Abstract
To improve the screening efficiency of high-yield neomycin sulfate (NM) Streptomyces fradiae strains after mutagenesis, a high-throughput screening method using streptomycin resistance prescreening (8 μg/mL) and a 24-deep well plates/microplate reader (trypan blue spectrophotometry) rescreening strategy was developed. Using this approach, we identified a high-producing NM mutant strain, Sf6-2, via six rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and screening. The mutant displayed a NM potency of 7780 ± 110 U/mL and remarkably stable genetic properties over six generations. Furthermore, the key components (soluble starch, peptone, and (NH4)2SO4) affecting NM potency in fermentation medium were selected using Plackett-Burman and optimized by Box-Behnken designs. Finally, the NM potency of Sf6-2 was increased to 10,849 ± 141 U/mL at the optimal concentration of each factor (73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively), and it exhibited about a 40% and 100% enhancement when compared with before optimization conditions and the wild-type strain, respectively. In this study, we provide a new S. fradiae NM production strategy and generate valuable insights for the breeding and screening of other microorganisms.
Highlights
Introductionneomycin sulfate (NM) 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
Neomycin sulfate (NM, the sulfate salt form of neomycin) was the first 2-deoxystreptaminecontaining aminoglycoside antibiotic discovered during Streptomyces fradiae fermentation [1]
Streptomycin resistance mutations are the most frequently used for screening high-yielding antibiotic strains, including actinorhodin (48-fold) in Streptomyces coelicolor A3(2) [35], fredericamycin (26-fold) in Strep
Summary
NM 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.
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