TABLE 2.
Transcriptional activation tools and methods for fungal biosynthetic gene clusters.
| Transcriptional activation method | Benefits | Drawbacks |
|---|---|---|
| Overexpression of BGC core gene | • Reliable transcriptional activation of the targeted gene | • Although transcription is activated, product formation is not ensured |
| • Limited genomic modulation needed | • Does not activate the complete BGC | |
| Modulation of BGC-specific TF | • Limited genomic modulation needed | • Often no cluster-specific TFs are present in a BGC |
| • Overexpression of positive regulator can upregulate entire BGCs | • Overexpression of such TF does not guarantee transcriptional activation of the entire BGC | |
| • Other co-activators, mediators or inducers might be needed for activation | ||
| Modulation of global regulators | • Limited genomic modulation needed | • Regulator needs to be identified |
| • Multiple BGCs are affected, resulting in higher chances for compound discovery | • Global regulator targets are often unknown | |
| • Modulation can be lethal | ||
| • Difficult to assign newly produced compounds to specific BGCs | ||
| Epigenome modulation | • Feeding of chemical modulators is easy to carry out | • Histone modifying enzymes have to be identified and engineered |
| • Multiple BGCs are affected, resulting in higher chances for compound discovery | • Modulation can be lethal | |
| • Difficult to assign newly produced compounds to specific BGCs | ||
| BGC refactoring | • Native regulatory system is bypassed | • Requires extensive DNA cloning and/or synthesis efforts |
| • Episomal delivery of BGCs can lift the burden of epigenetic repression | • Limited number of established promoters | |
| • Transcription relies solely on established promoters | ||
| • Fungal SM deficient strains are available | ||
| Heterologous expression in non-fungal host | • Established heterologous systems and regulation tools are broadly available | • Potential problems with codon usage, available precursors, cellular trafficking, RNA splicing and post-translational modifications |
| STF-based BGC regulation | • Native regulatory system is eliminated or bypassed | • Extensive DNA cloning and/or DNA synthesis effort required |
| • Transcription relies on an orthogonal regulatory system | • Genome editing or BGC refactoring is required | |
| • Modular features and scalable transcriptional regulation possible | • Validation (specificity, activity) of new STFs is necessary | |
| CRISPR-based BGC regulation | • Genome editing-free transcriptional activation or repression | • Extensive DNA cloning and/or DNA synthesis effort required |
| • Rapid library construction | • Genome editing or BGC refactoring is required | |
| • Various regulatory domains are available for transcriptional activation, repression or epigenetic modulation | • No established rules available for creating STF fusions | |
| • Preceding validation required (activity, specificity) |