## 3 Discovery of New Natural Products Using Ribosome Engineering  
Genome sequencing has revealed the vast potential to produce valuable secondary metabolites in _Streptomyces_. However, many BGCs are silent under standard fermentation conditions. Natural product synthesis in _Streptomyces_ is affected by many factors, including nutrient supply including carbon and nitrogen sources, cofactors, the expression of functional biosynthetic enzymes, drug resistance, and export mechanisms. Many approaches have been used in activating silent BGCs [71; 72], such as the \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Miscellaneous Products** & **Strain** & **Method** & **Mutation*** & **Fold/g/L)*** & **Year/Ref.** \\ \hline CITase & _P. agaridevans_ & Str, Rif & K5 GR, R485H & 1100 (1104 \(\pm\) 143 U/mL) & 2018 [60] \\ \hline \(\alpha\)-amylase & _B. subtilis_ & Str & K5 GR & 1.5 (4.0 U/mL) & 2006 [61] \\ \hline Xylanase & _S. viridochromogenes_ & Str & K5 GR & 1.14 (\(\pm\)60 U/mL) & 2013 [62] \\ \hline Vitamin B12 & _P. shoreamuli_ & Rif, Gen, Ery & H4 SY, H447R & 52.0 (\(\pm\) 3.9 k/H/OC600) & 2017 [63] \\ \hline \multirow{3}{*}{\(\varepsilon\)-poly-\(\lambda\)-Lysine} & _S. albulas_ & ARIT, Str, CS & -\({}^{c}\) & 1.71 (3.0) [4] & 2016 [64] \\  & _S. albulas_ & HT, Par & - & 1.45 (2.59) & 2017 [65] \\  & _S. albulas_ & Str, Gen, Rif & K108R & 1.75–2.39 (3.83) & 2017 [66] \\  & _S. albulas_ & Str & E8 SG & 1.79 (3.04) & 2019 [67] \\ \hline \multirow{3}{*}{L-Isoleucine} & _C.pyruvate_ & _frr_ & _fusA_ & & \\  & _glutamicin_ & Overexpression & - & 1.76 (28.5) & 2015 [68] \\ \hline \multirow{3}{*}{Butanol} & _Clostriainen_ & \multirow{3}{*}{Str} & \multirow{3}{*}{K43N} & \multirow{3}{*}{1.6 (16.5)} & \multirow{3}{*}{2017 [69]} \\  & _Saccharomyces_ & & & & \\ \cline{1-1} \cline{5-5}  & Ethanol & _K. variecia_ & Str & K43N & 1.3 (34) & 2015 [70] \\ \hline \hline \end{tabular}  
* The strains with the highest titer of _rpoB_ and _rpsL_ mutation. b The term “fold” was defined as the increased yield of the mutant strain to its parental strain, which was recalculated based on the cited articles. c The highest yield strain has no detected mutation within the _rpoB_ and _rpsL_ gene. d This is the highest yield of the \(\varepsilon\)-poly-L-lysine in shake flasks. CITase: cycloisomaltooligosaccharide glucanotransferaseer.  
\end{table} 
Table 3: Yield improvement of primary metabolites, proteins, and fuels by ribosome engineering. \"one strain many compounds\" approach [73; 74], occulturation [75; 76], manipulation of pleiotropic or pathway-specific regulators, genome mining [77; 78], and BGC refactoring [17]. In 2009, [PERSON] and coworkers performed massive antibiotic-resistance screening on 1068 actinomycetes from soil, using rifampicin or streptomycin [79]. They discovered that 43% (51/119) of the nonantibiotic-producing _Streptomyces_ strains from soil were able to commence antibacterial production. This could be due to the marked enhancement of previously undetectable antibiotic production or to triggering the biosynthesis of new antibiotics (Table 4). They subsequently isolated a series of novel macrocyclic piperidamycins (**32-34**) (Figure 5) from one soil strain _Streptomyces mauvecolor_ with either a single H437L mutation in _rpoB_ or double mutation in _rpoB_ (H437L) and _rspL_ (K88R). They proposed that the enhanced gene expression for piperidamycin biosynthesis in the mutants was due to the increased affinities of mutant RNA polymerases for their promoters, based on surface plasmon resonance analysis and in vitro synthesis of green fluorescent proteins. Fredericamycin A (**15**) is a uniquely structured aromatic polyketide compound with excellent antitumor activity. [PERSON] et al. [41] identified the mutation of R444H in _rpoB_ of the resistant _Streptomyces__somaliensis_ ZH66-RIF1 using a _rif_ screen with 300 \(\mu\)g/mL of rifampicin, which resulted in activating a previously silent gene cluster. After fermentation optimization, an optimized yield of 679.5 \(\pm\) 15.8 mg/L of **15** was obtained.