Mutant-type acetyltransferase Mpr1

US008 l 0 l 390B2
(12) United States Patent
Takagi
(54)
(10) Patent N0.:
(45) Date of Patent:
MUTANT-TYPE ACETYLTRANSFERASE
MPR1
(75) Inventor: Hiroshi Takagi, Ikoma (JP)
(73) Assignee: National University Corporation Nara
Institute of Science and Technology,
Nara (JP)
Notice:
Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 336 days.
(21) Appl. No.:
logue, AZetidine-2-Carboxylic Acid,” J. Biochem., vol. 133, pp.
charomyces cerevisiae,” Applied and Environmental Microbiology,
PCT N0.:
PCT/JP2007/053667
§ 371 (0X1)’
(2), (4) Date:
Mar. 24, 2009
(65)
Enzymes on L-Proline Accumulation and Freeze Tolerance in Sac
vol. 69, No. 11, pp. 6527-6532, 2003.
H. Takagi et al., “Kobo ni Hakken shita ‘Acetyl-ka Koso Mprl’ ni yoru
Atarashii Ko Sanka,” vol. 44, No. 2, pp. 83-84, Feb. 2006.
K. Iitani et al., “Random Hen’I Donyu ni yoru Kobo
Prior Publication Data
US 2010/0196987 A1
Aug. 5, 2010
Foreign Application Priority Data
Jul. 14, 2006
(JP) ............................... .. 2006-194365
Int. Cl.
C12N 9/12
C12N 1/00
C12N 15/00
C07H 21/04
Acetyltransferase Mprl no Sanka Stress Taisei Kojo,” Japan Society
for Bioscience, Biotechnology and Agrochemistry Kansai Shibu
Koenkai Koen Yoshishu, vol. 2006, p. 32(C13), Sep. 2006.
K.
Iitani et al.,
“Random Hen’I Donyu ni yoru Kobo
Acetyltransferase Mprl no Sanka Stress Taisei Kojo,” Japan Society
for Bioscience, Biotechnology and Agrochemistry Kansai Shibu
Koenkai Koen Yoshishu, vol. 2006, p. 32(C13), Sep. 2006. [With
(2006.01)
(2006.01)
(2006.01)
(2006.01)
English translation].
* cited by examiner
US. Cl. ................ .. 435/193; 435/254.1; 435/320.1;
536/23.2
(58)
plex species,”Yeast, vol. 19, pp. 1437-1445, 2002.
PCT Pub. Date: Jan. 17, 2008
(86)
(52)
AZetidine-2-carboxylic Acid,” J. Bio. Chem., vol. 276, No. 45, pp.
41998-42002, Nov. 9, 2001.
C. HoshikaWa et al., “A nonconserved Ala40l in the yeast RspS
ubiquitin ligase is involved in degradation of Gapl permease and
stress-induced abnormal proteins,” Proc. Natl. Acad. Sci., vol. 100,
No. 20, pp. 11505-11510, 2003.
Y. Kimura et al., “Polymorphism of the MPRI gene required for toxic
proline analogue resistance in the Saccharomyces cerevisiae com
PCT Pub. No.: WO2008/007475
Feb. 27, 2007
(51)
M. Shichiri et al., “A Novel Acetyltransferase Found in Sac
charomyces cerevisiae E1278b That Detoxi?es a Proline Analogue,
67-74, 2003.
M. Nomura et al., “Role the yeast acetyltransferase Mprl in oxidative
stress: Regulation of oxygen reactive species caused by a toxic
proline catabolism intermediate,” Proc. Natl. Acad. Sci., vol. 101,
No. 34, pp. 12616-12621, 2004.
W. Li et al., “Proline Biosynthesis in Saccharomyces cerevisiae:
Molecular Analysis of the PRO] Gene, Which Encodes y-Glutamyl
Kinase,” J. Bacteriol, vol. 174, No. 12, pp. 4148-4156, 1992.
Y. Terao et al., “Gene Dosage Effect of L-Proline Biosynthetic
PCT Filed:
(30)
Jan. 24, 2012
M. Nomura et al., “Characteristization of Novel Acetyltransferases
Found in Budding and Fission Yeasts That Detoxify a Proline Ana
12/309,271
(22)
(87)
US 8,101,390 B2
Field of Classi?cation Search ................ .. 435/193,
435/254.1, 320.1; 536/23.2
See application ?le for complete search history.
Primary Examiner * Tekchand Saidha
(74) Attorney, Agent, or Firm * Wenderoth, Lind & Ponack,
LLP
References Cited
(57)
ABSTRACT
The present invention provides a mutant-type acetyltrans
U.S. PATENT DOCUMENTS
yeast Wild-type Mprl represented by SEQ ID NO: 1, Wherein
(56)
ferase Mprl: Which comprises an amino acid sequence of a
2010/0196987 Al*
8/2010
Takagi ........................ .. 435/193
FOREIGN PATENT DOCUMENTS
JP
JP
9-234058
2006-067806
9/1997
3/2006
OTHER PUBLICATIONS
H. Takagi et al., “Saccharomyces cerevisiae E1278b Has Novel
Genes of the N-Aceytltransferase Gene Superfamily Required for
L-Proline Analogue Resistance,” J. Bacteriol, vol. 182, No. 15, pp.
4249-4256, 2000.
at least one amino acid at positions 63 to 65 and 117 of the
amino acid sequence is substituted and said mutant-type
acetyltransferase Mprl exhibits a higher antioxidant capacity
than the Wild-type Mprl. The mutant-type acetyltransferase
Mprl of the present invention exhibits a higher resistance to
oxidative stress compared to the Wild-type Mprl . The present
invention further provides a gene encoding the mutant-type
Mprl, a vector comprising the gene and a yeast transformed
With the gene.
8 Claims, 20 Drawing Sheets
US. Patent
Jan. 24, 2012
Sheet 1 0f20
US 8,101,390 B2
Figure l
9
CH3
Figure 2
P56
Oxida?ve mass
:
l
r’ M
éiumma'fe
\
N~Aaefyia?on
19567651 é- Mprlp -—-> X
PSC/éSA /
\
4
Pmiine
\ Mitochondria
;
4
t‘
‘if?
R05 Cyfopiasm
US. Patent
Jan. 24, 2012
Sheet 2 0f 20
US 8,101,390 B2
Figure 3
MP}?
PGAL_MPR
Error- Prone PCR
_
<- and
Digestion
HindIII
at M1111 _.
NM
restriction sites
Ligation
mt}?
Transformation into
E.coli JMIOQ
Construction of random
mutation library
__
JMIOQ
+
U S Patent
Jan. 24, 2012
Sheet 3 0f 20
iguxe 4A
A) Stressfree
KY8
AD
SD+Ura
US 8,101,390 B2
U S Patent
Jan. 24, 2012
Sheet 4 0f 20
US 8,101,390 B2
Figur@ QB
CK Y8 gDUra+A(¢/ml)
US. Patent
Jan. 24, 2012
Sheet 5 0f 20
US 8,101,390 B2
Figmre 4C
CZ) Heat shock
CM
@AD4
QAD~MFR
5W1
._
‘
US. Patent
Jan. 24, 2012
Sheet 6 0f 20
US 8,101,390 B2
Figuxre #111
D) Hydrogen peroxide
CKYQ
QADQ
13 MH 1
SD+Um+H,-O (m
US. Patent
Jan. 24, 2012
Sheet 7 0f 20
US 8,101,390 B2
US. Patent
Jan. 24, 2012
Sheet 8 0f 20
US 8,101,390 B2
Figure 6A
A) Stress-"free
CK‘Y’Q
pAD4 (water)
pz‘i'aD "'MPR
pAD~MP EPQ USE
pEE‘aDWIWKPRWFEELX L1 NV
pAD-P1APR~I léiéM
pAUWMPR-MEBZQV
S WUm
US. Patent
Jan. 24, 2012
Sheet 9 0f 20
US 8,101,390 B2
Figura 6B
EDAZQ:
‘1W8 SD+Um+AZ 0&1 mam)
pADé (vecmr)
pAD ""MPR
pAD "MP R"GMZS
WBDMMPR-FSSLXLI NV
pAD~MPR-I 184M
)
US. Patent
Jan. 24, 2012
Sheet 10 0f 20
US 8,101,390 B2
Figure: 6C
C) Heat sh0ck
CH. Y8
SWUYE?EOOCQh
W134 (vector)
pAD "MP R~F163E1
pf-ED *MPRWQ 179 R
wwxwpwmsull1W
pAD "MPRMI 13 4M
'
-
US. Patent
Jan. 24, 2012
Sheet 11 0120
US 8,101,390 B2
Figure 613
D) Hydrogen pemxide
CR ‘YE
pADé (vector)
p?D?VIPR
pAD ~00? R-KEB 33R
pAD “MP RMCEMSFS
pAD WMP R~F65Lf L1 17%’
p311!) "MPEP-I 184M
pAD-MPR-E2’2%V
$D+Um+HZOZKZ 111M)
US. Patent
Jan. 24, 2012
Sheet 12 0f 20
US 8,101,390 B2
Figure 7A
A) Stressfree
CK'YQ
Mm
pAD“ MFR
{11 AU MP Rmii?g R
SD+Um
US. Patent
Jan. 24, 2012
Sheet 13 0f 20
US 8,101,390 B2
Figure: 7B
5) ABC
Cm
QAQWMPRM $65M’ L1 WV
Maw
.
US. Patent
Jan. 24, 2012
Sheet 14 0120
US 8,101,390 B2
Figuzre "7C
C) Heat shock
{III/{Y8 SD+#U1‘50@, 1.511
13 Am
13 AD" MP R
QAD“MPR“K63 R
US. Patent
Jan. 24, 2012
Sheet 15 0120
US 8,101,390 B2
Figure 7;)
9) Hydrogen peroxide
YYJADMMPR
CKYB SD+TQ+HO§
pmwmwmww
@AUwfvfiPR» $551,111 111/"
.... “
1
'
US. Patent
Jan. 24, 2012
Sheet 16 0f 20
US 8,101,390 B2
Figuxe ‘7133
E) Ethanol
cyqyg
mm
QADM MFR
pADW I‘AFR”K63 R
SD+a+ETO(5%> ‘
US. Patent
Jan. 24, 2012
Sheet 17 0f 20
US 8,101,390 B2
Figure 8
ROS level after the hydrogen peroxide treatment
E 0min
[330mm
E3 60min
21|
o5
0o
pAD4
Figure 9
Survival rate after the hydrogen peroxide treatment
100
axe3wew82za5m.5u
dl 0
"+"pAD4
” ‘I’ "' pAD-MPR
“1m pAD-MPR-Kasn
2
Treatment time
3
(hr)
US. Patent
Jan. 24, 2012
Sheet 18 0120
US 8,101,390 B2
F1 gure 1 O
Mutant-type Mprl the?rms-ta:bilit},1 (4513)
HID
wild-‘typa
33m5i3.1w5?:
i
1-.
2°
25
35
25
Treatment time at 45°C (min)
Figure 11
Stability in the presence of hydrogen peroxide (0.5M)
1- c- - K63R
wW
Mm
m
H
3.3
W
w
.w WM +i_. F 565 |._L
“
"ii-1.1.2!" - .
.
ml-2‘b
m
.W
_
m
_
on0
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u
m
w
.
m
_
_
. .__
.Fir
H
. .
n
_
u
m
w
,.
_
u
1
vV
m
1D
15
20
Treatment time (min)
US. Patent
iii
E2;
21.:
m33%;?
Jan. 24, 2012
Sheet 19 0f 20
US 8,101,390 B2