Journal
MICROORGANISMS
Volume 10, Issue 1, Pages -Publisher
MDPI
DOI: 10.3390/microorganisms10010094
Keywords
neomycin sulfate (NM); Streptomyces fradiae; high-throughput screening; atmospheric and room temperature plasma (ARTP) mutagenesis; fermentation medium optimization
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Funding
- National Nature Science Foundation of China [31471615, 31871781, 31772081]
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A high-throughput screening method combined with mutagenesis and optimization was developed to improve the production of neomycin sulfate in Streptomyces fradiae. The study identified a high-producing mutant strain with significantly increased potency and stable genetic properties. This strategy provides valuable insights for the breeding and screening of other microorganisms.
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 mu g/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)(2)SO4) 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.
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