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Efficient enhancement of the antimicrobial activity of Chlamydomonas reinhardtii extract by transgene expression and molecular modification using ionizing radiation

Biotechnology for Biofuels and Bioproducts volume 17, Article number: 125 (2024) Cite this article

Abstract

Background

Ionizing radiation has been used for mutagenesis or material modification. The potential to use microalgae as a platform for antimicrobial production has been reported, but little work has been done to advance it beyond characterization to biotechnology. This study explored two different applications of ionizing radiation as a metabolic remodeler and a molecular modifier to enhance the antimicrobial activity of total protein and solvent extracts of Chlamydomonas reinhardtii cells.

Results

First, highly efficient transgenic C. reinhardtii strains expressing the plant-derived antimicrobial peptides, AtPR1 or AtTHI2.1, were developed using the radiation-inducible promoter, CrRPA70Ap. Low transgene expression was significantly improved through X-irradiation (12–50 Gy), with peak activity observed within 2 h. Protein extracts from these strains after X-irradiation showed enhanced antimicrobial activity against the prokaryotic bacterium, Pseudomonas syringae, and the eukaryotic fungus, Cryptococcus neoformans. In addition, X-irradiation (12 Gy) increased the growth and biomass of the transgenic strains. Second, C. reinhardtii cell extracts in ethanol were γ-irradiated (5–20 kGy), leading to molecular modifications and increased antimicrobial activity against the phytopathogenic bacteria, P. syringae and Burkholderia glumae, in a dose-dependent manner. These changes were associated with alterations in fatty acid composition. When both transgenic expression of antimicrobial peptides and molecular modification of bioactive substances were applied, the antimicrobial activity of C. reinhardtii cell extracts was further enhanced to some extent.

Conclusion

Overall, these findings suggest that ionizing radiation can significantly enhance the antimicrobial potential of C. reinhardtii through efficient transgene expression and molecular modification of bioactive substances, making it a valuable source of natural antimicrobial agents. Ionizing radiation can act not only as a metabolic remodeler of transgene expression in microalgae but also as a molecular modifier of the bioactive substances.

Background

Microalgae are microscopic photosynthetic organisms that are single-celled prokaryotic or eukaryotic microbial species commonly found in marine and freshwater environments, and have the ability to fix carbon dioxide [1]. They accumulate high-value bioactive substances with diverse biological activities, including complex organic compounds and primary and secondary metabolites [2,3,4,5,6]. These compounds include phytopigments, carbohydrates, proteins, lipids, polyphenols, phytosterols, and vitamins, among others. Microalgae-derived antimicrobial compounds have several potential applications in the pharmaceutical industry as functional food ingredients, antibiotics for aquaculture and animal health, and biopesticides for agriculture [7]. Various antimicrobial compounds in eukaryotic microalgae have demonstrated antibacterial, antifungal, and antiviral activities as natural antibiotics against human pathogens and aquaculture diseases [8]. In addition, aqueous and organic extracts, including fatty acids, exopolysaccharides, and phycobiliproteins from cyanobacterial strains, have shown antibacterial and antifungal activities against foodborne pathogens [9]. Despite the functionality of microalgal extracts, further exploration, engineering, and processing of potential antimicrobial compounds are required to commercially utilize microalgae in the nutraceutical and pharmaceutical industries [7].

Several approaches have been demonstrated to be effective for increasing or enhancing high-value bioactive substances in microalgal cells or extracts. First, cultivation strategies, including media composition and inducer treatments, can be optimized for biomass and metabolite production in microalgae. For example, a heterotrophic fed-batch growth strategy was used to produce high concentrations of ICAM-1, a bioactive human recombinant protein, in Chlamydomonas reinhardtii cells [10]. A significant increase in the C/N ratio of the culture medium leads to the efficient accumulation of exopolysaccharides and polyunsaturated fatty acids in Porphyridium purpureum [11]. Chemical or physical inducers, such as hydrogen peroxide, acetic acid, and ionizing radiation, enhance starch, lipid, and recombinant protein production in C. reinhardtii cells [12,13,14]. Second, the expression of exogenous genes allows microalgae to produce various bioactive substances. Microalgae are attractive platforms for producing high-value recombinant proteins due to their simple transformation system, low production cost, and ability to secrete proteins [15, 16]. With the help of genetic engineering, eukaryotic microalgae are promising producers of bioactive substances and have been widely applied in medicine, food, health products, cosmetics, and environmental protection [17, 18]. Finally, microalgal cells or extracts can be treated with chemical or physical agents, such as organic solvents, ultrasound, heat, and ionizing radiation, to increase the extraction of bioactive substances from the cells or enhance the functionality of bioactive substances in microalgal extracts. In practice, the efficiency of lipid extraction from microalgae depends on the type of organic solvent or mechanical method used [19]. The yield of microalgae-derived bioactive substances can be increased using various isolation or extraction methods developed for each compound [20]. In addition, ionizing radiation can be applied to microalgal extracts to enhance the functionality of bioactive substances. Ionizing radiation has been used to improve the physicochemical and biological properties of natural bioactive substances through molecular modifications [21,22,23,24].

Chlamydomonas is a single-celled eukaryotic green alga that has been studied for a variety of biotechnological applications, including recombinant protein production. The genetics of C. reinhardtii is well characterized owing to the sequencing of its nuclear, chloroplast, and mitochondrial genomes [25,26,27], and various transformation methods for it are well established [15, 16, 28]. However, transgene expression in microalgae is often inefficient or unstable due to transgene silencing mechanisms that specifically affect the exogenously inserted DNA [29, 30]. Several approaches have been suggested to overcome the problem of transgene expression in microalgae. (1) Optimal transgene constructs should be designed to promote transgene expression using appropriate promoters [30], introns [31, 32], and terminators [33], and optimizing the use of transgene codons [34]. (2) Utilizing chloroplasts rather than nuclei for transformation can be an alternative to overcome low nuclear transgene expression [35]. In both algae and higher plants, recombinant proteins accumulate at much higher levels when expressed in the chloroplast genome than when expressed in the nuclear genome [36]. (3) Mutant strains of microalgae that efficiently express nuclear transgenes can be selected and utilized for nuclear transformation [37]. The recombinant biopharmaceutical HIV antigen, p24, was efficiently expressed by introducing a codon-optimized P24 gene variant into the nuclear genome of a mutant strain of C. reinhardtii [38].

In this study, three strategies were applied to enhance the antimicrobial activity of C. reinhardtii cells and extracts using ionizing radiation. First, Arabidopsis thaliana Pathogenesis-Related 1 (AtPR1) and Thionin 2.1 (AtTHI2.1) genes, which are specifically involved in plant defense mechanisms against pathogen attack, were transformed into C. reinhardtii cells for efficient expression using the putative promoter of C. reinhardtii Replication Protein A 70A (CrRPA70A) gene, induced by ionizing radiation. Second, X-rays were used as inducers to enhance growth, biomass production, and transgene expression in AtPR1- or AtTHI2.1-overexpressing transgenic C. reinhardtii strains. Finally, molecular modification of bioactive substances using γ-rays was applied as an efficient method to enhance the antimicrobial activity of C. reinhardtii extracts. Here, we demonstrated that microalgae, such as C. reinhardtii, could serve as promising sources of antimicrobial bioactive substances, and that their antimicrobial activity can be efficiently enhanced by various strategies using ionizing radiation.

Results

Generation of transgenic Chlamydomonas reinhardtii strains overexpressing plant antimicrobial peptides

Pathogenesis-related (PR) and thionin (THI) peptides play crucial roles in plant defense mechanisms against certain microbial pathogens, including bacteria and fungi [39, 40]. PR1 is a well-known marker of the hypersensitive response (HR) or systemic acquired resistance (SAR), a defense mechanism in plants that provides broad resistance to a variety of pathogens [41, 42]. THI2.1, however, is an antimicrobial protein belonging to the small cationic peptide family that is induced through a signal transduction pathway different from that of PR1 [43,44,45]. To express exogenous antimicrobial peptides in C. reinhardtii, we selected the A. thaliana PR1 and THI2.1 genes, which encode PR1 and THI2.1 peptides, respectively, as transgenes. Chlamydomonas is a promising platform for expressing recombinant proteins, and efficient expression vectors with appropriate promoters, introns, and terminators have been developed for it; however, low transgene expression or silencing remains a challenge [30, 31, 33, 46]. To overcome this problem, we generated the novel expression vector construct, CrRPA70Ap-HSP70Ap-RBCS2p-AtPR1 (or AtTHI2.1)-pOpt_mVenus, which was designed to enhance transgene expression in C. reinhardtii upon exposure to ionizing radiation (Fig. 1). CrRPA70Ap is a putative promoter of CrRPA70A, a DNA damage response (DDR) gene that is strongly induced by ionizing radiation [12, 47]. The strong radiation-inducible promoter, CrRPA70Ap, was added to the original pOpt_mVenus vector [46], and the exogenous antimicrobial genes, AtPR1 or AtTHI2.1, then introduced into the modified vector. The final vector construct was transformed into wild-type C. reinhardtii CC-125 cells using a square-wave electroporation system, and a hygromycin-resistant transformant selected as the positive strain.

Fig. 1
figure 1

Expression construct of CrRPA70Ap-HSP70Ap-RBCS2p-AtPR1 (or AtTHI2.1)-pOpt_mVenus used for overexpressing the AtPR1 (or AtTHI2.1) gene in Chlamydomonas reinhardtii

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Radiation-induced expression of transgenes and DNA damage response genes after X-irradiation

Exposure to ionizing radiation induces transcriptional upregulation of genes involved in the DDR pathway in C. reinhardtii [47]. To evaluate the effect of the radiation-inducible promoter (CrRPA70Ap) on transgene expression, four transgenic strains (AtPR1-OE1/2 and AtTHI-OE1/2) carrying AtPR1 or AtTHI2.1 as a transgene were irradiated with 6–50 Gy X-rays, and the transcript levels of AtPR1 and AtTHI2.1 measured via reverse transcription-quantitative PCR (RT-qPCR) analysis. All transgenic strains showed no significant change in transgene expression after X-irradiation at 6 Gy, but after X-irradiation at 12–50 Gy, transgene expression significantly increased in a dose-dependent manner (Fig. 2). The activity of stress-inducible promoters can vary over time after stress treatment, as shown by the overexpression of the Oryza sativa CCCH-Tandem Zinc Finger Protein 5 gene using the stress-responsive OsNAC6 promoter [48]. Therefore, to determine the optimal time for maximum transgene expression, we examined the CrRPA70Ap-mediated expression of AtPR1 or AtTHI2.1 in the transgenic strains at various time points after X-irradiation. The transcript levels of both genes peaked 2 h after X-irradiation and then gradually decreased over time (Additional file 1: Fig. S1). Although X-irradiation at 6 Gy strongly induces the expression of various DDR genes in wild-type C. reinhardtii [12], it failed to activate the radiation-inducible promoter (CrRPA70Ap) for transgene expression in the transgenic strains tested in the current study (Fig. 2). To explain the low activity of CrRPA70Ap observed after X-irradiation at 6 Gy, we examined and compared the transcript levels of the two DDR genes, CrRPA70A and CrRAD51A, in wild-type and transgenic strains after X-irradiation at 6–50 Gy. The transcript levels of both DDR genes increased in a dose-dependent manner and were relatively lower in the transgenic strains than in the wild type (Fig. 3). Even X-irradiation at 6 Gy significantly increased the expression of both DDR genes in the wild-type but not in the transgenic strains. As the radiation sensitivity of C. reinhardtii cells has been suggested to be genotype-dependent [12], this may be strongly associated with the higher radiation dose required to activate the radiation-inducible promoter in the transgenic strains. These results demonstrate that X-irradiation can be used to induce CrRPA70Ap-mediated transgene expression, and that the specific dose range for this should be determined by the radiation sensitivity of transgenic strains.

Fig. 2
figure 2

Transgene expression in transgenic Chlamydomonas reinhardtii strains after different doses of X-irradiation. The AtPR1-OE1/2 or AtTHI-OE1/2 strains, respectively, harbor an CrRPA70Ap-HSP70Ap-RBCS2p-AtPR1-pOpt_mVenus or CrRPA70Ap-HSP70Ap-RBCS2p-AtTHI2.1-pOpt_mVenus expression construct. Mid-exponential phase cells were subjected to different doses of X-irradiation. XR6, XR12, XR25, and XR50 represent X-rays of 6, 12, 25, and 50 Gy, respectively. The cells were harvested 2 h after X-irradiation. All transcript levels were measured using reverse transcription-quantitative PCR and are shown relative to the mock using CrTUBA1 as an endogenous reference gene. Data represent the mean ± standard error (n = 3). Different letters indicate significant differences at p < 0.05 (one-way analysis of variance, followed by Tukey’s honestly significant difference test)

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Fig. 3
figure 3

Expression of the DNA damage response genes, CrRPA70A and CrRAD51A, in transgenic Chlamydomonas reinhardtii strains after different doses of X-irradiation. WT, wild type; AtTHI-OE1/2 or AtPR1-OE1/2, transgenic strains overexpressing AtTHI2.1 or AtPR1 genes, respectively. Mid-exponential phase cells were subjected to different doses of X-irradiation. XR6, XR12, XR25, and XR50 represent X-rays of 6, 12, 25, and 50 Gy, respectively. The cells were harvested 2 h after X-irradiation. All transcript levels were measured via reverse transcription-quantitative PCR and are shown relative to the mock using CrTUBA1 as an endogenous reference gene. Data represent the mean ± standard error (n = 6) from two independent experiments. Different letters indicate significant differences at p < 0.05 (one-way analysis of variance, followed by Tukey’s honestly significant difference test)

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X-irradiation enhances the expression of antimicrobial peptides in transgenic Chlamydomonas reinhardtii cells

The increased transcript levels of antimicrobial transgenes after X-irradiation, as shown in Fig. 2, were further supported by examining the protein levels of AtPR1 and AtTHI2.1 via immunoblotting analysis following X-irradiation. Immunoblots of AtPR1 and AtTHI2.1 proteins were obtained through SDS-PAGE, followed by detection of the 26.9-kD tagged protein, mVenus, using an anti-green fluorescent protein (GFP) antibody. Both proteins were expressed in each transgenic strain, but further increased in a dose-dependent manner after X-irradiation (Fig. 4). These results indicate that the AtPR1 and AtTHI2.1 transgenes were successfully expressed in the transgenic strains, and that X-irradiation enhanced the expression of these genes at both the transcript and protein levels. The increased accumulation of AtPR1 and AtTHI2.1 proteins in the transgenic strains was further confirmed in living cells by measuring the fluorescence signal of mVenus fused to AtPR1 and AtTHI2.1 proteins over time after X-irradiation. In the transgenic strains, the accumulation of AtPR1 and AtTHI2.1 proteins increased up to 2 h after X-irradiation at 12 Gy and then decreased, which was consistent with the transcriptional changes observed (Fig. 5A, Additional file 1: Fig. S1). Visualization of the accumulation of AtPR1 and AtTHI2.1 proteins in living cells using confocal fluorescence imaging revealed that these proteins accumulated as spots in the cytoplasm of AtPR1-OE1/2 and AtTHI-OE1/2 cells and significantly increased 2 h after X-irradiation (Fig. 5B, Additional file 1: Fig. S2). The accumulation of AtPR1 and AtTHI2.1 proteins in the cytoplasm seems to be partially supported by previous reports, in which transgenic C. reinhardtii cells containing the pOpt_mVenus vector showed strong fluorescence of mVenus proteins in the cytoplasm [49, 50]. These results demonstrate that the radiation-inducible CrRPA70Ap promoter is most active 2 h after X-irradiation and can successfully drive the production of recombinant antimicrobial proteins in transgenic C. reinhardtii strains.

Fig. 4
figure 4

Expression of AtPR1 or AtTHI2.1 proteins in transgenic Chlamydomonas reinhardtii strains after X-irradiation. WT, wild type; EV, a transgenic line with an empty vector and no transgene; AtPR1-OE1/2 or AtTHI-OE1/2, transgenic strains overexpressing AtPR1 or AtTHI2.1 genes, respectively. Mid-exponential phase cells were subjected to X-irradiation at a dose of 12 or 50 Gy. The cells were harvested 2 h after X-irradiation. Immunoblotting analysis of AtPR1 proteins in AtPR1-OE1/2 strains (a) or AtTHI in AtTHI-OE1/2 strains (b) was performed by detecting AtPR1- or AtTHI2.1-fused mVenus tagging proteins with the rabbit anti-green fluorescent protein (GFP) antibody. The mVenus is a genetically mutated protein with an F46L substitution in GFP derived from Aequorea victoria. α-Tubulin was used as a loading control

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Fig. 5
figure 5

In vivo and time-course expression of AtPR1 or AtTHI2.1 proteins in transgenic Chlamydomonas reinhardtii strains after X-irradiation. Mid-exponential phase cells were subjected to X-irradiation at a dose of 12 Gy. The cells of Mock, 30 M, 1H, 2H, 4H, and 8H were harvested before, as well as 30 min and 1, 2, 4, and 8 h after, X-irradiation, respectively. a Time-course expression levels of AtPR1 or AtTHI2.1 proteins in AtPR1-OE1/2 or AtTHI-OE1/2 strains after X-irradiation. The fluorescent signal values of the fluorescent reporter, mVenus, were obtained from cells using a microplate reader with 515 nm excitation and 527 nm emission wavelengths. The values were normalized against the cell density (OD750) to calculate the expression levels of AtPR1 or AtTHI2.1 proteins in the same cell number and then expressed relative to the mock. Data represent the mean ± standard error (n = 9) from two independent experiments. Invisible error bars are smaller than the symbol size. Different letters indicate significant differences at p < 0.05 (one-way analysis of variance, followed by Tukey’s honestly significant difference test). b Confocal micrographs showing the in vivo expression of AtPR1 or AtTHI2.1 proteins in AtPR1-OE1/2 or AtTHI-OE1/2 strains, respectively. Chlorophyll a (Chl a) or mVenus images were captured using excitation/emission wavelengths of 488/650 nm or 515/528 nm, respectively. White scale bars represent 5 μm

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X-irradiation enhances the antimicrobial activity of total protein in transgenic Chlamydomonas reinhardtii cells

PR1 is not only involved in plant defense mechanisms, such as HR or SAR, but also exhibits antimicrobial activity against sterol auxotrophic pathogens by sequestering sterols from the cell membrane [41, 42]. THI2.1 is only present in plants and exhibits potent antimicrobial activity against bacteria and fungi by disrupting microbial cell membranes, inducing the leakage of cellular contents, and ultimately inducing cell death [39, 43]. Thus, AtPR1 and AtTHI2.1 proteins can be used not only as part of a host defense mechanism, but also to directly inhibit microbial pathogens. To evaluate the antimicrobial activity of total protein in the four transgenic strains overexpressing AtPR1 or AtTHI2.1, we performed a spotting assay using the prokaryotic bacterium, Pseudomonas syringae pv. tomato (Pst). Surprisingly, the total proteins from all transgenic strains inhibited the growth of Pst cells at protein concentrations ranging from 30 to 50 µg mL−1, and this effect was more pronounced from 10 to 50 µg mL−1 when transgene expression was enhanced by irradiating the transgenic cells with 12 Gy X-rays (Fig. 6). The minimum inhibitory concentration (MIC), typically defined as the concentration required to inhibit at least 90% of microbial growth, against Pst, was estimated to be 70 µg mL−1 for total proteins from mock cells of the transgenic strains and 40 µg mL−1 for total proteins from X-irradiated cells. In addition, the antifungal activity of total protein from the transgenic strains was measured using the eukaryotic fungus, Cryptococcus neoformans (Cn). While the total protein from the transgenic AtPR1-OE1/2 strains did not show any antifungal activity against Cn cells, the protein from the transgenic AtTHI-OE1/2 inhibited Cn cell growth at protein concentrations ranging from 20 to 50 µg mL−1, and this effect was more pronounced from 10 to 50 µg mL−1 when transgene expression was enhanced by irradiating the transgenic cells with 12 Gy X-rays (Fig. 7). The MIC of total protein extracts from mock AtTHI-OE1/2 cells against Cn was 50 μg mL−1, while for X-irradiated AtTHI-OE1/2 cells, it was reduced to 30 μg mL−1. These results demonstrate that overexpression of AtPR1 or AtTHI2.1 proteins can significantly enhance the antibacterial and antifungal activity of the total protein extracts of C. reinhardtii cells against Pst and Cn, respectively. In addition, X-irradiation is thought to increase the proportion of AtPR1 or AtTHI2.1 proteins in the total protein extracts of transgenic cells, thereby enhancing their antimicrobial activity against Pst and Cn.

Fig. 6
figure 6

Antibacterial activity of algal proteins from wild-type and transgenic Chlamydomonas reinhardtii strains after X-irradiation. Cr, C. reinhardtii; Pst, Pseudomonas syringae pv. tomato; WT, wild type; EV, a transgenic line with an empty vector and no transgene; AtPR1-OE1/2 or AtTHI-OE1/2, transgenic strains overexpressing AtPR1 or AtTHI2.1 proteins, respectively. Mid-exponential phase cells were subjected to X-irradiation at a dose of 12 Gy. Total protein was extracted from C. reinhardtii cells 2 h after X-irradiation, mixed with Pst cells at 0.1 OD600, and then spotted onto a King’s B agar plate. Left and right images represent the antibacterial activity of total protein from the mock and X-irradiated C. reinhardtii strains, respectively

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Fig. 7
figure 7

Antifungal activity of algal proteins from wild-type and transgenic Chlamydomonas reinhardtii strains after X-irradiation. Cr, C. reinhardtii; Cn, Cryptococcus neoformans; WT, wild type; EV, a transgenic line with an empty vector and no transgene; AtPR1-OE1/2 or AtTHI-OE1/2, transgenic strains overexpressing AtPR1 or AtTHI2.1 proteins, respectively. Mid-exponential phase cells were subjected to X-irradiation at a dose of 12 Gy. Total protein was extracted from C. reinhardtii cells 2 h after X-irradiation, mixed with Cn cells at 0.8 OD600, and then spotted onto a YPD agar plate. Left and right images represent the antifungal activity of total protein from the mock and X-irradiated C. reinhardtii strains, respectively

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X-irradiation increases the biomass of wild-type and transgenic Chlamydomonas reinhardtii cells

Microalgae have several advantages in recombinant protein production, including a simple transformation system, low production cost, and the ability to secrete proteins [15, 16]. These advantages may be further improved using strong inducers that enhance transgene expression in microalgal cells or by increasing their biomass production. Ionizing radiation, such as X- and γ-rays, has been reported to enhance growth and photosynthesis, as well as chlorophyll, protein, starch, and lipid synthesis, in C. reinhardtii cells within a certain dose range of 3–12 Gy [12]. Therefore, the effect of X-irradiation on the transgenic cells was investigated in terms of fresh and dry weights of the same culture and compared to those of the wild type. As the wild type has been shown to be more sensitive to X-irradiation in terms of growth when compared to the transgenic strains [12], we applied the dose of 6 or 12 Gy to the wild-type and transgenic cells, respectively, for maximum growth stimulation. Not only did X-irradiation at 12 Gy induce transgene expression in the transgenic strains, it also significantly increased biomass production by 13–17% in fresh weight and 27–29% in dry weight (Table 1). Furthermore, these values for the transgenic strains were significantly higher than the 11% and 15% measured for the wild type. These results demonstrate that the biomass production of transgenic C. reinhardtii cells overexpressing AtPR1 or AtTHI2.1 proteins through the radiation-inducible promoter, CrRPA70Ap, can be further increased using X-rays as inducers. Within a certain dose range, X-irradiation can be efficiently applied to transgenic microalgal cells as a versatile inducer of both transgene expression and biomass production.

Table 1 Fresh and dry weights of wild-type and transgenic Chlamydomonas reinhardtii cells after X-irradiation
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γ-Irradiation increases the antibacterial activity of wild-type Chlamydomonas reinhardtii cell extracts

Natural bioactive substances have been extracted using a variety of organic solvents that are selected for their selectivity, solubility, cost, and safety [51]. In practice, methanol, ethanol, chloroform, hexane, and aqueous solvent systems have been used to prepare microalgal extracts that exhibit antimicrobial activity against a variety of bacteria, fungi, and viruses [52]. However, as the yield of microalgae-derived bioactive substances increases through different isolation or extraction methods developed for each compound [20], we tested whether the antimicrobial properties of C. reinhardtii cell extracts could be further enhanced through molecular modification of the bioactive substances via γ-irradiation at 1–20 kGy. The antibacterial activity of the extract against two phytopathogenic bacteria (Pst and Bg) was investigated using an agar well diffusion assay, which showed a significant dose-dependent increase after γ-irradiation at 1, 5, 10, and 20 kGy (Fig. 8, Additional file 1: Fig. S3). These results suggest that γ-irradiation could be a practical processing method for enhancing the antimicrobial activity and functionality of C. reinhardtii cell extracts.

Fig. 8
figure 8

Antibacterial activity of wild-type Chlamydomonas reinhardtii cell extracts against Pseudomonas syringae pv. tomato after γ-irradiation. NC, negative control (100% [v/v] dimethyl sulfoxide [DMSO]); PC, positive control (3 µg mL−1 gentamycin in DMSO); Mock or GR1/5/10/20, 100 mg mL−1 control or γ-irradiated cell extract in DMSO. GR1, GR5, GR10, and GR20 represents γ-irradiation at a dose rate of 1, 5, and 10 kGy h−1 for 1 h or 10 kGy h−1 for 2 h, respectively. Cell extracts were prepared via sonication and heating in 100% (v/v) ethanol and used for agar well diffusion assays. The size of the inhibition zone (cm) was calculated by subtracting the well diameter from the width of the clear inhibition area. Data represent the mean ± standard error (n = 6) from two independent experiments. Different letters indicate significant differences at p < 0.05 (one-way analysis of variance, followed by Tukey’s honestly significant difference test)

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Changes in fatty acid composition may contribute to enhancement of the antibacterial activity of Chlamydomonas reinhardtii cell extracts by γ-irradiation

The physicochemical and biological properties of natural bioactive substances after γ-irradiation are related to their molecular modifications [21,22,23,24]. As fatty acid or lipid extracts from microalgae have been shown to have antimicrobial activity against a variety of bacteria and fungi [52,53,54], we investigated the fatty acid composition of C. reinhardtii cell extracts after γ-irradiation using gas chromatography–mass spectrometry (GC–MS) analysis. Compared to the control, the irradiated extracts showed a markedly different fatty acid profile, with four additional peaks observed on the chromatogram obtained, indicating that the fatty acid composition was altered by γ-irradiation (Fig. 9). The additional peaks observed at 13, 14, 15, and 16 were identified as n-hexadecanoic acid (palmitic acid, 16:0), 9,12,15-octadecatrienoic acid (linolenic acid, 18:3), 9-octadecenoic acid (oleic acid, 18:1), and octadecanoic acid (stearic acid, 18:0), respectively (Table 2). In addition, the areas of peaks 2 (17:4), 5 (17:1), 14, 15, and 16 decreased or increased in a dose-dependent manner after γ-irradiation. These results suggest that the fatty acid composition of C. reinhardtii cell extracts is modified by γ-irradiation, which may enhance the antibacterial activity of the extracts. When further experimental explanations of the enhanced antibacterial activity of irradiated microalgal extracts are accumulated, microalgal cell extracts may become new promising antibacterial agents for industrial and biotherapeutic applications through the molecular modification of bioactive substances using ionizing radiation.

Fig. 9
figure 9

Chromatograms of the fatty acids from wild-type Chlamydomonas reinhardtii cell extracts after γ-irradiation obtained through gas chromatography (GC). Mock, control; GR1, GR5, GR10, and GR20 represent γ-irradiation at a dose rate of 1, 5, and 10 kGy h−1 for 1 or 10 kGy h−1 for 2 h, respectively. Cell extracts were prepared via sonication and heating in 100% (v/v) ethanol, methylated to produce fatty acid methyl esters, and used for GC–MS analysis. The numbered GC peaks were selected, quantified, identified using the mass spectral library of the National Institute of Standards and Technology, and then summarized in Table 2

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Table 2 Changes in the fatty acid composition of wild-type Chlamydomonas reinhardtii cell extracts after γ-irradiation
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Radiation-induced transgene expression and molecular modification contribute to the antibacterial activity of transgenic Chlamydomonas reinhardtii cell extracts

Total protein extracts from transgenic C. reinhardtii cells overexpressing AtPR1 and AtTH2.1 proteins showed potent antimicrobial activity against Pst and Cn (Figs. 6, 7). When considered together with the enhanced antibacterial activity of ethanol cell extracts observed after γ-irradiation (Fig. 8, Additional file 1: Fig. S3), it is worthwhile to compare the antibacterial activity of the ethanol cell extracts between wild-type and transgenic strains overexpressing AtPR1 and AtTH2.1 proteins. Expression of the exogenous antimicrobial peptides, AtPR1 or AtTHI2.1, was associated with an increased antibacterial activity against Pst and Burkholderia glumae (Bg) in cell extracts of the transgenic strains compared to that in the wild-type, and γ-irradiation significantly enhanced the antibacterial activity of wild-type and transgenic cell extracts (Fig. 10, Additional file 1: Fig. S4). Furthermore, transgene expression in the former and molecular modification in the latter had different effects on the antibacterial activity of the cell extracts depending on the pathogen (Pst or Bg). These results demonstrate that the combined effect of radiation-induced transgene expression and molecular modification can further enhance the antimicrobial activity of C. reinhardtii cell extracts, but this may be dependent on the pathogenic microbial strain. Both approaches could contribute substantially to the utilization of C. reinhardtii cell extracts as promising antimicrobial agents.

Fig. 10
figure 10

Antibacterial activity of wild-type and transgenic Chlamydomonas reinhardtii cell extracts against Pseudomonas syringae pv. tomato after γ-irradiation. NC, negative control (100% [v/v] dimethyl sulfoxide [DMSO]); PC, positive control (3 µg mL−1 gentamycin in DMSO); WT, wild-type; AtPR1-OE1/2 or AtTHI-OE1/2, transgenic strains overexpressing AtPR1 or AtTHI proteins; M or G, 100 mg mL−1 mock or γ-irradiated cell extract in DMSO. Cells were harvested at a pre-stationary phase (0.8 OD750) and irradiated with X-rays at 12 Gy to induce transgene expression. To protect the antimicrobial activity of AtPR1 or AtTHI2.1 proteins, cell extracts were prepared via sonication in 100% (v/v) ethanol without subsequent heating and used for agar well diffusion assays. The γ-irradiation of cell extracts was performed at a dose rate of 10 kGy h−1 for 2 h. The size of the inhibition zone (cm) was calculated by subtracting the well diameter from the width of the clear inhibition area. Data represent the mean ± standard error (n = 6) from two independent experiments. Different letters indicate significant differences at p < 0.05 (one-way analysis of variance, followed by Tukey’s honestly significant difference test)

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Discussion

X-irradiation enhances antimicrobial activity in transgenic Chlamydomonas reinhardtii strains by inducing efficient expression of exogenous antimicrobial proteins

Stress-inducible promoters have been used to overcome the low activity of constitutive promoters or to regulate transgene expression [55, 56]. In this study, the promoters of DDR genes, such as CrRPA70A, were found to be strongly induced in the short term after X-irradiation, leading to high expression of the antimicrobial transgenes, AtPR1 or AtTHI2.1 (Figs. 2, 3, Additional file 1: Fig. S1). Ionizing radiation induces the expression of DDR genes and causes chromatin remodeling and epigenetic changes, including histone modifications and DNA hypomethylation [57,58,59,60]. As epigenetic regulation that induces a permissive chromatin structure is associated with transgene expression [29, 30], radiation-induced chromatin remodeling and epigenetic changes may contribute to the alleviation of low transgene expression or silencing. Furthermore, the increased biomass of C. reinhardtii cells after X-irradiation at 6 or 12 Gy (Table 1) may be related to enhanced growth and metabolic activity due to stress priming through epigenetic regulation [12]. Indeed, unpublished data have shown that the growth-stimulating effect of ionizing radiation is mediated by reactive oxygen species (ROS) and is associated with the epigenetic regulation of growth-related genes. Within a certain dose range, X- or γ-irradiation may facilitate effective transgene expression by enhancing metabolic activity, such as protein and lipid synthesis, through ROS-mediated stress priming, but the higher biomass production observed in this study for transgenic strains compared to that of the wild-type requires an alternative explanation.

Recently, deep learning has been used to predict the structures and properties of peptides, thereby facilitating the design and utilization of antimicrobial peptides [61,62,63]. However, when producing exogenous recombinant peptides through transgene expression, it is necessary to validate their practical efficiency not only in terms of transcription, but also in terms of translation and post-translational modifications. In this study, high expression of the plant-derived antimicrobial genes, AtPR1 or AtTHI2.1, in the transgenic C. reinhardtii strains after X-irradiation was further confirmed at the protein and activity levels (Figs. 4, 5, 6, 7). PR1 and THI proteins are secreted into the cell wall via intracellular sorting or trafficking and enter the membrane of phytopathogens to form pores [41, 64, 65]. The accumulation of AtPR1-mVenus and AtTHI2.1-mVenus protein spots in the cytoplasm of transgenic C. reinhardtii cells may be due to this property of the PR1 and THI proteins rather than that of mVenus (Fig. 5B, Additional file 1: Fig. S2). The AtPR1 and AtTHI2.1 recombinant proteins produced in C. reinhardtii cells exhibited the expected antimicrobial activity, despite not being purified and having an additional mVenus tag. The fact that plant-derived antimicrobial peptides can be efficiently produced from microalgae using ionizing radiation, and that even crude extracts of total protein can exhibit potent antibacterial and antifungal activities, is of great significance for industrial applications.

γ-Irradiation enhances the antibacterial activity of Chlamydomonas reinhardtii cell extracts through molecular modification of bioactive substances

Although the antimicrobial activity of microalgal extracts has been recognized [8, 9], the composition and activity of bioactive substances they harbor can vary significantly depending on the extraction method used [19, 20]. In this study, we demonstrated the potent antimicrobial activity of C. reinhardtii cell extracts prepared through sonication and heat treatment in ethanol and then modified via γ-irradiation (Figs. 8, 10, Additional file 1: Figs. S3 and S4). The C. reinhardtii cell extract in ethanol showed a significantly enhanced antibacterial activity against phytopathogenic bacteria (Pst and Bg) after γ-irradiation. Ionizing radiation has been used to increase the content and functionality of natural bioactive substances and to improve their extraction efficiency [22, 24, 66, 67]. High-dose γ-rays of tens of kGy are known to modify the molecular structure of natural bioactive substances, enhancing their functionality or conferring new features [21, 23, 68]. Therefore, the dose-dependent increase in antibacterial activity observed suggests that γ-irradiation enhanced the antimicrobial activity of C. reinhardtii cell extracts through molecular modification of the bioactive substances contained within them.

A variety of bioactive substances, including fatty acids and polysaccharides, have been reported to be involved in the antimicrobial activity of microalgal extracts [8, 9], but it is generally difficult to identify the specific components responsible for bioactivity in crude extracts. Free fatty acids with more than 10 unsaturated and saturated carbons have shown antibacterial activity by disrupting bacterial cell membranes [69,70,71]. In this study, the antimicrobial activity of C. reinhardtii cell extracts was increased after γ-irradiation and correlated with changes in fatty acid composition (Fig. 9, Table 2). Levels of the fatty acids with 18 carbons, linolenic acid (18:3), oleic acid (18:1), and stearic acid (18:0), increased in a dose-dependent manner, whereas that of the polyunsaturated fatty acid, methyl 4,7,10,13-hexadecatetraenoate (17:4), decreased significantly. Ionizing radiation induces changes in the fatty acid composition of natural fats and increases lipid peroxide formation [72]. Polyunsaturated fatty acids are especially highly susceptible to radiation-induced lipid peroxidation [73] and can be destroyed by γ-irradiation at doses of 2–10 kGy [72]. Thus, polyunsaturated fatty acids in the triacylglycerols and phospholipids of C. reinhardtii cell extracts may have been converted into free fatty acids with higher antimicrobial activity through γ-irradiation-mediated molecular modification. Changes in fatty acid composition may be related to the increased antimicrobial activity of C. reinhardtii cell extracts observed after γ-irradiation, but further investigation of polysaccharides and other compounds is still needed.

Ionizing radiation is a powerful metabolic remodeler and molecular modifier that produces high-value bioactive substances

Ionizing radiation within a certain dose range increases the growth and metabolic activity of C. reinhardtii [12]. In this study, transgenic C. reinhardtii strains harboring CrRPA70Ap and AtPR1 or AtTHI2.1 not only showed increased transgene expression at the transcript and protein levels after X-irradiation at 12 Gy, but also increased biomass in terms of fresh and dry weights (Figs. 2, 4, and 5, Table 1). Thus, ionizing radiation is a powerful metabolic remodeler that can induce synergistic effects on antimicrobial peptide production in C. reinhardtii cells by increasing transgene expression and biomass. The optimal dose range for these synergistic effects can be estimated from the expression levels of DDR genes, which represent the radiation sensitivity of transgenic C. reinhardtii strains (Fig. 3). However, radiation-inducible promoter-mediated transgene expression reached a maximum within 2 h after X-irradiation (Fig. 5, Additional file 1: Fig. S1). Therefore, two independent X-irradiations with different doses may be required to efficiently increase both biomass and transgene expression in transgenic C. reinhardtii strains.

In this study, ionizing radiation was used not only as a metabolic remodeler of C. reinhardtii cells, but also as a molecular modifier to increase the antimicrobial activity of natural bioactive substances in C. reinhardtii cell extracts. After X- and γ-irradiation was used to, respectively, induce antimicrobial protein expression and cell extract modification, a synergistic effect of antimicrobial activity was observed in the transgenic cell extracts, but this was somewhat limited and variable depending on the pathogen tested (Fig. 10, Additional file 1: Fig. S3). This may be due to differences in the stability to heat treatment and γ-irradiation and effective treatment methods between antimicrobial peptides and fatty acids, which are potent bioactive substances. The potential synergistic effects of two or more bioactive substances seem to be limited or variable in crude cell extracts. Nevertheless, this study is the first to propose and practically validate multiple strategies to enhance the antimicrobial activity of microalgal extracts using ionizing radiation as a metabolic remodeler and molecular modifier, which has been used for mutation or material modifications. However, several limitations must be addressed before industrial application of the strategies. First, the epigenetic mechanisms behind ionizing radiation-mediated enhancement of transgene expression need elucidation. Second, specific antimicrobial bioactive substances produced in C. reinhardtii cell extracts through radiation-induced molecular modifications require further exploration. Finally, the strategies should be validated in other microalgal species and scaled up for industrial use.

Conclusions

In this study, we demonstrated that ionizing radiation, such as X- and γ-rays, can be used to increase the expression of transgenes in transgenic C. reinhardtii strains or to enhance the functionality of C. reinhardtii cell extracts. Therefore, the multistep application of ionizing radiation is expected to significantly improve microalgal productivity by increasing (1) cell growth and metabolism, (2) transgene expression, and (3) cell extract functionality. However, further research on other microalgae is needed to determine if ionizing radiation is a versatile tool that can increase the value of microalgae as a platform for the production of bioactive substances.

Methods

Microalgal strains and cultural conditions

The unicellular green alga, C. reinhardtii strain CC-125 (1.5 × 106 cells), was cultured in 50-mL liquid tris–acetate-phosphate (TAP) medium in 250-mL flasks [74] with shaking at 25 °C and 140 rpm under constant white light of 90–100 µmol photons m−2 s−1. In addition, four transgenic C. reinhardtii strains (AtPR1-OE1, AtPR1-OE2, AtTHI-OE1, and AtTHI-OE2) harboring AtPR1 or AtTHI2.1 genes, were cultivated under the same conditions to compare antimicrobial activity.

For these transgenic strains, the overexpression vector was modified to include an additional radiation-inducible promoter (CrRPA70Ap) from the original pOpt_mVenus vector, which contained the two promoters, HSP70Ap and RBCS2p [46]. Therefore, CrPRA70Ap and AtPR1 (or AtTHI2.1) sequences were cloned into the pOpt_mVenus vector at specific restriction sites after amplification of their cDNAs with gene-specific primers (Additional file 1: Table S1), constituting the final vector construct of CrRPA70Ap-HSP70Ap-RBCS2p-AtPR1 (or AtTHI2.1)-pOpt_mVenus, along with the hygromycin resistance gene cassette for selection (Fig. 1). The construct was transformed into C. reinhardtii cells using a versatile electroporation system (Gene Pulser II; Bio-Rad, Hercules, CA, USA), as previously described [75]. When the electroporation parameters were set to 500 V, 50 µF, and 800 Ω, the pulse duration was approximately 30 ms. After transformation, the cells were transferred into 10-mL TAP medium containing 40 mM sucrose and cultured in the dark without shaking for 14–16 h. Finally, the transgenic strains were selected by incubating the cells on solid TAP medium containing 12 µg mL−1 hygromycin under 5 µmol photons m−2 s−1 for 3–4 weeks.

X- or γ-irradiation

Mid-exponential phase C. reinhardtii cells with an approximate optical density of 0.6 measured at 750 nm (0.6 OD750) were harvested and prepared for X-irradiation, as previously described [12]. The cells were subjected to X-irradiation of 6 or 12 Gy for 22.2 or 44.4 min at 160 kV and 1 mA, or 25 or 50 Gy for 4.05 or 8.1 min at 160 kV and 10 mA, using a cabinet type X-ray machine (CP-160; Faxitron X-ray LLC, Lincolnshire, IL, USA). In addition, pre-stationary phase cells of 0.8 OD750 were harvested and cell extracts in ethanol prepared for γ-irradiation, as described in the antimicrobial activity assay of microalgal extracts below. The cell extracts were exposed to γ-rays at a dose rate of 1, 5, and 10 kGy h−1 for 1 h or 10 kGy h−1 for 2 h, which were generated from a 3 kCi 60Co source at the Advanced Radiation Technology Institute (Jeonbuk-do, Korea). The absorbed radiation dose for each sample was determined using a 5-mm diameter alanine dosimeter (Bruker Instruments, Rheinstetten, Germany), as described previously [76].

Gene expression analysis via reverse transcription-quantitative PCR

Gene expression analysis was performed using RT-qPCR, as previously described [12]. Cells (1 × 108) were harvested and resuspended in 1-mL TRIzol reagent (Invitrogen, Carlsbad, CA, USA), vortexed for 10 min, incubated for 5 min at 25 °C, and centrifuged for 10 min at 4 °C and 12,000g. The supernatant was mixed with 250 µL chloroform (Sigma-Aldrich, St. Louis, MO, USA) via vortexing for 2 min, combined with an equal volume of phenol–chloroform (1:1 v/v; Sigma-Aldrich) after centrifugation, mixed via vortexing for 2 min, and then centrifuged again. It was finally mixed with an equal volume of isopropanol, incubated for 45 min at 4 °C, and centrifuged for 20 min at 4 °C and 12,000g. The resultant RNA pellet was washed twice with 800 µL of 75% (v/v) ethanol, dissolved in DEPC-treated water, and used for RT-qPCR. The cDNA was synthesized from 1 μg of each RNA sample using oligo(dT) primers and a LaboPass cDNA Synthesis Kit (Cosmo Genetech, Seoul, Korea). Subsequent qPCR amplification cycle conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 57 °C for 10 s, and 72 °C for 1 min using the CFX Connect Real-Time PCR Detection System (Bio-Rad) with the iTaq Universal SYBR Green Supermix (Bio-Rad) and gene-specific primers (Table S1). The relative transcript levels of each gene were calculated between the mock and irradiated samples using the comparative CT method [77]. The expression data of the three biological replicates were normalized to that of the endogenous reference gene, CrTUBA1.

Protein expression analysis by immunoblot

Transgene expression in the transgenic strains was assessed at the protein level via immunoblotting analysis using total protein. For total protein extraction, cells (1 × 108) were harvested, resuspended, and shaken in 300 µL lysis buffer containing 60 mM DTT, 60 mM Na2CO3, 2% (w/v) sodium dodecyl sulfate (SDS), and 12% (w/v) sucrose for 20 min at 25 °C, as previously described [12]. The cell lysate was centrifuged at 4 °C and 10,000g for 1 min, and the supernatant mixed with five volumes of 100% (v/v) ice-cold acetone. The mixture was kept at − 20 °C for 2 h to facilitate protein precipitation and thereafter centrifuged at 4 °C and 5000g for 15 min. The resulting protein pellets were dissolved in distilled water and used for immunoblotting analysis after determining the protein concentration using the Bradford method [78]. Approximately 30-µg total protein was separated via 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The PVDF membrane was first incubated with rabbit anti-GFP antibody (1:5,000; Invitrogen) after blocking with 5% nonfat dry milk in 10 mM Tris, 166 mM NaCl, and 0.05% Tween 20 (pH 7.4). The membrane was then incubated with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000; Cell Signalling Technology, Danvers, MA, USA). Finally, immunoblots on the membrane were visualized using Pierce ECL Western Blotting Substrate reagents (Thermo Fisher Scientific, Waltham, MA, USA), and images captured using an iBright FL1000 imaging system (Thermo Fisher Scientific).

In vivo protein expression analysis via fluorimetry and confocal microscopy

In vivo protein expression analysis was performed using the transgenic C. reinhardtii cells exposed to X-rays of 12 Gy at a mid-exponential phase. 100-µL aliquots of cells were transferred into wells of a black 96-well plate (Corning Costar, Tewksbury, MA, USA). Fluorescence signals were read from the cells at 30 min, 1, 2, 4, and 8 h after X-irradiation using a microplate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland), with 515 nm excitation and 528 nm emission wavelengths. The signals were normalized to the cell density (OD750) and expressed relative to the mock sample. In addition, cells were allowed to settle in wells of a black 96-well plate for 5 min, and each 8-µL supernatant spread onto a glass slide for fluorescence microscopy. Fluorescent images were taken of the cells 2 h after X-irradiation using a confocal laser-scanning microscope (LSM800; Carl Zeiss, Oberkochen, Germany) equipped with excitation/emission wavelengths of 515/528 nm for mVenus or 488/650 nm for chlorophyll a.

Antimicrobial activity analysis of microalgal proteins via a spotting assay

The antimicrobial activity of microalgal proteins was visualized using a spotting assay with cells of the prokaryotic bacterium, Pst, or the eukaryotic fungus, Cn. Cells of Pst or Cn were incubated overnight in King’s B or YPD liquid medium at 28 °C or 30 °C, respectively, and subcultured in fresh liquid medium. Microalgal total proteins were prepared from wild-type and transgenic C. reinhardtii cells, as described in the protein expression analysis above. Total proteins were extracted from C. reinhardtii cells 2 h after 12 Gy X-irradiation, mixed with Pst cells at 0.1 OD600 or Cn cells at 0.8 OD600 at a volume ratio of 1:9, and 5 or 4 µL of the resulting mixtures, containing total protein concentrations of 10 − 50 μg mL−1, were then spotted onto King’s B or YPD agar plates, respectively. The antibacterial or antifungal activity of microalgal proteins from wild-type and transgenic C. reinhardtii cells was estimated as the minimum concentration of total protein that inhibited the growth of Pst or Cn cells.

Analysis of antimicrobial activity of microalgal extracts via an agar well diffusion assay

To prepare microalgal extracts, pre-stationary phase C. reinhardtii cells with 0.8 OD750 were harvested at 3000g for 15 min. The cell pellet was rinsed thrice with distilled water and thereafter resuspended and vortexed in 100% (v/v) ethanol (Sigma-Aldrich) at a ratio of 1 g:50 mL (w/v). The pellet suspension was sonicated in an ultrasonic bath cleaner (KSDD-0400; Korea Sonic, Hwasung, Korea) at an 80% frequency for 30 min to facilitate the release of cellular compounds, heated at 78 °C (the boiling temperature of ethanol) for 2 h using a Duran glass Jacketed Coil Condenser (Daihan Scientific, Wonju, Korea), and then centrifuged at 4000g for 10 min. The supernatant containing the ethanol extract of microalgal cells was irradiated with γ-rays of 1, 5, 10, and 20 kGy, as described above. This solution was transferred to a round-bottom flask and dried using a rotary evaporator (Rotavapor RII; Büchi Labortechnik, Flawil, Switzerland). Dried microalgal extracts were dissolved in 100% (v/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich) at a concentration of 100 mg mL−1 and sterilized through a 0.22 µm syringe filter (Millipore).

The antimicrobial activity of the agal extracts was determined by measuring the zone of inhibition against the prokaryotic phytopathogenic bacteria, Pst and Bg, using an agar well diffusion assay, as previously described [79], with slight modifications. Cells of Pst or Bg were incubated in King’s B or LB liquid medium at 28 °C or 37 °C, respectively, and sub-cultured in fresh liquid medium. The Pst or Bg suspension (500 µL of 0.1 OD600) was spread out onto King’s B or LB agar plates with a dimension of 100 × 15 mm, respectively, and 100 µL of the microalgal extract in DMSO added into pre-punched wells (8-mm diameter) of the plates. The plates containing microalgal extracts and Pst or Bg cells were incubated at 28 °C for 48 h or 37 °C for 24 h, respectively, to observe the inhibition of cell growth. A 100% (v/v) DMSO solution was used as a negative control, whereas 3 or 50 µg mL−1 gentamycin in DMSO served as a positive control to inhibit Pst or Bg, respectively. The size of the inhibition zone (cm) was calculated by subtracting the well diameter from the width of the clear inhibition area in the plate images using Microsoft PowerPoint.

Analysis of fatty acids via gas chromatography and mass spectrometry

Dried microalgal extracts were prepared for fatty acid analysis, as described in the analysis of antimicrobial activity of microalgal extracts above. Total lipid extraction was performed using 20 mg dried microalgal extracts, as described previously [80]. The microalgal extracts were suspended in 2-mL saponification reagent (7.5 M NaOH:CH3OH, 1:1 v/v), shaken manually for 30 s, and incubated at 100 °C for 30 min. To produce fatty acid methyl esters (FAMEs), the mixture was cooled, 4-mL methylation reagent (6 N HCl:CH3OH, 1:1 v/v) added, and thereafter incubated at 80 °C for 10 min. To extract FAMEs, 2.5-mL extraction solvent (hexane:methyl tert-butyl ether, 1:1 v/v) was added, and the mixture incubated with shaking for 10 min, followed by centrifugation at 1500g for 10 min. The upper hexane phase containing FAMEs was collected and washed with 6-mL washing solution (0.5 M NaOH).

The FAMEs were validated and quantified using a GC–MS system (7890A-5957C; Agilent Technologies, Santa Clara, CA, USA) equipped with a capillary GC column (HP-5MS Ultra Inert; Agilent Technologies), as described previously, with some modifications [81]. For GC analysis of the FAMEs, the oven temperature was initially maintained at 180 °C for 5 min, raised to 220 °C at a rate of 2 °C min−1, held for 5 min, raised to 300 °C at a rate of 80 °C min−1, and then held for 5 min. The helium carrier gas was maintained at a flow rate of 1 mL min−1. Mass spectrometry was carried out with a transfer line temperature of 280 °C, source temperature of 230 °C, quadrupole temperature of 150 °C, ionization potential of 70 eV, and mass range of m/z = 50–350 amu. Peaks in the GC chromatograms were identified using the mass spectral library of the National Institute of Standards and Technology (Gaithersburg, MD, USA).

Statistical analysis

The experimental data were subjected to a two-sample independent t-test or one-way analysis of variance, followed by Tukey’s honest significance difference test, using the statistical and graphical functions of R version 4.3.1 [82] and ggplot2 [83] in RStudio 2023.06.1 + 524 [84]. A p value < 0.05 was considered statistically significant.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. No datasets were generated or analysed during the current study.

Abbreviations

AtPR1 :

Arabidopsis thaliana pathogenesis-related 1

AtTHI2.1 :

Arabidopsis thaliana thionin 2.1

Bg :

Burkholderia glumae

Chl a :

Chlorophyll a

Cn :

Cryptococcus neoformans

CrRPA70A :

Chlamydomonas reinhardtii replication protein A 70A

DDR:

DNA damage response

DMSO:

Dimethyl sulfoxide

DW:

Dry weight

FAMEs:

Fatty acid methyl esters

FW:

Fresh weight

GC:

Gas chromatography

GFP:

Green fluorescent protein

HR:

Hypersensitive response

LB:

Luria–Bertani

MS:

Mass spectrometry

OD750 :

Optical density at 750 nm

PAGE:

Polyacrylamide gel electrophoresis

Pst :

Pseudomonas syringae Pv. tomato

PVDF:

Polyvinylidene fluoride

qPCR:

Quantitative polymerase chain reaction

ROS:

Reactive oxygen species

RT:

Reverse transcription

SAR:

Systemic acquired resistance

SDS:

Sodium dodecyl sulfate

TAP:

Tris–acetate-phosphate

YPD:

Yeast extract-peptone-dextrose

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Funding

This study was supported by the KAERI Institutional Program (Grant number 523310–24), Republic of Korea. This funding body did not play any role in the design of this study and collection, analysis, and interpretation of data, and in writing the manuscript.

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  1. Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup-si, Jeonbuk-do, 56212, Republic of Korea

    Shubham Kumar Dubey, Seung Sik Lee & Jin-Hong Kim

  2. Department of Radiation Science, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea

    Shubham Kumar Dubey, Seung Sik Lee & Jin-Hong Kim

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SKD performed the experiments and generated the raw data. SKD, SSL, and J-HK analyzed, interpreted, and visualized the data. J-HK conceived, designed, and supervised the study. SDK and J-HK wrote the manuscript. All authors critically reviewed the manuscript and approved the submitted version.

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Correspondence to Jin-Hong Kim.

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Additional file 1

: Table S1. Primer sequences used for reverse transcription-quantitative PCR. Parentheses represent Arabidopsis Genome Initiative numbers or Chlamydomonas reinhardtii open reading framenames, which are used to alphabetically list names temporarily attributed to an ORF in a sequencing project. CrTUBA1 was used as an endogenous reference gene. Fig. S1. Time-course gene expression of AtPR1 or AtTHI2.1 in transgenic Chlamydomonas reinhardtii lines after X-irradiation. Mid-exponential phase cells were subjected to X-irradiation at a dose of 12 Gy. The Mock, 30 M, 1H, 2H, 4H, 6H, 8H, and 12H cells were harvested before, as well as 30 min and 1, 2, 4, 6, 8, and 12 h after, X-irradiation, respectively. All transcript levels were measured using reverse transcription-quantitative PCR and are shown relative to the mock using CrTUBA1 as an endogenous reference gene. Data represent the mean ± standard error. Different letters indicate significant differences at p < 0.05. Fig. S2. In vivo expression of mVenus proteins in transgenic Chlamydomonas reinhardtii cells after X-irradiation. Mid-exponential phase cells were subjected to X-irradiation at a dose of 12 Gy. The cells of Mock and 2H were harvested before and 2 h after X-irradiation, respectively.Diagram of the expression construct CrRPA70Ap-HSP70Ap-pOpt_mVenus.Confocal micrographs showing the in vivo expression of mVenus proteins in transgenic C. reinhardtii cells containing the modified empty vector shown in, which includes the CrRPA70A promoter but no transgene. Clorophyll aor mVenus images were captured using excitation/emission wavelengths of 488/650 nm or 515/528 nm, respectively. White scale bars represent 5 μm. Fig. S3. Antibacterial activity of wild-type Chlamydomonas reinhardtii cell extracts against Burkholderia glumae after γ-irradiation. NC, negative control; PC, positive control; Mock or GR1/5/10/20, 100 mg mL−1 control or γ-irradiated cell extract in DMSO. GR1, GR5, GR10, and GR20 represent γ-irradiation at a dose rate of 1, 5, and 10 kGy h−1 for 1 h or 10 kGy h−1 for 2 h, respectively. Cell extracts were prepared via sonication and heating in 100%ethanol and used for agar well diffusion assays. The size of the inhibition zonewas calculated by subtracting the well diameter from the width of the clear inhibition area. Data represent the mean ± standard errorfrom two independent experiments. Different letters indicate significant differences at p < 0.05. Fig. S4. Antibacterial activity of wild-type and transgenic Chlamydomonas reinhardtii cell extracts against Burkholderia glumae after γ-irradiation. NC, negative control; PC, positive control; WT, wild-type; AtPR1-OE1/2 or AtTHI-OE1/2, transgenic lines overexpressing AtPR1 or AtTHI proteins; M or G, 100 mg mL−1 mock or γ-irradiated cell extract in DMSO. Cells were harvested at a pre-stationary phaseand irradiated with X-rays at 12 Gy to induce transgene expression. To protect the antimicrobial activity of AtPR1 or AtTHI2.1 proteins, cell extracts were prepared via sonication in 100%ethanol without subsequent heating and used for agar well diffusion assays. The γ-irradiation of cell extracts was performed at a dose rate of 10 kGy h−1 for 2 h. The size of the inhibition zonewas calculated by subtracting the well diameter from the width of the clear inhibition area. Data represent the mean ± standard errorfrom two independent experiments. Different letters indicate significant differences at p < 0.05.

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Dubey, S.K., Lee, S.S. & Kim, JH. Efficient enhancement of the antimicrobial activity of Chlamydomonas reinhardtii extract by transgene expression and molecular modification using ionizing radiation. Biotechnol Biofuels 17, 125 (2024). https://doi.org/10.1186/s13068-024-02575-5

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Biotechnology for Biofuels and Bioproducts

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