## 3 Semi-Synthetic and Molecular Activation of Silent BGCs
### Ribosome and Polymerase Engineering  
The regulation of BGC expression is likely to be complex in light of the metabolic burden placed on the cell following production. However, at the simplest level, one of the limitations to overexpression of antibiotics and natural products under culture conditions is the transcriptional and translational requirement on cells. Removing the perception of the burden has the effect of increasing production, while also changing the profile of compounds produced. Different from other \"pathway specific\" approaches, this generalized activation has been attributed to mutation at Lys-88 to either Glu or Arg in the ribosomal protein S12 of Streptomyces species, which enhances protein synthesis in stationary growth phase [80].  
Ribosome engineering as a concept emerged from the finding of abundant quantities of the blue-pigmented antibiotic actinorhodin in a strain of _S. lividans_ with an altered RpsL protein, a critical component of the A-site of the 30S ribosomal subunit involved in both tRNA selection [81]. This was in contrast to the majority of _S. lividans_ strains which normally do not produce antibiotics due to the dormancy of their antibiotic biosynthesis genes. Possibly circumventing the need for binding by the alarmone ppGpp, targeting the _rpsL_ gene in microbes has shown potential as an emerging method for the activation of silent gene clusters [60]. Screening for _rpsL_ mutants is relatively simple, based on the premise that rifampicin selective pressure will result in drug-resistant mutants. Already, considerable success has been reported, enhancing the yield of secondary metabolites in a wide range of structural classes, including polyketides, macrolides, aminoglycos, and nucleosides (reviewed in [60]). In one study, screening of 1068 actinomycetes isolated from soil, identified 6% of non-Streptomyces actinomycetes species and 43% of Streptomyces species acquiring the ability to synthesize antibacterial against _Staphylococcus aureus_ after a selection step that generated spontaneous _rpsL_ or _rpoB_ mutations [82]. Importantly, not alone was antibiotic production increased, novel antibiotics were also elicited through this approach, suggesting a broader applicability for activation of silent BGCs [82].  
_RpoB_ mutations have been widely effective in activating silent and poorly expressed secondary metabolite-BGCs at the transcriptional level in _S. griseus_, _S. coelicolor_, and _S. erythraea_[60]. The introduction of the _rpoB_ mutation S487L into a _Bacillus subtilis_ strain resulted in cells that overproduced an aminosugar antibiotic, 3,3'-neotrehalosadiamine (NTD), the production of which is dormant in the wild-type strain, suggesting applicability in a wider spectrum of organisms [83]. The observation that several actinomycetes possess two _rpoB_ genes, in contrast to the widely accepted consensus of the existence of a single RNA polymerase (RNAP) in bacteria, presents a new opportunity to develop this technology [60,84]. Two _rpoB_ paralogs, _rpoB_(S) and _rpoB_(R), provide _Nonomuraea_ sp. strain ATCC 39727 with two functionally distinct and developmentally regulated RNA polymerases. Heterologous expression of _rpoB_(R) in _S. lividans_ resulted in activation of cryptic antibiotic biosynthesis, both in the wild type 1326 strain, and also in a relaxed (rel) mutant strain KO-421, unable to produce ppGpp [85]. The capacity for ectopic activation in heterologous hosts further enhances the feasibility of this technology for use in natural product biodiscovery.