Scale-up production of and dietary supplementation with the recombinant antimicrobial peptide tilapia piscidin 4 to improve growth performance in Gallus gallus domesticus

Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.


Microorganisms and animals
The bacterial strain Pichia pastoris X-33 (Invitrogen, CA, USA) was used as the TP4 expression host. Escherichia coli BL21 (DE3) (Stratagene, La Jolla, Calif., USA) was used as a DNA donor. DNA encoding TP4 with a His-tag was ligated into the pPICZαA (cat. no. V195-20, Invitrogen, CA, USA) expression vector. Procedures involving these animals adhered to the requirements of National Pingtung University of Science and Technology (NPUST) regarding their health and welfare and were approved by the Institutional Animal Care and Use Committee of NPUST (NPUST-107-026).

Construction and transformation of the expression recombinant plasmid and selection of positive transformants
To further increase the protein expression of recombinant TP4 in P. pastoris, the codon-optimized TP4 gene was designed to match P. pastoris expression by artificial gene synthesis from Omics Bio (Taipei, Taiwan). The nucleotide sequence products were confirmed by sequencing using primers, and then the identified plasmid was digested with EcoRI and XbaI. After enzyme digestion, the insert was ligated into the pPICZαA vector. The pPICZαA plasmid ligated with TP4 was digested with PmeI to linearize the vector and then transformed into P. pastoris X-33 by electroporation. The P. pastoris X-33 strain integrated with recombinant pPICZαA-TP4 DNA was grown in YPD broth at 30˚C with 250 rpm agitation for 16 h. The resulting culture was inoculated into 30 mL of BMGY medium and incubated for 24 h at 30˚C. The P. pastoris culture was then harvested by 10 min of centrifugation at 3,000 xg. The cell pellet was resuspended in 20 mM phosphate buffer, sonicated (400 cycles with 3 sec on/3 sec off) on ice by using a Sonicator 3000 system (HEAT Systems Inc., Farmingdale, NY) and then centrifuged at 5,000 xg for 20 min at 4˚C. The recombinant protein in the pellet and supernatant was detected by SDS-PAGE and Western blotting using an anti-6x-His tag monoclonal antibody (Invitrogen, MA1-21315).

Scale-up production of recombinant protein in a 500-L fermenter
The stock was streaked on YPD agar plates with zeocin and incubated for two days at 30˚C. BMGY with PTM4 broth and zeocin (100 μg/mL) was inoculated with a single colony and incubated in a shaker at 30˚C and 150 rpm for two days. A second preculture consisting of 5% cultured broth was inoculated into YPD medium under the same conditions for one day. In this experiment, a 500-liter fermenter (BTF-C500L, Biotop Process & Equipment Inc, Nantou, Taiwan) was used with 350 liters of commercial medium. Fermentor cultivations were carried out by using basal salt medium and PTM1. During the fermentation period, the temperature was maintained at 30˚C, and the pH was maintained and monitored at 6.0 with the addition of 14.5% ammonia and 0.1 N H 2 SO 4 . During the cultivation phase, the yeast were allowed to grow until the glycerol was exhausted after 36.5 h. Subsequently, the glycerol fed-batch phase consisted of feeding 50% (w/v) glycerol (containing 12 mL/L PTM) into the fermenter. After 47 h of cultivation, the methanol induction phase was performed by feeding 100% methanol (containing 16 mL/L PTM) for 24 h to induce rTP4 production. The rTP4-expressing yeast broth was harvested and freeze dried. Before rTP4 mixing with fodder preparation, rTP4 expression in the fermented culture and spray-dried powder was confirmed by Western blot analysis, and the rTP4 concentration was measured by comparing with different concentrations of synthetic TP4 peptide.

Antimicrobial activity of rTP4
The antimicrobial activity of rTP4 was measured in cultures of Escherichia coli (BCRC 10675), Pseudomonas aeruginosa (ATCC 19660), Staphylococcus aureus (BCRC 10780), and Riemerella anatipestifer (RA9, CFC27, CFC437, RA3, CFC363 and RA16). The microorganisms were collected from a single expanded colony and stored at -80˚C. The microorganisms were inoculated in liquid broth and incubated at 37˚C on a rotary shaker at 150 rpm overnight. The microorganisms were then diluted into fresh medium (1:1000) and incubated at 37˚C on a rotary shaker. The bacterial cultures (10 4 CFU/ml) were mixed with 100 μl of an rTP4 solution and incubated at 37˚C overnight. A negative control consisted of supernatant from P. pastoris transformed with vector alone, and ampicillin (2 mg/mL) was used as a positive control. After a 24-h incubation, the culture broth was measured at a wavelength of 600 nm. The antimicrobial activity of rTP4 was tested with an inhibition zone assay at different treatment temperatures [23]. Experiments were performed in duplicate and repeated independently three times.

G. g. domesticus maintenance and dietary treatment
A total of 119 male and 119 female 2-day-old G. g. domesticus were equally divided into seven groups for different dietary treatments, including basal diet, Spiraline-A (Juily Pharmaceutical Co., Ltd. San Sia, Taipei Hsien, Taiwan) and rTP4 groups (0.75, 1.5, 3.0, 6.0 and 12%). Thirtyfour G. g. domesticus were housed in each cage with different treatment groups. The formulation of the basal diet and the composition analysis of the basal feed with additive in the early, middle and late stages are presented in S1 and S2 Tables. The chickens had ad libitum access to water and fodder for the duration of the trial from 3 to 36 days of age. The animals were kept in a 12:12 light-dark cycle at 18.4-31.2˚C in a cage with a constant environment. The rTP4 feed, fecal sludge and wastewater were not discarded directly into the environment. Statistical analysis was performed with Student's t-test when comparing two groups. Multiple group comparisons were performed by ANOVA in GraphPad Prism 9. � P � 0.05, �� P � 0.01, ��� P � 0.001, ���� P � 0.0001.

The analysis of the phagocytic activity and cell size and complexity assessments
The effect of rTP4 on phagocytic activity was studied with the use of GFP-tagged E. coli bacteria using flow cytometry. The phagocytosis of GFP-E. coli (ATCC 25922GFP™) was analyzed by monitoring the mean GFP fluorescence intensity of engulfed bacteria. RAW264.7 cells were preincubated with rTP4 in 10% FBS/DMEM with gentle shaking for 24 h at 37˚C. The rTP4-induced RAW264.7 macrophage cells were allowed to uptake and become infected with GFP-labeled E. coli (10 7 cfu/mL) at an infective concentration of 1:20 (MOI) for an additional 4 h. The change in phagocytic activity was assessed according to the change in fluorescence intensity. After rTP4 stimulation, cells were removed from the culture dishes using a cell scraper and then centrifuged and washed with PBS. The cells were resuspended in a PBS solution. Flow cytometer (Beckman Coulter) analysis was performed using forward (FSC: cell size) and side scatter (SSC: cell complexity) parameters.

Physiological analysis and histological procedures
During the experimental period, the feed intake, body weight and survival rate were measured. Weight gain (WG), protein efficiency ratio (PER), feed efficiency (FE), and survival percentage were calculated. After 36 days of the feeding trial, G. g. domesticus were euthanized, and blood serum samples were collected after centrifugation (3,000 ×g for 10 min) and stored at -80˚C for analysis. The specimens were fixed in 4% formaldehyde in 0.1 M sodium phosphate buffer (PBS, pH 7.2) at room temperature. Subsequently, they were rinsed in PBS and dehydrated in graded ethanol. Tissue sections were deparaffinized in xylene and hydrated using serial percentages of alcohol. The sections were stained with hematoxylin for 10 min and eosin for 5 min.

Gut microbiota analysis
The duodenum stool of G. g. domesticus fed with the basal, Spiraline-A, or rTP4 diet was dissected for genomic DNA extraction using an innuPREP stool DNA kit (Jena AG, Jena, Germany). The DNA concentration was measured by using a NanoDrop ND1000 spectrophotometer (NanoDrop1 Technologies), and DNA was stored at -20˚C until use. The V4 region of the 16S rRNA gene was amplified with the forward PCR primer 515F and reverse primer 806R. All samples were amplified on a Veriti 96-well Thermal Cycler (Applied Biosystems, Foster City, USA). PCR amplicon sequencing was performed by the Biotools Microbiome Research Center (New Taipei City, Taiwan). The sequences were then processed using QIIME software [24]. QIIME can perform taxonomic assignment at the genus level on the sequencing from the Biotools Microbiome Research Center.

Construction of a TP4 expression pPICZαA plasmid
DNA encoding TP4 with a 6xHis tag was inserted into the pPICZαA expression vector to obtain the α-Factor-TP4-6xHis expression cassette (Fig 1). The pPICZαA plasmid ligated with TP4 was digested with PmeI within the AOX1 region to linearize the pPICZαA vector, and then TP4 genes were transformed into the Pichia pastoris X-33 strain. Colonies were screened by plating on YPDS plates containing different concentrations of zeocin. Transformed P. pastoris X-33 colonies integrated with recombinant pPICZαA-TP4 DNA were then selected by colony hybridization by using an anti-6xHis tag antibody, and high-expression clones were picked for subsequent experiments (previous data). The transformants were confirmed by colony PCR analysis with AOX1 primers (previous data).

Construction of the TP4 expression pPICZαA plasmid and large-scale expression of recombinant TP4 in a 500-L fermenter
DNA encoding TP4 with a 6xHis tag was inserted into the pPICZαA expression vector to obtain the α-Factor-TP4-6xHis expression cassette, which was described in our previous report [25]. Recombinant TP4 was expressed in the methylotrophic yeast P. pastoris under the control of the AOX1 promoter and secreted into the broth using the α-factor pre-pro sequence with the STE13 gene for dipeptidyl aminopeptidase A (Fig 1). After the successful production of the rTP4 transformant, the cells were cultured in a 5-liter bench-top fermenter. The parameters of dissolved oxygen (DO), RPM, pH and agitation were optimized for rTP4 production, and the effect of induction time of rTP4 expression was evaluated (previous data). We enlarged the scale from a 5 to 500 L fermenter with a two-stage fed-batch cultivation. Five percent of a P. pastoris culture was inoculated into 350 L of BSM. The cultivation process is shown in Fig  2A. During 36.5 h of cultivation, glycerol content decreased with time, and we began to feed glycerol into the fermentation. When the P. pastoris transformant was growing to stationary phase, the highest viable count reached approximately 8.3×10 10 CFU/mL at 48 h of cultivation. At the same time, methanol inductions were carried out in which the viable count and wet weight of yeast showed a sharp decrease (Fig 2A). After 65 h of cultivation, the fermented broth was harvested and then freeze dried to powder. The rTP4 powder was confirmed by Western blot analysis, and the rTP4 concentration was measured at 1.07 mg/g by comparison with different concentrations of synthetic TP4 peptide (Fig 2B). rTP4 powder was used as a feed additive for dietary supplementation in G. g. domesticus.

Antimicrobial activity of rTP4 expression in P. pastoris
To evaluate the antimicrobial activity of the rTP4 protein, the culture was induced with methanol and mixed with equal volumes of diluted bacterial cultures of gram-positive and gram-negative pathogens. After 16 h of incubation, the culture medium was measured at a wavelength of 600 nm. Compared to P. pastoris with a vector control, which did not inhibit pathogen

PLOS ONE
growth, rTP4 showed high activity to inhibit pathogen growth (Fig 3). Meanwhile, the Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility testing of rTP4, and the results indicated that rTP4 had a larger diameter and a more visible zone against the tested strains than the control treatment. The antimicrobial peptide was most effective against the pathogens R. anatipestifer (RA3) and R. anatipestifer (CFC27) (Table 1A). Furthermore, a study of the thermal stability of rTP4 was performed against S. aureus (BCRC 10780), P. aeruginosa (ATCC 19660) and E. coli (BCRC 10675). The thermal stability of the antimicrobial peptide was tested at 40, 60, 80 or 100˚C for 5 min, and the selected pathogens were detected by the agar disk diffusion method. The negative control group was P. pastoris with the empty vector. The antimicrobial peptide rTP4 showed good temperature stability at high temperature, inhibiting S. aureus and E. coli growth. The P. aeruginosa strain was inhibited by the antibacterial activity of rTP4 with increasing temperature (Table 1B). The results showed that heat treatment of rTP4 did not significantly affect the antimicrobial ability at temperatures up to 100˚C.

Effects of rTP4 on cell size and complexity in a phagocytic activity assay
To evaluate rTP4 in vitro stimulation of macrophage differentiation, the RAW264.7 cell line was treated with rTP4 for 24 h. Flow cytometry analysis using cell size forward-scattered light (FSC) and side-scattered light (SSC) parameters was performed. The results showed that the cell size and complexity after GFP-E. coli phagocytosis by RAW264.7 cells without rTP4 treatment were similar to those of RAW264.7 cells alone (Fig 3A and 3B). After rTP4 stimulation, the immunogenicity increased with molecular size and complexity in the macrophage differentiation assay (Fig 3C). In addition, increased cell complexity may be associated with increased storage or secretory vesicles, such as lysosomes. The mean fluorescent intensity (MFI) showing phagocytosis of GFP-E. coli in RAW264.7 cells after treatment with rTP4 was higher than that in the other groups (Fig 3D).

rTP4 supplementation improves growth performance and histology
After 36 days of the experimental period, the growth performance of G. g. domesticus in percent weight gain (WG) and feed efficiency (FE) in 3.0% rTP4-fed animals was significantly higher than that in the basal diet group animals and similar to that in the Spiraline-A group animals ( Table 2). The protein efficiency ratios of G. g. domesticus in the 0.75% and 1.5% rTP4-fed animals were higher than those in the animals in other groups (Fig 4). The survival rate of animals fed 1.5% rTP4 was lower than that of the animals in other groups, which was caused by one chicken dying from disease and two chickens dying after pecking an infected carcass. Morphological measurements of the heart, liver, spleen, lung, kidney and intestine are given in Fig 5. Histological examination of tissues showed white blood cell aggregates in the coronary vein of the heart in the antibiotic group (Fig 5A, black arrow). This phenomenon was not found in the coronary veins of the 3% rTP4 group or basal diet group. Microscopic examination of tissue sections, including those of the heart, liver, spleen, lung, kidney and intestine, in the three groups was normal, and no other symptoms were observed. Microbial profiles of the duodenum in chickens with different treatments were determined using high-throughput sequencing of the bacterial 16S rRNA gene. The results showed that the duodenum was predominantly populated by Peptostreptococcaceae and Erysipelotrichaceae. Reduced total viable counts of Ruminococcaceae and Lachnospiraceae were also observed after feeding with rTP4 (Fig 6).

Discussion
Antibiotics are widely used to prevent pathogen infection and promote growth in poultry and livestock production [26]. However, many reports indicate that long-term use of low-dose antibiotics may lead to resistant microorganisms. For various reasons, the use of antibiotics has been banned or restricted for growth promotion in the poultry industry in many countries. Consequently, there is a high demand for antibiotic alternatives in the livestock industry. Table 1. The antimicrobial activity and effect of temperature of rTP4. The rTP4 was expressed by P. pastoris X-33 and supernatant were collected after induction and applied to disk diffusion assay. After incubation, the inhibition zone diameters were measured on the culture plate. Representative radial diffusion assays are shown. (A) The antimicrobial activity of rTP4. (B) The effect of temperature on rTP4 antimicrobial activity. Compared with traditional antibiotics, AMPs have been shown to have multiple biological functions in addition to antimicrobial activities, including antibacterial, antiviral, antiprotozoan and antifungal activities [27]. In other respects, antimicrobial peptides protect fish against infections caused by the fish pathogen Vibrio anguillarum and provide a concept of transgenic fish with antimicrobial peptides. Antimicrobial peptides may decrease fish bacterial disease [28]. Antimicrobial peptides (AMPs) also stimulate the innate immune system via immunomodulatory functions and modulate the intestinal microflora in animals and humans [5]. In addition, AMPs had positive effects on intestinal morphology, nutrient digestibility, the gut microbiota, and growth performance in animals. These AMPs have a strong potential for application as a feed additive as alternatives to antibiotics in swine and poultry production. To study the role of antimicrobial peptides and mass production as feed supplements by use in the livestock industry, we performed this research. Yeast expression systems are ideally suited for large-scale production and cost-effective expression of functional AMP proteins. According to the codon usage preference of P. pastoris, the mature TP4 cDNA was modified, synthesized and cloned into the pPICZαA vector for P. pastoris X-33 expression.

A. The inhibition zone diameter methodological analysis of the effect of fermentation supernatants
There are many antimicrobial peptides that have been expressed in P. pastoris and possess natural antibacterial activities, such as ABP-dHC-cecropin A human neutrophil peptide 1 (HNP1) and histidalin [29][30][31]. In this study, we successfully produced recombinant TP4 on a 500-liter scale and freeze dried it to use as a feed additive for dietary supplementation in broilers. In the second cultivation, the viable count declined quickly from 8.3×10 10 to 6.5×10 10 CFU/mL, and the wet weight declined from 275 to 166 g/L during 49 to 58 hours of cultivation. This effect is presumably caused by methanol-induced antimicrobial peptide production to inhibit host cell growth [32]. According to previous research, the antimicrobial peptide HKPLP derived from Hippocampus kuda Bleeker showed good heat stability in thermal stability analysis [33]. After high-temperature treatment, rTP4 expressed by P. pastoris was found to be very stable in vitro and still retained full antimicrobial activity against S. aureus (BCRC 10780) and E. coli (BCRC 10675). The thermal stability of rTP4 may be related to its high arginine content (24%, 5 arginine in 25 total residues) [34]. In a previous report, increased arginine content enhanced peptide insertion into the membrane and significantly increased antimicrobial activity [35]. In addition, the antibacterial activity of AMPs can be enhanced by modifying the structural properties of natural peptides [36][37][38]. In a similar report, the crude antimicrobial extract of Bacillus velezensis strains isolated from stingless bee products showed bactericidal activity against methicillin-resistant Staphylococcus aureus and was stable at various temperatures (40-80˚C) [39]. Furthermore, Plantaricin LD1 produced by Lactobacillus plantarum LD1 showed stability at high temperatures (100˚C for 20 min and 121˚C for 15 min under 15 psi) and inhibited bacteria such as Staphylococcus aureus, urogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri and Vibrio sp [40]. Additionally, cecropinXJ expressed in Escherichia coli retained high stability against Staphylococcus aureus over a temperature range from 4 to 100˚C [41].
These results show that compared to the basal diet, fodder supplementation with crude rTP4 can improve the growth of G. g. domesticus. Regarding the antibiotic group, the 1.5% rTP4 and 3.0% rTP4 groups were similar, indicating that the antimicrobial peptide rTP4 can be recommended as an alternative to antibiotics to promote growth performance in chickens. According to this study, the FCR of the basal diet group was around 2.0, the Spiraline-A (commercial) group was around 1.88, and the 3% rTP4-fed group was around 1.85. In our experience, typical FCRs for animals raised using commercial feeds and intensive production methods are as follows: beef cattle: 6.0-10.0, pigs: 2.7-5.0, farmed fish and shrimp: 1.0-2.4, and chickens: 1.7-2.0. The FCRs in this study range from 1.85 to 2.0, which is within the normal range for chickens. In previous research, similar to an antibiotic, the antimicrobial peptide cLF36 improved the immune cells, intestinal morphology, intestinal microbiome, junctional proteins, and growth performance in E. coli-challenged broilers [42]. Pigs fed AMP-P5 at 60 mg kg -1 in dietary supplementation showed enhanced growth, inhibited coliform growth and increased nutrient digestion [43]. Tilapia piscidin, a marine antimicrobial peptide, has antimicrobial and immune regulatory activities that make it a potentially valuable feed supplement in aquaculture. Dietary supplementation with tilapia piscidin could enhance intestinal health, antioxidant activity, and the immune response. Furthermore, it confers protection against pathogen infections in Nile tilapia [44]. Morphological measurements of the heart, liver, spleen, lung, kidney and intestine showed white blood cell aggregates in the coronary vein of the heart in the broilers of the antibiotic group. The phenomenon of white blood cell aggregates is associated with infection or inflammation in the body. The Spiraline-A antibiotic group contained streptomycin sulfate. Streptomycin is administered by intramuscular

PLOS ONE
injections in humans. Repeated injections of streptomycin can cause pain and inflammation [45]. Some reports have shown that the cecum mucosa of streptomycin-treated mice has mild inflammatory infiltration [46]. In the gut microbiota community richness analysis, the total viable counts of the Ruminococcaceae and Lachnospiraceae were reduced. Peptostreptococcaceae and Erysipelotrichaceae were enriched in the duodenum in the TP4 group compared with in the antibiotic and basal diet groups. A report showed an increase in Lachnospiraceae in WIRS mice, which is associated with human diseases, such as ulcerative colitis, celiac disease and Crohn's disease [47].

Conclusions
This study revealed that recombinant TP4 expressed by yeast has high antimicrobial activity and thermal stability. Therefore, rTP4 is available for feed application, as the antimicrobial peptide can withstand the high temperature used in pelleted feed procedure and still have strong antibacterial activity. These results showed that the antimicrobial peptide tilapia piscidin 4 can be used as a feed additive for the livestock industry.