*3.5. A. resinae KUC3009 Pigment Production in Culture Filtrate*

We previously reported that *A*. *resinae* KUC3009 produced considerable amounts of melanin in the culture filtrate. The melanin synthesized by *A. resinae* KUC3009 in PYG media exhibited a high similarity with *Sepia* melanin in terms of their nitrogen content (approximately 7%) and indole-based chemical structures [8]. *Sepia* melanin is a typical type of eumelanin synthesized through the Raper–Mason pathway. L-tyrosine is a precursor, enzymatically oxidized by tyrosinase for melanin formation. Tyrosinase catalyzes the substrate using two consecutive activities of tyrosine hydroxylase and L-DOPA oxidase, resulting in the formation of L-dopaquinone [51–53]. Multi-copper oxidases, such as laccase, also can exhibit the activity of L-DOPA oxidase and be responsible for melanin formation [54].

The melanin derived from *A. resinae* slightly differed from *Sepia* melanin due to the following points; *Sepia* melanin results from the reaction of tyrosinase and L-tyrosine. However, L-tyrosine was not used in the preparation of the PYG media, and this strain cannot utilize the L-tyrosine as a substrate for melanin production (data not shown). Additionally, tyrosinase genes of *A*. *resinae* searched through gene annotation are predicted to be partial and intracellular (Supplementary File, Table S3), suggesting its activity in the culture filtrate would be insignificant in melanin formation. Instead, we focused on determining whether the pigments in the culture filtrate were formed through the laccase-like activity of multi-copper oxidase. Those enzymes are also known to possess a wide range of substrate specificity for melanin formation, and four putative complete genes predicted to have signal peptides were identified in the *A. resinae* genome (Supplementary File, Table S4).

*A. resinae* cultures were assessed based on the fungal biomass, melanin production, glucose concentrations, and laccase-like activity in culture filtrates (Figure 5). The fungal biomass increased prior to the stationary phase as the cells consumed the glucose in the culture medium. At a certain point of the stationary phase, both autolysis and melanin formation in the cultivation media occurred (Figure 5A–C). Interestingly, there was a surge in laccase-like activity prior to the melanin formation (Figure 5D–F). Interestingly, laccaselike activity was proportional to fungal biomass and melanin production in the cultivation media, and its surge exhibited a time lag as the initial glucose concentration in the media increased. The former was likely that enzyme production would be proportional to the fungal biomass, whereas the latter was possibly attributed to a glucose-suppressive effect, which has been reported in previous studies [55–58]. These results suggest a potential link between laccase-like activity in the culture filtrate and the melanin production of *A. resinae*.

The secreted enzymes can oxidize the nearby phenolic compounds to form melanins, whereas melanin can be formed via oxidation by enzymes released from the fungus during autolysis [48]. To unveil the exact mechanism of melanin formation in *A. resinae*, we induced melanin formation while keeping the cells alive by supplementing them with copper ions. As a result, both media's fungal biomass increased with the incubation time, suggesting cells in both media were in the growth phase. However, the amount of melanin and laccase-like activity were significantly high only when supplemented with copper after 3 and 6 days of cultivation (Figure 6A,B). The results indicated that melanin formation in the culture filtrate could occur before the autolysis phase, suggesting secretion of the enzyme is a key determinant in melanin formation in media.

**Figure 5.** Effect of initial glucose concentration on the *A*. *resinae* cultivation profile. Fungal biomass and glucose concentration of media when cultivated at an initial glucose concentration of (**A**) 5 g/L, (**B**) 10 g/L, and (**C**) 20 g/L. Melanin production and laccase-like activity in culture filtrate when cultivated at an initial glucose concentration of (**D**) 5 g/L, (**E**) 10 g/L, and (**F**) 20 g/L.

**Figure 6.** Effect of copper addition on *A*. *resinae* cultivation profile. (**A**) Fungal biomass and total nitrogen concentration of media. (**B**) Melanin production and laccase-like activity in the culture filtrate. (**C**) FT-IR spectrum of melanin synthesized using the culture filtrate and L-DOPA.

The substrate for melanin formation was not confirmed in this study. However, we discovered that the number of nitrogen sources in the metal-supplemented media was significantly decreased even though fungal biomass in copper-supplemented media was lower than that of normal PYG media (Figure 6A,B). Considering that the nitrogen content accounted for 7% of purified melanins derived from *A*. *resinae*, this suggests that *A*. *resinae* used nitrogen-containing substrates to synthesize melanin. Moreover, the purified melanin derived from *A. resinae* exhibited features of indolic moieties, which was likely due to the oxidation of indole-containing substrates derived from media components or metabolites derived from the strain. After removing mycelia, the culture filtrate supplemented with 1 mM CuSO4, exhibiting laccase-like activity, could synthesize the melanin using L-DOPA as a substrate. Figure 6C indicates the FT-IR spectrum of melanin synthesized using culture filtrate and L-DOPA, which exhibited a similar spectrum with that of L-DOPA melanin catalyzed by other laccases [59]. They commonly have the band at around 3400 cm−<sup>1</sup> and peak at 1650 cm−<sup>1</sup> due to the stretching vibrations of–OH and–NH2 groups and the vibrations of aromatic rings, respectively. These properties are commonly found within the melanin consisting of indole-based constituents [60].

Therefore, based on the above-described results, melanin production would likely be favored by enhanced laccase-like activity in the culture filtrate. Moreover, the selection of carbon sources and their concentrations should be prioritized to avoid glucose repression. Additionally, characterizing the effect of environmental factors on laccase-like activity is crucial.
