Automating Plasmid Construction for High-Throughput Heterologous Expression of Fungal Gene Clusters.

Fungi are renowned for producing a diverse range of small-molecule natural products with applications in the food, pharmaceutical, and agrochemical industries. While bioinformatics can rapidly identify potential biosynthetic gene clusters (BGCs), their functional analysis remains a bottleneck, limiting the discovery of novel compounds. Advances in molecular techniques and genome sequencing now enable heterologous expression of biosynthetic pathways in hosts like Aspergillus oryzae, facilitating access to metabolites not achievable through traditional methods. However, given the high number of uncharacterized gene clusters, a high-throughput process is crucial for efficiently mining novel natural products. This chapter outlines a streamlined, high-throughput protocol using yeast homologous recombination and automated liquid handling to construct multigene plasmids for heterologous expression. While this chapter uses A. oryzae NSAR1 and the pTYGS series of expression vectors as an example, the core principles of this robotics-driven protocol are adaptable to other heterologous hosts, vectors, and organisms, providing a powerful tool for high-throughput discovery and engineering of natural products.