## 4 Discussion  
We reported a rapid screening method for high\(-\)producing NM mutant strains. The process consisted of three modules: a commercially available ARTP mutagenesis system, prescreening on streptomycin plates, and rescreening using \(24-\)deep well plates, and a new detection method (TB spectrophotometry using a microplate reader). This approach had multiple advantages: (1) the ARTP mutation system is superior to other conventional methods in some respects [8; 9]. When generating mutant strains, costs are greatly reduced and safety is considerably improved when compared with traditional physical and chemical mutagenesis. In addition, more high frequency random mutations, induced by reactive chemical species produced by the helium\(-\)based ARTP, could generate desirable stable genetic phenotypes in a simple to operate manner. (2) Using streptomycin plates for prescreening not only improved the screening efficiency, but also triggered \(S\). _fradiae_ to produce more NM. Currently, the application of streptomycin resistance has improved the biosynthesis and growth tolerance properties of diverse bacterial and fungal species [22; 23; 24]. (3) Rescreening based on \(24-\)deep well plates and TB spectrophotometry using a microplate reader greatly improved screening efficiency when compared with traditional screening methods (shake-flask fermentation combined with HPLC determination). Figure 6: Response surface plots showing the effects of (\(X_{1}\)) soluble starch, (\(X_{2}\)) peptone, and (\(X_{3}\)) (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) on NM potency. These advantages meant this method was well suited for high-producing NM mutant strain screening. After six rounds of breeding and screening, a high\(-\)producing NM mutant strain, _Sf_6-2 was identified and displayed an NM potency of 7780 \(\pm\) 110 U/mL. This equated to an increase of 45% when compared with the wild\(-\)type strain. In addition, these data demonstrated ARTP feasibility in generating high\(-\)yielding NM mutant strains. For mutant strains generated by iterative mutagenesis, typically the original fermentation medium is no longer suitable for product synthesis, and further component optimization is required. Therefore, key fermentation media factors (soluble starch, peptone, and (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\)) which impacted NM potency were screened using PB designs. Subsequently, these components were further optimized using Box-Behnken designs and used at the optimized concentrations of 73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively. Finally, the NM potency of _Sf_6-2 reached 10,849 \(\pm\) 141 U/mL and reflected an increase of 40% when compared with before optimization conditions, and a two-fold increase on the wild\(-\)type strain. In the future, three possible research directions can be taken to improve _S. fradiae_ NM potency: (1) The overexpression of neomycin C 5\({}^{\prime\prime\prime}\)-epimerase (NeoN) which is involved in the last step of NM biosynthesis [44]. The last step of NM biosynthesis is the epimerization of neomycin C to neomycin B (the main component of NM); thus neomycin C is generally considered the biosynthetic precursor of neomycin B (Figure 7). It was previously reported that epimerization was the reason why the antibacterial activity of neomycin C was lower than neomycin B, and thus, epimerization at C-5\({}^{\prime\prime\prime}\) of neomycin C may be a rate-limiting step in the entire biosynthetic pathway [45]. Therefore, _S. fradiae_ NM potency may be greatly improved by overexpressing NeoN. (2) Transcriptomic analysis could be conducted on the wild-type _S. fradiae_ GC 6010 strain and _S_f_6-2 to identify and classify differentially expressed genes into related metabolic pathways. This approach could identify possible mechanisms underpinning increased NM potency in _Sf_6-2. (3) Fermentation broth viscosity could be optimized as it affects antibiotic production [46; 47], but has received little research attention.