Application of hammerhead ribozymes in filamentous fungi

Metabolic engineering in filamentous fungi is a emerging field of research as many fungi produce high value primary and secondary metabolites. Ribozyme technology can be used as a tool for metabolic engineering to influence metabolic pathways and to knock down the expression of specific genes of interest. Hammerhead ribozymes can target virtually any mRNA sequence of choice and prevent gene expression on the post-transcriptional level. They are thus a versatile tool for timed and spatial elimination of unwanted gene products. As current research has only investigated the application of ribozymes in bacteria, yeast and mammalian cells, we decided to carry out a study on whether this technology can also function with filamentous fungi.We employed a sensitive, quantitative reporter-based model system as a proof of concept, using the Escherichia coli β-glucuronidase transcript (uidA) as the target mRNA and Aspergillus giganteus as the host. This system was used to validate the in vivo activities of seven different hammerhead ribozymes, which were selected by in silico analysis of the uidA mRNA. All ribozymes tested were able to reduce the reporter activity up to a maximum of 100%, demonstrating that ribozyme technology is indeed a useful tool in fungal metabolic engineering.