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Because the production of variecolin depends on the two enzymes terpene synthase (TS) and cytochrome P450 monooxygenase, fungal transformants carrying the genes encoding these two enzymes were selected. The TS gene, along with the amyB promoter (PamyB) and terminator (TamyB), was inserted into the pAdeA vector and then transformed into Aspergillus oryzae NSAR1. Because the pAdeA plasmid does not contain a promoter and a terminator, the TS gene was first amplified by PCR from the genomic DNA of A. aculeatus CBS 172.66 and introduced into SmaI site of the pTAex3 plasmid. The resultant pTAex3-TS plasmid was transformed into the Escherichia coli DH5α strain, and transformants were selected on an LB + ampicillin agar plate. Colony PCR was performed to verify the presence of the pTAex3-TS plasmid. The pTAex3-TS plasmid was then extracted and used as a template for amplification of the promoter-TC gene-terminator fragment by PCR. The PCR fragment was ligated into pAdeA digested by XbaI, to yield the pAdeA-TS plasmid.

1. Construction of the pAdeA-vrcA plasmid (BBa_K3834035)
Figure 1. Workflow of construction of the pAdeA-vrcA plasmid.

The VrcA gene encoding a terpene synthase (TS) (2861 bp in size; Figure 1A) was PCR amplified from genomic DNA of A. aculeatus CBS 172.66 and inserted by ligation into the pTAex3 vector and transformed into E. coli DH5α. Transformants (4) carrying the vrcA gene were screened by colony PCR (2957 bp, Figure 1B).

A DNA fragment harboring the amyB promoter (PamyB)-vrcA-amyB terminator (TamyB) was then amplified from the pTAex3-based plasmid (3723 bp, Figure 1C), inserted into the SmaI site of pAdeA plasmid and transformants containing the DNA fragment confirmed by colony PCR (2861 bp, Figure 1D).

2. Construction of the pTAex3-vrcB plasmid (BBa_K3834036).

The vrcB gene encoding a P450 monooxygenease (2075 bp, Figure 2A) was PCR amplified from genomic DNA of A. aculeatus CBS 172.66 and inserted into the pTAex3 plasmid and transformed into E. coli DH5α. Transformants carrying the vrcB gene was confirmed by colony PCR (2198 bp, Figure 2B).
Figure 2. Workflow of construction of the pTAex3-vrcB plasmid

3. Construction of the pTAex3-HR plasmid (BBa_K3834037) for CRISPR-Cas9-mediated transformation into the Aspergillus oryzae host

As described in the background information for biobrick BBa_K3834037, the sC locus facilitates efficient and specific integration of any “exogenous gene fragment” into the Aspergillus oryzae host chromosome. In addition, via CRISPR-Cas9-mediated homologous recombination (using a tailored gRNA that is homologous to the sC locus), the transformation efficiency of this biobrick (and any exogenous gene it carries) into Aspergillus is greatly enhanced and requires significantly less plasmid DNA.
Figure 3. Construction of the pTAex3-HR plasmid that contains the front sC locus (1064 bp in size) and rear sC locus (1007 bp in size).

Therefore, to construct the pTAex3-HR plasmid, the two arms of the sC locus were PCR amplified from the genomic DNA of A. oryzae (1007 bp and 1064 bp, Figure 3A) and then inserted into the pTAex3 plasmid to generate the pTAex3-HR construct which was digested with HindIII to confirm the authenticity of the construct (Figure 3B).

4. PCR amplification of P450 genes and cloning into the pTAex3-HR vector

PCR amplified P450 homologues from 11 fungal strains were successfully amplified by PCR (Figure 4) and individually inserted into the pTAex3-HR plasmid by homologous recombination, and E. coli recombinants confirmed by colony PCR (Figure 5).

Figure 4. PCR amplification of P450 gene homologues from genomic DNA of different fungal strains. AcldA-P450 from Aspergillus calidoustus, oblBAc from Aspergillus clavatus, AsphetP from Aspergillus heteromorphus, AspvioP from Aspergillus violaceus, oblBBm from Bipolaris maydis, Stl-P450 and Qnn-P450 from Emericella variecolor, PenariP from Penicillium arizonense, TalstiP from Talaromyces stipitatus, astA and astB from Talaromyces wortmannii, TalacellP1 and TalacellP2 from Talaromyces cellulolyticus, bscB, bscC, bscF and bscG from Pseudocercospora fijiensis.

Figure 5. Confirmation of P450 transformants (E. coli DH5α) by colony PCR

5. Fungal transformation

Plasmid DNA was purified from the different pTAex3-HR-P450 E. coli transformants (Figure 5) and introduced into the Aspergillus oryzae fungal host using a PEG-mediated transformation coupled with CRISPR-Cas9-based homologous recombination protocol (details are provided in the protocol section). Fungal transformants produced by two different pTAex3-HR-P450 constructs are shown in Figure 6.
Figure 6. Fungal transformants containing 2 different pTAex3-HR-P450 gene constructs. A: pTAex3-HR-Stlp. B: pTAex3-HR-AcldA.

6. Analysis of metabolites

Metabolites were extracted from various fungal transformants harboring different pTAex3-HR-P450 gene constructs for HPLC analysis. HPLC analyses was performed to examine the production of variecolin metabolites in various A. oryzae transformants. Analysis results indicated that the P450 monooxygenases of four different P450 transformants can successfully oxidize the hydrocarbon intermediate (with a 5-8-6-5-fused ring system) into various oxidation products. Although two of the four P450s exhibited the identical metabolite profile to that of the P450 from A. aculeatus, the other two afforded new metabolites, which were not present in the variecolin pathway. These metabolites were isolated via a series of chromatographic techniques, including flash chromatography and semipreparative HPLC. NMR and MS analyses were performed to determine the structures of the variecolin analogues. In addition to identifying two known metabolites (variecolin and variecolactone) (Figure 7) in our screens, we have also successfully identified three new variecolin analogues (1, 2, and 3) (Figure 8).
Figure 7. HPLC chromatograms of two known metabolites, variecolin and variecolactone. The chemical identities of the peaks are shown on the right.

Figure 8. HPLC chromatograms of novel fungal metabolites. 1: compound 313, 2: compound 161, and 3: compound 163 from A. oryzae transformants.

7. Evaluation of biological activities
7.1. Novel compounds do not show strong inhibition on tested bacterial strains

The in vitro antimicrobial activity of variecolin and variecolin analogues were tested on five different bacterial species including Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, and Escherichia coli. One of the variecolin analogues (compound 313) exhibited a minimum inhibitory concentration (MIC70) of 16 μg/mL on Staphylococcus aureus, compared to 7 μg/mL of variecolin.

Table 1. Anti-bacterial activities of purified secondary metabolites

7.2 Novel compounds significantly inhibit the growth of MCF-7 breast cancer cells
Figure 9. Percentage of cell viability versus the concentration of purified secondary metabolites.

The in vitro anticancer activity of variecolin and variecolin analogues was tested utilizing breast cancer cell MCF-7. The half maximal inhibitory concentration (IC50) of variecolin was 1.4±0.3 μM, and the new variecolin analogues exhibited anticancer properties with IC50 values of 8±3 μM, 31±9 μM, and 146±82 μM, respectively (Figure 9).

The inhibition of cell viability assay of breast cancer MCF-7 has shown that variecolin is the most efficient anticancer compound among all six compounds with IC50 value 1.4±0.3 μg/ml. The new vairecolin analogue 313 showed inhibition of cell viability with IC50 value 8±3 μg/ml, which is stronger than that of the existing variecolactone with IC50 value 22±8 μg/ml. The new variecolin analogue 161 and 163 also showed anti-cancer properties against the breast cancer MCF-7 but weaker than that of variecolin and the new variecolin analogue 313. The low IC50 values of variecolin and the new variecolin analogue 313 indicate that they might be effective drugs for treating cancer and are worth further researching.