Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.


Introduction
Nowadays, people are suffering from the multidrug-resistant (MDR) microorganisms caused by the overuse of antibiotics [1][2][3]. The demand of finding alternatives to replace traditional antibiotics is increasing. Antimicrobial peptides (AMPs) are one of the promising candidates due to their positive effect on killing drug-resistant microorganisms [4,5]. AMPs are peptides consisting of 5-300 amino acids and are widely distributed in various organisms [6][7][8][9]. In general, AMPs own a broad-spectrum antimicrobial activity [10,11], anti-cancer cell activity [12,13], and antiviral activity [14]. As one of the known AMPs, anti-lipopolysaccharide factors (ALFs) were firstly identified from the hemocytes of Limulus polyphemus [15]. ALFs have the lipopolysaccharide (LPS) binding domain (LBD), which can bind to LPS to exhibit antimicrobial activity. To date, several ALFs have been identified from Penaeus monodon [16][17][18]. Among them, ALFPm3 has a wider anti-bacteria spectrum

The Design of ALFPm3 Expression Cassette
The coding sequence of ALFPm3 gene from P. monodon (Genbank accession: JQ256520) was optimized according to the codon bias of C. reinhardtii. The signal peptide of ALFPm3 composed of 25 amino-acid residues in its N-terminal was removed. After optimization, the GC content of ALFPm3 gene was increased from 55.89% to 68.37% ( Figure 1). The ble gene was fused to the N-terminal of optimized ALFPm3 gene by Foot and Mouth Disease Virus (FMDV) self-cleavage 2A peptide. FMDV 2A peptide can cleavage between Glycine and Proline to form two independent peptides ( Figure 2). Moreover, a 6× His tag was added to the ALFPm3 for protein detection. The designed ALFPm3 gene expression cassette was then inserted into the pH124 vector resulting in the pH2A-Pm3 plasmid ( Figure 3). Mar. Drugs 2021, 19, x FOR PEER REVIEW 3 of 13

The Screening of Transgenic C. reinhardtii
The pH2A-Pm3 plasmid was introduced into C. reinhardtii using the glass-bead method. After 2 weeks of recovery, the green colonies were visible and transferred to new TAP agar medium containing 8 µg/mL zeocin and 100 µg/mL ampicillin. Here, more than 200 colonies were obtained and the transformation frequency was 3 × 10 −6 . Later, genomic PCR and RT-PCR targeting at both ble and ALFPm3 were conducted to screen positive transformants. PCR results suggested that the non-transgenic C. reinhardtii generated no target bands. The specific bands representing the ble and ALFPm3 genes, respectively, were found in positive transformants, contributing to the positive ratio of 27.27% at the genomic level ( Figure 4A). Moreover, the Ble-2A-Pm3-fused expression cassette could be detected in genomic DNA, too ( Figure 4B). Furthermore, both optimized ALFPm3 gene and Ble-2A-Pm3-fused gene in positive transformants were detected in the transcription level ( Figure 5). Finally, more than 50 positive transformants were evidenced at both a genomic and transcriptional level and named T-2APm3.

The Screening of Transgenic C. reinhardtii
The pH2A-Pm3 plasmid was introduced into C. reinhardtii using the glass-bead method. After 2 weeks of recovery, the green colonies were visible and transferred to new TAP agar medium containing 8 μg/mL zeocin and 100 μg/mL ampicillin. Here, more than 200 colonies were obtained and the transformation frequency was 3 × 10 −6 . Later, genomic PCR and RT-PCR targeting at both ble and ALFPm3 were conducted to screen positive transformants. PCR results suggested that the non-transgenic C. reinhardtii generated no target bands. The specific bands representing the ble and ALFPm3 genes, respectively, were found in positive transformants, contributing to the positive ratio of 27.27% at the genomic level ( Figure 4A). Moreover, the Ble-2A-Pm3-fused expression cassette could be detected in genomic DNA, too ( Figure 4B). Furthermore, both optimized ALFPm3 gene and Ble-2A-Pm3-fused gene in positive transformants were detected in the transcription level ( Figure 5). Finally, more than 50 positive transformants were evidenced at both a genomic and transcriptional level and named T-2APm3.

The Analysis of ALFPm3 Protein Production in Transgenic C. reinhardtii
The ALFPm3 peptide with 6 × His tag could be detected by immunoblot using anti-His antibody from total soluble protein (TSP) extracted from T-2APm3 transformants (Figure 6). The molecular weight of ALFPm3 protein was about 12 kD, and the fused protein of BLE-2A-ALFPm3 was 30.64 kD. Our results indicated that the ALFPm3 could separate from BLE-2A-ALFPm3-fused protein by the self-cleavage of FMDV 2A peptide and accounted for approximate 0.35% of the TSP, demonstrating that the ALFPm3 gene was successfully expressed in C. reinhardtii.

The Screening of Transgenic C. reinhardtii
The pH2A-Pm3 plasmid was introduced into C. reinhardtii using the glass-bead method. After 2 weeks of recovery, the green colonies were visible and transferred to new TAP agar medium containing 8 μg/mL zeocin and 100 μg/mL ampicillin. Here, more than 200 colonies were obtained and the transformation frequency was 3 × 10 −6 . Later, genomic PCR and RT-PCR targeting at both ble and ALFPm3 were conducted to screen positive transformants. PCR results suggested that the non-transgenic C. reinhardtii generated no target bands. The specific bands representing the ble and ALFPm3 genes, respectively, were found in positive transformants, contributing to the positive ratio of 27.27% at the genomic level ( Figure 4A). Moreover, the Ble-2A-Pm3-fused expression cassette could be detected in genomic DNA, too ( Figure 4B). Furthermore, both optimized ALFPm3 gene and Ble-2A-Pm3-fused gene in positive transformants were detected in the transcription level ( Figure 5). Finally, more than 50 positive transformants were evidenced at both a genomic and transcriptional level and named T-2APm3.

The Analysis of ALFPm3 Protein Production in Transgenic C. reinhardtii
The ALFPm3 peptide with 6 × His tag could be detected by immunoblot using anti-His antibody from total soluble protein (TSP) extracted from T-2APm3 transformants (Figure 6). The molecular weight of ALFPm3 protein was about 12 kD, and the fused protein of BLE-2A-ALFPm3 was 30.64 kD. Our results indicated that the ALFPm3 could separate from BLE-2A-ALFPm3-fused protein by the self-cleavage of FMDV 2A peptide and accounted for approximate 0.35% of the TSP, demonstrating that the ALFPm3 gene was successfully expressed in C. reinhardtii.

The Analysis of ALFPm3 Protein Production in Transgenic C. reinhardtii
The ALFPm3 peptide with 6 × His tag could be detected by immunoblot using anti-His antibody from total soluble protein (TSP) extracted from T-2APm3 transformants ( Figure 6). The molecular weight of ALFPm3 protein was about 12 kD, and the fused protein of BLE-2A-ALFPm3 was 30.64 kD. Our results indicated that the ALFPm3 could separate from BLE-2A-ALFPm3-fused protein by the self-cleavage of FMDV 2A peptide and accounted for approximate 0.35% of the TSP, demonstrating that the ALFPm3 gene was successfully expressed in C. reinhardtii.

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The ALFPm3 peptide with 6 × His tag could be detected by immunoblot using anti-His antibody from total soluble protein (TSP) extracted from T-2APm3 transformants (Figure 6). The molecular weight of ALFPm3 protein was about 12 kD, and the fused protein of BLE-2A-ALFPm3 was 30.64 kD. Our results indicated that the ALFPm3 could separate from BLE-2A-ALFPm3-fused protein by the self-cleavage of FMDV 2A peptide and accounted for approximate 0.35% of the TSP, demonstrating that the ALFPm3 gene was successfully expressed in C. reinhardtii. Figure 6. The immunoblot analysis of the ALFPm3 derived from C. reinhardtii. Anti-His Tag: protein extracts incubated with mouse anti-His tag antibody for detecting His tag protein. Anti-α- Figure 6. The immunoblot analysis of the ALFPm3 derived from C. reinhardtii. Anti-His Tag: protein extracts incubated with mouse anti-His tag antibody for detecting His tag protein. Anti-α-tublin: protein extracts incubated with mouse anti-α-tublin antibody for detecting α-tublin. N: negative control, P: positive control, a protein with 6 × His tag expressed by E. coli with a concentration of 3.4 µg/mL. A1 to A3: transgenic Chlamydomonas.

The Expression Analysis of ALFPm3 Gene in C. reinhardtii
By comparing the growth curves of T-2APm3 and wild-type, we found that the growth curve of T-2APm3 was identical to that of wild-type (Figure 7), showing that the introduction of the ALFPm3 gene did not affect the normal growth of C. reinhardtii. After 6 months of cultivation, ALFPm3 gene did not lose or silence, indicating that the target gene could be stably expressed in C. reinhardtii ( Figure 8). Moreover, heat shock significantly increased the transcription of the ALFPm3 gene, while the high light increased slightly ( Figure 9).

The Expression Analysis of ALFPm3 Gene in C. reinhardtii
By comparing the growth curves of T-2APm3 and wild-type, we found that the growth curve of T-2APm3 was identical to that of wild-type (Figure 7), showing that the introduction of the ALFPm3 gene did not affect the normal growth of C. reinhardtii. After 6 months of cultivation, ALFPm3 gene did not lose or silence, indicating that the target gene could be stably expressed in C. reinhardtii ( Figure 8). Moreover, heat shock significantly increased the transcription of the ALFPm3 gene, while the high light increased slightly ( Figure 9).

cALFPm3 Showed High Anti-Bacterial Activity
After small-scale cultivation, TSP containing ALFPm3 peptide was extracted from T-2APm3 transformants to evaluate the anti-bacterial activity. The results showed that the tublin: protein extracts incubated with mouse anti-α-tublin antibody for detecting α-tublin. N: negative control, P: positive control, a protein with 6 × His tag expressed by E. coli with a concentration of 3.4 μg/mL. A1 to A3: transgenic Chlamydomonas.

The Expression Analysis of ALFPm3 Gene in C. reinhardtii
By comparing the growth curves of T-2APm3 and wild-type, we found that the growth curve of T-2APm3 was identical to that of wild-type (Figure 7), showing that the introduction of the ALFPm3 gene did not affect the normal growth of C. reinhardtii. After 6 months of cultivation, ALFPm3 gene did not lose or silence, indicating that the target gene could be stably expressed in C. reinhardtii ( Figure 8). Moreover, heat shock significantly increased the transcription of the ALFPm3 gene, while the high light increased slightly ( Figure 9).

cALFPm3 Showed High Anti-Bacterial Activity
After small-scale cultivation, TSP containing ALFPm3 peptide was extracted from T-2APm3 transformants to evaluate the anti-bacterial activity. The results showed that the

The Expression Analysis of ALFPm3 Gene in C. reinhardtii
By comparing the growth curves of T-2APm3 and wild-type, we found that the growth curve of T-2APm3 was identical to that of wild-type (Figure 7), showing that the introduction of the ALFPm3 gene did not affect the normal growth of C. reinhardtii. After 6 months of cultivation, ALFPm3 gene did not lose or silence, indicating that the target gene could be stably expressed in C. reinhardtii ( Figure 8). Moreover, heat shock significantly increased the transcription of the ALFPm3 gene, while the high light increased slightly ( Figure 9).

cALFPm3 Showed High Anti-Bacterial Activity
After small-scale cultivation, TSP containing ALFPm3 peptide was extracted from T-2APm3 transformants to evaluate the anti-bacterial activity. The results showed that the

cALFPm3 Showed High Anti-Bacterial Activity
After small-scale cultivation, TSP containing ALFPm3 peptide was extracted from T-2APm3 transformants to evaluate the anti-bacterial activity. The results showed that the cALFPm3 at the final concentration of 0.77 µM could significantly inhibit the growth of Gram-negative bacteria such as E. coli Top10, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus within 12 h ( Figure 10). Notably, the antibacterial rate of cALFPm3 against Gram-negative bacteria was 1.4-7.2 times higher than 5 mg/mL ampicillin. Those Gram-negative bacteria, except V. anguillarum, hardly grew when treated with cALFPm3 within 24 h ( Figure 10). Meanwhile, the inhibition on the Gram-positive bacteria, such as Streptococcus agalactiae, Staphylococcus aureus, and Bacillus sp T2, was also observed within 12 h (Figure 11). For example, the antibacterial rate of cALFPm3 against S. aureus was 2.3 times higher than 5 mg/mL ampicillin ( Figure 11). The antibacterial activities of cALFPm3 are superior against both Gram-negative bacteria and Gram-positive bacteria and lasted for over 12 h at a concentration of 0.77 µM, which was far beyond the ampicillin (Figures 10 and 11). Hence, our studies provided a promising cALFPm3, which showed a broad antimicrobial spectrum and strong antimicrobial activity.

Mar. Drugs 2021, 19, x FOR PEER REVIEW 6 of 13
Gram-negative bacteria such as E. coli Top10, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus within 12 h ( Figure 10). Notably, the antibacterial rate of cALFPm3 against Gram-negative bacteria was 1.4-7.2 times higher than 5 mg/mL ampicillin. Those Gram-negative bacteria, except V. anguillarum, hardly grew when treated with cALFPm3 within 24 h ( Figure 10). Meanwhile, the inhibition on the Gram-positive bacteria, such as Streptococcus agalactiae, Staphylococcus aureus, and Bacillus sp T2, was also observed within 12 h (Figure 11). For example, the antibacterial rate of cALFPm3 against S. aureus was 2.3 times higher than 5 mg/mL ampicillin ( Figure 11). The antibacterial activities of cALFPm3 are superior against both Gram-negative bacteria and Gram-positive bacteria and lasted for over 12 h at a concentration of 0.77 μM, which was far beyond the ampicillin ( Figures  10 and 11). Hence, our studies provided a promising cALFPm3, which showed a broad antimicrobial spectrum and strong antimicrobial activity.

Discussion
In this study, we designed an expression cassette of Ble-2A-ALFPm3 to produce the ALFPm3 peptide in C. reinhardtii. With the help of 2A peptide, which is a self-cleavage peptide from FMDV [48,49], the ALFPm3 peptide was successfully expressed in C. reinhardtii and released from the Ble-2A-ALFPm3-fused protein with a yield of 0.35% of total soluble protein. In order to increase the expression level and prevent gene silencing, the ble gene was fused to the N-terminal of the ALFPm3 gene via the 2A peptide, according to Beth A Rasala et al. [41]. The 2A peptide system has been co-expressed with several proteins at a high level, such as the fluorescent protein targeted to various cellular subcompartments [50], secreted xylanases [41], Cpf1 endonuclease [51], and squalene synthase [52]. Here, the ALFPm3 gene was co-introduced into C. reinhardtii with the ble gene. With this expression strategy, we could express the mature peptide of ALFPm3 without its own signal peptide, which was essential for expression in cells. Finally, cALFPm3 could be released from the Ble-2A-ALFPm3 protein and showed a great antimicrobial activity against both Gram-negative bacteria and Gram-positive bacteria. Therefore, this expression strategy was suitable to produce antimicrobial peptides.
Research has indicated that the E. coli system could not be used for expressing active AMPs or high-antimicrobial activity peptide due to the lack of post-protein processing [23][24][25][26][27]. The P. pastoris system could over-glycosylate AMPs, which reduced the antimicrobial activity [28,29]. Moreover, a lot of materials and energy are required in both E. coli and P. pastoris system and a large number of harmful gases are produced, including CO2 [27][28][29]. On the other hand, with the advantages of rapid growth, high photosynthetic efficiency, and carbon fixation ability, C. reinhardtii as a green microalga has been regarded as a novel host for the production of high-value chemicals and recombinant proteins. Although the productivity was not very high in C. reinhardtii, cALFPm3 showed high antimicrobial activity at a low concentration. The yield of AMPs in C. reinhardtii could reach up to 262 mg/L, as reported by Dong et al. [53]. Moreover, the production of cALFPm3 owned the potentiality to increase its yield with a strong promoter, leaky codon, and Kozak sequence.
It is reported that the ALFPm3 derived from P. pastoris exhibited high antimicrobial activity, especially for Gram-negative bacteria at a low concentration (less than 3 μM) and the Gram-positive bacteria at a higher concentration (between 50 mM and 100 mM) [30].

Discussion
In this study, we designed an expression cassette of Ble-2A-ALFPm3 to produce the ALFPm3 peptide in C. reinhardtii. With the help of 2A peptide, which is a self-cleavage peptide from FMDV [48,49], the ALFPm3 peptide was successfully expressed in C. reinhardtii and released from the Ble-2A-ALFPm3-fused protein with a yield of 0.35% of total soluble protein. In order to increase the expression level and prevent gene silencing, the ble gene was fused to the N-terminal of the ALFPm3 gene via the 2A peptide, according to Beth A Rasala et al. [41]. The 2A peptide system has been co-expressed with several proteins at a high level, such as the fluorescent protein targeted to various cellular subcompartments [50], secreted xylanases [41], Cpf1 endonuclease [51], and squalene synthase [52]. Here, the ALFPm3 gene was co-introduced into C. reinhardtii with the ble gene. With this expression strategy, we could express the mature peptide of ALFPm3 without its own signal peptide, which was essential for expression in cells. Finally, cALFPm3 could be released from the Ble-2A-ALFPm3 protein and showed a great antimicrobial activity against both Gram-negative bacteria and Gram-positive bacteria. Therefore, this expression strategy was suitable to produce antimicrobial peptides.
Research has indicated that the E. coli system could not be used for expressing active AMPs or high-antimicrobial activity peptide due to the lack of post-protein processing [23][24][25][26][27]. The P. pastoris system could over-glycosylate AMPs, which reduced the antimicrobial activity [28,29]. Moreover, a lot of materials and energy are required in both E. coli and P. pastoris system and a large number of harmful gases are produced, including CO 2 [27][28][29]. On the other hand, with the advantages of rapid growth, high photosynthetic efficiency, and carbon fixation ability, C. reinhardtii as a green microalga has been regarded as a novel host for the production of high-value chemicals and recombinant proteins. Although the productivity was not very high in C. reinhardtii, cALFPm3 showed high antimicrobial activity at a low concentration. The yield of AMPs in C. reinhardtii could reach up to 262 mg/L, as reported by Dong et al. [53]. Moreover, the production of cALFPm3 owned the potentiality to increase its yield with a strong promoter, leaky codon, and Kozak sequence.
It is reported that the ALFPm3 derived from P. pastoris exhibited high antimicrobial activity, especially for Gram-negative bacteria at a low concentration (less than 3 µM) and the Gram-positive bacteria at a higher concentration (between 50 mM and 100 mM) [30]. Here, compared with P. pastoris-derived ALFPm3, the cALFPm3 showed a higher antimicrobial activity and a long-time effect on the tested bacteria within 24 h at a lower concentration of 0.77 µM. More encouragingly, most of the tested bacteria hardly grew when incubated with 0.77 µM cALFPm3 within 24 h, which is different from the bacteria that were recovered after 9 h when treated with ampicillin. Thus, it is possible to reduce the number of antibacterial substances and obtain better antimicrobial effects by using cALFPm3. Moreover, microalgae are easy to cultivate and processing and is recognized as a safe feed additive, showing its great application in the future.
In this work, an expression cassette of ALFPm3 was designed and expressed in C. reinhardtii with high antimicrobial activity. It provides a new way for producing AMPs with high activity and the potential use of AMPs in the aquaculture industry.

Algal Strain and Culture Conditions
C. reinhardtii cell wall-deficient strain CC-849 was purchased from the Chlamydomonas Resource Center (USA), and was cultivated mixotrophically in Tris-Acetate-Phosphate (TAP) liquid medium or TAP agar medium contained 100 µg/mL ampicillin (Biosharp, Anhui, China) at 25 • C under a continuous light intensity of 100 µE·m −2 ·s −1 . Transgenic Chlamy was selected on the TAP agar plate supplemented with 8 µg/mL zeocin (Invitrogen, CA, USA) and 100 µg/mL ampicillin. Algal cells were cultivated until the cell density reached 1 × 10 6 cell/mL. For the heat shock induction, cells were incubated at 40 • C for 20 min. For high light stress, cells were cultivated under the light intensity of 100, 200, 300, and 400 µE·m −2 ·s −1 for 30 min. After each treatment, cells were harvested by centrifugation at 5000× g for 5 min.

Plasmid Construction and Genetic Transformation
To improve the transformation frequency and enhance hetero-gene expression, the coding sequence of ALFPm3 (Genbank accession: JQ256520) was optimized according to the codon bias of C. reinhardtii. The optimized nucleotides were synthesized by AnHui General Biosystem Company (Anhui, China) and then inserted into pH124 vector to obtain the plasmid pH2A-Pm3 (Figure 3). This vector contains HSP70A-RBCS2 fusion promoter, which can be induced by high light and heat shock. The ble gene was fused to the N-terminal of the ALFPm3 gene via the FMDV self-cleavage 2A peptide. The constructed plasmid was proliferated in E. coli Top10 on an LB agar plate containing 100 µg/mL ampicillin.
The C. reinhardtii transformation was processed using glass-bead method according to recommendations [54]. Briefly, the algal cells were cultivated in 100 mL TAP liquid medium in an incubator shaker at the condition of 25 • C, 100 rpm, and continuous light intensity of 100 µE·m −2 ·s −1 until the cell density was reached to 1~2 × 10 6 cell/mL. Cells were harvested by centrifugation at 5000× g for 5 min and resolved with 300 µL TAP liquid medium to keep the final density of 2 × 10 8 cell/mL. Cell cultures were transferred to a new 1.5 mL microcentrifuge tube containing 300 mg glass beads (0.5 mm in diameter) and 2 µg linearized pH2A-Pm3 plasmid. The glass beads were blown gently and vortexed at the highest speed, 2500 rpm, for 25 s. Afterward, the supernatant was transferred to a new microcentrifuge tube with 10 mL TAP liquid medium, cultivated at 25 • C with a light intensity of 90 µE·m −2 ·s −1 , and shaken continuously at 100 rpm for 22 h. Finally, cells were harvested by centrifugation at 3000× g for 5 min, resolved with 100 µL TAP liquid medium, and spread on the TAP agar medium containing 8 µg/mL zeocin and 100 µg/mL ampicillin. They were cultivated at 25 • C in an incubator under a continuous light intensity of 100 µE·m −2 ·s −1 until the green colonies were visible. The number of colonies was recorded and counted as the transformation frequency. Transformation frequency = numbers of colonies/total recipient cells.

Genomic PCR and RT-PCR Analysis
The green colonies were copied to a new TAP agar medium containing 8 µg/mL zeocin and 100 µg/mL ampicillin, and cultivated for one week. The positive transformants were selected by genomic PCR as described, targeting the ble gene and ALFPm3 gene [55]. For genomic PCR, 1 × 10 5 cells were harvested and then resuspended in 50 µL EDTA solution (10 mM, pH 8.0), after which they were incubated in boiling water for 8 min, cooled down on ice for 1 min, and pelleted at 14,000× g for 1 min. Finally, one microliter of the extract was used in the 20 µL PCR reaction containing a set of primers (Table 1) and PCR reaction buffer according to the instruction (Mei5 Biotechnology Co., Ltd, Beijing, China). The PCR program was 95 • C for 5 min, 35 cycles of 95 • C for 1 min, 56 • C for 30 s, 72 • C for 30 s, and finally extended at 72 • C for 5 min. For RT-PCR, Total RNA was extracted by RNA fast 200 Kit (Fastagen, Shanghai, China) and 1 µg of the total RNA was used to synthesize the first strand of cDNA by PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Two microliter cDNA was used in a 20-µL PCR reaction containing a set of primers (Table 1) and reaction buffer according to the manufacturer's instructions (Mei5 Biotechnology Co.,Ltd, Beijing, China). For the RT-PCR, β-actin was chosen as an internal control ( Table 1). All PCR products were analyzed by 1% agarose gel electrophoresis. Table 1. Sequence of primers used in this study.

Protein Extraction and Immunoblot Analysis
To evaluate the performance of transgenic C. reinhardtii (T-2APm3) on the ALFPm3 production, total protein was extracted and processed on immunoblot analysis. Algal cells were cultivated in TAP liquid medium under constant light intensity of 100 µE·m −2 ·s −1 until a cell density of 1~2 × 10 6 cell/mL was reached. Cells were harvested from one milliliter culture and resuspended with 100 µL lysis buffer (Solution A:Solution B, 3:2, v/v) (Solution A: 1M Na 2 CO 3 120 µL, 1M DTT 120 µL, deionized water 960 µL, Cock tail 20 µL; Solution B: 5% SDS, 30% Sucrose). After being shaken at 1200 rpm under room temperature for 10 min, cells were frozen-thawed three times in liquid nitrogen with 3 min between each repetition. The supernatant was collected at 14,000× g for 10 min at 4 • C for the following western blot analysis. Briefly, 20 µg total protein was separated by 4-20% SDS-PAGE (Genscrip, New Jersey, USA) and then transferred to a PVDF membrane (0.2 µm, Millipore, Massachusetts, USA) activated by 100% methanol. The membrane was then blocked with blocking buffer (5% non-fat dry milk in TBST (10 mM Tris, 166 mM NaCl, pH 7.4, contained 0.05% Tween 20)) for 2 h at room temperature. The membrane was incubated with the mouse anti-His tag antibody (1:8000) (Agrisera, Vännäs, Sweden) in blocking buffer at 4 • C overnight right after washed three times with TBST. Afterward, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibody (1:10,000) (Beijing Biodragon, Beijing, China) at room temperature for 2 h, and washed with TBST for 3 times. Finally, the membrane was incubated in ECL substrate buffer (Solution A: Solution B, 1:40, v/v) (ThermoScientific, MA, USA) at room temperature for 3 min. The chemiluminescence signal was detected by Odyssey·Fc (Gene Company Limited, HK, China). The α-tublin was used as the internal control. The gray values referring protein content were analyzed by Image J software (NIH, Maryland, USA). For calculating the content of target protein, a protein contained 6 × His tag at the concentration of 3.4 µg/mL was used as the calibrator. The content of the target protein was calculated by comparing its gray value with that of calibrator.

Anti-Bacterial Assay
For the anti-bacterial assay, the ALFPm3 peptide was prepared as follows. The transgenic Chlamydomonas T-2APm3 was cultivated in 800 mL TAP liquid medium under a continuous light intensity of 100 µE·m −2 ·s −1 until the cell density reached 1~2 × 10 6 cell/mL. Cultures were centrifuged at 5000× g at 4 • C for 5 min, the supernatant was discarded, and pellets were resuspended with 10 mL lysis buffer (Solution A: Solution B is 3:2, v/v) (Solution A: 1M Na 2 CO 3 120 µL, 1M DTT 120 µL, deionized water 960 µL, Cock tail 20 µL. Solution B: 30% Sucrose) and shaken at 1200 rpm under room temperature for 10 min. Then cells were frozen and thawed three times in liquid nitrogen with 3 min between each repetition. The supernatant contained ALFPm3 peptide was obtained by centrifugation at 14,000× g for 10 min at 4 • C, followed by filtration with 0.22 µm filter tip. To investigate the antibacterial activity of peptide extracts, a total of six Gram-negative bacteria (E. coli Top 10, Vibrio vulnificus, Vibrio parahemolyticus, Vibrio harveyi, Vibrio anguillarum, and Vibrio alginolyticus) and three Gram-positive bacteria (Streptococcus agalactiae, Bacillus sp T2, Staphylococcus aureus) were tested. Before the assay, the tested bacteria were cultivated in 5 mL LB liquid medium under 37 • C with shaking at 200 rpm overnight. The 500 µL cultures were added into 5 mL fresh LB liquid medium and cultivated at 37 • C with shaking at 200 rpm for 1 h. Then, the bacteria cultures were diluted by 1000 times using fresh LB medium and 150 µL diluted cultures were mixed with 50 µL ALFPm3 peptide in 96-well plate. The final concentration of ALFPm3 peptide was 0.7669 µM. The extracts from wide type C. reinhardtii and 5 mg/mL ampicillin acted as negative and positive control, respectively. Finally, the mixture of bacteria and ALFPm3 was incubated at 37 • C and the OD600 was determined at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h, respectively. The growth rate (∆OD600) of the bacteria under different treatments was obtained by comparing the OD600 value with that at 0 h.

Expression Analysis of ALFPm3 in C. reinhardtii Transformants
To observe the expression of ALFPm3 in C. reinhardtii, the transgenic Chlamydomonas T-2APm3 was kept constant for 6 months and the total RNA of T-2APm3 was isolated and followed by RT-PCR with primers ALF3-F and ALF3-R. PCR products were analyzed by 1% agarose gel electrophoresis. The growth curves of non-transgenic C. reinhardtii and transgenic C. reinhardtii were measured to analyze the effect of ALFPm3 on algae growth. They were cultivated in 100 mL TAP liquid medium under continuous light intensity of 100 µE·m −2 ·s −1 with shaking. Cell numbers were counted every day via a hemocytometer measurement.

Statistical Analysis
Each experiment was conducted with three independent replicates. GraphPad Prism 5 was used to perform the statistical analysis and generate figures and tables. Student's t-test was used to calculate the statistical difference between treatments and p < 0.05 was considered as showing significant differences.