzymes, and its Crabtree-negative phenotype further enhances its capacity for high-density fermentation in minimal salt media when methanol is used as the sole carbon source [19]. In terms of cost, although bacteria can ferment waste compounds from the agro-industrial or fuel industries, methanol is also a low-cost carbon source. In addition, methanol, as a renewable and carbon–neutral single-carbon (C1) feedstock, has emerged as a promising alternative carbon source for green biomanufacturing because of its noncompetition with food resources and scalability for industrial production [20,21]. Consequently, we engineer K. phaffii to offer a sustainable platform for the production of pyomelanin, a downstream derivative of L-tyrosine. However, the complex methanol metabolism of the organism and the stringent regulation of precursor supply present significant challenges for the de novo biosynthesis of pyomelanin in this host. To date no studies have reported pyomelanin production using K. phaffii.

Furthermore, the absence of a standardized reference for pyomelanin has rendered the traditional absorbance-based quantification method insufficiently rigorous in this field [22]. In this study, for the first time, we present a modular framework for pyomelanin biosynthesis from methanol, dividing the process into two stages and three modules (Fig. 1). The two stages involve the efficient synthesis of HGA from methanol, followed by the promotion of HGA oxidative polymerization into pyomelanin using either alkali treatment or laccase catalysis. The three modules include central carbon metabolism, the shikimate pathway, and the HGA biosynthetic pathway. Changes in HGA production were accurately measured using high-performance liquid chromatography (HPLC), and the precise dry weight of pyomelanin was determined through direct weighing. Furthermore, the design concept underlying this newly proposed synthesis method offers a feasible and standardized framework for future research on pyomelanin biosynthesis.

In this study, we construct an engineered K. phaffii platform for the de novo biosynthesis of HGA by optimizing three parallel biosynthetic pathways. Afterwards, we establish a pyomelanin colour screening system (PCSS) for directed evolutionary modification of the first key enzyme 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), and semi rational design modification of the second key enzyme hydroxyphenylpyruvate dioxygenase (HPPD) in the biosynthetic pathway of pyomelanin. Through the combined application of metabolic and enzyme engineering strategies, we increase the production of HGA 66-fold. Afterward, we employ either alkali or laccase to promote the oxidative polymerization of HGA, generating high-yield pyomelanin-producing K. phaffii strains derived from high-HGA-producing strains. The resulting pyomelanin, is referred to as Pyo-NaOH and Pyo-Lac. Then, we conduct characterization experiments using Fourier-transform infrared (FTIR) spectroscopy, elemental analysis, thin-layer chromatography (TLC), and scanning electron microscopy (SEM). Next, we then conduct high-density fermentation of the top-producing strain (Pyo29) in a 5-L fermenter, achieving a pyomelanin titer of 70.5 ± 0.7 g/L with a specific pyomelanin yield of 0.63 ± 0.04 g/g DCW and a productivity of 0.37 ± 0.05 g/L/h; the carbon conversion ratio is 8.83%. These results significantly surpass the highest value previously reported in the literature [23]. Finally, we prepare a pyomelanin powder and functionally analysed the powder to demonstrate its application potential. Antioxidant and radiation-resistance evaluations suggest the potential application of pyomelanin in cosmetics. Moreover, we demonstrate that pyomelanin exhibits excellent performance in energy storage material development, showing high cycling stability as a negative electrode material for sodium-ion batteries. This work significantly advances the industrial potential of producing pyomelanin from renewable single-carbon (C1) methanol as the sole carbon source and highlights its broad application prospects.

Results
Construction of the HGA biosynthetic pathway
K. phaffii possesses a native pathway for HGA synthesis. First, the HGA-producing capability of the WT strain was tested. After 5 d of shake-flask fermentation, the production of HGA reached 25 ± 0.85 mg/L, but no visible colour change was observed in the supernatant, and the OD400 was only 0.006 (Fig. 2a). We then supplemented the culture medium with L-tyrosine at various concentrations; however, this did not significantly increase HGA production (Supplementary Fig. 1). These results suggest that the enzymes HPPD and TYRB, which catalyze the conversion of L-tyrosine to HGA in K. phaffii, exhibit low catalytic efficiency. Therefore, identifying alternative enzyme sources may provide a feasible strategy for improving HGA biosynthesis.

We next explored HPPD and TYRB genes from various sources and identified HPPD from Alcaligenes faecalis and TYRB from Yarrowia lipolytica as candidates that significantly enhanced HGA production. Compared with TYRB, the overexpression of HPPD had a greater effect on HGA yield, supporting reports in the literature that HPPD is a key enzyme in the HGA biosynthetic pathway (Fig. 2a). Multicopy integration of key enzymes has been shown to effectively increase target product yields [25]. Therefore, we further optimized AfHPPD expression through multicopy integration. Strain Pyo9, which carried three AfHPPD copies integrated into the genome at different loci (HIS4, INT1, and INT18), achieved the highest HGA yield of 64.1 ± 0.68 mg/L (Fig. 2b). Notably, compared with the WT strain, its supernatant developed a darker, light brown colouration, with an OD400 of 0.362 (Fig. 2d), which was likely due to self-oxidative polymerization of HGA.

Previous studies have shown that HPPD, like other dioxygenases, contains divalent metal ion active sites in its catalytic centre, which may influence HGA synthesis [26]. On this basis, we hypothesized that supplementation of the medium with specific metal ions could increase AfHPPD activity and improve the conversion of L-tyrosine to HGA. To test this hypothesis, we examined the effects of the addition of various metal ions, including K+, Cu2+, Fe2+, Mn2+, Na+, Zn2+, Mg2+, and Ca2+, during the fermentation of strain Pyo9. After 5 d of fermentation, most ions—except for Cu2+—showed no significant improvement in HGA production, and no noticeable colour change was observed in the supernatant of Pyo9. Interestingly, no detectable HGA production was observed with the addition of Cu2+; however, the OD400 increased significantly to 1.375 (Fig. 2c), and the supernatant became darker in colour, suggesting that more HGA underwent self-oxidative polymerization to form pyomelanin (Fig. 2d). To confirm that Cu2+ enhanced the catalytic activity of the AfHPPD enzyme and promoted the conversion of L-tyrosine to HGA, we recombined and purified AfHPPD and established eight standard enzyme reaction systems containing different metal ions (Fig. 2e). A reaction system without AfHPPD served as the control, and the amount of HGA produced after completion was used as the benchmark for relative enzyme activity. The results revealed that only the reaction system containing Cu2+ exhibited a noticeable colour change, indicating increased HGA production, whereas other metal ions had little to no effect on AfHPPD activity (Supplementary Fig. 2). Overall, the relative activity of the AfHPPD enzyme increased by 4.12-fold in the presence of Cu2+ (Fig. 2f). Interestingly, Seo D. et al. demonstrated that Cu2+ ions do not increase the catalytic activity of the HPPD enzyme of Ralstonia pickettii. However, the addition of Cu2+ to the culture medium of R. pickettii can increase the production of pyomelanin [8]. Therefore, we speculate that Cu2+ may both increase HPPD activity and promote the nonenzymatic oxidation of HGA. To verify this hypothesis, HGA standard powder was prepared as a solution, and Cu2+ was added to the solution. After shaking for 24 h, compared with the control solution, the solution with Cu2+ added showed a colour change, appearing brownish yellow, with an increase in the OD400 (Supplementary Fig. 3). This proves that Cu2+ can also promote the nonenzymatic oxidation of HGA.

Establishment of a rapid screening system for the directed evolution of DAHP synthase with the pyomelanin colour to improve the production of HGA
After the conversion of L-tyrosine to HGA was optimized, increasing the supply of L-tyrosine became the next goal. We first focused on the modification of the shikimate pathway because DAHP synthase is a key enzyme involved in this pathway, and its catalytic activity affects the metabolic flux of the shikimate pathway [27]. Therefore, we aimed to modify endogenous DAHP synthase in K. phaffii as a target to further increase the production of HGA.

On the basis of the characteristics of pyomelanin, the PCSS was established to evaluate both the changes in metabolic flux caused by the activity of upstream DAHPS and the level of pyomelanin colour (Fig. 3a). We first evaluated the feasibility of using the PCSS to screen DAHPS variants with high catalytic efficiency. Different concentrations of HGA resulted in different colour differences after 24 h in the 24-well plate, indicating excellent parallelism. Although the linear changes between low concentrations of HGA are not significant, a good linear relationship exists between high concentrations of HGA. The overall trend of change indicated that as the concentration of HGA increased, short-term oxidative polymerization gradually intensified the colour and increased the OD400 (Fig. 3b). In summary, strains corresponding to different mutation sites exhibited different colour change patterns with shorter fermentation times, suggesting the reliable usability of PCSS.

Then, variant libraries of the DAHPS1 and DAHPS3 enzymes were constructed using error-prone PCR, and extensive screening was conducted using the Pyo9 strain. The results demonstrated highly successful screening. For example, after 36 h of fermentation, colour differences were clearly observed in the 24-well plate (Fig. 3c). A microplate reader was used to determine the OD400 of the strains showing the darkest colour, and these strains were sequenced to determine the corresponding mutation sites. Variant plasmids were further constructed and transferred to strain Pyo9 for secondary validation of in vivo fermentation (Fig. 3a). Finally, we obtained a total of 2 optimal mutation sites. Compared with the WT, the K129R variant resulted in a 2.3-fold increase in HGA productio, and the E252K variant resulted in a 1.7-fold increase in HGA production (Fig. 3d).

To investigate the molecular mechanisms underlying the enhanced HGA production in these variants, the 3-dimensional structures of DAHPS1 of strain K129R and DAHPS3 of strain E252K were constructed using ARO3 (PDB: 7ykc) from S. cerevisiae as a template, and the structures were analyzed using MDS. First, the calculation results revealed that the root mean square deviation (RMSD) of DAHPS1K129R was significantly lower than that of the DAHPS1-WT, indicating that the mutation from lysine (K) to arginine (R) increased the stability of the overall structure of the enzyme, revealing the contribution of K129R to stability (Fig. 3f). In addition, we performed sequence alignment between two variants and ARO3 (Supplementary Fig. 4), and we were pleasantly surprised to note that spatial position 129 of DAHPS1 derived from K. phaffii corresponded to the amino acid at position 133 of ARO3 derived from S. cerevisiae, and R mutated from DAHPS1 corresponded exactly to R at position 133 of ARO3. This interesting phenomenon likely corresponds to an optimal solution problem where the observed amino acid residue, despite apparent structural conflicts or flexibility, may represent the best possible compromise between stability, function, and evolutionary constraints. Similarly, we aligned the DAHPS3 variant from the same source as that of DAHPS1 with ARO3 and reported that the amino acid at position 252 of ARO3 corresponding to its spatial position at 252 was the mutated glutamic acid (E), indicating that DAHPS3E252K had the same effect as that of DAHPS1 K129R (Fig. 3e).

Further improvement of HGA production through semi-rational design and modification of AfHPPD
To fully explore the potential of AfHPPD enzyme modification, we employed two semi-rational design strategies. First, since the crystal structure of AfHPPD was not yet available, we used AlphaFold2 for modelling. The reliability of the model was demonstrated by standard quality metrics and a Ramachandran plot (Supplementary Fig. 5). To investigate whether the model generated by AlphaFold2 can be used for further modification, we used Modeller for homology modelling. A template search using SWISS-MODEL identified only two HPPD crystal structures that satisfy the sequence similarity and coverage criteria for AfHPPD modelling: PfHPPD (Pseudomonas fluorescens, PDB: 1cjx) and AtHPPD (Arabidopsis thaliana, PDB: 8hza). The sequence alignment results revealed that compared with AtHPPD, PfHPPD is more suitable as a template (Supplementary Fig. 6). The model established by Modeller is similar to the model generated by AlphaFold2. Therefore, the model established by AlphaFold2 was used for the semirational design. The analysis revealed that the predicted Cu2+ binding site overlapped precisely with the Fe2+ site in the P. fluorescens structure. These results strongly supported the reliability of the modelled structures, which were used for subsequent analyses and calculations (Fig. 4a). First, we performed MSA of HPPD enzymes with sequences similar to those of AfHPPD to identify low-conservation sites as potential targets for modification. Using the residue-scanning function of FoldX, three candidate mutations, S48F, E314Y, and A287E, were selected (Supplementary Fig. 7). Second, PROSS and FoldX were used in combination to design additional modification sites. PROSS integrates phylogenetic sequence information with Rosetta-based modelling to enhance protein thermal stability. These algorithms were applied to calculate mutations that could improve the thermal stability of AfHPPD and to statistically analyze the resulting candidate sites. Three sites with ΔΔG values less than –1.0 were retained as candidate mutations: A281G, S224G, and D146P. Additionally, favourable residues identified from alanine scanning were further optimized using the residue-scanning function of FoldX, resulting in E219P, T164A, and K203L as additional candidates. In total, nine promising mutation sites (Fig. 4c) were selected, and the corresponding variants were purified for in vitro enzymatic validation and subsequent combination to generate AfHPPD variants with enhanced HGA production (Supplementary Fig. 8).

After the catalytic reactions, HGA production was used to assess the relative enzyme activity of the variants. Among the nine variants, E314Y exhibited the greatest increase in activity, with a 76.3% increase compared with that of the WT, followed by S224G, with a 43.4% increase. The activity of the remaining seven variants did not significantly differ from that of the WT. Afterwards, a double mutant combining E314Y and S224G was constructed, resulting in the E314Y/S224G variant, which displayed a 120% increase in enzyme activity relative to that of the WT (Fig. 4d). The stability of the E314Y/S224G variant was subsequently evaluated. The WT enzyme and E314Y/S224G variant were incubated at 30 °C, 40 °C, and 50 °C for 0–4 h, after which their residual activities were measured under optimal conditions. The WT enzyme retained 59.7%, 54.3%, and 32.7% of its initial activity at 30 °C, 40 °C, and 50 °C, respectively (Fig. 4e). In contrast, compared with the WT enzyme, the E314Y/S224G variant maintained more than 90% of its activity at all three temperatures after 4 h, demonstrating markedly enhanced thermal stability compared (Fig. 4f). These results strongly support both the rationale and success of our AfHPPD enzyme modifications.

To investigate the molecular basis for the enhanced HGA production, we analyzed structural differences between the E314Y/S224G variant and the WT enzyme. Docking studies showed that neither mutation site was located near the substrate (Fig. 4b). The molecular dynamics simulation results showed that the root mean square fluctuation (RMSF) of the S224G and E314Y enzymes was greater than that of the WT enzyme (Fig. 4g). This revealed an increase in flexibility near the mutant, which may be related to the increase in the enzymatic activity. In the analysis of the mechanism underlying the improvement in thermal stability, the RMSD of E314Y/S224G was greater than that of the WT enzyme at 300 K (Fig. 4h), which is contrary to the results of the in vitro stability experiments. To further investigate this, we performed molecular dynamics simulations at 350 K, and the results revealed that the RMSD of the E314Y/S224G enzyme were smaller than those of the WT enzyme (Fig. 4i). This explains why E314Y/S224G has better thermal stability than the WT enzyme does. We then calculated the 3D free energy landscape for each mutant and generated a 2-dimensional energy map based on the RMSD and radius of gyration (Rg) (Supplementary Fig. 9). Although the lowest energy point appeared in a region with a larger RMSD, its projection was concentrated, and the energy well was deeper, indicating that the mutant favoured a more stable conformational potential well. These findings align with the regional structural softening and local stability observed in the RMSF analysis (Fig. 4j). In summary, the E314Y/S224G variant exhibited both higher conformational stability and increased enzyme activity.

Construction of engineered K. phaffii to increase the synthesis of HGA through a metabolic flow push-and-pull strategy
Overall, 9 genes were modified (Fig. 5a). First, three mutations of the enzyme were combined with other recombinant genes to increase HGA production. The two mutations (i.e., DAHPS1K129R and DAHPS3E252K) were integrated into the chromosome of strain Pyo9 to obtain strains Pyo11 and Pyo12, respectively, resulting in increased HGA accumulation and higher OD400 values. Both DAHPS1K129R and DAHPS3E252K were subsequently combined with the overexpression of ARO7G141S (encoding chorismate mutase) to construct strain Pyo14, which produced 563.7 ± 2.71 mg/L HGA, with the OD400 reaching 3.53 (Fig. 5b).

In the metabolic pathway of yeast strains, an imbalance exists between the precursors PEP and E4P of the shikimate pathway, with PEP levels higher than those of E4P, which limits the synthesis of DAHP and, consequently, HGA [28]. To increase metabolic flux, genes involved in the pentose phosphate pathway were expressed. First, the transketolase gene TKIL1 was overexpressed in the genome of strain Pyo14 to generate strain Pyo15, achieving HGA production of 776.4 ± 3.53 mg/L, a 37.7 ± 1.25% increase compared with that of its parent strain Pyo14. A second copy of TKL1 was subsequently added to the INT18 locus of the genome of strain Pyo15 to obtain strain Pyo22. The results showed that strain Pyo22, carrying the TKL1 gene integrated into two different loci (INT1 and INT18), exhibited a sharp decrease in HGA production. This decrease may be due to the high expression of TKL1, a key enzyme in the pentose phosphate pathway, which caused excessive flux towards E4P and disrupted the balance with PEP production. Finally, the remaining genes of the pentose phosphate pathway were overexpressed in the genome of strain Pyo15 to generate different strains. Among these strains, Pyo21, which overexpresses the 6-phosphogluconate dehydrogenase gene ZWF1, showed a 9.7 ± 0.68% increase in HGA production, reaching 852.6 ± 7.34 mg/L (Fig. 5c). Notably, throughout the modification of the pentose phosphate pathway, the OD400 values of strains with different HGA production levels remained largely unchanged (Fig. 5c). We speculate that owing to the limited amount of dissolved oxygen in shake flasks, the efficiency of HGA self-oxidation and polymerization reached its maximum.

Furthermore, during methanol conversion, K. phaffii utilizes methanol through the XuMP pathway, providing the necessary precursors for cell growth [29]. However, formaldehyde, the first product of methanol metabolism, is toxic to cells. Therefore, enhancing the expression of enzymes involved in formaldehyde assimilation may improve methanol utilization efficiency and achieve detoxification [30]. In this study, overexpression of the phosphodihydroxyacetone synthase gene DAS2 in strain Pyo21 generated strain Pyo23, which produced 923.7 ± 10.27 mg/L HGA, representing an 8.3 ± 1.18% increase compared with that of strain Pyo21 (Fig. 5d). Although overexpression of DAS2 only slightly increased HGA production, cell growth was significantly improved (Supplementary Fig. 10), indicating that enhancing formaldehyde assimilation helps alleviate metabolic stress and promote cell growth.

The overexpression strategy of the above genes “pushed” metabolic flux towards the main branch of HGA production using methanol, while knocking out competing pathway genes could “pull” carbon flux from competing branches back to the main HGA synthesis pathway, thus further enhancing HGA production. We first knocked out the prephenate dehydratase gene pheA and the anthranilate synthase gene trpE to block the flow of intermediates towards L-phenylalanine and L-tryptophan synthesis, respectively. The results revealed that compared with the parent strain Pyo23, the strain with trpE knockout stopped growing, whereas the Pyo26 strain with pheA knockout exhibited reduced growth, as indicated by the OD600 (Supplementary Fig. 11). However, compared with that in the parent strain Pyo23, HGA production in the Pyo26 strain reached 1.27 ± 0.05 g/L, representing a 37.3% increase. Afterwards, we blocked the Ehrlich pathway to prevent tyrosol synthesis and obtained the Pyo24 and Pyo25 strains by knocking out the phenylpyruvate decarboxylase genes Aro10 and Pdc5, respectively. Compared with its parent, strain Pyo25 increased HGA production by only 6.7 ± 2.33%, whereas strain Pyo24 increased HGA production by 28.9 ± 3.45%, indicating that isoenzymes with different structures had varying effects on HGA synthesis. We subsequently constructed a double-gene knockout strain (Aro10 and PheA), Pyo27, which produced of 1.43 ± 0.038 g/L HGA (Fig. 5e).

Given the performance of strain Pyo27, we speculated that overexpression of an additional copy of the AfHPPD gene could further increase HGA production. This hypothesis was based on the observation that the upstream metabolic flux in strain Pyo27 was significantly greater than that in strain Pyo9, suggesting that an extra copy of AfHPPTD (integrated into the INT21 locus) might be needed to channel carbon flux effectively for HGA synthesis. Therefore, we overexpressed AfHPPD in the genome of strain Pyo27 to obtain strain Pyo28, which contains four copies of the AfHPPD gene. The results showed that HGA production in strain Pyo28 increased by 6.3 ± 2.15% compared with that in the parent strain Pyo27, providing strong evidence to support our hypothesis. Finally, we integrated the E314Y/S224G variant into the genome of strain Pyo27 to evaluate its potential for enhancing HGA production. The results revealed that compared with the parent strain, strain Pyo29, carrying the E314Y/S224G variant, achieved HGA production of 1.65 ± 0.04 g/L, representing a 15.4% increase (Fig. 5f).

Synthesis and manufacturing of pyomelanin from HGA oxidation polymerization via alkaline or laccase catalysis
We subsequently investigated the production of pyomelanin on the basis of HGA. Three approaches were used to achieve high yields of pyomelanin from HGA-producing yeast cells, self-oxidation of HGA exposed to air, alkali, or laccase (Fig. 6a). A comparison of the efficiency of these three methods in promoting oxidative polymerization, revealed that both alkali and laccase converted all the HGA (1.65 ± 0.04 g/L) into pyomelanin in the supernatant within 3 h, after which the OD400 of the supernatant reached 18.27. However, the process of self-oxidation polymerization of HGA exposed to air was very slow, and no decrease was detected in the production of HGA within 3 h of exposure to air (Fig. 6b). Following purification, the dried pyomelanin powders treated with alkali or laccase were designated as Pyo-NaOH and Pyo-Lac, respectively.

To analyze the functional characteristics of pyomelanin, high-density fermentation of strain Pyo29 was carried out in a 5-L fermenter using a fed-batch strategy for the preparation of pyomelanin samples. Two stages were evaluated during fermentation: cell growth and product accumulation. In the first stage, methanol induction was initiated when strain Pyo29 had grown for 36 h and the OD600 reached 160. In the second stage, methanol was added as the sole carbon source to activate transcription at the PAOXm promoter and simultaneously further increase the biomass. The fermentation was terminated after 168 h of induction, the OD600 reached 284.1 ± 1.18, and the HGA concentration increased to 50.3 ± 0.24 g/L. In addition, the OD400 reached 36.5 ± 0.37, which gradually increased throughout the entire induction process (Fig. 6c). This was consistent with the continuously darkened colour of the supernatant, which eventually turned completely black (Fig. 6d). During the entire fermentation process, a total of 340.92 ± 14.51 g of glycerol and 1.84 ± 0.17 kg of methanol were consumed. Next, all the fermentation broth was centrifuged, and either alkali or laccase was used to promote the polymerization of HGA in the supernatant. After 36 h, all the HGA was oxidized and polymerized into pyomelanin, and the OD400 reached 222.0 (Fig. 6e-f). Afterwards, we carried out acid precipitation treatment on the supernatant and obtained dry powder of pyomelanin after freeze drying (Fig. 6g). The precise dry weight of pyomelanin reached 70.5 ± 0.7 g/L. To our knowledge, this is the highest yield of pyomelanin that has been reported in the literature to date.

Structural and functional characterization of pyomelanin
Both Pyo-NaOH and Pyo-Lac were characterized, and their differences were investigated. We first conducted TLC analysis on Pyo-NaOH and Pyo-Lac dry powder prepared by the acid precipitation method, and no significant differences were observed in the chromatograms between Pyo-NaOH and Pyo-Lac before and after acid precipitation; a total of 4 bands were revealed for pyomelanin derived from the fermentation supernatant, and only two bands corresponding to pyomelanin were observed when it was dissolved in 0.1 M NaOH and subsequently precipitated with hydrochloric acid, indicating that the acidic environment may alter the polymerization form of pyomelanin (Fig. 7c). In summary, the results of the TLC analysis demonstrated no significant difference in the effects of the two methods (i.e., treatments with alkali or laccase) on the polymerization mode of pyomelanin. Elemental analysis revealed that the ratios of Pyo-Lac to Pyo-NaOH were C:H:O:N = 43.55:5.24:45.62:4.94 and C:H:O:N = 44.84:4.65:44.98:4.74, respectively. By calculating and comparing the ratios of elements, the chemical formulas of Pyo-Lac and Pyo-NaOH were determined to be the same, i.e., (C8H11O6N)n, indicating that the composition and elemental ratios of Pyo-NaOH and Pyo-Lac identical (Fig. 7b). However, that HGA does not contain nitrogen in its structure while pyomelanin does is confusing. Therefore, the nitrogen detected in the dry weight suggested that pyomelanin was possibly contaminated with secreted proteins. However, owing to the lack of a pyomelanin standard, its purity could not be determined. Interestingly, Lorquin et al. conducted an elemental analysis on the pyomelanin obtained by laccase-catalysed HGA-lactone and pyomelanin synthesized by Halomonas titanicae. The results also demonstrated the presence of nitrogen in pyomelanin, possibly because of the abundance of laccase extract or the components of the H. titanicae culture medium [22]. To further clarify this issue, we conducted oxidative polymerization of HGA standards using laccase and alkali. Two types of dry pyomelanin powders were subsequently prepared for elemental analysis, and the results revealed that the pyomelanin obtained after laccase treatment contained nitrogen, whereas nitrogen was not detected in the pyomelanin obtained after alkali treatment. These findings indicated that pyomelanin does not contain nitrogen in its structure and that the nitrogen detected in this study was inevitably introduced during yeast culture medium or laccase treatment.

Next, FTIR analysis of both Pyo-NaOH and Pyo-Lac revealed similar characteristic peaks in both samples. The absorption peaks at 3177 cm−1 and 3049 cm−1 were attributed to the stretching vibrations of –OH and –CH, respectively; the absorption peaks at 1713 cm−1 and 1419 cm−1 were attributed to the stretching vibrations of C=O and C=C, respectively; and the absorption peaks at 1066 cm−1 was attributed to the stretching vibration of C–O (Fig. 7a). To further analyse the chemical structural characteristics of pyomelanin, 13C CP-MAS solid-state nuclear magnetic resonance (NMR) analysis was performed on Pyo-NaOH and Pyo-Lac. The results revealed that the overall profile and main signal distribution of Pyo-NaOH and Pyo-Lac were highly similar, which is consistent with the spectral characteristics of amorphous polymer melanin substances (Supplementary Fig. 12) and highly consistent with the spectra of pyomelanin reported in the literature [22]. At δ 170–180 ppm, a clear wide blunt signal was observed in both samples, which can be attributed to the unpotonated carbonyl carbon (–COOH) in the carboxyl group, indicating that a certain proportion of the carboxyl structural units were still retained in the polymer structure. The aromatic carbon region (δ 110–160 ppm) was the most concentrated part of the spectrum, and the overall signal of Pyo-Lac in the aromatic region was greater than that of Pyo-NaOH. A strong broad peak appeared at approximately δ 120–130 ppm, corresponding to the C–H and C=C carbon signals in the aromatic ring, whereas the secondary signal in the δ 145–150 ppm region was attributed to the aromatic carbon connected by phenolic hydroxyl groups. This spectral characteristic indicates that the main chain skeleton of the sample is composed mainly of aromatic structural units. Notably, no clear independent signal peak was observed in the δ 160–165 ppm region, indicating that the samples do not contain aromatic ether bond (Car–O–Car) structures and that the polymerization process may be achieved mainly through aromatic carbon‒carbon bond (Car–CAr) coupling, which is highly consistent with the reported mechanism of pyomelanin polymerization in the literature. At δ 30–80 ppm, weak but distinguishable signals were detected in both samples, with peaks near δ 30–35 ppm attributed to aliphatic–CH2–carbon, and at δ 50–80 ppm, broad peak signals were observed in Pyo-Lac and Pyo-NaOH, which are typically attributed to aliphatic carbon (C–O type carbon) connected to oxygen atoms. In summary, Pyo-Lac and Pyo-NaOH both exhibit polymerization characteristics of melanin with aromatic carbon–carbon bond connections as the main and highly disordered structures according to solid-state nuclear magnetic resonance spectra, and their overall structures are highly similar. These results demonstrated that the pyomelanin produced by K. phaffii shares similar characteristics with pyomelanin derived from other sources [22, 31]. Finally, we observed both Pyo-NaOH and Pyo-Lac using SEM, which revealed similar morphological features. Surprisingly, variations in particle size and distribution uniformity were detected between Pyo-NaOH and Pyo-lac; i.e., compared with Pyo-Lac, Pyo-NaOH showed a more uniform and compact distribution, suggesting differences in both the properties and application prospects of these products (Fig. 7d).

Afterwards, the practical application value of pyomelanin was examined to determine the significance of its high yield. Both types of pyomelanin (i.e., Pyo-NaOH and Pyo-Lac) exhibited significant antioxidant capacity in a concentration-dependent manner, i.e., a higher concentration of pyomelanin resulted in a stronger ability to scavenge free radicals. However, at the same concentration, compared with Pyo-Lac, Pyo-NaOH had a 2-fold stronger ability to scavenge free radicals, i.e., the IC50 values of Pyo-NaOH and Pyo-Lac were approximately 80 μg/mL and 160 μg/mL, respectively (Fig. 7e). Pyomelanin was subsequently added to HaCaT cells exposed to UV irradiation. Regardless of the type of pyomelanin added, the relative viability of HaCaT cells increased, with cell viability increasing progressively as the pyomelanin concentration increased. No significant difference in the antiradiation effect was observed between Pyo-NaOH and Pyo-Lac (Fig. 7f). Notably, pyomelanin from bacteria has been widely reported. In particular, pyomelanin from Pseudomonas aeruginosa ΔhmgA has been well studied and characterized by several research groups [32-35]. Therefore, comparing the antioxidant and antiradiation properties of pyomelanin produced by K. phaffii with those from bacterial sources is necessary. Interestingly, Diaz Appella et al. demonstrated that the composition and physiological role of pyomelanin produced by P. aeruginosa from diverse lifestyles also differ [33]. A similar study also demonstrated significant differences in functional applications between water-soluble and water-insoluble pyomelanin from the same source [34]. These findings are similar to our results. Furthermore, almost all the microbial sources of pyomelanin exhibit excellent antioxidant and radiation resistance properties. However, the diversity of experimental cell models and evaluation methods make comparisons difficult. In summary, this study demonstrated that compared with pyomelanin produced by bacterial producers, the pyomelanin produced by K. phaffii has similar antioxidant and radiation resistance, which can supplement the biotechnology applications of pyomelanin.

In addition, when pyomelanin was used as a precursor, a simple high-temperature carbonization process was employed to prepare hard carbon for use as a negative electrode in sodium-ion batteries. Notably, the sodium storage performance of Pyo-NaOH was superior to that of Pyo-Lac, which was probably attributed to the tighter arrangement inside Pyo-NaOH, as indicated by SEM observations, facilitating the storage of sodium ions. The sodium storage performance of Pyo-NaOH is as follows: the preliminary thermogravimetric results indicated that a small mass loss of pyomelanin occurred at temperatures above 100 °C during the carbonization process, which was due to the detachment of adsorbed crystal water in the materials. When the temperature rose above 250 °C, the pyomelanin precursor began to undergo thermal decomposition and reconstruction, resulting in significant mass loss. The material then stabilized above 480 °C, yielding a final carbon content of 34.1% (Fig. 7g). The hard carbon material obtained after carbonization of pyomelanin exhibited a characteristic curve of amorphous carbon, corresponding to the (002) crystal plane characteristic peak near 23° and the (101) crystal plane characteristic peak near 44° (Fig. 7h). Finally, the electrochemical performance of the sodium-ion batteries was tested. At 0.1 C, the pyomelanin-derived hard carbon exhibited a reversible capacity of 266.51 mAh/g and an initial coulombic efficiency of 75.04%, demonstrating high cycling stability (Fig. 7i). In summary, the reversible capacities at 0.1 C, 0.2 C, 0.5 C, 1 C, and 2 C were 266.51 mAh/g, 252.94 mAh/g, 195.61 mAh/g, 117.6 mAh/g, and 80.74 mAh/g, respectively. Upon returning from 2 C to 0.1 C, the material maintained high-capacity retention (Fig. 7j). The results revealed that pyomelanin has excellent performance in the field of energy storage material preparation. Although the performance of pyomelanin hard carbon as a negative electrode for sodium-ion batteries is similar to that of some materials reported in the literature, these inexpensive biomass hard carbon materials (industrial pitch or rice husk) seem to have lower costs [36,37]. In particular, Tang et al. used waste wood as a hard carbon negative electrode and reported a high reversible capacity of 430 mAh/g [38]. Compared with these methods, the advantage of this study lies in the direct carbonization of pyomelanin to prepare hard carbon negative electrode materials, which is a simpler preparation process. Of course, applying pyomelanin to sodium-ion batteries is meaningful, and in the future, the preparation process of pyomelanin can be further regulated to obtain structurally unique precursors to enhance the performance of hard carbon negative electrodes in sodium-ion batteries.

Methods
Microbial strains and plasmids
The microbial plasmids and strains used in this study are listed in Supplementary Data 1 and 2, respectively, and the primer sequences are provided in Supplementary Data 3. K.phaffii GS115 (Invitrogen, Carlsbad, CA, USA) was used as the host strain, while Escherichia coli TOP10F’ (Invitrogen, Carlsbad, CA, USA) was used for plasmid construction and amplification. E. coli BL21(DE3) (Sangon Biotech, Shanghai, China) was used for protein expression. Considering the importance of genetic stability, the pZACH plasmid was utilized for gene overexpression integration. The Cre/loxP ZeoR marker recycling method [24] ensured the stability of every integration. The pENO1-Cas9 plasmid was used for gene knockout to avoid off-target effects. The plasmid pET-28a was used for protein expression and purification. The genes encoding heterologous tyrosine transaminase (TYRB) and hydroxyphenylpyruvate dioxygenase (HPPD) were codon-optimized and synthesized by Generay (Shanghai, China). The sequences of the synthesized HPPD gene and TYRB gene are listed in Supplementary Data 4. The sequences of the plasmids and integration sites used in this study are listed in Supplementary Data 5.

Culture media and conditions
All E. coli strains were cultured in lysogeny broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37 °C. Recombinant yeast strains were grown in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% glucose, and 2% peptone), buffered glycerol-complex medium (BMGY) [1% yeast extract, 1.34% YNB, 2% peptone, 0.1 mM potassium phosphate buffer (pH 6.0), and 1% glycerol], or buffered methanol-complex medium (BMMY) medium [1% yeast extract, 2% peptone, 1.34% YNB, 0.1 mM potassium phosphate buffer (pH 6.0), and 1% methanol] at 30 °C. Appropriate amounts of zeocin were added to the culture as needed. Standard HGA was purchased from Aladdin (Shanghai, China).

Cultivation of engineered yeast strains in shake flasks
For shake-flask fermentation of HGA production, single colonies of recombinant yeast were inoculated into 10 mL of BMGY medium in a 50 mL Erlenmeyer flask and cultured at 30 °C with shaking at 0.70 xg for 12–16 h. The seed culture was then transferred to 25 mL of BMMY medium with an initial OD600 of 1.0 and incubated under the same conditions for 120 h. Fresh methanol (1%, v/v) was added every 24 h, and 0.5 mM copper ions were added to the BMMY medium at the start of fermentation.

Quantification of homogentisic acid
HGA standard was purchased from Aladdin (Shanghai, China). The culture supernatant (as described in Cultivation of engineered yeast strains in shake flasks) was collected, centrifuged at 6000g for 5 min, and analysed by HPLC using a Thermo Scientific Syncronis C18 column (5 μm, 250 mm × 4.6 mm). The column temperature was maintained at 35 °C. The mobile phase consisted of phase A (water with 0.1% trifluoroacetic acid) and phase B (methanol with 0.1% trifluoroacetic acid) at a ratio of 7:3 (v/v). The flow rate was set to 0.7 mL/min, and HGA was detected at a wavelength of 292 nm with a retention time of 6.5 min.

Measurement of the absorbance value of pyomelanin
Owing to the lack of a pyomelanin standard, a semiquantitative analysis of pyomelanin production was performed using the absorbance-based method.

Construction and screening of DAHP synthase variant libraries
A methanol-induced promoter, Paox1-controlled DAHPS1, and DAHPS3 overexpression vector, pPICZA-Paox1-INT1, were constructed. The genes DAHPS1 and DAHPS3 were mutated via error-prone PCR on the basis of the template plasmid. The mutation conditions included low fidelity 2× Rapid Taq Master Mix (Vazyme, Nanjing, China) and 0.8 mM manganese ions. The amplified DNA product was transformed into E. coli TOP10F’ competent cells, and the E. coli transformants were cultivated in 5 mL of LB medium supplemented with zeocin and grown overnight at 37 °C and 0.45 xg for extraction of the mutant plasmids. The mutant plasmid libraries for DAHPS1 and DAHPS3 were subsequently transformed into electrocompetent K. phaffii Pyo9 cells. Colonies were first cultured in 24-deep-well plates containing 1000 μL of BMGY medium per well and incubated overnight at 30 °C with shaking at 7.16 xg. Afterwards, 200 μL of seed culture was transferred to a fresh 24-deep-well plate containing 1000 μL of BMMY medium per well and fermented for 36 h, with 1% (v/v) methanol added at the start and again after 24 h. Following centrifugation at 402.5 xg for 5 min, 200 μL of the supernatant was transferred to a 96-well microplate, and the OD400 was measured using a Biotek Synergy microplate reader (Agilent, Palo Alto, CA, USA).

Semirational design of HPPD variants and molecular dynamics simulations
Using Alcaligenes faecalis HPPD (AfHPPD) as a template, the BLAST protein alignment tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to retrieve 1000 homologous sequences, of which 974 sequences (75–95% similarity) were selected for further analysis. Multiple sequence alignment (MSA) was performed with MUSCLE (v5.2; Linux64). Mutational effects were assessed using FoldX (https://foldxsuite.crg.eu/), which involves the use of alanine scanning to evaluate residue-specific stability contributions and positional scanning to predict stabilizing mutations. In addition, PROSS (https://pross.weizmann.ac.il/step/pross-terms/) was employed to increase AfHPPD stability through evolutionary-guided sequence redesign, combining phylogenetic conservation with Rosetta-based energy minimization.

For AfHPPD enzyme studies, AlphaFold2 and Modeller homology modelling (PDB: 1CJX) were used to generate the structural model, which was subsequently refined through docking and molecular dynamics simulation (MDS). The structural model of the HPPD–4-HPPA complex was constructed using AutoDockTools 1.5.7, and SAVES v6.1 (https://saves.mbi.ucla.edu/) was used to generate a Ramachandran plot to evaluate structural quality. Default parameters were applied for all modelling procedures. The Binding modes selected for further analysis were based on the reference crystal structure (PDB: 5XGK). MDS was performed using CHARMM-GUI with the CHARMM36 force field, with the protein placed in a TIP3P water box supplemented with NaCl (in addition to counterions for charge neutralization). The simulations were run for 100 ns with a 2 fs time step. For the DAHPS variant studies, homology modelling was performed using Modeller with Saccharomyces cerevisiae ARO3 (PDB: 7YKC) as the template, and the docking and MDS procedures were conducted as described for AfHPPD.

In vitro AfHPPD enzyme assays
Both the wild-type (WT) and variant genes were cloned and inserted into the pET-28a vector and transformed into E. coli BL21(DE3). A single E. coli colony was inoculated into LB medium and grown overnight, followed by induction of protein expression with 0.5 mM IPTG for 18 h at 16 °C. Cells were harvested and resuspended in lysis buffer (50 mM HEPES, 200 mM NaCl, pH 8.0). Proteins in the clarified supernatant were purified using Ni+-affinity chromatography on an ÄKTA protein purification system, and protein concentrations were determined with a Bradford assay kit (Sangon Biotech, Shanghai, China).

An AfHPPD enzyme activity assay was conducted by measuring HGA production. The reaction mixture (500 μL) contained 50 mM phosphate buffer (pH 7.4), 1 mM CuSO4, 4 mM ascorbic acid, 1 mM 4-hydroxyphenylpyruvic acid (4-HPP), and 50 μg of AfHPPD enzyme. After mixing, the reaction was incubated at 30 °C for 10 min and then quenched with 100 μL of 10% trifluoroacetic acid. HGA levels were quantified by HPLC analysis.

Pyomelanin purification and characterization
After 5 d of fermentation at 30 °C, the culture broth was centrifuged at 1118 xg for 10 min, and the supernatant was collected. To promote oxidative polymerization, either 1 M NaOH or laccase (Sigma‒Aldrich SAE0050) was added until HPLC analysis confirmed the complete depletion of HGA, indicating full conversion to pyomelanin. The supernatant was then acidified to a pH of 2 with 1 M HCl and incubated at room temperature for 24 h. The resulting suspension was centrifuged at 1118 xg for 15 min, and the brown pellet was collected, washed twice with distilled water, and freeze-dried overnight to obtain pyomelanin powder.

FTIR analysis was performed to identify chemical bonds and functional groups and to clarify the composition of the unknown mixture. The dry pyomelanin pellet was scanned in the range of 500–4000 cm⁻1 using a Nicolet iS50 FTIR spectrometer, with a precision of less than 0.01 cm⁻1.

The CP-MAS method for 13C CP-MAS solid-state nuclear magnetic resonance (NMR) analysis was used. The instrument used was a JNM-ECZL600G from Japan Electronics, with a 1H resonance frequency of 599.57 MHz, a 3.2 mm solid rotor sample, and an HX dual resonance probe.

Elemental analysis was performed to classify different types of melanin on the basis of their composition, properties, and elemental ratios. Dry pyomelanin powder was analysed using a CHNS elemental analyser (Elementar UNICUBE, Germany). The samples were digested at 850–1150 °C, and the resulting gases were separated through a gas chromatography column with helium as the carrier gas for component detection.

TLC of pyomelanin was performed using a silica gel plate (Sigma‒Aldrich, F254, product number 15554) with dimensions of 20 cm × 20 cm. The developing solvent consisted of petroleum ether, ethyl acetate, absolute ethanol, ammonia water, and water at a ratio of 1:4:6:1:5 (v/v). A total of 15 mL of the developing solvent was added to the chromatography chamber and allowed to presaturate for 2 h. The purified pyomelanin sample was dissolved in 0.1 M NaOH, and a capillary tube was used to spot the sample onto premarked positions on the silica gel plate. After air-drying, the plate was placed in the chromatography chamber. Once the solvent front had risen sufficiently, the plate was removed and dried in an oven.

SEM images of the dry pyomelanin powder were captured using a Zeiss Gemini Sigma 300 FEG-SEM (Jena, Germany) at an appropriate working distance and an accelerating voltage (EHT) of 5.0 kV.

Fed-batch fermentation of the pyomelanin-producing strain Pyo29
High-density fermentation was performed using the highest HGA-producing strain, Pyo29, in a 5-L fermenter (T&J Bio-engineering, Shanghai, China). A single colony was first inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD medium and cultured with shaking at 0.70 xg and 30 °C for 24 h. Afterwards, approximately 4% of the seed culture (OD600=1.0) was transferred into a 500 mL Erlenmeyer flask containing 100 mL of YPD medium and cultured under the same conditions for approximately 18 h. Finally, 8% of this culture was inoculated into basal salt medium (BSM) in a 5-L fermenter for fed-batch fermentation at 30 °C.

The BSM contained 4% glycerol, 1.147% MgSO4, 0.413% KOH, 0.093% CaSO4·2H2O, 1.82% K2SO4, 2.67% H3PO4, and 0.435% PTM1. The PTM1 solution consisted of 0.05% CoCl2·6H2O, 0.6% CuSO4·5H2O, 6.5% FeSO4·7H2O, 0.002% H3BO3, 0.008% NaI, 0.5% H2SO4, 0.02% Na2MoO4·2H2O, 2% ZnCl2, 0.3% MnSO4·H2O, and 0.02% biotin. A 50% (w/v) glycerol solution containing 1.2% (v/v) PTM1 and a methanol solution containing 1.2% (v/v) PTM1 were used as feed solutions according to the reference protocol in the Pichia Fermentation Process Guidelines (Invitrogen, Carlsbad, CA, USA).

The fermentation parameters were as follows: a temperature of 30 °C, a pH of 5.5 (adjusted with 25% ammonia solution, v/v), and an air flow rate of 10 L/min. Dissolved oxygen (DO) levels were maintained at approximately 20–30%.

The fermentation process consisted of three phases. The batch growth phase (Phase I) was conducted in BSM to support initial cell growth and lasted approximately 24 h. After the glycerol was exhausted, the fed-batch phase (Phase II) was initiated by feeding limited amounts of glycerol at a speed of 15−30 mL/h to promote further cell growth. When the OD600 reached approximately 200, glycerol feeding was stopped to allow the cells to consume the remaining glycerol. Methanol was then introduced in the induction phase (Phase III). During this phase, the dissolved oxygen (DO) level was maintained at approximately 20–30%, the temperature was reduced to 25 °C, and the pH was adjusted to 6.0. The stirring speed was maintained as described above. Methanol was first added at a speed of 5 mL/h, with an increase of 1–2 mL/h every 5–8 h until the flow rate reached 20–25 mL/h. If no methanol accumulated, this flow rate was used throughout the entire remaining fermentation process. However, if necessary, this flow rate was adjusted appropriately according to the dissolved oxygen level. Next, 10 mL of 200 mM Cu2+ was supplemented every 18 h during the induction process. HGA productivity and cell density were monitored during the 5-day induction period. After fermentation, appropriate volumes of the culture were collected for centrifugation, followed by extraction and purification of pyomelanin. The purified product was freeze-dried to obtain a pyomelanin powder, and its dry weight was determined.

Analysis of the antioxidant performance of pyomelanin
The antioxidant capacity of the samples was evaluated on the basis of their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), a deep purple free radical whose maximum absorbance occurs at 517 nm. When antioxidants react with DPPH, DPPH is reduced to 2,2-diphenyl-1-hydrazine (DPPH-H), causing a colour change from dark purple to light yellow. DPPH and pyomelanin dry powder were dissolved in ethanol and DMSO, respectively. The final reaction mixture contained 900 μL of 0.2 g/L DPPH solution and 100 μL of pyomelanin at varying concentrations (50, 100, 150 and 200 μg/mL), and was incubated in the dark for 30 min. Each measurement was performed in triplicate. After incubation, the absorbance of the mixture was measured at 517 nm, and the DPPH radical scavenging activity (Zeta, %) was calculated as follows: radical scavenging activity (Zeta,%) = (A0 – AS – AP)/A0, where A0 is the absorbance of a mixture of 100 μL of DMSO and 900 μL of DPPH solution, AS is the absorbance of a mixture of 900 μL of DPPH solution and 100 μL of sample after 30 min of reaction, and AP is the absorbance of pyomelanin at the same concentration measured at 517 nm.

Analysis of the radiation resistance performance of pyomelanin
Human immortalized epidermal cells (HaCaT) were used to evaluate the radiation resistance of pyomelanin, with three experimental groups treated with different concentrations of pyomelanin. The control group was divided into two subgroups: (1) a light control, in which cells were exposed to ultraviolet (UV) irradiation for 3 h, and (2) a dark control, in which cells were kept in the dark for 3 h under the same conditions. Frozen-stored cells were thawed, passaged twice for stabilization, and seeded into 96-well plates, followed by overnight incubation in a CO2 incubator. After incubation, the culture medium was removed from each well, and different concentrations (0, 100, 500 and 1000 μg/mL) of pyomelanin solutions dissolved in DMSO were added. Cells were exposed to UVB irradiation at an intensity of 142 mJ/cm2 for 3 h, while the control group was maintained in a dark environment under the same conditions. Following irradiation, cell viability was assessed using the MTT assay, and the absorbance values for each well were measured with a microplate reader.

Analysis of the sodium storage performance of pyomelanin
The dry powder of pyomelanin obtained as described above was used as a precursor for hard carbon production. Carbonization was performed in a tube furnace, first at 400 °C for 3 h, followed by heating to 1300 °C for 2 h under an argon atmosphere at a heating rate of 3 °C/min. The resulting carbonized product was then ground into a fine powder.

The synthesized hard carbon was mixed with CMC and C65 in deionized water at a weight ratio of 90:5:5. The resulting slurry was uniformly coated onto copper foil and dried under vacuum at 100 °C for 12 h. The mass loading of the active materials ranged from approximately 1.5–2.0 mg/cm2.

Electrochemical measurements were performed using CR2025-type coin cells consisting of a hard carbon electrode, a separator (Whatman GF/F, DoDoChem), an electrolyte solution of 1.5 M NaPF₆ in DMC:EC:EMC (2:1:2, v/v/v; DoDoChem), and sodium metal (MTI Corporation) as the counter electrode. The coin cells were assembled in an argon-filled glove box. Galvanostatic charge–discharge cycling tests were conducted in the voltage range of 0.001–2.0 V using a LAND CT2001A system at current rates of 30 mA/g (0.1 C) and 150 mA/g (0.5 C).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors describe a strategy to enhance the biosynthetic yield of pyomelanin—a key raw material used in pharmaceuticals, cosmetics, bioremediation, and energy applications. To improve flux through the pathway, they engineered DAHPS and HPPD, two rate-limiting enzymes, thereby boosting overall production. The final strain achieved the highest pyomelanin titer reported to date. Collectively, the study presents a compelling, stepwise optimization of the production process. The data are robust and the conclusions well supported. Nevertheless, the manuscript still has several shortcomings, detailed below.\nLines 168-171: The manuscript states that the AfHPPD PDB structure from YASARA was used for structural analysis, but this is ambiguous. No experimental structure of AfHPPD has been deposited, so the authors presumably built a homology model. Clarify whether YASARA was used only for visualisation or also for modelling. If the latter, explain why an additional model was generated with MODELLER and AlphaFold2 (lines 417–418). For the AlphaFold2 model, report standard quality metrics (pLDDT, pTM, RMSD to template, etc.) instead of only the Ramachandran plot.\nLines 417-418: Crystal structure of PfHPPD was used as the template for modeling, yet the manuscript offers no justification for this choice. A sequence-alignment comparison of AfHPPD against all HPPD with available crystal structures should be presented to demonstrate that PfHPPD is the most appropriate template (highest identity, best coverage, conserved active-site geometry). Likewise, the final downstream analyses must specify which AfHPPD model (YASARA, MODELLER, or AlphaFold2) was adopted and provide the selection rationale.\nLines 462-464: The authors attribute the improved HGA production to a 0.03-nm decrease in the F338–4-HPPA distance in the variant. This displacement is within the coordinate error of a homology model and is indistinguishable from thermal noise; it is therefore insufficient grounds for claiming enhanced catalysis. In addition, the manuscript states that S224G acts as a “structural bridge,” but never defines what is being bridged or how glycine—lacking a side chain—could fulfill such a role. Provide (i) ensemble docking or MD-derived distance distributions to show the 0.03-nm shift is statistically significant, and (ii) a clear mechanistic explanation of the proposed S224G bridging function, supported by hydrogen-bond maps, etc.\nLines 467-472: The authors assert that a higher RMSD does not imply greater instability for the variant, yet lines 398–401 equate lower RMSD with enhanced structural stability. This is internally inconsistent. Clarify whether RMSD is being used as a proxy for stability in this study; if not, remove the stability claim. If it is, provide evidence—e.g., radius-of-gyration trends, hydrogen-bond counts, or free-energy calculations—to demonstrate that the variant’s increased RMSD does not compromise thermostability.\nFigure 2f: Did the authors test the effect of Mn2+? It would be better if the result of Mn2+ was added.\nOther minor issues:\n1. ‘de novo’ should be italic. Check this thoroughly.\n2. Line27, further synthesis of pyomelanin.\n3. Line81: constructed\n4. The legend of supplementary fig.6, the nine mutated enzymes were not from Komagataella phaffii.", "reviewer_2": "What are the noteworthy results?\nThis study engineered Komagataella phaffii for de novo pyomelanin production, achieving a titer of 70.5 g/L. Key advances include modular pathway optimization, establishment of a color-based screening system, and use of directed evolution and semi-rational design for enzyme improvement.\nWill the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nThis paper reports a titer of 70.5 g/L, representing a significant improvement in pyomelanin production than previously published values. Larroude et al. (doi: 10.3390/microorganisms9040838) reported a pyomelanin titer of 4.5 g/L, Lorquin et al. (https://doi.org/10.1038/s41598-021-87328-2) reported 0.55 g of pyomelanin per liter of medium, and Koch et al. (https://doi.org/10.3389/fmicb.2023.1233740) reported 1.48 g of pyomelanin per liter of medium. The later paper also comparing titer/yield of pyomelanin production in different organisms by different approaches. Thus, this work demonstrates a significant improvement in terms of pyomelanin titers. However, important information on the process are missing to obtain a complete picture of the fermentation process. First of all the description of the fermentation L254 ff. (methods) and L567 ff. (results) is not well described. How was glycerol and methanol fed and which feeding profile was used? How much methanol and glycerol was actually consumed?\nDoes the work support the conclusions and claims, or is additional evidence needed?\nAdditional evidence is needed.\nThe title states “de novo biosynthesis of pyomelanin from methanol,” but the engineered K. phaffii strains actually produce homogentisic acid (HGA) as the final metabolic product. Pyomelanin formation occurs non-enzymatically via oxidation/polymerization of HGA or enzymatically after laccase addition, rather than through an intracellular biosynthetic pathway.\nThe reported value of 70.5 g/L refers to the titer, not the yield (lines 33 and 95). Titer represents the final concentration of product in the culture medium (often as grams per liter), while yield describes the efficiency of conversion in reference to a given substrate (here: yield on methanol or total carbon source would be appropriate). Additional data on carbon input, substrate consumption, and productivity are needed to support the claims. How reproducible is this fermentation in the 5L setup?\nPurity of the reported pyomelanin is also not certain. Should pyomelanin contain N in its structure? Nitrogen detected in the dry weight suggests possible contamination with secreted proteins, meaning not all measured mass is pure pigment.\nAre there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nYes, this paper needs revision. It lacks sufficient quantitative and methodological detail to fully support its conclusions.\nThe authors should clarify how 70 g/L was achieved, meaning what carbon sources were used, feeding rates of methanol and glycerol, and the carbon conversion ratio.\nProductivity (g L⁻¹ h⁻¹) and specific yield (for example grams of pyomelanin per grams of cell dry weight) are missing and should be compared with literature values.\nSome results (for example the reported 6.3 % increase in HGA, lines 548 and 549) don’t have evidence of statistical significance.\nOD₄₀₀ readings are presented without justification that the signal detected is only pyomelanin.\nFigure 2 and related text imply inconsistent effects of the addition of copper ions, which may both enhance HPPD activity and promote non-enzymatic oxidation of HGA. This needs clarification.\nIt would be interesting to see the comparison of the native HPPD enzyme from Komagataella phaffii to the engineered one from this paper.\nThe English in general needs more revision to be clearer and gramatically correct.\nGiven these issues, the work needs significant revision before publication.\nIs the methodology sound? Does the work meet the expected standards in your field?\nNot fully. The experimental concept is interesting and the modular metabolic engineering strategy is promising, but methodological details are missing.\nThere is no clear genetic characterization of strains in this work. Number of integrated copies of some genes is mentioned (lines 338, 499, 501, 547, 700), but there is no information on the way that was achieved.\nNo plasmid sequences are provided in the supplementary material.\nThe lack of OD measurements during inoculation (lines 238 and 240), the vague description of adding copper “as needed” (lines 262 and 263) and “pyomelanin solutions of varying concentrations” (lines 291 and 292) make replication and data interpretation difficult.\nIs there enough detail provided in the methods for the work to be reproduced?\nNo. A lot of experimental details are missing or too vague to reproduce, as mentioned above.", "reviewer_4": "The manuscript “De novo biosynthesis of pyomelanin from methanol with engineered Komagataella phaffii and its characterizations” presents several interesting aspects, such as the production of pyomelanin in a yeast strain, the directed evolution of enzymes to improve biosynthetic processes, the metabolic push-pull strategy and manipulation of K. phaffii for pyomelanin production.\nHowever, we identified several major issues that need to be addressed:\nThe main observation that I have is that the pigment being produced, pyomelanin, is widely distributed among bacterial species, like those of the genus Pseudomonas, among others. It is not clear what the advantage would be of producing pyomelanin in a eukaryotic organism. On one hand, achieving high pigment production requires a rather complex fed-batch fermentation scheme, which is not inexpensive. Although methanol is a low-cost substrate, bacteria can utilize waste compounds from agro-industrial or fuel industries, for example. If the advantage lies in cost, this point has not been thoroughly discussed. Moreover, the fermentation lasts five days (example in line 205), which is quite long, and no comparison is made to yields obtained in other studies using fast-growing bacterial producers. In the Results section (line 320), no measurement uncertainty or deviation is indicated, which prevents assessing reproducibility. Considering the importance of genetic stability in organisms intended for biotechnological use, it would be highly relevant to discuss the stability of this yeast strain, which carries multiple genetic modifications, even the use of Crispr-Cas9 can lead to off-targets editions that should be analyzed. More importantly, while the reported yield of 1.65 g/L is high, productivity (yield per unit time) is not expressed, so it is unclear whether this microorganism is actually a better producer than bacterial systems, either natural producers or recombinant, fast growing strains. For example, line 573 states that fermentation was completed after 168 hours (seven days), yet this aspect is not discussed at all.\nRegarding the chemical characterization, we found significant issues:\nFirst, in the M-M section (line 210), the purification method described appears to yield poorly purified material, potentially containing impurities such as proteins and polysaccharides. These contaminants could have influenced some of the reported scavenging results. Moreover, the reported production values might be highly overestimated due to this purification protocol.\nOn the other hand, all experiments should have included, at least as a control, the best-characterized pyomelanin from Pseudomonas aeruginosa ΔhmgA. While it is true that commercial melanin from Sigma (derived from alkaline treatment of tyrosine) is available, the authors could have used P. aeruginosa ΔhmgA pyomelanin as reference, which has been well characterized by several research groups over time.\nFurthermore, in the M-M (lines 269 and 290), in the antioxidant and cell assays, it is not stated how the pyomelanin solutions were prepared, given that the natural pyomelanin is insoluble in water, buffer, or ethanol therefore is crucial to understand how the experiments were made. Additionally, there is a lack of controls with pyomelanins from bacterial. Antioxidant, cosmetic, and UV-protective properties of pyomelanin have already been extensively described in the literature, so many of the authors’ statements overlook the substantial existing body of work (lines 623–627). The characterization relies mainly on IR spectroscopy, which provides limited structural information for polymers as complex and heterogeneous as melanins. Even for pyomelanin, key structural differences have been revealed primarily by NMR analysis. Therefore, the claims made in lines 607–609 cannot supported by IR data alone.\nIn line 629, the authors discuss the use of pyomelanin for electrode fabrication in sodium-ion batteries. However, it is unclear what the advantage would be of using such an expensive compound for this purpose. Without a clear rationale, this part of the study lacks relevance. There are numerous inexpensive precursors for synthesizing hard carbon anodes for SIBs, including agricultural biomass/waste (lignin included), industrial pitch, and waste plastics (e.g., PET), all of which have demonstrated competitive performance (DOI: 10.1016/j.jechem.2018.01.025; DOI: 10.1038/s41467-023-39637-5; DOI: 10.1039/C5TA08601A).\nFinally, the Discussion contains numerous claims about the advantages and novelty of the study while overlooking prior research that had already addressed many of these points. For example, the comparison between laccase-synthesized and chemically synthesized pyomelanins (under alkaline conditions) has already been thoroughly studied, including NMR analysis, as reported in Lorquin et al., Scientific Reports (2021): 10.1038/s41598-021-87328-2. Given that this work is even cited by the authors themselves in the present manuscript, the claimed novelty is practically null and contradictory in this regard. Lorquin’s study provides a complete structural analysis with NMR data.\nLastly, the statement that this melanin could serve as a reference pyomelanin standard is not well justified, since naturally producing bacterial strains already exist and are routinely used as reference sources in various studies.\nSome important publications:\nRodríguez-Rojas A, Mena A, Martín S, Borrell N, Oliver A, Blázquez J. Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection. Microbiology (Reading). 2009 Apr;155(Pt 4):1050-1057. doi: 10.1099/mic.0.024745-0. PMID: 19332807.\nLorquin, F., Ziarelli, F., Amouric, A. et al. Production and properties of non-cytotoxic pyomelanin by laccase and comparison to bacterial and synthetic pigments. Sci Rep 11, 8538 (2021). https://doi.org/10.1038/s41598-021-87328-2\nLorquin F, Piccerelle P, Orneto C, Robin M, Lorquin J. New insights and advances on pyomelanin production: from microbial synthesis to applications. J Ind Microbiol Biotechnol. 2022 Jul 30;49(4):kuac013. doi: 10.1093/jimb/kuac013. PMID: 35482661; PMCID: PMC9338888.\nDiaz Appella MN, Kolender A, Oppezzo OJ, López NI, Tribelli PM. The structural complexity of pyomelanin impacts UV shielding in Pseudomonas species with different lifestyles. FEBS Lett. 2024 Nov;598(21):2702-2716. doi: 10.1002/1873-3468.15000. Epub 2024 Aug 16. PMID: 39152523.\nUrbaniak MM, Gazińska M, Rudnicka K, Płociński P, Nowak M, Chmiela M. In Vitro and In Vivo Biocompatibility of Natural and Synthetic Pseudomonas aeruginosa Pyomelanin for Potential Biomedical Applications. Int J Mol Sci. 2023 Apr 25;24(9):7846. doi: 10.3390/ijms24097846. PMID: 37175552; PMCID: PMC10178424.\nBen Tahar I, Kus-Liśkiewicz M, Lara Y, Javaux E, Fickers P. Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica. Biotechnol Prog. 2020 Mar;36(2):e2912. doi: 10.1002/btpr.2912. Epub 2019 Nov 4. PMID: 31525285.\nAl Khatib M, Costa J, Spinelli D, Capecchi E, Saladino R, Baratto MC, Pogni R. Homogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural Characterization and Antioxidant Activity. Int J Mol Sci. 2021 Feb 9;22(4):1739. doi: 10.3390/ijms22041739. PMID: 33572316; PMCID: PMC7916096.\nBayram S. Production, purification, and characterization of Streptomyces sp. strain MPPS2 extracellular pyomelanin pigment. Arch Microbiol. 2021 Sep;203(7):4419-4426. doi: 10.1007/s00203-021-02437-w. Epub 2021 Jun 14. PMID: 34128104.\nUrbaniak MM, Gazińska M, Rudnicka K, Płociński P, Nowak M, Chmiela M. In Vitro and In Vivo Biocompatibility of Natural and Synthetic Pseudomonas aeruginosa Pyomelanin for Potential Biomedical Applications. Int J Mol Sci. 2023 Apr 25;24(9):7846. doi: 10.3390/ijms24097846. PMID: 37175552; PMCID: PMC10178424.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to four reviewers’ comments (NCOMMS-25-76159A)\nDear Reviewers\nWe appreciate very much all your constructive comments and suggestions, which\ndefinitely gave us helpful guidance to improve our manuscript. We have now revised\nour manuscript accordingly, and for your reference, the changes in the revised\nmanuscript are highlighted in red. We have also provided a point-by-point response\n(below) with detailed explanations. We are willing to make further changes upon your\nrequest.\nResponses to Reviewer 1\nReviewer #1 (Remarks to the Author):\nThe authors describe a strategy to enhance the biosynthetic yield of pyomelanin—a key\nraw material used in pharmaceuticals, cosmetics, bioremediation, and energy\napplications. To improve flux through the pathway, they engineered DAHPS and HPPD,\ntwo rate-limiting enzymes, thereby boosting overall production. The final strain\nachieved the highest pyomelanin titer reported to date. Collectively, the study presents\na compelling, stepwise optimization of the production process. The data are robust and\nthe conclusions well supported. Nevertheless, the manuscript still has several\nshortcomings, detailed below.\nResponse: Thank you for the positive comments. We have now revised the manuscript\naccording to your suggestions.\nLines 168-171: The manuscript states that the AfHPPD PDB structure from YASARA\nwas used for structural analysis, but this is ambiguous. No experimental structure of\nAfHPPD has been deposited, so the authors presumably built a homology model.\nClarify whether YASARA was used only for visualization or also for modelling. If the\nlatter, explain why an additional model was generated with MODELLER and\nAlphaFold2 (lines 417–418). For the AlphaFold2 model, report standard quality metrics\n(pLDDT, pTM, RMSD to template, etc.) instead of only the Ramachandran plot.\nResponse: Thank you for the helpful comments. We apologize for the confusing\ndescriptions. We would like to indicate that YASARA was used only for visualization,\nnot for modelling. The revised description is shown below. In addition, we have now\nprovided the standard quality metrics of AlphaFold2 model in the revised\nsupplementary materials and here as well (below).\nLines 449-452: “First, since the crystal structure of AfHPPD was not yet available, we\nused AlphaFold2 for modelling. The reliability of the model was demonstrated by\nstandard quality metrics and a Ramachandran plot (Supplementary Fig. 5).”\nSupplementary Fig. 5. Reliability analysis of AlphaFold2 modelling. a Standard\nquality metrics of AlphaFold2 modelling based on atom-plddts. pTM = 0.93. RMSD =\n0.325. b Ramachandran plot of the structure of AfHPPD based on AlphaFold2, showing\nthat in 3-dimensional/1-dimensional analysis, at least 80% of the amino acid scores are\nequal to or higher than 0.1, and more than 90% of the amino acids are detected in\nappropriate positions.\nLines 417-418: Crystal structure of PfHPPD was used as the template for modeling, yet\nthe manuscript offers no justification for this choice. A sequence-alignment comparison\nof AfHPPD against all HPPD with available crystal structures should be presented to\ndemonstrate that PfHPPD is the most appropriate template (highest identity, best\ncoverage, conserved active-site geometry). Likewise, the final downstream analyses\nmust specify which AfHPPD model (YASARA, MODELLER, or AlphaFold2) was\nadopted and provide the selection rationale.\nResponse: Thank you for the helpful comments. As suggested, we have now revised\nthe manuscript (below) accordingly. In addition, we have added an additional figure\n(Supplementary Fig. 6) in the revised supplementary materials to demonstrate that\nPfHPPD is the most appropriate template.\nLines 452-460: “To investigate whether the model generated by AlphaFold2 can be\nused for further modification, we used Modeller for homology modelling. A template\nsearch using SWISS-MODEL identified only two HPPD crystal structures that satisfy\nthe sequence similarity and coverage criteria for AfHPPD modelling: PfHPPD\n(Pseudomonas fluorescens, PDB: 1cjx) and AtHPPD (Arabidopsis thaliana, PDB:\n8hza). The sequence alignment results revealed that compared with AtHPPD, PfHPPD\nis more suitable as a template (Supplementary Fig. 6). The model established by\nModeller is similar to the model generated by AlphaFold2. Therefore, the model\nestablished by AlphaFold2 was used for semirational design.”\nComparison with AfHPPD Coverage (%) Identify (%) Similarity (%)\nPfHPPD 99 84.27 91.29\nAtHPPD 97 28.10 45.10\nHPPD structure Binding site with metal ion\nAfHPPD H162-H241-E323\nPfHPPD H161-H240-E322\nAtHPPD H226-H308-E394\nSupplementary Fig. 6. Sequence alignment of PfHPPD, AtHPPD, and AfHPPD.\nCompared to AtHPPD, PfHPPD has a higher identify and similarity with AfHPPD. For\nthe active center, the important metal binding domains are analyzed. PfHPPD and\nAtHPPD bind Fe2+, while AfHPPD bind Cu2+. They are all composed of H-H-E motifs.\nLines 462-464: The authors attribute the improved HGA production to a 0.03-nm\ndecrease in the F338–4-HPPA distance in the variant. This displacement is within the\ncoordinate error of a homology model and is indistinguishable from thermal noise; it is\ntherefore insufficient grounds for claiming enhanced catalysis. In addition, the\nmanuscript states that S224G acts as a “structural bridge,” but never defines what is\nbeing bridged or how glycine—lacking a side chain—could fulfill such a role. Provide\n(i) ensemble docking or MD-derived distance distributions to show the 0.03-nm shift is\nstatistically significant, and (ii) a clear mechanistic explanation of the proposed S224G\nbridging function, supported by hydrogen-bond maps, etc.\nResponse: Thank you for the helpful comments. We have now conducted a significance\ntest on the distance distribution exported by MD. The results showed that the change in\ndistance distribution of mutant F338-4-HPPA was not significant. Therefore, we deleted\nthe relevant descriptions in the revised manuscript. The modified figure is also shown\nbelow. In addition, we apologize for the inappropriate description of the S224G acting\nas a “structural bridge.” We have now revised relevant description (below).\nLines 496-500: “The molecular dynamics simulation results showed that the root mean\nsquare fluctuation (RMSF) of the S224G and E314Y enzymes was greater than that of\nthe WT enzyme (Fig. 4g). This revealed an increase in flexibility near the mutant, which\nmay be related to the increase in the enzymatic activity.”\nLines 467-472: The authors assert that a higher RMSD does not imply greater instability\nfor the variant, yet lines 398–401 equate lower RMSD with enhanced structural stability.\nThis is internally inconsistent. Clarify whether RMSD is being used as a proxy for\nstability in this study; if not, remove the stability claim. If it is, provide evidence—e.g.,\nradius-of-gyration trends, hydrogen-bond counts, or free-energy calculations—to\ndemonstrate that the variant’s increased RMSD does not compromise thermostability.\nResponse: Thank you for the helpful comments. We are sorry for the inappropriate\ndescription. Yes, RMSD is being used as a proxy for stability in this study. However,\nthis is conflicting with the results demonstrated in in vitro stability experiments. To\nfurther investigate the cause of this discrepency, we supplemented molecular dynamics\nsimulations at temperature of 350 K. The revised manuscript and figure are shown\nbelow. In addition, we demonstrated the radius-of-gyration trends of E314Y/S224G and\nWT enzyme at 300 K and 350 K as evidence of improved thermal stability (Response\nFigure 1).\nLines 500-506: “In the analysis of the mechanism underlying the improvement in\nthermal stability, the RMSD of E314Y/S224G was greater than that of the WT enzyme\nat 300 K (Fig. 4h), which is contrary to the results of the in vitro stability experiments.\nTo further investigate this, we performed molecular dynamics simulations at 350 K, and\nthe results revealed that the RMSD of the E314Y/S224G enzyme were smaller than\nthose of the WT enzyme (Fig. 4i). This explains why E314Y/S224G has better thermal\nstability than the WT enzyme does.”\nFig. 4 Semi rational design and modification of AfHPPD enzyme. a Structural\nmodeling of AfHPPD. The HPPD of Pseudomonas fluorescens is represented in green,\nwhich serves as the template chain (PDB: 1CJX); the AfHPPD structure predicted by\nAlphaFold 2 is shown in yellow; and the AfHPPD structure obtained from homology\nmodeling was shown in blue. b Complex of AfHPPD and substrate 4-HPPA based on\nmolecular docking. c Schematic diagrams of 9 different mutation sites obtained under\ntwo semirational design strategies. d Enzyme activity assay of different variants. e and\nf Thermal stability assay of the wild type (WT) and variant E314Y/S224G at three\ndifferent temperatures. g. RMSF analyses of variant E314Y/S224G at 300 K. The WT\nand the variant E314Y/S224G are represented in blue and red, respectively. h RMSD\nanalyses of variant E314Y/S224G at 300 K. The WT and the variant E314Y/S224G are\nrepresented in blue and red, respectively. i RMSD analyses of variant E314Y/S224G at\nrespectively. j 3D free energy landscape (FEL) diagram. Data are means of 3 biological\nreplicates with error bars representing standard deviations.\nResponse Figure 1. Molecular dynamics simulations for AfHPPD and\nAfHPPDE314Y/S224G, showing the radius of gyration (Rg) at 300 K (left) and 350 K (right).\nFigure 2f: Did the authors test the effect of Mn2+? It would be better if the result of\nMn2+ was added.\nResponse: Thank you for the helpful comments. No, we did not test the effectiveness\nof Mn2+. As suggested, we have now tested the effect of Mn2+ on the catalytic activity\nof AfHPPD enzyme. The experimental details are as follows:\nAfHPPD enzyme was purified again and established two standard enzyme reaction\nsystems containing Cu2+ and Mn2+ (Response Figure 2a). A reaction system without\nmetal ion served as the control, and the amount of HGA produced after completion was\nused as the benchmark for relative enzyme activity. The results showed that only the\nreaction system containing Cu2+ exhibited a noticeable color change (Response Figure\n2b), indicating higher HGA production, whereas Mn2+ had little to no effect on AfHPPD\nactivity (Response Figure 2c). However, it was suspected that the above response may\nnot fully solve this problem. It has been reported that the addition of Mn2+ enhances\nthe production of pyomelanin. For example, Zhang et al. demonstrated that the\naddition of Mn2+ increased the yield of pyomelanin (J. Biosci. Bioeng., 2023, 135, 93-\n101). Therefore, we further conducted additional experiments to solve this problem.\nThe experimental details are as follows: HGA standard powder (Aladdin, Shanghai,\nChina) was prepared into a water solution with equal amounts of Mn2+ and Cu2+ added.\nAfter shaking for 24 h, the solution with added Mn2+ and Cu2+ showed a color change,\nappearing brownish yellow, with an increase in OD compared to the control\n400\n(Response Figure 2d). These results indicated that Mn2+ and Cu2+ could promote\nnonenzymatic oxidation of HGA, and the efficiency of Mn2+ is higher than that of Cu2+.\nIn summary, Cu2+ have a dual function of promoting nonenzymatic oxidation of HGA\nand enhancing the catalytic activity of AfHPPD enzyme, while Mn2+ can only promote\nnonenzymatic oxidation of HGA.\nResponse Figure 2 Comparison of the effects of Mn2+ and Cu2+ on AfHPPD enzyme\nactivity and HGA oxidative polymerization. a SDS-PAGE analysis of AfHPPD protein.\nLane M, the protein marker; lane 1, whole cell protein supernatant; lane 2, the purified\nAfHPPD protein. b Enzyme reaction system containing Cu2+ exhibits the darkest color\nafter the reaction is completed. c Effect of Mn2+ and Cu2+ on AfHPPD enzyme activity.\nd Effect of Mn2+ and Cu2+ on promoting nonenzymatic oxidation of HGA.\nOther minor issues:\nResponse: Thanks so much for reminding us of these issues. We have now carefully\nchecked the whole manuscript and corrected these issues accordingly.\n1. ‘de novo’ should be italic. Check this thoroughly.\nResponse: Revised as suggested in our manuscript.\n2. Line27, further synthesis of pyomelanin.\nResponse: As suggested, the “systhesis” has been corrected to “synthesis” in our\nrevised manuscript.\n3. Line81: constructed\nResponse: As suggested, the “constucted” has been corrected to “constructed” in our\nrevised manuscript.\n4. The legend of supplementary fig.6, the nine mutated enzymes were not from\nKomagataella phaffii.\nResponse: We apologize for this careless error, which is now corrected from\n“Komagataella phaffii” to “Alcaligenes faealis”.\nResponses to Reviewer 2\nReviewer #2 (Remarks to the Author):\nWhat are the noteworthy results?\nThis study engineered Komagataella phaffii for de novo pyomelanin production,\nachieving a titer of 70.5 g/L. Key advances include modular pathway optimization,\nestablishment of a color-based screening system, and use of directed evolution and\nsemi-rational design for enzyme improvement.\nResponse: Thank you for the positive comments on our manuscript. We have now\nprovided a point-by-point response to address your concerns. We apologize for our\nconfusing description on experimental details, and we have now carefully revised these\ndescriptions in the revised manuscript.\nWill the work be of significance to the field and related fields? How does it compare to\nthe established literature? If the work is not original, please provide relevant references.\nThis paper reports a titer of 70.5 g/L, representing a significant improvement in\npyomelanin production than previously published values. Larroude et al. (doi:\n10.3390/microorganisms9040838) reported a pyomelanin titer of 4.5 g/L, Lorquin et al.\n(https://doi.org/10.1038/s41598-021-87328-2) reported 0.55 g of pyomelanin per liter\nof medium, and Koch et al. (https://doi.org/10.3389/fmicb.2023.1233740) reported\n1.48 g of pyomelanin per liter of medium. The later paper also comparing titer/yield of\npyomelanin production in different organisms by different approaches. Thus, this work\ndemonstrates a significant improvement in terms of pyomelanin titers. However,\nimportant information on the process are missing to obtain a complete picture of the\nfermentation process. First of all the description of the fermentation L254 ff. (methods)\nand L567 ff. (results) is not well described. How was glycerol and methanol fed and\nwhich feeding profile was used? How much methanol and glycerol was actually\nconsumed?\nResponse: Thank you for the helpful comments. We appreciate very much for\nproviding us these valuable references. As suggested, we have now revised the\nmanuscript (below) as follows:\nLines 272-274: “After the glycerol was exhausted, the fed-batch phase (Phase II) was\ninitiated by feeding limited amounts of glycerol at a speed of 15−30 mL/h to promote\nfurther cell growth.”\nLines 279-282: “Methanol was first added at a speed of 5 mL/h, with an increase of 1–\n2 mL/h every 5–8 h until the flow rate reached 20–25 mL/h. If no methanol accumulated,\nthis flow rate was used throughout the entire remaining fermentation process. However,\nif necessary, this flow rate was adjusted appropriately according to the dissolved oxygen\nlevel.”\nLines 612-613: “During the entire fermentation process, a total of 340.92 ± 14.51 g of\ngof\nglycerol and 1.84 ± 0.17 kg of methanol were consumed.”\nDoes the work support the conclusions and claims, or is additional evidence needed?\nAdditional evidence is needed.\nResponse: We apologize for not providing additional evidence to support the\nconclusions and claims. As suggested, we have now provided detailed explanations.\nThe title states “de novo biosynthesis of pyomelanin from methanol,” but the\nengineered K. phaffii strains actually produce homogentisic acid (HGA) as the final\nmetabolic product. Pyomelanin formation occurs non-enzymatically via\noxidation/polymerization of HGA or enzymatically after laccase addition, rather than\nthrough an intracellular biosynthetic pathway.\nResponse: Yes, you are absolutely right! We have now revised the title of the\nmanuscript (below).\nRevised title: Biosynthesis of pyomelanin from methanol with engineered\nKomagataella phaffii and its characterizations.\nThe reported value of 70.5 g/L refers to the titer, not the yield (lines 33 and 95). Titer\nrepresents the final concentration of product in the culture medium (often as grams per\nliter), while yield describes the efficiency of conversion in reference to a given substrate\n(here: yield on methanol or total carbon source would be appropriate). Additional data\non carbon input, substrate consumption, and productivity are needed to support the\nclaims. How reproducible is this fermentation in the 5L setup?\nResponse: Thanks for your detailed explanations. We used two 5-L fermenters for\nhigh-density fermentation again (Response Figure 3). Based on the combined data from\nthe 5 L fermenter in our study, we averaged the fermentation results of the three 5-L\nfermenters. In summary, the fermentation results demonstrated high reproducibility. As\nsuggested, we have now revised the manuscript (below).\nLines 32-34: “After high-density fermentation in a 5-L fermenter for 204 h, strain\nPyo29 achieve the highest titer (70.5 ± 0.7 g/L).”\nLines 102-104: “achieving a pyomelanin titer of 70.5 ± 0.7 g/L with a specific\npyomelanin yield of 0.63 ± 0.04 g/g DCW and a productivity of 0.37 ± 0.05 g/L/h; the\ncarbon conversion ratio is 8.83%.”\nDuring the entire fermentation process, a total of 340.92 ± 14.51 g of glycerol and 1.84\n± 0.17 kg of methanol were consumed.\nResponse Figure 3 Fed-batch culture of strain Pyo29 in two 5-L fermenters.\nPurity of the reported pyomelanin is also not certain. Should pyomelanin contain N in\nits structure? Nitrogen detected in the dry weight suggests possible contamination with\nsecreted proteins, meaning not all measured mass is pure pigment.\nResponse: Thanks for your suggestive comments. We apologize for the lack of\ncorresponding evidence and adequate discussion. We have now performed additional\nrelevant experiments to address your concerns. The source data are shown below\n(Response Figure 4). We have now revised the manuscript accordingly (below).\nLines 637-652: “However, that HGA does not contain nitrogen in its structure while\npyomelanin does is confusing. Therefore, the nitrogen detected in the dry weight\nsuggested that pyomelanin was possibly contaminated with secreted proteins. However,\nowing to the lack of a pyomelanin standard, its purity could not be determined.\nInterestingly, Lorquin et al. conducted an elemental analysis on the pyomelanin\nobtained by laccase-catalysed HGA-lactone and pyomelanin synthesized by\nHalomonas titanicae. The results also demonstrated the presence of nitrogen in\npyomelanin, possibly because of the abundance of laccase extract or the components of\nthe H. titanicae culture medium [22]. To further clarify this issue, we conducted\noxidative polymerization of HGA standards using laccase and alkali. Two types of dry\npyomelanin powders were subsequently prepared for elemental analysis, and the results\nrevealed that the pyomelanin obtained after laccase treatment contained nitrogen,\nwhereas nitrogen was not detected in the pyomelanin obtained after alkali treatment.\nThese findings indicated that pyomelanin does not contain nitrogen in its structure and\nthat the nitrogen detected in this study was inevitably introduced during yeast culture\nmedium or laccase treatment.”\nResponse Figure 4 Elemental analysis of pyomelanin prepared by alkaline or laccase\noxidative polymerization of HGA standard.\nAre there any flaws in the data analysis, interpretation and conclusions? Do these\nprohibit publication or require revision?\nYes, this paper needs revision. It lacks sufficient quantitative and methodological detail\nto fully support its conclusions.\nResponse: We appreciate the opportunity you have given us to make revisions. We\napologize for the confusing description about quantitative and methodological detail.\nAs suggested, we have now revised the manuscript.\nThe authors should clarify how 70 g/L was achieved, meaning what carbon sources\nwere used, feeding rates of methanol and glycerol, and the carbon conversion ratio.\nResponse: Thanks for your suggestive comments. We apologize for the unclear\ndescriptions. We would like to indicate that throughout the fermentation process, we\nused two types of carbon sources, namely glycerol and methanol. The revises\ninformation is as follows:\nLines 272-274: “After the glycerol was exhausted, the fed-batch phase (Phase II) was\ninitiated by feeding limited amounts of glycerol at a speed of 15−30 mL/h to promote\nfurther cell growth.”\nLines 279-282: “Methanol was first added at a speed of 5 mL/h, with an increase of 1–\n2 mL/h every 5–8 h until the flow rate reached 20–25 mL/h. If no methanol accumulated,\nthis flow rate was used throughout the entire remaining fermentation process. However,\nif necessary, this flow rate was adjusted appropriately according to the dissolved oxygen\nlevel.”\nThe carbon conversion ratio reached 8.83%.\nIn summary, we have now provided these results in the revised manuscript (below).\nLines 101-104: “Next, we then conduct high-density fermentation of the top-producing\nstrain (Pyo29) in a 5-L fermenter, achieving a pyomelanin titer of 70.5 ± 0.7 g/L with a\nspecific pyomelanin yield of 0.63 ± 0.04 g/g DCW and a productivity of 0.37 ± 0.05\ng/L/h; the carbon conversion ratio is 8.83%. [23].”\nProductivity (g L⁻¹ h⁻¹) and specific yield (for example grams of pyomelanin per grams\nof cell dry weight) are missing and should be compared with literature values.\nResponse: Thanks for your suggestive comments. We apologize for not providing these\nresults, i.e., “A specific pyomelanin yield of 0.63 ± 0.04 g/g DCW and a productivity\nof 0.37 ± 0.05 g/L/h.” As suggested, we have now compared the ability of different\nstrains to produce pyomelanin in bioreactor (Response Table 1). We have also added a\ncomparison with the literature in the revised manuscript.\nResponse Table 1. Comparative analysis of the ability of Komagataella phaffii and\nothers producers to produce pyomelanin in bioreactor. Symbol “/” indicates no data\navailable.\nStrain Titer Yield (g/g DCW) Productivity References\n(g/L) (g/L/h)\nKomagataella 70.5 ± 0.7 0.63 ± 0.04 0.37 ± 0.05 This study\nphaffii\nFlavobacterium 6.07 ± 0.32 / 0.03 Lee H. S. et al.\nkingsejongi\nEscherichia 3.76 ± 0.30 / 0.04 Lee H. S. et al.\ncoli\nLee H. S. et al. Melanin biopolymer synthesis using a new melanogenic strain\nof Flavobacterium kingsejongi and a recombinant strain of Escherichia coli expressing\n4-hydroxyphenylpyruvate dioxygenase from F. kingsejongi. Microb. Cell Fact. 21:75\n(2022).\nSome results (for example the reported 6.3 % increase in HGA, lines 548 and 549) don’t\nhave evidence of statistical significance.\nResponse: Thanks for reminding us of lacking evidence of statistical significance. We\nhave now carefully checked the entire manuscript and corrected all results.\nOD readings are presented without justification that the signal detected is only\n400\npyomelanin.\nResponse: We apologize for the lacking this justification. We agree with you that OD\n400\nreadings does not fully represent pyomelanin. In our study, we only treated the OD\n400\nreadings as the relative value of changes in pyomelanin titer among different strains. In\naddition, we measured the OD of BMMY fermentation medium and the reading\n400\nremained stable in the range of 0.47–0.51. In fact, each OD reading in our manuscript\n400\nhas subtracted this value to enhance the correspondence between OD and pyomelanin.\nmay both enhance HPPD activity and promote non-enzymatic oxidation of HGA. This\nneeds clarification.\nResponse: Thanks for your suggestions. You are definitely right. HGA standard\npowder was prepared into a water solution with Cu2+ added. After shaking for 24 h, the\nsolution with Cu2+ showed a color change, appearing brownish yellow, with an increase\nin OD readings compared to the control (Response Figure 5). This indicates that Cu2+\n400\ncan also promote nonenzymatic oxidation of HGA. In summary, Cu2+ has shown a\ndual function of promoting non-enzymatic oxidation of HGA and enhancing the\ncatalytic activity of AfHPPD enzyme. We have now revised our manuscript as follows:\nLines 387-395: “Interestingly, Seo D. et al. demonstrated that Cu2+ ions do not increase\nthe catalytic activity of the HPPD enzyme of Ralstonia pickettii. However, the addition\nof Cu2+ to the culture medium of R. pickettii can increase the production of pyomelanin\n[8]. Therefore, we speculate that Cu2+ may both increase HPPD activity and promote\nthe nonenzymatic oxidation of HGA. To verify this hypothesis, HGA standard powder\nwas prepared as a solution, and Cu2+ was added to the solution. After shaking for 24 h,\ncompared with the control solution, the solution with Cu2+ added showed a colour\nchange, appearing brownish yellow, with an increase in the OD (Supplementary Fig.\n400\n3). This proves that Cu2+ can also promote the nonenzymatic oxidation of HGA.”\nResponse Figure 5 Effect of Cu2+ on promoting nonenzymatic oxidation of HGA.\nIt would be interesting to see the comparison of the native HPPD enzyme from\nKomagataella phaffii to the engineered one from this paper.\nResponse: Thanks for your suggestive comment. Yes, we also think it will be very\ninteresting. We will compare PpHPPD enzyme (Komagataella phaffii) and AfHPPD\n(Alcaligenes faecalis) from two perspectives (below).\nOn the one hand, the sequence alignment of PpHPPD and AfHPPD showed that the\ntwo enzymes have 32.93% identical residues and 48.64% similar residues (including\nconservative substitutions) within the alignment region. Although there are 14.2% gaps,\nreflecting insertion or deletion during evolution, the core functional region that binds\nto metal ions is conserved (Response Figure 6a).\nOn the other hand, previous manuscript explored HPPD genes from various sources\nand identified AfHPPD significantly enhanced HGA production than PpHPPD\n(Response Figure 6b). Then, we have added new experiments to further compare\nPpHPPD and AfHPPD. According to the standard of “2.8 In vitro AfHPPD enzyme\nassays” in the manuscript’s Materials and methods, we tested which metal ions could\nenhance the catalytic activity of PpHPPD enzyme. The results showed that Fe2+ could\nslightly enhance the activity of PpHPPD enzyme. And the results showed that the\nenhancement effect of Fe2+ on PpHPPD enzyme was inferior to that of Cu2+ on AfHPPD\nenzyme (Response Figure 6c). Therefore, thank you again for your question, which has\nstrengthened our determination to choose the AfHPPD enzyme for this study.\nResponse Figure 6 Comparison of AfHPPD enzyme and PpHPPD enzyme. a Sequence\nalignment of AfHPPD enzyme and PpHPPD enzyme. b,c In vitro and in vivo\ncomparison of AfHPPD enzyme and PpHPPD enzyme.\nThe English in general needs more revision to be clearer and gramatically correct.\nResponse: Thanks for reminding us! As suggested, the language has been thoroughly\nrevised with the Nature Research Editing Service provided by Nature Communications\naffiliates (Order#: 1P6M32SJ).\nGiven these issues, the work needs significant revision before publication.\nResponse: Thank you again for giving us the opportunity to revise and improve our\nmanuscript.\nIs the methodology sound? Does the work meet the expected standards in your field?\nNot fully. The experimental concept is interesting and the modular metabolic\nengineering strategy is promising, but methodological details are missing.\nResponse: We appreciate your positive evaluation on our work. As suggested, we\nprovided a detailed description of the methodological details in the revised manuscript\nand supplementary materials.\nThere is no clear genetic characterization of strains in this work. Number of integrated\ncopies of some genes is mentioned (lines 338, 499, 501, 547, 700), but there is no\ninformation on the way that was achieved.\nResponse: Sorry for the confusing description. In order to clearly describe the genetic\ncharacterization of strains, the plasmids and strains information table in the\nsupplementary materials have been modified. The details of the gene copy number\nintegration method have also been revised in the revised manuscript (below).\nLines 361-362: “Strain Pyo9, which carried three AfHPPD copies integrated into the\ngenome at different loci (HIS4, INT1, and INT18).”\nLines 531-533: “A second copy of TKL1 subsequently added to the INT18 locus of the\ngenome of strain Pyo15 to obtain strain Pyo22.”\nLines 533-534: “The results showed that strain Pyo22, carrying the TKL1 gene\nintegrated into two different loci (INT1 and INT18).”\nLines 578-579: “suggesting that an extra copy of AfHPPD (integrated into the INT21\nlocus) might be needed to channel carbon flux effectively for into HGA synthesis.\nLines 802-804: “In this study, the highest pyomelanin-producing strain, Pyo29, carried\nfour copies of the AfHPPD enzyme (integrated into HIS4, INT1, INT18 and INT21).”\nNo plasmid sequences are provided in the supplementary material.\nResponse: Thanks for reminding us! We have included the plasmids sequences and\nintegration sites used in this study in the supplementary materials (Table S5).\nThe lack of OD measurements during inoculation (lines 238 and 240), the vague\ndescription of adding copper “as needed” (lines 262 and 263) and “pyomelanin\nsolutions of varying concentrations” (lines 291 and 292) make replication and data\ninterpretation difficult.\nResponse: Sorry for the confusing description. We corrected these descriptions in the\nrevised manuscript (below).\nLines 254-257: “Afterwards, approximately 4% of the seed culture (OD =1.0) was\n600\ntransferred into a 500 mL Erlenmeyer flask containing 100 mL of YPD medium and\ncultured under the same conditions for approximately 18 h.”\nLines 282-283: “Next, 10 mL of 200 mM Cu2+ was supplemented every 18 h during the\ninduction process.”\nLines 310-312: “After incubation, the culture medium was removed from each well,\nand different concentrations (0, 100, 500 and 1000 μg/mL) of pyomelanin solutions\ndissolved in DMSO were added.”\nIs there enough detail provided in the methods for the work to be reproduced?\nNo. A lot of experimental details are missing or too vague to reproduce, as mentioned\nabove.\nResponse: Thanks for your suggestive comment. As mentioned above, we revised the\nmanuscript and supplementary materials carefully.\nResponses to Reviewer 4\nReviewer #4 (Remarks to the Author):\nThe manuscript “De novo biosynthesis of pyomelanin from methanol with engineered\nKomagataella phaffii and its characterizations” presents several interesting aspects,\nsuch as the production of pyomelanin in a yeast strain, the directed evolution of\nenzymes to improve biosynthetic processes, the metabolic push-pull strategy and\nmanipulation of K. phaffii for pyomelanin production.\nResponse: We appreciate your positive evaluation on our work!\nHowever, we identified several major issues that need to be addressed:\nResponse: Thanks for your suggestive comments. We here provide a point-by-point\nreply to address all your concerns (below).\nThe main observation that I have is that the pigment being produced, pyomelanin,\nis widely distributed among bacterial species, like those of the genus Pseudomonas,\namong others. It is not clear what the advantage would be of producing pyomelanin in\na eukaryotic organism. On one hand, achieving high pigment production requires a\nrather complex fed-batch fermentation scheme, which is not inexpensive. Although\nmethanol is a low-cost substrate, bacteria can utilize waste compounds from agro-\nindustrial or fuel industries, for example. If the advantage lies in cost, this point has not\nbeen thoroughly discussed.\nResponse: Thank you for the helpful comments. Yes, you are completely right. Almost\nall reports of pyomelanin are widely distributed among bacterial species. Especially,\npyomelanin from Pseudomonas aeruginosa ΔhmgA, which has been reported and well\ncharacterized by many research groups. In this study, we choose K.phaffii to produce\npyomelanin. On the one hand, the genetic background of K.phaffii is clear, and gene\nediting tools are constantly improving. On the other hand, many studies use K.phaffii\nas a cell factory for synthesizing various high-value compounds. This provides the\npossibility for K.phaffii to produce pyomelanin. Therefore, we have added more\ndiscussion to introducte K.phaffii to you in the revised manuscript (below).\nLines 57-69: “The methylotrophic yeast Komagataella phaffii is classified as a\n“Generally Recognized as Safe” (GRAS) organism by the U.S. Food and Drug\nAdministration (FDA) [15,16]. Many studies have demonstrated its potential as an\neffective host for the production of L-tyrosine-derived compounds, including\nresveratrol, naringenin, and norcoclaurine [17,18]. Compared with bacteria, K. phaffii\nhas advantages in terms of efficient and high-level expression of heterologous enzymes,\nand its Crabtree-negative phenotype further enhances its capacity for high-density\nfermentation in minimal salt media when methanol is used as the sole carbon source\n[19]. In terms of cost, although bacteria can ferment waste compounds from the agro-\nindustrial or fuel industries, methanol is also a low-cost carbon source. In addition,\nmethanol, as a renewable and carbon–neutral single-carbon (C1) feedstock, has\nemerged as a promising alternative carbon source for green biomanufacturing because\nof its noncompetition with food resources and scalability for industrial production\n[20,21]. Consequently, engineered K. phaffii is expected to offer a sustainable platform\nfor the production of pyomelanin, a downstream derivative of L-tyrosine.”\nMoreover, the fermentation lasts five days (example in line 205), which is quite long,\nand no comparison is made to yields obtained in other studies using fast-growing\nbacterial producers.\nResponse: Thanks for your suggestive comment. In order to better compare the\ndifferences in the levels of pyomelanin production between K. phaffii and bacterial\nproducers, we have listed a table to better present this data of titer, yield, and\nproductivity to you (below).\nResponse Table 2. Comparison of the ability of K. phaffii and bacterial producers to\nproduce pyomelanin in shaking flask.\nStrain Titer Productivity Reference\nKomagataella phaffii 5.11 ± 0.13 g/L 42.6 mg/L/h This study\nEscherichia coli 315.5 ± 12.9 mg/L 13.1 mg/L/h Seo et al.\nEscherichia coli 804.4 ± 5.9 mg/L 16.8 mg/L/h Zhang et al.\nEscherichia coli 213 mg/L 1.48 mg/L/h Bolognese et al.\nHalomonas titanicae 0.55 g/L 11.5 mg/L/h Lorquin et al.\nAspergillus niger 1.48 g/L 10.3 mg/L/h Koch et al.\nPseudomonas aeruginosa 1.79 ± 0.18 g/L 12.8 mg/L/h Urbaniak et al.\nYarrowia lipolytica 4.5 g/L 13.3 mg/L/h Larroude et al.\nSeo, D. et al. Heterologous production of pyomelanin biopolymer using 4-\nhydroxyphenylpyruvate dioxygenase isolated from Ralstonia pickettii in Escherichia\ncoli. Biochem. Eng. J. 157, 107548 (2020).\nZhang, Q. H. et al. Pyomelanin production via heterologous expression of 4-\nhydroxyphenylpyruvate dioxygenase (HPPD) and construction of HPPD inhibitor scr\neening model. J. Biosci. Bioeng. 135, 93-101 (2023).\nBolognese, F. et al. Bacterial melanin production by heterologous expression of 4\nhydroxyphenylpyruvate dioxygenase from Pseudomonas aeruginosa.\nInt. J. Biol. Macromol. 133, 1072-1080 (2019).\nLorquin, F. et al. Production and properties of non-cytotoxic pyomelanin by laccase and\ncomparison to bacterial and synthetic pigments. Sci. Rep. 11, 1–16 (2021).\nKoch, S. et al. Aspergillus niger as a cell factory for the production of pyomelanin,\na molecule with UVC radiation shielding activity. Front. Microbiol. 14, 1233740 (2023).\nUrbaniak, M. et al. In Vitro and In Vivo Biocompatibility of Natural and Synthetic\nPseudomonas aeruginosa Pyomelanin for Potential Biomedical Applications. Int. J.\nMol. Sci. 24,7846 (2023).\nIn the Results section (line 320), no measurement uncertainty or deviation is indicated,\nwhich prevents assessing reproducibility.\nResponse: Thanks for reminding us of this missing information. We have now carefully\nchecked the whole manuscript and provided missing information in results.\nConsidering the importance of genetic stability in organisms intended for\nbiotechnological use, it would be highly relevant to discuss the stability of this yeast\nstrain, which carries multiple genetic modifications, even the use of Crispr-Cas9 can\nlead to off-targets editions that should be analyzed.\nResponse: Thanks for your helpful suggestions. We have added a description and\ndiscussion on the genetic stability of K. phaffii in the revised manuscript (below).\nLines 121-124: “Considering the importance of genetic stability, the pZACH plasmid\nwas utilized for gene overexpression integration. The Cre/loxP ZeoR marker recycling\nmethod [24] ensured the stability of every integration. The pENO1-Cas9 plasmid was\nused for gene knockout to avoid off-target effects.”\nLines 816-820: “In addition, K. phaffii usually expresses exogenous genes through\nchromosome integration rather than through plasmid amplification. Its genetic stability\nis high in microbial expression systems, which ensures that strain Pyo29 is suitable for\nlong-term and large-scale fermentation production of pyomelanin [46,47].”\nMore importantly, while the reported yield of 1.65 g/L is high, productivity (yield per\nunit time) is not expressed, so it is unclear whether this microorganism is actually a\nbetter producer than bacterial systems, either natural producers or recombinant, fast\ngrowing strains. For example, line 573 states that fermentation was completed after 168\nhours (seven days), yet this aspect is not discussed at all.\nResponse: We apologize for the incomplete description. We have now provided\nappropriate description in the revised manuscript (below).\nLine 340-342: “After various remoulding strategies were implemented, we obtained a\nhigh-yield HGA-producing K. phaffii strain, Pyo29, with a titer of 1.65 ± 0.04 g/L,\nand a productivity of 13.8 mg/L/h.”\nWe apologize for any misunderstanding caused our inadequate discusson. In this study,\nwe divided the synthesis of pyomelanin into two distinct processes. First, we achieved\nefficient production of HGA from methanol. Second, we explored the oxidative\npolymerization of HGA into pyomelanin. The final strain Pyo29 can produce 1.65 ±\n0.04 g/L HGA at the shake flask level, which can then be oxidized and polymerized\ninto 5.11 g/L pyomelanin. However, we have not seen any reports on HGA titer by\nbacterial systems. The comparative analyis of the production capacity of pyomelanin in\nshaking flask is shown in the Response Table 2 (above). Then, as you mentioned, the\nhigh-density fermentation time in a 5-L fermentor was a total of 204 hours, and the\ninduction time was 168 hours. The comparison of the production capacity of\npyomelanin in bioreactor is shown in the Response Table 1 (above). In summary,\nregardless of shaking flask or bioreactor, the K. phaffii used in this study exhibited\nhigher pyomelanin titer and productivity (mg/L/h) than bacterial producers. In fact, we\nare very grateful to all the research teams involved in the production of pyomelanin by\nbacteria. These reports have provided excellent guidance for our study!\nRegarding the chemical characterization, we found significant issues:\nResponse: Thanks for your suggestive comments. We here now provide a point-by-\npoint response to address all your concerns (below).\nFirst, in the M-M section (line 210), the purification method described appears to yield\npoorly purified material, potentially containing impurities such as proteins and\npolysaccharides. These contaminants could have influenced some of the reported\nscavenging results. Moreover, the reported production values might be highly\noverestimated due to this purification protocol.\nResponse: Thanks for your suggestive comments. We aologize for the concern caused\nby lacking sufficient discussion and experimental evidence. We would like to address\nyour concerns in the following three aspects (below).\nFirstly, the purification method used in this study is acid precipitation, which is\nconsistent with most previous reports. Especially, we noticed that the publications that\nyou provided us also used the same method for the purification of pyomelanin.\nSecondly, we have been trying to determine the purity of pyomelanin. However, due to\nthe lack of standard and the fact that pyomelanin is a polymer, the detection of its purity\nis technically challenging. As you mentioned that our purified pyomelanin may contain\nproteins or polysaccharides. We speculated that your concerns are based on the\nelemental analysis of pyomelanin in our study. In fact, another reviewer also expressed\nthe same concerns. We would like to indicate that we obtained HGA standard to use in\noxidative polymerization treatment based on alkali or laccase. We conduct elemental\nanalysis after obtaining two types of pyomelanin. Our conclusion is that the structure\nof pyomelanin definitely does not contain nitrogen. The reason why pyomelanin\nproduced by K. phaffi contains nitrogen may come from the rich extract of laccase or\nthe components of the culture medium. Therefore, we agree with this reviewer on that\nthe purified pyomelanin in our study may contain a small amount of protein.\nInterestingly, our results are consistent with Lorquin’s report, which seems to be a\ncommon issue.\nFinally, we agree with this reviewer on the concern that the incorporation of nitrogen\ncan lead to an overestimation of the titer and yield of pyomelanin. However,\nconsidering that the nitrogen content of pyomelanin in this study is less than 5%, the\nprotein content is relatively low. Again, we would like to express our sincere gratitude\nto your constructive advice on the purity of pyomelanin. Indeed, the entire pyomelanin\nindustry should pay attention to the problem of purity, which is crucial for large-scale\nindustrial production and product applications. We now look forward to working\ntogether with other research groups involved in the production of pyomelanin by\nbacteria to establish a standardized purification process for pyomelanin.\nOn the other hand, all experiments should have included, at least as a control, the best-\ncharacterized pyomelanin from Pseudomonas aeruginosa ΔhmgA. While it is true that\ncommercial melanin from Sigma (derived from alkaline treatment of tyrosine) is\navailable, the authors could have used P. aeruginosa ΔhmgA pyomelanin as reference,\nwhich has been well characterized by several research groups over time.\nResponse: Yes, you are definitely right! We apologize for overlooking the reports by\nseveral research groups on the use of P. aeruginosa ΔhmgA to produce pyomelanin.\nFirstly, through our research and the reference you provided of pyomelanin based on\nPseudomonas aeruginosa ΔhmgA, we agree that many researchers have conducted\ndetailed characterization of pyomelanin produced by P. aeruginosa ΔhmgA. As you\nmentioned, this microbial-derived pyomelanin can serve as the standard for the entire\npyomelanin industry. Therefore, we agree with this reviewer on the argument that, as\none of the few cases of fungal production of pyomelanin, it is necessary to compare our\nresults with this standard pyomelanin. Secondly, as mentioned in our response to your\nquestion below, we have now provided comparison and discussion of P. aeruginosa\nΔhmgA in the revised manuscript regarding the characterization experiments of\npyomelanin, in order to draw readers' attention to the pyomelanin produced by P.\naeruginosa ΔhmgA. Finally, we would like to express our gratitude to all the research\ngroups studying the production and characterization of pyomelanin by P. aeruginosa\nΔhmgA for their contributions to the development of the pyomelanin industry. It is\nstrongly believed that these studies have significantly guided and improved our\nmanuscript. Most importantly, we would like to thank you for your suggestions and\ntolerance of our inappropriate descriptions in this study, as well as for taking your time\nto provide us with valuable references on the production and characterization of\npyomelanin by P. aeruginosa ΔhmgA. We believe that our manuscript is now\nsignificantly improved.\nFurthermore, in the M-M (lines 269 and 290), in the antioxidant and cell assays, it is\nnot stated how the pyomelanin solutions were prepared, given that the natural\npyomelanin is insoluble in water, buffer, or ethanol therefore is crucial to understand\nhow the experiments were made.\nResponse: We apologize for the confusing description. As you suggested, we have now\nadded more details in the revised manuscript (below).\nLines 293-294: “DPPH and pyomelanin dry powder were dissolved in ethanol and\nDMSO, respectively.”\nLines 310-312: “After incubation, the culture medium was removed from each well,\nand different concentrations (0, 100, 500 and 1000 μg/mL) of pyomelanin solutions\ndissolved in DMSO were added.”\nAdditionally, there is a lack of controls with pyomelanins from bacterial. Antioxidant,\ncosmetic, and UV-protective properties of pyomelanin have already been extensively\ndescribed in the literature, so many of the authors’ statements overlook the substantial\nexisting body of work (lines 623–627).\nResponse: Thanks so much for your critical comments. We have provided relevant\ncomparison and discussion in the revised manuscript (below).\nLines 704–719: “Notably, pyomelanin from bacteria has been widely reported. In\nparticular, pyomelanin from Pseudomonas aeruginosa ΔhmgA has been well studied\nand characterized by several research groups [32-35]. Therefore, comparing the\nantioxidant and antiradiation properties of pyomelanin produced by K. phaffii with\nthose from bacterial sources is necessary. Interestingly, Diaz Appella et al.\ndemonstrated that the composition and physiological role of pyomelanin produced by\nP. aeruginosa from diverse lifestyles also differ [33]. A similar study also demonstrated\nsignificant differences in functional applications between water-soluble and water-\ninsoluble pyomelanin from the same source [34]. These findings are similar to our\nresults. Furthermore, almost all the microbial sources of pyomelanin exhibit excellent\nantioxidant and radiation resistance properties. However, the diversity of experimental\ncell models and evaluation methods make comparisons difficult. In summary, this study\ndemonstrated that compared with pyomelanin produced by bacterial producers, the\npyomelanin produced by K. phaffii has similar antioxidant and radiation resistance,\nwhich can supplement the biotechnology applications of pyomelanin.”\nThe characterization relies mainly on IR spectroscopy, which provides limited\nstructural information for polymers as complex and heterogeneous as melanins.\nEven for pyomelanin, key structural differences have been revealed primarily by NMR\nanalysis. Therefore, the claims made in lines 607–609 cannot supported by IR data\nalone.\nResponse: Thanks so much for your suggestive comments. We have now conducted\n13C CP-MAS solid-state nuclear magnetic resonance analysis (NMR) on two\npyomelanin samples, Pyo-NaOH and Pyo-Lac. Then, we have provided the\nexplanations of NMR methods and results in the revised manuscript (below).\nLines 229-232: “The CP-MAS method for 13C CP-MAS solid-state nuclear magnetic\nresonance (NMR) analysis was used. The instrument used was a JNM-ECZL600G from\nJapan Electronics, with a 1H resonance frequency of 599.57 MHz, a 3.2 mm solid rotor\nsample, and an HX dual resonance probe.”\nLines 657-685: “To further analyse the chemical structural characteristics of\npyomelanin, 13C CP-MAS solid-state nuclear magnetic resonance (NMR) analysis was\nperformed on Pyo-NaOH and Pyo-Lac. The results revealed that the overall profile and\nmain signal distribution of Pyo-NaOH and Pyo-Lac were highly similar, which is\nconsistent with the spectral characteristics of amorphous polymer melanin substances\n(Supplementary Fig. 12) and highly consistent with the spectra of pyomelanin reported\nin the literature [22]. At δ 170–180 ppm, a clear wide blunt signal was observed in both\nsamples, which can be attributed to the unpotonated carbonyl carbon (–COOH) in the\ncarboxyl group, indicating that a certain proportion of the carboxyl structural units were\nstill retained in the polymer structure. The aromatic carbon region (δ 110–160 ppm)\nwas the most concentrated part of the spectrum, and the overall signal of Pyo-Lac in\nthe aromatic region was greater than that of Pyo-NaOH. A strong broad peak appeared\nat approximately δ 120–130 ppm, corresponding to the C–H and C=C carbon signals\nin the aromatic ring, whereas the secondary signal in the δ 145–150 ppm region was\nattributed to the aromatic carbon connected by phenolic hydroxyl groups. This spectral\ncharacteristic indicates that the main chain skeleton of the sample is composed mainly\nof aromatic structural units. Notably, no clear independent signal peak was observed in\nthe δ 160–165 ppm region, indicating that the samples do not contain aromatic ether\nbond (Car–O–Car) structures and that the polymerization process may be achieved\nmainly through aromatic carbon‒carbon bond (Car–CAr) coupling, which is highly\nconsistent with the reported mechanism of pyomelanin polymerization in the literature.\nAt δ 30–80 ppm, weak but distinguishable signals were detected in both samples, with\npeaks near δ 30–35 ppm attributed to aliphatic–CH –carbon, and at δ 50–80 ppm, broad\n2\npeak signals were observed in Pyo-Lac and Pyo-NaOH, which are typically attributed\nto aliphatic carbon (C–O type carbon) connected to oxygen atoms. In summary, Pyo-\nLac and Pyo-NaOH both exhibit polymerization characteristics of melanin with\naromatic carbon–carbon bond connections as the main and highly disordered structures\naccording to solid-state nuclear magnetic resonance spectra, and their overall structures\nare highly similar.”\nSupplementary Fig. 12 13C Cross-Polarization Magic Angle Spinning (CP-MAS)\nsolid-state NMR spectra of Pyo-NaOH and Pyo-Lac. The red and blue lines represent\nPyo-Lac and Pyo-NaOH, respectively.\nIn line 629, the authors discuss the use of pyomelanin for electrode fabrication in\nsodium-ion batteries. However, it is unclear what the advantage would be of using such\nan expensive compound for this purpose. Without a clear rationale, this part of the study\nlacks relevance. There are numerous inexpensive precursors for synthesizing hard\ncarbon anodes for SIBs, including agricultural biomass/waste (lignin included),\nindustrial pitch, and waste plastics (e.g., PET), all of which have demonstrated\ncompetitive performance (DOI: 10.1016/j.jechem.2018.01.025; DOI: 10.1038/s41467-\n023-39637-5; DOI: 10.1039/C5TA08601A).\nResponse: Thank you for the helpful comments. We appreciate very much your\nproviding us the three publications on sodium ion batteries. We have provided\nadditional comparative analysis of the application of pyomelanin as a sodium ion\nbattery in the revised manuscript (below). We have now cited and discussed these\nreferences you provided in our revised manuscript.\nLines 742-753: “Although the performance of pyomelanin hard carbon as a negative\nelectrode for sodium-ion batteries is similar to that of some materials reported in the\nliterature, these inexpensive biomass hard carbon materials (industrial pitch or rice husk)\nseem to have lower costs [36,37]. In particular, Tang et al. used waste wood as a hard\ncarbon negative electrode and reported a high reversible capacity of 430 mAh/g [38].\nCompared with these methods, the advantage of this study lies in the direct\ncarbonization of pyomelanin to prepare hard carbon negative electrode materials, which\nis a simpler preparation process. Of course, applying pyomelanin to sodium-ion\nbatteries is meaningful, and in the future, the preparation process of pyomelanin can be\nfurther regulated to obtain structurally unique precursors to enhance the performance\nof hard carbon negative electrodes in sodium-ion batteries.”\nFinally, the Discussion contains numerous claims about the advantages and novelty of\nthe study while overlooking prior research that had already addressed many of these\npoints. For example, the comparison between laccase-synthesized and chemically\nsynthesized pyomelanins (under alkaline conditions) has already been thoroughly\nstudied, including NMR analysis, as reported in Lorquin et al., Scientific Reports\n(2021): 10.1038/s41598-021-87328-2. Given that this work is even cited by the authors\nthemselves in the present manuscript, the claimed novelty is practically null and\ncontradictory in this regard. Lorquin’s study provides a complete structural analysis\nwith NMR data.\nResponse: Thanks for reminding us of this issue. We agree with this reviewer on these\narguments. We have now revised relevant description in the revised manuscript (below).\nIn addition, we have supplemented the NMR data to compare with the NMR results by\nLorquin’s study (above). Additionally, we greatly appreciate the research published by\nLorquin et al. for providing guidance to our work.\nLines 766-768: “Afterwards, we characterized the pyomelanin produced by K. phaffii\nand tested its performance in cosmetics. In addition, we first demonstrated its potential\nfor applications in emerging energy storage.”\nLines 824-827: “Interestingly, the comparison between laccase catalysis and chemically\nsynthesized pyomelanins (under alkaline conditions) has already been thoroughly\nstudied [22]. Therefore, this study referred to previous work to explore the structural\nand functional differences in the application of pyomelanin obtained through these two\nmethods.”\nLines 841-842: “Furthermore, this study explored the potential of pyomelanin in the\nnew energy storage field.”\nLastly, the statement that this melanin could serve as a reference pyomelanin standard\nis not well justified, since naturally producing bacterial strains already exist and are\nroutinely used as reference sources in various studies.\nResponse: Thanks for your kind clarification. Yes, you are definitely right! We have\nnow removed the description in the revised manuscript (below).\n“In addition, it is expected that this study of pyomelanin would be used as a guidance\nto further explore the functional application values of other types of melanin.”\nMore importantly, we are truly grateful for all previous reports on bacterial production\nof pyomelanin, which have provided strong guidance for our study.\nSome important publications:\nRodríguez-Rojas A, Mena A, Martín S, Borrell N, Oliver A, Blázquez J. Inactivation of\nthe hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction,\nstress resistance and increased persistence in chronic lung infection. Microbiology\n(Reading). 2009 Apr;155(Pt 4):1050-1057. doi: 10.1099/mic.0.024745-0. PMID:\n19332807.\nLorquin, F., Ziarelli, F., Amouric, A. et al. Production and properties of non-cytotoxic\npyomelanin by laccase and comparison to bacterial and synthetic pigments. Sci Rep 11,\n8538 (2021). https://doi.org/10.1038/s41598-021-87328-2\nLorquin F, Piccerelle P, Orneto C, Robin M, Lorquin J. New insights and advances on\npyomelanin production: from microbial synthesis to applications. J Ind Microbiol\nBiotechnol. 2022 Jul 30;49(4):kuac013. doi: 10.1093/jimb/kuac013. PMID: 35482661;\nPMCID: PMC9338888.\nDiaz Appella MN, Kolender A, Oppezzo OJ, López NI, Tribelli PM. The structural\ncomplexity of pyomelanin impacts UV shielding in Pseudomonas species with different\nlifestyles. FEBS Lett. 2024 Nov;598(21):2702-2716. doi: 10.1002/1873-3468.15000.\nEpub 2024 Aug 16. PMID: 39152523.\nUrbaniak MM, Gazińska M, Rudnicka K, Płociński P, Nowak M, Chmiela M. In Vitro\nand In Vivo Biocompatibility of Natural and Synthetic Pseudomonas aeruginosa\nPyomelanin for Potential Biomedical Applications. Int J Mol Sci. 2023 Apr\n25;24(9):7846. doi: 10.3390/ijms24097846. PMID: 37175552; PMCID:\nPMC10178424.\nBen Tahar I, Kus-Liśkiewicz M, Lara Y, Javaux E, Fickers P. Characterization of a\nnontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica. Biotechnol\nProg. 2020 Mar;36(2):e2912. doi: 10.1002/btpr.2912. Epub 2019 Nov 4. PMID:\n31525285.\nAl Khatib M, Costa J, Spinelli D, Capecchi E, Saladino R, Baratto MC, Pogni R.\nHomogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural\nCharacterization and Antioxidant Activity. Int J Mol Sci. 2021 Feb 9;22(4):1739. doi:\n10.3390/ijms22041739. PMID: 33572316; PMCID: PMC7916096.\nBayram S. Production, purification, and characterization of Streptomyces sp. strain\nMPPS2 extracellular pyomelanin pigment. Arch Microbiol. 2021 Sep;203(7):4419-\n4426. doi: 10.1007/s00203-021-02437-w. Epub 2021 Jun 14. PMID: 34128104.\nUrbaniak MM, Gazińska M, Rudnicka K, Płociński P, Nowak M, Chmiela M. In Vitro\nand In Vivo Biocompatibility of Natural and Synthetic Pseudomonas aeruginosa\nPyomelanin for Potential Biomedical Applications. Int J Mol Sci. 2023 Apr\n25;24(9):7846. doi: 10.3390/ijms24097846. PMID: 37175552; PMCID:\nPMC10178424.\nResponse: We appreciate very much your time and providing these important\npublications. We have now cited and discussed these references in our revised\nmanuscript. These reports provide valuable references for us to better understand\nbacterial production of pyomelanin. Finally, we are grateful for your guidance in this\nstudy.\nResponses to Reviewer 5\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the reviewer for the time and helpful comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have almost addressed all the issues. One remaining major issue: In response figure 2d, Mn2+ promoted nonenzymatic oxidation of HGA. Why in response figure 2b, no nonenzymatic oxidation phenonmenon was observed in the presence of Mn2+.\nAnother minor issue in supplementary figure 6: for the sequence alignment, the name for each sequence should be clear. In the present version, I don't know which sequence they used.", "reviewer_2": "The authors carefully revised the manuscript, clarified the methodology, improved the representation of their data and corrected some inconsistencies. Thus, the manuscript appears ready for publication.", "reviewer_4": "The manuscript has been substantially improved and is now supported by a much stronger scientific framework. The addition of key structural experiments significantly strengthens the main conclusions, and the discussion is more rigorous and better integrated with previously published data from multiple independent groups.\nThe purification of the material, however, remains inherently complex: although the nitrogen content is reported to be approximately 5%, the nitrogen-containing components may differ in molecular mass, and their contribution to the final purified melanin preparation is therefore not necessarily linear. However, this limitation reflects a broader methodological challenge in the field rather than a specific weakness of this study. Overall, and within my area of expertise all our questions were addressed.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to four reviewers’ comments (NCOMMS-25-76159B)\nDear Reviewers:\nWe appreciate very much all your constructive comments and suggestions, which\ndefinitely gave us helpful guidance to improve our manuscript. We have now revised\nour manuscript accordingly. For your reference, the changes in the revised manuscript\nare highlighted in red. We have also provided a point-by-point response (below) with\ndetailed explanations. We are willing to make further changes upon your request.\nResponses to Reviewer 1\nReviewer #1 (Remarks to the Author):\nThe authors have almost addressed all the issues.\nResponse: Thank you for the positive comments, especially your guidance in enzyme\nengineering and analysis.\nOne remaining major issue: In response figure 2d, Mn2+ promoted nonenzymatic\noxidation of HGA. Why in response figure 2b, no nonenzymatic oxidation\nphenonmenon was observed in the presence of Mn2+.\nResponse: Thank you for these helpful comments. We are sorry for any confusion\ncaused due to a lack of detailed explanation. As suggested, we have now provided\nadditional explanations (below) to clarify this issue.\nIn response to figure 2b, our aim is to test whether Mn2+ can enhance the catalytic\nactivity of AfHPPD enzyme. The substrate in the system is 4-hydroxyphenylpyruvic\nacid (4-HPP), and HGA is the product after the reaction is completed. The results\nshowed that the amount of generated HGA is trivial in the presence of Mn2+ and cannot\nbe nonenzymatically oxidized into pyomelanin; therefore, no color phenomenon can be\nobserved.\nIn response to figure 2d, our aim is to investigate whether Mn2+ can promote\nnonenzymatic oxidative polymerization of HGA. We prepared a low concentration\nHGA standard, which was shaken for 24 h. We observed a color change in the HGA\nwater solution with added Mn2+, indicating that Mn2+ can promote nonenzymatic\noxidative polymerization of HGA.\nThe in vitro AfHPPD enzyme assay method described in the manuscript is also provided\nhere for your reference:\nLines 210-215: “An AfHPPD enzyme activity assay was conducted by measuring HGA\nproduction. The reaction mixture (500 μL) contained 50 mM phosphate buffer (pH 7.4),\n1 mM CuSO , 4 mM ascorbic acid, 1 mM 4-hydroxyphenylpyruvic acid (4-HPP), and\n4\n50 μg of AfHPPD enzyme. After mixing, the reaction was incubated at 30 °C for 10 min\nand then quenched with 100 μL of 10% trifluoroacetic acid. HGA levels were quantified\nby HPLC analysis.”\nOverall, our results indicated that Mn2+ can promote the nonenzymatic oxidative\npolymerization of HGA into pyomelanin but cannot enhance the production of HGA\nbased on the substrate 4-HPP catalyzed by AfHPPD enzyme.\nResponse Figure 2 Comparison of the effects of Mn2+ and Cu2+ on AfHPPD enzyme\nactivity and HGA oxidative polymerization. a SDS-PAGE analysis of AfHPPD protein.\nLane M, the protein marker; lane 1, whole cell protein supernatant; lane 2, the purified\nAfHPPD protein. b Enzyme reaction system containing Cu2+ exhibits the darkest color\nafter the reaction is completed. c Effect of Mn2+ and Cu2+ on AfHPPD enzyme activity.\nd Effect of Mn2+ and Cu2+ on promoting nonenzymatic oxidation of HGA.\nAnother minor issue in supplementary figure 6: for the sequence alignment, the name\nfor each sequence should be clear. In the present version, I don't know which sequence\nthey used.\nResponse: Thank you for the helpful comments. We apologize for the unclear\ndescriptions. As suggested, we have now modified supplementary figure 6 in the\nrevised supplementary materials (below).\nComparison with AfHPPD Coverage (%) Identify (%) Similarity (%)\nPfHPPD 99 84.27 91.29\nAtHPPD 97 28.10 45.10\nHPPD structure Binding site with metal ion\nAfHPPD H162-H241-E323\nPfHPPD H161-H240-E322\nAtHPPD H226-H308-E394\nSupplementary Fig. 6. Sequence alignment of PfHPPD, AtHPPD, and AfHPPD.\nCompared to AtHPPD, PfHPPD has a higher identify and similarity with AfHPPD.\nFor the active center, the important metal binding domains are analyzed. PfHPPD and\nAtHPPD bind Fe2+, while AfHPPD bind Cu2+. They are all composed of H-H-E\nmotifs.\nResponses to Reviewer 2\nReviewer #2 (Remarks to the Author):\nThe authors carefully revised the manuscript, clarified the methodology, improved the\nrepresentation of their data and corrected some inconsistencies. Thus, the manuscript\nappears ready for publication.\nResponse: Thank you for your positive comments. We appreciate very much this\nreviewer for the recognition of our manuscript.\nResponses to Reviewer 4\nReviewer #4 (Remarks to the Author):\nThe manuscript has been substantially improved and is now supported by a much\nstronger scientific framework. The addition of key structural experiments significantly\nstrengthens the main conclusions, and the discussion is more rigorous and better\nintegrated with previously published data from multiple independent groups.\nThe purification of the material, however, remains inherently complex: although the\nnitrogen content is reported to be approximately 5%, the nitrogen-containing\ncomponents may differ in molecular mass, and their contribution to the final purified\nmelanin preparation is therefore not necessarily linear. However, this limitation reflects\na broader methodological challenge in the field rather than a specific weakness of this\nstudy. Overall, and within my area of expertise all our questions were addressed.\nResponse: Thank you for your positive comments on our revised manuscript,\nespecially the suggestions on characterization and purification of pyomelanin. Indeed,\nthe purification of melanin is a technical challenge faced by the entire industry. We\ndefinitely should spend more time and effort in our future research on melanin to try to\noptimize the purification scheme. In addition, as suggested, we have added addtional\ndiscussion in our revised manuscript to stimulate readers' interest and attract the\nattention of other melanin research groups (below).\nLines 832-833: “In fact, the purification of pyomelanin remains a technical challenge\nin the entire field.”\nLines 836-837: “and (3) the strategies to obtain purer pyomelanin for application\nresearch.”\nResponses to Reviewer 5\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank this reviewer for the time and helpful comments during the review\nof our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns."}, "author_response": "Responses to four reviewers’ comments (NCOMMS-25-76159C)\nDear Reviewers:\nWe appreciate very much all your constructive comments and suggestions, which\ndefinitely gave us helpful guidance to improve our manuscript. We have now revised\nour manuscript accordingly. For your reference, the changes in the revised manuscript\nare highlighted in red. We have also provided a point-by-point response (below) with\ndetailed explanations. We are willing to make further changes upon your request.\nResponses to Reviewer 1\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my concerns.\nResponse: Thank you for the positive comments."}]

Decision letter