iGEM 2021 — Team CityU_HK

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# Team:CityU HK/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:CityU HK/Engineering

Foxclore | Pages | Basic Grid

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* [VarieCure](https://2021.igem.org/Team%3ACityU_HK)
* Project
  + [Description](https://2021.igem.org/Team%3ACityU_HK/Description)
  + [Engineering](https://2021.igem.org/Team%3ACityU_HK/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ACityU_HK/Contribution)
  + [Implementation](https://2021.igem.org/Team%3ACityU_HK/Implementation)
* Labwork
  + [Results](https://2021.igem.org/Team%3ACityU_HK/Results)
  + [Protocols & Log](https://2021.igem.org/Team%3ACityU_HK/Protocol)
* [Parts](https://2021.igem.org/Team%3ACityU_HK/Parts)
* [Modelling](https://2021.igem.org/Team%3ACityU_HK/Model)* Human Practice
    + [Integrated Human Practice](https://2021.igem.org/Team%3ACityU_HK/Human_Practices)
    + [Education & Science Communication](https://2021.igem.org/Team%3ACityU_HK/Communication)
    + [Collaboration](https://2021.igem.org/Team%3ACityU_HK/Collaborations)
  * Team
    + [Member](https://2021.igem.org/Team%3ACityU_HK/Team)
    + [Attribution](https://2021.igem.org/Team%3ACityU_HK/Attributions)

![](https://static.igem.org/mediawiki/2021/4/44/T--CityU_HK--engineering-bg.png)

**Cycle 1 - Biosynthesis of Variecolin**

**Design – Search for the biosynthetic genes of variecolin**

The first step of this project comprised the search for relevant gene clusters responsible for variecolin production, utilizing bioinformatic tools. In silico analyses revealed that *Aspergillus aculeatus* ATCC 16872 (CBS 172.66) contains a putative biosynthetic gene cluster for variecolin, which encodes the chimeric terpene synthase consisting of prenyltransferase (PT) and terpene cyclase (TC) domains, the cytochrome P450 monooxygenase, and the ATP-binding cassette (ABC) transporter. The biosynthetic pathway of variecolin can be predicted as follows: The PT domain of the terpene synthase first catalyzes the condensation of one molecule of dimethylallyl pyrophosphate (DMAPP) and four molecules of isopentenyl pyrophosphate (IPP), to form a C25 geranylfarnesyl pyrophosphate (GFPP). The TC domain then cyclizes GFPP, to afford a hydrocarbon intermediate with a 5-8-6-5-fused ring system, which undergoes multiple cycles of oxidative reactions catalyzed by the P450 to produce variecolin.

![](https://static.igem.org/mediawiki/2021/f/f5/T--CityU_HK--pathway.png)

**Figure 1.** *Predicted biosynthetic pathways of variecolin.*

For heterologous production of variecolin, *Aspergillus oryzae* NSAR1 (*niaD-, sC, ΔargB, adeA*)[1] was used as an auxotrophic fungal host. A terpene synthase (TS) gene was introduced into the pAdeA vector that contains the *adeA* prototrophic marker for adenine synthesis, while diverse P450 gene constructs were prepared using the pTAex3 vector that harbors the *argB* prototrophic gene marker. The plasmids, pAdeA-TS and pTAex3-P450, were transformed into the auxotrophic *A. oryzae* NSAR1 recipient strain for variecolin production.

**Build – Introduction of TS and P450 genes**

![](https://static.igem.org/mediawiki/2021/3/35/T--CityU_HK--engineering2.png)

**More detailed information**

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 a pTAex3 vector that had been digested by *SmaI*. 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.

The pAdeA-TS plasmid was then introduced into the fungal host via PEG-mediated protoplast transformation. Because fungal protoplasts are sensitive to osmotic pressure, sorbitol is used to maintain a stable osmotic pressure during PEG-mediated protoplast transformation, to keep the protoplasts intact. Calcium is thought to open channels in the cytomembrane of protoplasts, facilitating the incorporation of the pAdeA-TS plasmid. Because pAdeA contains the *adeA* gene, *A. oryzae* transformants are adenine prototrophs, and can be selected using a selective medium lacking adenine. The metabolites of transformants were analyzed and characterized via gas chromatography–mass spectrometry (GC-MS). Transformants producing the hydrocarbon intermediate with a 5-8-6-5-fused ring system (Fig. 1) were used as host for subsequent transformation with diverse P450 monooxygenase gene constructs.

The pTAex3-P450 plasmid was then constructed by ligating the P450 gene into a pTAex3 vector. The successfully constructed pTAex3- P450 plasmid was introduced as described above, into the *A. oryzae/TC* strain by PEG-mediated protoplast transformation.

By transforming pAdeA-TS plasmid and pTAex3-P450 plasmid into the *Aspergillus oryzae* NSAR1 strain, the fungal transformant becomes adenine (*adeA⁺*) and arginine (*argB⁺*) prototrophs, respectively. Therefore, *Aspergillus oryzae* will become a prototroph to grow in minimum medium without adenine and arginine, and variecolin can be detected in the metabolites of the fungal host.

To confirm the variecolin production, the metabolites derived from the *A. oryzae* transformant which possesses the TS and P450 genes were analyzed through liquid chromatography–mass spectrometry (LC–MS).

**Test – Analysis of the product - variecolin**

HPLC and NMR analyses were performed to verify whether our biosynthetic approach of variecolin was successful.

HPLC was used to confirm the presence of variecolin in the metabolites. The result indicated successful synthesis of variecolin, and further indicated that two other variecolin analogues, varicolactone and emericolin A, were also present in the metabolites; the peak of emericolin A overlapped with the peak of an endogenous metabolite derived from the *A. oryzae* strain. Their structures were further validated by NMR analysis as well as their specific rotation values, and the NMR data of the three compounds were in good agreement with the reported data [2][3][4].

![](https://static.igem.org/mediawiki/2021/d/df/T--CityU_HK--HPLC-variecolin.jpg)

**Figure 2.**  *(A) HPLC chromatograms of the metabolites from the *A. oryzae* transformants with (i) TS and (ii) TS and P450. Asterisk indicates an endogenous metabolite that co-elutes with emericolin A. The chromatograms were monitored at 215 nm. (B) Structures of the metabolites.*

**Learn**

Transformation based on the random integration of target DNA requires a large amount of DNA and the screening of many transformants to find the most appropriate one. Because the introduction of individual P450 monooxygenase genes would be conducted in the next step, we employed a recently developed CRISPR-Cas9-mediated transformation method, coupled with homologous recombination, to facilitate the process.

**Cycle 2 - Biosynthesis of variecolin analogues**

**Design - Selection of P450 homologues using cblaster**

Because many fungal sesterterpenoid pathways adopt one or more P450 monooxygenases as a tailoring enzyme, we predicted that co-expression of a TS gene responsible for the synthesis of the variecolin scaffold and a homologue of variecolin synthase could yield a new variecolin analogue. Therefore, a number of fungal genomes containing P450 genes that are potentially associated with di-/sesterterpenoid pathways were selected in silico using the cblaster program [5]. A total of 17 cytochrome P450 genes were identified and selected for synthesis of novel variecolin analogues.

![](https://static.igem.org/mediawiki/2021/e/e1/T--CityU_HK--HPLC-cblaster.png)

**Figure 3.** *Part of the result of cblaster search.*

**Build - P450 gene transformation**

P450 genes were PCR amplified from genomic DNA of different fungi and separately ligated to pTAex3-HR (a vector that contains the *argB* prototrophic gene marker) to generate a series of pTAex3-HR-P450 plasmids. The pTAex3-HR-P450 plasmids were separately transformed into the *A. oryzae/TS* strain by PEG-mediated protoplast transformation with the help of the CRISPR-Cas9 system.

**CRISPR-Cas9 genome editing**[6]

The essential components of CRISPR-Cas9 genome editing include guide RNA and Cas9. The guide RNA is designed to target the *sC* locus of the *A. oryzae*NSAR1 strain. Guide RNA and Cas9 were added to the protoplast, causing the DNA double strand to break at the expected site. The double strand break can be repaired by homologous recombination using the pTAex3-HR-P450 plasmid. Because pTAex3-HR harbors the *argB* gene, and the resultant *A. oryzae* transformants become arginine prototroph, it can be selected on the transformation medium which lacks arginine. The introduction of the target P450 was verified by diagnostic PCR using a gene-specific primer, and an additional primer that attaches outside the homology arm used for the transformation.

**Test - Analysis of the possible variecolin analogues**

HPLC, LC-MS, and GC-MS analyses were performed to examine whether there were variecolin analogues in the *A. oryzae* transformants. Analysis results indicated that four of the tested P450s successfully converted the hydrocarbon product into oxidized metabolite(s). 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. A total of three new variecolin analogues (**1**, **2**, and **3**) were successfully obtained.

![](https://static.igem.org/mediawiki/2021/5/56/T--CityU_HK--new.png)

**Figure 4.** *HPLC chromatograms of the metabolites (**1**, **2**, and **3**）from the *A. oryzae* transformants with the artificial sesterterpenoid pathways.**1**: compound 313, **2**: compound 161, and **3**: compound 163.*

**Learn**

The antibacterial and anticancer activities of the three new variecolin analogues were discovered and compared with the biological activities of variecolin. The result shows the potentially therapeutic properties of variecolin analogues; their other biological activities can be tested in the future.

**Anti-bacterial properties**

The *in vitro* antimicrobial activity of variecolin and variecolin analogues were tested with several microbial species, including *Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis*, and *Escherichia coli*. One of the variecolin analogues 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*

![](https://static.igem.org/mediawiki/2021/b/bf/T--CityU_HK--enginew.png)

**Anticancer properties**

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 μM, and the new variecolin analogues exhibited anticancer properties with IC50 values of 8±3 μM, 31±9 μM, and 146±82(**Table 2**), respectively.

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 (**Table 2**). 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.

**Table 2.** *Anticancer activities of purified secondary metabolites*

![](https://static.igem.org/mediawiki/2021/3/3c/T--CityU_HK--anticancer.png)

![](https://static.igem.org/mediawiki/2021/4/48/T--CityU_HK--anticancer1.png)

**Figure 5.** *%cell viability versus the concentration of purified metabolites*

##### **References**

1. Jin, F. J., Maruyama, J. I., Juvvadi, P. R., Arioka, M., & Kitamoto, K. (2004). Development of a novel quadruple auxotrophic host transformation system by *argB* gene disruption using *adeA* gene and exploiting adenine auxotrophy in *Aspergillus oryzae*. *FEMS Microbiology Letters, 239*(1), 79-85.

2. Yoganathan, K., Rossant, C., Glover, R. P., Cao, S., Vittal, J. J., Ng, S., Huang, Y., Buss, A. D., & Butler, M. S. (2004). Inhibition of the human chemokine receptor CCR5 by variecolin and variecolol and isolation of four new variecolin analogues, emericolins A−D, from *Emericella aurantiobrunnea. Journal of Natural Products, 67*(10), 1681-1684.

3. Takahashi, H., Hosoe, T., Nozawa, K., & Kawai, K. I. (1999). Two new sesterterpenes from the ascomycetous fungus *Emericella purpurea. Journal of Natural Products, 62*(12), 1712-1713.

4. Yodsing, N., Lekphrom, R., Sangsopha, W., Aimi, T., & Boonlue, S. (2018). Secondary metabolites and their biological activity from *Aspergillus aculeatus* KKU-CT2. *Current Microbiology, 75*(5), 513-518.

5. Gilchrist, C. L., Booth, T. J., van Wersch, B., van Grieken, L., Medema, M. H., & Chooi, Y. H. (2021). cblaster: a remote search tool for rapid identification and visualization of homologous gene clusters. *Bioinformatics Advances, 1*(1), vbab016.

6. Wei, X., Matsuyama, T., Sato, H., Yan, D., Chan, P. M., Miyamoto, K., Uchiyama, M., & Matsuda, Y. (2021). Molecular and computational bases for spirofuranone formation in setosusin biosynthesis. *Journal of the American Chemical Society*, in press.

Copyright © 2021 - All Rights Reserved - [VarieCure](https://2021.igem.org/Team%3ACityU_HK)

Tech Supported by [CS Dept, CityU](https://www.cs.cityu.edu.hk/ "CityU CS")

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:CityU HK/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:CityU HK/Results

Foxclore | Pages | Basic Grid

![](https://static.igem.org/mediawiki/2021/b/b5/T--CityU_HK--varie-cure-revised.PNG)

* [VarieCure](https://2021.igem.org/Team%3ACityU_HK)
* Project
  + [Description](https://2021.igem.org/Team%3ACityU_HK/Description)
  + [Engineering](https://2021.igem.org/Team%3ACityU_HK/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ACityU_HK/Contribution)
  + [Implementation](https://2021.igem.org/Team%3ACityU_HK/Implementation)
* Labwork
  + [Results](https://2021.igem.org/Team%3ACityU_HK/Results)
  + [Protocols & Log](https://2021.igem.org/Team%3ACityU_HK/Protocol)
* [Parts](https://2021.igem.org/Team%3ACityU_HK/Parts)
* [Modelling](https://2021.igem.org/Team%3ACityU_HK/Model)* Human Practice
    + [Integrated Human Practice](https://2021.igem.org/Team%3ACityU_HK/Human_Practices)
    + [Education & Science Communication](https://2021.igem.org/Team%3ACityU_HK/Communication)
    + [Collaboration](https://2021.igem.org/Team%3ACityU_HK/Collaborations)
  * Team
    + [Member](https://2021.igem.org/Team%3ACityU_HK/Team)
    + [Attributions](https://2021.igem.org/Team%3ACityU_HK/Attributions)

![](https://static.igem.org/mediawiki/2021/3/30/T--CityU_HK--resultsbnfinal.png)

**Results**

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 *Xba*I, to yield the pAdeA-TS plasmid.

**1. Construction of the pAdeA-vrcA plasmid [(BBa\_K3834035)](http://parts.igem.org/Part%3ABBa_K3834035)**

![](https://static.igem.org/mediawiki/2021/5/5f/T--CityU_HK--resultfig1.png)

**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 *Sma*I 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)](http://parts.igem.org/Part%3ABBa_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**).

![](https://static.igem.org/mediawiki/2021/8/84/T--CityU_HK--resultfig2.png)

**Figure 2.** *Workflow of construction of the pTAex3-vrcB plasmid*

**3. Construction of the pTAex3-HR plasmid [(BBa\_K3834037)](http://parts.igem.org/Part%3ABBa_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.

![](https://static.igem.org/mediawiki/2021/0/02/T--CityU_HK--re3.png)

**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 *Hin*dIII 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**).

![](https://static.igem.org/mediawiki/2021/0/00/T--CityU_HK--re4.png)

**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.*

![](https://static.igem.org/mediawiki/2021/6/66/T--CityU_HK--0001.png)

**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**.

![](https://static.igem.org/mediawiki/2021/3/3d/T--CityU_HK--re6.png)

**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**).

![](https://static.igem.org/mediawiki/2021/6/63/T--CityU_HK--re7.png)

**Figure 7.** *HPLC chromatograms of two known metabolites, variecolin and variecolactone. The chemical identities of the peaks are shown on the right.*

![](https://static.igem.org/mediawiki/2021/0/02/T--CityU_HK--re8.png)

**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*

![](https://static.igem.org/mediawiki/2021/b/bf/T--CityU_HK--enginew.png)

*7.2 Novel compounds significantly inhibit the growth of MCF-7 breast cancer cells*

![](https://static.igem.org/mediawiki/2021/7/75/T--CityU_HK--contribution.png)

**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.

Copyright © 2021 - All Rights Reserved - [VarieCure](https://2021.igem.org/Team%3ACityU_HK)

Tech Supported by [CS Dept, CityU](https://www.cs.cityu.edu.hk/ "CityU CS")