## 4 Discussion  
The improvement of antibiotic production is critical procedure in industrial strain modification. In order to increase toyocamycin production, genome shuffling was applied to achieve the target phenotypes. Several published articles have indicated that genome shuffling could combine other genetic engineering methods to effectively improve product yield, including tylosin in _Streptomyces fradiae_[29], fructosyltransferase in _Aspergillus oryzae_[30], and succinic acid in _Actinobacillus succinogenes_[31]. For butanol fermentation, after chemical mutagenesis and genome shuffling, the new strain increased productivity and yield by 6.8% under high gravity conditions [32]. In our case, the solo antibiotic mutants of _S. diastatchromogenes_ were used as the parental strains and multiple rounds of genome shuffling were applied to generate mutants with diverse genotypes. The toyocamycin production increased each round by 15.4% to 57.9% by testing the toyocamycin production of several strains in comparison with the WT. Antibiotic resistance was a promising method to improve secondary metabolic pathways, which is called ribosome engineering. The antibiotics commonly used to screen mutants are kanamycin, gentamicin, rifampicin, streptomycin, fusidic acid, and so on. In addition, the product was also used for improving the product yield by increasing product-resistance. For example, enhancing pristinamycin-resistance in _S. pristinaspiralis_ would relieve feedback regulation of pristinamycin on its own biosynthesis [23]. However, in this study, we found the antibiotic-screening mutants could also increase product resistance, although the product was not used to screen mutants. A similar phenomenon was also observed in the _S. viridchromogenes_ mutants using streptomycin-screening but resulting in increasing avilamycin resistance after genome shuffling and ribosome engineering [22]. Although the mechanism of how the antibiotic resistance was correlated with product productivity was unclear, it is an interesting phenomenon to explain. The energy metabolic could be seen as the bridge to connect the primary metabolic and secondary metabolic pathway, which was at a high level to maintain the rapid growth rate at primary metabolism and a low level to stimulate the silent biosynthesis pathway when the nutrients were limited. A previous study showed that intracellular adenosine triphosphate (inATP) level is negatively correlated with tautomycin biosynthesis, which is regulated by a pathway-specific transcriptional regulator TmcN belonging to the large ATP-binding LuxR (LAL) family [33]. The ATPase domain of TmcN can sense the endogenous pool of ATP, then activate the silent pathway of tautomycetin [34]. In addition, a similar  
Figure 4: The intracellular ATP level and ATP hydrolysis rate in vitro in wild-type and its mutants. (**A**) The amount of inATP measured at 48 h; (**B**) the ATP hydrolysis rate of wild-type SD1628 and its mutants. phenomenon existed in other strains. For example, the generation of an artificial ATP deficit triggers antibiotic production in _S. lividans_[35]. The antibiotic production is correlated with highly active oxidative metabolism in _S. coelicolor_ M145 [25]. Although extensive research has been conducted to characterize the transcriptional level of the biosynthesis pathway, the shifted energy metabolic could play a significant role in secondary metabolites, especially for the pathway controlled by an ATP-binding LuxR transcriptional regulator. In this study, ToyA is the specific regulation factor in toyocamycin synthesis clusters, which was found to activate the transcription of toyocamycin biosynthetic genes. ToyA is an ATP dependent enzyme, belonging to the large ATP-binding regulators of the luxR family (LAL) family, due to the well-conserved motifs of Walker A and Walker B and high homology to the ATPase domain. In addition, the ToyA has a C-terminal DNA binding domain which may bind to the toyocamycin cluster to regulate its transcription [36].