BACKGROUND
bacteriocins are antimicrobial peptides synthesized in the ribosome and secreted into medium to establish a competitive advantage in their environment by eliminating competitors to gain resources  <cit> .
bacteriocins are generally classified in terms of size, structure, and modifications.
class i bacteriocins are lantibiotics.
class ii bacteriocins consist of small peptides that do not contain modified residues.
class iii bacteriocins usually are large and heat-labile proteins  <cit> .
the well-known bacteriocin is nisin, a class i bacteriocin, which is widely used in commerce  <cit> .
recently, many reports clearly indicate that bacteriocins of class iia have greater potential as antimicrobial agents  <cit>  with a narrower inhibitory spectrum to listeria strains than nisin  <cit> .
listeria, the most common pathogen in food, can lead the host to suffer from serious diseases such as enteritis, sepsis, meningitis and abortion  <cit> .
the mortality rate caused by listeriosis is between  <dig> and 30%  <cit> .
additionally, some strains of l. monocytogenes easily acquire resistance to many antibiotics  <cit> .
to control food contamination and listeriosis effectively, more or better anti-listerial drugs are needed.
enterocin a , with many antimicrobial merits, is a class iia bacteriocin that was first isolated from enterococcus faecium ctc <dig> in the mid-1990s.
its mature form is composed of  <dig> amino acids with two disulfide bridges  <cit> .
it shows high activity, particularly against listeria species at nanomolar concentrations  <cit> .
the native enta has proven to effectively inhibit l. monocytogenes in fermented foods  <cit> .
however, the low levels of bacteriocins secreted from natural strains do not meet the requirements of the industrial scale and have limited its application to study stages thus far.
therefore, various heterologous expressions were attempted in lactic acid bacteria, escherichia.
coli  and yeast , but their actual production levels were not desirable and left room for improvement.
pichia pastoris is considered to be a promising system because the target protein can be directly secreted into culture medium.
it was reported that the production and bactericidal titer of enterocin p expressed by p. pastoris x- <dig> was  <dig> - and 16-fold higher , respectively, than that from the native e. faecium p <dig>  <cit> ; in fact, even though the level of  <dig>  μg/ml of recombinant enterocin a expressed by p. pastoris  <cit>  was still too low for its industrial production and end application, it demonstrates the potential to increase its productivity to be as high as possible and to further easily characterize its purification and properties.
however, there are only few studies at the modification of bacteriocin genes, such as gene synthesis or codon optimization, which is considered as a promising technique for increasing protein expression level  <cit> ; thus, further work with this system is necessary to achieve an increased protein expression level of target gene.
due to the high anti-lister activity of enta and its low yield either in native strain and recombinant expression system, the enta gene was optimized by the preferential codon usage of p. pastoris and was expressed into medium as recombinant enta .
the purification of renta from ferment supernatant was tried by four methods including gel filtration chromatography, then the antimicrobial activity, proteolytic sensibility and stabilities of heat, ph and salt of purified renta were examined.
RESULTS
construction and transformation of the expression vector
compared to naturally occurring enta, the base codons coding for  <dig> residues  in total  <dig> amino acids were optimized by the preferential codon usage of p. pastoris .
the gc content of the full target sequence increased from  <dig> % to  <dig> %.
the gene sequence of the optimized enta was synthesized and inserted into ppiczαa between xhoi and xbai sites .
the expression vector ppiczαa-enta was transferred into competent e. coli dh5α cells.
resulting transformants were confirmed by pcr and dna sequencing.
correct plasmid and control vector ppiczαa were linearized by pmei and transferred into competent p. pastoris x- <dig> cells by electroporation.
positive transformations were screened and confirmed by colony pcr.figure  <dig>
construction of the expression plasmid ppicz
α
a-enta.
a, the nucleotide sequence of enta and its corresponding amino acid sequence.
the upper line indicates the wild-type enta gene sequence.
the middle line is the codon-optimized enta gene sequence.
optimized codons are underlined with boldface type.
the lower line represents the amino acid sequence of enta.
the termination codon is marked by an asterisk.
b, map of the recombinant plasmid ppiczαa-enta.
c, electrophoretic analysis of the recombinant vector containing the enta gene.
lane  <dig>  dna marker; lane  <dig>  ppiczαa-enta digested by xhoi and xbai.
expression of renta in shake flasks and at the fermenter level
the heterologous expression of renta in p. pastoris x- <dig> was induced by methanol at the concentration of  <dig> % and analyzed by agar diffusion and tricine-sds-page.
p. pastoris x- <dig> containing the empty ppiczαa vector was used as a negative control.
as shown in figure 2a, after 12 h of methanol induction, the antibacterial activity of the supernatants of p. pastoris x- <dig>  was observed.
its antibacterial activity reached maximum with  <dig>  au/ml after 24 h of methanol induction.
however, the antimicrobial activity decreased from  <dig> to 72 h. no antibacterial activity was detected in the supernatants of p. pastoris x- <dig> .
the results of the maldi-tof ms for fermentation supernatants indicated that the molecular weight of renta was  <dig> .1 da, which was consistent with its theoretical value of  <dig>  da .figure  <dig>
expression and purification of renta.
a, total secreted protein level and antimicrobial titer of the fermentation supernatants of recombinant p. pastoris at the shake-flask level .
b, antimicrobial activity of the fermentation supernatants of recombinant p. pastoris at the fermenter level.
1– <dig>  50 μl supernatant taken at  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and 90 h of induction, respectively;  <dig>  1 μg ampicillin.
c, the total secreted protein level and antimicrobial titer in the fermenter level .
d, tricine-sds-page analysis of renta secreted in the fermentation supernatant of p. pastoris cultures at the fermenter level.
lane m, 5 μl molecular mass standards ; lanes 1– <dig>  20 μl supernatant taken at  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 90 h of induction, respectively.
e, maldi-tof map of renta.
f, purification and identification of renta.
lane  <dig>  purified renta ; lane m, 5 μl molecular mass standards .
lane  <dig>  10 μl of renta supernatant taken at 24 h of induction.
to increase the production of renta, high-density fermentation of the recombinant yeast was performed using a 5-l fermenter.
although the total supernatant protein and biomass reached 365 mg/l and 343 g/l after induction for 90 h, the maximal antimicrobial activity was 51200 au/ml  after induction for 24 h , which was 8-fold higher than that found at the shake-flask level.
figures 2b and d clearly showed that renta was rapidly degraded after 72 h of induction.
moreover, the expression of renta in the fermenter could be detected directly by coomassie blue staining , while its expression in the shake-flask could only be detected by silver staining .
purification of renta
the renta was purified from the ferment supernatant after a 24-h induction in a 5-l fermenter.
the bacteriocin activity of  <dig>  × 105 au/mg with a  <dig> -fold increase was obtained after gel filtration.
the purified renta was analyzed by tricine-sds–page and showed a band at  <dig>  kda representing the target protein band , corresponding with its theoretical molecular weight.
antimicrobial spectrum of renta
only l. ivanovii atcc <dig>  e. faecalis cgmcc <dig>  and e. faecalis cgmcc <dig>  were sensitive to renta in the  <dig> tested strains.
other gram-positive bacteria, such as e. faecium cgmcc <dig> , s. aureus atcc <dig>  s. epidermidis atcc <dig>  b. licheniformis cgmcc <dig> , and b. coagulans cgmcc <dig> , were found to be resistant to renta.
all of the gram-negative bacteria strains were resistant to renta in this assay .
the mic and mbc of renta against l. ivanovii atcc <dig> were 20 ng/ml and 80 ng/ml, respectively, and were lower than those of ampicillin .table  <dig>
antimicrobial spectrum of renta
strains
antimicrobial activity
+
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
note: “+” refers to positive antimicrobial activity ; “-” refers to negative antimicrobial activity .
in-vitro killing curve assay
the time-killing kinetics curve showed that the amount of l. ivanovii atcc <dig> increased from  <dig>  log10cfu/ml to  <dig>  log10cfu/ml within 10 h in the absence of renta.
the decrease in the counts of l. ivanovii atcc <dig> varied considerably depending on the concentration of renta.
for example, the maximum viability loss , which was approximately  <dig>  log <dig> cfu/ml , was reached within 2 h in 1 × mic of renta.
the 2 × mic of renta could cause approximately  <dig>  log <dig> cfu/ml viability loss  within 6 h. moreover, the mvl of l. ivanovii treated by renta at 4 × mic was approximately  <dig>  log <dig> cfu/ml  within 4 h. although renta could inhibit the growth of l. ivanovii atcc <dig>  the survivors resumed growth at 1× and 2 × mic of renta and 2 × mic ampicillin for l. ivanovii atcc <dig> after mvl was achieved .
however, l. ivanovii atcc <dig> treated by 4 × mic of renta did not show re-growth within 10 h, revealing that  <dig> ng/ml renta could effectively inhibit the growth of pathogenic bacteria for an extended time.figure  <dig>
time-kill curves of renta.
l. ivanovii atcc <dig> was incubated in the presence of medium alone or in the presence of 1×, 2×, or 4× mic of renta.
ampicillin of 2 × mic was used as a positive control.
three duplicate observations were made; bars represent the standard error of the mean.
effects of ph, temperature, proteolytic enzymes and nacl on the activity of renta
as shown in figure 4a, renta was highly stable at a wide range of ph values.
the activity of renta was maintained completely within a ph range of 2– <dig> at 37°c for 12 h and was 75% retained even after incubation at a ph of  <dig> for 12 h. furthermore, the antimicrobial activity of renta was not affected by heat treatment at  <dig>   <dig>   <dig> and 100°c for 1 h under acid conditions  .
the residual activity decreased to 20% at a ph of  <dig> at 80°c, to 50% at a ph of  <dig>   <dig> at 100°c, and to 10% at a ph of  <dig> at 100°c.
in addition, the antimicrobial activity of renta was completely abolished by pepsin and trypsin treatment, but it retained  <dig> % of initial antimicrobial activity after papain treatment at 37°c for 1 h .figure  <dig>
effects of ph, temperature and proteolytic enzymes on the renta activity.
a, ph stability of renta.
purified renta was incubated in buffers with a ph range from  <dig> to  <dig> at 37°c for 12 h. the initial activity of the sample in a buffer with a ph of  <dig> was described as 100% activity.
b, thermal stability of enta.
purified renta was incubated in buffers with a ph range from  <dig> to  <dig> at temperatures of  <dig>   <dig>   <dig>  and 100°c for 1 h. the initial activity of the sample in a buffer with a ph of  <dig> was described as 100% activity.
c, proteolysis resistance of renta.
purified renta was incubated with pepsin, papain and trypsin at 37°c for 4 h. the residual antimicrobial activity of samples was tested after the ph was readjusted to ph  <dig>  with sodium phosphate buffer.
the antimicrobial activity of renta against l. ivanovii atcc <dig> was slightly enhanced by the addition of  <dig> and 50 mm nacl .
the lowest amount of  <dig>  log <dig> cfu/ml was observed with a treatment of renta  in 25 mm nacl .
the other treatments, from  <dig> – 400 mm nacl, had no significant effect on the bactericidal ability of renta .
in the controls without renta, growth was not influenced by nacl  .figure  <dig>
effect of nacl concentration on the activity of renta.
control: l. ivanovii atcc <dig> was incubated in the absence of renta.
4 × mic: l. ivanovii atcc <dig> was incubated in the presence of renta at 4 × mic.
discussion
bacteriocin has attracted attention in recent years for its potential application as a food preservative and therapeutic antimicrobial agent  <cit> .
however, low production of these bacteriocins by native strains cannot meet the requirements of commercial applications.
moreover, some enterococci strains were recognized as opportunistic pathogens associated with lots of infections  <cit> .
attempts to produce bacteriocins by using safe heterologous hosts have been undertaken in recent years  <cit> , including some typical expression systems such as e. coli, l. lactis, and p. pastoris.
although e. coli and l. lactis are widely used in heterologous protein expression because of their easy operation and safety  <cit> , they are not suitable for bacteriocins due to toxicity to the host  <cit>  and low recovery percentages from the fusion protein  <cit> .
many bacteriocins, such as enterocin p  <cit> , hiracin jm <dig>  <cit> , enterocin l <dig>  <cit> , pediocin pa- <dig>  <cit>  and enta  <cit> , have been expressed as active forms in p. pastoris, but their expression levels remained low .
it is known that codon optimization is a useful strategy to increase the yield of target protein during heterogeneous expression.
many antimicrobial peptides, such as plectasin  <cit> , nz <dig>  <cit>  and agplectasin  <cit> , were expressed with high production through codon-usage optimization in our laboratory.
in addition, divercin v <dig>  a class iia bacteriocins was also expressed through this system  <cit> .
these cases encouraged us to use codon optimization to break through the bottleneck of low yield in heterologous expression of enta.
the total protein level in the supernatant reached 180 mg/l with the activity of  <dig>  au/ml at 24 h of induction in 5-l fermenter level  after the gene was optimized.
although the yield of target peptide was still low, and even lower than 280 mg/l as the highest result of expression in case of enterocin l <dig> in p. pastoris  <cit> , it was much higher than that of pediocin pa- <dig> , enterocin p , divercin v <dig>  and enta  expressed in e. coli and l. lactis  <cit> .
furthermore, the production of renta increased  <dig> -times compared with its native sequence expressed in p. pastoris , which indicated codon optimization is a good tool to enhance expression efficiency and level in p. pastoris, and at the same time, it also left a large room to improve in future work at the similar aim and technical scheme.
however, the maximal activity of renta in the supernatant was reached at an early stage  of induction .
this is similar to previous results in which the highest level of renta was reached at 36 h. an even earlier peak of renta at 6 h was observed in other yeasts such as kluyveromyces lactis and hansenula polymorpha  <cit> .
obviously, its final successful application suffered from this strong decomposition in the supernatant at an earlier period of expression related to the possible disruption of renta to host cells and the proteolysis of the target protein.
the latter situation was reported in “collagen-like” bacteriocin with a high cleavage by collagenase  <cit> .
however, the exact mechanism of the above described early degradation and its solution should be further studied.
a series of methods, such as ion exchange chromatography , hydrophobic exchange chromatography , and gel filtration , were attempted to purify renta in this study.
only gel filtration could purify renta with a yield of  <dig>  mg/l  after attempts with sp ff, cm ff, and phenyl hp in which almost all renta was lost in the penetration peak  due to unknown reasons.
although there are different antibacterial spectrums between class iia bacteriocins, they consistently have particularly high antibacterial activity against listeria — the most common pathogen in food — at nanomolar concentrations  <cit> .
the mics of purified native enta from e. faecium t <dig> against listerias ranged from  <dig> to 120 ng/ml  <cit> .
similarly, renta also showed a narrow antibacterial spectrum  including l. ivanovii atcc <dig>  and with a low mic value of 20 ng/ml, it is approximately 20-fold lower than that of ampicillin .
the re-growth after mvl achievement was a common phenomenon when the listeria was treated with bacteriocins such as enta, pediocin, sakacin a and enterococcin efs <dig> in relatively low concentrations   <cit> , but we found no re-growth after mvl within 10 h when 4 × mic rena was used with the listeria , indicating that higher concentrations of rena are essential to inhibit the multiplication of listeria.
the bactericidal activity and overall structure of pediocin pa- <dig> and piscicolin  <dig> were well maintained at higher temperatures  <cit> .
the native enta was stable at 100°c and acidic ph conditions  <cit> .
we found that renta also exhibited high stability under a wide range of temperatures  and ph levels  .
these properties were potentially due to the higher cysteine content of the antimicrobial peptides  <cit> , similar to the enta containing four cysteine residues.
in addition, the antimicrobial activity of some bacteriocins  was significantly enhanced with the addition of nacl from  <dig> to  <dig>  m  <cit> .
however, the activity of renta against listeria was enhanced only at low nacl concentrations .
despite the unknown mechanisms of the above differential effects, the high stability of renta over wide ranges of temperature, ph, and nacl concentration supports its use as a food preservative and drug candidate.
due to the high content of basic and aromatic amino acids in class iia bacteriocins, pediocin pa- <dig>  enterocin b, plantaricin  <dig> and native enta were very sensitive to the digestive proteases trypsin and pepsin  <cit> .
similarly, the purified renta, with  <dig> % basic amino acids and  <dig> % aromatic amino acids, was inactivated with trypsin and pepsin .
this high sensitivity to digestive proteases of renta contributes to its safety in foods and drugs, during and after oral administration.
CONCLUSIONS
in conclusion, renta, as an antimicrobial agent with merit, could selectively kill important and pathogenic listeria and retain bio-activity over a wide range of ph values, temperature and nacl concentrations.
these excellent antibacterial properties make it a potential candidate as a food preservative and therapeutic antimicrobial agent.
renta was successfully expressed in p. pastoris x- <dig> at the highest level of  <dig>  au/ml and was purified through a gel filtration column.
this yeast system may be a feasible technological approach to produce renta as a potent anti-listeria agent after further optimization.

 bacteriocins are antimicrobial peptides synthesized in the ribosome and secreted into medium to establish a competitive advantage in their environment by eliminating competitors to gain resources. bacteriocins are generally classified in terms of size, structure, and modifications. class i bacteriocins are lantibiotics. class ii bacteriocins consist of small peptides that do not contain modified residues. class iii bacteriocins usually are large and heat-labile proteins. the well-known bacteriocin is nisin, a class i bacteriocin, which is widely used in commerce. recently, many reports clearly indicate that bacteriocins of class iia have greater potential as antimicrobial agents with a narrower inhibitory spectrum to listeria strains than nisin. listeria, the most common pathogen in food, can lead the host to suffer from serious diseases such as enteritis, sepsis, meningitis and abortion. the mortality rate caused by listeriosis is between and 30%. additionally, some strains of l. monocytogenes easily acquire resistance to many antibiotics. to control food contamination and listeriosis effectively, more or better anti-listerial drugs are needed. enterocin a, with many antimicrobial merits, is a class iia bacteriocin that was first isolated from enterococcus faecium ctc in the mid-1990s. its mature form is composed of amino acids with two disulfide bridges. it shows high activity, particularly against listeria species at nanomolar concentrations. the native enta has proven to effectively inhibit l. monocytogenes in fermented foods. however, the low levels of bacteriocins secreted from natural strains do not meet the requirements of the industrial scale and have limited its application to study stages thus far. therefore, various heterologous expressions were attempted in lactic acid bacteria, escherichia. coli and yeast, but their actual production levels were not desirable and left room for improvement. pichia pastoris is considered to be a promising system because the target protein can be directly secreted into culture medium. it was reported that the production and bactericidal titer of enterocin p expressed by p. pastoris x- was - and 16-fold higher, respectively, than that from the native e. faecium p; in fact, even though the level of  μg/ml of recombinant enterocin a expressed by p. pastoris was still too low for its industrial production and end application, it demonstrates the potential to increase its productivity to be as high as possible and to further easily characterize its purification and properties. however, there are only few studies at the modification of bacteriocin genes, such as gene synthesis or codon optimization, which is considered as a promising technique for increasing protein expression level; thus, further work with this system is necessary to achieve an increased protein expression level of target gene. due to the high anti-lister activity of enta and its low yield either in native strain and recombinant expression system, the enta gene was optimized by the preferential codon usage of p. pastoris and was expressed into medium as recombinant enta. the purification of renta from ferment supernatant was tried by four methods including gel filtration chromatography, then the antimicrobial activity, proteolytic sensibility and stabilities of heat, ph and salt of purified renta were examined.  construction and transformation of the expression vector compared to naturally occurring enta, the base codons coding for residues in total amino acids were optimized by the preferential codon usage of p. pastoris. the gc content of the full target sequence increased from % to %. the gene sequence of the optimized enta was synthesized and inserted into ppiczαa between xhoi and xbai sites. the expression vector ppiczαa-enta was transferred into competent e. coli dh5α cells. resulting transformants were confirmed by pcr and dna sequencing. correct plasmid and control vector ppiczαa were linearized by pmei and transferred into competent p. pastoris x- cells by electroporation. positive transformations were screened and confirmed by colony pcr.figure construction of the expression plasmid ppicz α a-enta. a, the nucleotide sequence of enta and its corresponding amino acid sequence. the upper line indicates the wild-type enta gene sequence. the middle line is the codon-optimized enta gene sequence. optimized codons are underlined with boldface type. the lower line represents the amino acid sequence of enta. the termination codon is marked by an asterisk. b, map of the recombinant plasmid ppiczαa-enta. c, electrophoretic analysis of the recombinant vector containing the enta gene. lane dna marker; lane ppiczαa-enta digested by xhoi and xbai. expression of renta in shake flasks and at the fermenter level the heterologous expression of renta in p. pastoris x- was induced by methanol at the concentration of % and analyzed by agar diffusion and tricine-sds-page. p. pastoris x- containing the empty ppiczαa vector was used as a negative control. as shown in figure 2a, after 12 h of methanol induction, the antibacterial activity of the supernatants of p. pastoris x- was observed. its antibacterial activity reached maximum with  au/ml after 24 h of methanol induction. however, the antimicrobial activity decreased from to 72 h. no antibacterial activity was detected in the supernatants of p. pastoris x-. the results of the maldi-tof ms for fermentation supernatants indicated that the molecular weight of renta was.1 da, which was consistent with its theoretical value of  da.figure expression and purification of renta. a, total secreted protein level and antimicrobial titer of the fermentation supernatants of recombinant p. pastoris at the shake-flask level. b, antimicrobial activity of the fermentation supernatants of recombinant p. pastoris at the fermenter level. 1– 50 μl supernatant taken at and 90 h of induction, respectively; 1 μg ampicillin. c, the total secreted protein level and antimicrobial titer in the fermenter level. d, tricine-sds-page analysis of renta secreted in the fermentation supernatant of p. pastoris cultures at the fermenter level. lane m, 5 μl molecular mass standards; lanes 1– 20 μl supernatant taken at and 90 h of induction, respectively. e, maldi-tof map of renta. f, purification and identification of renta. lane purified renta; lane m, 5 μl molecular mass standards. lane 10 μl of renta supernatant taken at 24 h of induction. to increase the production of renta, high-density fermentation of the recombinant yeast was performed using a 5-l fermenter. although the total supernatant protein and biomass reached 365 mg/l and 343 g/l after induction for 90 h, the maximal antimicrobial activity was 51200 au/ml after induction for 24 h, which was 8-fold higher than that found at the shake-flask level. figures 2b and d clearly showed that renta was rapidly degraded after 72 h of induction. moreover, the expression of renta in the fermenter could be detected directly by coomassie blue staining, while its expression in the shake-flask could only be detected by silver staining. purification of renta the renta was purified from the ferment supernatant after a 24-h induction in a 5-l fermenter. the bacteriocin activity of  × 105 au/mg with a -fold increase was obtained after gel filtration. the purified renta was analyzed by tricine-sds–page and showed a band at  kda representing the target protein band, corresponding with its theoretical molecular weight. antimicrobial spectrum of renta only l. ivanovii atcc e. faecalis cgmcc and e. faecalis cgmcc were sensitive to renta in the tested strains. other gram-positive bacteria, such as e. faecium cgmcc, s. aureus atcc s. epidermidis atcc b. licheniformis cgmcc, and b. coagulans cgmcc, were found to be resistant to renta. all of the gram-negative bacteria strains were resistant to renta in this assay. the mic and mbc of renta against l. ivanovii atcc were 20 ng/ml and 80 ng/ml, respectively, and were lower than those of ampicillin.table antimicrobial spectrum of renta strains antimicrobial activity + - + + - - - - - - - - - - - - - note: “+” refers to positive antimicrobial activity; “-” refers to negative antimicrobial activity. in-vitro killing curve assay the time-killing kinetics curve showed that the amount of l. ivanovii atcc increased from log10cfu/ml to log10cfu/ml within 10 h in the absence of renta. the decrease in the counts of l. ivanovii atcc varied considerably depending on the concentration of renta. for example, the maximum viability loss, which was approximately log cfu/ml, was reached within 2 h in 1 × mic of renta. the 2 × mic of renta could cause approximately log cfu/ml viability loss within 6 h. moreover, the mvl of l. ivanovii treated by renta at 4 × mic was approximately log cfu/ml within 4 h. although renta could inhibit the growth of l. ivanovii atcc the survivors resumed growth at 1× and 2 × mic of renta and 2 × mic ampicillin for l. ivanovii atcc after mvl was achieved. however, l. ivanovii atcc treated by 4 × mic of renta did not show re-growth within 10 h, revealing that ng/ml renta could effectively inhibit the growth of pathogenic bacteria for an extended time.figure time-kill curves of renta. l. ivanovii atcc was incubated in the presence of medium alone or in the presence of 1×, 2×, or 4× mic of renta. ampicillin of 2 × mic was used as a positive control. three duplicate observations were made; bars represent the standard error of the mean. effects of ph, temperature, proteolytic enzymes and nacl on the activity of renta as shown in figure 4a, renta was highly stable at a wide range of ph values. the activity of renta was maintained completely within a ph range of 2– at 37°c for 12 h and was 75% retained even after incubation at a ph of for 12 h. furthermore, the antimicrobial activity of renta was not affected by heat treatment at and 100°c for 1 h under acid conditions. the residual activity decreased to 20% at a ph of at 80°c, to 50% at a ph of at 100°c, and to 10% at a ph of at 100°c. in addition, the antimicrobial activity of renta was completely abolished by pepsin and trypsin treatment, but it retained % of initial antimicrobial activity after papain treatment at 37°c for 1 h.figure effects of ph, temperature and proteolytic enzymes on the renta activity. a, ph stability of renta. purified renta was incubated in buffers with a ph range from to at 37°c for 12 h. the initial activity of the sample in a buffer with a ph of was described as 100% activity. b, thermal stability of enta. purified renta was incubated in buffers with a ph range from to at temperatures of and 100°c for 1 h. the initial activity of the sample in a buffer with a ph of was described as 100% activity. c, proteolysis resistance of renta. purified renta was incubated with pepsin, papain and trypsin at 37°c for 4 h. the residual antimicrobial activity of samples was tested after the ph was readjusted to ph  with sodium phosphate buffer. the antimicrobial activity of renta against l. ivanovii atcc was slightly enhanced by the addition of and 50 mm nacl. the lowest amount of log cfu/ml was observed with a treatment of renta in 25 mm nacl. the other treatments, from – 400 mm nacl, had no significant effect on the bactericidal ability of renta. in the controls without renta, growth was not influenced by nacl.figure effect of nacl concentration on the activity of renta. control: l. ivanovii atcc was incubated in the absence of renta. 4 × mic: l. ivanovii atcc was incubated in the presence of renta at 4 × mic. discussion bacteriocin has attracted attention in recent years for its potential application as a food preservative and therapeutic antimicrobial agent. however, low production of these bacteriocins by native strains cannot meet the requirements of commercial applications. moreover, some enterococci strains were recognized as opportunistic pathogens associated with lots of infections. attempts to produce bacteriocins by using safe heterologous hosts have been undertaken in recent years, including some typical expression systems such as e. coli, l. lactis, and p. pastoris. although e. coli and l. lactis are widely used in heterologous protein expression because of their easy operation and safety, they are not suitable for bacteriocins due to toxicity to the host and low recovery percentages from the fusion protein. many bacteriocins, such as enterocin p, hiracin jm, enterocin l, pediocin pa- and enta, have been expressed as active forms in p. pastoris, but their expression levels remained low. it is known that codon optimization is a useful strategy to increase the yield of target protein during heterogeneous expression. many antimicrobial peptides, such as plectasin, nz and agplectasin, were expressed with high production through codon-usage optimization in our laboratory. in addition, divercin v a class iia bacteriocins was also expressed through this system. these cases encouraged us to use codon optimization to break through the bottleneck of low yield in heterologous expression of enta. the total protein level in the supernatant reached 180 mg/l with the activity of  au/ml at 24 h of induction in 5-l fermenter level after the gene was optimized. although the yield of target peptide was still low, and even lower than 280 mg/l as the highest result of expression in case of enterocin l in p. pastoris, it was much higher than that of pediocin pa-, enterocin p, divercin v and enta expressed in e. coli and l. lactis. furthermore, the production of renta increased -times compared with its native sequence expressed in p. pastoris, which indicated codon optimization is a good tool to enhance expression efficiency and level in p. pastoris, and at the same time, it also left a large room to improve in future work at the similar aim and technical scheme. however, the maximal activity of renta in the supernatant was reached at an early stage of induction. this is similar to previous results in which the highest level of renta was reached at 36 h. an even earlier peak of renta at 6 h was observed in other yeasts such as kluyveromyces lactis and hansenula polymorpha. obviously, its final successful application suffered from this strong decomposition in the supernatant at an earlier period of expression related to the possible disruption of renta to host cells and the proteolysis of the target protein. the latter situation was reported in “collagen-like” bacteriocin with a high cleavage by collagenase. however, the exact mechanism of the above described early degradation and its solution should be further studied. a series of methods, such as ion exchange chromatography, hydrophobic exchange chromatography, and gel filtration, were attempted to purify renta in this study. only gel filtration could purify renta with a yield of  mg/l after attempts with sp ff, cm ff, and phenyl hp in which almost all renta was lost in the penetration peak due to unknown reasons. although there are different antibacterial spectrums between class iia bacteriocins, they consistently have particularly high antibacterial activity against listeria — the most common pathogen in food — at nanomolar concentrations. the mics of purified native enta from e. faecium t against listerias ranged from to 120 ng/ml. similarly, renta also showed a narrow antibacterial spectrum including l. ivanovii atcc and with a low mic value of 20 ng/ml, it is approximately 20-fold lower than that of ampicillin. the re-growth after mvl achievement was a common phenomenon when the listeria was treated with bacteriocins such as enta, pediocin, sakacin a and enterococcin efs in relatively low concentrations, but we found no re-growth after mvl within 10 h when 4 × mic rena was used with the listeria, indicating that higher concentrations of rena are essential to inhibit the multiplication of listeria. the bactericidal activity and overall structure of pediocin pa- and piscicolin were well maintained at higher temperatures. the native enta was stable at 100°c and acidic ph conditions. we found that renta also exhibited high stability under a wide range of temperatures and ph levels. these properties were potentially due to the higher cysteine content of the antimicrobial peptides, similar to the enta containing four cysteine residues. in addition, the antimicrobial activity of some bacteriocins was significantly enhanced with the addition of nacl from to  m. however, the activity of renta against listeria was enhanced only at low nacl concentrations. despite the unknown mechanisms of the above differential effects, the high stability of renta over wide ranges of temperature, ph, and nacl concentration supports its use as a food preservative and drug candidate. due to the high content of basic and aromatic amino acids in class iia bacteriocins, pediocin pa- enterocin b, plantaricin and native enta were very sensitive to the digestive proteases trypsin and pepsin. similarly, the purified renta, with % basic amino acids and % aromatic amino acids, was inactivated with trypsin and pepsin. this high sensitivity to digestive proteases of renta contributes to its safety in foods and drugs, during and after oral administration.  in conclusion, renta, as an antimicrobial agent with merit, could selectively kill important and pathogenic listeria and retain bio-activity over a wide range of ph values, temperature and nacl concentrations. these excellent antibacterial properties make it a potential candidate as a food preservative and therapeutic antimicrobial agent. renta was successfully expressed in p. pastoris x- at the highest level of  au/ml and was purified through a gel filtration column. this yeast system may be a feasible technological approach to produce renta as a potent anti-listeria agent after further optimization. 
moreover, it showed high stability under a wide ph range and maintained full activity after 1 h of treatment at 80°c within a ph range of 2– its antimicrobial activity was enhanced at and 50 mm nacl, while 100–400 mm nacl had little effect on the bactericidal ability of renta. furthermore, it showed high stability under wide ranges of conditions, which could be potential as the new candidate of anti-listeria agent. the highest anti-listeria activity reached  au/ml from 180 mg/l of total protein after 24 h of induction in a 5-l fermenter. recombinant enterocin a, purified by gel filtration chromatography, showed very strong activity against listeria ivanovii atcc with a low mic of 20 ng/ml. the gene sequence of enterocin a was optimized based on preferential codon usage in pichia pastoris to increase its expression efficiency. enterocin alisteria ivanoviiantimicrobial activitypichia pastorisexpressionissue-copyright-statement© the author 2014 the enta was successfully expressed in p. pastoris, and this feasible system could pave the pre-industrial technological path of renta as a competent candidate as an anti-listeria agent. enterocin a is a classic iia bacteriocin isolated firstly from enterococcus faecium ctc with selective antimicrobial activity against listeria strains. the present work describes high production of enterocin a through codon optimization strategy and its character study.
 enterocin a is a classic iia bacteriocin isolated firstly from enterococcus faecium ctc with selective antimicrobial activity against listeria strains. however, the application of enterocin a as an anti-listeria agent has been limited due to its very low native yield. the present work describes high production of enterocin a through codon optimization strategy and its character study.  the gene sequence of enterocin a was optimized based on preferential codon usage in pichia pastoris to increase its expression efficiency. the highest anti-listeria activity reached  au/ml from 180 mg/l of total protein after 24 h of induction in a 5-l fermenter. recombinant enterocin a, purified by gel filtration chromatography, showed very strong activity against listeria ivanovii atcc with a low mic of 20 ng/ml. in addition, the renta killed over 99% of tested l. ivanovii atcc within 4 h when exposed to 4 × mic. moreover, it showed high stability under a wide ph range and maintained full activity after 1 h of treatment at 80°c within a ph range of 2– its antimicrobial activity was enhanced at and 50 mm nacl, while 100–400 mm nacl had little effect on the bactericidal ability of renta.  the enta was successfully expressed in p. pastoris, and this feasible system could pave the pre-industrial technological path of renta as a competent candidate as an anti-listeria agent. furthermore, it showed high stability under wide ranges of conditions, which could be potential as the new candidate of anti-listeria agent. keywords enterocin alisteria ivanoviiantimicrobial activitypichia pastorisexpressionissue-copyright-statement© the author 2014 