Tạo dòng biểu hiện nisin trên bề mặt tế bào nấm men saccharomyces cerevisiae bằng dung hợp với gen mã hóa ∝ agglutinin

Tap chi Cong nghe Sinh hpc 13(2): 379-385, 2015

TAO DoNG BIEU HIEN NISIN TREN BE MAT TE BAO NAM MEN SACCHAROMYCES
CEREVISIAE BANG DUNG HOP VefI GEN MA HOA a-AGGLUTININ
Nguyen Hien Nghia, Thai Ngoc Anh, Nguyen Quynh Hwang, Ng8 Thi CAm Nhung, Nguyen Tri Nhan,
TrAn Linh Thtrac, D#ng Thi Phtrong Thao
Truerng Dgi hpc Khoa hpc to nhien, Dgi hpc Quac gia Theinh pha Ho Chi Minh
Ngay nhiin bai: 19.5.2015
Ngay nhfin ding: 30.6.2015
TOM TAT
Nisin duqc biet den nhu bacteriocin ci ph6 khang khuan remg va irc the cac vi khan gay benh tren ngueri
va ging vet nhu Staphylococcus aureus, Listeria sp. Ben canh Q6, mic di Nisin thu nhttn tir djch len men
ching Lactococcus lactis ttr nhien a duqc ling dung remg rii trong c8ng nghiep bao quan thuc phim, tuy
nhien cac chi ng to nhien bieu hien Nisin veri ham !yang thap, do di hien nay nghien cuu trong va ngosi mac
Escherechia colt va nam men Saccharomyces cerevisiae (S.
huang tai vies. san xat Nisin tai to hqp
cerevisiae). NM= tim ra met cach tiep cent mdri di thu nhCin va ling dung bacteriocin trong brio quitn thuc phim
cling nhu tic the vi khuin gay benh trong chin nu8i gia sic va nu8i trong thug hai san. Trong bai bao nay,
chin toi trinh bay chilli ltrqc bleu hien Nisin tren be mit to bao am men S. cerevisiae bang phuong phip
dung hqp gene nisin v6i gene a-agglutinin. Deng to bac) nem men nay se duqc thu nhan va huding tdri lam
nguyen lieu cho nghien cuu ling dung trong bao quan thuc phitm ding nhu trong cac the phim irc the vi sinh
vitt gaY bOnh•
Tir khda: Agglutinin, bacteriocin, cong ngh

m(sit, hui,nh (pang. nisin. pICAS-1, Saccharomyces cerevisiae

MO DAU

kiem soat tic titian gay benh cna vi sinh v#t trong
tucmg lai (Yang et al., 2014).

Bacteriocin la nheng peptide hay protein khang
khan, do vi khan nham Grain am hoc Gram
duong sinh ra. Bacteriocin cilia vi khan Gram
ducmg, d#t biet la cilia nhom vi khuan lactic hay con
goi la nhom vi IchuAn LAB (lactic acid bacteria)
duqc nghien tiro va irng dung King trong nhieu linh
Arc nhu c8ng nghiep bao quan thuc phim, y ductc vA
probiotic (Chen, Hoover 2003). Nisin la bacteriocin
thuec nhom lantibiotic, duqc sinh tong hop bfri
Lactococcus lactis (L. lactis), peptide truang thanh
china 34 amino acid, khei ltrqng pha'n tin 3.3kDa,
Nisin to nhien c6 kha nang khang vi khuin lactic,
Bacillus sp, Listeria sp va Micrococcus sp. (Zendo et
al., 2003; Fukao et al., 2008). Ben canh de, nhieu
thin nghiem in vitro cho thAy lantibiotic c6 khA Wing
khang cac vi khan gay berth ten nguai va deng vet
bao gem ca cac vi khuAn khang khang sinh nhu
Staphylococcus aureus khang methicillin (MRSA),
Enterococcus faecalis khang vancomycin (VRE)
(Piper et al., 2009). Ca the tic deng mkt bacteriocin
'chic vei thuoc khang sinh th8ng thuang, c6 xu the
di{C trong tren cac loci vi khan gay benh, nen ching
co the coi nhu met ngu6n mei, dAy flan' Wing cho skr

Vi khan lactic ke ca sin phAm ctia chimg nhu
bacteriocin duqc ding nhtln an toil' va duqc 1mg
dung nhieu trong c8ng nghiep thuc phim. Tuy nhien,
sir bleu hien him Wong thip la nhtrqc diem dm cac
ching sinh bacteriocin to nhien, do de nhieu nghien
dm dA huang den bieu hien bacteriocin tai to hqp

trong cac he thong to bao chit khic nhau nhu
Escherichia colt (E. colt), Saccharomyces cerevisiae
(S. cerevisiae), LAB (Chen et al., 2012; Mierau,
Kleerebezem, 2005; Reenen et al., 2003). Nim men
S. cerevisiae duqc ang **mg nhieu trong cong nghiep
thuc phAm va duce cong nh#n la an town GRAS
(generally regarded as safe), nhieu nghien ciru ve san
xuet bacteriocin tai to hqp ta S. cerevisiae dA thanh
c8ng nhu Pediocin (Schoeman et al., 1999),
Enterocin L50 (Basanta et al., 2009). Song song d6,
nhfmg thAnh cong ye cling nghe be met nem men dA
ma ra met huang phat trien mei cho ding nghe
protein tai to hqp. Cong nghe be met to bao nem
men la phuang phap bieu hien protein muc lieu bing
dung hop gene nhAm dung hqp protein muc tieu yeti
protein tren ming to bao nem men nhu FLO1, Cwps
va a-/a-Agglutinin (Ueda, Tanaka, 2000). Trong bai
379

11


Nguyen Hieu Nghia et al.
bao nay, chilli* toi trinh bay cac ket qua thief ke too
clang to bao nam men S. cerevisiae mang dung hop
gene bacteriocin vbi a-Agglutinin va bieu hien Nisin
tren be met to bao nam men.
VAT LIEU VA PHtJONG PHAP
Chung
Escherichia coli

DH5a [F,
0801acZAM15, recAl, endAl, hsdR17 (rk, Ink+),
phoA, supE44,
thi-1, gyrA96, relA 1] (Takara,
Nhet) dung lam 4 brio chic de nhan ban plasmid.
Chung
Saccharomyces cerevisiae
W303
[MATaIMATa {leu2-3,112 trpl-1 can 1-100 ura3-1
ade2-1 his3-11,15)] dung de bleu hien protein dung
hqp len be met.
Vector pICAS-1 (PTN.Cong nghe sinh hoc phan
dr, Truemg Dai, hoc Khoa hoc tv nhien, DHQGHCM) ducrc thiet ke de lam vector tai to hqp mang
gene nisin, co kich tit& 6457bp, mang gene khang
ampicilin giup sang 19c d6ng 4 bao vi khan mang
vector, gene ma hem protein sinh tong hqp
tryptophan giup sang 19c cac &rig nam men mang
vector trong mei trueng khuyet dueng tryptophan,
promoter GADPH (glyceraldehyde-3-phosphate
dehydrogenease) kiem sat bieu hien beri glucose,
trinh tier tiet SSS (secrection signal sequence) giap
protein, chuyen vi qua mang, trinh tv gin neo GPI
giup On ming to bac), met Oa 3' a-Agglutinin la
trinh ter dung h9p yen Nisin.
Ciu truc vector tai to hqp pICAS-nisin
Trinh tv gene nisin duce tei tru hoa cac codon
bang phan mem thiet ke gene Snap Gene 2.6.2
(www.snapgene.com) nhArn tucmg thick trong to bao
chit S. cerevisiae. Gene nisin dtrqc khuech dai bang
phin img PCR veri cep mei dee hieu cho gene BglIIN (GGA AGA TCT CCA TGT CTA CTA AAG

ATT TTA ATT TGG) va XhoI-N (CCG CTC GAG
CGG ATG GTG ATG GTG ATG GTG TTT AGA
AAC ATG AAC AGA AC, va duqc cher) vao vector
pICAS-1. San pham not duqc hoa bien nap vao to
bao E. coli DH5a va duce sang 19c tren mei trueng
LB agar (trypton 1%, NaCI 1%, cao nam men 0.5%)
co
khang sinh
Ong de 100 µg/ml.
The . bien nap duvc kiem tra bang phan irng PCR
khuan lac veri cep mei BglII-N va
Plasmid
tai to hop duqc kiem tra bing phan img cat veri
enzyme Bell va XhoI va kiem tra sv hien dien cua
trinh tv gene tren palsmid bang PCR vei cep mei
BglII-N va XhoI-N.
380

Tao deng 4 bao nAm men S. cerevisiae W303
mang vector tai to hqp pICAS-nisin
Vector pICAS-nisin duqc hien nap vao to bao
nAm men S. cerevisiae W303 bang phuang phap
lithium acetate (Gietz, Schiestl 2007). San pham bien
nap duqc sang 19c tren mei tnreng khuyet clueing
tryptophan (SD-: leucin 30mg, Histidin 20mg,
Adenin 20mg, Uracin 20mg, Yeast nitrogene base
6.7g, Glucose 20g va ntrac cat du Hit). Cac khuAn
lac MQC duqc tren moi tnrerng khuyet duang tien
hanh tach chiet geneome (Rose et al., 1990) va PCR
kiem tra veri cep mei BglII-N vi Xhol-N dee hieu

cho gene nisin.
De kiem tra sir bleu hien Nisin tren be met to
bio nAm men tai to hop, cac chting nAm men tai to
hop sau khi duvc PCR kiem tra tien hanh lai mien
djch hujrnh quang(Tang et al., 2008) tren be met to
bio veri khang the so cap la khang the don deng
khang Histidin tir chuet Anti-His Antibody (GE
Healthcare, Thy Si) ti le 1:1000 then gian u 1 gib va
khang khang the thin cap la khang the da dor% gin
hujmh quang Goat anti-Mouse, Alexa Fluor 488
(Life Technologies, My) ti le 1:3000, thei gian ti 1
gib. Cfic 4 bito sau khi nhuem ducfc quan sat dueri
kinh hien vi hujrnh quang btrec song kich thich
488nm.
KET QUA VA THAO LUAN
Toi tru va thiet ke dung hqp gene Mint bieu hi?n
Nisin teen be met 4 ban nam men
Trong
, nghien ciru nay, Nisin duet bieu hien tren
be mot to bao nAm men bang dung hop veri met nira
cau tnic a —Agglutinin, cAu trite dung hqp gene nay
duce dieu hoa bleu hien bei promoter GADPH (Hinh
IA). Theo nghien ciru cua Ueda va Tanaka (2000),
a-Agglutinin ding da dirge sir dung film met protein
neo de Neu hien protein ngoai lai tren be met to bac)
A
nam men.
Nisin. la peptide c6 nguen gee tir vi khtffin L.
lactis, nham tei tru gene ma, hoa cho peptide nay
trong

thong to bio chit nam men, chimg ten sir
ding phan mem thiet ke gene Snap Gene 2.6.2 yen
be thong so ve codon ma hofi cua nam men S.
cerevisiae de chuyen el& cac codon kho nhen dien va
cac codon khong dich ma tren gene nisin dm L.
lactis thank cac codon djch ma cho ding amino acid
cna nam men. Khi so sinh codon ma h6a cho Nisin
tir vi khan L. lactis vei codon ma hoa cua nAm men
S. cerevisiae, ket qua cho thay chi co 57% codon


Tap chi Cong ngh0 Sinh kw

13(2): 379-385, 2015

ten tong s6 57 codon c6 de ttrcrng thich 100% vai
ma di truyen cua nam men S. cerevisiae. 14% codon
c6 de Wang thich tir 50-70% va 28% codon c6 de
twang thich nho han 50% (Hinh 1B). Sau khi tai tru

trinh ur gene ma hoa Nisin, ket qua phan tich cho
thay do 96% codon twang thich 100% voi codon ma
hod ctia nam men S. cerevisiae, chi c6 4% codon c6
de ttrcmg thich 50-70% (Hinh IC).

Trinh tkr
neo GPI

A
Promoter

GADPH
I
SSS

Dich ma

411111

W— Neo GPI

B Ten gen: Nisin - Lacto
Ngu6n g6c: Lactococcus lactis
Bang ma so sanh: Saccharomyces cerevisiae
Tryc y: m6i tuang quan
nh6 han 70% (mau xanh), nh6 han 50% (mau dO)
3333 3 3 3 3 3

mice

s s 3 33g ;33 3333 3

°S° 333

1 1 1 1 1 1 1 1 1 1,1 1 1 1 1 1 1 1 1 1 1 1
118:116FILDLVEVII/LTDSGASTRISISLIPOC

VLIIGCALR A CA C4VPIV:.%

C Ten gen: Nisin - Yeast
Ngu6n g6c: Dirac hen d6i tir Nisin-Lacto

Bang ma so sanh: Saccharomyces cerevisiae
Tryc y: m6i tuang quan
nho han 70% (mau xanh), nh6 han 50% (mau do)

:::::::: Ess

33333 333:33333333333333338338333.333333333

too - 11000,111,11101001111
111
1111

so —

0

Hinh 1. Tai vu va thiet ke dung hgp gen nham bieu hien Nisin trOn be mat to bao nem men S. cerevisiae. (A): Cau trtic gen
dung hcrp protein. SSS: secrection signal sequence; GADPH: glyceraldehyde-3-phosphate dehydrogenase; GPI:
glycosylphosphatidylinositol. (B): So sanh bang ma di truyan cue trinh ti,r gen nisin ma hda tit, L. lactis \Idyl nam men S.
cerevisiae. (C): So earth bang ma di truyan sau khi tai trinh tio, gen nisin vai nam men S. cerevisiae.

381


Nguyen Hieu Nghia et al.
Cau frac vector tai

to hop pICAS-nisin

Gene nisin duqc thu nhan bang phan Cmg PCR,

sau do dtrqc cat bang hai enzyme cat giai hen BgiII
va •XhoI. Ket qui dien di tren gel agarose 1%, san
phi= PCR cho mOt vach kich thu6c khoang 200bp
nhu da thiet ke (Hinh 2A, gieng 3). Song theo do,
vector pICAS-1 cling duqc cat ma wing bang hai
enzyme BglII va Xhol cho mOt vach c6 kich thu6c
khoing. 6500bp tren ban dien di (Hinh 2B, gieng 4).
San phim gene va plasmid sau khi cat ducc not lei
va Wen nap vao to bao E. colt DH5a.
Cac d6ng 4 bao E. colt DH5a tai to hop duqc
sang loc tren moi tnrang LB agar ampicilin va kiem
tra bang PCR khuan lac vai cap mai Bg1117N va
Xhol-N dac hieu cho gene nisin. Ket qua kiem tra
cho they mOt so clang to bao E. coil cho san pham

A

PCR mOt vach twang ducmg chimg ducmg (Hinh 2B,
gieng 2, giong 3, gieng 5-7), dial d6 chimg to cac
thing to bac) nay mang gene mac heti. BEn canh do,
cac plasmid tai to hqp tir cac dong tai to hqp nay
duqc PCR vai cap moi Bg111-N/XhoI-N cling cho
san pham PCR twang to chimg duang (Hinh 2C,
gieng 2-4).
Cu6i ding, cac plasmid tai to hqp duqc cat bang
hai enzyme Bg111 va Xhol va giai trinh hr d8 khing
dinh trinh ti,r cling nhu si,r do khung dich ma. Ket
qua dien di san pham cat cho they plasmid tai to hop
sau khi cat bang hai enzyme da cho hai vach, mOt
vach co kich thu6c khoing 6500bp tucmg ducrng

kich plasmid pICAS-1 duqc cat va mOt vach co kich
thu6c khoang 200bp ttrang, dung kich thuac gene
nisin (Hinh 2D, gieng 2, gieng 3). Nhu vay, plasmid
tai to hcp pICAS- nisin de duqc too thanh ding.

by

6000 —
3000 —

1000 —

250 —

C by
6000 —
3000 —

1000 —

250 —

D

by

1 2 3 4 5

6000 —
3000 —

1000 —

250 —

Hinh 2. Chen gen nisin vao vector pICAS1 va kiem tra vector tai t6 hap pICAS-nisin. (A): Thu nhan gen nisin va plasmid
pICAS-1. 1. Thang DNA 1 kbp; 2. Chung am phan Ong PCR; 3. San pham PCR gen sau khi cat; 4. Plasmid sau khi cat; 5.
Plasmid Utah bang phvang phap SDS kiem. (B): PCR khan lac E. coil DH5a/pICAS-nisin. 1. Thang DNA 1 kbp; 2-7. San
pham PCR khan IOC E. coil DH5a khac nhau; 8. ChOng am phan Ong PCR; 9. Chirng &rang phan Ong PCR. (C): PCR
plasmid tai td hap E. coil DH5a/pICAS-nisin. 1. Thang DNA 1 kbp; 2-4. San pham PCR plasmid khac nhau; 5. Chirng dtrang
phan Ong PCR; 6. Chirng am phan Ong PCR. (13): Cat plasmid tai t6 hop pICAS-nisin bang BgllI va Xhol. 1. Thang DNA 1
kbp; 2-4. San pham cat plasmid khac nhau; 5. Plasmid pICAS-1.

382


Tap chi Cong nghe Sinh hac 13(2): 379-385, 2015

B

A

Scarevistao W303

SD- (Trp-)

Tryptophan

SD- (Trp-)

C

S.C.enovislas W303/
pICAS-nisin

D

nisin 6xHistidine

Promote

GPI

Xhol-N
us

,

.

Wit nil a-agglutinin

IPCR

Pha tai

Pha sing
S.Convisloo W303

1 2 3 4 5 6 7 8
600030001000-

250—

Pha sang

Pha tit

s.centvislas W3031pICAS-nisin
Hlnh 3. Sang loc clang to bao nem men mang vector tai t6 hqp pICAS-nisin. (A): Nguyen tic sang lqc tren mei trtreng
khuyet clueing. (B): Ket qua Wen nap vector pICAS-nisin vao to bao nem men S. cerevisiae W303. (C): Kiem tra skr hien dien
die dung hop gen trong to bao nem men. 1. Thang DNA 100bp; 2. Kik qua phan irng PCR v6i khulen la DNA be gen cua
nem men; 3. Dung hqp gen; 4-9. Keit qua phan itng PCR v&i khu6n la DNA be gen tit cac khuan lac nem men tai 16 hqp
sang loc dttqc tren mei trtteng
(D): Kiem tra bleu hien gen nisin tren be met te bao bang nhuem mien djch huynh quang
yeti khang the khang Histidin. Pha sang le anh sang trAng va pha t6i anh sang huynh quang bulfrc song 488 nm.

Tao dung t4 bite nem men S. cerevisiae W303

trong thi nghie. m chuyen dung hqp gene nisin - a -

mang vector di to hqp pICAS-nisin ye bieu hien
Nisin tren be mat to bao

agglutinin va bie.0 hien Nisin ten be mat to bao la to
bao nem men S. cerevisiae W303 mang d6t bien

re bao cha nem men S. cerevisiae ductc sir dung

khuyet &rang tryptophan, khang phat trien dtrqc tren
mai trtremg SD- (yeast minimal media minus

383


Nguyen Hieu Nghia et al.
tryptophan). 'Chi 4 bio dim men W303 tiep nh#n
vector tai to hqp pICAS-nisin, trinh to gene ma hem
protein sinh tong hqp tryptophan tren vector duqc sat
nh#p vio bil) gene ciut nam men, do v#y d6ng 4 NW
nay co khi nang sinh twang tren moi tru6ng SD"
(Hinh 3A). Cic ket qui time nghiem cia chum; t8i
cho thiy, sau khi bien nap vector pICAS-nisin van 4
bao nam men S. cerevisiae W303, chin toi di thu
duqc mgt so khuAn lac nam men phit trien tren
m8itru6ng SD" (Hinh 3B).
Cic deng 4 bac) nam men tai to hqp nay tiep tVc
duqc kiem tra su tai to hop cis gene nisin vac) b0
gene nam men. Ket qua cho thAy trong 5/30 deng
nam men phit trien duqc tren m6i truing SD", da
duqc chen gene nisin vio b0 gene the hien qua lc&
qui PCR duang tinh khi sir dting DNA b0 gene cis
cfic deong nam men nay lim khu8n mau trong phan
1mg PCR veri cep mOi d#c hieu cho gene nisin (Hinh
3C, gieng 4-7).
Nhu trinh bay tren hinh 3C, gene nisin duqc
ket hqp them 6 codon ma hoi cho cic amino acid
histidine tram khi duqc dung hqp veri mCit nira cau
trite a-Agglutinin. Viec dung hqp veri the histidine
nham mtic dich nh#n dien chat peptide Nisin tren
be m#t to bao dim men thong qua phuang thirc
nhuOrn mien dich huynh quang veri khing the

khing histidine. icet qua thuc nghiem cho they
dual inh sing huynh quang, &mg to bao S.
cerevisiae W303/pICAS-nisin cho tin hien huynh
quang teen be met 4 bio. Tin hieu nay kh8ng quan
sit ductc tren deng to bao S. cerevisiae W303
(Hinh 3D).
Nisin vi vi sinh sinh tong hqp Nisin da va clan§
duqc sir ding rOng di b nhieu clang Ichic nhau: Che
phi= probiotic hay clang peptide khing khuin
(Deegan et al., 2006). Nhieu nghien cim truck day
di cling ve ve cic ket qua bieu hien Nisin tai to hqp
tren cic he thOng 4 bao chu L. lactis (Mierau,
Kleerebezem, 2005). Tuy nhien, Nisin tai
hqp
thutmg duvc tong hop va tiet ra ben ngoii mei
truemg. Vai ham luqng bleu hien kh8ng cao, viec thu
nhan nisin tir m8i truemg nuoi ciy slip khong it kh6
khan va la nguyen nihan lam giim hieu qua cia quit
trinh sin xuat nisin tai to hq?. Nghien clru cis chang
toi la nghien ciru ditu tien bieu hien nisin tai tto hop 6
hang thii dinh tren be met to bio nAm men. Ket qua
thuc nghiem. khing djnh chimg Vol di thanh cling
trong viec bieu hien Nisin be met to bio nam men S.
cerevisiae.
384

KET LUAN
Trong nghien ciru. nay, chimg toi da cAu true
thanh cong vector tai to hop pICAS-nisin nham bieu
hien Nisin dung hqp 3'a-Agglutinin tren be met to

bao nAm men S. cerevisiae. Dong theri biz& du sang
lqc duqc deng 4 bao am men S. cerevisiae W303
mang vector pICAS-nisin. Dong dim men tai to hqp
nay li nguyen lieu ca bin cho thir nghiem hoot tinh
Nisin khi duqc dung hqp vi c0 djnh tren ming to
bao, cAng nhu nghien cuu (mg ding chung trong bio
quirt thuc phew vi probiotic.
TAI LI$U THAN' KHAO
Basanta A, Herren C, Gutierrez J, Criado R, Herandez
PE, Cintas LM (2009) Development of bacteriocinogeneic
strains of Saccharomyces cerevisiae heterologuously
expressing and secret the leaderless EnterocinL50A and
L5OB from Enterococcus faecium L50. App! Environ
Microbiol 75: 2382-2392.
Bimboim HC, Doly J (1979) A rapid alkaline procedure
for screening recombinant plasmid DNA. Nucleic Acids
Res 7: 1513-1523.
Chen H, Hoover DG (2003) Bacteriocin and their Food
Application. Comprehensive Reviews in Food Science and
Food Safety 2: 98-100.
Chen H, Tian F, Li S, Xie Y, Zhang H, Chen W (2012)
Cloning and heterologous expression of a bacteriocin
sakacin P from Lactobacillus sakei in Escherichia coll.
App! Microbiol Biotechnol 94: 1061 - 1068.
Deegan LH, Cotter PD, Hill C, Ross P (2006)
Bacteriocins: biological tools for bio-preservation and
shelf-life extension. Int Dairy J 16: 1058-1071.
Fukao M, Obits T, Yoneyama F, Kohda D, Zendo
T, Nakayama J, Sonomoto K (2008) Complete Covalent
Structure of Nisin Q, New Natural Nisin Variant,

Containing Post-Translationally Modified Amino Acids.
Biosci Biotechnol Biochem 72: 1750- 1755.
Gietz RD, Schiestl RH (2007) High-efficiency yeast
transformation using the LiAc/SS carrier DNA/PEG
method. Nat Protoc 2: 31-34.
Klaenhammer TR (1993) Genetics of bacteriocins
produced by lactic acid bacteria. FEMS Microbiol Rev 12:
39-85.
Mierau 1, Kleerebezem M (2005) 10 year of nisincontrolled gene expression system (NICE). Appl Microbiol
Biotechnol 68:705-717.
Piper C, Draper LA, Cotter PD, Ross RP, Hill C (2009) A
comparison of the activities of lactacin 3147 and nisin
against drug-resistant Staphylococcus aureus and


Tap chi Cong nghe Sinh hoc 13(2): 379-385, 2015
Enterococcus species. J Antimicrob Chemother 64: 546-551.
Reenen CAV, Chikindas ML, Zyl WHV, Dicks LMT
(2003) Characterization and heterologous expression of a
class Ifa bacteriocin, Plantaricin 423 from Lactobacillus
plantarum 423 in Saccharomyces cerevisiae. Int J Food
Microbiol 81: 29-40.
Rose MD, Winston F, Hieter P (1990) Methods in Yeast
Genetics. A Laboratory Course Manual. Cold Spring
Harbor, NY.
Schoeman H, Vivier MA, Toit MD, Dicks LM, Pretorius
IS (1999) The development of bactericidal yeast strains by
expressing the Pediococcus acidilactici Pediocin Gene
(pedA) in Saccharomyces cerevisiae. Yeast 15: 647-656.
Tang YQ, Han SY, Hong ZH, Wu L, Ueda M, Wang XN,

Lin Y (2008) Construction of cell surface-engineered
yeasts displaying antigene to detect antibodies by
immunofluorescence and yeast-ELISA. Appl Microbiol
Biotechnol 79: 1019-1026.
Ueda M, Tanaka A (2000) Genetic immobilization of
proteins on the yeast cell surface. Biotechnol Adv 18: 121-140.
Yang SC, Lin CH, Sung CT, Fang JY (2014) Antibacterial
Activities of Bacteriocin Application in Foods and
Pharmaceuticals. Front Microbiol 5: 1-10.
Zendo T, Fukao M, Ueda K, Higuchi T, Nakayama
J, Sonomoto K (2003) Identification of the Lantibiotic
Nisin Q, a New Natural Nisin Variant Produced by
Lactococcus lactis 61 - 14 Isolated from a River in Japan.
Biosci Biotechnol Biochem 67: 1616-1619.

CLONING AND EXPRESSION NISIN ON SACCAROMYCES CEREVISIAE CELL
SURFACE BY FUSION WITH a-AGGLUTININ GENE
Nguyen Hien Nghia, Thai Ngoc Anh, Nguyen Quynh Huong, Ngo Thi Cam Nhung, Nguyen Tri Nhan,
Tran Linh Thuoc, Dang TM Phuong Thao23
University of Science, Vietnam National University in Ho Chi Minh
SUMMARY
Nisin is a bacteriocin which has a board antibacteria spectrum and inhibiting pathogeneic bacteria in
humans and animals, such as Staphylococcus aureus, Listeria sp. In addition, althought Nisin was obtained
from fermenttion broth of Lactococcus lactis that have been widely applied in food preservation industry, but
native strains secreted naturally Nisin with low levels. Hence, the domestic and foreign researches currently
perform to increase Nisin expression level by recombinant techniques in many host cells as Escherichia coli or
Saccharomyces cerevisiae (S. cerevisiae). In order to find a new approach to applications in food preservation
as well as collect new strains inhibiting pathogeneic bacteria in cattle and aquaculture. We will show strategy
in which we aim to express Nisin on S. cerevisiae cell - surface by fusion nisin gene with a-agllutinin gene.

The yeast cell lines are collected as the raw material for applied research in preservation food as well as in the
preparations inhibiting pathogeneic microorganisms.
Keywords: Agglutinin, bacteriocin, fluoresent, nisin, pICAS-1, Saccharomyces cerevisiae, yeast surface

23 Author of correspondence: E-mail:

385