Modelling of Cordycepin Production by an Engineered Aspergillus oryzae Under Different Substrates

Abstract

Given the therapeutic potential of bioactive cordycepin in medical and healthcare products, precision fermentation using an engineered strain of Aspergillus oryzae was performed to enhance cordycepin production. To understand and predict the dynamics of cell growth and cordycepin production in this fungal strain, mathematical modeling of submerged fermentation was applied. The effects of different nitrogen sources (yeast extract, peptone, (NH₄)₂SO₄, NH₄Cl, NaNO₃, and KNO₃) and carbon sources (glucose and cassava starch hydrolysate, CSH) on cell growth and cordycepin production were evaluated under submerged fermentation conditions. The results showed that organic nitrogen sources significantly enhanced biomass formation and cordycepin production compared with inorganic nitrogen sources. Among them, yeast extract provided the best performance, yielding the highest biomass (13.63–15.99 g/L) and cordycepin titer (1.24–1.72 g/L). In contrast, nitrate-based nitrogen sources supported cell growth but resulted in negligible cordycepin production. Under optimized conditions in a bioreactor, both glucose and CSH supported fungal growth, although CSH promoted higher biomass formation while glucose favored cordycepin biosynthesis. The kinetic model demonstrated that the growth of engineered A. oryzae was well described by the logistic growth model (R² > 0.88). The cordycepin production profiles were well fitted by the Luedeking–Piret model (R² > 0.99), indicating a mixed growth-associated product with kinetic constants α and β representing growth-associated and non-growth-associated production, respectively. Overall, the developed kinetic model provides a quantitative framework for describing cell growth, substrate utilization, and cordycepin formation, offering guidance for process optimization and scale-up of cordycepin production in engineered fungal systems.

1. Introduction

Cordycepin (3′-deoxyadenosine) is a bioactive purine nucleoside with a wide range of pharmacological activities including antitumor, antidiabetic, anti-inflammatory, antimicrobial, antiviral, and immunomodulatory properties [1,2,3,4,5,6,7]. These properties attribute cordycepin to gain attention for increasing the body’s immunity and reducing the risk of various non-communicable diseases (NCDs), such as diabetes, cardiovascular disease, hypertension, and cancer. Consequently, it is widely used as a functional ingredient in health supplements [8]. However, most commercial cordycepin products are currently derived from the whole fruiting body of Cordyceps spp., which have relatively low cordycepin yield (1–8 mg/g cell) and require a long production period (18–75 days) [9,10,11,12,13,14]. To enhance cordycepin productivity, strain improvement and production processes have been extensively studied in both native and nonnative cordycepin producers. Among them, engineered A. oryzae strains capable of producing high levels of extracellular cordycepin in a shorter cultivation period are of great interest as potent strains for the commercial production of high-purity cordycepin [15]. The extracellular cordycepin produced by A. oryzae is highly stable under extreme atmospheres, enabling simplified downstream processing through straightforward cell separation and reduced purification requirements [16]. In addition, A. oryzae has notable metabolic flexibility in utilizing a wide range of carbon sources, making it a promising scalable host for the industrial production of cordycepin [17,18]. We systematically investigated the ability of the engineered A. oryzae strain to utilize different carbon sources for cordycepin production. This strain is capable of metabolizing a broad range of carbon sources including C5, C6, and C12 sugars. Among the carbon sources evaluated, glucose was the most efficient substrate for cordycepin production [15]. However, the effects of different organic and inorganic nitrogen sources on cordycepin production by A. oryzae have not been thoroughly investigated. Nitrogen sources play a crucial role in cell growth and regulation of nucleoside synthesis in Cordyceps spp. [19,20]. Previous studies on cordycepin-producing strains have predominantly reported that organic nitrogen sources such as peptone and yeast extract are the most suitable for cordycepin production [10,21,22,23].

However, to develop cordycepin production using an engineered A. oryzae strain with industrial relevance, the growth behavior and cordycepin production in relation to nutrient assimilation and fermentation parameters should be thoroughly investigated. Such studies provide insight into the dynamics of cell growth and cordycepin production throughout the fermentation process. Mathematical modeling is a valuable tool for describing microbial fermentation phenomena and forecasting system-level behaviors, including cell growth, substrate utilization, and product formation [24,25]. These models play crucial roles in the optimization and design of fermentation processes for various bioproducts. Mathematical models have been developed to describe the growth of A. oryzae and its production of intracellular lipids [17,18], as well as the cell growth, substrate consumption, and ethanol production by Saccharomyces cerevisiae during high-gravity fermentation of sweet sorghum juice [26]. In addition, a mathematical model was established to describe the transient heat and mass transfer during the freeze-drying process of Cordyceps militaris [27]. Although mathematical modeling is essential for understanding and predicting cell dynamics, there are currently no reports describing models specifically developed for cordycepin production in either native (Cordyceps spp.) or engineered production systems, such as the engineered A. oryzae AoCordy-T1 strain. Previous studies on cordycepin production in these strains have primarily focused on strain engineering and medium optimization, rather than on quantitative modeling [11,14,15,16,23].

The development of a dedicated mathematical model for Aspergillus oryzae AoCordy-T1 is essential for accurately describing both growth- and non-growth-associated cordycepin production, optimizing fermentation strategies, and enabling reliable process scale-up and performance prediction. Accordingly, this study aimed to develop a systematic kinetic model to quantitatively characterize cell growth, substrate utilization, and cordycepin production in engineered A. oryzae cultivated under submerged fermentation with different nitrogen sources. Given the critical structural and functional roles of nitrogen in biological systems, various nitrogen sources [yeast extract, peptone, (NH₄)₂SO₄, NH₄Cl, NaNO₃, and KNO₃] were selected as key variables. In addition, glucose and cassava starch hydrolysate (CSH) were used as carbon sources to evaluate their effects on cell growth and cordycepin production.

2. Materials and Methods

2.1. Fungal Strain and Cultivation

An engineered A. oryzae strain capable of producing cordycepin (AoCordy-T1) [15] was employed in this work. Spores were obtained by cultivating the fungal strain on polished rice at 30 °C for 5 days, followed by suspension in 0.05% (v/v) Tween 80 solution. To determine the optimal nitrogen source for cordycepin production, the spore suspension (final concentration: 1 × 10⁶ spores/mL) was used to inoculate 50 mL of semi-synthetic medium (SM) [28] containing 1 g/L adenine in Erlenmeyer flasks.

For stirred-tank bioreactor studies (Biostat B-DCU, Satorius, Göttingen, Germany), a mycelial inoculum was used and prepared by culturing the fungal spore in optimal medium [16] for 24 h at 30 °C with agitation speed 200 rpm and gas flow rate 1.0 vvm. Approximately 10% (v/v) active mycelial cells were used.

2.2. Cassava Starch Hydrolysate Preparation

Cassava starch hydrolysate (CSH) was obtained via enzymatic hydrolysis of cassava starch using α-amylase (4.0 × 10⁴ U/mL; iKnowZyme, Bangkok, Thailand) and glucoamylase (2.0 × 10⁵ U/mL; iKnowZyme, Thailand), using a procedure modified from the protocol described by Wei et al. [29]. A CSH medium, containing a final glucose concentration of 3% (v/v), was prepared by diluting the prepared CSH (30% v/v) in culture medium.

2.3. Growth and Cordycepin Production of the Cordycepin-Producing Strain Under Different Nitrogen Sources

The growth and cordycepin production of the A. oryzae engineered strain were investigated by submerged fermentation, and the kinetic parameters of the fungal fermentation were assessed. The culture medium was modified by varying the nitrogen sources, including yeast extract, peptone, (NH₄)₂SO₄, NH₄Cl, NaNO₃ and KNO₃ at a concentration of 5 g/L. Cultivation of the recombinant strains was carried out in 250 mL Erlenmeyer flasks containing 50 mL of culture medium supplemented with 1 g/L adenine, using 4% (w/v) glucose as the carbon source. Cultures were inoculated with a spore suspension at a final concentration 1 × 10⁶ spores/mL and incubated at 30 °C and 200 rpm for 4 days. For determinations of dry cell weight (DCW), glucose consumption, and the production of cordycepin, samples were collected at 24 h intervals.

To obtain comparable results among experimental sets, the kinetic parameters of fungal fermentation were determined from time zero (t₀) to a time point (t_x) corresponding to the maximum concentration of cordycepin (C_P).

2.4. Batch Fermentation of A. oryzae Engineered Strain in Lab-Scale Bioreactor

The A. oryzae engineered strain was cultivated in a 5 L stirred tank bioreactor with an initial working volume of 3 L of production medium using yeast extract as the nitrogen source. Active mycelial cells at 10% (v/v) were used as the inocula. Fungal growth and cordycepin production were compared between glucose and CSH, which were used as carbon substrates. The cultures were operated at temperature of 30 °C, agitation rate of 200 rpm and aeration rate of 1.0 vvm for 4 days. Samples were collected every 24 h to measure dry cell weight (DCW), glucose concentration, and cordycepin production.

2.5. Analytical Procedures

2.5.1. Determination of Fungal Biomass and Residual Glucose Concentration

Dry cell weight or biomass concentration was determined by filtering mycelial cells through Miracloth (EMD Chemicals, Gibbstown, NJ, USA). Subsequently, the fungal cells were dried in a hot-air oven at 60 °C until reaching a constant weight. Then, biomass concentration was reported as grams of dry cell weight per liter (g DCW/L).

Residual glucose in the fermented broths was analyzed using a high-performance liquid chromatograph (HPLC, Ultimate 3000; Thermo Scientific, Waltham, MA, USA) equipped with a refractive index detector (RID, RefractoMax 521; Thermo Scientific and an Aminex^TM HPX-87H ion-exclusion column (Bio-Rad Laboratories, Hercules, CA, USA). Separation was performed under isocratic conditions using 5 mM H₂SO₄ as the mobile phase [15], and residual glucose concentrations were determined based on comparisons with a calibration curve generated using a glucose standard.

2.5.2. Determination of Purine Nucleosides by HPLC-UV

The concentrations of cordycepin and adenine in cell-free fermentation broth of the engineered A. oryzae strain were determined using HPLC equipped with a diode array detector (DAD, VH-D10; Thermo Scientific) and a reversed-phase C18 column (5 µm, 4.6 mm × 150 mm), following a previously described method [15]. Cordycepin and adenine were identified by comparing their retention times and UV absorption spectra at 260 nm with those of authentic standards (Sigma-Aldrich, Saint Louis, MO, USA). Quantification was performed using calibration curves constructed from known concentrations of the respective compounds (R² > 0.990).

2.5.3. Statistical Analysis

All experimental data are reported as the mean values obtained from three independent experiments. Statistical analyses were performed using Duncan’s multiple range test with the Statistical Package for the Social Sciences (SPSS) software version 11.5 for Windows, and values were considered statistically significant at p < 0.05.

2.5.4. Calculation of the Fermentation Kinetic Parameters

The volumetric rates of biomass production (Q_X), substrate consumption (Q_S) and cordycepin production (Q_P) were calculated as follows:

$${\mathit{Q}}_{\mathrm{X}}=\frac{C_{\mathrm{X},2}-C_{\mathrm{X},1}}{t_2-t_1}$$

(1)

$${\mathit{Q}}_{\mathrm{S}}=-\frac{C_{\mathrm{S},2}-C_{\mathrm{S},1}}{t_2-t_1}$$

(2)

$${\mathrm{Q}}_P={\displaystyle \frac{C_{\mathrm{P},2}-C_{\mathrm{P},1}}{t_2-t_1}},$$

(3)

where $C_{X,1}$ and $C_{X,2}$ represent the concentrations of biomass at the initial time (t = 0) and at the time of the highest cordycepin concentration (t = 4), respectively; $C_{S,1}$ and $C_{S,2}$ represent the corresponding glucose concentrations; and $C_{P,1}$ and $C_{P,2}$ represent the corresponding cordycepin concentrations at these time points.

Specific rates were determined by normalizing the corresponding volumetric rates to the average biomass concentration, as described below:

$$\mu ={\displaystyle \frac{1}{\left(\frac{C_{\mathrm{X},1}+C_{\mathrm{X},2}}{2}\right)}}{Q}_{\mathrm{X}}$$

(4)

$$q_{\mathrm{S}}={\displaystyle \frac{1}{\left(\frac{C_{\mathrm{X},1}+C_{\mathrm{X},2}}{2}\right)}}{Q}_{\mathrm{S}}$$

(5)

$$q_P={\displaystyle \frac{1}{\left(\frac{C_{\mathrm{X},1}+C_{\mathrm{X},2}}{2}\right)}}{Q}_P$$

(6)

Yield coefficients were determined based on glucose consumption, with the biomass yield (Y_X/S) defined as the amount of biomass formed per unit of glucose consumed, and the cordycepin yield (Y_P/S) defined as the amount of cordycepin produced per unit of glucose consumed, as follows:

$$Y_{\mathrm{X}/\mathrm{S}}={\displaystyle \frac{C_{\mathrm{X},2}-C_{\mathrm{X},1}}{C_{\mathrm{S},1}-C_{\mathrm{S},2}}}$$

(7)

$$Y_{\mathrm{P}/\mathrm{S}}={\displaystyle \frac{C_{\mathrm{P},2}-C_{\mathrm{P},1}}{C_{\mathrm{S},1}-C_{\mathrm{S},2}}}$$

(8)

2.6. Kinetic Modeling of Mycelial Growth and Cordycepin Production

The growth profile of the A. oryzae engineered strains was evaluated using a kinetic model. A logistic equation was used to describe the growth rates (dC_X/dt) of the fungal cells, as follows:

$$\frac{\mathrm{d}{\mathrm{C}}_{\mathrm{X}}}{\mathrm{dt}} = {\mathrm{\mu }}_{\max }{\mathrm{C}}_{\mathrm{X}}\left(1- \frac{{\mathrm{C}}_{\mathrm{X}}}{{\mathrm{C}}_{\mathrm{Xm}}}\right)$$

(9)

where µ_max represents the maximum specific growth rate, C_X is the biomass concentration, and C_Xm corresponds to the maximum biomass concentration.

Theglucose consumption rate (dC_S/dt) is influenced by fungal growth rate, biomass maintenance, and cordycepin production as described by the following equation:

$$\frac{\mathrm{d}{\mathrm{C}}_{\mathrm{S}}}{\mathrm{dt}} = -\left[\left(\frac{1}{{\mathrm{Y}}_{\mathrm{X}/\mathrm{S}}}\right)\left(\frac{\mathrm{d}{\mathrm{C}}_{\mathrm{X}}}{\mathrm{dt}}\right)+{ \mathrm{m}}_{\mathrm{S}}{\mathrm{C}}_{\mathrm{X}}+\left(\frac{1}{{\mathrm{Y}}_{\mathrm{P}/\mathrm{S}}}\right)\left(\frac{\mathrm{d}{\mathrm{C}}_{\mathrm{P}}}{\mathrm{dt}}\right)\right]$$

(10)

where Y_X/S is the biomass yield of the substrate, m_S is the biomass maintenance coefficient, and Y_P/S is the cordycepin yield from the substrate. The Y_X/S value was 0.452 g/g, which was estimated from calculations based on the molecular formula of the biomass (CH_1.79 O_0.5 N_0.2) when glucose was used as the carbon source [18]. The Y_P/S value was 0.835 g/g, which was the maximum theoretical yield obtained from the calculation of the carbon balance between cordycepin production and glucose consumption, assuming 100% carbon conversion efficiency and no carbon loss to CO₂ evolution.

The cordycepin production rate (dC_P/dt) depended on the biomass concentration according to the Luedeking-Piret equation:

$$\frac{\mathrm{d}{\mathrm{C}}_{\mathrm{P}}}{\mathrm{dt}} =(\mathrm{\alpha }\mathrm{\mu }+\mathrm{\beta }){\mathrm{C}}_{\mathrm{X}}$$

(11)

where α represents the product formation coefficient associated with fungal growth, while β is the product formation coefficient independent of fungal growth.

The experimental results were used to estimate the kinetic parameters of the proposed fermentation models (Equations (9)–(11)), including µ_max, C_Xm, m_S, α, and β, by fitting the models to the experimental data using Berkeley Madonna 10 software for Windows. The performance of the model was assessed based on the coefficient of determination (R²), such that values close to 1.0 indicate a strong correlation between the predicted and experimental data, as described previously [30]. R² values were calculated using the following equation:

$${\mathit{R}}^{\mathit{2}}=\frac{{\sum ({\mathrm{C}}_{\mathrm{cal}}-{\mathrm{C}}_{\exp ,ave})}^2}{{\sum ({\mathrm{C}}_{\mathrm{cal}}- {\mathrm{C}}_{\exp ,\mathrm{ave}})}^2+\sum {({\mathrm{C}}_{\mathrm{cal}}- {\mathrm{C}}_{\exp })}^2}$$

(12)

where C_cal is the concentration predicted using the model, C_exp is the experimentally determined concentration, and C_exp,ave represents the mean of all experimental concentration values obtained using the variables examined.

3. Results and Discussion

3.1. Growth and Cordycepin Production by Engineered A. oryzae Strain Under Different Nitrogen Sources

Cordycepin production using various nitrogen sources was examined by cultivating an engineered A. oryzae strain in SM supplemented with 1 g/L adenine. The engineered strain utilized all tested nitrogen sources for growth (Supplementary Figure S1). Glucose was rapidly consumed when the culture was grown in medium containing KNO₃ and NaNO₃. However, the glucose consumed was utilized solely for growth, and cordycepin production was not observed. Therefore, cultures containing yeast extract, peptone, (NH₄)₂SO₄ or NH₄Cl promoted cordycepin production (Supplementary Figure S1 and Figure 1). With organic nitrogen sources (yeast extract and peptone), biomass concentrations reached 15.988–16.593 g/L, and the highest cordycepin titer (1.125–1.248 g/L) was obtained after 4 d of cultivation. The inorganic ammonium salts ((NH₄)₂SO₄ and NH₄Cl) supported moderate biomass levels (7.521–8.011 g/L) and cordycepin production (0.709–0.734 g/L). In contrast, nitrate salts (NaNO₃ and KNO₃) supported cell growth but resulted in negligible cordycepin production, with biomass and cordycepin titers of approximately 10 g/L and 0.021 g/L, respectively (

Table 1. Kinetic parameters of the glucose cultures of the recombinant of A. oryzae using different nitrogen sources.

Kinetic Parameters	Nitrogen Sources
	Yeast Extract	Peptone	(NH4)2SO4	NH4Cl	NaNO3	KNO3
Concentration (g/L)
CX	16.593 ± 0.140 A	15.988 ± 0.334 A	7.521 ± 0.030 C	8.011 ± 0.157 C	10.645 ± 0.898 B	10.095 ± 1.153 B
CP	1.248 ± 0.007 A	1.125 ± 0.104 A	0.709 ± 0.009 B	0.734 ± 0.010 B	0.021 ± 0.003 C	0.020 ± 0.005 C
Volumetric rates (g/L d)
QX	4.098 ± 0.035 A	3.947 ± 0.083 A	1.830 ± 0.007 C	1.953 ± 0.039 C	2.611 ± 0.225 B	2.474 ± 0.288 B
QS	9.120 ± 0.078 B	8.471 ± 0.072 B	5.537 ± 0.227 C	5.390 ± 0.265 C	10.000 ± 0.000 A	10.000 ± 0.000 A
QP	0.312 ± 0.002 A	0.281 ± 0.026 A	0.177 ± 0.002 B	0.184 ± 0.002 B	0.005 ± 0.001 C	0.005 ± 0.001 C
Specific rates (g/g d)
μ (/d)	0.488 ± 0.000 A	0.488 ± 0.000 A	0.474 ± 0.000 C	0.476 ± 0.000 C	0.481 ± 0.002 B	0.480 ± 0.002 B
qS	1.086 ± 0.000 C	1.047 ± 0.013 C	1.434 ± 0.053 B	1.314 ± 0.090 B	1.851 ± 0.153 A	1.955 ± 0.219 A
qP	0.037 ± 0.000 B	0.035 ± 0.004 B	0.046 ± 0.000 A	0.045 ± 0.000 A	0.001 ± 0.000 C	0.001 ± 0.000 C
Yield (g/g)
YX/S	0.449 ± 0.000 A	0.466 ± 0.006 A	0.331 ± 0.012 B	0.363 ± 0.025 B	0.261 ± 0.022 C	0.247 ± 0.029 C
YP/S	0.034 ± 0.000 A	0.033 ± 0.003 A	0.032 ± 0.001 A	0.034 ± 0.002 A	0.001 ± 0.000 D	0.000 ± 0.000 D

^{A, B, C, D} Values marked with different superscript letters in the same row for each strain are significantly different (p < 0.05). Calculated from the cultures grown for 0−4 days. C_X, biomass titer; C_P, cordycepin titer; Q_X, biomass production rate; Q_S, substrate consumption rate; Q_P, cordycepin production rate; q_S, specific rate of substrate consumption; q_P, specific rate of cordycepin production; Y_X/S, biomass yield on the substrate and Y_P/S, cordycepin yield on the substrate

). These findings are consistent with previous reports showing that complex organic nitrogen sources such as yeast extract and peptone are more favorable for cordycepin production than inorganic nitrogen sources in fungal systems, including C. militaris cultivation [10,11,14,21,22,23]. The kinetic parameters summarized in Table 1 further demonstrate that the nitrogen source markedly influenced cell growth, substrate utilization, and cordycepin production in recombinant A. oryzae cultures.

Organic nitrogen sources (yeast extract and peptone) supported higher biomass and cordycepin production (C_X and C_P) than inorganic nitrogen sources and were associated with higher growth and product formation rates (µ, Q_X, and Q_P). In contrast, ammonium salts ((NH₄)₂SO₄ and NH₄Cl) supported moderate growth and production, whereas nitrate salts (NaNO₃ and KNO₃) resulted in limited biomass accumulation and negligible cordycepin formation despite relatively high substrate consumption rates. Yield coefficients (Y_X/S and Y_P/S) further confirmed that organic nitrogen sources enabled more efficient substrate conversion into biomass and product.

The differences among nitrogen sources can be explained by variations in carbon-to-nitrogen (C:N) balance and nutrient complexity. Nitrogen availability is known to influence the distribution of metabolic flux between biomass formation and metabolite production. Accordingly, the elevated levels of cordycepin observed in cultures supplemented with yeast extract may be attributed to the complex composition of this nutrient source, which provides not only organic nitrogen but also vitamins, trace elements, and small amounts of carbon compounds. These findings suggest that cordycepin production in engineered A. oryzae is governed not only by nitrogen type but also by the overall nutritional balance and cellular metabolic state, thereby highlighting the importance of C:N interactions in the optimization of fermentation conditions.

3.2. Growth and Cordycepin Production by Engineered A. oryzae Strain from Glucose and CSH in Lab-Scale Bioreactor

In this study, the growth and cordycepin production of the engineered A. oryzae strain were evaluated in a lab-scale bioreactor. Cultivation using 40 g/L glucose, 5 g/L yeast extract, and 1.0 g/L adenine was used as the control and compared with the optimized condition (30 g/L glucose, 9.8 g/L yeast extract, and 1.5 g/L adenine) previously reported for cordycepin production in shake-flask cultivation [16]. The profiles of biomass growth, glucose consumption, and cordycepin production by the engineered A. oryzae strain cultivated in a 5 L bioreactor under control and optimal conditions using different carbon sources are illustrated in Figure 2, and the kinetic parameters are illustrated in

Table 2. Kinetic parameters of the cultivation of the recombinant of A. oryzae by using glucose and CSH as carbon sources in 5 L bioreactor.

Kinetic Parameters	Carbon Sources
	Glucose-40 *	Glucose-30 **	CSH **
Concentration (g/L)
CX	8.467 ± 0.523 C	13.632 ± 0.271 B	15.277 ± 0.000 A
CP	1.538 ± 0.002 B	1.721 ± 0.018 A	1.567 ± 0.008 B
Volumetric rates (g/L/d)
QX	1.962 ± 0.131 C	3.164 ± 0.068 B	3.504 ± 0.000 A
QS	7.053 ± 0.194 B	7.545 ± 0.000 A	7.641 ± 0.052 A
QP	0.371 ± 0.001 B	0.412 ± 0.004 A	0.367 ± 0.002 B
Specific rates (g/g d)
μ (/d)	0.432 ± 0.004 A	0.433 ± 0.001 A	0.424 ± 0.000 B
qS	1.554 ± 0.047 A	1.033 ± 0.019 B	0.924 ± 0.006 C
qP	0.082 ± 0.005 A	0.056 ± 0.002 B	0.044 ± 0.000 C
Yield (g/g)
YX/S	0.278 ± 0.011 C	0.419 ± 0.009 B	0.459 ± 0.003 A
YP/S	0.053 ± 0.002 A	0.055 ± 0.001 A	0.048 ± 0.001 B

* Cultivation under control conditions of 40 g/L glucose, 5 g/L yeast extract and 1.0 g/L adenine. ** Cultivation under optimal condition at 30 g/L glucose, 9.8 g/L yeast extract and 1.5 g/L adenine. ^{A, B, C} Values marked with different superscript letters in the same row for each strain are significantly different (p < 0.05). Calculated from the cultures grown for 0−4 days. CSH is cassava starch hydrolysate.

. The final biomass of the control in the bioreactor (8.467 g/L) was lower than that at the flask scale (16.593 g/L). This may be due to the effect of shear stress from the bioreactor impeller on the fungal mycelia [31].

The lower biomass observed in the bioreactor, accompanied by increased cordycepin production, may be attributed to differences in oxygen and nutrient transfer under controlled bioreactor conditions. Improved oxygen availability and environmental control can redirect metabolic flux toward secondary metabolite production rather than biomass accumulation [32,33]. Given that cordycepin has been characterized as a mixed growth-associated product, improved metabolic activity under optimized bioreactor conditions could promote higher product formation despite a slightly lower biomass. These findings suggest that scale-up does not necessarily compromise reproducibility but may instead enhance the efficiency of cordycepin production.

Under optimal conditions, both commercial glucose and CSH supported cell growth and cordycepin production compared to the control conditions. As summarized in Table 2, cultivation under the optimal condition with glucose significantly increased biomass concentration (C_X = 13.63 g/L) and cordycepin concentration (C_P = 1.72 g/L), which were approximately 61% and 12% higher than those under the control condition (C_X = 8.47 g/L and C_P = 1.54 g/L), respectively. Additionally, the use of CSH as an alternative carbon source under optimal condition resulted in the highest biomass concentration (C_X = 15.28 g/L) and volumetric biomass production rate (Q_X = 3.50 g/L d), indicating superior support for cell growth because of the presence of additional nutrients in this hydrolysate [29,34,35]. The glucose consumption rates (Q_S) were comparable between glucose and CSH under optimal conditions. However, cultures grown on CSH exhibited a lower cordycepin formation rate (Q_P) than those grown on glucose, indicating more efficient substrate utilization for biomass formation than for cordycepin biosynthesis. This result is consistent with the yield analysis, in which CSH showed the highest biomass yield (Y_X/S), whereas the product yield (Y_P/S) was the lowest.

These findings indicate that the engineered A. oryzae strain can efficiently utilize both glucose and CSH for cordycepin production. While glucose appears to support a pronounced precursor flux toward cordycepin biosynthesis, CSH provides additional nutrients that promote biomass formation, leading to enhanced cell growth but a relatively lower product yield. The ability of the engineered strain to produce cordycepin using CSH highlights the potential of low-cost agro-industrial feedstocks as alternative carbon sources for the sustainable production of high-value functional compounds.

3.3. Kinetic Modeling of Fungal Growth and Cordycepin Production in Shake Flask

The fermentation parameters of the recombinant fungal strain, using yeast extract as the best nitrogen source, were subjected to kinetic modeling for growth and cordycepin production. The kinetic equations for cell growth, glucose consumption, and cordycepin production (Equations (9)–(11); see Section 2) were fitted to the experimental data. The values of the best fit model parameters (µ_max, C_Xm, m_S, α and β) are presented in

Table 3. The model parameters of the growth and cordycepin production of the engineered A. oryzae estimated by fitting the models to experimental data by using various nitrogen sources in shake flask.

Kinetic Models		Parameters	Nitrogen Sources
			Yeast Extract	Peptone	Inorganic Ammonium Salts
Cell growth	$\frac{d{C}_{\mathrm{X}}}{\mathit{dt}}\mathrm{=}{\mathrm{\mu }}_{\max }{C}_{\mathrm{X}}\left(1-\frac{C_{\mathrm{X}}}{C_{\mathrm{Xm}}}\right)$	µmax (d−1)	2.85	2.85	2.85
		CXm (g/L)	16.742	13.857	7.057
		R2	0.894	0.929	0.945–0.977
Glucose consumption	${\displaystyle \frac{d{C}_{\mathrm{S}}}{\mathit{dt}}}=-\left[\left({\displaystyle \frac{1}{Y_{\mathrm{X}/\mathrm{S}}}}\right)\left({\displaystyle \frac{d{C}_{\mathrm{X}}}{\mathit{dt}}}\right)+m_{\mathrm{S}}{C}_{\mathrm{X}}+\left({\displaystyle \frac{1}{Y_{\mathrm{P}/\mathrm{S}}}}\right)\left({\displaystyle \frac{d{C}_{\mathrm{P}}}{\mathit{dt}}}\right)\right]$	mS (g/g d)	0.00054	0.058	0.296
		YX/S (g/g)	0.452	0.452	0.452
		YP/S (g/g)	0.835	0.835	0.835
		R2	0.934	0.934	0.965–0.984
Cordycepin production	$\frac{d{C}_P}{\mathit{dt}}= (\mathrm{\alpha }\mathrm{\mu }+\mathrm{\beta })C_{\mathrm{X}}$	α (g/g)	0.065	0.065	0.058
		β (g/g d)	0.0055	0.0055	0.017
		R2	0.964	0.978	0.996–0.998

. The high coefficients of determination (R² > 0.89) across all models indicated a good fit between the experimental data and the model predictions (Figure 1), suggesting that the growth of recombinant A. oryzae could be well described by a logistic model when glucose was used as the carbon source. The maximum specific growth rate (µ_max) was identical across all nitrogen sources at 2.85 d⁻¹, indicating that the growth of the strain was not strongly affected by nitrogen type. This value was higher than that reported for the parental A. oryzae BCC7051 strain (2.58–2.62 d⁻¹) and comparable to that of the genetically engineered Δags1 strain (2.85 d⁻¹), which exhibited altered mycelial morphology due to deletion of the ags1 gene involved in cell wall synthesis [17,18]. These results indicate that the genetic modification did not adversely affect fungal growth, further supporting the suitability of A. oryzae BCC7051 as a host for metabolite production. However, the maximum biomass concentration (C_Xm) varied depending on the nitrogen source. Yeast extract could support the highest biomass accumulation (16.74 g/L), followed by peptone (13.86 g/L), while inorganic ammonium salts resulted in markedly lower biomass formation (7.06 g/L). In addition, the effect of various nitrogen sources on fungal growth and the maintenance coefficient for substrate consumption (m_S) increased significantly when inorganic ammonium salts were used, suggesting higher energy expenditure for cellular maintenance compared with organic nitrogen sources. This behavior implied inefficient substrate utilization under inorganic nitrogen conditions. In contrast, yeast extract exhibited the lowest m_S value, reflecting a more efficient metabolic utilization of glucose for growth and cordycepin formation.

According to the Luedeking–Piret equation, cordycepin was characterized as a mixed growth-associated product under all nitrogen sources. Although the growth-associated production coefficient (α) was similar for yeast extract and peptone, the non-growth-associated coefficient (β) was notably higher in cultures supplied with inorganic ammonium salts. These results suggest that cordycepin production under inorganic nitrogen conditions tends to occur predominantly during the stationary phase, whereas under organic nitrogen conditions it is more closely associated with active cell growth.

3.4. Kinetic Modeling of Fungal Growth and Cordycepin Production in Fermenter

To further evaluate the scalability of the engineered A. oryzae strain and the influence of cultivation conditions, its growth and cordycepin production were investigated under controlled and optimized conditions using glucose and CSH as carbon sources in fermenter-scale experiments. The behavior observed in all the experiments was analyzed by comparing the experimental data with kinetic model simulations following the same approach applied at the shake-flask scale. The estimated kinetic parameters are presented in

Table 4. The model parameters of the engineered A. oryzae culture estimated by fitting the models to experimental data in fermenter using glucose and cassava starch hydrolysate as carbon sources.

Kinetic Models		Parameters	Glucose *	Glucose **	CSH **
Cell growth	$\frac{d{C}_{\mathrm{X}}}{\mathit{dt}}={\mathrm{\mu }}_{\max }{C}_{\mathrm{X}}\left(1-\frac{C_{\mathrm{X}}}{C_{\mathrm{Xm}}}\right)$	µmax (d−1)	1.743	1.980	1.210
		CXm (g/L)	7.616	11.470	15.750
		R2	0.876	0.904	0.930
Glucose consumption	${\displaystyle \frac{d{C}_{\mathrm{S}}}{\mathit{dt}}}=-\left[\left({\displaystyle \frac{1}{Y_{\mathrm{X}/\mathrm{S}}}}\right)\left({\displaystyle \frac{d{C}_{\mathrm{X}}}{\mathit{dt}}}\right)+{ m}_{\mathrm{S}}{C}_{\mathrm{X}}+\left({\displaystyle \frac{1}{Y_{\mathrm{P}/\mathrm{S}}}}\right)\left({\displaystyle \frac{d{C}_{\mathrm{P}}}{\mathit{dt}}}\right)\right]$	mS (g/g d)	0.300	0.142	0.011
		YX/S (g/g)	0.452	0.452	0.452
		YP/S (g/g)	0.835	0.835	0.835
		R2	0.877	0.982	0.896
Cordycepin production	$\frac{d{C}_{\mathrm{P}}}{\mathit{dt}}= (\mathrm{\alpha }\mathrm{\mu }+\mathrm{\beta })C_{\mathrm{X}}$	α (g/g)	0.158	0.147	0.115
		β (g/g d)	0.021	0.0041	0.0051
		R2	0.997	0.995	0.928

* Cultivation under control condition of 40 g/L glucose, 5 g/L yeast extract and 1.0 g/L adenine. ** Cultivation under optimal condition at 30 g/L glucose, 9.8 g/L yeast extract and 1.5 g/L adenine. CSH is cassava starch hydrolysate.

, and the agreement between the experimental data and model predictions is shown in Figure 2. The profiles of fungal growth, glucose consumption, and cordycepin production were well fitted by the kinetic models (R² > 0.88), indicating that the growth of the recombinant A. oryzae could be adequately described by a logistic model when glucose and CSH were used as carbon sources. According to the Luedeking–Piret equation, cordycepin was characterized as a mixed growth-associated product, consistent with observations at the shake-flask scale. From a bioprocess perspective, mixed growth-associated behavior is advantageous, as it enables cordycepin production throughout cultivation rather than being restricted to either the exponential or stationary growth phase. This characteristic provides flexibility for process optimization, since both biomass accumulation and post-growth metabolic activity contribute to the overall product yield of engineered A. oryzae strain.

Fermenter-scale cultivation using glucose as the carbon source under controlled conditions exhibited lower growth than shake-flask cultivation because of shear stress on the fungal mycelia. Consequently, lower growth parameters (µ_max = 1.74 d⁻¹; C_Xm = 7.62 g/L) and a higher biomass maintenance coefficient (m_S = 0.300 g/g d) were observed. However, cultivation in the fermenter favored cordycepin production over shake-flask cultivation. Consequently, higher cordycepin production coefficients (α and β) of 0.158 g/g and 0.021 g/g d, respectively, were obtained. These values were approximately 2.43- and 38.18-fold higher than those observed in the shake-flask cultures. The maximum specific growth rate (µ_max) varied with cultivation conditions and carbon source, with the highest value observed under optimized glucose conditions (1.980 d⁻¹). Therefore, the cultivation under optimal condition using glucose increased both µ_max and C_Xm compared with the control condition, demonstrating that medium optimization significantly enhanced cell growth.

Notably, the use of CSH under optimal condition resulted in the highest biomass concentration (15.75 g/L), despite exhibiting the lowest µ_max. This indicates that CSH promotes sustained biomass accumulation during cultivation [29]. The biomass maintenance coefficient (m_S) decreased noticeably under optimized and CSH conditions, reflecting improved substrate utilization efficiency for fungal growth. The α and β values for glucose (0.147 g/g and 0.0041 g/g d) and CSH (0.115 g/g and 0.0051 g/g d) under optimized conditions were slightly lower than control condition (0.158 g/g and 0.021 g/g d), indicating that carbon flux was preferentially directed toward biomass formation rather than secondary metabolite biosynthesis. However, cordycepin production remained significant in all cultivations, demonstrating the feasibility of using CSH as an alternative carbon source. The kinetic and production parameters obtained in this study are consistent with previous reports on engineered A. oryzae for cordycepin production. Jeennor et al. (2023) [15] reported titers of 1.02–1.37 g/L in shake-flask cultures, while Anantayanon et al. (2025) [16] achieved approximately 1.72 g/L after medium optimization using DOE analysis. The maximum titer obtained in this study (1.24–1.72 g/L) is therefore comparable to these previous results, confirming the robustness of the engineered A. oryzae production platform.

Although no mathematical models have yet been specifically developed to describe cordycepin production in either native Cordyceps species or engineered systems, kinetic modeling approaches based on Logistic growth and Luedeking–Piret models have been widely applied to describe biomass growth and secondary metabolite production in microbial fermentations, including lovastatin-producing Aspergillus terreus [33] and penicillin-producing Penicillium chrysogenum [36]. In such systems, logistic models effectively describe sigmoidal biomass growth, whereas the Luedeking–Piret equation differentiates between growth-associated and non-growth-associated product formation.

In this study, a kinetic framework integrating the logistic growth model and the Luedeking–Piret equation was established to quantitatively describe biomass growth, substrate utilization, and cordycepin production in engineered A. oryzae. Unlike previous studies [17,18] focusing mainly on fungal growth or lipid production, this model specifically addresses cordycepin biosynthesis and was validated across different substrates and cultivation scales. This work therefore provides the first quantitative framework for predicting cordycepin production in an engineered A. oryzae system.

4. Conclusions

In this study, cordycepin production by engineered A. oryzae AoCordy-T1 was evaluated under submerged fermentation at both shake-flask and controlled bioreactor scales. Among the nitrogen sources tested, yeast extract was identified as the most effective for cordycepin production, while cassava starch hydrolysate also supported substantial biomass accumulation and demonstrated potential as an alternative carbon source. A kinetic model integrating logistic growth kinetics with the Luedeking–Piret equation was developed to describe fungal growth and cordycepin formation. Collectively, these findings provide a quantitative framework for understanding cordycepin fermentation and support future process optimization and scalable production.