## 1 Introduction  
Neomycin sulfate (NM, the sulfate salt form of neomycin) was the first 2-deoxy streptramine-containing aminoglycoso antibiotic discovered during _Streptomyces fradiae_ fermentation [1]. NM is widely used as a broad spectrum, water soluble antibiotic that inhibits Gram-negative and Gram-positive bacteria. NM also has a narrow therapeutic range due to potential nephrotoxicity and ototoxicity issues, but its use as a treatment for hepatic encephalopathy and hepatocellular carcinoma [2], human immunodeficiency virus [3], human genetic diseases [3], and catheter-associated urinary tract infections [4] has seen demand rapidly increase in recent years. Natural selection is the main driving force of biological evolution; however, mutation frequencies are typically very low, especially for changes in specific phenotypes [5]. Therefore, artificial random mutation methods, including physical and chemical mutagenesis, comprising alkylating agents (ethyl methane-sulfonate and nitrosoguanidine) [6], azides (sodium and potassium azides) [7], conventional radiation (X-ray and \(\gamma\)-ray) [7], and heavy ion beams [6], have been widely used to increase mutation rates and derive desired phenotypes [8]. However, the tools for these random mutagenesis methods are disadvantageous in terms of poor operability and high safety risks due to chemical and physical mutagens at high toxicity or radiation levels. The atmospheric and room temperature plasma (ARTP) mutagenesis system uses radiofrequency atmospheric-pressure glow discharge (RF-APGD) plasma and acts on organisms under different operating parameters. This incurs DNA damage which is subsequently repaired by the SOS (\"Save Our Soul\") system leading to DNA alterations [8; 9]. ARTP technology generates high mutation frequencies, is simple and safe, and is widely used for bacteria [10; 11; 12; 13], fungi [14; 15], and microalgae [16; 17; 18]. On the other hand, mutations in strains due to streptomycin resistance are closely related to their ability to produce antibiotics [19]. Specifically, ppGpp (guanosine 5\({}^{\prime}\)-diphosphate 3\({}^{\prime}\)-diphosphate) accumulation, triggered by stringent responses, is thought to play key roles in antibiotic biosynthesis initiation, whereas mutant strains generating streptomycin resistance allow for antibiotic production/initiation without ppGpp requirements [20; 21]. Therefore, introducing mutations conferring streptomycin resistance is widely used in bacteria and _Streptomyces_ sp. for improving antibiotic and other metabolite production [22; 23; 24]. In the early years, metabolite/antibiotic screening was conducted on single colonies, which after physical or chemical mutagenesis, were randomly selected from agar plates, fermented in shake-flask and processed. Such traditional screening methods generated large workloads, were time-consuming, and had low efficiency and high costs, and were gradually eliminated over time. Currently, high-throughput screening technologies based on microplate (24/48/96/384-deep well plates) technologies are widely used for screening mutant strains, biologically active substances, and candidate drugs [25; 26; 27] due to their similar characteristics to automatic parallel microreactors. In addition, spectrophotometry based microplate readers have huge advantages when compared with cylinder plate and high-performance liquid chromatography (HPLC) methods in terms of antibiotic identification, as they process large numbers of simultaneous samples and reduce reagent costs and use. Therefore, high-throughput screening technologies combined with microplate readers can greatly improve the screening efficiency of mutant strains after mutagenesis. In this study, we used multiple strategies to improve the screening efficiency of high-producing NM mutant strains. First, we used ARTP technology on wild-type _S. fradiue_ to generate large numbers of mutant strains. We then prescreened strains on streptomycin plates using correlations between resistance mutations and antibiotic biosynthesis.