Improved Natamycin Production in Streptomyces gilvosporeus Through Mutagenesis and Enhanced Nitrogen Metabolism

Abstract

Natamycin is a polyene macrocyclic antibiotic extensively used in food, medical, and agricultural industries. However, its high production cost and low synthetic efficiency fail to meet the growing market demand. Therefore, enhancing the production of natamycin-producing strains is crucial for achieving its industrial-scale production. This study systematically evaluated 16 mutagenesis methods and identified atmospheric and room temperature plasma mutagenesis combined with 2-deoxyglucose tolerance screening as the optimal strategy for enhancing natamycin production. A high-yield mutant strain, AG-2, was obtained, achieving an 80% increase in natamycin production (1.53 g/L) compared to the original strain. Metabolic analysis revealed that glycolysis and the pentose phosphate pathway were enhanced in AG-2, while the tricarboxylic acid cycle was weakened, significantly increasing the supply of precursors such as acetyl-CoA, methylmalonyl-CoA, and the reducing power of NADPH. Additionally, overexpression of the nitrogen metabolism regulatory gene glnR promoted the supply of glutamate and glutamine, further increasing natamycin production in AG-2 to 1.85 g/L. In a 5 L fermenter, the engineered strain AG-glnR achieved a final natamycin production of 11.50 g/L, 1.67 times higher than the original strain. This study is the first to combine mutagenesis with nitrogen metabolism regulation, effectively enhancing natamycin production and providing a novel approach for the efficient synthesis of other polyene antibiotics.

Keywords: 2-deoxyglucose; ARTP mutagenesis; Streptomyces gilvosporeus; natamycin; nitrogen metabolism regulation.

Figure 1

The chemical structure of natamycin.

Figure 2

Different strategies for screening natamycin high-producing mutants. (a) Strategies based on UV mutagenesis; (b) strategies based on NTG mutagenesis; (c) strategies based on DES mutagenesis; and (d) strategies based on ARTP mutagenesis. In each method, the strains were mutagenized and then coated onto plates containing varying concentrations of streptomycin, 2-DG, KH₂PO₄, and LiCl. The plates were then incubated at 28 °C for 8 d. Natamycin production and dry cell weight (DCW) of mutants were measured after 96 h of shake flask fermentation. Each fermentation experiment was performed in triplicate, and the results were expressed as the mean of three independent replicates, with error bars representing the standard deviation. * indicates significance at p < 0.05, ** indicates significance at p < 0.01, and *** indicates significance at p < 0.001.

Figure 3

Screening for natamycin-producing strains using ARTP mutagenesis combined with 2-DG. (a) Workflow of ARTP mutagenesis combined with 2-DG tolerance screening; (b) agar diffusion method to assess the antimicrobial activity of the mutants; (c) natamycin production and dry cell weight (DCW) of the five highest-yielding mutants obtained on plates with different phosphate concentrations; (d) comparison of the inhibition zone diameter between S. gilvosporeus ATCC 13326 and S. gilvosporeus AG-2; and (e) HPLC analysis of natamycin production in S. gilvosporeus ATCC 13326 and S. gilvosporeus AG-2. Fermentation was repeated three times. Data are presented as the mean of three independent cultures, with error bars representing standard deviation.

Figure 4

Physiological differences between S. gilvosporeus ATCC 13326 and S. gilvosporeus AG-2. (a) Mycelial morphology; (b) natamycin production; (c) dry cell weight (DCW); (d) specific production rate; and (e) productivity.

Figure 5

Key genes and metabolic changes in the natamycin biosynthetic pathway in the natamycin high-producing strain AG-2. Red indicates upregulated genes/metabolites, while blue indicates downregulated genes/metabolites. The red numbers represent the fold changes in upregulated genes, and the blue numbers represent the fold changes in downregulated genes.

Figure 6

Effect of nitrogen metabolism on natamycin production by S. gilvosporeus AG2. (a) Exogenous addition of glutamate at 24, 36, and 48 h on natamycin production of AG-2; (b) exogenous addition of glutamine at 24, 36, and 48 h on natamycin production of AG-2. DCW: dry cell weight.

Figure 7

Enhancement of nitrogen metabolism increases natamycin production in AG2. (a) Effect of GlnR overexpression and inhibition on natamycin production. (b) Comparison of natamycin production between S. gilvosporeus AG2-glnR and the parental strain S. gilvosporeus ATCC 13326 in 5 L bioreactors. (c) Comparison of dry cell weight (DCW) between S. gilvosporeus AG2-glnR and the parental strain S. gilvosporeus ATCC 13326 in 5 L bioreactors.