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

Area of Science:

• Biotechnology
• Synthetic Biology
• Natural Product Discovery

Background:

• Fungi produce diverse small-molecule natural products vital for various industries.
• Bioinformatics identifies potential biosynthetic gene clusters (BGCs), but functional analysis is a bottleneck.
• Heterologous expression in hosts like Aspergillus oryzae facilitates access to novel metabolites.

Purpose of the Study:

• To develop a streamlined, high-throughput protocol for the functional analysis of fungal biosynthetic gene clusters (BGCs).
• To enable efficient mining of novel natural products through heterologous expression.
• To provide a adaptable robotics-driven platform for natural product discovery.

Main Methods:

• Utilized yeast homologous recombination for constructing multigene plasmids.
• Employed automated liquid handling for high-throughput plasmid assembly.
• Demonstrated protocol using Aspergillus oryzae NSAR1 and pTYGS expression vectors.

Main Results:

• Developed a robotics-driven protocol for efficient construction of expression plasmids.
• Facilitated high-throughput heterologous expression of fungal biosynthetic pathways.
• Established a adaptable system for natural product discovery.

Conclusions:

• The developed protocol significantly accelerates the discovery and engineering of fungal natural products.
• This high-throughput approach overcomes limitations in functional BGC analysis.
• The adaptable methodology supports broader applications in natural product research across different organisms and hosts.