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Construction of G. oxydans NH-10 sArDH disruption mutant and preliminary study on the catalytic characteristics of the mutant

Gluconobacter oxydans NH-10中NAD+型阿拉伯糖醇脱氢酶基因缺失菌株的构建



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ChineseJournalofBioprocessEngineering
Vol.10No.1
Jan.2012
doi:10.3969/j.issn.1672-3678.2012.01.007
OPQI
:2011-05-10
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(20103221120007);
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(CX09B_143Z)
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GluconobacteroxydansNH10
l
NAD+
´
‹ŒÄ£ŽcKœº@D‰f˜
©
 
›

–ª«

¬
 
­

®¨;pK5 LM8¯;56 °±75;
®¨
210009)
7
 
h

묥¦1!Ò
GluconobacteroxydansNH10
5Ó§¬

ÔÕÖ1Á

×ز1
RÏÎ
t¶³
NAD+
Ù

ÔÕÖ1ÁÚÛ©XŸÎã—a

ز134/

ÔÕÖ1Á

Üf̧Ø1Á³ÃÝ

š¦û¡•Ð¼¸Þò³ª­®
G.oxydansNH10NAD+
Ù

ÔÕÖ1ÁÚÛ©
(sArDH)
¼¸Þòß}€

PCR
ÝÞBà
:sArDH
¼¸¶

€•ÐÎáâãäåê켸æç

ßy
sArDH
¼¸Þòß}­®/Æ

<
aèéìêrBà

ß}¶ëìí


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à

ß}€wt¶34

ز1—

ÔÕÖ1Á³ôõ¨

ö—aØ1Á³—JEC¯Š

ijk

묥¦1!Ò
;sArDH;
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lmnop
:Q78    
6qrst
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:1672-3678(2012)01-0037-05
ConstructionofGluconobacteroxydansNH10sArDHgenedisruption
mutantanditscatalyticcharacteristics
ZHOUPeng,ZHUHongyang,XUHong
(StateKeyLaboratoryofMaterialsOrientedChemicalEngineering,
ColegeofFoodScienceandLightIndustry,NanjingUniversityofTechnology,Nanjing210009,China)
Abstract:Inapreviousstudy,aGluconobacteroxydansNH10tobescreenedabletoproducexylitolfrom
Darabitol.However,solubleNAD+dependentDarabitoldehydrogenasealsoexistedinthisstrain,and
reversiblyconvertedDxylulosetoDarabitol,thusmakingacertaininfluenceontheproductionofxyli
tol.ThispaperconstructedasArDHgenedisruptionmutantofG.oxydansNH10bytheprincipleofho
mologousrecombination.PCRanalysisshowedthesArDHgenewascompletelyreplacedbyKanamycin
resistantgene.Biologicalcharacteristicanalyseconfirmedthatnodiferenceswereobservedinmycelial
morphologyandthegrowthproperties.Thecatalyticcharacteristicsofthemutantrestingcelswerestud
ied.Comparedwiththewildstrain,noreversiblereactionofDxylulosereducetoDarabitolwasfoundin
thesArDHmutants,andmuchmorexylitolwasobtained.
Keywords:Gluconobacteroxydans;sArDH;genedisruption
  
9-deD‹cZ
(G.oxydans)
Õo¬%9E Íf)C;Z

×Üÿ‹cZÖ

8ܘõö$C
`¼Exyh

!YD

w

¹Fí9-ú1D

R
"

“4l|ö)ÙÚE*P
[1-2]。Suzuki
þ
[3]
‡
ÂdDcZ×r+“G/ŽE

tuvDwxy
h
(mArDH)
âË;²Dwxyh
(XDH),
“¶ù
pH
í
50~60
EklÀ

!Y

tuvDwf-
ú²Dw
。Adachi
þ
[4]
Õ
G.suboxydansIFO3257
r
‰-6G/Ž
ArDH,
©@7hM;’½ñ6ø,
³´

‡Â!hHa
PQQ,
â9-

tuvDw^
€

²1D

í¹âÎë*
。Cheng
þ
[5]

Acetobactersuboxydans

G.oxydansCGMCC1110
Z_r‰-©ij6
NAD(P)
HaEtuvDwx
yh
(sArDH),
‡Â!h+“Ü<=’r

×ܖP
xyh¦X

âY

tuvDw9-ú

²1
D

>âY

²1Dóäí

tuvDw

@AғC3ã\V¿

_
G.oxydansNH10,
âf-

tuvDw^€²Dw

ë*X¹ÆÖ

Òr

)ë*E½ñ

Ëf-EœmtuvDw
õ™öҞ

ϘÕ8Ü!Zr+“
sArDH;
½o
DE³´‡C!h@

tuvDwE
Kmí607
mmol/L,
@

²1D
Kmí364mmol/L
[6],
R"
Q0Y-

²1Dóäí

tuvDw

@f-
^€¹FF)

í62]²Dw@

tuvDw
Ef-=

Y|}
sArDH
z{Z_

©@7Íë<
=Y-f-E°;½ñ³´

m
1 G.oxydansNH10
@Dl‘Y

‹ŒÄ£C’Ä£‰}“
Fig.1 SchematicrepresentationofxylitolproductionfromDarabitolbyG.oxydans
1 
vw%xy
1.1 
@D%ýg
E.coliJM109,E.coliHB101,G.oxydansNH10,
pSUP202
’è
,pRK2013
’èˆ8C3ãïí

pET 28a(+)
’èÕJ
Takara
vw

1.2 
mnK%z{
G.oxydansNH10
cåQ
(g/L):
deD
50,
ˆ
œÐ
30,
ÑÒÐ
5,D
tuvDw
10;
”²cåQò
~ö{°
20g/L。G.oxydansNH10
“
30℃
À
200r/min

3d。
x‚cZ67`SZ_P
LB
cåQ

Ú¬8
€\’™£
(μg/mL):Kanamycin(Kan)25,Ampi
cilin(Amp)100,Cefotaxime5。
  TaqDNA
Žh
、T4
(Rh

“¹;Ñ、¨
;ú‹h
(CIAP)、
Ú¬8€、DNA
Ô÷‰-2 
o

§Ÿa2 oˆÕJ
Takara
vw

2mŽúš
˜B8»k§hS€m4ÀUBö“vwFú

1.3 
€xy
x‚cZƒ7N<=E¹ºf-6ðáE
DNA
€Åì>çDE
[7]。
gæ/Ž
(tripa
rentalconjugation)
RŽfgÍ.>çDE
[8]。
1.4 
ӟ~W%
PCR
Ž
141 
2m45
÷
G.oxydans621H
QRSíZ–

45
sArDH
abBp|z
(L)
Àz
(R)
2m

|z2m
L1:
5′TATGAATTCCCTCTTGAAAACCTATCATAGC3′
(EcoRΙh<{v);L2:5′CTGTTTATGTAAGCCT
CGAGAAACTTGAAGTCC3′(XhoΙh<{v)。À
z2m
R1:5′AATAAACAAATAGCTCGAGAAAA
TGGCCGGGAAG3′(XhoΙh<{v);R2:5′ATG
AATTCATGGCGACTGTCGAACTCAAG3′(EcoRΙ
h<{v
)。
÷
pET 28a(+)
íZ–45
Kan
8;Q
R2m
,F1:5′GGACTTCAAGTTTCTCGAGGCTTAC
ATAAACAG3′(XhoΙh<{v);F2:5′CTTCCCG
GCCATTTTCTCGAGCTATTTGTTTATT3′(XhoΙh
<{v
)。
142 
ðá
PCR

50μL²øEë*kl:94℃ 2min;94℃
30s,45~65℃ 30s,72℃ 1~4min,
ìa
30
¡

83
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!
10
"
 
72℃ 10min。
143 

PCR

ë*p{Dxž

!oDE²ø
:10×PCRbuf
er25μL,Mg2+2μL,dNTP(Ú20mmol/L)2μL,
Î`ÓÔ÷Z–

Z–

Ú
2μL,LADNApoly
merase05μL, ddH2Oóë*J²Ìí 25μL。
xžÀB
:95℃
Ô»
4min;94℃ 55s,65℃40s,
72℃ 1min,
ìa

¡
;72℃
I;
2min。
“!oDEQW|Ê
25μLë*m’,²ø
ÆÀ
:10×PCRbufer5μL,Mg2+ 2μL,dNTP(Ú
20mmol/L)4μL,Î`ÓÔ÷Z–1|E2mZ
–

ÀE2mÚ
2μL,LADNApolymerase1μL,
ddH2Oóë*J²Ìí50μL。xžÀB:95℃Ô
»
4min;94℃50s,55~60℃40s,72℃1min,
ì
a
30
¡
;72℃
I;
10min。
1.5 G.oxydansNH10
Kœ9•
sArDH
–K—˜
¤‰f˜%ó‹
1.5.1 

pSUP202sArDH::Kan
E|}
÷
NH10
QRSíZ–

pÇP2m
L1/L2,
R1/R2

sArDH
QRE|ÀzÔ÷
L、R;
P2m
F1/F2
Õ
pET28a(+)
QRSrxž
Kan
8;Ô÷


sArDH
QRzú‚ðPEh<{v

*P`Ó
PCR
Í.Y


Kan
Ô÷(R

Ï<
KR
Ô÷

Y


KR
Ô÷(R

Ï<
LKR
Ô÷


pSUP202
_
EcoRΙhpSUP202sArDH::Kan。
(R^mf-ó
E.coli
JM109
ƒ7N<=r

“õ
Amp

Kan
E
LB
˜–
|ÕV`S­

1.5.2 G.oxydansNH10sArDH
ïX²E|}
Yõz{ïXe²
pSUP202sArDH::Kan
E
E.coliJM109
ìíG²ZPÜgæRŽ

7²Zí
G.oxydansNH10。
z { ï X e ² “ Ì L Z
pRK2013
EÖLÀ½Ê
G.oxydansNH10,

Kan
QRETrQRÔ÷3Ž¿
G.oxydansNH10

^²|

x¿
Cefotaxime、Kan
\VgæRŽ­


Vx¿
PCR
xžë*Vµ
sArDH
QRz{Z

1.6 
º@‰0ˆ[g
YòêZ²™Ez{Zšß€ZÜ
GAra
•²
cåQrcå
,30℃、200r/min

48h,
þ&o
÷ë93«˜
OD660,÷²˜Z²€;&½。
1.7 
™š&aY
Darabitol
‰ôV
Ycå
24h
E
G.oxydansNH10
6z{ZÜ
8000
r/min
±š
10min,
©P
100mmol/LPBS
Jû•
(pH
6.0)
±”

¡

ab<=

s¸ž3
1g
»Z²Êù
ö
10mL30g/LD
tuvDw
,100mmol/LPBS

•
(pH6.0)

20g/LCaCO3E50mLg1`r,30℃、
220r/min
Àë*
9h
V


5%
ƒw
,30℃
ÀÍÈë
*
27h,
3«˜µœm^m™

HPLC
kl
:^
¯’í
Aglient1200;^
¯¬í
ShodexSugarSP0810;
¬Ý
80℃;
 
1mL/min;
 
ãòí‰ç

²˜€ír[׌²˜€

2 
‚ƒ%n¯
2.1 
º—˜k¤
pSUP202sArDH::Kan
‰f˜
÷
G.oxydansNH10
J
DNA
íZ–
,PCR

<¿
sArDH
|ÀzÔ÷


R,
ˆí
800bp。
÷
pET28a(+)
íZ–
,PCR

900bp
E
Kan
Ô÷

x¿`Ó
PCR,
÷
K1

R2
í2m|}
KR
Ô÷

Æ
÷
L1、K2
í2m|}
LKR
Ô÷

—~/æýrÔ÷
x£í
2600bp,
š˜B/æò˜

P
EcoRΙh<LKRÔ÷e² pSUP202½ñ
(Rë*

f-ƒ7N
E.coliJM109,
“öù™
Amp

Kan
E˜–|(V`S­

x¿
EcoRΙãh
<3‡

/æ‹Ö
2。


âï
:EcoRΙh<`S
’è!/<À:
2600bp
x£EÔ÷

št#/æ
ò˜

nôË_úÿàY
LKR
Ô÷(R¿6
pSUP202
e²|

M—DNA
rs¦

1—pSUP202sArDH:KanEcoRI
h<
m
2 pSUP202sArDH::Kan
‰ch›m
Fig.2 IdentificationofthepSUP202sArDH::Kan
withEcoRΙdigestion
2.2 sArDH
Kœº—˜¤‰PsArDH
QRz{ïX²E|}8gæRŽC
93 
!

# _
 

:GluconobacteroxydansNH10
r
NAD+
stuvDwxyhQRz{Z_E|}
Â,
G²Zˆíõ`S’è
pSUP202sArDH::Kan
E
E.coliJM109,
7²Zí
G.oxydansNH10,
ÌLZÕ
õÌL’è
pRK2013
E
E.coliHB101。
x¿
Cefo
taxime、Kan
8;\VgæRŽ­

í6½oD‡C
sArDH
QRË_Dz{

P
L1、K2
6
Kan
QR2m

÷
sArDH
z{ïX²
DNA
íZ–

x¿
PCR


/æ‹Ö
3。


âï

xž´
1700

900bp
E
Ô÷

8"‡ôúÿà|}69-deD‹cZ
NH
10sArDH
QRz{Z
NSA18(NH10sArDH::Kan)。
1—
2m
L1/K2PCR
E^m

2—
2m
K1/K2PCR
E^m
;M—DNA
rs¦
m
3 PCR
ó‹
sArDH
Kœº—˜¤
Fig.3 PCRproductstoconfirmsArDHdisruption
2.3 
º@‰0ˆ[œ
Y
G.oxydansNH10
߀Z_šz{ZE¿ˆ
cå•)ó
OD600í10,÷5%ER¬™fR¿Wù
E
100mL
õò*8€\E
GAra
cåQr
,30℃、
220r/min

48h,
²˜Z²E€;&½

Ö
4),
8
Ö

âï

z{Z_
G.oxydansNH10sArDH::Kan
NSA18
šß€Z_
G.oxydansNH10
€;89Q
oC

R"âAí
sArDH
QREz{©¹F)9-
deD‹cZ“
GAra
cåQrE€;

m
4 
0@%º—˜@Õ
GAra
mnKl‰0ˆ–—
Fig.4 GrowthofthewildandmutantstrainNSA18
inGAramedium
2.4 sArDH
Õ
G.oxydansNH10
‘Y

‹Œ
Ä£0ž’Ä£‰RÜ
  
í6%&z{Z_
NSA18
šß€Z_Íë<
=Y-°;EáÇ

YcåúØE<=à
1.7
rE
Í.Y-

tuvDw^²Dw

/æ‹i
1。
®
1 sArDH
Kœºž‘Y

‹ŒÄ£C’Ä£‰¡U
Table1 EfectsonDxylitolproductionofNSA18andNH10
Z_
œmš^m
/(g·L-1)

tuvDw

²1D ²Dw
0h 9h 27h 0h 9h 27h 0h 9h 27h
߀Z
30 2.1 13.7 0 270 7.1 0 1.7 7.8
z{Z
NSA18 30 2.2 2.1 0 26.9 15.5 0 1.9 10.1
  
8i

âï

šß€ZòÃ
,sArDH
Ez{@
ïXZ_Y-9-

tuvDwE!ŠF)¹
x

Íë<=f-

tuvDw
9h,D
tuv
Dw@r9^m

²1Df-=ˆRú
95%,
i
ô

tuvDwE]Ÿ9-ù¼HaÜG/Ž
E

uvDwxyh

êë˕r€ú61™E
²Dw

nô²Dwxyhh:;¹]

f-
9h
V

Î˕r~
5%
ƒw

FP<=J¯^€o
µ™EóäŠ
NADH,
â2]²DwxyhEh
:

QöFܲDwEÌÙ

)f-E½ñ

ß
€ZrEtuvDwõ™ôý|7

»ß€Zr
+“E
sArDH
!/FPƒw^€E
NADH,
Y

²1Dóäú

tuvDw

Hf-ë*Î
¹FܲDwÌÙEÍνñ
,27h
²Dwõ™
ïí
7.8g/L,D
tuvDw@²Dwf-=í
26%;
(z{Zr¹+“

²1DÎf€ú

04
€
 
m
 

 
4
 
¿
 
À
  
!
10
"
 
tuvDwEë*

x¿~
5%
ƒw
,27h
ë²
Dwõ™í
10.1g/L,D
tuvDw@²Dwf
-=í
34%,
“oµÀ|2]6²DwEõ™

8Ü!f-ë*rEó,h²DwxyhHaÜ
NADH,
BC߀Zz{Z@

tuvDwE
3²f-=ˆ¹]

R"“f-¿Àr2GÙ
E
NADH
Õ2]3²f-=Eó,

3 
‚
 
)
x¿êh`S

z{69-deD‹cZ‰^²
|E
sArDH
QR

HâË;
NAD(P)
Has

tu
vDwxyhj:

x¿
PCR
3‡\VÏ<`SZ
_

@7½ñf-

tuvDw^²DwE&½½
ñ6pq

/æiô
:sArDH
QRz{Z_“óä

²1D^²Dw¿Àr

¹ûö߀ZY

²1D
óäí

tuvDwE!Š

R"òÃ6À
,D
t
uvDw@²DwEf-=:]

M¼6q

[1] MatsushitaK,YakushiT,TakakiY,etal.Generationmechanism
andpurificationofaninactiveformconvertibleinvivototheac
tiveformofquinoproteinalcoholdehydrogenaseinGluconobacter
oxydans[J].JBacteriol,1995,177:65526559.
[2] ChengH,JiangN,ShenA,etal.Molecularcloningandfunctional
expressionofDarabitoldehydrogenasegenefromGluconbobacter
oxydansinEscherichiscoli[J].FEMSMicrobiolLet,2005,252:
3542.
[3] SuzukiS,SugiyamaM,MiharaY,etal.Novelenzymaticmethod
fortheproductionofxylitolfromDarabitolbyG.oxydans[J].
BiosciBiotechnolBiochem,2002,66:26142620.
[4] AdachiO,FujiY,GhalyMF,etal.Membraneboundquinopro
teinDarabitoldehydrogenaseofGluconobactersuboxydansIFO
3257:aversatileenzymefortheoxidativefermentationofvarious
ketoses[J].BiosciBiotechnolBiochem,2001,65(12):
27552762.
[5] ChengH,LiZ,JiangN,etal.Cloning,purificationandcharacter
izationofanNADdependentDarabitoldehydrogenasefromace
ticacidbacterium,Acetobactersuboxydans[J].ProteinJ,2009,
28:263272.
[6] 
Ú£˜

½{B

u–í
.GluconobacteroxydansNH10
r–P

tuvDwxyhE³´
[J].
ru€m4ÀgŽ
,2010,
30(11):3943.
[7] SambrookJ,FritsehE,ManiatisT.Molecularcloning:alaboratory
manual[M].Kanazawa:ColdSpringHarborLaboratoryPress,1989.
[8] HolscherT,GorischH.Knockoutandoverexpressionofpyrolo
quinolinequininebiosyntheticgenesinGluconobactersuboxydans
621H[J].JBacteriol,2006,188:
櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒
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