Safety evaluation of an extension of use of the food enzyme endo-polygalacturonase from the genetically modified Aspergillus oryzae strain AR-183.

The food enzyme endo-polygalacturonase ((1 → 4)-α-d-galacturonan glycanohydrolase EC 3.2.1.15) is produced with the genetically modified Aspergillus oryzae strain AR-183 by AB ENZYMES GmbH. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in three food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining four processes. Dietary exposure was up to 0.087 mg TOS/kg body weight (bw) per day in European populations. When combined with the NOAEL reported in the previous opinion (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 11,494. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of an extension of use of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR-962.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus oryzae strain AR-962 by AB Enzymes GmbH. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for uses in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in three food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining four processes. Dietary exposure was up to 0.575 mg TOS/kg body weight (bw) per day in European populations. When combined with the NOAEL reported in the previous opinion (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 1739. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Modulating flavanone compound for reducing the bitterness and improving dietary fiber, physicochemical properties, and anti-adipogenesis of green yuzu powder by enzymatic hydrolysis.

Yuzu (Citrus junos Sieb.) is a peel-edible fruit with a pleasant aroma, but its bitter taste can impact consumer appeal. In this study, an efficient enzymatic method reduced bitterness in green yuzu powder (GYP). Cellulase KN and naringinase from Aspergillus oryzae NYO-2 significantly decreased naringin and neohesperidin content by over 87 %, while increasing total dietary fiber and soluble dietary fiber by up to 10 % and 51 %, respectively. Insoluble dietary fiber decreased by up to 22 %. Cellulose, hemicellulose, lignin, and pectin contents in enzyme-treated YP decreased by 1.15-2.00-fold, respectively. Enzyme-treated GYP exhibited improved physicochemical properties, including enhanced solubility, oil-holding capacity, and water swelling capacities. 3T3-L1 cells treated with cellulase-treated GYP and naringinase-treated GYP showed lower lipid accumulation and higher lipolysis capability than GYP, along with decreased fatty acid synthase contents. These findings suggest that enzyme-treated GYP holds potential as a functional ingredient in the food industry.

Double knockout of two target genes via genome co-editing using a nitrate transporter gene nrtA and a putative thiamine transporter gene thiI as selectable markers in Aspergillusoryzae.

Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, thiI gene was found as a selectable marker for such genome co-editing in the filamentous fungus Aspergillus oryzae, while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in A. oryzae. In this study, we focused on the membrane transporter gene nrtA (AO090012000623), which promotes uptake of nitrate (NO3-). It is known that, in nrtA knockout strain, chlorate (ClO3-), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene wA was attempted using both nrtA- and wA-specific single-guide RNAs via genome co-editing with KClO3 supplementation in A. oryzae laboratory strain RIB40 and industrial strain KBN616. Resultantly, wA knockout mutant was generated, and nrtA was identified as a selectable marker. Moreover, this genome co-editing system using nrtA was compatible with that using thiI, and thus, a double knockout mutant of two target genes wA and yA was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As nrtA homologs have been found in several industrial Aspergillus species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli.

Functional Characterization of Sesquiterpene Synthases and P450 Enzymes in Flammulina velutipes for Biosynthesis of Spiro [4.5] Decane Terpene.

Flammulina velutipes, a popular edible mushroom, contains sesquiterpenes with potential health benefits. We characterized 12 sesquiterpene synthases and one P450 enzyme in F. velutipes using Aspergillus oryzae as a heterologous expression system, culminating in the biosynthesis of 16 distinct sesquiterpene compounds. An enzyme encoded by the axeB gene responsible for the synthesis of the spiro [4.5] decane compound axenol was discovered, and the mechanism of spirocycle formation was elucidated through quantum mechanical calculations. Furthermore, we delineated the role of a P450 enzyme colocated with AxeB in producing the novel compound 3-oxo-axenol. Our findings highlight the diverse array of sesquiterpene skeletons and functional groups biosynthesized by these enzymes in F. velutipes and underscore the effectiveness of the A. oryzae system as a heterologous host for expressing genes in the Basidiomycota genome. These insights into the biosynthesis of bioactive compounds in F. velutipes have significant implications for functional food and drug development.

Identification of cheese rancidity-related lipases in Aspergillus oryzae AHU 7139.

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB, tglA, and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because ΔtglA and ΔmdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A.oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor.

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae.

DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5'-CGGCTAATGCGG-3', and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

Construction of self-cloning Aspergillus oryzae strains with high production of multiple biomass-degrading enzymes on solid-state culture.

Filamentous fungi produce numerous industrially important enzymes. Among them, Aspergillus oryzae-derived enzymes are widely used in various fermentation applications. In this study, we constructed self-cloning strains that overproduce multiple biomass-degrading enzymes under the control of a strong promoter of α-amylase-coding gene (amyB) using the industrial strain A. oryzae AOK11. Two strains (strains 2-4 and 3-26) were introduced with different combinations of genes encoding xylanase (xynG1), phytase (phyA), pectin lyase (pelA), and polygalacturonase (pgaB). These strains had at least one copy of each enzyme gene derived from the expression cassette in the genome. The transcription levels of enzyme-coding genes introduced were more than 100-fold higher than those in the parent strain. Reflecting the high transcription levels, the activities of the enzymes derived from the expression cassettes of these two strains were significantly higher than those of the parent strain in both liquid and solid-state cultures. Even in ventilated solid-state cultures that were scaled up using mechanical equipment for practical applications, the two strains showed significantly higher enzyme activity than the parent strain. These results indicate that these strains constructed using a safe self-cloning technique represent industrially valuable practical strains that can be used in the food and livestock industries.

Transcriptional and post-transcriptional regulation of genes encoding secretory proteins in Aspergillus oryzae.

Aspergillus oryzae, also known as the yellow koji mold, produces various hydrolytic enzymes that are widely used in different industries. Its high capacity to produce secretory proteins makes this filamentous fungus a suitable host for heterologous protein production. Amylolytic gene promoter is widely used to express heterologous genes in A. oryzae. The expression of this promoter is strictly regulated by several transcription factors, whose activation involves various factors. Furthermore, the expression levels of amylolytic and heterologous genes are post-transcriptionally regulated by mRNA degradation mechanisms in response to aberrant transcriptional termination or endoplasmic reticulum stress. This review discusses the transcriptional and post-transcriptional regulatory mechanisms controlling the expression of genes encoding secretory proteins in A. oryzae.

Aspergillus oryzae PrtR alters transcription of individual peptidase genes in response to the growth environment.

Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors. KEY POINTS: • Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR • However, several genes are regulated negatively by PrtR • PrtR optimizes transcription of peptidase genes in response to culture conditions.

Class VI G protein-coupled receptors in Aspergillus oryzae regulate sclerotia formation through GTPase-activating activity.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in eukaryotes that sense and transduce extracellular signals into cells. In Aspergillus oryzae, 16 canonical GPCR genes are identified and classified into nine classes based on the sequence similarity and proposed functions. Class VI GPCRs (AoGprK-1, AoGprK-2, and AoGprR in A. oryzae), unlike other GPCRs, feature a unique hybrid structure containing both the seven transmembrane (7-TM) and regulator of G-protein signaling (RGS) domains, which is not found in animal GPCRs. We report here that the mutants with double or triple deletion of class VI GPCR genes produced significantly increased number of sclerotia compared to the control strain when grown on agar plates. Interestingly, complementation analysis demonstrated that the expression of the RGS domain without the 7-TM domain is sufficient to restore the phenotype. In line with this, among the three Gα subunits in A. oryzae, AoGpaA, AoGpaB, and AoGanA, forced expression of GTPase-deficient mutants of either AoGpaA or AoGpaB caused an increase in the number of sclerotia formed, suggesting that RGS domains of class VI GPCRs are the negative regulators of these two GTPases. Finally, we measured the expression of velvet complex genes and sclerotia formation-related genes and found that the expression of velB was significantly increased in the multiple gene deletion mutants. Taken together, these results demonstrate that class VI GPCRs negatively regulate sclerotia formation through their GTPase-activating activity in the RGS domains. KEY POINTS: • Class VI GPCRs in A. oryzae regulate sclerotia formation in A. oryzae • RGS function of class VI GPCRs is responsible for regulation of sclerotia formation • Loss of class VI GPCRs resulted in increased expression of sclerotia-related genes.

Comparative pangenome analysis of Aspergillus flavus and Aspergillus oryzae reveals their phylogenetic, genomic, and metabolic homogeneity.

Aspergillus flavus and Aspergillus oryzae are closely related fungal species with contrasting roles in food safety and fermentation. To comprehensively investigate their phylogenetic, genomic, and metabolic characteristics, we conducted an extensive comparative pangenome analysis using complete, dereplicated genome sets for both species. Phylogenetic analyses, employing both the entirety of the identified single-copy orthologous genes and six housekeeping genes commonly used for fungal classification, did not reveal clear differentiation between A. flavus and A. oryzae genomes. Upon analyzing the aflatoxin biosynthesis gene clusters within the genomes, we observed that non-aflatoxin-producing strains were dispersed throughout the phylogenetic tree, encompassing both A. flavus and A. oryzae strains. This suggests that aflatoxin production is not a distinguishing trait between the two species. Furthermore, A. oryzae and A. flavus strains displayed remarkably similar genomic attributes, including genome sizes, gene contents, and G + C contents, as well as metabolic features and pathways. The profiles of CAZyme genes and secondary metabolite biosynthesis gene clusters within the genomes of both species further highlight their similarity. Collectively, these findings challenge the conventional differentiation of A. flavus and A. oryzae as distinct species and highlight their phylogenetic, genomic, and metabolic homogeneity, potentially indicating that they may indeed belong to the same species.

Monaspin B, a Novel Cyclohexyl-furan from Cocultivation of Monascus purpureus and Aspergillus oryzae, Exhibits Potent Antileukemic Activity.

Natural products are a rich resource for the discovery of innovative drugs. Microbial cocultivation enables discovery of novel natural products through tandem enzymatic catalysis between different fungi. In this study, Monascus purpureus, as a food fermentation strain capable of producing abundant natural products, was chosen as an example of a cocultivation pair strain. Cocultivation screening revealed that M. purpureus and Aspergillus oryzae led to the production of two novel cyclohexyl-furans, Monaspins A and B. Optimization of the cocultivation mode and media enhanced the production of Monaspins A and B to 1.2 and 0.8 mg/L, respectively. Monaspins A and B were structurally elucidated by HR-ESI-MS and NMR. Furthermore, Monaspin B displayed potent antiproliferative activity against the leukemic HL-60 cell line by inducing apoptosis, with a half-maximal inhibitory concentration (IC50) of 160 nM. Moreover, in a mouse leukemia model, Monaspin B exhibited a promising in vivo antileukemic effect by reducing white blood cell, lymphocyte, and neutrophil counts. Collectively, these results indicate that Monaspin B is a promising candidate agent for leukemia therapy.

Synthetic Biology Tools for Engineering Aspergillus oryzae.

For more than a thousand years, Aspergillus oryzae has been used in traditional culinary industries, including for food fermentation, brewing, and flavoring. In recent years, A. oryzae has been extensively used in deciphering the pathways of natural product synthesis and value-added compound bioproduction. Moreover, it is increasingly being used in modern biotechnology industries, such as for the production of enzymes and recombinant proteins. The investigation of A. oryzae has been significantly accelerated through the successive application of a diverse array of synthetic biology techniques and methodologies. In this review, the advancements in biological tools for the synthesis of A. oryzae, including DNA assembly technologies, gene expression regulatory elements, and genome editing systems, are discussed. Additionally, the challenges associated with the heterologous expression of A. oryzae are addressed.

Copper ion (Cu2+) is involved in the transcription of the tyrosinase-encoding melB gene of Aspergillus oryzae in solid-state culture.

In Aspergillus oryzae, the tyrosinase-encoding gene melB causes undesirable browning of sake and sake lees. This gene is known to be expressed specifically in solid-state culture; however, its expression mechanisms remain unknown. Here, we evaluated the possible factors affecting the transcription of melB and found that the copper ion (Cu2+) significantly enhanced the transcription level of melB in solid-state culture.

Aspergillus oryzae α-l-rhamnosidase: Crystal structure and insight into the substrate specificity.

The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to ß-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to ß-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat /Km ) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6 -barrel with the active site located in a deep cleft and two ß-sheet domains were also present in the N- and C-terminal sites of the catalytic domain. Additionally, five asparagine-attached N-acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.

Plant polysaccharide degradation-related enzymes in Aspergillus oryzae.

Plants synthesize large amounts of stored and structural polysaccharides. Aspergillus oryzae is used in traditional Japanese fermentation and produces many types of plant polysaccharide degradation-related enzymes. The carbohydrate-active enzymes of A. oryzae are important in the fermentation process and biotechnological applications. Because plant polysaccharides have a complex structure, cooperative and synergistic actions of enzymes are crucial for the degradation of plant polysaccharides. For example, the cooperative action of isoprimeverose-producing oligoxyloglucan hydrolase, ß-galactosidase, and α-xylosidase is important for the degradation of xyloglucan, and A. oryzae coordinates these enzymes at the expression level. In this review, I focus on the plant polysaccharide degradation-related enzymes identified in A. oryzae.

Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform.

BACKGROUND: Cordycepin (3'-deoxyadenosine) is an important bioactive compound in medical and healthcare markets. The drawbacks of commercial cordycepin production using Cordyceps spp. include long cultivation periods and low cordycepin yields. To overcome these limitations and meet the increasing market demand, the efficient production of cordycepin by the GRAS-status Aspergillus oryzae strain using a synthetic biology approach was developed in this study. RESULTS: An engineered strain of A. oryzae capable of cordycepin production was successfully constructed by overexpressing two metabolic genes (cns1 and cns2) involved in cordycepin biosynthesis under the control of constitutive promoters. Investigation of the flexibility of carbon utilization for cordycepin production by the engineered A. oryzae strain revealed that it was able to utilize C6-, C5-, and C12-sugars as carbon sources, with glucose being the best carbon source for cordycepin production. High cordycepin productivity (564.64 ± 9.59 mg/L/d) was acquired by optimizing the submerged fermentation conditions. CONCLUSIONS: This study demonstrates a powerful production platform for bioactive cordycepin production by A. oryzae using a synthetic biology approach. An efficient and cost-effective fermentation process for cordycepin production using an engineered strain was established, offering a powerful alternative source for further upscaling.