Our iGEM team has the following contributions for the iGEM community and future iGEM teams:
1. Establishing a synthetic biology platform to produce variecolin
Our iGEM project has established Aspergillus oryzae as a fungal synthetic biology platform (providing some basic protocols and biobricks) for gene expression, for production of the natural product, variecolin, as well as a means to derivatize variecolin (with various P450 biobricks) to produce an array of natural product derivatives as a possible source of therapeutics. The platform is useful for future iGEM teams wishing to use Aspergillus as a fungal host and for projects on natural products.
2. Design, construct and experimentally tested a series of new basic and composite biobricks for the iGEM registry
In developing Aspergillus oryzae as a new fungal synthetic biology platform, we have designed, constructed, and experimentally tested a series of new basic and composite biobricks for the iGEM registry. These biobricks could be useful to future iGEM teams working on similar projects related to natural products.
An example of a new biobrick for generic use in gene cloning is described below:
(BBa_K3834037) Biobrick sC(front)_PamyB_TamyB_sC(rear): The composite biobrick (as shown in Figure 1) takes advantage of an sC locus to facilitate the 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 homologous to the sC locus), transformation of this biobrick harboring any exogenous gene (e.g. Stl-P450 and acldA-P450) into the fungal host is highly efficient and requires significantly less plasmid DNA.
Figure 1. Genetic circuit of sC(front)_PamyB_(exogenous gene)_TamyB_sC(rear).
3. Application of the in silico modelling protocol to other natural products
In this project, several computer modelling techniques were used to evaluate (1) the anticancer properties, and (2) interactive effects of two natural products (variecolin and variecolactone) with three anticancer drug targets: human aromatase (CYP19A1), human topoisomerase II (Topo II), and cyclin-dependent protein kinase II (CDK-2).
Topo II and CDK-2 are reported to serve important roles in cytotoxicity (Cummings et al., 1993; Benson et al., 2005). The modelling data we obtained was corroborated by data from anticancer bioassay experiments in this project, which indicated that the computer modelling protocols used for this project could potentially be applied to novel natural products to evaluate their mechanisms of action as well as provide useful insights into their potential anticancer properties.