S161: Development of expression platforms in yeast and filamentous fungi for stable introduction of multiple gene copies and complex metabolic pathways

The rapid amassment of sequence information, including fully sequenced genomes, increases the potential of moving genes and pathways into fungal cell factories for gene analysis and characterization, and for production of new compounds. However, heterologous expression requires the creation of DNA vectors, which is often a major bottleneck in strain construction. Here we present efficient methods based on uracil-excision based cloning (USER-cloning) that simplifies single gene transfer, pathway reconstruction and gene amplification. All genes are integrated by iterative gene targeting into defined integration sites that support high expression. In the yeast, Saccharomyces cerevisiae, we have developed a platform for integration of more than 20 genes, hence, supporting reconstruction of even very complex metabolic pathways. The genes are integrated into clusters allowing for subsequent strain development via sexual crosses. Moreover, a variation of the platform allows for controlled gene amplification. Successful reconstruction of a pathway, containing 8 genes from Arabidopsis thaliana, for production of health-promoting glucosinolates in S. cerevisiae will be presented as an example. A similar system has been developed for filamentous fungi using Aspergillus nidulans as a model. In this case, we demonstrate functionality of the system by transferring the entire 25 kb gene cluster responsible for geodin production in A. terreus into the genome of A. nidulans triggering geodin production in this host. Moreover, to demonstrate the possibility for performing large scale experiments, more than 50 megasynthases from different fungi have been transferred into this expression platform, setting the stage for detection of novel secondary metabolites.