Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization

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.

Bioprocess Optimization for Enhanced Production of L-asparaginase via Two Model-Based Experimental Designs by Alkaliphilic Streptomyces fradiae NEAE-82

Background: L-asparaginase is one of the most widely used chemotherapeutic agents for the treatment of a variety of lymphoproliferative disorders and particularly acute lymphoblastic leukemia. Due to increased applications of L-asparaginase in several industrial fields including food processing and medical fields, its production needs to be increased to several folds. Objective: The aim was (i) to identify the significant factors which affect L-asparaginase production by Streptomyces fradiae NEAE-82 and (ii) to achieve higher production of L-asparaginase. Methods: Sixteen assigned factors and three dummy factors were screened using Plackett-Burman experimental design to determine the most important factors for the production of L-asparaginase by Streptomyces fradiae NEAE-82. Results: L-asparagine was determined to be the most significant positive independent factor (P-value 0.0092) affecting L-asparaginase production by Streptomyces fradiae NEAE-82 followed by pH and NaCl with significant P-values of 0.0133 and 0.0272; respectively. These factors were further optimized by Box-Behnken experimental design. The optimized fermentation conditions, which resulted in the maximum L-asparagine activity of 53.572 UmL-1 are g/L: dextrose 4, L-asparagine 15, KNO3 2, MgSO4.7H2O 0.5, K2HPO4 1, FeSO4.7H2O 0.02, NaCl 0.2, ZnSO4 0.01 and inoculum size 2 %, v/v for 7 days incubation at temperature 37°C, agitation speed 100 rpm, pH 8.5. Conclusion: A total of 3.41-fold increase in the production of L-asparaginase was achieved in the medium after statistical improvement (53.572 UmL-1) as compared to the unoptimized basal medium used prior to the application of Plackett-Burman (15.704 UmL-1).