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Article

Heterologous Expression and Adaptive Evolution of ε-Poly-lysine Synthase Gene in Corynebacterium glutamicum

by
Long Pan
1,2,3,
Yihang Chen
2,
Aimei Liao
1,2,3,
Yinchen Hou
3,4 and
Jihong Huang
1,3,*
1
School of Food and Pharmacy, Xuchang University, Xuchang 461000, China
2
Henan Provincial Key Laboratory of Biological Processing and Nutritional Function of Wheat, School of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China
3
Food Laboratory of Zhongyuan, Luohe 462300, China
4
College of Food and Biological Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450044, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(1), 11; https://doi.org/10.3390/fermentation11010011
Submission received: 23 November 2024 / Revised: 19 December 2024 / Accepted: 25 December 2024 / Published: 2 January 2025
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
ε-Poly-L-lysine (ε-PL) is a natural preservative that has excellent properties such as high safety, good antibacterial effect, wide antibacterial spectrum, and high temperature resistance compared to other food preservatives. At present, the main production strain of ε-PL is Streptomyces albulus (S. albulus). Due to the large amount of mycelium and by-products during fermentation, its production cost is much higher than other food preservatives, which seriously hinders the application of ε-PL in the food industry. Corynebacterium glutamicum (C. glutamicum) is a food safety strain that is widely used in the fermentation industry to produce various amino acids. Its ability to produce high amounts of L-lysine can provide sufficient precursor substances for the synthesis of ε-PL, making it an ideal strain for the heterologous expression of ε-PL synthase genes (pls). In this experiment, a recombinant C. glutamicum capable of synthesizing ε-PL and exhibiting certain physiological resistance to ε-PL was obtained by amplifying pls and heterologous expression in C. glutamicum for the first time. Further optimization of the fermentation temperature, initial pH, and inoculation amount of the recombinant strain resulted in an increase in the ε-PL fermentation yield from 0.12 g/L to 0.22 g/L. Finally, through adaptive evolution of the recombinant strain, the ε-PL tolerance of the recombinant strain was increased to 1.3 g/L, and the yield of ε-PL ultimately reached 0.34 g/L, which increased by 54.55% compared to the initial strain. The recombinant C. glutamicum constructed in this study can significantly shorten the fermentation cycle, reduce bacterial volume and the synthesis of secondary metabolites, which is beneficial for the separation and purification of products, thereby further reducing the production cost of ε-PL and accelerating the process of replacing chemical food preservatives with natural food preservatives.

1. Introduction

ε-Poly-L-lysine (ε-PL) is an amino acid polymer polymerized by 25–35 lysine [1]. ε-PL exhibits multi-cationic properties in environments ranging from acidic to slightly alkaline due to numerous free amino groups along its main chain. Additionally, it demonstrates potent antibacterial effects against a broad range of microorganisms, encompassing most Gram-positive and Gram-negative bacteria as well as fungi, yeasts, and certain viruses [2], where the minimum inhibitory concentration (MIC) for these microorganisms were 10–50 µg/mL, 50–200 µg/mL, 100–300 µg/mL and 10–50 µg/mL, respectively. Moreover, this polymer is biodegradable and non-toxic, which has led to its regular use as a natural and safe food preservative in the food industry for many years, particularly in countries such as Japan, Korea, the USA, and China [3]. However, the high cost of production continues to be a significant barrier to the broader application of this natural antimicrobial agent and highly functional material.
To address the increasing demand for ε-PL across various sectors, extensive research has been conducted to enhance its production methods [4,5,6]. For instance, to overcome the issue of end-product feedback inhibition during ε-PL production, a resin-based in situ product removal technique was employed. This approach successfully increased ε-PL production to 23.4 g/L within 192 h in a 5-L fermenter [7]. Furthermore, to reduce energy consumption during ε-PL production, airlift bioreactors [8], immobilized cells with repeated fed-batch cultures [9], and solid-state fermentation [10] have been employed instead of the popular approach of using free cells in a jar fermenter. In addition, pH control has also been used to improve metabolites during fermentation. Kahar et al. [11] reported that no ε-PL is produced when the pH is maintained above 5.0 during fermentation, although this pH range is beneficial for cell growth. Conversely, a pH range of 3.5 to 4.5 is much more conducive to ε-PL accumulation. Accordingly, a two-stage pH control strategy was developed, which significantly enhanced ε-PL production, reaching levels of 48.3 g/L. This approach optimizes the conditions for both cell growth and ε-PL synthesis by adjusting the pH at different stages of the fermentation process. In the research of Ren et al., a pH shock strategy was used to improve the production of ε-PL [12]. Although this strategy could effectively enhance the production of ε-PL, it also led to a significant increase in bacterial biomass, which can complicate the control of dissolved oxygen (DO) in industrial applications. The higher bacterial density can consume more oxygen, making it challenging to maintain the optimal DO levels necessary for efficient fermentation.
As early as 2014, Geng et al. had already achieved the expression of the ε-PL synthase gene in Streptomyces lividans, but it was difficult to achieve industrial production [13]. In 2020, Claudine et al. successfully achieved heterologous expression of the ε-PL synthase gene in Bacillus subtilis by optimizing its codon, and then synthesized ε-PL in vitro through whole cell catalysis [14]. Although this method greatly shortens the fermentation cycle, the exogenous addition of L-lysine not only increases the difficulty of the process, but also poses certain challenges with regard to the purification of the product. Ultimately, the lower yield of ε-PL still cannot meet the requirements of its industrial production.
As an important industrial production strain, C. glutamicum is not only used for producing amino acids and other compounds, but also for expressing exogenous proteins due to its significant advantages [15]. It belongs to the order Actinobacteria and genus Actinobacterium, and is an aerobic, non-spore producing Gram-positive bacterium with guanine and cytosine contents of approximately 56%. C. glutamicum is non-pathogenic and can grow rapidly, making it a widely considered safe production strain [16]. C. glutamicum is a strain capable of producing L-lysine, and its fermentation product serves as a precursor for the synthesis of ε-PL, providing a continuous supply of polymeric monomers for the biosynthesis of ε-PL. In 2024, Egbune et al. reported that a yield of L-lysine of 242 g/L could be achieved in industrial production by C. glutamicum [17]. Its ability to produce high levels of L-lysine provides sufficient precursor substances for the synthesis of ε-PL. By the heterologous expression of ε-PL in C. glutamicum, it is possible to effectively utilize C. glutamicum to efficiently synthesize ε-PL in high yields providing new ideas for the construction of recombinant strains for producing ε-PL.
In this study, we first extracted the genome of S. albulus, and by amplifying it by PCR, the ε-PL synthase gene (pls) was obtained. The purified fragment was then linked to the linear expression vector pXMJ19 to obtain the recombinant plasmid pXMJ19-pls. The recombinant plasmid pXMJ-19-pls was introduced into the competent cells of C. glutamicum through electroporation to achieve the heterologous expression of pls in C. glutamicum. After measuring the yield of ε-PL through shake flask fermentation, fermentation optimization was carried out to determine the optimal temperature, initial pH, and inoculation amount for fermentation. Finally, through adaptive evolution, the tolerance of recombinant C. glutamicum to ε-PL was improved, thereby enhancing its ability to ferment and produce ε-PL.

2. Materials and Methods

2.1. Microorganism, Plasmids, and Primers

The strain of S. albulus IFO 14147 and C. glutamicum CICC 10064 was purchased from the China Industrial Microbial Culture Collection and Management Center (CICC). The Escherichia coli DH5α and pXMJ19 plasmids were purchased from Wuhan Miaoling Biotechnology Co., Ltd., Wuhan, China. Primers for amplifying pls genes were synthesized by Suzhou Junji Biotechnology Co., Ltd., Suzhou, China (Table 1).

2.2. Culture and Fermentation Media Composition

The LB medium (g/L): peptone, 10; yeast extract, 5; NaCl, 10, (1.6% agar was added when it was a solid culture medium) with the initial pH of 7.5 adjusted by 1 M NaOH solution and/or 1 M H2SO4.
The M3G [18] medium as the seed medium was used for Streptomyces albulus cultivation in this study, which contained (g/L): glucose, 50; yeast extract, 5; (NH4)2SO4, 10; KH2PO4, 1.36; K2HPO4·3H2O, 0.8; MgSO4·7H2O, 0.5; ZnSO4·7H2O, 0.04; and FeSO4·7H2O, 0.03, with the initial pH of 6.8 adjusted by 1 M NaOH solution and/or 1 M H2SO4.
Two rings of bacterial cells were scraped from the solid culture medium, inoculated in a 250 mL shake flask with 50 mL seed medium, and cultivated at 30 °C in a rotary shaker (HYL-C, Qiangle Experimental Co., Ltd., Taicang, China) with 200 rpm for 24 h. Then, 8% of the seeds were transferred to fresh medium and incubated at 37 °C and 200 rpm on a rotating shaker for 72 h. These cultures were used for all fermentations in this study.
The fermentation medium [19] for ε-PL adopted by our previous study was slightly modified as follows (g/L): glucose, 60; (NH4)2SO4, 10; beef extract, 10; KH2PO4, 4; MgSO4·7H2O, 0.8; and FeSO4·7H2O, 0.05, with the initial pH of 6.8 adjusted by 1 M NaOH solution and/or 1 M H2SO4.
All media components were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) with analytical and biochemical grades. All media were sterilized in an autoclave at 121 °C for 20 min.

2.3. Construction of the Recombinant Strains

To construct heterologous expression strains of C. glutamicum, the partial DNA fragments of the target genes (pls) were amplified by PCR, and then cloned into the Xba I and EcoR I sites of the pXMJ19 plasmid by using the ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech, Nanjing, China) to construct recombinant plasmids. Recombinant plasmids and empty plasmids were transformed into competent cells of C. glutamicum by electroporation.

2.4. Single Factor Optimization of Shake Flask Culture of Recombinant Bacterial Strains

In the optimization of fermentation conditions using a recombinant strain, a single-factor experiment was employed to fine-tune key parameters. The main factors considered were inoculum volume, fermentation temperature, initial pH, and shaker speed. Specifically:
-
Inoculum volume: 2%, 4%, 6%, 8%, and 10%.
-
Fermentation temperature: 26°C, 28°C, 30°C, 32°C, and 34°C.
-
Initial pH: 6.6, 6.8, 7.0, 7,2, 7.4.
-
Shaker speed: 140, 160, 180, 200, and 220 rpm.
After 72 h of fermentation, the ε-PL content in the fermentation broth was measured to determine the optimal conditions for each parameter

2.5. Adaptive Evolution of Recombinant Strain

The recombinant strain was selected as the original strain for adaptive evolution. Based on the principle of survival of the fittest, an adaptive evolution experiment was selected to screen for a recombinant strain that could tolerate high concentrations of ε-PL as the concentration of ε-PL in the culture medium continued to increase. Firstly, we selected a single colony of recombinant strain stored at 4 °C and transferred it to the LB liquid medium containing 10 mg/L chloramphenicol. This was then incubated at 30 °C with 200 rpm for 12 h for activation. The activated bacterial solution was then inoculated into LB liquid medium with a concentration of 0.1 g/L ε-PL at a inoculation rate of 5%, and then evenly coated with 200 μL of bacterial solution onto solid LB medium of the same concentration. The strain grown in the solid culture medium was used as the adaptive evolution passaging strain, and a concentration gradient of 0.05 g/L ε-PL was used to streak the strain onto the solid culture medium and select the strain onto liquid culture medium for adaptive evolution. The cultivation conditions were both 30 °C and 200 rpm. This continued in sequence until the strain no longer grew.

2.6. Analytical Methods

The ε-PL concentration that was determined was appropriately revised based on the method described by Itzhaki [20]. Briefly, the supernatant was diluted at suitable multiple with pH 6.90 phosphate buffer solution, and then 2.0 mL of diluent was mixed with 2.0 mL 1 mM methyl orange solution. After vortex mixing, the precipitation reaction was incubated at 30 °C for 30 min in a shaker at 200 rpm. Subsequently, the mixture was centrifuged at 4500× g for 15 min, and the supernatant was diluted 20-fold with pH 6.90 phosphate buffer solution and its absorbance was measured at 465 nm.
The ε-PL in the fermentation liquid was analyzed by MALDI-TOF/MS with an Autoflex 2 mass spectrometer (Bruker Daltonics, Billerica, MA, USA). As a matrix, 2,5-dihydroxybenzoic acid was used, which was used by Nishikawa et al. [21].
The determination of cell membrane fatty acid composition refers to the following method, which was previously reported by Wang et al. [22]. Mycelia were harvested by centrifugation at 3000× g for 15 min, and the pellet was washed three times with distilled water. The fatty acids in the cells (40–50 mg in wet weight) were saponified and methylated. The methyl ester mixtures were determined by gas chromatography (TSQ 8000, Triple Quadrupole GC-MS/MS, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Supelco SP-2560 capillary column (100 m × 0.25 mm × 0.20 μm). Helium was used as the carrier gas with a flow rate of 1.2 mL/min. The column temperature program was as follows: initial temperature 150 °C for 3 min; 170 C at 3 °C/min and maintained for 5 min; the 240 °C at 3 °C temperature was set at 240 °C, and the electronic energy was 70 eV. The scanning quality scope was recorded as 35–400 amu. Fatty acids were identified by the MIDI microbial identification system [23]. Minor fatty acids (<0.6% of the total) were not considered. The data were expressed as a relative percentage of each fatty acid compared to the total area. The relative content of fatty acid components is expressed as the molar ratio of each component in the total fatty acids, and the fatty acid unsaturation ratio (UFA/SFA) was calculated using the following formulas. For each condition, three repetitions of three independent experiments were considered.
UFA/SFA = (Σ Relative content of unsaturated fatty acids)/(Σ Relative content of saturated fatty acids)
The bacterial growth was determined by measuring the optical density at 600 nm (U-2000 Spectrophotometer, Hitachi, Tokyo, Japan) [24].

2.7. Statistical Analysis

Each value reported was the mean of three replications, and the results were expressed as the mean ± standard deviation (mean ± S.D.). Statistical analysis was performed using the t-test in GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) to distinguish differences between the experimental groups and the control group. A p-value < 0.05 was considered statistically significant. A histogram was generated using Origin 2024 software (OriginLab, Northampton, MA, USA).

3. Results

3.1. Construction of Recombinant C. glutamicum

In this study, the pls gene was amplified by PCR through the genome of S. albulus IFO 14147 (Figure 1a), and plasmid pXMJ19 (Figure 1b) was connected to the pls gene after enzyme digestion. The recombinant plasmid was validated by double enzyme digestion, and the band size was correct, as shown in Figure 1c. Then, the constructed recombinant plasmid was transferred to the competent cells of C. glutamicum. After screening with resistance plates, single colonies were selected with pls gene primers for PCR verification, and the obtained band shown in Figure 1d was the pls gene, indicating that the recombinant C. glutamicum-pXMJ19-pls was successfully constructed.

3.2. Fermentation Yield Determination and Mass Spectrometry Identification of Recombinant Strains

The yield of ε-PL in the fermentation broth was measured, which reached 0.12 g/L, indicating a successful fermentation process for ε-PL production. To further explore the characteristics of the synthesized ε-PL, its molecular weight and polymerization degree were analyzed using time-of-flight mass spectrometry (MS), a technique that provides high precision in molecular analysis. As shown in Figure 2, the relative molecular weight distribution of ε-PL produced by the fermentation of recombinant C. glutamicum-pXMJ19-pls ranged from 3605 to 4284 Da, with the highest concentration observed at 3861 Da. This molecular weight distribution closely matched that of the standard ε-PL sample, confirming the identity of the synthesized product. Furthermore, based on the aggregation degree formula calculation, the aggregation degree distribution of ε-PL ranged from 25 to 30, with the majority of the polymer population concentrated at a value of 28. This finding suggests that the polymerization process in recombinant C. glutamicum-pXMJ19-pls resulted in a well-defined distribution of polymer chain lengths. Taken together, these results indicate that C. glutamicum-pXMJ19-pls is capable of efficiently utilizing glucose as a substrate to synthesize ε-PL, demonstrating its ability to functionally express this biopolymer. This provides strong evidence for the potential application of this engineered strain in the industrial production of ε-PL.

3.3. Single Factor Experiment of the Fermentation by Recombinant C. glutamicum-pXMJ19-pls

3.3.1. Effect of Inoculum Volume on ε-PL Production

The inoculum volume plays a pivotal role in determining the efficiency and outcome of the fermentation process. If the inoculum volume is too small, it can lead to slow cell growth during the early stages, extending the lag phase and ultimately resulting in lower productivity. Conversely, an excessively large inoculum volume can promote overly rapid cell growth, which may quickly deplete essential nutrients or cause a dissolved oxygen (DO) deficiency in the fermentation medium. These issues can negatively affect metabolic activity and lead to a decline in overall productivity [25]. In this study, the inoculation amount of C. glutamicum-pXMJ19-pls was optimized by testing a range of values including 2%, 4%, 6%, 8%, and 10%. As shown in Figure 3a, the highest production of ε-PL was obtained when the inoculation amount of C. glutamicum-pXMJ19-pls was 6%. This finding suggests that the inoculation amount plays a crucial role in maximizing ε-PL production, with 6% being the most effective concentration. Further increases in inoculum size did not result in higher ε-PL production, indicating a saturation effect. Therefore, the 6% inoculation amount was determined to be the optimal condition for ε-PL synthesis in the tested fermentation process, which could potentially be applied in future industrial-scale fermentations to enhance the yield and efficiency.

3.3.2. Effect of Fermentation Temperature on ε-PL Production

The results of optimizing the fermentation temperature for ε-PL production by C. glutamicum-pXMJ19-pls are presented in Figure 3b. The study was conducted with an inoculation amount of 6%, and fermentation lasted for 72 h. The findings revealed that the fermentation temperature had a significant impact on ε-PL production, with the highest yield observed at a temperature of 30 °C. This result indicates that 30 °C is the optimal temperature for maximizing ε-PL production under the tested conditions.

3.3.3. Effect of Initial pH on ε-PL Production

The initial pH is a crucial factor that can significantly influence the growth of mycelia and the overall fermentation process. To evaluate its impact on ε-PL production, the effects of different initial pH levels were systematically tested, with the results presented in Figure 3c. As the initial fermentation pH increased, the production of ε-PL gradually improved, reaching its highest level at pH 7.0. However, further increases in the initial fermentation pH beyond this point resulted in a noticeable decline in ε-PL production. Based on these findings, it was concluded that the optimal initial fermentation pH for maximizing ε-PL production is 7.0.

3.3.4. Effect of Shaker Speed on ε-PL Production

The shaker speed is a critical parameter that significantly influences the dissolved oxygen levels in the fermentation broth, which in turn affects the respiration, growth, and metabolic activity of bacterial cells. As such, it is essential to optimize the shaking speed for the recombinant strain during flask fermentation to achieve maximum efficiency. The results, presented in Figure 3d, demonstrate that ε-PL production gradually increased as the shaker speed was raised, reaching its peak at 200 rpm. Beyond this point, further increases in shaker speed did not result in any additional enhancement in ε-PL production, suggesting a saturation effect. Therefore, 200 rpm was identified as the optimal shaker speed for maximizing the ε-PL yield under the experimental conditions.

3.4. Adaptive Evolution and Fermentation Level Analysis of C. glutamicum-pXMJ19-pls

Adaptive evolution experiments were conducted on C. glutamicum-pXMJ19-pls, where the content of ε-PL in the culture medium was continuously increased to gradually enhance its tolerance to ε-PL, thereby improving the ability of C. glutamicum-pXMJ19-pls to produce ε-PL. Adaptive evolution occurred simultaneously in liquid and solid media, mainly by observing the growth status of recombinant C. glutamicum-pXMJ19-pls in solid media with different concentrations of ε-PL. The final adaptive evolution result showed that C. glutamicum-pXMJ19-pls can tolerate a concentration of ε-PL up to 1.3 g/L. As shown in Figure 4, the ability of C. glutamicum-pXMJ19-pls to produce ε-PL gradually increased with the increase in ε-PL concentration during this process, ultimately reaching 0.34 g/L. Compared to the non-adapted recombinant strain, its yield increased by 19.05%.

3.5. The Effect of Adaptive Evolution on the Growth of Recombinant Bacterial Strains and the Saturation of Cell Membrane Fatty Acids

The effects of adaptive evolution on the growth of recombinant bacterial strains and the saturation of cell membrane fatty acids are shown in Table 2. The biomass and membrane unsaturation of strains expressing heterologous pls showed no significant difference to those of strains containing empty plasmids, indicating that the expression of pls has no significant effect on the growth of C. glutamicum. With the increases in the concentration of ε-PL during adaptive evolution, the biomass of recombinant strains showed a trend of first increasing and then decreasing, indicating that the improvement of their ability to tolerate ε-PL contributed to the synthesis of ε-PL to some extent, which is consistent with the results of most adaptive evolution studies [26,27,28]. With the improvement in the tolerance ability of recombinant strains, the unsaturation rate of cell membrane fatty acids was significantly increased, which is consistent with the improvement in the ε-PL synthesis ability, indicating that the increase in ε-PL production may be the result of changes in cell membrane permeability. More ε-PL is released outside the cell after intracellular synthesis, thereby increasing the production of ε-PL.

4. Discussion

ε-PL as a natural food preservative, has a certain antibacterial effect. Therefore, the tolerance of recombinant strains seriously affects their production of ε-PL during fermentation. Further research is needed to improve the strain tolerance in the future. This experiment first amplified the pls from S. albulus and then connected it to the linear expression vector pXMJ19 to obtain the recombinant plasmid pXMJ19-pls. Subsequently, it was transformed into the competent state of C. glutamicum, successfully achieving the heterologous expression of pls in C. glutamicum and obtaining a recombinant C. glutamicum-pXMJ19-pls that was capable of synthesizing ε-PL and exhibited a certain physiological resistance to ε-PL. The recombinant C. glutamicum-pXMJ19-pls was achieved by the direct fermentation synthesis of ε-PL using glucose for the first time. By optimizing the fermentation conditions, the optimal fermentation conditions were obtained, and the yield of ε-PL fermentation increased by 83.33% (from 0.12 g/L to 0.22 g/L).
Adaptive evolution is a commonly used experimental method in laboratories, which involves long-term domestication under certain environmental pressures to select mutant strains with specific physiological functions and improve microbial resistance to external pressures [29,30]. It can be used to produce special biological products and increase microbial metabolism. The purpose of adaptive evolution is generally to increase the strain yield by improving the strain tolerance. So far, adaptive evolution techniques have been widely used for the targeted screening of high-performance strains, which typically select certain inhibitory substances as stress factors during fermentation such as substrates or by-products [31]. Compared with rational engineering strategies and targeted modifications for specific enzymes, the advantage of laboratory adaptive evolution is that it allows many different genes and regulatory regions to simultaneously have nonintuitive beneficial mutations, providing more opportunities for microbial modification [32]. In this study, by continuously increasing the content of ε-PL in the culture medium, the tolerance of recombinant C. glutamicum-pXMJ19-pls to ε-PL was gradually improved, and its ability to produce ε-PL was continuously enhanced (Table 2). Finally, when the tolerance concentration of ε-PL reached 1.3 g/L, the production of ε-PL reached by C. glutamicum-pXMJ19-pls-B5 was 0.34 g/L, which increased by 54.55% compared to before evolution (0.22 g/L). The biomass of recombinant C. glutamicum-pXMJ19-pls also increased to a certain extent with the improvement of tolerance ability. Compared with the strains without adaptive evolution, the increase in biomass reached a significant level (p < 0.05).
The cell membrane plays an important role in microbial material transport, energy metabolism, biosynthesis, and intracellular microenvironment stability [33]. The cell membrane responds to different environmental stimuli by changing the composition of its fatty acid components, thereby ensuring the survival of cells under stress [34]. The change in fatty acid composition in the cell membrane led to an increase in the ratio of unsaturated fatty acids to saturated fatty acids from 11.74 to 18.56 (Table 2), which was increased significantly (p < 0.05). The ratio of saturated fatty acids to unsaturated fatty acids is an important factor in regulating cell membrane permeability [35]. This discovery suggests that it is feasible to increase the cell tolerance and thus increase ε-PL production by altering cell membrane permeability.
Although this study achieved the direct fermentation of recombinant C. glutamicum-pXMJ19-pls to produce ε-PL, from the current level of shake flask fermentation, the ε-PL production of the adaptively evolved recombinant C. glutamicum-pXMJ19-pls B5 still did not reach the gram level. This is still a considerable gap compared to the fermentation yield of the existing ε-PL producing strain S. albulus (the highest yield reached 4.41 g/L in shake flasks by Wang et al. [36]). In the current situation where its tolerance cannot be further improved, adjusting the composition of the culture medium and supplementing nutrients to increase the biomass of the fermentation strain may be a supplementary method for improving its fermentation yield in the future.

5. Conclusions

In conclusion, we successfully achieved the synthesis of ε-PL by C. glutamicum-pXMJ19-pls through heterologous expression, resulting in a yield of 0.12 g/L. Through the optimization of the fermentation process, the yield was further increased to 0.22 g/L. Additionally, adaptive evolution was applied to enhance the ε-PL tolerance of the recombinant strain, which reached a maximum tolerance of 1.3 g/L. This improvement allowed the corresponding ε-PL yield to increase to 0.34 g/L, representing a 183.3% increase over the initial yield of 0.12 g/L. These results indicate that adaptive evolution is an effective method for significantly improving the yield of ε-PL in industrial microbial fermentation, demonstrating its potential for enhancing production efficiency at a larger scale.

Author Contributions

Conceptualization, J.H.; Methodology, writing—review and editing, L.P. and Y.C.; Investigation, A.L.; Data curation, Y.H.; Writing—original draft preparation, L.P.; Supervision, J.H.; Project administration, J.H.; Funding acquisition, J.H. and L.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Open Competition Research Projects of Xuchang University (2022JBGS11), the High-Level Talents Research Fund of HAUT (Grant No. 2020BS064), the Henan Province Youth Science Fund Project (232300421266), and the Central Government Guides the Local Science and Technology Development Special Fund (Z20221341069).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts interests.

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Fermentation 11 00011 g001
Figure 1. Construction of recombinant plasmids and the screening and identification of recombinant strain C. glutamicum-pls: (a) pls gene; (b) pXMJ19 plasmids; (c) double enzyme digestion of recombinant plasmids; (d) identification of recombinant C. glutamicum-pXMJ19-pls.
Figure 1. Construction of recombinant plasmids and the screening and identification of recombinant strain C. glutamicum-pls: (a) pls gene; (b) pXMJ19 plasmids; (c) double enzyme digestion of recombinant plasmids; (d) identification of recombinant C. glutamicum-pXMJ19-pls.
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Figure 2. The result of the mass spectra of the 1 g/L ε-PL standard (a) and in fermentation broth (b).
Figure 2. The result of the mass spectra of the 1 g/L ε-PL standard (a) and in fermentation broth (b).
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Figure 3. Optimization of the fermentation conditions of C. glutamicum-pXMJ19-pls to produce ε-PL at the flask cultivation level: (a) inoculation volume; (b) fermentation temperature; (c) initial pH; (d) shaker speed.
Figure 3. Optimization of the fermentation conditions of C. glutamicum-pXMJ19-pls to produce ε-PL at the flask cultivation level: (a) inoculation volume; (b) fermentation temperature; (c) initial pH; (d) shaker speed.
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Figure 4. The ε-PL production of different strains after adaptation and evolution (A: strain with empty plasmids; B: C. glutamicum-pXMJ19-pls; B1: strain that can tolerate a concentration of 0.3 g/L ε-PL; B2: strain that can tolerate a concentration of 0.6 g/L ε-PL; B3: strain that can tolerate a concentration of 0.9 g/L ε-PL; B4: strain that can tolerate a concentration of 1.2 g/L ε-PL; B5: strain that can tolerate a concentration of 1.3 g/L ε-PL).
Figure 4. The ε-PL production of different strains after adaptation and evolution (A: strain with empty plasmids; B: C. glutamicum-pXMJ19-pls; B1: strain that can tolerate a concentration of 0.3 g/L ε-PL; B2: strain that can tolerate a concentration of 0.6 g/L ε-PL; B3: strain that can tolerate a concentration of 0.9 g/L ε-PL; B4: strain that can tolerate a concentration of 1.2 g/L ε-PL; B5: strain that can tolerate a concentration of 1.3 g/L ε-PL).
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Table 1. Bacterial strains and plasmids used in this study.
Table 1. Bacterial strains and plasmids used in this study.
Bacterial Strains/Plasmids/PrimersFunctionSource
Bacterial strains
S. albulus IFO 14147Provide genomic templatesCICC
C. glutamicum CICC 10064Heterologous expression of host bacteriaCICC
C. glutamicum-pXMJ19C. glutamicum with empty plasmidConstructed in this study
C. glutamicum-pXMJ19-plsC. glutamicum heterologous expression with plsConstructed in this study
E. coli DH5αHost bacteria for cloneWuhan Miaoling Biotechnology Co., Ltd.
Plasmids
pXMJ19Used for heterologous expression vector of C. glutamicumWuhan Miaoling Biotechnology Co., Ltd.
pXMJ19-plsHeterologous expression of pls genes in Corynebacterium glutamicumConstructed in this study
Primers
pls-FtgcctgcaggtcgactctagaATGTCCAGCCCGCTGCTGSuzhou Junji Biotechnology Co., Ltd.
pls-RcaaaacagccaagctgaattcTGCCGCAGCACCACCTTCSuzhou Junji Biotechnology Co., Ltd.
Table 2. Biomass and membrane unsaturation of different bacterial strains.
Table 2. Biomass and membrane unsaturation of different bacterial strains.
Strains *Biomass (OD600)Membrane Unsaturation (%)
A2.68 ± 0.23 d12.63 ± 1.15 c
B2.52 ± 0.21 de11.74 ± 1.69 c
B12.76 ± 0.13 c12.88 ± 0.52 c
B22.98 ± 0.11 ab14.51 ± 1.76 b
B33.14 ± 0.17 a16.14 ± 2.03 ab
B42.94 ± 0.09 ab17.33 ± 1.42 a
B52.82 ± 0.12 b18.56 ± 2.13 a
* A: C. glutamicum with empty plasmids; B: C. glutamicum-pXMJ19-pls; B1: strain that can tolerate a concentration of 0.3 g/L ε-PL; B2: strain that can tolerate a concentration of 0.6 g/L ε-PL; B3: strain that can tolerate a concentration of 0.9 g/L ε-PL; B4: strain that can tolerate a concentration of 1.2 g/L ε-PL; B5: strain that can tolerate a concentration of 1.3 g/L ε-PL. (The letters a, b, c, d, e represent significant differences between different data).
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Pan, L.; Chen, Y.; Liao, A.; Hou, Y.; Huang, J. Heterologous Expression and Adaptive Evolution of ε-Poly-lysine Synthase Gene in Corynebacterium glutamicum. Fermentation 2025, 11, 11. https://doi.org/10.3390/fermentation11010011

AMA Style

Pan L, Chen Y, Liao A, Hou Y, Huang J. Heterologous Expression and Adaptive Evolution of ε-Poly-lysine Synthase Gene in Corynebacterium glutamicum. Fermentation. 2025; 11(1):11. https://doi.org/10.3390/fermentation11010011

Chicago/Turabian Style

Pan, Long, Yihang Chen, Aimei Liao, Yinchen Hou, and Jihong Huang. 2025. "Heterologous Expression and Adaptive Evolution of ε-Poly-lysine Synthase Gene in Corynebacterium glutamicum" Fermentation 11, no. 1: 11. https://doi.org/10.3390/fermentation11010011

APA Style

Pan, L., Chen, Y., Liao, A., Hou, Y., & Huang, J. (2025). Heterologous Expression and Adaptive Evolution of ε-Poly-lysine Synthase Gene in Corynebacterium glutamicum. Fermentation, 11(1), 11. https://doi.org/10.3390/fermentation11010011

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