Potential of Aspergillus oryzae as a biosynthetic platform for indigoidine, a non-ribosomal peptide pigment with antioxidant activity

Sarocha Panchanawaporn; Chanikul Chutrakul; Sukanya Jeennor; Jutamas Anantayanon; Nakul Rattanaphan; Kobkul Laoteng

doi:10.1371/journal.pone.0270359

Abstract

The growing demand for natural pigments in the industrial sector is a significant driving force in the development of production processes. The production of natural blue pigments, which have wide industrial applications, using microbial systems has been gaining significant attention. In this study, we used Aspergillus oryzae as a platform cell factory to produce the blue pigment indigoidine (InK), by genetic manipulation of its non-ribosomal peptide synthetase system to overexpress the indigoidine synthetase gene (AoinK). Phenotypic analysis showed that InK production from the engineered strain was growth associated, owing to the constitutive control of gene expression. Furthermore, the initial pH, temperature, and glutamine and MgSO₄ concentrations were key factors affecting InK production by the engineered strain. The pigment secretion was enhanced by addition of 1% Tween 80 solution to the culture medium. The maximum titer of total InK was 1409.22 ± 95.33 mg/L, and the maximum productivity was 265.09 ± 14.74 mg/L·d. Moreover, the recombinant InK produced by the engineered strain exhibited antioxidant activity. These results indicate that A. oryzae has the potential to be used as a fungal platform for overproduction of extracellular non-ribosomal peptide pigments.

Citation: Panchanawaporn S, Chutrakul C, Jeennor S, Anantayanon J, Rattanaphan N, Laoteng K (2022) Potential of Aspergillus oryzae as a biosynthetic platform for indigoidine, a non-ribosomal peptide pigment with antioxidant activity. PLoS ONE 17(6): e0270359. https://doi.org/10.1371/journal.pone.0270359

Editor: Daniel Cullen, USDA Forest Service, UNITED STATES

Received: March 6, 2022; Accepted: June 8, 2022; Published: June 23, 2022

Copyright: © 2022 Panchanawaporn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting information files.

Funding: 1. Chanikul Chutrakul (Grant No. RSA6280066) from Thailand Science Research and Innovation (TSRI), Thailand. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 2. Kobkul Laoteng (Grant No. P19-52263) from Targeted Research Grant of Functional Ingredients and Food Innovation, National Science and Technology Development Agency (NSTDA), Thailand. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Pigments are widely used in food, cosmetic, and textile industries to produce diverse colored products. The colorant market has been continually expanding worldwide [1–3]. However, because of concerns of the impact of synthetic colorants on health and environment, some synthetic colorants are banned. Evidence indicated that these synthetic colorants caused hyper allergenicity, carcinogenicity, and other toxicological problems [4, 5]. Plants and microorganisms are the main sources of natural pigments. However, the production of plant pigments has several drawbacks, such as land-use requirements, plant diseases, weather-dependent yields, and post-harvest processing. Microbial strains capable of biosynthesis of pigments provide a valuable alternative and are readily accessible for commercial production. Simple and feasible fermentation and downstream processes for microbial pigment production offer advantages over traditional pigment production from plant sources [1, 6, 7]. Red, orange, and yellow are common pigment shades produced by bacteria, yeast, and microalgae [8]. However, there is a high demand for other colors of spectra, such as blue shades, becuase of the scarcity of stable natural blue pigments in the market [9].

Indigoidine [5,5’-diamino-4,4’-dihydroxy-3,3’-diazadiphenoguinone-(2,2’)] is a water-insoluble dark blue pigment belonging to the pyridine class, which consists of two L-glutamine residues. However, the intrinsic mechanisms involved in the production of indigoidine remain elusive. Further research is required to elucidate the potential commercial applications of indigoidine. Indigoidine acts as both an antimicrobial substance and cryoprotectant [10, 11]. Based on the two-pyridone ring structure with carbonyl and amino groups, it has been postulated that the pigment possesses antiradical and antioxidant activities [12, 13]. Indigoidine is naturally produced by several groups of bacteria during the non-growth phase of cultivation [10, 13–17].

Indigoidine synthetase (INDS), a member of the non-ribosomal peptide synthetase (NRPS) family, biosynthesizes indigoidine by catalyzing the thioesterification of the two L-glutamines. In fact, the post-translational modification process is required for the conversion of inactive apo-form of INDS to the active holo-form. This evidence is accessed by introducing a coenzyme A-derived moiety into the thiolation (T)-domain of the enzyme with actions of a co-factor and 4’-phosphopantetheinyl transferase (PPTase) activity [17, 18]. When the INDS is become active, the biosynthesis of indigoidine is initiated by cyclization of a single molecule of glutamine through an intramolecular amide bond. The amino acid intermediate is then dehydrogenated, most likely by oxidation. Finally, the two modified cyclic glutamines condense to form the pigment [13, 19]. However, the pigment production yield from exotic microbial strains is comparatively low, which is impractical for industrial production. Genetic recombination improved the rate of indigoidine production in diverse microbes such as Escherichia coli, Corynebacterium glutamicum, and yeasts [16, 18, 20–22]. Among these genetic modified strains, a high titer of indigoidine was acquired by the overexpression of INDS and other relevant genes in conjunction with optimized fermentation conditions. However, some host systems may not be suitable for producing dipeptide pigments, because of the absence of a complex NRPS system. Aditionally, most pigments are intracellular metabolite products that require downstream processing, including cell disruption and extraction processes.

Aspergillus oryzae is a filamentous fungus that is generally recognized as safe (GRAS) [23]. A. oryzae has an amino acid precursor pool and a complete NRPS system; therefore, this fungus has been successfully used to produce functional peptides, such as β-lactam antibiotics [24]. Similar to other eukaryotes, this filamentous fungus is the host of choice for improving metabolite biosynthesis by self-tolerance mechanisms. These mechanisms include the transport of specific compounds to storage organelles such as vacuoles, modification of metabolites to generate less toxic forms, and cellular release of metabolites [25, 26]. In addition, studies have been conducted on the effects of nonionic surfactants, such as polyethylene glycol (PEG), Triton X, Tween, and Tergitol, on cell membrane structure, particularly on membrane integrity and permeability. These studies led to the development of the process for production of extracellular metabolites from filamentous fungi [27–31]. Recently, a fungal platform for oligopeptide production from A. oryzae has been established, by genetic manipulation of the endogenous NRPS system [32]. However, pigment production by using an NRPS-assisted mechanism in filamentous fungi has not yet been established.

Using a gene-editing approach, indigoidine production using A. oryzae was investigated in this study. The engineered strain of A. oryzae was constructed by overexpressing the gene encoding INDS, and the pigment production yield was evaluated under different variables in fungal fermentation. Furthermore, the pigment permeability with regard to extracellular production and cell morphology was investigated. This study explored the non-ribosomal peptide (NRP) biopigment overproduction by a filamentous fungal system through synthetic biology and physiological manipulation.

Materials and methods

Microbial strains and cultivations

The pyrG-deficient strain A. oryzae BCC 7051 was used as the recipient in this study [32]. The fungal strain was maintained on Czapek Dox medium supplemented with 0.5% (w/v) uridine and 0.2% (w/v) uracil. Spore inoculum was prepared by cultivating the fungal cells on rice medium at 30°C for 5–7 d; 0.05% (v/v) Tween 80 solution was used to harvest the spores. Spore suspension at a final concentration of 2 × 10⁶ spores/mL was inoculated into a 250-mL Erlenmeyer flask. The flask contained 50 mL of a semi-synthetic medium (SM), consisting of 4.0% (w/v) glucose, 0.5% (w/v) yeast extract, 0.02% (w/v) NH₄Cl, 0.24% (w/v) KH₂PO₄, 0.05% (w/v) MgSO₄ · 7H₂O, 0.01% (w/v) CaCl₂ · 2H₂O, 0.0015% (w/v) FeCl₃ · 7H₂O, 0.001% (w/v) MnSO₄ · H₂O, and 0.008% (w/v) ZnSO₄ · 7H₂O) [33]. Cultures were incubated at 30°C in a rotary shaker at 200 rpm. Mycelial cells were harvested at different time points to determine biomass, residual glucose, and blue pigment concentrations.

E. coli DH5α (supE44, ΔlacU169, (Φ80, lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi1, relA1) (Thermo Fisher Scientific, USA) was used as the recipient for propagation of the constructed recombinant plasmids. E. coli transformant was grown in Luria–Bertani (LB) medium containing 100 mg/L ampicillin at 37°C with shaking at 200 rpm.

Saccharomyces cerevisiae strain INVSCI (MATα, his3-Δ1, leu2, trp1-289, ura3-52, MAT, his3-Δ1, leu2, trp1-289, ura3-52) (Invitrogen, USA) was used for plasmid construction. The yeast was grown in either a complete medium, YPD (consisting of 1.0% (w/v) bacto-yeast extract, 2.0% (w/v) bacto-peptone, and 2.0% (w/v) glucose), or a selective medium, SD (consisting of 0.67% (w/v) bacto-yeast nitrogen base and 2.0% (w/v) glucose). Both media were supplemented with L-tryptophan (20 mg/L), L-histidine hydrochloride (20 mg/L), and L-leucine (30 mg/L). Yeast cultures were incubated at 30°C with shaking at 200 rpm.

Construction of the indigoidine-producing strain of A. oryzae (AoInK)

The 4134-bp DNA sequence of A. oryzae (AoinK) was designed from the coding region of the ScindC gene of Streptomyces chromofuscus (S1 Table). The design was based on the general rule of RNA stability and the codon usage of A. oryzae using the OptimumGene^™ algorithm by Genscript (Piscataway, USA). We synthesized the AoinK fragment using Genscript and then subcloned it into the pUC57 plasmid. DNA fragments, including the gpdA promoter of A. nidulans (AnPgpdA), AoinK, nos3 terminator of Agrobacterium tumefaciens, and AfpyrG marker, were prepared by PCR, using Platinum^™Taq DNA Hi Fidelity polymerase (Invitrogen, USA). Using 20-bp overlapping primer sets (S2 Table), 4 DNA fragments and EcoRI-linearized pPNGB plasmid [34] were co-transformed into S. cerevisiae cells using the PEG/lithium acetate method [35]. The resulting plasmid, pAoInK-pyrG, was extracted from the yeast transformant using Zymoprep^™ Yeast Plasmid Miniprep II (Zymo Research, USA) and then shuttled into E. coli for propagation. Restriction enzyme analysis and DNA sequencing were performed to verify the accuracy of plasmid construction. The construction scheme for the pAoInK-pyrG plasmid is shown in S1 Fig.

The constructed plasmid (1–3 μg) was transformed into the pyrG-deficient strain of A. oryzae using the PEG-mediated method (PMT) [36–38] with a modification to the enzyme cocktail concentration. Fungal protoplast cells were prepared and suspended in ice-cold sorbitol / Tris–HCl / CaCl₂ solution until they were used for DNA transformation. The transformed protoplasts were regenerated in potato dextrose broth with 1.2 M sorbitol for 2 h. The protoplasts were then immediately plated on Czapek Dox medium without nutrient supplementation. After incubation at 30°C for 10–14 d, the transformant colonies were selected based on the dark blue phenotype. Spore reisolation was conducted to obtain a pure colony of the AoInK strain.

Submerged fermentation of AoInK strain

For submerged fermentation, we used SM as the basal medium and performed fungal cultivation for 5 d. Initial pHs (4.5, 5.5, 6.5, and 7.5) were the variables for indigoidine production by the AoInK strain. Furthermore, the effects of culture temperatures (25°C, 30°C, and 35°C) were further investigated by using the optimal initial pH value. Subsequently, under the optimal conditions of temperature and initial pH, we further investigated the effects of L-glutamine concentrations (2, 5, and 10 mM) and MgSO₄ concentrations (2, 5, 10, and 50 mM), for optimization of indigoidine production by the AoInK strain.

Effect of nonionic surfactants on extracellular pigment production

The effects of polyethylene glycol-4000 (PEG-4000), Tergitol NP-40, TritonX-100, and Tween 80 (Sigma-Aldrich, USA) at different concentrations (1% (w/v), 3% (w/v), and 5% (w/v)) were investigated. Each preparation was individually added to the culture medium. Fungal cultures were grown at 25°C for 5 d with shaking at 200 rpm. The following fermentation parameters were assessed: biomass yield (g/L), InK yield on glucose (Y_p/S, mg/g), InK yield on biomass (Y_p/x, mg/g), and InK productivity (mg/L·d).

Determination of biomass titer and residual glucose concentration

The culture samples were filtered to separate the mycelial cells and fermented broth through Miracloth (Merck Millipore, Germany). Mycelial cells were hot-air-dried until their weight was constant. The residual glucose concentration in the fermented broth was measured using a high-performance liquid chromatography (HPLC) column (Ultimate 3000, Thermo, USA) equipped with a refractive index detector and an Aminex^® HPX-87H ion exclusion column (300 × 7.8 mm, 9-μm particle size, Bio-Rad Laboratories, USA), under isocratic mode with 18 mM H₂SO₄ solution as the mobile phase at a flow rate of 0.6 mL/min at 60°C for 30 min. Residual glucose concentration was calculated using a calibration curve of glucose standard at concentrations of 0.1–10.0 mg/mL.

Extraction and quantification of indigoidine

Fresh fungal mycelia (50 mg) were ground with liquid nitrogen and then subjected to extraction of indigoidine by adding 1 mL of dimethyl sulfoxide (DMSO) with sonication, as previously described [18]. For the extracellular indigoidine extraction, the cell-free broth was diluted with 100% (v/v) DMSO solution. The prepared samples were subjected to quantification of indigoidine using a spectrophotometer (Jasco V-730BIO) at a wavelength of 612 nm. A calibration curve for calculating pigment concentration was generated using an indigoidine standard with known concentrations (ViabLife, PRC).

To analyze the indigoidine pigment, 10 μL of the sample dissolved in DMSO was subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) column (Ultimate 3000, Thermo, USA) equipped with a diode-array detector (DAD) and C-18 Acclaim^™120 column (4.6× 250 mm, 5-mm particle size, Bio-Rad Laboratories, USA). The gradient system was set up according to a previous report [32], with a slight modification to methanol concentration gradient. The pigment was monitored at 600 nm with a running time of 20 min and then compared with the standard.

Determination of total phenolic content (TPC) and antioxidant activity

TPC was determined calorimetrically using a modified Folin-Ciocalteau (FC) method [39]. The cell-free broth (0.5 mL) was added to 0.2 mL of FC reagent, and the sample was allowed to stand at room temperature for 10 min. Thereafter, 0.6 mL of 20% sodium carbonate was added to the mixture, followed by incubation at 40°C for 30 min. The absorbance measurement was performed at 765 nm. Deionized water was used as the blank, and FC reagent was used as the control. Gallic acid was used as a standard to generate a calibration curve, and the TPC was expressed as grams of gallic acid equivalents per liter.

The antioxidant activity was evaluated by using a DPPH free-radical-scavenging assay according to a previous report [40]. The cell-free broth (0.5 mL) was mixed with 1.0 mL of 0.1 mM DPPH. The decolorization of DPPH was measured as the decrease in absorbance at 517 nm. Trolox (6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as a standard. The DPPH free-radical-scavenging activity was calculated as follows: where A0 is the light absorbance of the DPPH control sample, A1 is the light absorbance of the reaction mixture sample, and A2 is the light absorbance of the sample with deionized water (no DPPH). The half- maximal inhibitory concentration (IC₅₀, μg/mL) was determined from the plot of DPPH-scavenging activity against InK concentrations.

Image analysis of fungal morphology

Using a bright-field microscope, the mycelial morphology and intracellular pigment distribution of the AoInK culture treated with Tween 80 were compared with those of the untreated culture. Fungal mycelia that were grown for 1, 3, and 5 d were image analyzed using an Olympus BX53/DP73 microscope coupled with licensed CellSens Standard software. A mycelial sample from the recipient (non-pigmented strain) was used as a control.

Statistical analysis

Statistical analysis was performed using SPSS version 11.5 software for Windows (SPSS Inc., USA). Each experiment was performed in triplicate. The results are presented as mean values with standard deviation (mean ± SD). The data were subjected to one-way analysis of variance (ANOVA), and compared using Duncan’s multiple range test (DMRT) with statistically significant difference (p-value < 0.05).

Results

Overexpression of the indigoidine synthetase gene in A. oryzae

The 4134-bp codon-optimized sequence (AoinK) designed from the ScindC was functionally overexpressed in A. oryzae under the constitutive control of AnPgpdA promoter, generating the AoInK strain. The functional activity of the InK multi-functional synthetase was clearly observed as a dark blue colony of the engineered strain grown on solid agar, as shown in Fig 1A (left panel). After submerged fermentation, the fungal culture appeared dark blue, as shown in Fig 1B (left panel), in contrast to the control (recipient strain). A clear brilliant blue solution of DMSO extract of mycelial cells of the AoInK strain was also observed, as shown in Fig 1C, exhibiting a spectral peak with visible light absorption at 612 nm (Fig 1D. RP-HPLC analysis indicated that the indigoidine pigment was detected only in the AoInK culture, as shown in S2 Fig. Pigment production during growth of the engineered strain in the basal SM medium showed that the total InK titer increased with the increase in biomass titer. The maximal titer (287 mg/L) was obtained in the 5-d culture, in which glucose was exhausted (S3 Fig).

Download:

Fig 1. Phenotypic characteristics of the engineered AoInK strain and InK production.

Colony growth of the engineered strain (left) and recipient (right) on Czapek Dox agar for 5 d (A). Submerged cultures of the engineered strain with dark blue color (left panel) and the recipient as a control (right panel) (B). The color of DMSO extract of AoInK mycelia (left panel) in comparison with that of the control (right panel) (C). Ultraviolet-visible spectrum of the indigoidine extract of the AoInK strain (D).

https://doi.org/10.1371/journal.pone.0270359.g001

Indigoidine production from the AoInK strain at different cultivation conditions

At different initial pH conditions for fungal cultivation, a dark blue pigment was observed in all cultures of the engineered strain of A. oryzae. The optimal condition for total InK production by the AoInK strain was pH 6.5–7.5, yielding an indigoidine titer of 365–408 mg/L, which is approximately 34–41% higher than the titer of the culture with an optimal pH of 5.5 for biomass production (241 mg/L indigoidine) as shown in Fig 2A. At initial pH 6.5, the InK titer of the AoInK culture was significantly improved when grown at 25°C, showing a two-fold increase (853 mg/L) compared to the pigment production at 30°C, as shown in Fig 2B. However, the InK titer decreased markedly when grown at 35°C.

Download:

Fig 2. Effect of cultivation conditions on cell growth and InK production of the AoInK strain.

All fungal cultures were grown in selective medium (SM) with agitation at 200 rpm for 5 d. Cultures grown with varying pH values at 30°C (A). Fungal cultures grown at initial pH 6.5 at varying temperatures (B). Fungal cultures grown at initial pH 6.5 and then supplemented with varying concentrations of glutamine and incubated at 25°C (C). Fungal cultures supplemented with 2 mM glutamine, grown at initial pH 6.5, and supplemented with varying MgSO₄ concentrations (D). Dry biomass (gray circle) and intracellular and extracellular InK titers (white and black bars, respectively) are shown. Letters above the symbol and inside the bars indicate the significant difference in dry biomass and total InK titers (p < 0.05), respectively.

https://doi.org/10.1371/journal.pone.0270359.g002

Glutamine is a direct precursor for the biosynthesis of InK. The effect of glutamine supplementation on pigment production in the engineered strain was investigated by growing the fungus in a basal medium (pH 6.5) at 25°C. Although InK was generated in the culture without glutamine supplementation, pigment production was significantly enhanced by the addition of optimal concentrations of glutamine (2 and 5 mM), as shown in Fig 2C. Although biomass production was enhanced with increase in glutamine concentrations, an overly high glutamine concentration (10 mM) negatively contributed to the InK titer.

Magnesium (Mg²⁺) is a co-factor involved in the conversion of INDS to its active form. The concentration of this divalent cation may affect pigment production by the AoInK strain. The fungal cultures were supplemented with various MgSO₄ concentrations. The results of experiments with varying MgSO₄ concentrations showed that there was not much difference in biomass titers among the cultures with different MgSO₄ concentrations. However, the addition of 2 mM MgSO₄ was sufficient for pigment production, as clearly illustrated by the intracellular indigoidine titer (Fig 2D). Notably, a substantial amount of indigoidine accumulated in the fungal cells, with the ratio of intracellular and extracellular indigoidine concentrations varying from 5:1 to 24:1, depending on the cultivation conditions (Fig 2).

Effect of nonionic surfactants on extracellular production of indigoidine

The effect of nonionic surfactants on InK pigment secretion by the AoInK cells was studied. As shown in Fig 3, the extracellular InK titers of the AoInK cultures with 2 mM glutamine were enhanced by all the nonionic surfactants that were evaluated. Triton X-100 and Tween 80 solutions could promote extracellular InK titers compared to other surfactants. The amount of secreted InK was dependent on the concentration of nonionic surfactants. High concentration of Triton X-100 (3% and 5%) negatively affected total pigment (intracellular and extracellular indigoidine) titers and biomass, whereas the maximal biomass and total InK titers were detected in the fungal culture treated with 3% Tween 80 (Fig 3). Consequently, a 5–6-fold increase in extracellular pigment was achieved by the addition of 1% and 3% Tween 80 solution as compared with the untreated culture. Supplementation with Triton X-100 at a low concentration (1%) also induced extracellular pigment secretion from mycelial cells of the AoInK strain.

Download:

Fig 3. Cellular growth and extracellular InK production of AoInK cultures treated with nonionic surfactants.

Dry biomass (gray circle) and intracellular and extracellular InK titers (white and black bars, respectively) of the AoInK cultures treated with different nonionic surfactants. Letters above the symbol and inside the bars indicate significant difference in dry biomass and the total InK titers (p < 0.05), respectively.

https://doi.org/10.1371/journal.pone.0270359.g003

The cell growth and pigment production profiles of the AoInK strain were analyzed. When cultures were supplemented with 2 mM glutamine and 1% Tween 80, the total indigoidine titer increased with the increase in cultivation time. The maximal level of total indigoidine (1409.22 ± 95.33 mg/L) was found at the end of the growth phase (5-d cultivation, Fig 4), indicating that this metabolite was growth associated. The biomass titer of the engineered strain was lower than that of the recipient strain. However, the growth profiles of both cultures were similar (S4 Fig). The pigment production yields of the AoInK culture supplemented with 2 mM glutamine and 1% Tween 80 were 37.66 ± 1.23 mg/g glucose and 90.29 ± 5.63 mg/g dry biomass which were 4–5-fold higher than those of the basal SM culture (7.17±1.20 mg/g glucose and 20.36±1.99 mg/g dry biomass, respectively). Moreover, the InK productivity of the culture grown under the optimized condition was 265.09 ± 14.74 mg/L·d which was also an approximate 4–5-fold increase when compared to culture grown in basal SM medium (57.37 ± 9.58 mg/L·d).

Download:

Fig 4. Cellular growth and extracellular InK production profiles of AoInK cultures treated with Tween 80.

Fungal cultures were grown in SM medium supplemented with 2 mM glutamine and 1% Tween 80 at 25°C, 200 rpm. The samples were taken at different cultivation times for analyses. Dry biomass (gray circle), residual glucose concentration (white square), and intracellular and extracellular InK titers (white and black bars, respectively) are shown. Letters above the bars indicate significant differences in total InK titer (p < 0.05).

https://doi.org/10.1371/journal.pone.0270359.g004

TPC and antioxidant activity of InK from the AoInK strain

With regard to the proposed antiradical and antioxidant properties of InK, as described above, the TPC and antioxidant activity of the 1% Tween 80-treated and untreated cultures of the AoInK strain were investigated. Results of the investigation into the antiradical and antioxidant properties based on the structural features of InK showed that the TPC of both cultures increased with the cultivation time (Fig 5A). The TPC of the Tween 80-treated culture was higher than that of the culture without the nonionic treatment, which corresponded to the pigment titers. The highest TPC (0.31 ± 0.01 g/L) was obtained in the Tween 80-treated culture grown for 5 d. In contrast, an extremely low TPC was measured for the recipient strain, which was unable to produce InK. The pigment that was extracted from the 5-d culture with Tween 80 treatment showed the highest scavenging rate (90.68 ± 3.07%, Fig 5B). The IC₅₀ value representing the concentration of InK that caused 50% neutralization of DPPH radicals was 38.89 ± 1.56 μg/mL (S5A Fig); this value was closely related to the value of the indigoidine standard (39.20 ± 0.06 μg/mL, S5B Fig). These results suggest that the recombinant InK produced by the AoInK strain showed antioxidant activity and that the inhibitory scavenging efficiency of this pigment was reliable for the commercial InK product.

Download:

Fig 5. Total phenolic content and antioxidant activity of InK derived from the engineered strain.

Total phenolic content of InK in cell-free broth of AoInK cultures grown at different cultivation times (A). Scavenging activity of InK (B) in cell-free broth of AoInK cultures grown at different cultivation times. Smooth and dotted lines indicate the cultures of AoInK and recipient strains, respectively. Black circles represent 1% Tween-treated cultures; white circles represent untreated cultures. Letters above the circles indicate the significant differences in measured values (p < 0.05).

https://doi.org/10.1371/journal.pone.0270359.g005

Image analysis of the indigoidine-producing strain of A. oryzae

Through image analysis under a bright field microscope, blue pigment granules were observed in the mycelial cells of AoInK strain in both Tween 80-treated and untreated cultures (Fig 6). High pigment accumulation, as illustrated by color intensity, was clearly detected in the 1% Tween 80-treated culture grown for 5 d (Fig 6, panel A), when compared with the untreated culture (Fig 6, panel B). The hyphal surface was attached by dark blue granules (black arrows), particularly in the AoInK culture with 1% Tween 80 treatment. This result indicated that pigment secretion occurred from the mycelial cells along the growth stages, which coincided with the InK titers, as shown in Fig 4. It seems that secretion might occur from the sub-apical hyphae (Fig 6A and 6B, day 1). Swollen hyphae with vacuoles inside (red arrows) were observed only in the pigment-producing strain without Tween 80 treatment (Fig 6B, days 3 and 5), but were not detected in the Tween 80 treatment throughout the cultivation period (Fig 6A).

Download:

Fig 6. Image analysis of the mycelia of AoInK cultures treated with 1% Tween 80.

Mycelial cultures of AoInk and recipient strains were grown at 25°C, 200 rpm, and the samples were taken at different cultivation times for image analysis using the Olympus BX53/DP73 bright field microscope (100× magnification). Tween 80-treated mycelia of the AoInK strain (A). Untreated mycelia of the AoInK strain (B). Black arrows indicate blue pigment granules attached to cell surface, and red arrows indicate swollen hyphae containing vacuoles. Tween 80-treated mycelial cells of the recipient culture (C). Untreated mycelial cells of the recipient culture (D). Data represent three independent experiments.

https://doi.org/10.1371/journal.pone.0270359.g006

Discussion

The NRPS system has been discovered in diverse microbial species through the genetic evolution of bacteria to higher eukaryotes via horizontal gene transfer [41–43] and is involved in cell growth and adaptation [44]. Recently, the development of oligopeptide production in A. oryzae using the NRPS system has been reported, exploring the potential of this complex system for gene editing in filamentous fungi [32]. In this study, the nrps gene (ScindC) encoding the mega-synthetase for indigoidine biosynthesis was chosen as the target for indigoidine overexpression in A. oryzae. The engineered strain was able to synthesize the dipeptide pigment during cultivation, verifying the functional NRPS machinery of this fungus to support foreign dipeptide pigment production. However, the concomitant increase in InK and biomass titers during cell growth (Fig 4) indicated that this dipeptide pigment was a growth-related metabolite in the AoInK strain. This was a result of constitutive gene expression under the control of AnPgpdA promoter. The intracellular pigment product might slightly affect cell growth, as indicated by the low biomass titer compared with the recipient (S4 Fig). The dipeptide is small and does not form misfolded structures or toxic aggregates when compared to high-molecular weight proteins [45]. This suggests that the dipeptide pigment metabolite does not have a direct negative impact on fungal growth. Furthermore, the chemical structure of the pigment indicates its involvement in free radical scavenging. The presence of hydroxyl and/or amino group(s) in phenolic, enolic, or anilinic compounds (as presented in the InK structure) has been proven for hydroxyl-radical-scavenging activity [12]. Our results verified the biological function of the recombinant pigment produced by the engineered strain of A. oryzae and showed that the antioxidant activity was retained.

The production of dipeptide pigment in A. oryzae can be improved through physiological manipulation. In fact, the optimal cultivation conditions for heterologous production of targeted metabolites are species dependent, underlying the metabolic regulation in host cells. Usually, acidic conditions are preferred for A. oryzae growth [46] but not for indigoidine production by the AoInK strain, in which the ionic balance might contribute to the activities of enzymes involved in pigment biosynthesis. Comparable result has been previously reported for Penicillium purpurogenum [47]. The significant effect of low temperature on the InK titer of the engineered strain may be explained by the fact that regulation of fungal secondary metabolism is reliable for low-temperature-responsive transcriptional factors and biosynthetic enzyme stability [48]. The supplementation of high glutamine concentrations in the culture might shift the metabolic flux toward biomass production rather than pigment biosynthesis. We suggest that glutamine should be allocated at an optimal level to maximize the InK production yield. The role of a divalent cation agent (Mg²⁺) in pigment production by the AoInK strain was also explored. We found that Mg²⁺ is involved in the regulation of secondary metabolism, consistent with a previous report [49]. Mg²⁺ is required for 4ʹ-PPTase activity to convert inactive INDS to the active form for metabolite biosynthesis. We postulate that magnesium is a co-factor ion for the activation of INDS, based on our finding that the optimal concentration of magnesium (2 mM) was required to enhance pigment production by the engineered strain. A magnesium concentration lower than the optimal level might be insufficient for 4ʹ-PPTase activity, and reduced production may result from the feedback inhibition of phosphopantetheinylation, when the magnesium concentration is excessive.

Although the blue pigment mostly accumulated in the cells, its secretion corresponding to the increase in total InK content might be attributed to overproduction, particularly in the 5-d culture. In filamentous fungi, the mechanism controlling metabolite secretion, particularly that of secondary metabolites, has not been elucidated. A scheme for secondary metabolite production and secretion by filamentous fungi has been previously reported [50]. Studies have proven that the secondary metabolite biosynthesis particularly in filamentous fungi is generated in endosomes or peroxisomes, wherein synthetases and other tailor-made enzymes exist. Apart from the transport system via plasma membrane transporters, the secretion of certain metabolites can be coupled with the classical secretory route through exocytosis, as reported for yeasts and filamentous fungi [25, 51, 52]. It is likely that the InK secretion found in the engineered strain of A. oryzae might be involved in exocytosis, as observed when the pigment was overproduced (Fig 6). The dark blue protrusions observed in the engineered strain of A. oryzae might be a result of the attachment of InK to the cell surface, similar to a previous report describing the secretion mechanism of aflatoxin and pathogenicity-related effectors via exocytosis [52–54].

Modification of the cell membrane structure is an alternative strategy to enhance production by releasing the generated protein/enzymes across the membrane. Nonionic surfactants play a role in cell membrane integrity and permeability [55, 56]. Our study demonstrated that Tween 80 enhanced total pigment production (Fig 3), which might be explained by the dual action of the nonionic surfactant. Oleic acid (C18:1 n-9), which is a constituent of Tween 80, is an alternative carbon source to support growth of the AoInK strain, thus leading to pigment overproduction [57, 58]. The enhanced pigment secretion observed was also a result of the amphipathic characteristics of Tween 80, which was attributed to the permeability of the cytoplasmic membrane, as previously described [27, 59]. The structural features of InK do not appear to be toxic to fungal cells. However, the low biomass of the AoInK strain compared to that of the recipient might have resulted from the leverage of metabolic energy for cell growth and production of the colored dipeptide. Pigment secretion is also a cellular mechanism that sustains fungal growth and reduces stress, as shown by the morphological alteration of hyphae (Fig 6).

The results of this study present a potent approach to employing A. oryzae as a platform for producing indigoidine through synthetic biology. In contrast to some bacterial and yeast cell factories [16, 22], this fungal chassis cell has a complete NRPS system that permits a feasible option for efficient synthesis of the dipeptide pigment indigoidine. Biosynthesis can be achieved with minimal manipulation by the constitutive overexpression of the single AoinK gene, as proven in this study. Furthermore, the AoInK strain was cultivated in various conditions (varying pH, temperature, nutrient concentration, and nonionic surfactant treatment); thus, our findings reveal the robust features of the AoInK strain, facilitating bioprocess development with no behavioral trade-offs affecting the InK yield. Secretion system existing in this fungus is a promising machinery for the extracellular production of foreign pigments. We suggest that this fungal system is a promising platform for manufacturing bioactive pigments as well as other extracellular secondary metabolites, including NRP compounds, demonstrating a simple harvesting process and feasibility for large-scale industrial production.

Supporting information

S1 Fig. Plasmid map of pAoInK-pyrG containing the AoinK expression cassette and pyrG selectable marker (A), and enzyme restriction analysis of the recombinant plasmid (B).

Lane 1 indicates the 2.3, 7.2 and 7.6-kb DNA fragments digested by EcoRI and NotI. Lane 2 indicates 0.9-, 6.7- and 9.4-kb DNA fragments digested by NotI and SgsI. Lane M is Thermo Scientific GeneRuler 1 kb DNA Ladder.

https://doi.org/10.1371/journal.pone.0270359.s001

(DOCX)

S2 Fig. Analysis of indigoidine in the AoInK strain.

Reversed-phase high performance liquid chromatography (RP-HPLC) analysis of indigoidine derived from the AoInK and recipient strains compared with the indigoidine (InK) standard. Arrows indicate chromatographic peaks of indigoidine with retention times (Peaks A and B).

https://doi.org/10.1371/journal.pone.0270359.s002

(DOCX)

S3 Fig. Profiling of cell growth and InK production of the AoInK culture grown in the basal SM medium.

Fungal cultures were grown at 30°C with shaking at 200 rpm. Dry biomass (grey circle), residual glucose (white circle), intracellular and extracellular InK titers (white and black bars, respectively) are shown. Letters above the bars indicate significant difference in the total InK titers (p < 0.05).

https://doi.org/10.1371/journal.pone.0270359.s003

(DOCX)

S4 Fig. Profiling of cell growth of the AoInK and recipient strains.

Fungal cultures were grown in the SM medium supplemented with 2 mM glutamine and 1% Tween 80 at 25°C, 200 rpm. The samples were taken at different cultivation times for analyses. Dry biomass titers of the AoInK (grey circle) and recipient (white circle) strains are shown. Residual glucose concentrations of the AoInK and recipient strains are represented by grey and white squares, respectively. Letters above the circles indicate significant difference in cell biomass of both strains (p < 0.05). The experiments were carried out in triplicates.

https://doi.org/10.1371/journal.pone.0270359.s004

(DOCX)

S5 Fig. Determination of IC₅₀ values of InK derived from the AoInK strain (A) and the InK standard (B).

The IC₅₀ value was determined from the plot of DPPH scavenging activity (%) against InK concentrations.

https://doi.org/10.1371/journal.pone.0270359.s005

(DOCX)

S1 Table. The codon-optimized sequence of AoinK gene.

https://doi.org/10.1371/journal.pone.0270359.s006

(DOCX)

S2 Table. Overlapping primer sets used for amplifying specific DNA fragments by PCR.

https://doi.org/10.1371/journal.pone.0270359.s007

(DOCX)

References

1. Kalra R, Conlan XA, Goel M. Fungal as a potential source of pigments: Harnessing filamentous fungi. Front Chem. 2020;8:369. pmid:32457874
- View Article
- PubMed/NCBI
- Google Scholar
2. Martins N, Roriz C, Morales P, Barros L, Ferreira ICFR. Food colorants: Challenges, opportunities and current desires of agro-industries to ensure consumer expectations and regulatory practices. Trends Food Sci Technol. 2016;52:1–15.
- View Article
- Google Scholar
3. Sigurdson GT, Tang P, Giusti MM. Natural colorants: Food colorants from natural sources. Annu Rev Food Sci Technol. 2017;8(1):261–80. pmid:28125346
- View Article
- PubMed/NCBI
- Google Scholar
4. Kobylewski S, Jacobson MF. Toxicology of food dyes. Int J Occup Environ Health. 2012;18(3):220–46. pmid:23026007
- View Article
- PubMed/NCBI
- Google Scholar
5. Price PJ, Suk WA, Freeman AE, Lane WT, Peters RL, Vernon ML, et al. In vitro and in vivo indications of the carcinogenicity and toxicity of food dyes. Int J Cancer. 1978;21(3):361–7. pmid:631937
- View Article
- PubMed/NCBI
- Google Scholar
6. Narsing Rao MP, Xiao M, Li W-J. Fungal and bacterial pigments: Secondary metabolites with wide applications. Front Microbiol. 2017;8:1113. pmid:28690593
- View Article
- PubMed/NCBI
- Google Scholar
7. Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: Current trends and future perspectives. J Food Sci Technol. 2015;52(8):4669–78. pmid:26243889
- View Article
- PubMed/NCBI
- Google Scholar
8. Tanuka S, Barrow CJ, Deshmukh SK. Microbial pigments in the food Industry—Challenges and the way forward. Front Nutr. 2019;6:7. pmid:30891448
- View Article
- PubMed/NCBI
- Google Scholar
9. Santos MCD, Bicas JL. Natural blue pigments and bikaverin. Microbiol Res. 2021;244:126653. pmid:33302226
- View Article
- PubMed/NCBI
- Google Scholar
10. Cude WN, Mooney J, Tavanaei AA, Hadden MK, Frank AM. The production of the antimicrobial secondary metabolite indigoidine contributes to competitive surface colonization in the marine roseobacter Phaeobacter sp. strain Y4I. Appl Environ Microbiol. 2012;78(14):4771–80. pmid:22582055
- View Article
- PubMed/NCBI
- Google Scholar
11. Day PA, Villalba MS, Herrero OM, Arancibia LA, Alvarez HM. Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments. Antonie Leeuwenhoek. 2017;110: 415–28. pmid:27915412
- View Article
- PubMed/NCBI
- Google Scholar
12. Ali HM, Abo-Shady A, Eldeen HAS, Soror HA, Shousha WG, Abdel-Barry OA, et al. Structural features, kinetics and SAR study of radical scavenging and antioxidant activities of phenolic and anilinic compounds. Chem Cent J. 2013;7:53. pmid:23497653
- View Article
- PubMed/NCBI
- Google Scholar
13. Reverchon S, Rouanet C, Expert D, Nasser W. Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity. J Bacteriol. 2002;184(3):654–65. pmid:11790734
- View Article
- PubMed/NCBI
- Google Scholar
14. Gromek SM, Suria AM, Fullmer MS, Garcia JL, Gogarten JP, Nyholm SV, et al. Leisingera sp. JC1, a bacterial isolate from Hawaiian bobtail squid eggs, produces indigoidine and differentially inhibits Vibrios. Front Microbiol. 2016;87(7). pmid:27660622
- View Article
- PubMed/NCBI
- Google Scholar
15. Kong L, Xu G, Liu X, Wang J, Tang Z, Cai YS, et al. Divergent biosynthesis of C-nucleoside minimycin and indigoidine in bacteria. iScience. 2019;20:430–40. pmid:31816530
- View Article
- PubMed/NCBI
- Google Scholar
16. Xu F, Gage D, Zhan J. Efficient production of indigoidine in Escherichia coli. J Ind Microbiol Biotechnol. 2015;42:1149–55. pmid:26109508
- View Article
- PubMed/NCBI
- Google Scholar
17. Yu D, Xu F, Valiente J, Wang S, Zhan J. An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue. J Ind Microbiol Biotechnol. 2013;40:159–68. pmid:23053349
- View Article
- PubMed/NCBI
- Google Scholar
18. Wehrs M, Gladden JM, Liu Y, Platz L, Prahl J-P, Moon J, et al. Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products in R. toruloides to include non-ribosomal peptides. Green Chem. 2019;21:3394–406.
- View Article
- Google Scholar
19. Pang B, Chen Y, Gan F, Yan C, Jin L, Gin JW, et al. Investigation of indigoidine synthetase reveals a conserved active-site base residue of non-ribosomal peptide synthetase oxidases. J Am Chem Soc. 2020; 142:10931–5. pmid:32510939
- View Article
- PubMed/NCBI
- Google Scholar
20. Ghiffary MR, Prabowo CPS, Sharma K, Yan Y, Lee SY, Kim HU. High-level production of the natural blue pigment indigoidine from metabolically engineered Corynebacterium glutamicum for sustainable fabric dyes. ACS Sustain Chem Eng. 2021;9(19):6613–22.
- View Article
- Google Scholar
21. Müller M, Auslander S, Auslander D, Kemmer C, Fussenegger M. A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine. Metab Eng. 2012;14:325–35. pmid:22543310
- View Article
- PubMed/NCBI
- Google Scholar
22. Wehrs M, Prahl J-P, Moon J, Li Y, Tanjore D, Keasling JD, et al. Production efciency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb Cell Fact. 2018;17:193. pmid:30545355
- View Article
- PubMed/NCBI
- Google Scholar
23. Barbesgaard P, Heldt-Hansen HP, Diderichsen B. On the safety of Aspergillus oryzae: A review. Appl Microbiol Biotechnol. 1992;36:569–72. pmid:1368061
- View Article
- PubMed/NCBI
- Google Scholar
24. Marui J, Ohashi-Kunihiro S, Ando T, Nishimura M, Koike H, Machida M. Penicillin biosynthesis in Aspergillus oryzae and its overproduction by genetic engineering. J Biosci Bioeng. 2010;110:8–11. pmid:20541108
- View Article
- PubMed/NCBI
- Google Scholar
25. Martín JF. Transport systems, intracellular traffic of intermediates and secretion of β‑lactam antibiotics in fungi. Fungal Biol Biotechnol. 2020;7(6).
- View Article
- Google Scholar
26. Shitan N. Secondary metabolites in plants: transport and self-tolerance mechanisms. Biosci Biotechnol Biochem. 2016;80(7):1283–93. pmid:26940949
- View Article
- PubMed/NCBI
- Google Scholar
27. El-Batal AI, Al Tamie MSS. Optimization of melanin production by Aspergillus oryzae and incorporation into silver nanoparticles. Der Pharm Lett. 2016;8(2):315–33.
- View Article
- Google Scholar
28. Hu Z, Zhang X, Wu Z, Qi H, Wang Z. Perstraction of intracellular pigments by submerged cultivation of Monascus in nonionic surfactant micelle aqueous solution. Appl Microbiol Biotechnol. 2012;94(1):81–9. pmid:22228260
- View Article
- PubMed/NCBI
- Google Scholar
29. Lee H, Lee YM, Heo YM, Hong J-H, Jang S, Ahn BJ, et al. Optimization of fungal enzyme production by Trichoderma harzianum KUC1716 through surfactant-induced morphological changes. Mycobiology. 2017;45(1):48–51. pmid:28435355
- View Article
- PubMed/NCBI
- Google Scholar
30. Morales-Oyervides L, Oliveira J, Sousa-Gallagher M, Méndez-Zavala A, Montañez JC. Perstraction of intracellular pigments through submerged fermentation of Talaromyces spp. in a surfactant rich media: A novel approach for enhanced pigment recovery. J Fungi. 2017;3(3):1–12. pmid:29371551
- View Article
- PubMed/NCBI
- Google Scholar
31. Reese ET, Maguire A. Surfactants as stimulants of enzyme production by microorganisms. Appl Microbiol. 1969;17(2):242–45. pmid:5813298
- View Article
- PubMed/NCBI
- Google Scholar
32. Chutrakul C, Panchanawaporn S, Jeennor S, Anantayanon J, Laoteng K. Promoter exchange of the cryptic non-ribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae. J Microbiol. 2022;60(1):47–56. pmid:34751906
- View Article
- PubMed/NCBI
- Google Scholar
33. Laoteng K, Jitsue S, Dandusitapunth Y, Cheevadhanarak S. Ethanol-induced changes in expression profiles of cell growth, fatty acid and desaturase genes of Mucor rouxii. Fungal Genet Biol. 2008;45:61–7. pmid:17532653
- View Article
- PubMed/NCBI
- Google Scholar
34. Punya J, Tachaleat A, Wattanachaisaereekul S, Haritakun R, Boonlarppradab C, Cheevadhanarak S. Functional expression of a foreign gene in Aspergillus oryzae producing new pyrone compounds. Fungal Genet Biol. 2013;50:55–62. pmid:23174282
- View Article
- PubMed/NCBI
- Google Scholar
35. Pahirulzaman KA, Williams K, Lazarus CM. A toolkit for heterologous expression of metabolic pathways in Aspergillus oryzae. Meth Enzymol. 2012;517:241–60. Epub 2012/10/23. pmid:23084942.
- View Article
- PubMed/NCBI
- Google Scholar
36. Chutrakul C, Jeennor S, Panchanawaporn S, Cheawchanlertfa P, Suttiwattanakul S, Veerana M, et al. Metabolic engineering of long chain-polyunsaturated fatty acidbiosynthetic pathway in oleaginous fungus for dihomo-gammalinolenic acid production. J Biotechnol. 2016;218:85–93. pmid:26686314
- View Article
- PubMed/NCBI
- Google Scholar
37. Malardier L, Daboussi MJ, Julien J, Roussel F, Scazzocchio C, Brygoo Y. Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum. Gene. 1989;78:147–56. pmid:2670677
- View Article
- PubMed/NCBI
- Google Scholar
38. Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW. Transformation by integration in Aspergillus nidulans. Gene. 1983;26:205–21. pmid:6368319
- View Article
- PubMed/NCBI
- Google Scholar
39. Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 1999;299:152–78.
- View Article
- Google Scholar
40. Arora DS, Chandra P. Antioxidant Activity of Aspergillus fumigatus. ISRN Pharmacol. 2011;2011:619395. pmid:22084718
- View Article
- PubMed/NCBI
- Google Scholar
41. Brakhage AA. Molecular regulation of β-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev. 1998;62(3):547–85. pmid:9729600
- View Article
- PubMed/NCBI
- Google Scholar
42. Brakhage AA, Al-Abdallah Q, Tuncher A, Sprote P. Evolution of beta-lactam biosynthesis genes and recruitment of trans-acting factors. Phytochemistry. 2005;66:1200–10. pmid:15950251
- View Article
- PubMed/NCBI
- Google Scholar
43. Kück U, Bloemendal S, Teichert I. Putting fungi to work: harvesting a cornucopia of drugs, toxins, and antibiotics. PLoS Pathogens. 2014;10(3): e1003950. pmid:24626260
- View Article
- PubMed/NCBI
- Google Scholar
44. Keller NP. Fungal secondary metabolism: Regulation, function and drug discovery. Nat Rev Microbiol. 2019;17(3):167–80. pmid:30531948
- View Article
- PubMed/NCBI
- Google Scholar
45. Wang Q, Zhong C, Xiao H. Genetic engineering of filamentous fungi for efficient protein expression and secretion. Front Bioeng Biotechnol. 2020;8. pmid:32322579
- View Article
- PubMed/NCBI
- Google Scholar
46. Gomi K. Aspergillus: Aspergillus oryzae. In: Batt CA, Tortorello ML, editors. Encyclopedia of food microbiology. 1. Poland: Elsevier; 2014. p. 92–6.
47. Méndez A, Pérez C, Montañéz JC, Martnez G, Aguilar CN. Red pigment production by Penicillium purpurogenum GH2 is influenced by pH and temperature. J Zhejiang Univ Sci B (Biomedicine & Biotechnology). 2011;12(12):961–68. pmid:22135144
- View Article
- PubMed/NCBI
- Google Scholar
48. Lind AL, Smith TD, Saterlee T, Calvo AM, Rokas A. Regulation of secondary metabolism by the velvet complex Is temperature-responsive in Aspergillus. G3 (Bethesda). 2016;6(12):4023–33. pmid:27694115
- View Article
- PubMed/NCBI
- Google Scholar
49. Thines E, Aguirre J, Foster AJ, Deising HB. Genetics of phytopathology: Secondary metabolites as virulence determinants of fungal plant pathogens. Esser K, Lüttge U, Beyschlag W, Murata J, editors: Springer, Berlin, Heidelberg; 2006.
50. Cairns TC, Zheng X, Zheng P, Sun J, Meyer V. Turning inside out: Filamentous fungal secretion and its applications in biotechnology, agriculture, and the clinic. J Fungi. 2021;7. pmid:34356914
- View Article
- PubMed/NCBI
- Google Scholar
51. Camacho E, Vij R, Chrissian C, Prados-Rosales XR, Gil D, O’Meally RN, et al. The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans. J Biol Chem. 2019 294(27):10471–89. pmid:31118223
- View Article
- PubMed/NCBI
- Google Scholar
52. Kistler HC, Broz K. Cellular compartmentalization of secondary metabolism. Front Microbiol. 2015;6. pmid:25709603
- View Article
- PubMed/NCBI
- Google Scholar
53. Chanda A, Roze LV, Linz JE. A possible role for exocytosis in aflatoxin export in Aspergillus parasiticus. Eukaryot Cell. 2010;9(11): 1724–27. pmid:20870882
- View Article
- PubMed/NCBI
- Google Scholar
54. Shoji J, Kikuma T, Kitamoto K. Vesicle trafficking, organelle functions, and unconventional secretion in fungal physiology and pathogenicity. Curr Opin Microbiol. 2010;20:1–9. pmid:24835421
- View Article
- PubMed/NCBI
- Google Scholar
55. Miozzari GF, Niederberger P, Hotter R. Permeabilization of microorganisms by Triton X-100. Anal Chem. 1978;90:220–33. pmid:365019
- View Article
- PubMed/NCBI
- Google Scholar
56. Zhao F, Yu J. L-asparaginase released from Escherichia coli cells with K₂HPO₄ and 2% (w/v) Triton X-100. Biotechnol Prog. 2001;17:490–4. pmid:11386870
- View Article
- PubMed/NCBI
- Google Scholar
57. Castro FF, Pinheiro ABP, Gerhardt ECM, Oliveira MAS, Barbosa-Tessmann IP. Production, purification, and characterization of a novel serine-esterase from Aspergillus westerdijkiae. J Basic Microbiol. 2018;58:131–43. pmid:29193163
- View Article
- PubMed/NCBI
- Google Scholar
58. Li Q, Lei Y, Hu G, Lei Y, Dan D. Effects of Tween 80 on the liquid fermentation of Lentinus edodes. Food Sci Biotechnol. 2018;27(4):1103–9. pmid:30263840
- View Article
- PubMed/NCBI
- Google Scholar
59. Pardo AG. Effect of surfactants on cellulase production by Nectria catalinensis. Curr Microbiol. 1996;33:275–8. pmid:8824176
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Kalra R, Conlan XA, Goel M. Fungal as a potential source of pigments: Harnessing filamentous fungi. Front Chem. 2020;8:369. pmid:32457874
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Martins N, Roriz C, Morales P, Barros L, Ferreira ICFR. Food colorants: Challenges, opportunities and current desires of agro-industries to ensure consumer expectations and regulatory practices. Trends Food Sci Technol. 2016;52:1–15.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref3] 3. Sigurdson GT, Tang P, Giusti MM. Natural colorants: Food colorants from natural sources. Annu Rev Food Sci Technol. 2017;8(1):261–80. pmid:28125346
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Kobylewski S, Jacobson MF. Toxicology of food dyes. Int J Occup Environ Health. 2012;18(3):220–46. pmid:23026007
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Price PJ, Suk WA, Freeman AE, Lane WT, Peters RL, Vernon ML, et al. In vitro and in vivo indications of the carcinogenicity and toxicity of food dyes. Int J Cancer. 1978;21(3):361–7. pmid:631937
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Narsing Rao MP, Xiao M, Li W-J. Fungal and bacterial pigments: Secondary metabolites with wide applications. Front Microbiol. 2017;8:1113. pmid:28690593
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: Current trends and future perspectives. J Food Sci Technol. 2015;52(8):4669–78. pmid:26243889
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Tanuka S, Barrow CJ, Deshmukh SK. Microbial pigments in the food Industry—Challenges and the way forward. Front Nutr. 2019;6:7. pmid:30891448
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Santos MCD, Bicas JL. Natural blue pigments and bikaverin. Microbiol Res. 2021;244:126653. pmid:33302226
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Cude WN, Mooney J, Tavanaei AA, Hadden MK, Frank AM. The production of the antimicrobial secondary metabolite indigoidine contributes to competitive surface colonization in the marine roseobacter Phaeobacter sp. strain Y4I. Appl Environ Microbiol. 2012;78(14):4771–80. pmid:22582055
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref11] 11. Day PA, Villalba MS, Herrero OM, Arancibia LA, Alvarez HM. Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments. Antonie Leeuwenhoek. 2017;110: 415–28. pmid:27915412
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref12] 12. Ali HM, Abo-Shady A, Eldeen HAS, Soror HA, Shousha WG, Abdel-Barry OA, et al. Structural features, kinetics and SAR study of radical scavenging and antioxidant activities of phenolic and anilinic compounds. Chem Cent J. 2013;7:53. pmid:23497653
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref13] 13. Reverchon S, Rouanet C, Expert D, Nasser W. Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity. J Bacteriol. 2002;184(3):654–65. pmid:11790734
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref14] 14. Gromek SM, Suria AM, Fullmer MS, Garcia JL, Gogarten JP, Nyholm SV, et al. Leisingera sp. JC1, a bacterial isolate from Hawaiian bobtail squid eggs, produces indigoidine and differentially inhibits Vibrios. Front Microbiol. 2016;87(7). pmid:27660622
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref15] 15. Kong L, Xu G, Liu X, Wang J, Tang Z, Cai YS, et al. Divergent biosynthesis of C-nucleoside minimycin and indigoidine in bacteria. iScience. 2019;20:430–40. pmid:31816530
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref16] 16. Xu F, Gage D, Zhan J. Efficient production of indigoidine in Escherichia coli. J Ind Microbiol Biotechnol. 2015;42:1149–55. pmid:26109508
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref17] 17. Yu D, Xu F, Valiente J, Wang S, Zhan J. An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue. J Ind Microbiol Biotechnol. 2013;40:159–68. pmid:23053349
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Wehrs M, Gladden JM, Liu Y, Platz L, Prahl J-P, Moon J, et al. Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products in R. toruloides to include non-ribosomal peptides. Green Chem. 2019;21:3394–406.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref19] 19. Pang B, Chen Y, Gan F, Yan C, Jin L, Gin JW, et al. Investigation of indigoidine synthetase reveals a conserved active-site base residue of non-ribosomal peptide synthetase oxidases. J Am Chem Soc. 2020; 142:10931–5. pmid:32510939
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref20] 20. Ghiffary MR, Prabowo CPS, Sharma K, Yan Y, Lee SY, Kim HU. High-level production of the natural blue pigment indigoidine from metabolically engineered Corynebacterium glutamicum for sustainable fabric dyes. ACS Sustain Chem Eng. 2021;9(19):6613–22.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref21] 21. Müller M, Auslander S, Auslander D, Kemmer C, Fussenegger M. A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine. Metab Eng. 2012;14:325–35. pmid:22543310
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Wehrs M, Prahl J-P, Moon J, Li Y, Tanjore D, Keasling JD, et al. Production efciency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb Cell Fact. 2018;17:193. pmid:30545355
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref23] 23. Barbesgaard P, Heldt-Hansen HP, Diderichsen B. On the safety of Aspergillus oryzae: A review. Appl Microbiol Biotechnol. 1992;36:569–72. pmid:1368061
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref24] 24. Marui J, Ohashi-Kunihiro S, Ando T, Nishimura M, Koike H, Machida M. Penicillin biosynthesis in Aspergillus oryzae and its overproduction by genetic engineering. J Biosci Bioeng. 2010;110:8–11. pmid:20541108
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Martín JF. Transport systems, intracellular traffic of intermediates and secretion of β‑lactam antibiotics in fungi. Fungal Biol Biotechnol. 2020;7(6).
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref26] 26. Shitan N. Secondary metabolites in plants: transport and self-tolerance mechanisms. Biosci Biotechnol Biochem. 2016;80(7):1283–93. pmid:26940949
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref27] 27. El-Batal AI, Al Tamie MSS. Optimization of melanin production by Aspergillus oryzae and incorporation into silver nanoparticles. Der Pharm Lett. 2016;8(2):315–33.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref28] 28. Hu Z, Zhang X, Wu Z, Qi H, Wang Z. Perstraction of intracellular pigments by submerged cultivation of Monascus in nonionic surfactant micelle aqueous solution. Appl Microbiol Biotechnol. 2012;94(1):81–9. pmid:22228260
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref29] 29. Lee H, Lee YM, Heo YM, Hong J-H, Jang S, Ahn BJ, et al. Optimization of fungal enzyme production by Trichoderma harzianum KUC1716 through surfactant-induced morphological changes. Mycobiology. 2017;45(1):48–51. pmid:28435355
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref30] 30. Morales-Oyervides L, Oliveira J, Sousa-Gallagher M, Méndez-Zavala A, Montañez JC. Perstraction of intracellular pigments through submerged fermentation of Talaromyces spp. in a surfactant rich media: A novel approach for enhanced pigment recovery. J Fungi. 2017;3(3):1–12. pmid:29371551
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref31] 31. Reese ET, Maguire A. Surfactants as stimulants of enzyme production by microorganisms. Appl Microbiol. 1969;17(2):242–45. pmid:5813298
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref32] 32. Chutrakul C, Panchanawaporn S, Jeennor S, Anantayanon J, Laoteng K. Promoter exchange of the cryptic non-ribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae. J Microbiol. 2022;60(1):47–56. pmid:34751906
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref33] 33. Laoteng K, Jitsue S, Dandusitapunth Y, Cheevadhanarak S. Ethanol-induced changes in expression profiles of cell growth, fatty acid and desaturase genes of Mucor rouxii. Fungal Genet Biol. 2008;45:61–7. pmid:17532653
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref34] 34. Punya J, Tachaleat A, Wattanachaisaereekul S, Haritakun R, Boonlarppradab C, Cheevadhanarak S. Functional expression of a foreign gene in Aspergillus oryzae producing new pyrone compounds. Fungal Genet Biol. 2013;50:55–62. pmid:23174282
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref35] 35. Pahirulzaman KA, Williams K, Lazarus CM. A toolkit for heterologous expression of metabolic pathways in Aspergillus oryzae. Meth Enzymol. 2012;517:241–60. Epub 2012/10/23. pmid:23084942.
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref36] 36. Chutrakul C, Jeennor S, Panchanawaporn S, Cheawchanlertfa P, Suttiwattanakul S, Veerana M, et al. Metabolic engineering of long chain-polyunsaturated fatty acidbiosynthetic pathway in oleaginous fungus for dihomo-gammalinolenic acid production. J Biotechnol. 2016;218:85–93. pmid:26686314
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref37] 37. Malardier L, Daboussi MJ, Julien J, Roussel F, Scazzocchio C, Brygoo Y. Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum. Gene. 1989;78:147–56. pmid:2670677
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref38] 38. Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW. Transformation by integration in Aspergillus nidulans. Gene. 1983;26:205–21. pmid:6368319
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref39] 39. Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 1999;299:152–78.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref40] 40. Arora DS, Chandra P. Antioxidant Activity of Aspergillus fumigatus. ISRN Pharmacol. 2011;2011:619395. pmid:22084718
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref41] 41. Brakhage AA. Molecular regulation of β-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev. 1998;62(3):547–85. pmid:9729600
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref42] 42. Brakhage AA, Al-Abdallah Q, Tuncher A, Sprote P. Evolution of beta-lactam biosynthesis genes and recruitment of trans-acting factors. Phytochemistry. 2005;66:1200–10. pmid:15950251
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref43] 43. Kück U, Bloemendal S, Teichert I. Putting fungi to work: harvesting a cornucopia of drugs, toxins, and antibiotics. PLoS Pathogens. 2014;10(3): e1003950. pmid:24626260
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref44] 44. Keller NP. Fungal secondary metabolism: Regulation, function and drug discovery. Nat Rev Microbiol. 2019;17(3):167–80. pmid:30531948
View Article
PubMed/NCBI
Google Scholar

[168] View Article

[169] PubMed/NCBI

[170] Google Scholar

[ref45] 45. Wang Q, Zhong C, Xiao H. Genetic engineering of filamentous fungi for efficient protein expression and secretion. Front Bioeng Biotechnol. 2020;8. pmid:32322579
View Article
PubMed/NCBI
Google Scholar

[172] View Article

[173] PubMed/NCBI

[174] Google Scholar

[ref46] 46. Gomi K. Aspergillus: Aspergillus oryzae. In: Batt CA, Tortorello ML, editors. Encyclopedia of food microbiology. 1. Poland: Elsevier; 2014. p. 92–6.

[ref47] 47. Méndez A, Pérez C, Montañéz JC, Martnez G, Aguilar CN. Red pigment production by Penicillium purpurogenum GH2 is influenced by pH and temperature. J Zhejiang Univ Sci B (Biomedicine & Biotechnology). 2011;12(12):961–68. pmid:22135144
View Article
PubMed/NCBI
Google Scholar

[177] View Article

[178] PubMed/NCBI

[179] Google Scholar

[ref48] 48. Lind AL, Smith TD, Saterlee T, Calvo AM, Rokas A. Regulation of secondary metabolism by the velvet complex Is temperature-responsive in Aspergillus. G3 (Bethesda). 2016;6(12):4023–33. pmid:27694115
View Article
PubMed/NCBI
Google Scholar

[181] View Article

[182] PubMed/NCBI

[183] Google Scholar

[ref49] 49. Thines E, Aguirre J, Foster AJ, Deising HB. Genetics of phytopathology: Secondary metabolites as virulence determinants of fungal plant pathogens. Esser K, Lüttge U, Beyschlag W, Murata J, editors: Springer, Berlin, Heidelberg; 2006.

[ref50] 50. Cairns TC, Zheng X, Zheng P, Sun J, Meyer V. Turning inside out: Filamentous fungal secretion and its applications in biotechnology, agriculture, and the clinic. J Fungi. 2021;7. pmid:34356914
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

[ref51] 51. Camacho E, Vij R, Chrissian C, Prados-Rosales XR, Gil D, O’Meally RN, et al. The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans. J Biol Chem. 2019 294(27):10471–89. pmid:31118223
View Article
PubMed/NCBI
Google Scholar

[190] View Article

[191] PubMed/NCBI

[192] Google Scholar

[ref52] 52. Kistler HC, Broz K. Cellular compartmentalization of secondary metabolism. Front Microbiol. 2015;6. pmid:25709603
View Article
PubMed/NCBI
Google Scholar

[194] View Article

[195] PubMed/NCBI

[196] Google Scholar

[ref53] 53. Chanda A, Roze LV, Linz JE. A possible role for exocytosis in aflatoxin export in Aspergillus parasiticus. Eukaryot Cell. 2010;9(11): 1724–27. pmid:20870882
View Article
PubMed/NCBI
Google Scholar

[198] View Article

[199] PubMed/NCBI

[200] Google Scholar

[ref54] 54. Shoji J, Kikuma T, Kitamoto K. Vesicle trafficking, organelle functions, and unconventional secretion in fungal physiology and pathogenicity. Curr Opin Microbiol. 2010;20:1–9. pmid:24835421
View Article
PubMed/NCBI
Google Scholar

[202] View Article

[203] PubMed/NCBI

[204] Google Scholar

[ref55] 55. Miozzari GF, Niederberger P, Hotter R. Permeabilization of microorganisms by Triton X-100. Anal Chem. 1978;90:220–33. pmid:365019
View Article
PubMed/NCBI
Google Scholar

[206] View Article

[207] PubMed/NCBI

[208] Google Scholar

[ref56] 56. Zhao F, Yu J. L-asparaginase released from Escherichia coli cells with K₂HPO₄ and 2% (w/v) Triton X-100. Biotechnol Prog. 2001;17:490–4. pmid:11386870
View Article
PubMed/NCBI
Google Scholar

[210] View Article

[211] PubMed/NCBI

[212] Google Scholar

[ref57] 57. Castro FF, Pinheiro ABP, Gerhardt ECM, Oliveira MAS, Barbosa-Tessmann IP. Production, purification, and characterization of a novel serine-esterase from Aspergillus westerdijkiae. J Basic Microbiol. 2018;58:131–43. pmid:29193163
View Article
PubMed/NCBI
Google Scholar

[214] View Article

[215] PubMed/NCBI

[216] Google Scholar

[ref58] 58. Li Q, Lei Y, Hu G, Lei Y, Dan D. Effects of Tween 80 on the liquid fermentation of Lentinus edodes. Food Sci Biotechnol. 2018;27(4):1103–9. pmid:30263840
View Article
PubMed/NCBI
Google Scholar

[218] View Article

[219] PubMed/NCBI

[220] Google Scholar

[ref59] 59. Pardo AG. Effect of surfactants on cellulase production by Nectria catalinensis. Curr Microbiol. 1996;33:275–8. pmid:8824176
View Article
PubMed/NCBI
Google Scholar

[222] View Article

[223] PubMed/NCBI

[224] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Microbial strains and cultivations

Construction of the indigoidine-producing strain of A. oryzae (AoInK)

Submerged fermentation of AoInK strain

Effect of nonionic surfactants on extracellular pigment production

Determination of biomass titer and residual glucose concentration

Extraction and quantification of indigoidine

Determination of total phenolic content (TPC) and antioxidant activity

Image analysis of fungal morphology

Statistical analysis

Results

Overexpression of the indigoidine synthetase gene in A. oryzae

Indigoidine production from the AoInK strain at different cultivation conditions

Effect of nonionic surfactants on extracellular production of indigoidine

TPC and antioxidant activity of InK from the AoInK strain

Image analysis of the indigoidine-producing strain of A. oryzae

Discussion

Supporting information

S1 Fig. Plasmid map of pAoInK-pyrG containing the AoinK expression cassette and pyrG selectable marker (A), and enzyme restriction analysis of the recombinant plasmid (B).

S2 Fig. Analysis of indigoidine in the AoInK strain.

S3 Fig. Profiling of cell growth and InK production of the AoInK culture grown in the basal SM medium.

S4 Fig. Profiling of cell growth of the AoInK and recipient strains.

S5 Fig. Determination of IC50 values of InK derived from the AoInK strain (A) and the InK standard (B).

S1 Table. The codon-optimized sequence of AoinK gene.

S2 Table. Overlapping primer sets used for amplifying specific DNA fragments by PCR.

References

S5 Fig. Determination of IC₅₀ values of InK derived from the AoInK strain (A) and the InK standard (B).