Many valuable microbes are not usually evolved to produce desired products and this necessitates the need to improve/maximize their metabolic and regulatory networks through genetic engineering. The main aim of our research in Mario's laboratory of yeast synthetic biology is to re-engineer some key metabolic pathways from filamentous fungi into Saccharomyces cerevisiae (yeast) cells in other to enhance its abilities to synthesis natural products (NPs). Metabolic pathways associated with NPs are usually encoded into clusters of genes (biosynthetic gene clusters—BGCs) while the traditional methods for the integration of genes into the yeast genome rely on homologous recombination at the loci of auxotrophic markers.  Our project is designed to establish a reliable protocol for the integration of multiple genes into the yeast genome in a single “shot” through CRISPR-Cas9 or CRISPR-Cas12a system with which allows multiple-integration protocol. This will allow us to build a different type of synthetic gene circuits such as complex digital transcription networks for application as biosensors, molecular classifiers, and DNA computing.

      We are considering a pilot comparative study by assembling in yeast, the “natural” and a “retrosynthetic” gene cluster for a well-known NPs from different filamentous fungi, which grow in extreme conditions. We are also looking at a different way to enhancing their production. In this case, new transcriptional activators for these NPs are designed via the CRISPR-dCas9 system, where dCas9 means “deficient Cas9” i.e. a Cas9 stripped of its nuclease activity. Guide RNA molecules would be designed such that they bind only in the proximity of the target promoter without any off-target effects that could lead even to cell death. This issue might force us to consider other nuclease-deficient proteins (e.g. dCas12a) that bind the DNA in the presence of protospacer adjacent motifs (PAMs) distinct from those recognized by dCas9 (NGG and NAG). Finally, activation of transcription is optimized by fusing different activation domains, such as VPR and VP64, to the chosen deficient Cas protein.