## 2 Ribosome/RNA Polymerase Engineering Approach  
\"Ribosome engineering\" was coined in 1996, when streptomycin was used to induce the mutation of _rpsL_ (gene encoding ribosomal protein S12) in _Streptomyces_ _lividans_, resulting in the activation of actinorhodin production. The point mutation occurred by changing of Lys-88 to Glu [25]. The nucleotide guanosine 5'-diphosphate 3'-diphosphate (ppGpp) plays an important role by responding to nutrient limitation, and initiating the biosynthesis of antibiotics, whereas ppGpp binds to RNA polymerase [26]. ppGpp is synthesized by ppGpp synthase (RelA), which is a subunit of the ribosome. The mutation of ribosomal protein S12 by streptomycin leads to an increase in the amount of ppGpp. As a result, the secondary metabolite production is enhanced or activated [27]. Challenging _Streptomyces_ with streptomycin often introduces the mutation in ribosomal protein S12 in the spontaneous resistant strain. Additionally, it often introduces mutation in _rsmG_ (encoding for 165 rRNA methyltransferase) in the resistant strain. The mutation in 165 rRNA methyltransferase can also enhance the S-adenosyl methionine (SAM) synthetase activity and the transcription level of secondary metabolism; which results in activating the cryptic pathway [28; 29]. Similarly, rifampicin was used to activate the production of actinorhodin in _Streptomyces_ _coelicolor_ A3(2) and _S. lividans_. It was found that rifampicin induced mutation in _rpoB_ (encoding the \(\beta\)-subunit of RNA polymerase). This mutation makes RNA polymerase mimic the function of ppGpp-RNA polymerase bound form, which leads to activation of the cryptic pathway [30; 31; 32]. Similarly, induction of combined drug-resistant mutations for different antibiotics such as streptomycin, gentamicin, and rifampicin was used to continuously increase the production of antibiotics in a stepwise manner in _S. coelicolor_. The single, double, and triple mutants displayed a remarkable increase in the production of ActII-ORF4 (a pathway-specific regulatory protein) in the same hierarchical order as observed for the increase in the production titer of actinorhodin [33]. Further, the ribosome engineering strategy was used to activate antibiotic production from a large number of actinomycetes using streptomycin, rifampicin, or gentamicin [34]. In the test of different actinomycetes strains, two out of seven _Streptomyces_ strains (efficiency 29%), and five out of 61 non-_Streptomyces_ strains (efficiency 8%) were activated for secondary metabolites production. The technique was also applied to soil-isolated strains, where antibiotic production in 51 _Streptomyces_ strains and 15 non-_Streptomyces_ strains were activated successfully, among 119 _Streptomyces_ strains and 234 non-_Streptomyces_ strains, with the efficiency of 43% and 6%, respectively [34]. Similarly, rifampicin was used to induce the mutation in _rpoB_ in _S. griseus_, _S. coelicolor_, _S. antibioticus_, _S. lavendulae_, _Streptomyces_, _Sacharopolyspora erythraea_ (_Sac. erythraea_) and _Amyclotapsis orientalis_ (_Amy. orientalis_) [34]. This led to enhanced production of streptomycin (_S. griseus_), actinorhodin (_S. coelicolor_), actinomycin (_S. antibioticus_), formycin (_S. lavendulae_), erythromycinomycin (_Sac. erythraea_) and vancomycin (_Amy. orientalis_). Transcriptional level analysis of cryptic pathway in _S. griseus_, _S. coelicolor_, and _Sac. erythraea_ by real-time PCR showed that the cryptic pathway was activated in the _rpoB_ and _rsmG_ strains, 3 to 70-fold increase in transcription [35]. Rifampicin was used for the activation of fredericamycin production in deep sea-derived _Streptomyces__somaliensis_[36]. The efficiency of different antibiotics, such as rifampicin, streptomycin, paromomycin, erythromycinycin, and gentamicin were compared for enhancing antibiotic production and activating the silent gene in _Streptomyces__diastotechnogenes_.