## 4 Discussion
The results in this study provide a primary insight into the energy change in different mutants, indicating that the mutants with higher ATP hydrolysis rate generated higher production of toyocamycin. We will conduct further research to reveal the energetic shift between primary and secondary metabolism in the future. Despite the ATP signal factor, other factors have included intracellular signals such as SAM for the stimulation of silent genes; the translation efficiency exerts a positive impact on the secondary metabolites production. There was a _rsmG_ mutation harbored in all four strains after genome shuffling, which make the SAM-dependent 16S rRNA methyltransferase inactive. The mutations result in the absence of m\({}^{7}\)G modification in 16S rRNA, stimulating the expression of SAM synthetase markedly by perception of the change in 16S rRNA and catalyzing the synthesis of SAM from methionine and ATP [37]. It has been proven that SAM is an effective intracellular signal in _Streptomyces_ and _Bacillus_[38]. For example, the _rsmG_ mutants in _S. coelicolor_ improve the antibiotic actinorhodin production by improving the SAM level [39; 40]. In addition, the overexpression of SAM synthetase via multicopy plasmid or addition of SAM to the medium display leads to enhanced antibiotics in _S. lividans_, _S. griseus_, _S. griseofalueva_, and _S. coelicolor_[41; 42; 40]. In _B. subtilis_, the intracellular SAM activates the bacilsyn pathway by mutants in the enzyme involved in te SAM-recycling pathway [43]. However, although R1-31, R1-41, R2-8 and R3-33 have a similar mutant in the _rsmG_, the inactivation of RsmG was not the only factor that improved the toyocamycin production, especially with the genome shuffling method applied. For example, genome shuffling was applied using the ribosome engineering mutant as a starting strain for the enhanced production of tiancium-A, which lead to the deficiency of _rsmG_ in the strains after genome shuffling. However, the knockouts of _rsmG_ in the wild-type could not lead to the improvement of production of tiancium-A [44]. Therefore, the _rsmG_ mutant was not the only reason explaining why the secondary metabolites production were enhanced. There may exist other mutants which were not detected in the genome of potential strains and mutants. The highest toyocamycin production was obtained by R3-33, which contains another mutant in _rpsL_ (encoding the 16S ribosomal RNA), improving the translation efficiency by enhancing the ribosome stability over that of wild-type. The mutants in _rpsL_ contribute to higher protein synthesis during the stationary phases and higher secondary metabolites production. This phenomenon has been proven in several strains, including _Streptomyces_ spp., _B. subtilis_, _Erwinia carotivora_, and _E. coli_[45; 46]. In conclusion, this study achieved a higher toyocamycin production by genome shuffling and antibiotic screening. Apart from the solo mutants in ribosome, genome shuffling could lead to a comprehensive shift in energy metabolic and intracellular signal synthesis. The mutants saw an incredible increase in ATP-hydrolysis rate, which primarily reveals that intercellular energy metabolic flux change could effectively stimulate the transcription of silent genes by the ATP-related pathway regulator. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/](https://www.mdpi.com/article/10.3390/)]([https://www.mdpi.com/article/10.3390/](https://www.mdpi.com/article/10.3390/)) fermentation8100535/s1, Table S1: The primers used in this study. **Author Contributions:** Conceptualization, Y.S. and X.S. ; methodology, Z.Z. ; software, X.Z. ; validation, J.Y., Y.S. and Z.Z. ; formal analysis, X.Z. ; investigation, Y.S. ; resources, X.S. ; data curation, Y.S. ; writing--original draft preparation, Y.S. ; writing--review and editing, X.S. and X.Y. ; supervision, X.Y. ; project administration, X.Y. ; funding acquisition, Y.S and X.Y. All authors have read and agreed to the published version of the manuscript.