Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins...

Research ArticleCloning and Expression of Synthetic Genes Encodingthe Broad Antimicrobial Spectrum Bacteriocins SRCAM 602OR-7 E-760 and L-1077 by Recombinant Pichia pastoris

Sara Arbulu Juan J Jimeacutenez Loreto Guacutetiez Luis M CintasCarmen Herranz and Pablo E Hernaacutendez

Departamento de Nutricion Bromatologıa y Tecnologıa de los Alimentos Facultad de VeterinariaUniversidad Complutense de Madrid (UCM) Avenida Puerta de Hierro sn 28040 Madrid Spain

Correspondence should be addressed to Pablo E Hernandez ehernanvetucmes

Received 30 July 2014 Accepted 2 November 2014

Academic Editor J Eleazar Barboza-Corona

Copyright copy 2015 Sara Arbulu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocinsSRCAM 602 OR-7 E-760 and L-1077 by recombinant Pichia pastoris Synthetic genes matching the codon usage of P pastoriswere designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vectorpPICZ120572A The recombinant derived plasmids were linearized and transformed into competent P pastoris X-33 and the presenceof integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts The antimicrobialactivity expected in supernatants of the recombinant P pastoris producers was purified using a multistep chromatographicprocedure including ammonium sulfate precipitation desalting by gel filtration cation exchange- hydrophobic interaction- andreverse phase-chromatography (RP-FPLC) However a measurable antimicrobial activity was only detected after the hydrophobicinteraction and RP-FPLC steps of the purified supernatants MALDI-TOF MS analysis of the antimicrobial fractions eluted fromRP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected MALDI-TOFTOF MSanalysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragmentsnot related to the amino acid sequence of the cloned bacteriocins

1 Introduction

The antimicrobial peptides (AMPs) are broad spectrumsmallmolecular weight compoundswith antagonistic activityagainst bacteria viruses and fungi Among them bacteri-ocins form a widely studied and well-characterized groupof ribosomally synthesized peptides produced by bacteriaand those produced by lactic acid bacteria (LAB) attractconsiderable interest primarily as natural food preservativesbut also with great interest in exploring their applicationas therapeutic antimicrobial agents [1ndash3] Most LAB bac-teriocins are synthesized as biologically inactive precursorsor prepropeptides containing an N-terminal extension thatis cleaved off during export to generate their biologicallyactive ormature formThemature peptides are often cationicamphiphilic molecules that are generally classified into two

main classes the lantibiotics or class I bacteriocins that con-sist of modified bacteriocins and the class II or nonmodifiedbacteriocins [4 5]The class II bacteriocins have been furtherdivided into several subgroups from which the class IIa orpediocin-like bacteriocins show a strong antilisterial activityand the N-terminal consensus sequence YGNGV(X)C [5]

Considering the low amount of AMPs obtained fromtheir direct purification from natural producers and the ele-vated production costs of chemical synthesis the biologicalproduction of bacteriocins by heterologous microbial hostsmay provide an opportunity for their production in largeamounts and with higher specific antimicrobial activity [6ndash8] Furthermore the production of bacteriocins by yeastsmay have some advantages over bacterial cells regardingspecific posttranscriptional and posttranslational modifica-tions [9] Yeasts are also cost-effective producers with large

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 767183 11 pageshttpdxdoiorg1011552015767183

2 BioMed Research International

production and high yields of the desired protein [10] Pichiapastoris (currently reclassified as Komagataella pastoris) isbeing used as heterologous producer of bacteriocins becauseof its ability to produce large amounts of properly foldedand biologically active bacteriocins [8 11 12] P pastorisalso produces disulfide-bonded and glycosylated proteinswhich are crucial features for functionality [13] The use ofsynthetic genes may also constitute a successful approachfor heterologous production and functional expression ofbacteriocins by recombinant yeasts when the DNA sequenceencoding the bacteriocin is not available or difficult to obtain[12] Furthermore the use of synthetic genes matching thecodon usage of the host microorganism can have a significantimpact on gene expression levels and protein folding [14]

Several bacteriocins with broad antimicrobial spectrumhave been identified from chicken commensal bacteriaincluding bacteriocin SRCAM602 produced by Paenibacilluspolymyxa [15 16] bacteriocins OR-7 and L-1077 producedby Lactobacillus salivarius [17 18] and bacteriocins E-760 and E 50-52 produced by Enterococcus spp [19 20]All these bacteriocins have been reported to be activeagainst Gram-positive and Gram-negative bacteria includingCampylobacter spp reducing Campylobacter colonization inpoultry and considered potentially useful towards on-farmcontrol of this foodborne human pathogen [21] Most animalstudies suggest that these bacteriocins considerably reduceC jejuni colonization in chicken intestine and thus mayreduce Campylobacter spp infections in humans [17 19 20]However to our knowledge none of the genes encoding thesebacteriocins have been sequenced so far Accordingly in thisstudy we report the use of synthetic genes designed from thepublished amino acid sequence of the mature bacteriocinsSRCAM 602 OR-7 E-760 and L-1077 and with adaptedcodon usage for expression by P pastoris their cloning intothe protein expression vector pPICZ120572A and their expressionby recombinant P pastoris X-33

2 Materials and Methods21 Microbial Strains and Plasmids Microbial strains andplasmids used in this study are listed in Table 1 Enterococcusfaecium T136 and Pediococcus damnosus CECT4797 weregrown in MRS broth (Oxoid Ltd Basingstoke UK) at 32∘CP pastoris X-33 (Invitrogen SA Barcelona Spain) wascultured in YPDmedium (Sigma-Aldrich Inc St Louis MOUSA) at 30∘C with shaking (200ndash250 rpm) Escherichia coliJM109 (Promega WI USA) was grown in LB broth (Sigma-Aldrich) at 37∘C with shaking (250 rpm) Listeria monocyto-genes CECT4032 was grown in LB at 37∘C and Salmonellatyphimurium CECT443 was grown in TSB (Oxoid) at 37∘CCampylobacter jejuni ATCC33560 and C jejuni NCTC11168were grown in BHI supplemented with 1 defibrinated horseserum (BD Bioscience CA USA) at 37∘C in microaerophilicconditions E coli O157H7 was grown in LB at 37∘C withshaking (250 rpm) Yersinia ruckeri LMG3279 was grown inTSB at 28∘C Zeocin (Invitrogen) was added when neededat concentrations of 25 100 or 1000 120583gmL Strains citedas CECT belong to the Coleccion Espanola de CultivosTipo (Valencia Spain) ATCC to the American Type Culture

Collection (Rockville MD USA) and NCTC to the NationalCollection of Type Cultures (London UK)

22 Basic Genetic Techniques and Enzymes The publishedamino acid sequences of mature bacteriocins SRCAM 602OR-7 E-760 and L-1077 were used as templates to design thenucleotide sequence of the synthetic genes srcam602 or-7 e-760 and l-1077 matching the codon usage of P pastoris X-33These synthetic genes contained a 51015840-nucleotide appendixincluding aXhoI restriction site and a 31015840-nucleotide appendixincluding the termination of translation codon (TAA) andthe NotI restriction site All synthetic genes were supplied byGeneArt (Life Technologies Paisley UK) DNA restrictionenzymes were supplied by New England BioLabs (IpswichMA USA) Ligations were performed with the T4 DNA lig-ase (Roche Molecular Biochemicals Mannheim Germany)E coli JM109 cells were transformed as described by thesupplier Competent P pastoris X-33 cells were obtainedas recommended by the supplier and electroporation ofcompetent cells was performed as previously described [22]Electrocompetent cells were transformed with a Gene Pulserand Pulse Controller apparatus (Bio-Rad Laboratories Her-cules CA USA)

23 PCR Amplification and Nucleotide Sequencing Oligonu-cleotide primers were obtained from Sigma-Genosys Ltd(Cambridge UK) PCR amplifications were performed in50 120583L reaction mixtures containing 1 120583L of purified DNA70 pmol of each primer and 1U of Platinum Pfx DNAPolymerase (Invitrogen) in a DNA thermal cycler Techgene(Techne Cambridge UK) The PCR-generated fragmentswere purified by a NucleoSpin Extract II Kit (Macherey-Nagel GmbH amp Co Duren Germany) for cloning andnucleotide sequencing Nucleotide sequencing of the purifiedPCR products was performed using the ABI PRISM BigDyeTerminator cycle sequence reaction kit and the automaticDNA sequencerABI PRISMmodel 377 (Applied BiosystemsFoster City CA USA) at the Unidad de Genomica Facultadde Ciencias Biologicas Universidad Complutense de Madrid(UCM) Madrid Spain

24 Cloning of the srcam602 or-7 e-760 and l-1077 SyntheticGenes in P pastoris X-33 and Antimicrobial Activity of theTransformants The primers and inserts used for construc-tion of the recombinant plasmids are listed in Table 2Derivatives of plasmid pPICZ120572Awere constructed as followsprimers S602-F S071-F and SARP-R were used for PCRamplification from plasmids pMATSRCAM602 pMATOR-7 pMATE-760 and pMATL-1077 of nucleotide fragments inframe with the S cerevisiae 120572-factor secretion signal withoutthe Glu-Ala spacer adjacent to the Kex2 protease cleavagesite fused to the srcam602 or-7 e-760 and l-1077 syntheticgenes Digestion of the above cited fragments with the XhoI-NotI restriction enzymes permitted ligation of the result-ing R-SRCAM602 R-OR7 R-E760 and R-L1077 nucleotidefragments of 136- 181- 242- and 167-bp respectively intopPICZ120572A digested with the same enzymes to generate theplasmid-derived vectors pSRCAM602 pOR-7 pE-760 andpL-1077 respectively CompetentE coli JM109 cells were used

BioMed Research International 3

Table1Ba

cterialstrains

andplasmidsu

sedin

thisstu

dy

Strain

orplasmid

Descriptio

naSource

andor

referenceb

Strains

Enterococcus

faecium

T136

EnterocinAandBprod

ucerM

PApo

sitivec

ontro

lDNBT

APediococcusd

amnosusC

ECT4

797

MPA

indicatorm

icroorganism

CECT

Escherich

iacoliJM

109

Selectionof

recombinant

plasmids

Prom

ega

Pichiapasto

risX-

33Yeastp

rodu

cer

Invitro

genLifeTechno

logies

Plasmids

pMA-T

Amprcarrie

rofsyntheticgenes

GeneA

rtLifeTechno

logies

pPICZ120572

AZe

orintegrativ

eplasm

idcarrying

thes

ecretio

nsig

nalsequencefrom

theS

cerevisiae120572-fa

ctor

prepropeptidea

ndfunctio

nalsitesfor

integrationatthe51015840

AOX1

locuso

fPpastoris

X-33

Invitro

genLifeTechno

logies

pMAT

SRCA

M602

AmprpMA-Tplasmid

carrying

thesrcam

602synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

OR-7

AmprpMA-Tplasmid

carrying

theo

r-7synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

E-760

AmprpMA-Tplasmid

carrying

thee

-760

synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

L-1077

AmprpMA-Tplasmid

carrying

thel-10

77synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pSRC

AM602

pPICZ120572

Aderiv

ativew

iththesrcam

602synthetic

gene

Thiswork

pOR-7

pPICZ120572

Aderiv

ativew

iththeo

r-7synthetic

gene

Thiswork

pE-760

pPICZ120572

Aderiv

ativew

iththee

-760

synthetic

gene

Thiswork

pL-1077

pPICZ120572

Aderiv

ativew

iththel-10

77synthetic

gene

Thiswork

a Amprampicillinresis

tanceZe

orzeocinresistance

b DNBT

AD

epartamento

deNutric

ion

Brom

atologıayTecnologıade

losA

limentosFacultadde

Veterin

ariaU

niversidad

Com

plutense

deMadrid

(Madrid

Spain)CE

CTC

oleccion

Espano

lade

CultivosT

ipo

ValenciaSpain)


Table2Prim

ersa

ndPC

Rprod

uctsused

inthisstu

dy

Prim

ersPC

Rprod

ucts

orbacteriocins

Nucleotides

equence(51015840-31015840

)ord

escriptio

nAmplificatio

nsPrim

ers

S602-F

GCC

ATGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM602R-E7

60R

-L1077

S071-F

GTC

CAGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM

602R-E7

60R

-L1077R

-OR7

SARP

-RAG

GTA

CCAT

AAG

TTGCG

GCC

GC

R-OR7

ALFA-

FTA

CTAT

TGCC

AGCA

TTGCT

GC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

3AOX1-R

GCA

AAT

GGCA

TTCT

GAC

ATCC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

PCRprod

ucts

R-SR

CAM602

136-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticsrc

am602gene

with

theP

pastoris

codo

nusage

R-OR7

181-b

pXh

oIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticor-7

gene

with

theP

pastoris

codo

nusage

R-E7

60242-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

ynthetice-760gene

with

theP

pastoris

codo

nusage

R-L1077

167-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticl-1077gene

with

theP

pastoris

codo

nusage

Bacteriocins

BacSRC

AM602(aminoacid

sequ

ence)

ATYY

GNGLY

CNKQ

KHYT

WVDWNKA

SREIGKI

TVNGWVQ

HBa

cSRC

AM602(Ppastoris

codo

nusage)

gctacttactacggtaacggtctttactgtaacaagcagaagcact

acacttg

ggttg

actggaacaaggcttccagagagatcggtaag

atcactgttaacggttg

ggttc

aaca

BacO

R-7(aminoacid

sequ

ence)

KTYY

GTN

GVHCT

KNSLWGKV

RLKN

MK

YDQNTT

YMGRL

QDILLG

WAT

GAFG

KTFH

BacO

R-7(Ppastoris

codo

nusage)

aagacttactacggaactaacggtgttcactgtactaagaattcctt

gtggggtaaggttagattg

aagaacatgaagtacgaccagaaca

ctactta

catgggtagattg

caggacatcttgttg

ggttg

ggctact

ggtgctttc

ggtaagacttttcat

BacE-760

(aminoacid

sequ

ence)

NRW

YCNSA

AGGVG

GAAV

CGLA

GYV

GE

AKE

NIAGEV

RKGWGMAG

GFT

HNKA

CKS

FPGSG

WASG

BacE-760

(Ppastoris

codo

nusage)

aacagatggtactgtaactccgctgctggtggtgttg

gtggtgct

gctgtttgtggtttggctggtta

tgttg

gtgaggctaaagaaaacat

tgctggtgaggttagaaagggttggggtatggctggtggtttc

actcataacaaggcttg

taagtccttcccaggttctggttgggcttctggt

BacL-1077(aminoacid

sequ

ence)

TNYG

NGVG

VPD

AIM

AGIIKL

IFIFNIRQGY

NFG

KKAT

BacL-1077(Ppastoris

codo

nusage)

actaactacggtaacggtgttggtgttccagacgctattatggctg

gtatcatcaagttgatcttcatcttcaacatcagacagggtta

caac

ttcggtaagaaggctact


for cloning and vector propagation and the resulting transfor-mants were confirmed by PCR amplification and sequencingof the inserts Subsequently the SacI-linearized pSRCAM602pOR-7 pE-760 and pL-1077 vectors were transformed intocompetent P pastoris X-33 cells yielding zeocin resistantderivatives on YPD agar supplemented with zeocin (100and 1000120583gmL) and sorbitol (1M) The presence of theintegrated synthetic genes in the transformed yeast cells wasconfirmed by PCR and DNA sequencing of the inserts

The antimicrobial activity of individual P pastoris X-33SRCAM602 P pastoris X-33OR-7 P pastoris X-33E-760and P pastoris X-33L-1077 was screened by a streak-on-agar test (SOAT) Briefly the P pastoris transformants werestreaked onto BMMY buffered methanol complex medium(1 yeast 2 peptone 100mM potassium phosphate (pH6) 134 yeast nitrogen base (YNB) without amino acids4 times 10minus5 biotin 05 methanol) agar and grown at 30∘Cto induce production of the bacteriocins After incubationof the plates at 30∘C during 24 h 40mL of MRS soft-agar containing 105 cfumL of the indicator microorganismPediococcus damnosus CECT4797 was added to the platesthat were further incubated at 32∘C for 24 h inhibition halosvisualization

25 Purification of the Antimicrobial Activity of Supernatantsfrom the Recombinant Yeasts The antimicrobial activity ofsupernatants from P pastoris X-33 and P pastoris X-33(pPICZ120572A) and the recombinant P pastorisX-33SRCAM602P pastoris X-33OR-7 P pastoris X-33E-760 and P pastorisX-33L-1077 was purified using a previously described proce-dure [12] Briefly supernatants from early stationary phase05 L cultures of the recombinant yeasts grown in BMMYbuffered methanol complex medium at 30∘C were precipi-tated with ammonium sulfate desalted by gel filtration andsubjected to cation exchange-chromatography followed byhydrophobic interaction-chromatography and reverse phase-chromatography in a fast protein liquid chromatographysystem (RP-FPLC) (GE Healthcare Barcelona Spain) Theantimicrobial activity of the purified fractions was evaluatedagainst Pediococcus damnosus CECT4797 by the microtiterplate assay (MPA)

26 Mass Spectrometry Analysis of Purified SupernatantsFor determination of the molecular mass of peptides insupernatants of the recombinant yeasts the eluted puri-fied fractions with antimicrobial activity were subjectedto matrix-assisted laser desorptionionization time-of-flightmass spectrometry (MALDI-TOF MS) Briefly 1 120583L sampleswere spotted onto a MALDI target plate and allowed to air-dry at room temperature Then 04 120583L of a 3mgmL of 120572-cyano-4-hydroxy-transcinnamic acid matrix (Sigma) in 50acetonitrile were added to the dried peptide to digest spotsand allowed again to air-dry at room temperature MALDI-TOF MS analyses were performed in a 4800 ProteomicsAnalyzer MALDI-TOFTOF mass spectrometer (AppliedBiosystems Framingham MA) operated in 1 KV reflectormode All mass spectra were calibrated externally using astandard peptide mixture (AB Sciex MA USA)

To determine the amino acid sequence of the purifiedpeptides the eluted purified fractions with antimicrobialactivity were further subjected to MALDI-TOFTOF tandemmass spectrometry The samples were reduced alkylateddigested with trypsin [23] and analysed in a 4800 ProteomicsAnalyzer MALDI-TOFTOF mass spectrometer (AppliedBiosystems) The acquisition method for MS analysis was1 KV reflector positive mode Peptides from the MS spec-tra were manually selected for fragmentation analysis Theacquisition method for MSMS analysis was MSMS-1 KVin reflector positive mode with CID for fragmentation Thecollision gas was atmospheric and the precursor mass win-dow was plusmn5DaThe plate model and default calibration wereoptimized for theMSMS spectra processingThe parametersused to analyze the data were signal to noise = 20 resolutiongt6000 For protein identification the de novo amino acidsequence from the fragmentation spectra of selected peptideswas performed using the De Novo tool software (AppliedBiosystems) and tentative sequences were manually checkedand validated Homology searches of the deduced amino acidsequences were performed through the NCBIBlast All massspectrometry (MS) analyses were performed at theUnidad deProteomica Facultad de FarmaciaUniversidadComplutensede Madrid (UCM) Madrid Spain

27 Antimicrobial Activity of Purified Supernatants fromthe Recombinant Yeasts The antimicrobial activity of puri-fied supernatants from the recombinant P pastoris X-33SRCAM602 P pastoris X-33OR-7 P pastoris X-33E-760and P pastoris X-33L-1022 was evaluated against Listeriamonocytogenes CECT4032 E coli O157H7 Yersinia ruckeriLMG3279 Campylobacter jejuni ATCC33560 and C jejuniNCTC11168 by using a MPA as previously described [12]

3 Results31 Cloning of Synthetic Genes Encoding Bacteriocins andTheir Expression by Recombinant P pastoris The cloning ofPCR-amplified fragments from plasmids encoding syntheticgenes designed from the published amino acid sequence ofthe mature bacteriocins SRCAM 602 OR-7 E-760 and L-1077 into the protein expression vector pPICZ120572A resultedin the recombinant plasmids pSRCAM602 pOR-7 pE-760and pL-1077 (Table 1) Similarly transformation of the lin-earized plasmids into competent P pastoris X-33 permittedisolation of the P pastoris X-33SRCAM602 (srcam602) PpastorisX-33OR-7 (or-7)P pastorisX-33E-760 (e-760) andPpastorisX-33L-1077 (l-1077) recombinants However none ofthe recombinant yeasts showed direct antimicrobial activityagainst P damnosus CECT4797 even those recombinantyeasts selected for their high zeocin resistance (1000120583gmL)Colonies of P pastoris X-33 and P pastoris X-33 (pPICZ120572A)were used as bacteriocin-negative controls to discard thepossibility that the antimicrobial activity possibly observedwas due to metabolites other than bacteriocins

32 Purification of the Antimicrobial Activity of Supernatantsfrom the Recombinant P pastoris andMass Spectrometry Anal-ysis When supernatants from P pastorisX-33SRCAM602 P


100

90

80

70

0

10

20

30

40

60

50

1006683536640492732141501

Inte

nsity

()

33888135

3372231234054204

33563608

33262061

12 + 4

Mass (mz)

(a)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

652456194714380929041999

4287

21671641

24692393

30944207

33728801

33896047

28084270

29682549

47090483

50382788

Mass (mz)

(b)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100298183633744912645799

335333206313

54511987

49676523 64456704 70819937

Mass (mz)

(c)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100308183633744912645799

385218930446

19090667

24790750360197256

3583737534952290

49746992

Mass (mz)

(d)

Figure 1 Mass spectrometry analysis of purified supernatants from P pastoris X-33SRCAM602 (a) P pastoris X-33OR-7 (c) P pastoris X-33E-760 (b) and P pastoris X-33L-1077 (d) eluted after RP-FPLC Numbers indicate the molecular mass in daltons of most of the observedpeptide fragments

pastorisX-33OR-7P pastorisX-33E-76 andP pastorisX-33L-1077 were purified by a multistep chromatographic proce-dure only eluted fractions after the hydrophobic interaction-chromatography step showed full antimicrobial activityagainst Pediococcus damnosus CECT4797 as the indicatormicroorganism (Table 4) MALDI-TOFMS analysis of elutedfractions after the RP-FPLC step showed that supernatantsfrom P pastoris X-33SRCAM602 showed a major peptidefragment of 33888Da (Figure 1(a)) However the purifiedsupernatant from P pastoris X-33OR-7 showed a majorpeptide fragment of 30944Da and peptide fragments ofmajor and minor molecular mass (Figure 1(b)) Similarlya large display of peptide fragments of different molecularmasses was observed in the purified supernatants from Ppastoris X-33E-760 (Figure 1(c)) and P pastoris X-33L-1077(Figure 1(d)) MALDI-TOF MSMS spectrometry analysis oftrypsin-digested peptides from purified supernatants fromall recombinant P pastoris determined that none of themost probable or de novo amino acid sequence of theevaluated peptide fragments matched the expected aminoacid sequence of the bacteriocins SRCAM 602 OR-7 E-760 and L-1077 (Table 5) However some of the determinedpeptide fragments were homologous to those observed inproteins from ABC-transporter systems histidine kinaseprotein family peptidase C1 superfamily and nucleosomebinding proteins

33 Antimicrobial Activity of Purified Supernatants fromthe Recombinant Yeasts The purified supernatants from

recombinant P pastoris constructed for expression ofthe bacteriocins SRCAM 602 OR-7 E-760 and L-1077showed antimicrobial activity against Pediococcus damnosusCECT4797 but did not display a measurable antimicrobialactivity against L monocytogenesCECT4032 E coliO157H7Y ruckeri LMG3279 C jejuni ATCC33560 and C jejuniNCTC11168 when evaluated by a microtiter plate assay(MPA) The purified supernatants from P pastoris X-33 andP pastorisX-33 (pPICZ120572A) did not show antagonistic activityneither against Pediococcus damnosus CECT4797 nor againstany of the indicator bacteria cited above

4 DiscussionTo circumvent the proliferation of emerging pathogenicand antibiotic-resistant bacteria bacteriocins produced byLAB emerge as natural antimicrobial peptides with potentialapplications in food preservation livestock protectionand medical applications [1 24] However the high costof synthetic bacteriocin synthesis their low yields and theproduction of potential virulence factors from some naturalbacterial producers drive the exploration of microbialsystems for the biotechnological production of bacteriocinsby heterologous LAB and yeasts [6 8] Furthermore the useof synthetic genes may become a useful tool for productionand functional expression of bacteriocins by heterologousmicrobial hosts [12]

The development of heterologous expression systemsfor bacteriocins may offer a number of advantages over


Table 3 Amino acid sequence of cloned bacteriocins and other class IIa bacteriocins

Bacteriocin Amino acid sequence Number of amino acidsSRCAM 602 ATYYGNGLYCNKQKHYTWVDWNKASREIGKITVNGWVQH 39

OR-7 KTYYGTNGVHCTKNSLWGKVRLKNMKYDQNTTYMGRLQDILLGWATGAFGKTFH 54

E-760 NRWYCNSAAGGVGGAAVCGLAGYVGEAKENIAGEVRKGWGMAGGFTHNKACKSFPGSGWASG 62

E 50-52 TTKNYGNGVCNSVNWCQCGNVWASCNLATGCAAWLCKLA 39L-1077 TNYGNGVGVPDAIMAGIIKLIFIFNIRQGYNFGKKAT 37EntP ATRSYGNGVYCNNSKCWVNWGEAKENIAGIVISGWASGLAGMGH 44

EntA TTHSGKYYGNGVYCYKNKCTVDWAKATTCIAGMSIGGFLGGAIPGKC 47

HirJM79 ATYYGNGLYCNKEKCWVDWNQAKGEIGKIIVNGWVNHGPWAPRR 44

SakA ARSYGNGVYCNNKKCWVNRGEATQSIIGGMISGWASGLAGM 41Underlined the class IIa N-terminal consensus sequence YGNGV(X)C

Table 4Antimicrobial activity of fractions generated during purification of supernatants fromP pastorisX-SRCAM602P pastorisX-33OR-7P pastoris X-33E-760 and P pastoris X-33L-1077 grown in BMMY with methanol

Strain Antimicrobial activity (BUmL) of the purified fractionsa

SN AS GF SE OE RP-FPLCP pastoris X-33SRCAM602 NA NA NA NA 12800 1106531P pastoris X-33OR-7 NA NA 127 32 1391 10027P pastoris X-33E-760 NA NA NA NA 687 14442P pastoris X-33L-1077 NA NA 35 23 2069 13213Most of the data are mean from two independent determinations in triplicateaAntimicrobial activity against Pediococcus damnosus CECT4797 as determined by microtiter plate assay (MPA) BU bacteriocin units NA no activityPurification fractions SN supernatant AS ammonium sulfate precipitation GF gel filtration SE Sepharose fast flow eluate OE Octyl Sepharose eluate RP-FPLC reversed-phase eluate

native systems facilitating the control of bacteriocin geneexpression or achieving higher production levels Although anumber of yeast platforms have been used for the productionof peptides and proteins including bacteriocins [10 11 25]the use of synthetic genes has been only barely exploredfor their expression by recombinant yeasts [12] In thisstudy the protein expression vector pPICZ120572A containing anstrong and inducible promoter and the Kex2 signal cleavagesite for processing of fusion proteins [26] has been usedto drive the expression of synthetic genes encoding themature bacteriocins SRCAM 602 OR-7 E-760 and L-1077by recombinant P pastoris X-33 derivatives

Initial results with P pastoris X-33SRCAM602 P pastorisX-33OR-7 P pastoris X-33E-760 and P pastoris X-33L-1077determined that none of the recombinant yeasts encodingsynthetic genes for expression of the cloned bacteriocinsshowed antimicrobial activity when individual colonies werescreened by a streak-on-agar test (SOAT) Highly variableyields of secreted proteins have been achieved using the Ppastoris expression system and cases of low secretory yieldsor complete failure in protein production have also beenreported [3 27 28] A number of factors may affect the pro-duction of foreign peptides by heterologous yeasts including

copy number integration of the expression vectors in theyeast DNA mRNA stability errors in mRNA translationuncoordinated rates of protein synthesis folding and translo-cation and undesired proteolysis of heterologous proteinsby resident proteases or by proteases in the extracellularspace being secreted cell-wall associated or released into theculture medium as a result of cell disruption [29ndash31] Theuse of the P pastoris expression system for overproductionof peptides and proteins is known to be somewhat hamperedby its unpredictable yields of production of heterologousproteins which is now believed to be caused in part bytheir varied efficiencies to traffic through the host secretionmachinery [3 28]

The amino acid sequence following the Kex2 secretionsignal may also interfere with the secretion of fused peptidesor proteins by recombinant P pastoris Furthermore theyields of many recombinant proteins seem to be influencedby the Kex2 P11015840 site residue [3] In this study the Kex2 P11015840site residues formature SRCAM602 OR-7 E-760 and L-1077were the amino acids A K N and T respectively (Table 3)However the cloning in P pastoris of the bacteriocins ente-rocin A (EntA) and enterocin P (EntP) with the Kex2 P11015840 siteresidues A and T respectively showed an overproduction of


Table 5 Results obtained by MALDI-TOFTOFMS analysis of the eluted RP-FPLC fractions from purified supernatants of the recombinantP pastoris X-33 derivatives

Bacteriocin Predicted molecular mass (Da) Trypsin-digested precursor (Da) Amino acid sequence (MSMS analysis)

SRCAM 602 4630192665150095161497

SANALRPPTGDKENAAKASSVPARKTGGNRAVSGAGEIAAR

OR-7 62151 Deficient signal mdash

E-760 61798 116663157968

VGNPLHGIFGRSLSAYMFFANEQR

L-1077 40026 163876171873

IVGSQAGIGEYLFERLVELSEQELVDCER

both bacteriocins over their natural producers and an intenseantimicrobial activity when colonies of the recombinantyeasts were evaluated by the SOAT [11 22]

The use of a multistep chromatographic procedurefor purification of the expected antimicrobial activity insupernatants of the recombinant P pastoris determinedthat only eluted fractions after the hydrophobic interaction-chromatography step showed full antimicrobial activity(Table 4) probably due to removal of antimicrobialinhibitors disaggregation of the bacteriocins or changesin conformation of the bacteriocins in the hydrophobicsolvent The antimicrobial activity of the supernatantfrom P pastoris X-33SRCAM602 was much higher thanthat of the rest of the recombinant P pastoris Whilebeing interesting this was not an unexpected observationsince purification of the circular bacteriocin garvicin MLproduced by Lactococcus garvieae DCC43 showed a higherantibacterial activity and a broader antimicrobial spectrumas it was increasingly purified [32] However MALDI-TOFMS analysis of the purified supernatants from P pastorisX-33SRCAM602 P pastorisX-33OR-7 P pastorisX-33E-760and P pastoris X-33L-1077 mostly showed a large displayof peptide fragments of different molecular mass thandeduced from the calculated molecular mass of the clonedbacteriocins (Figure 1) The different molecular mass of theresulting peptide fragments may suggest the existence oftruncated bacteriocins the interaction of the bacteriocinswith unknown biological compounds or the bacteriocinsbeing subjected to posttranslational modifications (PTM)such as phosphorylation acetylation methylation oxidationformylation disulfide bond formation and N-linked andO-linked glycosylation [8 33] The presence of cysteineresidues in the bacteriocins SRCAM 602 OR-7 and E-760would permit the formation of disulfide bridges but alsopermits its oxidation glutathionylation and cysteinylationThe absence in all cloned bacteriocins of attachment sites forN-linkages precludes its N-glycosylation but the presence ofthreonines and serines makes the bacteriocins sensitive toO-glycosylation [8 12 33]

However MALDI-TOF MSMS analysis of trypsin-digested peptides from purified supernatants from all recom-binant P pastoris determined that none of the evaluated pep-tide fragments matched the expected amino acid sequence

of the bacteriocins SRCAM 602 OR-7 E-760 and L-1077(Table 5) From the results obtained it may be suggestedthat very low yields of secreted andor purified bacteriocinsare obtained after cloning of synthetic genes encoding thebacteriocins SRCAM 602 OR-7 E-760 and L-1077 in Ppastoris This observation was also not unexpected becausebacteriocins cloned into Saccharomyces cerevisiae P pastorisKluyveromyces lactis Hansenula polymorpha and Arxulaadeninivorans have been produced with variable successregarding their production secretion and functional expres-sion [11] Furthermore since one of the main bottlenecks inrecombinant protein production is the inability of foreignpeptides to reach their native conformation in heterologousyeast hosts it could also happen that incorrectly foldedSRCAM 602 OR-7 E-760 and L-1077 are accumulated inthe endoplasmic reticulum (ER) of recombinant P pastorisactivating the unfolded protein response (UPR) and theER-associated degradation (ERDA) of the misfolded bacte-riocins leading to persistent ER stress conditions causingmuch lower efficiencies to traffic through the host secretionmachinery apoptosis and cell death [8 28 34 35] In anycase the inconsistent secretory productivity among recom-binant proteins has always been a major obstacle for routineapplication of P pastoris as an eukaryotic protein expressionsystem in both research and industry [3]

The purified supernatants from the recombinant P pas-toris constructed for expression of the bacteriocins SRCAM602 OR-7 E-760 and L-1077 showed a measurable antimi-crobial activity against Pediococcus damnosus CECT4797(Table 4) but not against LmonocytogenesCECT4032 E coliO157H7 Y ruckeri LMG3279 C jejuni ATCC33560 and CjejuniNCTC11168 One of the remarkable features of bacteri-ocins is that they are very potent being active in nanomolarconcentrations thereby surpassing by about 1000-fold theactivity ofAMPsproduced by eukaryotic cells [36]One of themajor reasons for this extreme potency is that bacteriocinsapparently recognize specific receptors on target cells whilethe interactions between AMPs and microorganisms aremostly nonspecific Furthermore the target receptor for classIIa bacteriocins has been identified as proteins of the sugartransportermannose phosphotransferase system (Man-PTS)with the most potent receptors being those found in Listeriaspp [37 38] However the very low yields of secreted or


purified bacteriocins in supernatants of the recombinant Ppastorismay be responsible for their nondetected antilisterialactivity

It could be also hypothesized that peptide fragmentsaggregated to or coeluting with the purified bacteriocinsmay be responsible for the antimicrobial activity of theeluates against the sensitive indicator Pediococcus damnosusCECT4797 but not against any other of the indicator bacteriatested Many proteins contain encrypted within their pri-mary structure bioactive peptides with antimicrobial activityfollowing hydrolytic release from the native molecule [3940] Incorrect disulfide bond formation misfolding of thesecreted bacteriocin and extensive PTMs were suggested tobe responsible for the lower antilisterial activity and thenonmeasurable antimicrobial activity against Gram-negativebacteria of the bacteriocin E 50-52 (BacE50-52) producedby recombinant P pastoris X-33BE50-52S and K lactisGG799BE50-52S [12] This bacteriocin originally producedby Enterococcus faeciumB-30746 was also reported to displaya high and broad antimicrobial activity againstGram-positiveand Gram-negative bacteria including Campylobacter spp[20 41]

The correct processing secretion and functional expres-sion of the bacteriocins EntP [22] hiracin JM79 (HirJM79)[42] and EntA [11] produced by recombinant yeasts contrastwith the low biological activity of the sakacin A (SakA)and the chimera EntPSakA produced by recombinant Ppastoris and K lactis producers [8] Misfolding of SakA andEntPSakA and induction of the yeastsrsquo UPR may be respon-sible for apoptosis in recombinant P pastoris producers ofSakA and for extensive PTMs in recombinant P pastorisand K lactis producers of SakA and EntPSakA [8] Theseresults obtained by our research group also contrast withthe low antimicrobial activity against Pediococcus damnosusCECT4797 and the absence of antimicrobial activity againstGram-positive and Gram-negative bacteria of the purifiedsupernatants from recombinant P pastoris encoding themature bacteriocins SRCAM 602 OR-7 E-760 and L-1077

Nevertheless it should be also worth to notice thatbacteriocins SRCAM 602 [16] OR-7 [17] E-760 [19] andL-1077 [18] although being reported as bacteriocins witha broad antimicrobial activity against Gram-positive andGram-negative microorganisms including Campylobacterspp have not been fully characterized at their biochemicaland genetic level the genetic identification of their struc-tural and adjacent genes has not been yet reported andtheir molecular masses deduced from their reported aminoacid sequences were not identical to the experimentallydetermined molecular mass from the purified bacteriocins[16ndash19] Furthermore recent reports suggest that bacte-riocin SRCAM 602 previously reported to be producedby Paenibacillus polymyxa NRRL B-30509 and claimed tobe responsible for inhibition of C jejuni could not bedetected in the purified supernatants of the producer strainwhile the srcam602 structural gene was not found via aPCR-based approach using degenerate nucleotide primersor by genomic sequencing of the bacteriocin producer[43] Similarly Bacillus circulans (now Paenibacillus terrae)NRRLB-30644 previously reported to produce SRCAM1580

a bacteriocin active against C jejuni [16] has been recentlysuggested not to produce this bacteriocin and no geneticdeterminants for its productionwere shown Instead the anti-Campylobacter activity of this strain is due to the productionof the lipopeptide tridecaptin A1 whereas this strain alsoproduces the novel lantibiotic paenicidin B active againstGram-positive bacteria [44]

Although the cloning in recombinant yeasts of syntheticgenes encoding bacteriocins drives the production antimi-crobial activity and specific antimicrobial activity of thecloned bacteriocins in the absence of dedicated immunity andsecretion proteins [12] these results contrast with the negli-gible antimicrobial activity against Gram-positive andGram-negative bacteria of purified supernatants from recombinantP pastoris encoding the bacteriocins SRCAM 602 OR-7 E-760 and L-1077 Accordingly since production of bacteri-ocins from synthetic bacteriocin genes is difficult to predictfurther efforts should be performed for a more efficientgenetically engineered production and functional expressionof other bacteriocin synthetic genes or their chimeras byheterologous producer yeastsThedesign of further and novelsuccessful genetic approaches for production and functionalexpression of bacteriocins by yeasts would facilitate theirbiotechnological applications as natural antimicrobial agentsin food animal husbandry and medicine

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thiswork was partially supported by Project AGL2012-34829from Ministerio de Economıa y Competitividad (MINECO)andProjectAGL2009-08348 from theMinisterio deCiencia eInnovacion (MICINN) by Grant GR35-10A from the BSCH-UCM and by Grants S2009AGR-1489 and S2013ABI-2747from the Comunidad de Madrid (CAM) S Arbulu holdsa fellowship (FPI) from the MINECO J J Jimenez and LGutiez held a fellowship from the MICINN and the Minis-terio de Educacion y Ciencia (MEC) Spain respectively

References

[1] C T Lohans and J C Vederas ldquoDevelopment of class IIabacteriocins as therapeutic agentsrdquo International Journal ofMicrobiology vol 2012 Article ID 386410 13 pages 2012

[2] P D Cotter R P Ross and C Hill ldquoBacteriocins-a viablealternative to antibioticsrdquo Nature Reviews Microbiology vol 11no 2 pp 95ndash105 2013

[3] S Yang Y Kuang H Li et al ldquoEnhanced production ofrecombinant secretory proteins in Pichia pastoris by optimizingKex2 P1rsquo siterdquo PLoS ONE vol 8 no 9 Article ID e75347 2013

[4] P D Cotter C Hill and R P Ross ldquoFood microbiologybacteriocins developing innate immunity for foodrdquo NatureReviews Microbiology vol 3 no 10 pp 777ndash788 2005

[5] D Drider G Fimland Y Hechard L M McMullen andH Prevost ldquoThe continuing story of class IIa bacteriocinsrdquo


Microbiology and Molecular Biology Reviews vol 70 no 2 pp564ndash582 2006

[6] J Borrero J J Jimenez L Gutiez C Herranz L M Cintas andP E Hernandez ldquoProtein expression vector and secretion signalpeptide optimization to drive the production secretion andfunctional expression of the bacteriocin enterocin A in lacticacid bacteriardquo Journal of Biotechnology vol 156 no 1 pp 76ndash86 2011

[7] N S Parachin K C Mulder A A B Viana S C Dias andO L Franco ldquoExpression systems for heterologous productionof antimicrobial peptidesrdquo Peptides vol 38 no 2 pp 446ndash4562012

[8] J J Jimenez J Borrero D B Diep et al ldquoCloning productionand functional expression of the bacteriocin sakacin A (SakA)and two SakA-derived chimeras in lactic acid bacteria (LAB)and the yeasts Pichia pastoris and Kluyveromyces lactisrdquo Journalof Industrial Microbiology amp Biotechnology vol 40 no 9 pp977ndash993 2013

[9] M Gao and Z Shi ldquoProcess control and optimization forheterologous protein production by methylotrophic pichiapastorisrdquo Chinese Journal of Chemical Engineering vol 21 no2 pp 216ndash226 2013

[10] MAhmadMHirzH Pichler andH Schwab ldquoProtein expres-sion in Pichia pastoris recent achievements and perspectivesfor heterologous protein productionrdquoApplied Microbiology andBiotechnology vol 98 pp 5301ndash5317 2014

[11] J Borrero G Kunze J Jimenez et al ldquoCloning productionand functional expression of the bacteriocin enterocin Aproduced by Enterococcus faecium T136 by the Yeasts PichiapastorisKluyveromyces lactisHansenula polymorpha andArx-ula adeninivoransrdquo Applied and Environmental Microbiologyvol 78 no 16 pp 5956ndash5961 2012

[12] J J Jimenez J Borrero L Gutiez et al ldquoUse of syntheticgenes for cloning production and functional expression of thebacteriocins enterocin A and bacteriocin E 50-52 by Pichiapastoris andKluyveromyces lactisrdquoMolecular Biotechnology vol56 no 6 pp 571ndash583 2014

[13] R A J Darby S P Cartwright M V Dilworth and R M BillldquoWhich yeast species shall i choose Saccharomyces cerevisiaeversus Pichia pastorisrdquo Methods in Molecular Biology vol 866pp 11ndash23 2012

[14] F Oberg J Sjohamn M T Conner R M Bill and K HedfalkldquoImproving recombinant eukaryotic membrane protein yieldsin Pichia pastoris the importance of codon optimization andclone selectionrdquoMolecular Membrane Biology vol 28 no 6 pp398ndash411 2011

[15] N J Stern E A Svetoch B V Eruslanov et al ldquoPaenibacilluspolymyxa purified bacteriocin to control Campylobacter jejuniin chickensrdquo Journal of Food Protection vol 68 no 7 pp 1450ndash1453 2005

[16] E A Svetoch N J Stern B V Eruslanov et al ldquoIsolation ofBacillus circulans and Paenibacillus polymyxa strains inhibitoryto Campylobacter jejuni and characterization of associatedbacteriocinsrdquo Journal of Food Protection vol 68 no 1 pp 11ndash17 2005

[17] N J Stern E A Svetoch B V Eruslanov et al ldquoIsolation of aLactobacillus salivarius strain and purification of its bacteriocinwhich is inhibitory to Campylobacter jejuni in the chicken gas-trointestinal systemrdquo Antimicrobial Agents and Chemotherapyvol 50 no 9 pp 3111ndash3116 2006

[18] E A Svetoch B V Eruslanov V P Levchuk et al ldquoIsolationof Lactobacillus salivarius 1077 (NRRL B-50053) and character-ization of its bacteriocin including the antimicrobial activityspectrumrdquoApplied and Environmental Microbiology vol 77 no8 pp 2749ndash2754 2011

[19] J E Line E A Svetoch B V Eruslanov et al ldquoIsolationand purification of enterocin E-760 with broad antimicrobialactivity against Gram-positive and Gram-negative bacteriardquoAntimicrobial Agents andChemotherapy vol 52 no 3 pp 1094ndash1100 2008

[20] E A Svetoch B V Eruslanov V V Perelygin et al ldquoDiverseantimicrobial killing by Enterococcus faecium E 50-52 bacteri-ocinrdquo Journal of Agricultural and Food Chemistry vol 56 no 6pp 1942ndash1948 2008

[21] E A Svetoch and N J Stern ldquoBacteriocins to control Campy-lobacter spp in poultry-a reviewrdquo Poultry Science vol 89 no 8pp 1763ndash1768 2010

[22] J Gutierrez R Criado M Martın C Herranz L M Cintasand P E Hernandez ldquoProduction of enterocin P an antilisterialpediocin-like bacteriocin from Enterococcus faecium P13 inPichia pastonsrdquoAntimicrobial Agents andChemotherapy vol 49no 7 pp 3004ndash3008 2005

[23] A Shevchenko A Loboda W Ens and K G StandingldquoMALDI quadrupole time-of-flight mass spectrometry a pow-erful tool for proteomic researchrdquo Analytical Chemistry vol 72no 9 pp 2132ndash2141 2000

[24] World Health Organization (WHO) ldquoAntimicrobial resis-tance global report on surveillance 2014rdquo httpwwwsearowhointthailandpublications20139789241564748en

[25] E Boer G Steinborn G Kunze and G Gellissen ldquoYeastexpression platformsrdquo Applied Microbiology and Biotechnologyvol 77 no 3 pp 513ndash523 2007

[26] JM Cregg J L Cereghino J Shi andD R Higgins ldquoRecombi-nant protein expression in Pichia pastorisrdquoApplied Biochemistryand BiotechnologymdashPart B Molecular Biotechnology vol 16 no1 pp 23ndash52 2000

[27] O J Burrowes G Diamond and T C Lee ldquoRecombinantexpression of pleurocidin cDNA using the Pichia pastorisexpression systemrdquo Journal of Biomedicine and Biotechnologyvol 2005 no 4 pp 374ndash384 2005

[28] K R Love T J Politano V Panagiotou B Jiang T A Stadheimand J C Love ldquoSystematic single-cell analysis of pichia pastorisreveals secretory capacity limits productivityrdquo PLoS ONE vol 7no 6 Article ID e37915 2012

[29] H A Kang E-S Choi W-K Hong et al ldquoProteolytic stabilityof recombinant human serumalbumin secreted in the yeast Sac-charomyces cerevisiaerdquo Applied Microbiology and Biotechnologyvol 53 no 5 pp 575ndash582 2000

[30] M W T Werten and F A De Wolf ldquoReduced proteolysis ofsecreted gelatin and Yps1-mediated 120572-factor leader processingin a Pichia pastoris kex2 disruptantrdquoApplied and EnvironmentalMicrobiology vol 71 no 5 pp 2310ndash2317 2005

[31] Z Ni X Zhou X Sun Y Wang and Y Zhang ldquoDecrease ofhirudin degradation by deleting the KEX1 gene in recombinantPichia pastorisrdquo Yeast vol 25 no 1 pp 1ndash8 2008

[32] J Borrero D A Brede M Skaugen et al ldquoCharacterizationof garvicin ML a novel circular bacteriocin produced by Lac-tococcus garvieae DCC43 isolated from mallard ducks (Anasplatyrhynchos)rdquo Applied and Environmental Microbiology vol77 no 1 pp 369ndash373 2011


[33] Y Zhao and O N Jensen ldquoModification-specific proteomicsstrategies for characterization of post-translational modifica-tions using enrichment techniquesrdquo Proteomics vol 9 no 20pp 4632ndash4641 2009

[34] B Gasser M Saloheimo U Rinas et al ldquoProtein folding andconformational stress inmicrobial cells producing recombinantproteins a host comparative overviewrdquoMicrobial Cell Factoriesvol 7 article 11 2008

[35] A Stolz and D H Wolf ldquoEndoplasmic reticulum associatedprotein degradation a chaperone assisted journey to hellrdquoBiochimica et Biophysica ActamdashMolecular Cell Research vol1803 no 6 pp 694ndash705 2010

[36] E Breukink I Wiedemann C van Kraaij O P Kuipers H-GSahl and B de Kruijff ldquoUse of the cell wail precursor lipid II bya pore-forming peptide antibioticrdquo Science vol 286 no 5448pp 2361ndash2364 1999

[37] D B Diep M Skaugen Z Salehian H Holo and I F NesldquoCommonmechanisms of target cell recognition and immunityfor class II bacteriocinsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 104 no 7 pp 2384ndash2389 2007

[38] M Kjos J Borrero M Opsata et al ldquoTarget recognition resis-tance immunity and genome mining of class II bacteriocinsfrom Gram-positive bacteriardquoMicrobiology vol 157 no 12 pp3256ndash3267 2011

[39] M Phelan A Aherne R J FitzGerald and N M OrsquoBrienldquoCasein-derived bioactive peptides biological effects industrialuses safety aspects and regulatory statusrdquo International DairyJournal vol 19 no 11 pp 643ndash654 2009

[40] G D BrandM T QMagalhaes M L P Tinoco et al ldquoProbingprotein sequences as sources for encrypted antimicrobial pep-tidesrdquo PLoS ONE vol 7 no 9 Article ID e45848 2012

[41] E A Svetoch V P Levchuk V D Pokhilenko et al ldquoInac-tivating methicillin-resistant Staphylococcus aureus and otherpathogens by use of bacteriocins OR-7 and E 50-52rdquo Journal ofClinical Microbiology vol 46 no 11 pp 3863ndash3865 2008

[42] J Sanchez J Borrero B Gomez-Sala et al ldquoCloning andheterologous production of hiracin JM79 a Sec-dependentbacteriocin produced by Enterococcus hirae DCH5 in lacticacid bacteria and Pichia pastorisrdquo Applied and EnvironmentalMicrobiology vol 74 no 8 pp 2471ndash2479 2008

[43] C T Lohans Z Huang M J Van Belkum et al ldquoStructuralcharacterization of the highly cyclized lantibiotic paenicidin Avia a partial desulfurizationreduction strategyrdquo Journal of theAmerican Chemical Society vol 134 no 48 pp 19540ndash195432012

[44] C T LohansM J VanBelkum S A Cochrane et al ldquoBiochem-ical structural and genetic characterization of tridecaptin A

1

an antagonist of Campylobacter jejunirdquo ChemBioChem vol 15no 2 pp 243ndash249 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


production and high yields of the desired protein [10] Pichiapastoris (currently reclassified as Komagataella pastoris) isbeing used as heterologous producer of bacteriocins becauseof its ability to produce large amounts of properly foldedand biologically active bacteriocins [8 11 12] P pastorisalso produces disulfide-bonded and glycosylated proteinswhich are crucial features for functionality [13] The use ofsynthetic genes may also constitute a successful approachfor heterologous production and functional expression ofbacteriocins by recombinant yeasts when the DNA sequenceencoding the bacteriocin is not available or difficult to obtain[12] Furthermore the use of synthetic genes matching thecodon usage of the host microorganism can have a significantimpact on gene expression levels and protein folding [14]

Several bacteriocins with broad antimicrobial spectrumhave been identified from chicken commensal bacteriaincluding bacteriocin SRCAM602 produced by Paenibacilluspolymyxa [15 16] bacteriocins OR-7 and L-1077 producedby Lactobacillus salivarius [17 18] and bacteriocins E-760 and E 50-52 produced by Enterococcus spp [19 20]All these bacteriocins have been reported to be activeagainst Gram-positive and Gram-negative bacteria includingCampylobacter spp reducing Campylobacter colonization inpoultry and considered potentially useful towards on-farmcontrol of this foodborne human pathogen [21] Most animalstudies suggest that these bacteriocins considerably reduceC jejuni colonization in chicken intestine and thus mayreduce Campylobacter spp infections in humans [17 19 20]However to our knowledge none of the genes encoding thesebacteriocins have been sequenced so far Accordingly in thisstudy we report the use of synthetic genes designed from thepublished amino acid sequence of the mature bacteriocinsSRCAM 602 OR-7 E-760 and L-1077 and with adaptedcodon usage for expression by P pastoris their cloning intothe protein expression vector pPICZ120572A and their expressionby recombinant P pastoris X-33

2 Materials and Methods21 Microbial Strains and Plasmids Microbial strains andplasmids used in this study are listed in Table 1 Enterococcusfaecium T136 and Pediococcus damnosus CECT4797 weregrown in MRS broth (Oxoid Ltd Basingstoke UK) at 32∘CP pastoris X-33 (Invitrogen SA Barcelona Spain) wascultured in YPDmedium (Sigma-Aldrich Inc St Louis MOUSA) at 30∘C with shaking (200ndash250 rpm) Escherichia coliJM109 (Promega WI USA) was grown in LB broth (Sigma-Aldrich) at 37∘C with shaking (250 rpm) Listeria monocyto-genes CECT4032 was grown in LB at 37∘C and Salmonellatyphimurium CECT443 was grown in TSB (Oxoid) at 37∘CCampylobacter jejuni ATCC33560 and C jejuni NCTC11168were grown in BHI supplemented with 1 defibrinated horseserum (BD Bioscience CA USA) at 37∘C in microaerophilicconditions E coli O157H7 was grown in LB at 37∘C withshaking (250 rpm) Yersinia ruckeri LMG3279 was grown inTSB at 28∘C Zeocin (Invitrogen) was added when neededat concentrations of 25 100 or 1000 120583gmL Strains citedas CECT belong to the Coleccion Espanola de CultivosTipo (Valencia Spain) ATCC to the American Type Culture

Collection (Rockville MD USA) and NCTC to the NationalCollection of Type Cultures (London UK)

22 Basic Genetic Techniques and Enzymes The publishedamino acid sequences of mature bacteriocins SRCAM 602OR-7 E-760 and L-1077 were used as templates to design thenucleotide sequence of the synthetic genes srcam602 or-7 e-760 and l-1077 matching the codon usage of P pastoris X-33These synthetic genes contained a 51015840-nucleotide appendixincluding aXhoI restriction site and a 31015840-nucleotide appendixincluding the termination of translation codon (TAA) andthe NotI restriction site All synthetic genes were supplied byGeneArt (Life Technologies Paisley UK) DNA restrictionenzymes were supplied by New England BioLabs (IpswichMA USA) Ligations were performed with the T4 DNA lig-ase (Roche Molecular Biochemicals Mannheim Germany)E coli JM109 cells were transformed as described by thesupplier Competent P pastoris X-33 cells were obtainedas recommended by the supplier and electroporation ofcompetent cells was performed as previously described [22]Electrocompetent cells were transformed with a Gene Pulserand Pulse Controller apparatus (Bio-Rad Laboratories Her-cules CA USA)

23 PCR Amplification and Nucleotide Sequencing Oligonu-cleotide primers were obtained from Sigma-Genosys Ltd(Cambridge UK) PCR amplifications were performed in50 120583L reaction mixtures containing 1 120583L of purified DNA70 pmol of each primer and 1U of Platinum Pfx DNAPolymerase (Invitrogen) in a DNA thermal cycler Techgene(Techne Cambridge UK) The PCR-generated fragmentswere purified by a NucleoSpin Extract II Kit (Macherey-Nagel GmbH amp Co Duren Germany) for cloning andnucleotide sequencing Nucleotide sequencing of the purifiedPCR products was performed using the ABI PRISM BigDyeTerminator cycle sequence reaction kit and the automaticDNA sequencerABI PRISMmodel 377 (Applied BiosystemsFoster City CA USA) at the Unidad de Genomica Facultadde Ciencias Biologicas Universidad Complutense de Madrid(UCM) Madrid Spain

24 Cloning of the srcam602 or-7 e-760 and l-1077 SyntheticGenes in P pastoris X-33 and Antimicrobial Activity of theTransformants The primers and inserts used for construc-tion of the recombinant plasmids are listed in Table 2Derivatives of plasmid pPICZ120572Awere constructed as followsprimers S602-F S071-F and SARP-R were used for PCRamplification from plasmids pMATSRCAM602 pMATOR-7 pMATE-760 and pMATL-1077 of nucleotide fragments inframe with the S cerevisiae 120572-factor secretion signal withoutthe Glu-Ala spacer adjacent to the Kex2 protease cleavagesite fused to the srcam602 or-7 e-760 and l-1077 syntheticgenes Digestion of the above cited fragments with the XhoI-NotI restriction enzymes permitted ligation of the result-ing R-SRCAM602 R-OR7 R-E760 and R-L1077 nucleotidefragments of 136- 181- 242- and 167-bp respectively intopPICZ120572A digested with the same enzymes to generate theplasmid-derived vectors pSRCAM602 pOR-7 pE-760 andpL-1077 respectively CompetentE coli JM109 cells were used


Table1Ba

cterialstrains

andplasmidsu

sedin

thisstu

dy

Strain

orplasmid

Descriptio

naSource

andor

referenceb

Strains

Enterococcus

faecium

T136

EnterocinAandBprod

ucerM

PApo

sitivec

ontro

lDNBT

APediococcusd

amnosusC

ECT4

797

MPA

indicatorm

icroorganism

CECT

Escherich

iacoliJM

109

Selectionof

recombinant

plasmids

Prom

ega

Pichiapasto

risX-

33Yeastp

rodu

cer

Invitro

genLifeTechno

logies

Plasmids

pMA-T

Amprcarrie

rofsyntheticgenes

GeneA

rtLifeTechno

logies

pPICZ120572

AZe

orintegrativ

eplasm

idcarrying

thes

ecretio

nsig

nalsequencefrom

theS

cerevisiae120572-fa

ctor

prepropeptidea

ndfunctio

nalsitesfor


AOX1

locuso

fPpastoris

X-33

Invitro

genLifeTechno

logies

pMAT

SRCA

M602

AmprpMA-Tplasmid

carrying

thesrcam

602synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

OR-7

AmprpMA-Tplasmid

carrying

theo

r-7synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

E-760

AmprpMA-Tplasmid

carrying

thee

-760

synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

L-1077

AmprpMA-Tplasmid

carrying

thel-10

77synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pSRC

AM602

pPICZ120572

Aderiv

ativew

iththesrcam

602synthetic

gene

Thiswork

pOR-7

pPICZ120572

Aderiv

ativew

iththeo

r-7synthetic

gene

Thiswork

pE-760

pPICZ120572

Aderiv

ativew

iththee

-760

synthetic

gene

Thiswork

pL-1077

pPICZ120572

Aderiv

ativew

iththel-10

77synthetic

gene

Thiswork


tanceZe

orzeocinresistance

b DNBT

AD

epartamento

deNutric

ion

Brom


losA

limentosFacultadde

Veterin

ariaU

niversidad

Com

plutense

deMadrid

(Madrid

Spain)CE

CTC

oleccion

Espano

lade

CultivosT

ipo

ValenciaSpain)


Table2Prim

ersa

ndPC

Rprod

uctsused

inthisstu

dy

Prim

ersPC

Rprod

ucts

orbacteriocins

Nucleotides

equence(51015840-31015840

)ord

escriptio

nAmplificatio

nsPrim

ers

S602-F

GCC

ATGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM602R-E7

60R

-L1077

S071-F

GTC

CAGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM

602R-E7

60R

-L1077R

-OR7

SARP

-RAG

GTA

CCAT

AAG

TTGCG

GCC

GC

R-OR7

ALFA-

FTA

CTAT

TGCC

AGCA

TTGCT

GC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

3AOX1-R

GCA

AAT

GGCA

TTCT

GAC

ATCC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

PCRprod

ucts

R-SR

CAM602

136-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticsrc

am602gene

with

theP

pastoris

codo

nusage

R-OR7

181-b

pXh

oIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticor-7

gene

with

theP

pastoris

codo

nusage

R-E7

60242-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

ynthetice-760gene

with

theP

pastoris

codo

nusage

R-L1077

167-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticl-1077gene

with

theP

pastoris

codo

nusage

Bacteriocins

BacSRC

AM602(aminoacid

sequ

ence)

ATYY

GNGLY

CNKQ

KHYT

WVDWNKA

SREIGKI

TVNGWVQ

HBa

cSRC

AM602(Ppastoris

codo

nusage)


acacttg

ggttg


atcactgttaacggttg

ggttc

aaca

BacO

R-7(aminoacid

sequ

ence)

KTYY

GTN

GVHCT

KNSLWGKV

RLKN

MK

YDQNTT

YMGRL

QDILLG

WAT

GAFG

KTFH

BacO

R-7(Ppastoris

codo

nusage)


gtggggtaaggttagattg


ctactta

catgggtagattg

caggacatcttgttg

ggttg

ggctact

ggtgctttc

ggtaagacttttcat

BacE-760

(aminoacid

sequ

ence)

NRW

YCNSA

AGGVG

GAAV

CGLA

GYV

GE

AKE

NIAGEV

RKGWGMAG

GFT

HNKA

CKS

FPGSG

WASG

BacE-760

(Ppastoris

codo

nusage)


gtggtgct


tgttg

gtgaggctaaagaaaacat


actcataacaaggcttg


BacL-1077(aminoacid

sequ

ence)

TNYG

NGVG

VPD

AIM

AGIIKL

IFIFNIRQGY

NFG

KKAT

BacL-1077(Ppastoris

codo

nusage)



caac

ttcggtaagaaggctact











100

90

80

70

0

10

20

30

40

60

50

1006683536640492732141501

Inte

nsity

()

33888135

3372231234054204

33563608

33262061

12 + 4

Mass (mz)

(a)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

652456194714380929041999

4287

21671641

24692393

30944207

33728801

33896047

28084270

29682549

47090483

50382788

Mass (mz)

(b)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100298183633744912645799

335333206313

54511987

49676523 64456704 70819937

Mass (mz)

(c)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100308183633744912645799

385218930446

19090667

24790750360197256

3583737534952290

49746992

Mass (mz)

(d)


























SRCAM 602 4630192665150095161497



E-760 61798 116663157968


L-1077 40026 163876171873
















Acknowledgments


References
















































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Ba

cterialstrains

andplasmidsu

sedin

thisstu

dy

Strain

orplasmid

Descriptio

naSource

andor

referenceb

Strains

Enterococcus

faecium

T136

EnterocinAandBprod

ucerM

PApo

sitivec

ontro

lDNBT

APediococcusd

amnosusC

ECT4

797

MPA

indicatorm

icroorganism

CECT

Escherich

iacoliJM

109

Selectionof

recombinant

plasmids

Prom

ega

Pichiapasto

risX-

33Yeastp

rodu

cer

Invitro

genLifeTechno

logies

Plasmids

pMA-T

Amprcarrie

rofsyntheticgenes

GeneA

rtLifeTechno

logies

pPICZ120572

AZe

orintegrativ

eplasm

idcarrying

thes

ecretio

nsig

nalsequencefrom

theS

cerevisiae120572-fa

ctor

prepropeptidea

ndfunctio

nalsitesfor


AOX1

locuso

fPpastoris

X-33

Invitro

genLifeTechno

logies

pMAT

SRCA

M602

AmprpMA-Tplasmid

carrying

thesrcam

602synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

OR-7

AmprpMA-Tplasmid

carrying

theo

r-7synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

E-760

AmprpMA-Tplasmid

carrying

thee

-760

synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pMAT

L-1077

AmprpMA-Tplasmid

carrying

thel-10

77synthetic

gene

with

theP

pastoris

codo

nusage

GeneA

rtLifeTechno

logies

pSRC

AM602

pPICZ120572

Aderiv

ativew

iththesrcam

602synthetic

gene

Thiswork

pOR-7

pPICZ120572

Aderiv

ativew

iththeo

r-7synthetic

gene

Thiswork

pE-760

pPICZ120572

Aderiv

ativew

iththee

-760

synthetic

gene

Thiswork

pL-1077

pPICZ120572

Aderiv

ativew

iththel-10

77synthetic

gene

Thiswork


tanceZe

orzeocinresistance

b DNBT

AD

epartamento

deNutric

ion

Brom


losA

limentosFacultadde

Veterin

ariaU

niversidad

Com

plutense

deMadrid

(Madrid

Spain)CE

CTC

oleccion

Espano

lade

CultivosT

ipo

ValenciaSpain)


Table2Prim

ersa

ndPC

Rprod

uctsused

inthisstu

dy

Prim

ersPC

Rprod

ucts

orbacteriocins

Nucleotides

equence(51015840-31015840

)ord

escriptio

nAmplificatio

nsPrim

ers

S602-F

GCC

ATGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM602R-E7

60R

-L1077

S071-F

GTC

CAGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM

602R-E7

60R

-L1077R

-OR7

SARP

-RAG

GTA

CCAT

AAG

TTGCG

GCC

GC

R-OR7

ALFA-

FTA

CTAT

TGCC

AGCA

TTGCT

GC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

3AOX1-R

GCA

AAT

GGCA

TTCT

GAC

ATCC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

PCRprod

ucts

R-SR

CAM602

136-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticsrc

am602gene

with

theP

pastoris

codo

nusage

R-OR7

181-b

pXh

oIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticor-7

gene

with

theP

pastoris

codo

nusage

R-E7

60242-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

ynthetice-760gene

with

theP

pastoris

codo

nusage

R-L1077

167-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticl-1077gene

with

theP

pastoris

codo

nusage

Bacteriocins

BacSRC

AM602(aminoacid

sequ

ence)

ATYY

GNGLY

CNKQ

KHYT

WVDWNKA

SREIGKI

TVNGWVQ

HBa

cSRC

AM602(Ppastoris

codo

nusage)


acacttg

ggttg


atcactgttaacggttg

ggttc

aaca

BacO

R-7(aminoacid

sequ

ence)

KTYY

GTN

GVHCT

KNSLWGKV

RLKN

MK

YDQNTT

YMGRL

QDILLG

WAT

GAFG

KTFH

BacO

R-7(Ppastoris

codo

nusage)


gtggggtaaggttagattg


ctactta

catgggtagattg

caggacatcttgttg

ggttg

ggctact

ggtgctttc

ggtaagacttttcat

BacE-760

(aminoacid

sequ

ence)

NRW

YCNSA

AGGVG

GAAV

CGLA

GYV

GE

AKE

NIAGEV

RKGWGMAG

GFT

HNKA

CKS

FPGSG

WASG

BacE-760

(Ppastoris

codo

nusage)


gtggtgct


tgttg

gtgaggctaaagaaaacat


actcataacaaggcttg


BacL-1077(aminoacid

sequ

ence)

TNYG

NGVG

VPD

AIM

AGIIKL

IFIFNIRQGY

NFG

KKAT

BacL-1077(Ppastoris

codo

nusage)



caac

ttcggtaagaaggctact











100

90

80

70

0

10

20

30

40

60

50

1006683536640492732141501

Inte

nsity

()

33888135

3372231234054204

33563608

33262061

12 + 4

Mass (mz)

(a)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

652456194714380929041999

4287

21671641

24692393

30944207

33728801

33896047

28084270

29682549

47090483

50382788

Mass (mz)

(b)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100298183633744912645799

335333206313

54511987

49676523 64456704 70819937

Mass (mz)

(c)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100308183633744912645799

385218930446

19090667

24790750360197256

3583737534952290

49746992

Mass (mz)

(d)


























SRCAM 602 4630192665150095161497



E-760 61798 116663157968


L-1077 40026 163876171873
















Acknowledgments


References
















































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Prim

ersa

ndPC

Rprod

uctsused

inthisstu

dy

Prim

ersPC

Rprod

ucts

orbacteriocins

Nucleotides

equence(51015840-31015840

)ord

escriptio

nAmplificatio

nsPrim

ers

S602-F

GCC

ATGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM602R-E7

60R

-L1077

S071-F

GTC

CAGAG

CTCG

AAT

TCTC

GAG

AAAAG

R-SR

CAM

602R-E7

60R

-L1077R

-OR7

SARP

-RAG

GTA

CCAT

AAG

TTGCG

GCC

GC

R-OR7

ALFA-

FTA

CTAT

TGCC

AGCA

TTGCT

GC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

3AOX1-R

GCA

AAT

GGCA

TTCT

GAC

ATCC

pPICZ120572

Aam

plificatio

nfragmentincluding

thec

lonedsynthetic

gene

PCRprod

ucts

R-SR

CAM602

136-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticsrc

am602gene

with

theP

pastoris

codo

nusage

R-OR7

181-b

pXh

oIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticor-7

gene

with

theP

pastoris

codo

nusage

R-E7

60242-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

ynthetice-760gene

with

theP

pastoris

codo

nusage

R-L1077

167-bp

XhoIN

otIfragm

entcon

tainingthe120572

-factor

Kex2

signalcleavage

fusedto

them

atures

yntheticl-1077gene

with

theP

pastoris

codo

nusage

Bacteriocins

BacSRC

AM602(aminoacid

sequ

ence)

ATYY

GNGLY

CNKQ

KHYT

WVDWNKA

SREIGKI

TVNGWVQ

HBa

cSRC

AM602(Ppastoris

codo

nusage)


acacttg

ggttg


atcactgttaacggttg

ggttc

aaca

BacO

R-7(aminoacid

sequ

ence)

KTYY

GTN

GVHCT

KNSLWGKV

RLKN

MK

YDQNTT

YMGRL

QDILLG

WAT

GAFG

KTFH

BacO

R-7(Ppastoris

codo

nusage)


gtggggtaaggttagattg


ctactta

catgggtagattg

caggacatcttgttg

ggttg

ggctact

ggtgctttc

ggtaagacttttcat

BacE-760

(aminoacid

sequ

ence)

NRW

YCNSA

AGGVG

GAAV

CGLA

GYV

GE

AKE

NIAGEV

RKGWGMAG

GFT

HNKA

CKS

FPGSG

WASG

BacE-760

(Ppastoris

codo

nusage)


gtggtgct


tgttg

gtgaggctaaagaaaacat


actcataacaaggcttg


BacL-1077(aminoacid

sequ

ence)

TNYG

NGVG

VPD

AIM

AGIIKL

IFIFNIRQGY

NFG

KKAT

BacL-1077(Ppastoris

codo

nusage)



caac

ttcggtaagaaggctact











100

90

80

70

0

10

20

30

40

60

50

1006683536640492732141501

Inte

nsity

()

33888135

3372231234054204

33563608

33262061

12 + 4

Mass (mz)

(a)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

652456194714380929041999

4287

21671641

24692393

30944207

33728801

33896047

28084270

29682549

47090483

50382788

Mass (mz)

(b)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100298183633744912645799

335333206313

54511987

49676523 64456704 70819937

Mass (mz)

(c)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100308183633744912645799

385218930446

19090667

24790750360197256

3583737534952290

49746992

Mass (mz)

(d)


























SRCAM 602 4630192665150095161497



E-760 61798 116663157968


L-1077 40026 163876171873
















Acknowledgments


References
















































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











100

90

80

70

0

10

20

30

40

60

50

1006683536640492732141501

Inte

nsity

()

33888135

3372231234054204

33563608

33262061

12 + 4

Mass (mz)

(a)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

652456194714380929041999

4287

21671641

24692393

30944207

33728801

33896047

28084270

29682549

47090483

50382788

Mass (mz)

(b)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100298183633744912645799

335333206313

54511987

49676523 64456704 70819937

Mass (mz)

(c)

Inte

nsity

()

100

90

80

70

0

10

20

30

40

60

50

100308183633744912645799

385218930446

19090667

24790750360197256

3583737534952290

49746992

Mass (mz)

(d)


























SRCAM 602 4630192665150095161497



E-760 61798 116663157968


L-1077 40026 163876171873
















Acknowledgments


References
















































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















SRCAM 602 4630192665150095161497



E-760 61798 116663157968


L-1077 40026 163876171873
















Acknowledgments


References
















































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Acknowledgments


References
















































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































1









Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins...

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Transcript of Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins...