DIVERSIFY: A Fungal Multispecies Gene Expression Platform.

Recent sequencing of numerous fungal species revealed large repertoires of putative biotechnologically relevant genes and secondary metabolite gene clusters. However, often the commercial potential of these species is impeded by difficulties to predict host physiological and metabolic compatibility with a given product, and lack of adequate genetic tools. Consequently, most heterologous production is performed in standard hosts where genetic tools and experience are in place. However, these species may not be suitable for all products. To increase chances of successful heterologous production, we have created a flexible platform, DIVERSIFY, for multispecies heterologous gene expression. This reduces the workload to construction of a single gene expression cassette, used to transform all DIVERSIFY strains in order to identify the optimal cell factory host. As proof of principle of the DIVERSIFY concept, we present the first version of our platform, DIVERSIFY 1.0, which we have successfully used for the production of three proteins and a metabolite in four different Aspergilli species, and for the identification of the best producer for each of the products. Moreover, we show that DIVERSIFY 1.0 is compatible with marker-free gene targeting induced by the CRISPR nucleases Cas9 and MAD7.