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Article

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

by
Siyu Tong
1,2,†,
Chen Zhang
1,2,†,
Zhanyang Zhang
1,2,
Huawei Zeng
1,2,
Bingyue Xin
1,2,
Mingtao Zhao
1,2,
Deyin Zhao
1,2,
Xin Zeng
1,2,* and
Fei Zhang
1,2,*
1
Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China
2
College of Life Sciences, Huaibei Normal University, Huaibei 235000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(8), 1868; https://doi.org/10.3390/microorganisms13081868 (registering DOI)
Submission received: 11 July 2025 / Revised: 8 August 2025 / Accepted: 8 August 2025 / Published: 10 August 2025
(This article belongs to the Special Issue Microbial Manufacture of Natural Products)
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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.

1. Introduction

ε-PL is an amino acid polymer composed of 25–35 L-lysine residues linked through dehydration condensation between the α-carboxyl and ε-amino groups [1]. As a biodegradable polymer with broad-spectrum antimicrobial activity, excellent water solubility, a wide pH tolerance range, and high biological safety, ε-PL has been widely adopted as a safe and environmentally friendly bioactive compound across the food, pharmaceutical, and personal care industries [2]. In the food sector, ε-PL effectively inhibits Penicillium expansum on apples and Botrytis cinerea on various fruits and vegetables, thereby reducing spoilage and extending shelf life [3,4]. Its combination with tea polyphenols enhances the microbial stability and sensory quality of meat products [5,6], while synergy with licoricidin demonstrates potent anti-biofilm activity [7]. In pharmaceuticals, the cationic nature and biofilm-penetrating capacity of ε-PL enable its application in drug and gene delivery systems [8], including micellar formulations for glioma gene therapy [9], adhesive materials for wound closure [10], and self-degradable medical glues [11]. In the personal care field, ε-PL combined with FP and domipramine exhibits strong antimicrobial and anti-halitosis effects, as confirmed in clinical trials [12]. Despite its promising functionalities, the industrial and commercial application of ε-PL is constrained by its high production cost (approximately USD 200/kg). Consequently, improving fermentation efficiency to reduce manufacturing costs remains a major focus of both academic and industrial research.
ε-PL is a secondary metabolite primarily synthesized by Streptomyces albulus under acidic conditions. The efficient biosynthesis of ε-PL necessitates the concurrent fulfillment of three key factors: highly active Pls (ε-PL synthetase, a member of the non-ribosomal peptide synthetase (NRPS) family), elevated intracellular ATP levels, and rapid provision of lysine precursors. To enhance the yield and efficiency of ε-PL fermentation, research efforts have focused on the following three aspects: (i) construction of efficient ε-PL-producing strains through random mutagenesis and directed evolution, including mutation screening [13], genome shuffling [14], and ribosome engineering [15]; (ii) metabolic enhancement of the key biosynthetic pathways involved in ε-PL synthesis using advanced genetic engineering methods [16,17]; and (iii) optimization of media formulations and precise control of culture parameters [18,19,20,21,22].
However, current strategies primarily follow the paradigm of “meeting the substrate requirements for ε-PL synthesis at either macroscopic or microscopic levels”, but they often overlook a fundamental question: “Why do ε-PL-producing strains actively synthesize this product?” In other words, “What is the physiological significance of ε-PL synthesis for the producing strains?” Compared to humans, the producing strains inherently possess the mechanisms that govern why and how they synthesize ε-PL efficiently. Therefore, only by comprehending the physiological significance of ε-PL synthesis for the producing strains and stimulating their intrinsic driving force can we achieve comprehensive metabolic enhancement.
Given that an acidic shock triggers excessive production of ε-PL [23,24,25,26,27], the potential physiological function of ε-PL in producing strains—to neutralize the surrounding acidic environment through alkaloid production—has attracted growing attention. However, other than the acidic stimulus, no other intrinsic driving forces have been identified over the past decade. Interestingly, ε-PL-producing strains predominantly belong to the actinomycetes, which exhibit a slower growth rate compared to most bacteria and have less efficient mycelium and spore dispersal capabilities than molds. This may indicate that ε-PL, a broad-spectrum bacteriostatic agent, enables these Streptomyces species to compete with other microorganisms for territory and nutrients. We hypothesize that the intrinsic drive to produce ε-PL stems from the need to resist invasion by competing microorganisms.
In this study, we found that certain other microorganisms could significantly enhance ε-PL production, particularly in Streptomyces species. To understand the underlying mechanisms, we conducted systematic physiological analyses, examining intracellular messengers, mycelial morphology, gene transcription, key enzymes in ε-PL production, and intracellular precursor pools.

2. Materials and Methods

Microorganisms and culture media. The strains utilized in this study were acquired from the China Center of Industrial Culture Collection (CICC), China General Microbiological Culture Collection Center (CGMCC), and American Type Culture Collection (ATCC). Detailed information is provided in Table 1. For the pre-activation of all microorganisms, an agar slant medium was used, consisting of 10 g/L glucose, 5 g/L yeast extract powder, 5 g/L peptone, and 20 g/L agar, with the initial pH adjusted to 7.5 using 2 M NaOH. This same medium was employed for spore preparation of the ε-PL-producing strain Streptomyces albulus IFO14147. A modified LB medium was used for bacterial cultures, containing 10 g/L glucose, 10 g/L tryptone, 5 g/L yeast extract powder, 0.5 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.04 g/L ZnSO4·7H2O, and 0.03 g/L FeSO4·7H2O, with the initial pH adjusted to 7.0 using 2 M NaOH. A modified YPD medium was used for fungal growth, composed of 40 g/L glucose, 10 g/L yeast extract powder, 20 g/L peptone, 0.5 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.04 g/L ZnSO4·7H2O, and 0.03 g/L FeSO4·7H2O, with the initial pH adjusted to 6.5 using 2 M NaOH. For the growth of Streptomyces species, a GYS medium was applied, which contains 60 g/L glucose, 10 g/L yeast extract, 20 g/L soy peptone, 5 g/L NaCl, and 5 g/L MgSO4·7H2O, with the initial pH adjusted to 7.5 using 6 M NaOH. Medium 3G (M3G) was specifically designed to support seed cultivation and formal ε-PL production, comprising 60 g/L glucose, 5 g/L yeast extract, 10 g/L (NH4)2SO4, 1.36 g/L KH2PO4, 0.8 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.04 g/L ZnSO4·7H2O, and 0.03 g/L FeSO4·7H2O, with the initial pH adjusted to 6.8 using 2 M NaOH. To prevent the Maillard reaction, glucose in all media was sterilized separately during autoclave treatment at 121 °C for 20 min.
ε-PL production after the addition of other microorganisms. The activation of strains was conducted on agar slant medium at 30 °C for 2–10 days, with the exception of B. cinerea, which was activated at 24 °C for 3–5 days. For the preparation of crude microbial extracts, inducing bacteria listed in Table 1 were cultured in a modified LB medium at 28 °C and 200 rpm for 15–24 h in a rotary shaker; inducing fungi from Table 1 were cultured in a modified YPD medium at 28 °C and 180 rpm for 2–4 days under the same conditions; Streptomyces species from Table 1 were incubated in a GYS medium at 28 °C and 200 rpm for 1–3 days in a rotary shaker. After cultivation, the broth was centrifuged at 6000× g for 10 min. The sediments (biomass) were used as the inducing microorganisms after sterilization at 115 °C for 15 min. The ε-PL-producing strain, S. albulus IFO 14147, was inoculated on an agar slant medium and incubated at 30 °C for 9 days until spore formation. For the seed pre-culture, 80 mL of M3G medium (in 500 mL flasks) was inoculated with one loop of spores (approximately 5 × 105 spores) and incubated at 200 rpm and 30 °C in a rotary shaker for 24 h. To initiate ε-PL production, 8% of the seed broth was transferred into 30 mL of M3G (in 250 mL flasks). The flasks were incubated at 200 rpm and 30 °C for 16 h, during which the pH naturally dropped to 4.0. Subsequently, a mixture of the prepared crude microbial extracts (harvested from former cultures) and citrate buffer (a final concentration in the broth of 10 g/L, pH 4.0 adjusted using 6 M NaOH) was added. The total culture time was set to 48 h, after which the final ε-PL titer in the broth was measured.
Primary extraction of microbial signal mixture and its influence on ε-PL production. Microbial extract mixtures were obtained from the biomass of selected fungi and actinomycetes that exhibited a positive effect on ε-PL production [28]. The fermentation broth was centrifuged at 5000× g for 10 min, and the resulting sediment was washed twice with deionized water to isolate the biomass. The biomass was then suspended in a 75% ethanol solution, ground with quartz sand, and centrifuged again at 5000× g for 20 min to separate the supernatant (Sn) and sediment (Sd). Ethanol was removed from the extract by evaporation, and the remaining solution was divided into four equal parts. Each part was sequentially extracted using ethyl acetate, chloroform, butyl alcohol, and petroleum ether. The organic phases were collected using a separating funnel and evaporated to remove the organic solvents. The resulting extracts were designated as fraction E (ethyl acetate extract), fraction C (chloroform extract), fraction B (butyl alcohol extract), and fraction P (petroleum ether extract). The effects of these crude microbial extracts on ε-PL production were evaluated via exogenous addition at 24 h in 250 mL flasks.
ε-PL production after the addition of S. gilvosporeus extracts in a 5 L fermenter. The impact of the S. gilvosporeus extracts on ε-PL production was evaluated through fed-batch cultures conducted in a 5 L jar fermenter (Baoxing Corp., Shanghai, China) with a working volume of 3 L. Profiles of fed-batch cultures without S. gilvosporeus extract addition (CK, control group) were compared to those with the extract addition (EG, experimental group). In the experimental group, crude extracts of S. gilvosporeus were added at a concentration of 36 g wet cells/L. The fermentation parameters were set as described previously [29]. The fermenter was equipped with two turbine agitators and automated control systems for temperature, dissolved oxygen (DO), and broth pH. Cultures were initiated after inoculation of the seed broth at an 8% ratio and maintained at 30 °C. Agitation rates ranged from 200 to 800 rpm, and aeration was set at 1.0 vvm to maintain DO levels at 30% throughout the culture period. When the broth pH naturally dropped to 4.0, 12.5% (w/v) NH3·H2O was automatically added to maintain a constant pH of 4.0. When the residual glucose concentration decreased to below 10 g/L, 70% of the sterilized glucose was automatically supplemented to maintain a concentration range of 5–15 g/L. Fed-batch cultures were terminated at 60 h, after which the impact of microbial extracts might be affected by multiple reasons. Broth samples were collected at specific time points for analysis of ε-PL titer and the DCW.
Impacts of S. gilvosporeus extracts on the morphology of S. albulus IFO 14147. The effects of S. gilvosporeus extracts on the morphology of S. albulus IFO 14,147 were assessed through macroscopic and microscopic observations. Macroscopically, the colony morphology on Petri plates was evaluated by comparing plates with and without the addition of fraction E extracts at a final concentration of 36 g wet cells/L. Microscopically, mycelial morphology in submerged cultures was examined using scanning electron microscopy (SEM). For macroscopic analysis, Petri plates containing agar slant medium supplemented with fraction E were inoculated with diluted spore suspensions of S. albulus IFO 14147. Colony morphology was observed at 3 and 6 days post-inoculation and compared to control plates without added extracts. For microscopic analysis, samples from submerged cultures were collected at 45 h with the addition of extracts at 24 h. These samples were fixed with glutaraldehyde, buffered with phosphate buffer, dehydrated with ethanol, and then observed using SEM (Sigma 300, ZEISS, Oberkochen, Germany). Cell viability was assessed using a CTC (5-cyano-2, 3-ditolyl tetrazolium chloride) Rapid Staining Kit (Dojindo Laboratories, Kumamoto, Japan), as previously described [29], and images of mycelial morphology after CTC staining were captured using a fluorescence microscope (BX51, Olympus, Tokyo, Japan).
RNA extraction and transcriptomic profiling. The most pronounced transcriptional divergence between CK and EG cultures occurred between 36 and 60 h. Thus, 36 h mycelial pellets were selected for RNA-seq analysis. Triplicate cultures were harvested at this time point, centrifuged (7000× g, 1 min), washed, flash-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using established protocols [30], and its quality was assessed via agarose gel electrophoresis and an Agilent (Santa Clara, CA, USA) 2100 bioanalyzer to ensure integrity and purity. For library construction, mRNA was isolated from non-coding rRNA, reverse transcribed into cDNA, and processed with end-repair and adapter ligation. Sequencing was performed on the Illumina platform to yield 150bp paired-end reads, followed by alignment to the Streptomyces albulus IFO14147 genome (CP104098.1) [31]. Differentially expressed genes (DEGs) were identified with Log2(EG/CK) > 1 (p < 0.05) through DESeq2, and functional enrichment analysis of these DEGs was conducted using GO and KEGG annotations to elucidate the underlying mechanisms of the fermentation enhancement derived from the addition of Streptomyces extract. Priority was given to transcriptional alterations in lipid catabolism and energy metabolism pathways for functional analysis.
Transcriptional performances of key genes in ε-PL biosynthesis. To explore the transcriptional levels of ε-PL biosynthesis-related genes, qRT-PCR was utilized to analyze the key genes using the samples collected at 42 h from the CK and EG fed-batch cultures in a fermenter. The total RNA extraction from S. albulus IFO 14,147 was carried out following the protocol reported previously [29]. The transcription of genes N1H47_11860, N1H47_11275, N1H47_14640, N1H47_21215, and N1H47_34205 was quantified via real-time fluorescent PCR (ABI Stepone plus, Applied Biosystems, USA) with SG Fast qPCR Master Mix (High Rox, Bio Basic Inc., Canada). The primer sequences for these genes are detailed in Table S4.
Activity assay of key enzymes and electron transport system in ε-PL biosynthesis. For the measurement of key enzymes and ETS, the broth samples were withdrawn from the fermenter at 36 h, 42 h, 48 h, 54 h, and 60 h. The cell extract preparation and activity assay of phosphoenolpyruvate carboxylase, pyruvate kinase, citrate synthase, aspartate kinase, and ETS were conducted following the procedures described previously [31,32,33,34,35].
Metabolomic profiling via UPLC-ESI-MS/MS. Metabolic variations between CK and EG were analyzed using ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). Cell samples were harvested from triplicate 42 h CK and EG cultures. Intracellular metabolites were quantified on a Vanquish UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC® HSS T3 column (150 × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA) and a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) employing ESI ionization, following established protocols [30]. Mass Profiler Professional 13.0 (Agilent, Santa Clara, CA, USA) facilitated metabolite profile analysis, with compound identification based on retention time and mass spectra using a laboratory-developed database. Multivariate statistical analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were performed using SIMCA 14.1 (Umetrics AB, Umeå, Sweden) to resolve inter-group metabolic disparities [36]. The Z-score was calculated to visualize the metabolic profiling.
Analytical methods. After centrifugation of the fermentation broth at 5000× g for 10 min, the clarified supernatant was subjected to quantitative analysis of ε-PL production and residual glucose content. Simultaneously, the pelleted biomass was collected for DCW determination. All analytical procedures, including ε-PL quantification, glucose consumption monitoring, and DCW measurement, were performed according to the standardized methods established in our prior research [29]. The concentration of ε-poly-L-lysine (ε-PL) was determined by diluting the fermentation supernatant with 0.7 mM sodium phosphate buffer (pH 7.0). A 2 mL aliquot of this diluted sample was mixed with 2 mL of 1 mM methyl orange solution. The mixture was allowed to react at 30 °C for 30 min, followed by centrifugation at 4500× g for 15 min. The resulting supernatant was diluted 20-fold with the same phosphate buffer, and its absorbance was measured at 465 nm using a spectrophotometer. The concentration of ε-PL was determined by referring to a previously established standard curve depicting the correlation between A465 and the concentration of ε-PL. The determination of dried cell weight (DCW) was accomplished using a gravimetric method that relied on the weight difference in filter paper. A 10 mL sample of the fermentation broth was withdrawn from the 5 L fermenter and centrifuged at 4500× g for 10 min. The resulting biomass pellet was washed twice with distilled water and then filtered through pre-weighed filter paper (Φ 7 cm, medium speed, SCRC) that had been previously dried at 105 °C. Subsequently, the filter paper with the biomass was dried at 105 °C until a constant weight was achieved and then reweighed. The DCW was calculated by measuring the weight difference before and after filtration.
Calculations. The average values of the specific cell growth rate and the specific ε-PL formation rate were calculated in the following manner:
Average   specific   cell   growth   rate =   1 1 2   [ c X t 2 + c X t 1 ] × c ( X ) t 2 − c ( X ) t 1 t 2 − t 1
Average   specific   ε - PL   formation   rate = 1 1 2   [ c X t 2 + c X t 1 ]   × c ( P ) t 2 − c ( P ) t 1 t 2 − t 1
The above parameters are mean values over a time range of 12 h, where t, c(X), and c(P) represent the culture time (h), DCW (g/L), and ε-PL titer (g/L) at the t h, respectively.
Statistical processing. All experimental data in this work were derived from three or more independent replicates. Data visualization was executed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA), while statistical evaluations, including significance testing, were conducted through SPSS 23.0 (IBM Corp, Armonk, NY, USA). The quantitative results are expressed as mean values with standard deviations (mean ± SD, n ≥ 3), with p-values below 0.05 considered statistically significant. For all statistically significant comparisons, the corresponding effect sizes (Cohen’s d > 0.8) indicated large effect magnitudes.

3. Results

ε-PL production after the addition of heterogeneous microorganisms’ extracts. The effect of extracts from different microorganisms (bacteria, fungi, and actinomycetes) on ε-PL production by S. albulus was systematically evaluated (Figure 1A). Among these, extracts from several Streptomyces species proved most effective, yielding a more than 1.8-fold increase in ε-PL titers compared to other microbial groups. Certain fungal species, including B. cinerea, A. niger, and P. chrysogenum, also elevated ε-PL titers. Conversely, none of the tested bacterial strains improved ε-PL biosynthesis. Among the fungi, A. niger was the top performer, yielding 2.3 g/L, while S. gilvosporeus was the most effective actinomycete, producing 3.4 g/L. Thus, extracts from various Streptomyces species are particularly effective at enhancing ε-PL biosynthesis, with those from S. gilvosporeus showing the most pronounced effect. To better understand the impact of microbial signals on ε-PL biosynthesis, we extracted biomass from strains (Figure 1B) that positively influenced ε-PL production and assessed their effects on ε-PL synthesis (Figure 1C,D). The data suggested that potential microbial signals from fungal and actinomycete biomass were soluble in 75% ethanol (Sn). Furthermore, these potential microbial signals were detectable in ethyl acetate (extract E) and butyl alcohol (extract B), but not in chloroform (extract C) or petroleum ether (extract P). These findings suggest that the functional signals from fungi and actinomycetes are likely small molecules. Notably, intriguing differences were observed between extracts from fungi and those from actinomycetes. For fungi, higher ε-PL titers were detected in extract B, while extract E yielded greater ε-PL concentrations for actinomycetes (Figure 1C,D). These observations underscore the structural distinctions in functional signals between fungi and actinomycetes, indicating that fungal signals exhibit higher polarity compared to those of actinomycetes.
ε-PL fermentation profile after addition of S. gilvosporeus biomass extract. The S. gilvosporeus extracts induced a marked increase in intracellular H2O2 and Ca2+ levels over the subsequent 5-h period (Figure 2A,B), potentially triggering extensive physiological modifications via ROS- and Ca2+-dependent regulatory pathways. Furthermore, the experimental group (EG) treated with the S. gilvosporeus extracts exhibited higher ε-PL concentrations (Figure 2C) and increased dried cell weight (DCW) (Figure 2D) compared to the control group (CK). Additionally, Figure 2E,F shows a sequential enhancement in cell growth between 24 and 36 h of cultivation, followed by an increase in ε-PL biosynthesis from 36 to 60 h. The significant improvement in ε-PL biosynthesis upon stimulation via the S. gilvosporeus extracts warrants further investigation into the underlying physiological mechanisms.
Cell morphology of S. albulus after S. gilvosporeus extract addition. As shown in Figure 3A, macroscopic colonies exposed to the S. gilvosporeus extracts were significantly larger and showed a pronounced tendency to aggregate centrally, an effect that was particularly evident by day 6. Similarly, in liquid culture, the extracts induced denser and more organized microscopic mycelial structures with increased excretion (Figure 3B). The CTC assay confirmed this enhanced metabolic state, showing that the extracts substantially increased mycelial respiratory activity over an extended period (42–54 h) (Figure 3C).
Transcriptome performance after S. gilvosporeus extract addition. As shown in Figure 4A, the S. gilvosporeus extracts induced a significant transcriptional response, upregulating 296 genes and downregulating 216 genes. Many of these changes were substantial (Log2FoldChange > 4, −log10(p-value) > 5), suggesting that the extracts activated numerous previously dormant genes. GO clustering analysis (Figure 4B) of the top 30 differentially expressed genes indicated that the S. gilvosporeus extracts activated global catabolic pathways, particularly those involving the hydrolysis of phosphoric esters and diesters. Specifically, three of the seven genes encoding phospholipase were significantly upregulated (Table 2). KEGG cluster analysis (Figure 4C) showed that the top 30 significantly differentially expressed genes were concentrated in pathways for signal transduction (e.g., quorum sensing and two-component systems), ABC transporters, and the metabolism of nicotinate, nicotinamide, and glycerophospholipids, among other processes. Notably, all subunits (A–F, H) of NADH-quinone oxidoreductase in the EG displayed significantly higher transcription levels compared to the CK, although F0F1 ATP synthase in the EG did not show substantial transcriptional changes (Table 3). These findings suggest that S. albulus may be regulating itself to boost energy production via intensive cell respiration, primarily through lipid hydrolysis and oxidation.
Transcriptional and enzymatic performance of key genes in ε-PL biosynthesis after S. gilvosporeus extract addition. To elucidate the impact of S. gilvosporeus extracts on ε-PL biosynthesis, a comprehensive analysis integrating gene transcription and enzyme activity was conducted, as shown in Figure 5. The extracts enhanced the metabolic pathways specific to ε-PL production. Notably, increased activity of glucose-6-phosphate dehydrogenase was observed in the EG during the 42–60 h period. This pathway is crucial for NADPH generation, a key cofactor that facilitates both cell growth and ε-PL synthesis. The extract also significantly upregulated the transcription of key genes in the Embden–Meyerhof–Parnas (EMP) pathway, including N1H47_11860 (encoding phosphofructokinase) and N1H47_11275 (encoding pyruvate kinase). Phosphofructokinase is a key regulator of glycolytic flux, while pyruvate kinase links glycolysis to the TCA cycle by catalyzing its final step. This metabolic enhancement was confirmed by higher pyruvate kinase activity in the EG (42–54 h). The increased glycolytic flux was subsequently channeled into the TCA cycle, as evidenced by elevated transcription of the citrate synthase gene (N1H47_14640) and increased activity of the enzyme itself. Since citrate synthase catalyzes the first committed step of the TCA cycle—the conversion of oxaloacetate and acetyl-CoA into citrate—this upregulation directly facilitated greater NADH production. Concurrently, the activities of the electron transport system (ETS) were elevated in the EG during the 42–60 h period, indicating efficient ATP production from NADH. The ETS is crucial for oxidative phosphorylation, where NADH is used to drive ATP synthesis, supplying the energy necessary for ε-PL biosynthesis. Notably, phosphoenolpyruvate carboxykinase, a crucial enzyme for replenishing intermediates in the TCA cycle, showed significant activity improvement in the EG throughout the entire time range (36–60 h), ensuring timely replenishment of TCA cycle metabolite pools. By converting oxaloacetate into phosphoenolpyruvate, this enzyme facilitates the gluconeogenesis pathway, supporting the supply of carbon skeletons. This facilitated the efficient conversion of carbon skeletons (oxaloacetate) into L-lysine via aspartate kinase and subsequently into ε-PL through ε-PL synthase. In the EG, the upregulated transcription of N1H47_21215 (aspartate kinase) and N1H47_34205 (ε-PL synthase), along with higher aspartate kinase activity during the 36–54 h period, ensured the high efficiency of these metabolic processes. The overall metabolic enhancement induced by S. gilvosporeus extracts laid a solid foundation for rapid cell growth and improved ε-PL production.
Effect of S. gilvosporeus extracts on the metabolic pools of intermediates in pathways for ε-PL biosynthesis. Principal component analysis (PCA) and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) were employed to assess the data quality of the non-targeted metabolomic assay using a UPLC-ESI-MS system. The score plot (Figure 6A) demonstrates a distinct separation between the EG and the CK. The two principal components derived from the PCA model accounted for 38.2% of the total variance. Furthermore, the OPLS-DA score scatter plots (Figure 6B) revealed a clear distinction between EG and CK. As illustrated in Figure 6C and Table S1, the S. gilvosporeus extracts (EGs) induced significant metabolic changes in the EMP pathway, the diaminopimelic acid pathway (DAP), and the intracellular composition of energy cofactors and amino acids. Within the EMP pathway, the concentrations of most metabolites increased following extract addition, exemplified by glucose 6-phosphate and glyceraldehyde 3-phosphate. The observed decrease in fructose-1,6-diphosphate is likely attributable to its rapid consumption for downstream metabolite synthesis. This metabolic reprogramming of the EMP pathway under EG conditions probably enhanced the supply of carbon skeletons for subsequent energy metabolism and product synthesis. In contrast, the metabolite pools in the TCA cycle and DAP changed minimally in response to the extract. A significant shift also occurred in the energy cofactor pools: low-energy molecules (ADP, NAD+) increased, while their high-energy counterparts (ATP, NADH) decreased. This trend was also observed for NADP+ and its reduced form, NADPH.
Interestingly, the extracts from S. gilvosporeus not only enhanced ε-PL production but also significantly modulated the composition of other secondary metabolites (Table S2). Specifically, treatment with the extracts led to a more than 2-fold increase in the biosynthesis of doxorubicin, apramycin, and neomycin. Conversely, the production of several other metabolites: clindamycin, bekanamycin, virginiamycin, dihydrostreptomycin, etamycin, rifamycin, streptomycin, and antimycin A (more than 2-fold lower). The affected metabolites belong to a diverse range of antibiotic classes, including macrolides (doxorubicin), aminoglycosides (neomycin, bekanamycin, and streptomycin), lincosamides (clindamycin), streptogramins (virginiamycin and etamycin), ansamycins (rifamycin), and others (antimycin A). This broad-spectrum effect suggests that the extract triggers a complex, large-scale reprogramming of secondary metabolism in S. albulus, diverting resources and regulatory control across multiple biosynthetic pathways.

4. Discussion

The production of secondary metabolites by Streptomyces species is highly regulated through complex and precise systems at the transcriptional level. Elicitors play a significant role in these regulations. These elicitors are typically categorized as chemical compounds (metals, rare earth elements, dimethyl sulfoxide, ethanol, and nanoparticles) [37,38,39,40,41,42,43,44] and environmental stressors (temperature shifts, pH shock, and dissolved oxygen levels) [45,46,47,48,49]. Recently, stress derived from heterogeneous microorganisms has been identified as a novel elicitor that intricately regulates the biosynthesis of secondary metabolites. The influence varies depending on the species of microorganisms. Generally, fungi secrete metabolic compounds that affect antibiotic synthesis in Streptomyces. For example, rimocidin production can be improved by adding broth and cells of Saccharomyces cerevisiae and F. oxysporum f. sp. cucumerinum due to increased transcription of genes [50]. Some fungi can generate elicitors that promote the biosynthesis of natamycin by S. natalensis HW-2, possibly due to an increased precursor supply through the regulation of amino acid metabolism [51,52,53]. However, heterogeneous Streptomyces influence the production of secondary metabolites through mycelia–mycelia physical interaction [54]. Interestingly, in this study, a heterogeneous Streptomyces, S. gilvosporeus, was also capable of secreting certain extract molecules to induce overproduction of ε-PL in S. albulus, similar to fungi [28], although they might operate through different physiological mechanisms.
In contrast to the effects of fungal extracts (e.g., B. cinerea) [28], the ε-PL-producing strain exhibits a distinct response to extracts from closely related Actinomycetes species (e.g., S. gilvosporeus) (Table S3). Specifically, (i) the variation in suitable solvents (butyl alcohol for B. cinerea and ethyl acetate for S. gilvosporeus) underscores the structural divergence of extract molecules derived from these two organisms; (ii) the extracts from S. gilvosporeus demonstrate a more potent inducing effect (3.42 ± 0.27 g/L ε-PL yield) compared to those from B. cinerea (1.88 ± 0.21 g/L ε-PL yield); (iii) the extracts from S. gilvosporeus lead to denser mycelial pellets, while the extracts from B. cinerea promote more robust and aggregated pellets, suggesting a potential physical confrontation between the ε-PL-producing strain and molds upon receiving the B. cinerea extracts; (iv) the B. cinerea extracts globally upregulate enzymes involved in ε-PL biosynthesis, while the S. gilvosporeus extracts specifically enhance the rate-limiting enzymes in the ε-PL biosynthetic pathways, indicating that the S. gilvosporeus extracts can induce more precise metabolic regulation in the ε-PL-producing strain; (v) the S. gilvosporeus extracts upregulate four phospholipase C genes and three phospholipase D genes, in contrast to the single phospholipase C gene upregulated by the B. cinerea extracts, demonstrating that the S. gilvosporeus extracts lead to stronger phospholipid hydrolysis, thereby increasing membrane fluidity and facilitating the transmembrane transport of nutrients, products, and extracts; and (vii) the B. cinerea extracts result in higher levels of ATP, NADH, and NADPH (and lower ADP, NAD+, and higher NADP+), whereas the S. gilvosporeus extracts induce lower ATP, NADH, and NADPH (and higher ADP, NAD+, and NADP+). Given the enhanced cell respiration of S. albulus IFO14147 observed under both B. cinerea and S. gilvosporeus extracts, the S. gilvosporeus extracts may indicate a greater demand for energy, which is likely utilized in ε-PL biosynthesis.
The coordinated physiological responses to S. gilvosporeus extract suggest a global reprogramming of metabolism in S. albulus. Enhanced expression and activity of key enzymes in glycolysis, the TCA cycle, and the pentose phosphate pathway indicate metabolic rerouting toward precursor supply and energy regeneration. The significant elevation of phosphoenolpyruvate carboxykinase, combined with increased intracellular glutamic acid and cysteine, supports improved carbon and nitrogen flux and potential oxidative stress adaptation. Notably, the reduction in aspartate and lysine pools likely reflects their enhanced consumption in ε-PL synthesis. These data support the hypothesis that the extract functions as a signaling molecule, triggering elevated fluxes in multiple metabolic nodes.
ε-PL is a secondary metabolite produced by S. albulus, known for its strong H+ absorption and antimicrobial activity [55]. Since it is not essential for growth, ε-PL likely serves a specific physiological role. Studies show that ε-PL synthesis increases under acidic pH conditions to counteract high extracellular H+ levels [13,14,15,16]. This research also identifies potential microbial signals from fungi or actinomycetes as a key factor influencing ε-PL production. These inducing effects are complex, depending on microbial groups and physiological changes. Compared to our previous study [28], this study reveals that actinomycete extracts have a stronger impact on ε-PL production than fungal extracts.
Despite the apparent complexity of physiological changes, a discernible pattern can be identified. In response to microbial invasions in a low-dimensional ecosystem, cells shift into a hyper-efficient state. Research on E. coli and B. subtilis has revealed that the regulation of intracellular Ca2+ homeostasis and the perception of extracellular signals mediated by Ca2+ are of crucial significance in the biosynthesis of secondary metabolites [56]. Analogously, a comparable regulatory mechanism might exist in Streptomyces species. In these organisms, fluctuations in intracellular Ca2+ levels could potentially modulate the reprogramming of secondary metabolic networks by targeting key nodes within metabolic pathways [52]. Additionally, reactive oxygen species (ROS) can modulate secondary metabolites synthesis by altering the availability of precursor molecules, increasing the supply for ε-PL production while concurrently decreasing it for other secondary metabolites [36]. Regulation of this state involves the elevation of intracellular secondary messengers, such as H2O2 and Ca2+, which signal via the quorum-sensing system. This signaling leads to the formation of aggregated colonies that enhance resistance to external threats and potentially trigger comprehensive physiological modifications via ROS- and Ca2+-dependent regulatory pathways.
The above studies reveal that the active synthesis of ε-PL aims to eliminate surrounding microorganisms to protect nutrients and space. This disclosure of the intrinsic driving force for ε-PL production offers a wide range of possibilities for developing more efficient strategies to increase ε-PL production through the co-cultivation of multiple microorganisms, which has significant theoretical and practical implications for industrial ε-PL production.

5. Conclusions

This study demonstrates that actinomycete extracts can markedly enhance ε-PL production in Streptomyces albulus. Integrated multi-omics and morphological analyses suggest that S. gilvosporeus extracts can trigger a distinct physiological shift involving metabolic reprogramming and stress-response activation. These findings underscore the regulatory potential of interspecies interactions and offer a promising ecological strategy for improving ε-PL biosynthesis in industrial applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13081868/s1, Table S1: Relative fold change of compounds involved in ε-PL biosynthesis by S. albulus after S. gilvosporeus signal addition according to UPLC-ESI-MS analysis. Note: ns indicates no statistical significance (p ≥ 0.05); * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. Table S2: Relative fold change of compounds involved in other secondary metabolites biosynthesis by S. albulus after S. gilvosporeus signal addition according to UPLC-ESI-MS analysis. Note: ns indicates no statistical significance (p ≥ 0.05); * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. Table S3: Comparison of the physiological response under different interspecies signaling. Table S4: Primer pairs sequences for quantitative real-time PCR (qRT-PCR) assay.

Author Contributions

Conceptualization, X.Z. and F.Z.; Validation, S.T., C.Z., Z.Z. and M.Z.; Formal analysis, H.Z., B.X. and D.Z.; Writing—original draft, S.T., X.Z. and F.Z.; Writing—review & editing, X.Z. and F.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (grant no. 32372308); Science Fund for Outstanding Young Scholars of Anhui Province (2408085Y019); the Key Project to Cultivate Outstanding Young Teachers in Anhui Province (YQZD2023038); University Natural Science Research Project in Anhui Province (2023AH050327 and 2023AH050339); and the National Innovation and Entrepreneurship Program for College Students (202310373033).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available in [Streptomyces noursei strain IFO14147 chromosome, complete genome] at [https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP104098.1] [NZ_CP104098.1]. Accessed on 30 June 2025.

Conflicts of Interest

The authors declare no conflict of interest.

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Microorganisms 13 01868 g001
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 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.
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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 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.
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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 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.
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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 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.
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Microorganisms 13 01868 g005
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 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.
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Microorganisms 13 01868 g006
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.
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.
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Table 1. Strains used in this study.
Table 1. Strains used in this study.
Strains RoleStrainsOrigin
ε-PL-producing strainStreptomyces albulus IFO14147CICC 11022
Inducing strain (bacteria)Escherichia coliCICC 10389
Bacillus subtilisCICC 10002
Micrococcus luteusCICC 10269
Bacillus thuringiensisCICC 10061
Corynebacterium glutamicumCICC 20182
Pseudomonas aeruginosaCICC 10419
Inducing strain (fungi)Botrytis cinereaCGMCC 3.3790
Aspergillus nigerCICC 40102
Monascus purpureusCICC 40942
Aspergillus oryzaeCGMCC 3.7084
Penicillium chrysogenumCGMCC 3.15725
Saccharomyces cerevisiaeCICC 1302
Inducing strain (actinomycetes)Streptomyces gilvosporeusATCC 13326
Streptomyces coelicolorCGMCC 4.3587
Streptomyces lividansCGMCC 4.7169
Streptomyces diastatochromogenesCICC 11011
Streptomyces atratusCICC 11048
Streptomyces aquilusCICC 11055
Table 2. Comparison of gene transcription related to lipid catabolism between cultures of EG (experiment group, with S. gilvosporeus extract supplement) and CK (control group, without extract supplement).
Table 2. Comparison of gene transcription related to lipid catabolism between cultures of EG (experiment group, with S. gilvosporeus extract supplement) and CK (control group, without extract supplement).
Gene IDLog2(EG/CK)p-ValueDescription
N1H47_007001.487592314.55 × 10−4Phospholipase D
N1H47_295153.2183366432.46 × 10−12Phospholipase C
N1H47_181604.3709543131.21 × 10−15Phospholipase C
N1H47_18130−0.1157596490.819Phospholipase D
N1H47_33410−0.1057151750.827Phospholipase C
N1H47_33420−0.283924150.584Phospholipase C
N1H47_39580−0.0372355960.954Phospholipase D
Table 3. Comparison of gene transcription related to energy metabolism between cultures of EG (experiment group, with S. gilvosporeus extract supplement) and CK (control group, without extract supplement).
Table 3. Comparison of gene transcription related to energy metabolism between cultures of EG (experiment group, with S. gilvosporeus extract supplement) and CK (control group, without extract supplement).
Gene IDLog2(EG/CK)p-ValueDescription
N1H47_179801.400.001NADH-quinone oxidoreductase subunit D
N1H47_179951.360.001NADH-quinone oxidoreductase subunit A
N1H47_179701.250.003NADH-quinone oxidoreductase subunit F
N1H47_179851.200.004NADH-quinone oxidoreductase subunit C
N1H47_179901.200.004NADH-quinone oxidoreductase subunit B
N1H47_179751.150.006NADH-quinone oxidoreductase subunit E
N1H47_179601.010.015NADH-quinone oxidoreductase subunit H
N1H47_262600.240.561F0F1 ATP synthase subunit beta
N1H47_262550.280.503F0F1 ATP synthase subunit gamma
N1H47_262400.070.860F0F1 ATP synthase subunit B
N1H47_262450.010.985F0F1 ATP synthase subunit delta
N1H47_262350.110.789F0F1 ATP synthase subunit C
N1H47_262650.380.363F0F1 ATP synthase subunit epsilon
N1H47_262300.030.936F0F1 ATP synthase subunit A
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Tong, S.; Zhang, C.; Zhang, Z.; Zeng, H.; Xin, B.; Zhao, M.; Zhao, D.; Zeng, X.; Zhang, F. Physiological Insights into Enhanced Epsilon-Poly-l-Lysine Production Induced by Extract Supplement from Heterogeneous Streptomyces Strain. Microorganisms 2025, 13, 1868. https://doi.org/10.3390/microorganisms13081868

AMA Style

Tong S, Zhang C, Zhang Z, Zeng H, Xin B, Zhao M, Zhao D, Zeng X, Zhang F. Physiological Insights into Enhanced Epsilon-Poly-l-Lysine Production Induced by Extract Supplement from Heterogeneous Streptomyces Strain. Microorganisms. 2025; 13(8):1868. https://doi.org/10.3390/microorganisms13081868

Chicago/Turabian Style

Tong, Siyu, Chen Zhang, Zhanyang Zhang, Huawei Zeng, Bingyue Xin, Mingtao Zhao, Deyin Zhao, Xin Zeng, and Fei Zhang. 2025. "Physiological Insights into Enhanced Epsilon-Poly-l-Lysine Production Induced by Extract Supplement from Heterogeneous Streptomyces Strain" Microorganisms 13, no. 8: 1868. https://doi.org/10.3390/microorganisms13081868

APA Style

Tong, S., Zhang, C., Zhang, Z., Zeng, H., Xin, B., Zhao, M., Zhao, D., Zeng, X., & Zhang, F. (2025). Physiological Insights into Enhanced Epsilon-Poly-l-Lysine Production Induced by Extract Supplement from Heterogeneous Streptomyces Strain. Microorganisms, 13(8), 1868. https://doi.org/10.3390/microorganisms13081868

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