Physiological Insights into Enhanced Epsilon-Poly-l-Lysine Production Induced by Extract Supplement from Heterogeneous Streptomyces Strain

Abstract

Epsilon-poly-l-lysine (ε-PL) is a potent antimicrobial agent, but strategies to enhance its biosynthesis remain limited due to insufficient understanding of its physiological regulation. This study explores the interaction between Streptomyces albulus and heterogeneous microbial extracts, with a focus on actinomycete-derived signals. The S. gilvosporeus extract induces the highest ε-PL production (3.4 g/L), exceeding the control by 2.6-fold and outperforming B. cinerea by 1.8-fold. Multi-omics analyses combined with morphological and biochemical profiling reveal that the induced state is characterized by intensified central carbon flux, enhanced lipid turnover, elevated respiratory activity, and cofactor regeneration, alongside suppression of competing secondary pathways. Morphological alterations, including denser mycelial aggregation and compact colony structures, accompany these metabolic shifts. Compared to B. cinerea, S. gilvosporeus elicits more pronounced stress adaptation and metabolic reprogramming in S. albulus. These findings suggest that interspecies interactions can activate intrinsic aggression resistance mechanisms, thereby driving ε-PL biosynthesis through a previously unrecognized physiological route.

Keywords: Streptomyces albulus; Streptomyces gilvosporeus; interspecies signaling; systemic biological analysis; ε-poly-l-lysine.

Figure 1

Inducing effect of microorganisms on ε-PL production. (A) ε-PL production after addition of heterogeneous microorganisms (dead cells); (B) primary extraction procedure of microbial signal mixture; (C) effects of fungi extracts on ε-PL production; (D) influences of actinomycetes extracts on ε-PL production. Mean ± SD, n ≥ 3, biological replicates. *** indicates p < 0.001.

Figure 2

Changes in intracellular secondary messengers (H₂O₂ and Ca²⁺) and profiles of fermentation parameters in ε-PL production upon addition of S. gilvosporeus extracts. (A) Intracellular Ca²⁺ content upon addition of S. gilvosporeus extracts. (B) Intracellular H₂O₂ content upon the addition of S. gilvosporeus extracts. (C) Profiles of ε-PL concentrations in cultures with (EG) or without (CK) addition of S. gilvosporeus extracts. (D) Profiles of dried cell weight (DCW) in cultures with (EG) or without (CK) addition of S. gilvosporeus extracts. (E) Average specific ε-PL formation rate in cultures with (EG) or without (CK) addition of S. gilvosporeus extracts. (F) Average specific cell growth rate in cultures with (EG) or without (CK) addition of S. gilvosporeus extracts. Mean ± SD, n ≥ 3, technical replicates. *** indicates p < 0.001.

Figure 3

Morphological comparison between cultures with (EG) or without (CK) addition of S. gilvosporeus extracts. (A) Morphology of Petri plate colony in agar medium. (B) Morphology of mycelia and mycelial pellets in submerged culture with M3G medium via SEM. (C) CTC staining images of mycelial pellets in submerged culture with M3G medium at 42 h, 48 h, and 54 h.

Figure 4

Transcriptome analysis of cell samples from cultures with (EG) or without (CK) S. gilvosporeus extract addition. (A) Volcano diagrams revealing a comparison of the number of differentially expressed genes in cultures between EG vs. CK. (B) The top 30 significantly enriched (padj < 0.05) GO terms in “EG vs. CK” comparison. (C) The top 30 significantly enriched (padj < 0.05) KEGG pathways in “EG vs. CK” comparison. Mean ± SD, n ≥ 3, technical replicates.

Figure 5

Effects of S. gilvosporeus extract addition (24 h) on the transcription of key genes, intracellular activities of key enzymes in ε-PL biosynthesis metabolism. The gene transcription in CK (without S. gilvosporeus extract addition) was set as 1 (dot lines), and those genes’ relative transcription in EG (with S. gilvosporeus extract addition) was calculated based on the levels of CK. Mean ± SD, n ≥ 3, technical replicates. *** indicates p < 0.001, ** indicates p < 0.001.

Figure 6

Metabolome analysis of cell samples from cultures with (EG) or without (CK) S. gilvosporeus extract supplement. (A) PCA score plot of CK and EG. (B) OPLS-DA score plot of “EG vs. CK” comparison group. (C) Heat map for Z-score of important intermediate metabolites in ε-PL biosynthesis metabolism. Relative content of significantly different metabolites calculated according to the OPLS-DA model (p < 0.05). Mean ± SD, n ≥ 6, technical replicates.