## 1 Introduction  
The _Streptomyces_ produces various kinds of antibiotics, including polykeptides, nucleosides, pyrrolidines, and so on, which have attracted interest for their antitumor, antiparasitic, and immunosuppressant functions [1; 2; 3; 4]. The metabolic pathways of these antibiotics have been comprehensively studied [5]. There are complex regulation networks to _Streptomyces_, and many intracellular compounds involve the switch from primary to secondary metabolisms during fermentation, such as S-adenosylmethionine (SAM), ppGpp, and triacylglycer breeds [6; 7; 8]. Therefore, the strategies to change the metabolism extensively often result in higher secondary metabolites production and more efficient fermentation productivity [9; 10].  
Toyocamycin belongs to the nucleoside's antibiotic division and exhibits various biological activities, such as antitumor, antibacterial, and antifungal activities [11; 12]. Nowadays, the toyocamycin could be regarded as a promising agricultural antibiotic in controlling plant disease. Toyocamycin can be generated by _Streptomyces diastastochronogenes_ 1628, isolated from the soil. The production of wild-type _S. diastochronogenes_ is just 59.64 mg/L, which is lower than the demand of large-scale agricultural applications [11]. Therefore, a series of modification by genetic engineering strategies have been applied to improve toyocamycin production. The structural genes _toyG_ and _toyF_ were overexpressed to enhance the synthesis pathway intensity [13]. The transcriptional level and translate rate were improved by optimized promoters and overexpressing the ribosome recycling factor, respectively [14; 15]. The oxygen uptake rate was increased by overexpression of hemoglobin [14]. However, the highest production by metabolic engineering technology was 489.7 mg/L, which still faces difficulty in industrial application. Therefore, more efficient methods are necessary to enhance the production of toyocamycin.  
Ribosome engineering has been proven to be an effective way to activate the secondary metabolic pathway and silent genes by spontaneous mutation [16]. The beneficial mutants were screened out by drug resistance (streptomycin, rifampin, paromomycin, and so on), leading to the common mutant in ribosome S12 protein (encoded by _rpsL_ gene, high streptomycin resistant), 16S rRNA Methyltransferase (encoded by _rmsG_, low streptomycin resistant) and RNA polymerase \(\beta\)-subunit (encoded by _rpoB_ gene, rifampicin resistant) [6; 17]. In order to enhance the toyocamycin production, an effective genome shuffling method was applied. The genome shuffling was a powerful method for the rapid evolution of strains toward desirable phenotypes [18]. It accelerates directed evolution through recursive recombination of improved progenies. By genome shuffling, interstrain or interspecific hybrids could be generated. Therefore, genome shuffling could be an ideal method to obtain the optimal phenotype after ribosome engineering, especially when several rounds of antibiotic screening have been employed and mutants with high production of secondary metabolites have been obtained. Furthermore, the genome shuffling could conveniently combine with other genetic engineering methods, such as screening beneficial traits from genome shuffling and UV irradiation or chemical agents [19]. However, the genome shuffling has not been used in combination with ribosome engineering to enhance toyocamycin production.  
Therefore, in this study, genome shuffling and an antibiotic screening strategy were combined to improve toyocamycin production. The difference in genotype, growth rate, toyocamycin production, and resistance were determined between the wild type, parental strains, and recombinant strains. The comparison of genetic mutants in the structural gene expression level was analyzed to explain the reason for higher production. Furthermore, intracellular ATP and ATP hydrolysis rates were detected to reveal the complex energy metabolic change in mutants.