## 3 Semi-Synthetic and Molecular Activation of Silent BGCs
### Awakening the Activator  
A recurring theme from the genomics based discovery of BGCs is the co-occurrence of genes encoding transcriptional regulatory proteins suggesting a programmable activation or repression of BGC expression. Many secondary metabolite BGCs contain one or more genes that encode transcription factors that potentially transcribe all the genes of the cluster. Of the BGCs characterized to date, a co-occurrence of the LysR-Type Transcriptional Regulator (LTTR) family with BGCs has been described for a diverse spectrum of bacterial organisms [86, 87, 88]. Known to control the expression of metabolic and virulence related functions in a broad spectrum of microbial species, these regulators have dual domains typically consisting of an \(N\)-terminal DNA binding domain and a _C_-terminal co-inducer or ligand binding domain (Figure 3A and [89]). Capable of binding to DNA in the absence of a signal, they nonetheless remain inactive and expression is not achieved. Upon interaction with a co-inducer ligand or substrate, a conformational change ensues, with the formation of tetrameric structures and subsequent activation of expression at target promoters (Figure 3B). The capacity to interact with promoters in the absence of signal is an interesting feature as it typically retains the promoter in the off-state, as is the case with silent BGCs. Therefore, identifying an activating signal has the potential to change the regulator to the on-state leading to activation of the BGC and production of the bioactive compound. In some cases, LysR proteins may act as repressors of expression whereby deactivation of the protein is required for elicitation of biosynthesis. ThnI has been shown to be required for synthesis of the \(\beta\)-lactam antibiotic thienamycin in _Streptomyces cattleya_[87], while another LysR protein, designated ORF-L16, is encoded within the spinomycin biosynthetic cluster of _Saccharomyces spinosa_[88]. The oxazolomycin BGC of _S. albus_ encodes the OzmR LysR transcriptional regulator [90]. As with many of the LysR proteins identified to date, the role of this protein in the synthesis of oxazolomycin A, a peptide-polyketide hybrid compound containing a unique spiro-linked \(\beta\)-lactone/\(\gamma\)-lactam, a 5-substituted oxazole ring, is unknown. However, the co-occurrence of LysR proteins within bioactive gene clusters strongly suggests a role in their transcriptional regulation. Intriguingly, LTTRs were found to be most abundant among the Actinobacteria, Proteobacteria and Firmicutes (Figure 3C), the three phyla that account for the majority of natural product biosynthetic potential [91]. A recent molecular phylogenetic analysis of LTTR proteins from the Proteobacteria pathogen _P. aeruginosa_ revealed clustering of the LTTR repertoire into nine independent clades [92]. Importantly, clustering of either the DNA binding domain or the co-inducer binding domain resulted in conservation of the clades indicating that some degree of structural similarity may exist within the activating signal. Extending such an approach within the genomes of BGCs-rich organisms may yield clues on the activating signals that activate the LTTR regulated module, providing hints on the experimental approaches to follow to express the BGCs. Of course, several other classes of transcriptional regulator have also been described in association with BGCs. Several reports describe the co-occurrence of _Streptomyces_ antibiotic regulatory protein (SARP) family proteins with BGCs. These include the _aur1_ polyketide gene cluster involved in biosynthesis of the angiycline-like antibiotic auricin in _S. aureofaciens_[93], ThuU which is required for cephalosporcin C biosynthesis in _S. cattleya_[87], as well as several other _Streptomyces_ sp. related clusters [94, 95, 96, 97]. The TetR and AraC families are also represented, with many regulatory systems co-occurring within the large BGCs [98]. In addition to the pathway specific activation of BGCs, some global regulators of secondary metabolite production have been identified.