Fluorescent based screening of heterologously-expressed polycistronic genes in eukaryotes
Filamentous fungi produce an inestimable number of high-value natural products (NPs). NPs are synthetized by metabolic pathways, whose genes are normally clustered in the genome. These clusters usually contain one or several central biosynthetic genes encoding large multidomain proteins belonging to the polyketide synthase and/or non-ribosomal peptide synthetase protein families. These enzymes represent a sort of signature that can be exploited to identify putative gene clusters in every sequenced genome. However, the majority of the computationally identified clusters are not expressed under standard laboratory conditions, requiring molecular engineering to induce the production of unknown NPs.
The heterologous expression of entire biosynthetic clusters can be a feasible solution to decrypt unknown fungal NPs. The parallel expression of eukaryotic genes is normally limited by the presence of regulatory elements (promoters and terminators); consequently, the expression of clustered genes can happen only when all promoters are activated together. This problem can be avoided by expressing a group of genes as a polycistron under the control of a single inducible promoter. This method was implemented in the last years, obtaining a toolbox to study eukaryotic gene clusters. In particular, one of the major problems encountered when working with large polycistronic genes is the screening of positive transformants. Plasmid vectors are ectopically integrated in the genome; thus, the majority of transformants (>95%) present a fragmented polycistronic construct unable to express the entire biosynthetic pathway. In order to have a faster and reliable screening method, we developed a fluorescence based selection system using a split-gfp marker. This methodology permits the fast selection of those transformants having complete clusters correctly expressed.