Efforts towards the heterologous expression of echinodol and congeners in the host Aspergillus oryzae

This study aimed to produce small-molecule natural products, specifically the pentacyclic triterpenoids echinodol and echinodone, from the basidiomycete Echinodontium tinctorium using the heterologous host Aspergillus oryzae. Previous studies have shown echinodol and echinodone demonstrate antiproliferative activity and therefore, echinodol isolation would potentially contribute to the field of anticancer drug discovery and development. The genome of E. tinctorium was retrieved from the 1000 Fungal Genome Project and analyzed using antiSMASH Fungi 7.1 to identify biosynthetic gene clusters (BGCs) containing terpene synthase and cytochrome P450 enzymes with potential for pentacyclic triterpenoid production. Scaffold 69 BGC was selected based on its gene sequences, which showed potential for producing terpene(s) according to limited homology data obtained through NCBI Blast. Gene sequences from Scaffold 69 were obtained using SnapGene, and gene amplification from genomic DNA was performed using PCR. The yeast homologous recombination technique was employed to construct plasmids containing genes from E. tinctorium. After multiple trials of yeast homologous recombination, genes 29scaffold69, 31scaffold69, and 30scaffold69 were successfully inserted into amy site of pTYGS-Arg and pTYGS-Ade plasmids respectively. However, there were problems in inserting more than one gene into pTYGS plasmids. In future experiments, the three constructs built in this study with single gene insertion i.e. pTYGS-Arg29scaffold69, pTYGS-Arg31scaffold69, and pTYGS-Ade30scaffold69 can be used for heterologous expression in A. oryzae. While challenges persist, the findings obtained from this study will be extremely valuable in refining these techniques and broadening their application across various areas of biotechnology and pharmaceutical research.