Skip to main content

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production



The filamentous fungus Aspergillus oryzae is widely used for secondary metabolite production by heterologous expression; thus, a wide variety of promoter tools is necessary to broaden the application of this species. Here we built a procedure to survey A. flavus genes constitutively highly expressed in 83 transcriptome datasets obtained under various conditions affecting secondary metabolite production, to find promoters useful for heterologous expression of genes in A. oryzae.


To test the ability of the promoters of the top 6 genes to induce production of a fungal secondary metabolite, ustiloxin B, we inserted the promoters before the start codon of ustR, which encodes the transcription factor of the gene cluster responsible for ustiloxin B biosynthesis, in A. oryzae. Four of the 6 promoters induced ustiloxin B production in all tested media (solid maize, liquid V8 and PDB media), and also ustR expression. Two of the 4 promoters were those of tef1 and gpdA, which are well characterized in A. oryzae and A. nidulans, respectively, whereas the other two, those of AFLA_030930 and AFLA_113120, are newly reported here and show activities comparable to that of the gpdA promoter with respect to induction of gene expression and ustiloxin B production.


We newly reported two sequences as promoter tools for secondary metabolite production in A. oryzae. Our results demonstrate that our simple strategy of surveying for constitutively highly expressed genes in large-scale transcriptome datasets is useful for finding promoter sequences that can be used as heterologous expression tools in A. oryzae.


The filamentous fungus Aspergillus oryzae has been traditionally used in Japanese fermentation industries to produce sake, shoyu and miso, as well as in enzyme production industries. A. oryzae has also been used as a host for production of fungal secondary metabolites, e.g., cyclopiazonic acid [1] and 1,3,6,8-tetrahydroxynaphthalene [2], mainly because A. oryzae scarcely produces secondary metabolites that could otherwise confound the isolation of target compounds [3]. Many genetic tools have been developed for Aspergillus oryzae; e.g., constitutive and inducible promoters as described below, auxotrophic (pyrG [4], argB [5], niaD [6], sC [7] and adeA [8]) and dominant (amdS [9] and ptrA [10]) selective markers, a marker recycling system [11], a quadruple auxotrophic transformation system [12], and genome editing systems [13, 14]. These tools facilitate simultaneous integration of several genes into the fungal genome, which is necessary for heterologous production of fungal secondary metabolites because usually several genes are involved in their biosynthesis. A. oryzae NSAR1, the quadruple auxotrophic strain (argB, niaD, sC and adeA) [12], is used to produce fungal secondary metabolites by simultaneously introducing two to nine genes for biosynthesis of such compounds as pleuromutilin [15], paxilline [16], terretonin [17], helvolic acid [18], menisporopsin A [19] and asperipin-2a [20]. A variety of basidiomycete terpenes have been successfully produced in A. oryzae by heterologous expression of their respective biosynthetic genes using the genome-editing system [21].

Although the number of promoters that can be used for heterologous expression in filamentous fungi is limited in comparison with that in the yeast Saccharomyces cerevisiae, where a well-established set of promoters covers virtually all patterns of expression [22], promoter tools have been developed for filamentous fungi including Trichoderma reesei [23], A. niger [24], Penicillium chrysogenum [25] and Ustilago maydis [26]. In A. oryzae, the maltose-inducible promoter of the Taka-amylase A gene (amyB) [27,28,29] is often used for heterologous expression [16, 20, 30], as are the thiamine-inducible promoter of the thiamine thiazole synthase gene (thiA) [31] and the constitutive promoter of the translation-elongation factor 1α (tef1) [32]. The glaA promoter of the glucoamylase A gene was originally characterized in A. niger [33], and was then also used in A. oryzae for secondary metabolite production and gene functional analyses [28, 34, 35]. The native A. oryzae promoter of the oxidoreductase gene, kojA, which is involved in kojic acid biosynthesis successfully induced the expression of the polyketide synthase gene (wA) and production of the respective polyketide, YWA1 [36]. Whilst mainly native promoters are used for heterologous expression in filamentous fungi [37], in S. cerevisiae, universal expression systems for fungal genes comprising a set of synthetic promoters and transcription factors have been recently developed to synthesize a wide range of fungal natural products [38,39,40]. However, because A. oryzae possesses a variety of proteins and secretion systems for proteins and low-molecular-weight compounds that differ from those in S. cerevisiae [41,42,43], finding additional promoters that would be functional in A. oryzae is important for the use of this species as a heterologous expression host in addition to S. cerevisiae.

Whilst A. oryzae almost never produces secondary metabolites except kojic acid [44], A. flavus produces quite a few secondary metabolites, including aflatoxin, a strong carcinogen, and cyclopiazonic acid, which is toxic in large amounts. A. oryzae was once proposed to be reduced to an A. flavus subspecies because of its 100% DNA complementarity with A. flavus [45], but was retained as a separate species due to economic and food safety concerns [46]. Georgianna et al. [47] investigated the transcriptomic pattern of A. flavus NRRL3357 under 28 different conditions affecting secondary metabolite production. They classified the 55 putative secondary metabolite biosynthesis (SMB) genes encoding polyketide synthases, non-ribosomal peptide synthetases and terpene synthases into four clades according to their expression patterns, and found that the SMB genes in the two clades are expressed at lower levels in A. oryzae than in A. flavus [47]. Ehrlich et al. [48] reported that some putative SMB genes in A. flavus are absent or expressed at significantly lower levels than in A. oryzae. These reports indicate that the genes necessary to produce secondary metabolites are likely to be expressed in A. flavus rather than A. oryzae, and thus A. flavus is likely to be suitable as a potential source of usable promoters that will activate secondary metabolism genes in A. oryzae.

Transcriptome datasets are used to identify constitutive promoters in bacteria [49] and plants [50]. Oda et al. [51] used transcriptome datasets collected under three different conditions to find sorbitol-inducible promoters in A. oryzae. Sibthorp et al. [52] used transcriptome datasets obtained under five different culture conditions for the global identification of promoters in A. nidulans. Here we extracted information about constitutively highly expressed A. flavus genes by analyzing the 75 publicly available [47] and 8 newly obtained transcriptome datasets generated under 32 conditions affecting secondary metabolite production. To the best of our knowledge, no such large-scale dataset analysis has been used to find constitutive promoters in filamentous fungi, at least in A. oryzae or A. flavus. We examined whether the promoter sequences of the 6 prioritized genes would enhance downstream gene transcription and secondary metabolite production in A. oryzae by inserting them just before the start codon of A. oryzae ustR, the gene encoding the transcription factor of the ustiloxin B biosynthetic gene cluster [34]. Ustiloxin B, a fungal secondary metabolite, is a toxic cyclic peptide originally isolated from the plant pathogenic fungus Ustilaginoidea virens [53, 54]. The biosynthetic gene cluster for ustiloxin B has been identified in A. flavus, revealing that ustiloxin B belongs to a relatively new class of fungal secondary metabolites, ribosomally synthesized and post-translationally modified peptides (RiPPs) [34]. A. oryzae does not produce the compound but possesses a gene cluster identical to that in A. flavus except the lack of an approximately 2 kb upstream region of ustR [34]. When the lacking promoter region of ustR is compensated with the glaA promoter, A. oryzae starts to produce ustiloxin B [35]. Therefore, we can efficiently assess the activity of a sequence as a promoter from ustiloxin B production by an A. oryzae transformant in which the target sequence is inserted before ustR.

Results and discussion

We ranked the 13,481 genes of A. flavus by the median of their expression ranks among the 83 (8 in-house and 75 publicly available) transcriptome datasets to identify constitutively highly expressed genes (Fig. 1, Additional file 1: Table S1). We included the in-house data generated under the conditions where ustiloxin B was produced, because ustiloxin B production by A. flavus and the corresponding biosynthetic pathway were not known when the publicly available datasets were published. To obtain the in-house data, we cultured the A. flavus ustROE and control strains in V8 vegetable juice (V8) or potato dextrose broth (PDB) liquid medium, where the strains produced ustiloxin B [34]. The publicly available data were obtained under 28 different conditions affecting secondary metabolite production, such as maize and wheat culture, for A. flavus NRRL3357 and the deletion and overexpression mutants of laeA, a global secondary metabolism regulator of gene expression [55], with A. oryzae RIB40 used as a control [47].

Fig. 1
figure 1

The procedure for computational prioritization of A. flavus genes from 83 transcriptome datasets. Processes are shown in rectangles with corners

We selected the top 6 genes for ustR expression test using their 5′-untranslated region (UTR) sequences as promoters (P1–P6; Table 1). P1, P2 and P5 have been reported as promoter tools for robust transcription of downstream genes in A. oryzae (P1 [32] and P5 [56]) or A. nidulans (P2 [57, 58]). The gene containing P4 (AFLA_113120) is indispensable in A. flavus for normal fungal growth and development, aflatoxin biosynthesis and seed colonization [59], but the promoter sequence has not been tested as a gene expression tool. P3 drives AFLA_014570, which is annotated to encode a conserved hypothetical protein.

Table 1 Top 13 genes constitutively highly expressed in 83 transcriptome datasets

To assess the degree of gene transcription activity induced by the promoters, we measured the ustR expression levels (relative to those of the tubulin transcript) in the transformants cultured in V8 and PDB liquid media. The relative expression level of ustR was highest in the P1 transformant, followed by those in the P4, P2, P6 (V8) or P6, P2, P4 (PDB) and P5 transformants (Fig. 2). P1 is the well-characterized promoter of tef1 [32] and P2 is that of gpdA reported in A. nidulans [57, 58]; accordingly, the P1 and P2 transformants showed respective ≈six- and ≈three-fold relative expression levels of ustR against tubulin. The P3 transformant showed negligible relative expression levels of ustR in both V8 and PDB media. The P5 transformant showed ≈1- and ≈0.3-fold relative expression levels of ustR against tubulin in V8 and PDB media, respectively, which are the smallest levels next to P3. P5 reportedly increase the relative mRNA abundance of a β-glucuronidase (GUS) gene from Escherichia coli in comparison with that of 18S rRNA to ≈1.4 at 30 °C in DP medium (2% dextrin, 1% polypeptone, 0.5% KH2PO4, and 0.05% MgSO4·7H2O) in A. oryzae [56]. Because the media and the standard genes are different, it is difficult to compare the induction efficiency of P5 between the current and previous studies.

Fig. 2
figure 2

Relative ustR transcript levels (normalized to those of the tubulin transcript) in A. oryzae transformants with promoters P1 to P6 fused to ustR after 3-day culture in liquid PDB and V8 media. The error bars represent the standard errors of the three replicates in a sample. *p < 0.05, **p < 0.01 by paired t-test against the control pyrG revertant

We tested ustiloxin B productivity by the transformants under three different conditions, i.e., solid cracked maize and liquid V8 and PDB media. Ustiloxin B production by A. flavus was also confirmed in solid cracked maize [34]. Ustiloxin B was produced by the transformants with the tested promoters except P3 in the cracked maize solid culture (Fig. 3a). The largest yield was > 220 mg/kg in the P1 transformant, followed by the transformants with P6, P2, P5 and P4; the latter two transformants had identical yields.

Fig. 3
figure 3

Ustiloxin B production by A. oryzae transformants with promoters P1 to P6 fused to ustR in (a) solid maize medium for 14 days, b V8 liquid medium for up to 10 days, and c PDB liquid medium for up to 20 days. The error bars represent the standard errors of the three replicates. The error bars of P1 are drawn in dashed line in b, c to distinguish them from others. In a, *p < 0.05, **p < 0.01 by paired t-test against the control pyrG revertant

In V8 liquid culture, ustiloxin B started to be produced on the 3rd day by the transformants with P1, P2, P4 and P6, but the yield did not increase after that (Fig. 3b). The yield tended to be unstable, with large differences among replicates. Unlike in the maize culture, the P1 transformant had the lowest yield (ca. 10 mg/L), whereas the yields were similar among the transformants with P2, P4 and P6 (ca. 15 mg/L). The P5 transformant did not produce ustiloxin B in liquid V8 culture, unlike in solid maize culture. The transformant with P3 did not produce ustiloxin B at all, either in V8 liquid culture or in maize culture.

In PDB liquid culture, ustiloxin B started to be produced by the P1, P2, P4 and P6 transformants on the 6th day, which was 3 days later than in V8 liquid culture. However, the transformants kept producing ustiloxin B up to around the 18th day; the highest yield (almost 120 mg/L) was achieved by the P6 transformant, followed by that with P1 and by those with P2 and P4. The transformant with P5 started to produce the compound on the 14th day, but the maximum yield was only around 6 mg/L at the 18th day, much lower than those achieved by the other four transformants. The P3 transformant did not produce ustiloxin B at all.

The order of the ustiloxin B production in PDB liquid medium was concordant with that of relative ustR expression in PDB liquid medium (Fig. 3c), as well as in solid maize culture except that the P4 and P5 transformants showed nearly identical ustiloxin B production (Fig. 3a). In the V8 medium, the ustiloxin B production by the P1 transformant was the lowest among those of the P1, P2, P4 and P6 transformants, even though the P1 transformant showed the highest relative ustR expression in the V8 medium. No ustiloxin B production by the P3 transformant was observed in any of the three media tested, in accordance with the almost 0 relative ustR expression in the P3 transformant. Total fungal cell weight did not differ among transformants cultured in either of the liquid media and was on average around 150 mg in V8 and 1.2 g in PDB cultures (Figure S2). We did not measure ustiloxin B yield or cell weight at 20 days in V8 liquid medium because the yield plateaued in 10 days; the slow production of the compound in PDB medium might have allowed high yield.

In summary, our results show that 4 promoters (P1, P2, P4 and P6) among 6 selected by our survey of 83 transcriptome datasets worked well to enhance the transcription of the key gene for fungal secondary metabolite production and the production of such a compound. P1 and P2 have been already reported and are widely used as constitutive promoters [32, 57]. P1 or the tef1 promoter reportedly induces to produce S. cerevisiae proteins (41 kDa) at ≈ 100 mg/L in glucose medium [32], which yield is comparable to our result of ustiloxin B (Mw 645.2) production at ≈ 100 mg/L in PDB liquid medium, 18 days (Fig. 3c). P2 or the gpdA promoter reportedly induces the expression of the endogenous gene, amdS, encoding acetamidase up to 30-fold in A. nidulans [58], whereas our result showed ≈four-fold relative expression level of ustR against tubulin in V8 medium (Fig. 2). In the 83 datasets used in this study, the average gene expression value of the β-tubulin gene (AFLA_051840) is 11.5, whereas that of an acetamidase gene homologous to A. nidulans amdS (AFLA_036780) is 6.6. The β-tubulin gene showed the 1.7-fold relative expression level against the amdS-homolog gene, suggesting that the gene induction activity of P2 was at the one-quarter weaker level in our study than in the previous report. P4 and P6 were newly identified in this study and showed useful activity in terms of both gene induction and secondary metabolite production. Their activities were comparable to those of P2, the well-characterized promoter of gpdA in A. nidulans [57, 58]. The P4 and P6 sequences were listed in Table S2. P3 did not induce either gene expression or compound production. The gene corresponding to P3 (AFLA_014570) is annotated as a “conserved hypothetical protein”; thus, a more informative annotation might require investigation of its coding sequence and other elements. The P5 promoter showed low gene transcription activity especially in PDB medium, resulting in no or scarce ustiloxin B production in V8 or PDB liquid media, contrary to a previous report that P5 greatly enhanced the transcription and translation efficiency of GUS mRNA in A. oryzae [56]. The P5 promoter comes from a gene (AFLA_052860) for a chaperon or heat shock protein; P5 might not be suitable for the culture conditions at 30 °C used for secondary metabolite production.

We combined the publicly available datasets (GSE15435) with in-house datasets (GSE136041) to prioritize the constitutively highly expressed genes. By using large-scale analysis of 83 transcriptome datasets obtained under 32 different culture conditions, we were able to stabilize the prioritized gene list (Additional file 1: Table S3). When we used only GSE136041, which was obtained under 4 conditions where ustiloxin B was produced, only the tef1 promoter was selected among the tested 6 promoters, and neither of the two new promoters (P4 and P6) was detected.

We did not test the 5′-UTR sequences shorter than 950 bp, even if the corresponding genes were constitutively highly expressed according to our survey. As our results have validated our surveying strategy for finding useful promoters for heterologous expression in A. oryzae, these shorter 5′-UTR sequences might be also worth testing, as short promoters make the construct small and convenient for transformation.


In this study, we showed that 4 promoters (P1, P2, P4 and P6) out of the 6 tested are suitable to enhance gene transcription for fungal secondary metabolite production in A. oryzae. To the best of our knowledge, P4 and P6 (5′-UTRs of AFLA_113120 and AFLA_030930, respectively) have not been previously reported as useful promoters. The performances of P4 and P6 in induction of the expression of a downstream gene and ustiloxin B production were comparable to those of P2, which is an well-characterized constitutive promoter of gpdA in A. nidulans [57, 58]. The identification of P4 and P6 shows that our simple ranking strategy using large sets of transcriptome data obtained under conditions affecting secondary metabolite production was able to prioritize genes whose promoter regions can be useful for enhancing translation of genes of interest under certain conditions in A. oryzae.


Fungal strains

Aspergillus oryzae NS4DLDP (RIB40 ΔligD::ptrA niaD ΔpyrG::sC of A. nidulans) [44] was used as the parental strain to construct the transformants in which the selected 6 different promoter sequences were inserted before ustR (NCBI Gene ID 5,995,877).

A. flavus ustROE strain along with the pyrG marker revertant as a control, which were previously constructed from the A. flavus CA14 Δku70 ΔpyrG strain [34], were used for the microarray assay as described below. In the ustROE strain, the constitutive tef1 promoter was inserted before the start codon of ustR (composed of NCBI Gene IDs 7917921 and 7917922).

The genome information with gene annotations of A. flavus NRRL3357 (NCBI acc. nos. EQ963472.1–EQ963493.1) was applied to genes of A. flavus CA14 derivatives and A. oryzae RIB40 used in the publicly available transcriptome data (GSE15435), as well as for the design of the microarray slide and primers.

Microarray assay

DNA microarray assay was performed with a one-color method as described previously [60]. Briefly, 105 conidia of A. flavus ustROE or the pyrG revertant strain were inoculated into 30 mL of V8 (20v/v% V8 juice [Campbell’s, Camden, NJ] containing 0.3w/v% CaCO3) or PDB (BD Biosciences, Franklin Lakes, NJ) liquid medium in a 100-mL flask and cultured for 2 days at 30 °C, 160 rpm. RNA was extracted from collected hyphae by using Isogen (Nippon Gene, Tokyo, Japan) and cDNA labeled with Cy3 was prepared by using a CyScribe cDNA Post-labeling Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturers’ instructions. The labeled cDNA mixture was hybridized at 42 °C for 15 h with a custom array slide designed for A. flavus on Agilent eArray (Agilent ID 052932; (Agilent, Santa Clara, CA) and, the slide was scanned on GenePix 4200A (Molecular Devices, San Jose, CA). The obtained data were normalized with the Agilent GeneSpring software. Transcriptome data obtained under four different conditions with two biological replicates for each, i.e., 8 samples in total, was submitted as a series to NCBI Gene Expression Omnibus (acc. no. GSE136041) ( We added the 8 in-house datasets to the survey in this study because they were obtained under conditions where we confirmed the ustiloxin B production.

Gene list

The GSE136041 microarray data and publicly available data obtained under 28 different conditions affecting secondary metabolite production (GSE15435, 75 sets in total) [47] were combined and used to prioritize the A. flavus genes constitutively highly expressed under conditions affecting secondary metabolite production conditions (Fig. 1). Genes were ranked according to their intensity values within each of the 83 sets and then re-ordered according to the median of the ranks among all 83 datasets (Additional file 1: Table S1). The median of the ranks of a gene was the 42nd number in the list of 83 ranks sorted in ascending order. The genes whose maximum ranks among the 83 datasets were greater than 1000 were excluded because promoters of such genes were not likely to work constitutively by overviewing the list. The genes whose 5′-UTRs to the next upstream genes were shorter than 950 bp were also excluded, taking into account minimal regulatory spaces [61, 62]. The top 6 genes were then chosen for experimental examination (Table 1).

Transformant construction

The selected 6 promoter sequences (each ≈1 kb; Table 1) were inserted upstream of A. oryzae ustR by homologous recombination using pyrG as the selective marker (Figure S1) as previously described [34]. Briefly, DNA constructs for transformation were prepared by concatenating the 1-kb 5′-UTR of A. oryzae ustR, A. nidulans pyrG, each selected promoter sequence, and 1 kb from the start codon of A. oryzae ustR via fusion PCR [63] using the primers listed in Additional file 1: Table S4. Approximately 1 µg of each DNA construct was transformed into A. oryzae NS4DLDP protoplasts using a PEG-mediated method. Three to five independent single colonies were screened by PCR amplification of the loci outside the pyrG marker and the candidate promoter sequence by using the primer pair 5′-TACTCCGTAAGTAATGCTCG-3′ and 5′-TGTCCGTCTTCATTACACTTC-3′.

Metabolite analysis

Ustiloxin B was analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The transformants and control pyrG revertant (1 × 105 conidia each) were inoculated into 30 mL of V8 or PDB liquid medium supplemented with 70 mM (NH4)2SO4 in 100-mL flasks with a baffle and were incubated at 30 °C, 165 rpm rotation for 10 days for V8 cultures and 20 days for PDB cultures. Conidia were also inoculated in 50-mL glass vials each containing 2.5 g cracked maize kernels and 1.5 mL sterile water for 14 days. From V8 and PDB cultures, 100 µL supernatant was taken every 1 or 2 days and reacted with 200 µL ethyl acetate on a rotator for 2 h at room temperature. Solid maize cultures were extracted with 5 mL of 70% acetone, the acetone was evaporated, and then the residual water fraction was reacted with an equal amount of ethyl acetate for 2 h at room temperature on a rotator. After centrifugation at 21,130 × g for 10 min, 5 µL aliquots of the water phase were filtered through a 0.22-µm filter (P/N SLLGH04NL, Merck Millipore) and separated in a water–acetonitrile gradient (98:2 for 0.5 min and then a linear change to 20:80 for 3.5 min) at a flow rate of 0.4 mL/min on a 2.1 × 50 mm Acquity UPLC BEH C18 column, 1.7 µm (Waters, Milford, MA) in an LC–MS/MS system (Acquity UPLC H class and Xevo TQD, Waters). Three biological replicates were measured per sample. The ions of m/z 646 [M + H]+, expected for ustiloxin B (C26H39N5O12S, exact mass 645.23), were selected for MS/MS fragmentation, and the MS/MS chromatograms were analyzed to estimate the amounts of ustiloxin B from the peak areas at 2.0 min with the TargetLynx software (Waters).

Quantitative PCR analyses of ustR

Transformants with P1 to P6 were inoculated as for metabolite analysis (except that only PDB medium was used) and cultured at 30 °C, 165 rpm for 3 days. Approximately 50 mg of mycelia was collected and homogenized with 300 μL of zirconia beads (0.5 mm diameter) and 1 mL of Isogen II (Nippon Gene) at 7 m/s for 1 min twice with a 10 s interval on a Shakeman6 homogenizer (Biomedical Science, Tokyo, Japan). Total RNA was extracted according to the manufacturer’s instructions of Isogen II. Chromosomal DNA was removed from 10 μg total RNA by treatment with RNase-Free DNase I (New England Biolabs, Ipswich, MA), and the resulting samples were used as templates for cDNA synthesis using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Inc., Shiga, Japan). cDNA samples (2 μL; ≈6 ng/μL) were used for quantitative real-time PCR with a Kapa SYBR Fast qPCR Kit for Roche Light Cycler (Kapa Biosystems, Wilmington, MA) on a LightCycler 480 System II (Roche, Penzberg, Germany). Primers for ustR were 5′-cacagtcacctatatctacg-3′ and 5′-ggactgcatgttcttactt-3′, and those for the tubulin gene, used as an internal standard (NCBI Gene ID 5997350), were 5′-gaaactccacctccatcca-3′ and 5′-atctcgtccataccctcacc-3′. PCR conditions were initial incubation at 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 1 s. The CT values were evaluated using the second derivative maximum method with the instrument software for 3 biological replicates per sample, each with 3 technical replicates. The CT values of the ustR and tubulin genes were converted to cDNA amounts according to the standard curves evaluated from serially diluted PCR amplicons using the above primers for each gene and genomic DNA of A. oryzae NS4DLDP as a template. The molar amount of ustR was normalized to that of tubulin for each replicate, and then averaged per sample.

Availability of data and materials

The gene list in order of the median of expression ranks in 83 transcriptome datasets is provided as the supplementary data of Additional file 1: Table S1. The procedure of constructing the transformants were described in Additional file 2: Figures S1 and S2, together with the primer list in Additional file 1: Table S3. The in-house transcriptome data is available in NCBI Gene Expression Omnibus (acc. no. GSE136041).





Liquid chromatography − tandem mass spectrometry


Potato dextrose broth


Ribosomally synthetized and post-translationally modified peptide


Secondary metabolite biosynthesis


Untranslated region


V8 vegetable juice


  1. Seshime Y, Juvvadi PR, Tokuoka M, Koyama Y, Kitamoto K, Ebizuka Y, et al. Functional expression of the Aspergillus flavus PKS-NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid. Bioorg Med Chem Lett. 2009;19(12):3288.

    Article  CAS  PubMed  Google Scholar 

  2. Fujii I, Mori Y, Watanabe A, Kubo Y, Tsuji G, Ebizuka Y. Heterologous expression and product identification of Colletotrichum lagenarium polyketide synthase encoded by the PKS1 gene involved in melanin biosynthesis. Biosci Biotechnol Biochem. 1999;63(8):1445.

    Article  CAS  PubMed  Google Scholar 

  3. Oikawa H. Reconstitution of biosynthetic machinery of fungal natural products in heterologous hosts. Biosci Biotechnol Biochem. 2020;84:433.

    Article  CAS  PubMed  Google Scholar 

  4. Mattern IE, Unkles S, Kinghorn JR, Pouwels PH, van den Hondel CAMJ. Transformation of Aspergillus oryzae using the A. niger pyrG gene. Mol Gen Genet. 1987;210(3):460.

    Article  CAS  PubMed  Google Scholar 

  5. Gomi K, Iimura Y, Hara S. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric Biol Chem. 1987;51(9):2549.

    CAS  Google Scholar 

  6. Unkles SE, Campbell EI, de Ruiter-Jacobs YMJT, Broekhuijsen M, Macro JA, Carrez D, et al. The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: a convenient and general selection system for filamentous fungal transformation. Mol Genet Genomics. 1989;218(1):99.

    Article  CAS  Google Scholar 

  7. Yamada O, Lee BR, Gomi K. Transformation system for Aspergillus oryzae with double auxotrophic mutations, niaD and sC. Biosci Biotechnol Biochem. 1997;61(8):1367.

    Article  CAS  Google Scholar 

  8. Jin FJ, Maruyama J, Juvvadi PR, Arioka M, Kitamoto K. Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts. Biosci Biotechnol Biochem. 2004;68:656.

    Article  CAS  PubMed  Google Scholar 

  9. Gomi K, Kitamoto K, Kumagai C. Transformation of the industrial strain of Aspergillus oryzae with the homologous amdS gene as a dominant selectable marker. J Ferment Bioeng. 1992;74(6):389.

    Article  CAS  Google Scholar 

  10. Kubodera T, Yamashita N, Nishimura A. Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: Cloning, characterization and application as a dominant selectable marker for transformation. Biosci Biotechnol Biochem. 2000;64(7):1416.

    Article  CAS  PubMed  Google Scholar 

  11. Mizutani O, Masaki K, Gomi K, Iefuji H. Modified Cre-LoxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue. Appl Environ Microbiol. 2012;78(12):4126.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jin FJ, Maruyama J, Juvvadi PR, Arioka M, Kitamoto K. Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae. FEMS Microbiol Lett. 2004;239(1):79.

    Article  CAS  PubMed  Google Scholar 

  13. Katayama T, Tanaka Y, Okabe T, Nakamura H, Fujii W, Kitamoto K, et al. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol Lett. 2016;38(4):637.

    Article  CAS  PubMed  Google Scholar 

  14. Katayama T, Nakamura H, Zhang Y, Pascal A, Fujii W, Maruyama J. Forced recycling of an AMA1-based genome-editing plasmid allows for efficient multiple gene deletion/integration in the industrial filamentous fungus Aspergillus oryzae. Appl Environ Microbiol. 2019;85(3):e01896–e1918.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Alberti F, Khairudin K, Venegas ER, Davies JA, Hayes PM, Willis CL, et al. Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives. Nat Commun. 2017;8(1):1.

    Article  CAS  Google Scholar 

  16. Tagami K, Liu C, Minami A, Noike M, Isaka T, Fueki S, Shichijo Y, et al. Reconstitution of biosynthetic machinery for indole-diterpene paxilline in Aspergillus oryzae. J Am Chem Soc. 2013;135(4):1260.

    Article  CAS  PubMed  Google Scholar 

  17. Matsuda Y, Iwabuchi T, Wakimoto T, Awakawa T, Abe I. Uncovering the unusual d-ring construction in terretonin biosynthesis by collaboration of a multifunctional cytochrome P450 and a unique isomerase. J Am Chem Soc. 2015;137:3393.

    Article  CAS  PubMed  Google Scholar 

  18. Lv JM, Hu D, Gao H, Kushiro T, Awakawa T, Chen GD, et al. Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis. Nat Commun. 2017.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Bunnak W, Wonnapinij P, Sriboonlert A, Lazarus CM, Wattana-Amorn P. Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases. Org Biomol Chem. 2019;17:374.

    Article  CAS  PubMed  Google Scholar 

  20. Ye Y, Ozaki T, Umemura M, Liu C, Minami A, Oikawa H. Biomolecular chemistry heterologous production of asperipin-2a: Proposal for sequential oxidative macrocyclization. Org Biomol Chem. 2019;17:39.

    Article  CAS  Google Scholar 

  21. Nagamine S, Liu C, Nishishita J, Kozaki T, Sogahata K, Sato Y, et al. Ascomycete Aspergillus oryzae is an efficient expression host for production of basidiomycete terpenes by using genomic DNA sequences. Appl Environ Microbiol. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Chang DTH, Huang CY, Wu CY, Wu WS. YPA: An integrated repository of promoter features in Saccharomyces cerevisiae. Nucleic Acids Res. 2011;39:647.

    Article  CAS  Google Scholar 

  23. Fitz E, Wanka F, Seiboth B. The promoter toolbox for recombinant gene expression in Trichoderma reesei. Front Bioeng Biotechnol. 2018;6:1.

    Article  Google Scholar 

  24. Fleiner A, Dersch P. Expression and export: Recombinant protein production systems for Aspergillus. Appl Microbiol Biotechnol. 2010;87:1255.

    Article  CAS  Google Scholar 

  25. Polli F, Meijrink B, Bovenberg RAL, Driessen AJM. New promoters for strain engineering of Penicillium chrysogenum. Fungal Genet Biol. 2016;89:62.

    Article  CAS  PubMed  Google Scholar 

  26. Basse CW, Farfsing JW. Promoters and their regulation in Ustilago maydis and other phytopathogenic fungi. FEMS Microbiol Lett. 2006;254:208.

    Article  CAS  PubMed  Google Scholar 

  27. Tada S, Gomi K, Kitamoto K, Takahashi K, Tamura G, Hara S. Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae. Mol Gen Genet. 1991;229:301.

    Article  CAS  PubMed  Google Scholar 

  28. Tsuchiya K, Tada S, Gomi K, Kitamoto K, Kumagai C, Tamura G. Deletion analysis of the Taka-amylase A Gene promoter using a homologous transformation system in Aspergillus oryzae. Biosci Biotechnol Biochem. 1992;56:1849.

    Article  CAS  PubMed  Google Scholar 

  29. Kanemori Y, Gomi K, Kitamoto K, Kumagai C, Tamura G. Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system. Biosci Biotechnol Biochem. 2005;63(1):180.

    Article  Google Scholar 

  30. Ye Y, Minami A, Igarashi Y, Izumikawa M, Umemura M, Nagano N, et al. Unveiling the biosynthetic pathway of the ribosomally synthesized and post-translationally modified peptide ustiloxin B in filamentous fungi. Angew Chemie Int Ed. 2016.

    Article  Google Scholar 

  31. Shoji JY, Maruyama JI, Arioka M, Kitamoto K. Development of Aspergillus oryzae thiA promoter as a tool for molecular biological studies. FEMS Microbiol Lett. 2005;244:41.

    Article  CAS  PubMed  Google Scholar 

  32. Kitamoto N, Matsui J, Kawai Y, Kato A, Yoshino S, Ohmiya K, et al. Utilization of the TEF1-alpha gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae. Appl Microbiol Biotechnol. 1998;50(1):85.

    Article  CAS  PubMed  Google Scholar 

  33. Boel E, Hansen MT, Hjort I, Høegh I, Fiil NP. Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger. EMBO J. 1984;3:1581.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Umemura M, Nagano N, Koike H, Kawano J, Ishii T, Miyamura Y, et al. Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide ustiloxin B in Aspergillus flavus. Fungal Genet Biol. 2014;68:23.

    Article  CAS  PubMed  Google Scholar 

  35. Yoshimi A, Umemura M, Nagano N, Koike H, Machida M, Abe K. Expression of ustR and the golgi protease KexB are required for ustiloxin B biosynthesis in Aspergillus oryzae. AMB Express. 2016;6(1):9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Tamano K, Kuninaga M, Kojima N, Umemura M, Machida M, Koike H. Use of the kojA promoter, involved in kojic acid biosynthesis, for polyketide production in Aspergillus oryzae: implications for long-term production. BMC Biotechnol. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Mojzita D, Rantasalo A, Ja J. Gene expression engineering in fungi. Curr Opin Biotechnol. 2019;59:141.

    Article  CAS  PubMed  Google Scholar 

  38. Rantasalo A, Landowski CP, Kuivanen J, Korppoo A, Reuter L, Koivistoinen O, et al. A universal gene expression system for fungi. Nucleic Acids Res. 2018;46:e111.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Harvey CJB, Tang M, Schlecht U, Horecka J, Fischer CR, Lin H, et al. HEx: A heterologous expression platform for the discovery of fungal natural products. Sci Adv. 2018;4:eaar5459.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Machens F, Balazadeh S, Mueller-Roeber B, Messerschmidt K. Synthetic promoters and transcription factors for heterologous protein expression in Saccharomyces cerevisiae. Front Bioeng Biotechnol. 2017.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, et al. Genome sequencing and analysis of Aspergillus oryzae. Nature. 2005;438:1157.

    Article  PubMed  Google Scholar 

  42. Hoang H, Maruyama J, Kitamoto K. Modulating endoplasmic reticulum-Golgi cargo receptors for improving secretion of carrier-fused heterologous proteins in the filamentous fungus Aspergillus oryzae. Appl Environ Microbiol. 2015;81:533.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Chanda A, Roze LV, Kang S, Artymovich KA, Hicks GR, Raikhel NV, et al. A key role for vesicles in fungal secondary metabolism. Proc Natl Acad Sci. 2009;106:19533.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Marui J, Yamane N, Ohashi-Kunihiro S, Ando T, Terabayashi Y, Sano M, et al. Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)2Cys6 transcriptional activator and induced by kojic acid at the transcriptional level. J Biosci Bioeng. 2011;112(1):40.

    Article  CAS  PubMed  Google Scholar 

  45. Kurtzman CP, Smiley MJ, Robnett CJ, Wicklow DT. DNA relatedness among wild and domesticated species in the Aspergillus flavus group. Mycologia. 1986;78(6):955.

    Article  Google Scholar 

  46. Machida M, Yamada O, Gomi K. Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res. 2008;15:173.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Georgianna DR, Fedorova ND, Burroughs JL, Dolezal AL, Bok JW, Horowitz-Brown S, et al. Beyond aflatoxin: Four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters. Mol Plant Pathol. 2010;11(2):213.

    Article  CAS  PubMed  Google Scholar 

  48. Ehrlich KC, Mack BM. Comparison of expression of secondary metabolite biosynthesis cluster genes in Aspergillus flavus, A. parasiticus, and A. oryzae. Toxins. 2014;6:1916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ouyang Q, Wang X, Zhang N, Zhong L, Liu J, Ding X, et al. Promoter screening facilitates heterologous production of complex secondary metabolites in Burkholderiales strains. ACS Synth Biol. 2020;9:457.

    Article  CAS  PubMed  Google Scholar 

  50. Hernandez-garcia CM, Finer JJ. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014;217–218:109.

    Article  PubMed  CAS  Google Scholar 

  51. Oda K, Terado S, Toyoura R, Fukuda H, Kawauchi M, Iwashita K. Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter. Biosci Biotechnol Biochem. 2016;80:1792.

    Article  CAS  PubMed  Google Scholar 

  52. Sibthorp C, Wu H, Cowley G, Wong PWH, Palaima P, Morozov IY, et al. Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters. BMC Genomics. 2013.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Koiso Y, Natori M, Iwasaki S, Sato S, Sonoda R, Fujita Y, Yaegashi H, et al. Ustiloxin: A phytotoxin and a mycotoxin from false smut balls on rice panicles. Tetrahedron Lett. 1992;33:4157.

    Article  CAS  Google Scholar 

  54. Koiso Y, Li Y, Iwasaki S, Hanaoka K, Kobayashi T, Sonoda R, et al. Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens. J Antibiot. 1994;47(7):765.

    Article  CAS  Google Scholar 

  55. Bayram Ö, Krappmann S, Ni M, Bok JW, Helmstaedt K, Yu J, et al. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science. 2008;320:1504.

    Article  CAS  PubMed  Google Scholar 

  56. Koda A, Bogaki T, Minetoki T, Hirotsune M. 5′ untranslated region of the hsp12 gene contributes to efficient translation in Aspergillus oryzae. Appl Microbiol Biotechnol. 2006;70(3):333.

    Article  CAS  PubMed  Google Scholar 

  57. Punt PJ, Dingemanse MA, Kuyvenhoven A, Soede RDM, Pouwels PH, van den Hondel CA. Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene. 1990;93(1):101.

    Article  CAS  PubMed  Google Scholar 

  58. Punt PJ, Van Biezen N, Conesa A, Albers A, Mangnus J, Van Den Hondel C. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol. 2002;20(5):200.

    Article  CAS  PubMed  Google Scholar 

  59. Chang PK, Zhang Q, Scharfenstein L, Mack B, Yoshimi A, Miyazawa K, et al. Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection. Appl Microbiol Biotechnol. 2018;102(12):5209.

    Article  CAS  PubMed  Google Scholar 

  60. Tamano K, Sano M, Yamane N, Terabayashi Y, Toda T, Sunagawa M, et al. Transcriptional regulation of genes on the non-syntenic blocks of Aspergillus oryzae and its functional relationship to solid-state cultivation. Fungal Genet Biol. 2008;45(2):139.

    Article  CAS  PubMed  Google Scholar 

  61. Chen W, Wei W, Lercher MJ. Minimal regulatory spaces in yeast genomes. BMC Genomics. 2011;12:320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Roy S, Kagda M, Judelson HS. Genome-wide prediction and functional validation of promoter motifs regulating gene expression in spore and infection stages of Phytophthora infestans. PLoS Pathog. 2013;9:13.

    Article  CAS  Google Scholar 

  63. Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, et al. Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc. 2006;1(6):3111.

    Article  CAS  PubMed  Google Scholar 

Download references


We thank Dr Koichi Tamano and Tomoko Ishii, AIST, for advice and discussion.


This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants 17H05456, 24710234) and by the Medical Mycology Research Center, Chiba University.

Author information

Authors and Affiliations



MU designed the study, performed experiments, analyzed data and wrote manuscript. LVD & KK performed culture and qPCR experiments. TO validated qPCR data. GT analyzed transcriptome data and ranked genes in order by expression. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Maiko Umemura.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Table S1.

Genes listed in order of the median of expression ranks in 83 transcriptome datasets. Table S2. The two nucleotide sequences confirmed to have promoter activities in this study. Table S3. Median of ranks and expression rank of top 13 genes in Table 1 evaluated from 83, 75 and 8 transcriptome datasets. Table S4. Primers for construction of transformants.

Additional file 2: Figure S1.

Construction of transformants for promoter activity test. Figure S2. Fungal cell weight after 10 days in V8 and 20 days in PDB liquid medium culture.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Umemura, M., Kuriiwa, K., Dao, L.V. et al. Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production. Fungal Biol Biotechnol 7, 3 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: