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1 {\b \fs32 Heterologous}{\b \fs32 production}{\b \fs32 of}{\b \fs32 polyunsa
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b \fs32 in}{\b\i \fs32 Saccharomyces}{\b\i \fs32 cerevisiae}{\b \fs32 causes}
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{\phpg\posx1131\pvpg\posy4364\absw3099\absh463 \i \f10 \fs19 \cf1 \i \f10 \fs19
\cf1 Rawisara Ruenwai{\dn006 \fs13 2,3 }
\par}{\phpg\posx1131\pvpg\posy4364\absw3099\absh463 \sl-279 \i \f10 \fs19 \cf1 S
upapon Cheevadhanarak{\dn006 \fs13 2,3 }\par
}
{\phpg\posx2922\pvpg\posy4364\absw8802\absh242 \i \f10 \fs19 \cf1 \i \f10 \fs19
\cf1 , Andrea Neiss {\dn006 \fs13 1}, Kobkul Laoteng {\dn006 \fs13 4}, Wanwipa
Vongsangnak {\dn006 \fs13 1}, Arastoo Badoei Dalfard {\dn006 \fs13 1}*, \par
}
{\phpg\posx3610\pvpg\posy4644\absw3904\absh211 \i \f10 \fs19 \cf1 \i \f10 \fs19
\cf1 , Dina Petranovic {\dn006 \fs13 1} and Jens Nielsen{\dn006 \fs13 1 }\par
}
{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \f10 \fs11 \cf1 \f10 \fs11 \cf1
1{\fs16 Department}{\fs16 of}{\fs16 Chemical}{\fs16 and}{\fs16 Biological}
{\fs16 Engineering,}{\fs16 Chalmers}{\fs16 University}{\fs16 of}{\fs16 Tech
nology,}{\fs16 Gothenburg,}{\fs16 Sweden }
\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-230 \f10 \fs11 \cf1 {\
dn006 2}{\fs16 School}{\fs16 of}{\fs16 Bioresources}{\fs16 and}{\fs16 Techn
ology,}{\fs16 King}{\fs16 Mongkut's}{\fs16 University}{\fs16 of}{\fs16 Tech
nology}{\fs16 Thonburi,}{\fs16 Bangkok,}{\fs16 Thailand }
\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-229 \f10 \fs11 \cf1 {\
dn006 3}{\fs16 Pilot}{\fs16 Plant}{\fs16 Development}{\fs16 and}{\fs16 Trai
ning}{\fs16 Institute,}{\fs16 King}{\fs16 Mongkut's}{\fs16 University}{\fs16
of}{\fs16 Technology}{\fs16 Thonburi,}{\fs16 Bangkhuntien,}{\fs16 Bangkok,
}
\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-230 \f10 \fs11 \cf1 \f

i90 {\fs16 Thailand }


\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-229 \f10 \fs11 \cf1 {\
dn006 4}{\fs16 Biochemical}{\fs16 Engineering}{\fs16 and}{\fs16 Pilot}{\fs16
Plant}{\fs16 Research}{\fs16 and}{\fs16 Development}{\fs16 Unit,}{\fs16 N
ational}{\fs16 Center}{\fs16 for}{\fs16 Genetic}{\fs16 Engineering}{\fs16 a
nd }
\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-230 \f10 \fs11 \cf1 \f
i90 {\fs16 Biotechnology,}{\fs16 National}{\fs16 Sciences}{\fs16 and}{\fs16
Technology}{\fs16 Development}{\fs16 Agency}{\fs16 at}{\fs16 King}{\fs16 Mo
ngkut's}{\fs16 University}{\fs16 of}{\fs16 Technology}{\fs16 Thonburi, }
\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-230 \f10 \fs11 \cf1 \f
i90 {\fs16 Bangkhuntien,}{\fs16 Bangkok}{\fs16 ,}{\fs16 Thailand }\par
}
{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \f10 \fs16 \cf1 \f10 \fs16 \cf1
Due to their health benefits there is much interest in developing microbial proc
esses for efficient
\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 pro
duction of polyunsaturated fatty acids (PUFAs). In this study we co-expressed{\i
Mucor}{\i rouxii}{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }} }{\dn006 \
fs11 12}\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 and
{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }} }{\dn006 \fs11 6}-desaturase
genes in{\i Saccharomyces}{\i cerevisiae}, which resulted in a yeast strain th
at accumu\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 lat
ed linoleic acid and{\f40 {\field{\*\fldinst SYMBOL 103 \\f Symbol }}}-linoleni
c acid in the different lipid species. Additionally, the strain contained
\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 hig
her levels of phospholipids and lower levels of ergosterol than the reference st
rain. Integrated
\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 ana
lysis of the transcriptome revealed decreased expression of genes involved in er
gosterol biosyn\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 the
sis, but more unexpectedly it also pointed towards attenuated activity of the ub
iquitin-protea\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 som
e system and a reduced oxidative stress response.{\i In}{\i vitro} and{\i in}
{\i vivo} measurements showed
\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 red
uced levels of all three proteasomal activities and also increased lev
els of reactive oxidative
\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 spe
cies in the PUFA-producing strain. Overall our results clearly show that PUFAs i
n yeast can be
\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 det
rimental for several key cellular pathways, such as the oxidative stress respons
e and proteaso\par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 mal
activity, suggesting that the membrane composition is of vital importance for t
hese processes. \par
}
{\phpg\posx8784\pvpg\posy7211\absw2130\absh488 \f10 \fs13 \cf1 \f10 \fs13 \cf1 R
eceived 21 September 2010
\par}{\phpg\posx8784\pvpg\posy7211\absw2130\absh488 \sl-199 \f10 \fs13 \cf1 Revi
sed
2 November 2010
\par}{\phpg\posx8784\pvpg\posy7211\absw2130\absh488 \sl-199 \f10 \fs13 \cf1 Acce
pted 10 November 2010 \par
}
{\phpg\posx8718\pvpg\posy9358\absw1674\absh307 \b \f10 \fs13 \cf1 \b \f10 \fs13

\cf1 Supporting information


\par}{\phpg\posx8718\pvpg\posy9358\absw1674\absh307 \sl-199 \b \f10 \fs13 \cf1 \
fi496 available online \par
}
{\phpg\posx1131\pvpg\posy10342\absw6412\absh154 \b \f10 \fs16 \cf1 \b \f10 \fs16
\cf1 Keywords:{\b0 Desaturase}{\b0 }{\b0 Lipid}{\b0 biosynthesis}{\b0 }{\b0
PUFAs}{\b0 }{\b0 yeast}{\b0 }{\b0 Stress}{\b0 response }\par
}
{\phpg\posx1131\pvpg\posy11013\absw165\absh203 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 1 \par
}
{\phpg\posx1491\pvpg\posy11013\absw1440\absh203 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 Introduction \par
}
{\phpg\posx1131\pvpg\posy11481\absw4620\absh574 \f20 \fs17 \cf1 \f20 \fs17 \cf1
The yeast{\i
Saccharomyces}{\i
cerevisiae} is a widely
\par}{\phpg\posx1131\pvpg\posy11481\absw4620\absh574 \sl-229 \f20 \fs17 \cf1 use
d model organism for studying eukaryal cellu\par}{\phpg\posx1131\pvpg\posy11481\absw4620\absh574 \sl-229 \f20 \fs17 \cf1 lar
and molecular principals [1]. This yeast is also \par
}
{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \b \f10 \fs13 \cf1 \b \f10 \fs1
3 \cf1 Correspondence:{\b0 Professor}{\b0 Jens}{\b0 Nielsen,}{\b0 Department
}{\b0 of}{\b0 Chemical}{\b0 and }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-199 \b \f10 \fs13 \cf1
{\b0 Biological}{\b0 Engineering,}{\b0 Chalmers}{\b0 University}{\b0 of}{\b
0 Technology, }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-199 \b \f10 \fs13 \cf1
{\b0 SE-412}{\b0 96}{\b0 Gothenburg,}{\b0 Sweden }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-199 \b \f10 \fs13 \cf1
E-mail:{\b0 nielsenj@chalmers.se }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-199 \b \f10 \fs13 \cf1
Fax:{\b0 +46-31-772-3801 }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-200 \par\b \f10 \fs13
\cf1 Abbreviations: AZE{\b0 ,}{\b0 \fs10
L}{\b0 -azetidine-2-carboxylic}{\b0
acid;} GLA{\b0 ,}{\b0 \f40 {\field{\*\fldinst SYMBOL 103 \\f Symbol }}}{\b
0 -linolenic}{\b0 acid; }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-200 \b \f10 \fs13 \cf1
HNE{\b0 ,}{\b0 4-hydroxy-2-nonenal;} LA{\b0 ,}{\b0 linoleic}{\b0 acid;} PUFA
s{\b0 ,}{\b0 polyunsaturated}{\b0 fatty }
\par}{\phpg\posx1131\pvpg\posy12672\absw5342\absh1568 \sl-199 \b \f10 \fs13 \cf1
{\b0 acids;} ROS{\b0 ,}{\b0 reactive}{\b0 oxidative}{\b0 species;} UPS{\b0 ,
}{\b0 ubiquitin-proteasome}{\b0 system }\par
}
{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \f20 \fs17 \cf1 \f20 \fs17 \cf1
an industrial workhorse used for the production of
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 fu
els, chemicals, food ingredients and pharmaceu\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 ti
cals [2-4]. It is particularly well known for its role
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 as
a cell factory for bioethanol production, where
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 it
s high ethanol tolerance allows for efficient pro\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 du
ction, reaching very high titers.Also a number of
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 re
combinant proteins (i.e.,hormones and vaccines)
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 ar
e being commercially produced by{\i S.}{\i cerevisiae},

\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 an


d here efficient protein production, folding and
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 se
cretion are of major importance [5].There is also
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 mu
ch interest in exploiting yeast as a production \par
}
{\phpg\posx6148\pvpg\posy14072\absw4792\absh308 \f10 \fs13 \cf1 \f10 \fs13 \cf1
* Current address: Department of Biology, Faculty of Science, Shahid
\par}{\phpg\posx6148\pvpg\posy14072\absw4792\absh308 \sl-199 \f10 \fs13 \cf1 \fi
112 Bahenar University of Kerman, Kerman, Iran \par
}
{\phpg\posx1131\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
{\phpg\posx10545\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
343 \par
}
\sect\sectd\pard\plain
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140

05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451401fffd9

}}\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \b \f10 \fs16 \cf1 \b \f10


\fs16 \cf1 Biotechnology
\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \f
i801 Journal \par
}
{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Bi
otechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \f20 \fs17 \cf1 \f20 \fs17 \cf1
organism for nutraceuticals, including polyunsatu\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ra
ted fatty acids (PUFAs), as this can lead to scala\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 bl
e production of pure and defined oils compared
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 wi
th fish oils.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \f
i283 Lipid metabolism has a central role in the phys\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 io
logy of the cell, e.g., the plasma membrane com\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 po
sition,which influences tolerance to high ethanol
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 co
ncentrations and other types of stress [6]. Lipid
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 me
tabolism also influences the membrane compo\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 si
tion of the endoplasmic reticulum (ER), which is
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ce
ntral to protein processing pathways [7]. The
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pr
ocesses of lipid metabolism are highly conserved
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 be
tween{\i S.}{\i cerevisiae} and mammals, such as mouse
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 an
d human [8]. For example, several key regulatory
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 el
ements involved in transcriptional regulation of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ge
nes involved in sterol biosynthesis and fatty acid
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 bi
osynthesis are found to be similar in yeast and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ma
mmals. The yeast Snf1p, like its mammalian or\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
olog AMP-activated protein kinase (AMPK), reg\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ul
ates acetyl-CoA carboxylation,which is a key step
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in
fatty acid biosynthesis [9]. In addition, the feed\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ba
ck regulation of unsaturated fatty acid synthesis
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
rough proteolytic activation of transcription fac\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 to
rs like Mga2p and Spt23p resembles in function
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e mammalian sterol regulatory element binding
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pr
oteins (SREBPs) [10]. Despite these similarities

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in


the regulatory mechanisms between yeast and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ma
mmalian cells,{\i S.cerevisiae} does,however,have a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 qu
ite different lipid composition as it contains no
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 PU
FAs in its membranes.The presence of PUFAs in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e membranes of yeast was previously shown to
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pl
ay important roles in cellular stress response to
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 va
rious stimuli, e.g., resulting in increased resist\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 an
ce to alkaline pH [11], oxidative stress [12], and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 fr
eezing and salt stress [13].
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \f
i283 Lipid metabolism is also of great interest in nu\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tr
itional studies and it has been shown that fish oils,
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 wh
ich are rich in PUFAs such as eicosapentaenoic
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ac
id (EPA, 20:5{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12
5,8,11,14,17 } ) and docosahexaenoic
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 ac
id (DHA, 22:6{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 4,7,
10,13,16,19 } ), have a number of pos\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 it
ive effects on health, including reduced risk of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ca
rdiovascular disease, cancer, neurodegeneration
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 an
d various other diseases [14-18]. In a recent
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 st
udy by our group [19] it was found that mice fed a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 he
rring-based diet that is rich in PUFAs resulted
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 no
t only in reduced expression of genes associated
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 wi
th cholesterol biosynthesis, but also reduced ex\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pr
ession of genes involved in oxidative stress re\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 sp
onse, protein folding and protein degradation in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 di
fferent tissues. It would be interesting to deter\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 mi
ne whether some of these responses to the pres\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 en
ce of PUFAs are conserved in eukaryotic cells.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \f
i283 As it is both of interest to produce PUFAs in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ye
ast and this serves as a good model organism for
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 eu
karyotes, we wanted to study the effect of PUFA

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pr


oduction in yeast. Therefore, to obtain a yeast \par
}
{\phpg\posx6148\pvpg\posy1591\absw4620\absh160 \f20 \fs17 \cf1 \f20 \fs17 \cf1 s
train that contains PUFAs, especially linoleic \par
}
{\phpg\posx6148\pvpg\posy1780\absw1868\absh429 \f20 \fs17 \cf1 \f20 \fs17 \cf1 a
cid (LA, C18:2{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12
9,12 }
\par}{\phpg\posx6148\pvpg\posy1780\absw1868\absh429 \sl-230 \f20 \fs17 \cf1 C18:
3{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 6,9,12 }\par
}
{\phpg\posx8017\pvpg\posy1780\absw2751\absh222 \f20 \fs17 \cf1 \f20 \fs17 \cf1 )
and{\f40
{\field{\*\fldinst SYMBOL 103 \\f Symbol }}}-linolenic acid (
GLA, \par
}
{\phpg\posx7141\pvpg\posy2051\absw3627\absh160 \f20 \fs17 \cf1 \f20 \fs17 \cf1 )
in its membranes, we co-expressed \par
}
{\phpg\posx6148\pvpg\posy2240\absw264\absh222 \f40 \fs17 \cf1 \f40 \fs17 \cf1 {\
field{\*\fldinst SYMBOL 68 \\f Symbol }}{\f20 \fs12 12 }\par
}
{\phpg\posx6412\pvpg\posy2240\absw4356\absh222 \f20 \fs17 \cf1 \f20 \fs17 \cf1 and{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 6}- desa
turases from{\i Mucor}{\i rouxii} in{\i S. }\par
}
{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \i \f20 \fs17 \cf1 \i \f20 \fs17
\cf1 cerevisiae{\i0 .}{\i0 The}{\i0 resulting}{\i0 yeast}{\i0 strain}{\i0
was}{\i0 analyzed }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 in}{\i0 terms}{\i0 of}{\i0 its}{\i0 lipidome}{\i0 and}{\i0 tran
scriptome}{\i0 so}{\i0 that }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 we}{\i0 could}{\i0 quantify}{\i0 the}{\i0 fluxes}{\i0 through}{\i0 th
e}{\i0 fatty}{\i0 acid }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 biosynthetic}{\i0 pathways}{\i0 and}{\i0 global}{\i0 transcriptiona
l }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 response}{\i0
to}{\i0
production}{\i0
of}{\i0
LA}{\i0
and}{\
i0
GLA.}{\i0 These }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 analyses}{\i0 showed}{\i0 that}{\i0 several}{\i0 biological}{\i0
processes }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 linked}{\i0 to}{\i0 the}{\i0 ubiquitin-proteasome}{\i0 system}{\i0
(UPS) }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 and}{\i0 oxidative}{\i0 stress}{\i0 response}{\i0 were}{\i0 affected}{
\i0 at}{\i0 the }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 transcriptional}{\i0 level,}{\i0 and}{\i0 this}{\i0 led}{\i0 us}{\i0
to}{\i0 perform}{\i0 fol- }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 low-up}{\i0 analysis}{\i0 of}{\i0 proteasomal}{\i0 activity}{\i0 and}{
\i0 cell}{\i0 vi- }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 ability.}{\i0 Based}{\i0 on}{\i0 this}{\i0 we}{\i0 were}{\i0 able}{\i
0 to}{\i0 draw}{\i0 conclu- }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1

{\i0 sions}{\i0 on}{\i0 the}{\i0 effects}{\i0 of}{\i0 PUFAs}{\i0 synthesis


}{\i0 and}{\i0 incor- }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 poration}{\i0 into}{\i0 membranes}{\i0 on}{\i0 cell}{\i0 viabilit
y}{\i0 and}{\i0 cell }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 sensitivity}{\i0
towards}{\i0
oxidative}{\i0
and}{\i0
p
roteotoxic }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 stress.}{\i0 Clearly}{\i0 our}{\i0 results}{\i0 have}{\i0 significant}
{\i0 implica- }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 tions}{\i0 on}{\i0 the}{\i0 use}{\i0 of}{\i0 yeast}{\i0 as}{\i0
a}{\i0 production}{\i0 host}{\i0 for }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 production}{\i0 of}{\i0 PUFAs.They}{\i0 may}{\i0 also}{\i0 partly}{\i0
explain }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 why}{\i0 the}{\i0 lack}{\i0 of}{\i0 PUFAs}{\i0 in}{\i0 the}{\i0
lipidome}{\i0 of} S. cere\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
visiae{\i0 makes}{\i0 it}{\i0 a}{\i0 very}{\i0 robust}{\i0 organism}
{\i0 for}{\i0 various }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 industrial}{\i0 processes.}{\i0 Furthermore,}{\i0 considering}{\i0 the
}
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 similarity}{\i0
in}{\i0
transcriptional}{\i0
response}{\i0
between }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 yeast}{\i0 and}{\i0 mouse}{\i0 cells,}{\i0 our}{\i0 results}{\i0 may}
{\i0 provide}{\i0 new }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 molecular}{\i0 insights}{\i0 into}{\i0 the}{\i0 influence}{\i0 of}{\i0
PUFAs}{\i0 on }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 cellular}{\i0 processes}{\i0 in}{\i0 eukaryotic,}{\i0 including}{\i
0 mam- }
\par}{\phpg\posx6148\pvpg\posy2511\absw4620\absh5128 \sl-229 \i \f20 \fs17 \cf1
{\i0 malian,}{\i0 cells. }\par
}
{\phpg\posx6148\pvpg\posy8689\absw342\absh611 \b \f10 \fs21 \cf1 \b \f10 \fs21 \
cf1 2
\par}{\phpg\posx6148\pvpg\posy8689\absw342\absh611 \sl-230 \par\b \f10 \fs21 \cf
1 {\fs19 2.1 }\par
}
{\phpg\posx6508\pvpg\posy8689\absw2624\absh611 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 Materials and methods
\par}{\phpg\posx6508\pvpg\posy8689\absw2624\absh611 \sl-230 \par\b \f10 \fs21 \c
f1 \fi112 {\fs19 Strains}{\fs19 and}{\fs19 plasmids }\par
}
{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \f20 \fs17 \cf1 \f20 \fs17 \cf1
Detailed plasmids and yeast strain used is summa\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 riz
ed in Table 1 and Fig. S1 (see Supplementary
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 Inf
ormation).
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-230 \par\f20 \fs17 \cf1
{\b \f10 \fs19 2.2}{\b \f10 \fs19
Batch}{\b \f10 \fs19 cultivation }
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-230 \par\f20 \fs17 \cf1

Strains were cultivated in 100 mL medium in


\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 500
mL baffled shake flasks for propagation. For
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 the
pre-culture, a minimal medium with glucose as
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 sol
e carbon source was used [20]. The cultures
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 wer
e incubated for 24 h at 30C, with shaking at
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 150
rpm (model 3033, GFL, Burgwedel, Germany).
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-292 \f20 \fs17 \cf1 Whe
n the absorbance (A{\dn006 \fs12 600} ) reached 5-6, 1 mL of
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-172 \f20 \fs17 \cf1 the
culture was used as inoculum for the batch cul\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 tiv
ations. Batch cultivations were carried out in
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 bio
reactors (BIOSTAT {\up006 \fs12 }B, B. Braun Biotech, Ger\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-230 \f20 \fs17 \cf1 man
y) with a working volume of 1 L of a defined
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 med
ium [20] - the initial glucose concentration was
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 20
g/L. The temperature of the cultivations was
\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 con
stantly kept at 30C, and the pH was automati\par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 cal
ly controlled at 5.0 by addition of 2 N KOH. The \par
}
{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
44 \par
}
{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
\sect\sectd\pard\plain
\pgwsxn11905\pghsxn15817
{\phpg\posx0\pvpg\posy0\absw11900\absh15820 \overlay
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}}\par}{\phpg\posx1131\pvpg\posy444\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13
\cf1 Biotechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx8787\pvpg\posy444\absw2250\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 ww
w.biotechnology-journal.com \par
}
{\phpg\posx1131\pvpg\posy1600\absw4372\absh731 \b \f10 \fs13 \cf1 \b \f10 \fs13
\cf1 Table 1.{\b0 The}{\b0 list}{\b0 of}{\b0 yeast}{\b0 strains}{\b0 and}
{\b0 plasmids}{\b0 used}{\b0 in}{\b0 this}{\b0 study }
\par}{\phpg\posx1131\pvpg\posy1600\absw4372\absh731 \sl-166 \par\b \f10 \fs13 \c
f1 \fi1870 {\fs15 Characteristics }
\par}{\phpg\posx1131\pvpg\posy1600\absw4372\absh731 \sl-166 \par\b \f10 \fs13 \c
f1 {\fs15 Plasmids }\par
}
{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \f10 \fs15 \cf1 \f10 \fs15 \cf1
pESC-TRP
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-219 \f10 \fs15 \cf1 YEp
356
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-219 \f10 \fs15 \cf1 pE9
12-TRP
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-220 \par\f10 \fs15 \cf1

pYE96-URA
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-220 \par\f10 \fs15 \cf1
{\b Strains }
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-219 \f10 \fs15 \cf1 CEN
.PK 113-9D
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-220 \f10 \fs15 \cf1 LM
001-REF
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-219 \f10 \fs15 \cf1 LM
002-D12
\par}{\phpg\posx1131\pvpg\posy2472\absw1107\absh2125 \sl-220 \f10 \fs15 \cf1 LM
003-D126 \par
}
{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \f10 \fs15 \cf1 \f10 \fs15 \cf1
Yeast epitope tagging vectors with{\i TRP1} marker
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-219 \f10 \fs15 \cf1 Yea
st episomal vector with{\i URA3} marker
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-219 \f10 \fs15 \cf1 Yea
st episomal vector carrying the construct of{\i M.}{\i rouxii}{\f40 {\field{\
*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs10 12}-desaturase gene fused
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-220 \f10 \fs15 \cf1 dow
nstream to the{\i M.}{\i rouxii}{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol
}}}{\up006 \fs10 9}-desaturase promoter
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-220 \f10 \fs15 \cf1 Yea
st episomal vector carrying the construct of{\i M.}{\i rouxii}{\f40 {\field{\
*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs10 6}-desaturase gene fused
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-220 \f10 \fs15 \cf1 dow
nstream to the{\i M.}{\i rouxii}{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol
}} }{\up006 \fs10 9}-desaturase promoter
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-219 \par\f10 \fs15 \cf1
MATa;{\i ura3-52;}{\i HIS3;}{\i LEU2;}{\i trp1-289;}{\i MAL2-8;}{\i SUC2
}
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-220 \f10 \fs15 \cf1 CEN
.PK 113-9D with empty plasmids pESC-TRP and YEp356
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-219 \f10 \fs15 \cf1 CEN
.PK 113-9D with plasmids YEp356 and pE912-TRP
\par}{\phpg\posx3001\pvpg\posy2472\absw6152\absh2125 \sl-220 \f10 \fs15 \cf1 CEN
.PK 113-9D with plasmids pE912-TRP and pYE96-URA \par
}
{\phpg\posx9351\pvpg\posy1919\absw911\absh143 \b \f10 \fs15 \cf1 \b \f10 \fs15 \
cf1 Reference \par
}
{\phpg\posx9351\pvpg\posy2472\absw1197\absh937 \f10 \fs15 \cf1 \f10 \fs15 \cf1 S
tratagene
\par}{\phpg\posx9351\pvpg\posy2472\absw1197\absh937 \sl-219 \f10 \fs15 \cf1 [60]
\par}{\phpg\posx9351\pvpg\posy2472\absw1197\absh937 \sl-220 \f10 \fs15 \cf1 [61]
\par}{\phpg\posx9351\pvpg\posy2472\absw1197\absh937 \sl-220 \par\f10 \fs15 \cf1
Kobkul Laoteng{\up006 \fs10 a) }\par
}
{\phpg\posx9351\pvpg\posy3987\absw942\absh762 \f10 \fs15 \cf1 \f10 \fs15 \cf1 Pe
ter Ktter{\up006 \fs10 b) }
\par}{\phpg\posx9351\pvpg\posy3987\absw942\absh762 \sl-220 \f10 \fs15 \cf1 This
study
\par}{\phpg\posx9351\pvpg\posy3987\absw942\absh762 \sl-219 \f10 \fs15 \cf1 This
study
\par}{\phpg\posx9351\pvpg\posy3987\absw942\absh762 \sl-220 \f10 \fs15 \cf1 This
study \par
}
{\phpg\posx1131\pvpg\posy5024\absw10164\absh408 \f10 \fs12 \cf1 \f10 \fs12 \cf1

a) Biochemical Engineering and Pilot Plant Research and Development Unit, Nation
al Center for Genetic Engineering and Biotechnology (BIOTEC) at King Mongkut's
\par}{\phpg\posx1131\pvpg\posy5024\absw10164\absh408 \sl-159 \f10 \fs12 \cf1 \fi
137 University of Technology Thonburi, Thailand.
\par}{\phpg\posx1131\pvpg\posy5024\absw10164\absh408 \sl-160 \f10 \fs12 \cf1 b)
Institut fr Mikrobiologie, der Johann Wolfgang Goethe-Universitt Frankfurt, German
y. \par
}
{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \f20 \fs17 \cf1 \f20 \fs17 \cf1
agitation was 800 rpm and the bioreactors were
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 aer
ated with air (1.0 L/min). The concentration of
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-146 \par\f20 \fs17 \cf1
CO{\dn006 \fs12 2} and O{\dn006 \fs12 2} in the exhaust gas was determined us
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-172 \f20 \fs17 \cf1 ing
a gas analyzer (MUX-100, B. Braun Biotech).
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
{\b \f10 \fs19 2.3}{\b \f10 \fs19
Chemostat}{\b \f10 \fs19 cultivations }
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
After diauxic growth, strains were cultivated in
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 aer
obic carbon-limited chemostat cultures with a
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 wor
king volume of 1.0 L defined medium contain\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 ing
10 g/L glucose.The cultures were switched from
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 the
initial batch phase to continuous mode with a
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 dil
ution rate of 0.10/h. Cultivations were carried
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 out
as described above.The off-gas was directed to
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-292 \f20 \fs17 \cf1 a g
as analyzer (MUX-100) for measurement of CO{\dn006 \fs12 2 }
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \f20 \fs17 \cf1 and
O{\dn006 \fs12 2}. The dissolved oxygen tension in the cul\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-172 \f20 \fs17 \cf1 tur
es was measured using a Mettler Toledo polaro\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 gra
phic electrode. At steady state, the dissolved
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 oxy
gen tension was greater than 70%. After steady
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 sta
te was reached, the cell samples for biomass, ex\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 tra
cellular metabolites, lipids, and transcriptome
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 ana
lyses were collected.
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
{\b \f10 \fs19 2.4}{\b \f10 \fs19
Shake}{\b \f10 \fs19 flask}{\b \f10 \fs1
9 cultivation }
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
The yeast recombinant strains were grown in
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 100
mL synthetic minimal medium (SD) comprising
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 20.
0 g/L glucose, and 6.7 g/L bacto-yeast nitrogen
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 bas
e without amino acid, at 30C with shaking at

\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 200


rpm.When the cultures reached the logarithmic
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-292 \f20 \fs17 \cf1 pha
se (OD{\dn006 \fs12 600 } =2.0-3.0), the cells were harvested for
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-172 \f20 \fs17 \cf1 det
ermination of biomass, fatty acids and lipids.
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
{\b \f10 \fs19 2.5}{\b \f10 \fs19
Biomass}{\b \f10 \fs19 measurement }
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
Biomass dry weight was determined by filtering a
\par}{\phpg\posx1131\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 kno
wn volume of culture broth, approximately \par
}
{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \f20 \fs17 \cf1 \f20 \fs17 \cf1
10 mL, through a dried, pre-weighed nitrocellulose
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 fil
ter (Gelman Science, MI, USA) with a pore size
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 0.4
5 m. The residue was washed twice with dis\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 til
led water.The filter was dried to constant weight
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 in
a microwave oven at 150 W for 10 min, cooled
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 dow
n in a desiccator, and weighed.
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
{\b \f10 \fs19 2.6}{\b \f10 \fs19
Analyses}{\b \f10 \fs19 of}{\b \f10 \fs1
9 lipids}{\b \f10 \fs19 and}{\b \f10 \fs19 fatty}{\b \f10 \fs19 acids }
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-230 \par\f20 \fs17 \cf1
Total lipids were extracted by the procedure of
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 Fol
ch{\i et}{\i al.} [21] from 0.2 g freeze-dried yeast cells.
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 Hep
tadecanoic
acid, C17:0-PC
and
C17:0-TAG
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 (Si
gma-Aldrich AS, Denmark) were added as inter\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 nal
standards. The resulting lipid extract was fur\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 the
r fractionated by thin-layer chromatography
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 (TL
C). Lipid solution was applied to the TLC plate
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 (Si
lica gel 60, 20{\f40 } 20 cm, 0.5 mm layer, Merck, Ger\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 man
y) and developed with a solvent system hep\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 tan
e:2-propanol:acetic
acid
(95:5:1).
Individual
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 lip
ids were visualized by spraying with 0.2% 2,7\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 dic
hlorofluoresceine in ethanol and identified by
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-292 \f20 \fs17 \cf1 com
parison of their R{\dn006 \fs12 f} values with known stan\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-172 \f20 \fs17 \cf1 dar
ds. The bands corresponding to individual lipid
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 cla
sses, i.e. triacylglycerol (TAG), phospholipid
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 (PL
) and free fatty acids (FFA) were scraped off

\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 and


directly transmethylated for GC analysis [22].
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 The
fatty acid methyl esters (FAMEs) were sepa\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 rat
ed on a 60-m Supelco SP-2380 column (Sigma\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 Ald
rich AS, Brndby, Denmark) in a HP 6890 gas
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 chr
omatograph (GC), in split mode using helium as
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 a
carrier gas. GC settings were: injector tempera\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 tur
e 260C, split ratio 11:1, carrier flow 1.2 mL/min,
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 det
ector temperature 270C, air flow in detector
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 300
mL/ min, hydrogen flow 35 mL/min. FAMEs
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 wer
e separated using a temperature gradient pro\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 gra
m starting at 70C and rising to 160C at
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh7612 \sl-229 \f20 \fs17 \cf1 30C/
min; hereafter the temperature was raised to \par
}
{\phpg\posx1131\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
{\phpg\posx10545\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
345 \par
}
\sect\sectd\pard\plain
\pgwsxn11905\pghsxn15817
{\phpg\posx0\pvpg\posy0\absw11900\absh15820 \overlay
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140

05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140

05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451401fffd9
}}\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \b \f10 \fs16 \cf1 \b \f10
\fs16 \cf1 Biotechnology
\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \f
i801 Journal \par
}
{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Bi
otechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \f20 \fs17 \cf1 \f20 \fs17 \cf1
200C at 1.5C/min and the oven temperature was
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ma
intained at 200C for 15 min before the temper\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 at
ure was raised to 225C at 30C/min. The final
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
mperature was maintained for 10 min. FAMEs
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 we
re identified by comparing with authentic stan\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 da
rds (Supelco, USA).The fatty acid contents were
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 de
termined based on the area of chromatographic
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pe
aks, compared with that of the internal standard.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.7}{\b \f10 \fs19
Ergosterol}{\b \f10 \fs19 extraction}{\
b \f10 \fs19 and}{\b \f10 \fs19 analysis }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 Total ergosterol content was extracted from 0.1 g
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 dr
ied yeast cells.The cell pellet was resuspended in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 4
mL 0.2 N HCl in a 15-mL falcon tube and heated
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in
a water bath at 85C for 1 h and allowed to cool
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 to
room temperature. After centrifuging for 10 min
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 at
5000 rpm, the supernatant was removed, and the
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ce
ll pellet was resuspended in 2 mL methanol con\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ta
ining 0.2% pyrogallol and 1 mL 4 N KOH and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tr
ansferred to a 14-mL glass vial sealed with a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 PT
FE-lined screw cap, heated at 85C for 2 h for

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 sa


ponification. Following incubation, glass vials
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 we
re allowed to cool to room temperature. Ergos\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
rol was then extracted by addition of 5 mL hep\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ta
ne followed by vigorous vortex mixing for at least
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 2
min. After 2 h at room temperature during which
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e heptane layer clarified, the lower phase was
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tr
ansferred to a new glass vial for HPLC analysis.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 Qu
antitative
determination
of
ergosterol
was
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ca
rried out by reversed-phase HPLC (Hewlett\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 Pa
ckard
HP
1090
chromatograph
series
II
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 eq
uipped with diode array detector and auto-injec\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 to
r). Ergosterol was separated using a Develosil
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 co
lumn (C30-UG-5, Nomura Chemicals, Japan)
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 wi
th methanol and acetonitrile (90:10) as the mo\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 bi
le phase at a flow rate of 0.1 mL/min.The analy\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 si
s was performed at 40C and monitored by UV de\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
ction at 280 nm. The amount of ergosterol is de\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
rmined
using
absolute
calibration
curves
of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 kn
own ergosterol concentrations (10-250 mg/L er\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 go
sterol, Sigma, USA).
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.8}{\b \f10 \fs19
Sampling}{\b \f10 \fs19 and}{\b \f10 \f
s19 RNA}{\b \f10 \fs19 isolation }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 Samples of the cultures for whole-genome analysis
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (1
0 mL) were withdrawn rapidly and transferred to
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 50
-mL tubes containing 35-40 mL crushed ice. In
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 do
ing so, the sample temperature fell below 2C in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 le
ss than 10 s. The cells were swiftly pelleted (at
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 45
00 rpm, 0C for 5 min), frozen instantaneously in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 li
quid nitrogen, and stored at -80C. After thawing
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e samples on ice, total RNA extraction was per\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 fo

rmed with the Qiagen RNeasy Mini Kit (QIAGEN,


\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 De
nmark), according to the protocol for isolation of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 to
tal RNA from yeast. The quality and quantity of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e total RNA extracted were determined using an \par
}
{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \f20 \fs17 \cf1 \f20 \fs17 \cf1
Agilent 2100 bioanalyzer (Agilent Technologies,
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 US
A).The total RNA was stored at -80C until fur\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
er processing.
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.9}{\b \f10 \fs19
Probe}{\b \f10 \fs19 preparation}{\b \f
10 \fs19 and}{\b \f10 \fs19 hybridization }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 The mRNA extraction, cDNA synthesis, cRNA syn\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
esis and labeling were performed as described in
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e Affymetrix GeneChip{\up006 \fs12 } Expression Analysis
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 Te
chnical Manual. cRNA (15 g) was hybridized to
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 Ye
ast Genome 2.0 Arrays (Affymetrix, USA).Wash\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in
g and staining of arrays were performed using
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e GeneChip Fluidics Station 400, and scanning
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 wa
s done using the Affymetrix {\up006 \fs12 } GeneChip{\up006 \fs12 } Scan\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 ne
r (Affymetrix, USA).
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.10}{\b \f10 \fs19
Analysis}{\b \f10 \fs19 of}{\b \f10 \f
s19 transcriptome}{\b \f10 \fs19 data }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 Affymetrix CEL-data files were preprocessed us\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in
g the statistical language and environment R ver\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 si
on 2.3.1 (R Development Core Team). The probe
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in
tensities were performed for background correc\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ti
on by the robust multiarray average method using
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 on
ly perfect match (PM) probes [23]. Normaliza\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ti
on was subsequently performed using the Qs\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pl
ine algorithm [24]. Gene expression indices were
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ca
lculated from the PM probes with the median\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 po
lish summary method [23].All statistical prepro\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ce
ssing methods were used by invoking them

\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th


rough the affy package [25]. Statistical analysis
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 wa
s applied to determine genes subjected to differ\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 en
tial transcriptional regulation. The limma pack\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ag
e [26] was used to perform moderated Student's
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 {\
i t-}tests between all of the possible pair-wise com\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pa
risons within the three different categories. Em\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pi
rical Bayesian statistics were used to moderate
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e standard errors within each gene and Benjami\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ni
-Hochberg's method was employed for multiple
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
sting [27]. A cut-off value of adjusted{\i p} value
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 <0
.01 was set to assess statistical significance. Gene
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ex
pression data was deposited at the Gene Expres\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 si
on
Omnibus
(GEO)
database
with
accession
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 nu
mber GSE20155 (platform), GSM505688-505696
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (s
amples).
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.11}{\b \f10 \fs19
Enriched}{\b \f10 \fs19 Gene}{\b \f10
\fs19 Ontology}{\b \f10 \fs19 analysis }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 To determine significantly enriched Gene Ontology
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (G
O) process terms, the Reporter Gene Ontologies
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 [2
8] and BiNGO 2.31 add-in to Cytoscape version
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 2.
6 [29] were applied. For BiNGO analysis, we used
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 a
hypergeometric test to determine which cate\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 go
ries were significantly represented. This signifi\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ca
nce value was adjusted for multiple hypotheses
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
sting using the Benjamini and Hochberg False
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 Di
scovery Rate (FDR) correction ({\i p}<0.05). Gene \par
}
{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
46 \par
}
{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
\sect\sectd\pard\plain
\pgwsxn11905\pghsxn15817

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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a00ffd9
}}\par}{\phpg\posx1131\pvpg\posy444\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13
\cf1 Biotechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx8787\pvpg\posy444\absw2250\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 ww
w.biotechnology-journal.com \par
}
{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \f20 \fs17 \cf1 \f20 \fs17 \cf1
annotation was analyzed using Yeast 2.0 Arrays
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 Af
fymetrix (revision 13 March 2009).
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.12}{\b \f10 \fs19
Detection}{\b \f10 \fs19 of}{\b \f10 \
fs19 reactive}{\b \f10 \fs19 oxygen}{\b \f10 \fs19 species }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \par\f20 \fs17 \cf
1 Reactive oxygen species (ROS) were detected with
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 di
hydrorhodamine 123. Strains were grown in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 tu
bes in synthetic drop-out medium without uracil
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 an
d tryptophan (SD-Ura-Trp medium) overnight
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 on
a horizontal shaker at 200 rpm and 30C.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-292 \f20 \fs17 \cf1 Ov
ernight cultures were diluted to{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }
}}OD{\dn006 \fs12 600} = 0.2 in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-172 \f20 \fs17 \cf1 6
mL of SD-Ura-Trp medium and shaken at
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-292 \f20 \fs17 \cf1 20
0 rpm and 30C. On specific days 1.0{\f40
{\field{\*\fldinst SYMBO

L 68 \\f Symbol }}}OD{\dn006 \fs12 600 }


\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-167 \f20 \fs17 \cf1 (~
2{\f40 } 10{\up006 \fs12 7} cells) were harvested. Cells were resus\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pe
nded in 1 mL 50 mM sodium citrate buffer,
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pH
5.0, containing 2% glucose and 50 M dihy\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 dr
orhodamine 123 (1 L 50 mM stock solution in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 DM
SO). Cells were incubated in the dark for
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 15
-30 min at room temperature and then pelleted.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 Th
e pellet was resuspended in 40 L 50 mM sodi\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 um
citrate buffer, pH 5.0, containing 2% glucose;
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 10
L of the suspension was transferred on a mi\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 cr
oscope slide and covered with a cover slip. Sam\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pl
es were analyzed with DIC and YFP filters of the
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 fl
uorescent microscope Leica DMI4000 B. At least
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 30
0 cells of each culture were counted and the per\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ce
ntage of ROS-positive cells was calculated.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs19 2.13}{\b \f10 \fs19
Survival}{\b \f10 \fs19 assays }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \par\f20 \fs17 \cf
1 Survival of LM 001-REF and LM 003-D126 was de\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 te
rmined by spot assays on plates after incubation
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-146 \par\f20 \fs17 \cf
1 with H{\dn006 \fs12 2}O {\dn006 \fs12 2} or{\fs12
L}-azetidine-2-carbox
ylic acid (AZE)
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-172 \f20 \fs17 \cf1 af
ter a defined period of incubation.The addition of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 AZ
E, an analogue of proline that incorporates into
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pr
oteins, changes protein structure and function
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 an
d imposes a stress to the cell [30, 31]. Strains
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 we
re grown in tubes in SD-Ura-Trp medium
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ov
ernight on a horizontal shaker at 200 rpm and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-292 \f20 \fs17 \cf1 30C
. Overnight cultures were diluted to{\f40 {\field{\*\fldinst SYMBOL 68 \\
f Symbol }}}OD{\dn006 \fs12 600 }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-172 \f20 \fs17 \cf1 =
0.2 in 6 mL SD-Ura-Trp medium and shaken at
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-292 \f20 \fs17 \cf1 20
0 rpm and 30C until{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}OD{\dn006 \f
s12 600} of ~1.0.Before treat\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-172 \f20 \fs17 \cf1 me
nt, all cultures were adjusted to the same

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-292 \f20 \fs17 \cf1 {\


f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}OD{\dn006 \fs12 600} . Subsequen
tly, 1 mL of each culture was
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-172 \f20 \fs17 \cf1 tr
ansferred to a new tube with a final concentration
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-146 \par\f20 \fs17 \cf
1 of 20 mM H {\dn006 \fs12 2}O {\dn006 \fs12 2} or 30 mMAZE,and incubated for
3 h
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-172 \f20 \fs17 \cf1 at
200 rpm and 30C. Control cultures were left un\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 tr
eated. Cells were pelleted and resuspended in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 1
mL fresh SD-Ura-Trp medium and diluted four
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ti
mes
in
10-fold
series
(10{\up006 \fs12 -1}, 10{\up006 \fs12 -2},
10{\up006 \fs12 -3}, 10{\up006 \fs12 -4}).
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \f20 \fs17 \cf1 Al
iquots (3.5 L) of the non-diluted culture and of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ea
ch dilution were spotted onto a SD-Ura-Trp
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pl
ate. Growth was recorded after 2-3 days of incu\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ba
tion at 30C. For the long-term incubation exper\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 im
ent,cells were harvested on specific days and the \par
}
{\phpg\posx1131\pvpg\posy14200\absw4089\absh292 \f20 \fs17 \cf1 \f20 \fs17 \cf1
density of the culture was adjusted to{\f40 {\field{\*\fldinst SYMBOL 68 \\f Sy
mbol }}}OD{\dn006 \fs12 600 }\par
}
{\phpg\posx5270\pvpg\posy14241\absw480\absh160 \f20 \fs17 \cf1 \f20 \fs17 \cf1 =
1.0. \par
}
{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \f20 \fs17 \cf1 \f20 \fs17 \cf1
Cells were then diluted and spotted as described
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 abo
ve.
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \par\f20 \fs17 \cf1
{\b \f10 \fs19 2.14}{\b \f10 \fs19
Proteasome}{\b \f10 \fs19 activity}{\b
\f10 \fs19 measurements }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-230 \par\f20 \fs17 \cf1
Luminescence activity measurements of the chy\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 mot
rypsin-, caspase- and trypsin-like proteolytic
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 act
ivities of the 20S proteasome were performed
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 usi
ng the Proteasome-Glo{\up006 \fs12 TM} Cell-Based Assay
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-230 \f20 \fs17 \cf1 (Pr
omega GmbH, Germany) according to the man\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 ufa
cturer's instructions. Cells were either treated
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 wit
h 30 mM AZE for 3 h or left untreated before
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 har
vest. SD-Ura-Trp medium (100 L) containing
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 dif
ferent numbers (20000, 40000 and 80000) of ex-

\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 pon


ential cells were mixed with 100 L of the re\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 spe
ctive Cell-Based Reagent and incubated for
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 20
min at room temperature. Luminescence was
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 rec
orded using the FLUOstar Galaxy plate reader
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 fro
m BMG Labtechnologies. Every sample was
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 mea
sured in duplicate. To exclude the possibility
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 tha
t the detected signal resulted from non-protea\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 som
e cleavage of the aminoluciferin substrates by
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 oth
er proteases, in a previous experiment some
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 sam
ples were incubated with the proteasome in\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 hib
itor epoxomicin for 1 h before measurement.
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 Sin
ce no significant luminescence was detected in
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 the
presence of epoxomicin, the signals detected in
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 the
samples without epoxomicin could be com\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 ple
tely attributed to the activity of the 20S protea\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh6163 \sl-229 \f20 \fs17 \cf1 som
e. \par
}
{\phpg\posx6148\pvpg\posy8919\absw342\absh611 \b \f10 \fs21 \cf1 \b \f10 \fs21 \
cf1 3
\par}{\phpg\posx6148\pvpg\posy8919\absw342\absh611 \sl-230 \par\b \f10 \fs21 \cf
1 {\fs19 3.1 }\par
}
{\phpg\posx6508\pvpg\posy8919\absw2130\absh611 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 Results
\par}{\phpg\posx6508\pvpg\posy8919\absw2130\absh611 \sl-230 \par\b \f10 \fs21 \c
f1 \fi112 {\fs19 Strain}{\fs19 construction }\par
}
{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \f20 \fs17 \cf1 \f20 \fs17 \cf1
We co-expressed{\f40
{\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs
12 12}- and{\f40
{\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12
6}-desaturases of{\i
M. }
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 {\i
rouxii} in{\i S.}{\i cerevisiae} using 2{\f40 {\field{\*\fldinst SYMBOL 109 \
\f Symbol }}}-based plasmids (re\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 sul
ting strain called LM 003-D126). The reference
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 str
ain in all experiments in this study is LM 001\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 REF
, which contains the same plasmids but without
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 the
genes for{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 12}and{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 6}-desaturas
es (Table 1).

\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-230 \par\f20 \fs17 \cf1


{\b \f10 \fs19 3.2}{\b \f10 \fs19
Fluxes}{\b \f10 \fs19 in}{\b \f10 \fs19
lipid}{\b \f10 \fs19 metabolism }
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-230 \par\f20 \fs17 \cf1
The recombinant yeast strain LM 003-D126 ex\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 pre
ssing the{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 12}and{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 6}- desa
turases of{\i M.}{\i rouxii }
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-230 \f20 \fs17 \cf1 was
found to produce LA and GLA and incorporate
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 the
se into both the phospholipid and storage lipids
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 lik
e triacylglycerols and sterol esters (Table 2).The
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 PUF
As producing LM 003-D126 strain and the ref\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 ere
nce strain were grown under both glucose-lim\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 ite
d conditions using chemostat cultures and glu\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 cos
e-excess growth conditions using shake flask
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 cul
tures (Supporting information S1;Tables S1 and
\par}{\phpg\posx6148\pvpg\posy9830\absw4620\absh4130 \sl-229 \f20 \fs17 \cf1 S2)
. A substantially higher level (more than 30- \par
}
{\phpg\posx1131\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
{\phpg\posx10545\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
347 \par
}
\sect\sectd\pard\plain
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{\*\shppict {\pict\jpegblip\picw595 \pich791
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{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
48 \par
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{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
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2800a28a2800a28a2800a28a2800a28a2800a28a2800a28a2800a28a2800a28a2800a28a2800a28a
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}}\par}{\phpg\posx1131\pvpg\posy444\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13
\cf1 Biotechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx8787\pvpg\posy444\absw2250\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 ww
w.biotechnology-journal.com \par
}
{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \f20 \fs17 \cf1 \f20 \fs17 \cf1
fold) of storage lipids was found under glucose-ex\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ce
ss growth conditions,i.e.shake flasks (Fig.1).The
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ph
ospholipid content was significantly higher in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e LM 003-D126 strain, under both carbon-excess
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 an
d carbon-limiting conditions. Based on the GC\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 MS
measurement of the fatty acids in the different
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 li
pid species inside the cells,we calculated the flux\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 es
through the biosynthetic pathways leading to
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e different fatty acids for both growth conditions
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (F
ig.1).Consistent with the increased phospholipid
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 le
vels in LM 003-D126 strain, there was a substan\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ti
ally higher flux through the committed step of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 li
pid biosynthesis,i.e.,the conversion of acetyl-CoA
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 to
malonyl-CoA catalyzed by acetyl-CoA carboxy\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 la
se (Acc1p) in the PUFA-producing yeast. Inter\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 es
tingly, this increased flux was associated with a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 si
gnificantly decreased level of oleic acid (C18:1{\f40 {\field{\*\fldinst SYMBOL
68 \\f Symbol }}}{\up006 \fs11 9})
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 in
the LM 003-D126 strain.{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\
up006 \fs11 9}-Desaturase activity
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 of
{\i S.}{\i cerevisiae} has been reported to be strongly in\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 hi
bited by its products [32], and{\i OLE1} transcription
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 is
repressed by unsaturated fatty acids, especially
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 un
saturated fatty acids containing{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }

}}{\up006 \fs11 9} double bonds


\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 [3
3].
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 {\b \f10 \fs18 3.3}{\b \f10 \fs18
Transcriptome}{\b \f10 \fs18 analysis }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \par\f20 \fs17 \cf
1 To further evaluate the global transcriptional effect
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 of
producing
PUFAs
in
yeast,
we
performed
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ge
nome-wide transcriptome analysis and found
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
at the production of PUFAs results in a large
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tr
anscriptional response. We found that 808 genes
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ha
d a significant transcriptional change, with a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 fa
lse discovery rate of 0.01. We also analyzed the
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tr
anscriptome of a strain only expressing{\f40 {\field{\*\fldinst SYMBOL 68
\\f Symbol }}}{\up006 \fs11 12}-de\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-230 \f20 \fs17 \cf1 sa
turase (Table 1) under the same conditions, but
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 fo
r this strain no significant genes were identified
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (S
upporting information S1; Table S3). Among the
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 80
8 significantly changed genes, 313 genes were
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 up
-regulated and 495 genes down-regulated (Sup\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 po
rting information S1; Tables S4 and S5). The
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tr
anscriptome data were used to identify key GO
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
rms using the so-called reporter algorithm and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 Bi
NGO analysis.Table 3 lists some of the key terms
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 id
entified (a complete list is given in Supporting in\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 fo
rmation S2;Table S6).We found that a large num\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 be
r of mitochondrial genes had altered expression
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 in
response to the presence of PUFAs in the mem\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 br
anes, and also that genes associated with specif\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ic
processes of energy metabolism were affected,
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 e.
g. genes involved in proton transport, the tricar\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 bo
xylic acid (TCA) cycle, cellular respiration and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 AT
P synthesis-coupled proton transport. Further\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 mo
re,a large number of genes involved in stress re-

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 sp


onse were down-regulated. Finally, many genes
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 as
sociated with lipid metabolism were also down\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 re
gulated, e.g., genes associated with sterol metab- \par
}
{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \f20 \fs17 \cf1 \f20 \fs17 \cf1
olism, fatty acid biosynthesis and{\f40 {\field{\*\fldinst SYMBOL 98 \\f Sy
mbol }}}-oxidation. In\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 ter
estingly, there was generally a down-regulation
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 of
genes associated with lipid metabolism even
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 tho
ugh there was an increased production of fatty
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 aci
ds in the PUFA-producing strain. To get further
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 ins
ight into the effect of PUFAs on lipid biosynthe\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 sis
, we made a simple model of lipid metabolism
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 and
mapped both mRNA data and lipid data onto
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 thi
s model using Cytoscape (Fig. 2). From this mod\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 el
it was clear that many biosynthetic genes in the
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 erg
osterol pathway were down-regulated, and that
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 man
y of the down-regulated genes were under reg\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 ula
tion by the transcription factor Upc2p, which is
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 bou
nd to the ER membrane and is proteolytically
\par}{\phpg\posx6148\pvpg\posy1550\absw4620\absh3095 \sl-229 \f20 \fs17 \cf1 act
ivated when the ergosterol level is low. \par
}
{\phpg\posx6148\pvpg\posy5260\absw342\absh177 \b \f10 \fs18 \cf1 \b \f10 \fs18 \
cf1 3.4 \par
}
{\phpg\posx6620\pvpg\posy5260\absw4792\absh384 \b \f10 \fs18 \cf1 \b \f10 \fs18
\cf1 Expression of PUFAs increases ROS accumu\par}{\phpg\posx6620\pvpg\posy5260\absw4792\absh384 \sl-229 \b \f10 \fs18 \cf1 l
ation and sensitivity towards oxidative stress \par
}
{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \f20 \fs17 \cf1 \f20 \fs17 \cf1
Surprisingly, the transcriptome analysis revealed
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 dow
n-regulation of many genes involved in the de\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 fen
se against oxidative stress (Table 3).We therefore
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 mea
sured the levels of ROS in the PUFA-producing
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 str
ain (LM 003-D126) and compared it to the refer\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 enc
e.ROS normally accumulate over time and can be
\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 fou

nd in greater concentrations in older cells.There\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 for


e, to assess the ROS accumulation in the PUFA\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 pro
ducing strain, the cells were grown in SD-Ura\par}{\phpg\posx6148\pvpg\posy5961\absw4620\absh2023 \sl-229 \f20 \fs17 \cf1 Trp
medium (200 rpm and 30C) and samples were \par
}
{\phpg\posx6148\pvpg\posy8715\absw5092\absh128 \b \f10 \fs13 \cf1 \b \f10 \fs13
\cf1 Table 3.{\b0 Combined}{\b0 list}{\b0 of}{\b0 GO}{\b0 terms}{\b0 iden
tified}{\b0 by}{\b0 GO}{\b0 and}{\b0 BiNGO}{\b0 analysis }\par
}
{\phpg\posx6148\pvpg\posy9034\absw850\absh143 \b \f10 \fs15 \cf1 \b \f10 \fs15 \
cf1 GO terms \par
}
{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \f10 \fs10 \cf1 \fi1797 \f10 \fs
10 \cf1 a)
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-153 \f10 \fs10 \cf1 {\f
s15 Mitochondrial}{\fs15 translation }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-163 \f10 \fs10 \cf1 \fi
1896 a,b)
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-153 \f10 \fs10 \cf1 {\f
s15 Sterol}{\fs15 biosynthetic}{\fs15 process }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-163 \f10 \fs10 \cf1 \fi
1058 a)
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-153 \f10 \fs10 \cf1 {\f
s15 Mitochondrion }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-163 \f10 \fs10 \cf1 \fi
1418 a)
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-153 \f10 \fs10 \cf1 {\f
s15 Fungal-type}{\fs15 cell}{\fs15 wall }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Protein}{\fs15 localization}{\fs15 to}{\fs15 pre- }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-219 \f10 \fs10 \cf1 {\f
s15 autophagosomal}{\fs15 structure}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Lipid}{\fs15 biosynthetic}{\fs15 process}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-219 \f10 \fs10 \cf1 {\f
s15 Propionate}{\fs15 metabolic}{\fs15 process}{\up006 a,b) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Glutathione}{\fs15 transferase}{\fs15 activity}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Response}{\fs15 to}{\fs15 toxin}{\up006 a,b) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Phosphorus}{\fs15 metabolic}{\fs15 process}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Proton}{\fs15 transport}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Tricarboxylic}{\fs15 acid}{\fs15 cycle}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Cellular}{\fs15 respiration}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 ATP}{\fs15 synthesis}{\fs15 coupled}{\fs15 proton }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-219 \f10 \fs10 \cf1 {\f
s15 transport}a)
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f
s15 Cell}{\fs15 wall}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-219 \f10 \fs10 \cf1 {\f
s15 Fatty}{\fs15 acid}{\f40 \fs15 {\field{\*\fldinst SYMBOL 98 \\f Symbol }}}{
\fs15 -oxidation}{\up006 a) }

\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-220 \f10 \fs10 \cf1 {\f


s15 Mitochondrion}{\fs15 organization}{\up006 a) }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-163 \f10 \fs10 \cf1 \fi
1331 a,b)
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-153 \f10 \fs10 \cf1 {\f
s15 Response}{\fs15 to}{\fs15 stress }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-165 \par\f10 \fs10 \cf1
{\fs12 a)}{\fs12 Based}{\fs12 on}{\fs12 reporter}{\fs12 GO}{\fs12 analysis
. }
\par}{\phpg\posx6148\pvpg\posy9568\absw2349\absh4360 \sl-160 \f10 \fs10 \cf1 {\f
s12 b)}{\fs12 Based}{\fs12 on}{\fs12 BiNGO}{\fs12 analysis. }\par
}
{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \b \f10 \fs15 \cf1 \fi176 \b \f1
0 \fs15 \cf1 Up-/down\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-220 \b \f10 \fs15 \cf1
regulated genes
\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-166 \par\b \f10 \fs15 \
cf1 \fi395 {\b0 39/0 }
\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-220 \b \f10 \fs15 \cf1
\fi395 {\b0 1/23 }
\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-220 \b \f10 \fs15 \cf1
\fi268 {\b0 270/164 }
\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-220 \b \f10 \fs15 \cf1
\fi353 {\b0 25/15 }
\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-219 \b \f10 \fs15 \cf1
\fi438 {\b0 0/3 }
\par}{\phpg\posx8425\pvpg\posy9034\absw1362\absh1831 \sl-220 \par\b \f10 \fs15 \
cf1 \fi395 {\b0 3/23 }\par
}
{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \f10 \fs15 \cf1 \fi127 \f10 \fs1
5 \cf1 5/0
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
127 0/3
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
42 11/17
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
84 12/6
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
84 10/5
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
84 10/5
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
84 22/4
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-219 \f10 \fs15 \cf1 \fi
127 9/3
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \par\f10 \fs15 \cf1
\fi84 16/7
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
127 0/5
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-220 \f10 \fs15 \cf1 \fi
84 16/8
\par}{\phpg\posx8736\pvpg\posy11127\absw541\absh2521 \sl-219 \f10 \fs15 \cf1 19/
111 \par
}
{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \b \f10 \fs15 \cf1 \b \f10 \fs15
\cf1 No. of genes
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi47 in GO term
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-166 \par\b \f10 \fs15 \
cf1 \fi362 {\b0 81 }

\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1


\fi362 {\b0 28 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi277 {\b0 1028 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 91 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-219 \b \f10 \fs15 \cf1
\fi404 {\b0 4 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \par\b \f10 \fs15 \
cf1 \fi362 {\b0 50 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi404 {\b0 5 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi404 {\b0 7 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 66 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 35 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 41 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 29 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 50 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-219 \b \f10 \fs15 \cf1
\fi362 {\b0 27 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \par\b \f10 \fs15 \
cf1 \fi362 {\b0 54 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 11 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-220 \b \f10 \fs15 \cf1
\fi362 {\b0 40 }
\par}{\phpg\posx9839\pvpg\posy9034\absw1029\absh4405 \sl-219 \b \f10 \fs15 \cf1
\fi319 {\b0 152 }\par
}
{\phpg\posx1131\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
{\phpg\posx10545\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
349 \par
}
\sect\sectd\pard\plain
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}}\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \b \f10 \fs16 \cf1 \b \f10
\fs16 \cf1 Biotechnology
\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \f
i801 Journal \par
}

{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Bi


otechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx1131\pvpg\posy9811\absw10679\absh668 \b \f10 \fs13 \cf1 \b \f10 \fs13
\cf1 Figure 2.{\b0 A}{\b0 small}{\b0 cytoscape}{\b0 metabolic}{\b0 networ
k}{\b0 of}{\b0 lipid}{\b0 biosynthesis}{\b0 in}{\b0 yeast.}{\b0 The}{\b0
network}{\b0 was}{\b0 overlaid}{\b0 with}{\b0 mRNA}{\b0 expression}{\b0 an
d}{\b0 lipid}{\b0 metabolite}{\b0 data}{\b0 for }
\par}{\phpg\posx1131\pvpg\posy9811\absw10679\absh668 \sl-99 \par\b \f10 \fs13 \c
f1 {\b0 the}{\b0 \f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\b0\up006 \fs
9 6}{\b0 \f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\b0\up006 \fs9 12}{\
b0 strain}{\b0 (LM}{\b0 003-D126)}{\b0 relative}{\b0 to}{\b0 the}{\b0 ref
erence}{\b0 strain.}{\b0 The}{\b0 metabolic}{\b0 nodes}{\b0 are}{\b0 color
ed}{\b0 according}{\b0 to}{\b0 a}{\b0 log}{\b0 -ratio}{\b0 color}{\b0 sca
le}{\b0 of}{\b0 \f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\b0\up006 \fs
9 6}{\b0 \f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\b0
strain}{\b0 r
elative }
\par}{\phpg\posx1131\pvpg\posy9811\absw10679\absh668 \sl-199 \b \f10 \fs13 \cf1
{\b0 to}{\b0 the}{\b0 reference}{\b0 strain}{\b0 (see}{\b0 node}{\b0 symbo
l}{\b0 and}{\b0 color}{\b0 key).}{\b0 Gray}{\b0 coloring}{\b0 indicates}{\
b0 lack}{\b0 of}{\b0 a}{\b0 measurement}{\b0 for}{\b0 that}{\b0 node.}{\b
0 The}{\b0 interactions}{\b0 between}{\b0 genes}{\b0 and }
\par}{\phpg\posx1131\pvpg\posy9811\absw10679\absh668 \sl-199 \b \f10 \fs13 \cf1
{\b0 metabolites}{\b0 are}{\b0 represented}{\b0 by}{\b0 solid}{\b0 edges.}{
\b0 Interactions}{\b0 between}{\b0 proteins}{\b0 and}{\b0 genes}{\b0 are}{
\b0 represented}{\b0 by}{\b0 dotted}{\b0 edges. }\par
}
{\phpg\posx9740\pvpg\posy9994\absw105\absh122 \f10 \fs9 \cf1 \f10 \fs9 \cf1 12 \
par
}
{\phpg\posx8228\pvpg\posy10093\absw55\absh122 \f10 \fs9 \cf1 \f10 \fs9 \cf1 2 \p
ar
}
{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \f20 \fs17 \cf1 \f20 \fs17 \cf1
taken over prolonged cultivation times (Fig.3A).Un\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 de
r these conditions, the cells were chronologically
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 ag
ing and starving, and were stationary (not divid\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 in
g), but a certain percentage of the cells were still
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 al
ive. In line with the transcriptional decrease in de\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 fe
nse mechanisms against ROS, we observed a
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 st
rong accumulation of ROS in the LM 003-D126
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 st
rain: on day 6 all LM 003-D126 cells stained with
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 di
hydrorhodamine 123 were ROS positive, whereas
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 in
the reference strain only about 40% showed rho\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 da
mine fluorescence (Fig. 3A). Consequently, no
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 ce
lls containing PUFAs survived more than 6 con\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 ti
nuous days of incubation (Fig. 3B). In addition, it
\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 wa

s clearly observed that the PUFA- expressing cells


\par}{\phpg\posx1131\pvpg\posy11021\absw4620\absh3058 \sl-229 \f20 \fs17 \cf1 we
re much smaller after 6 days of incubation in com- \par
}
{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \f20 \fs17 \cf1 \f20 \fs17 \cf1
parison to the reference cells (Fig. 3A, lower panel).
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 As
clearly shown in Fig. 3A, ROS accrued during
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 ch
ronological ageing in the reference strain as well
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 as
in the LM 003-D126 cells, but the presence of
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 PU
FAs in the latter accelerated the ROS accumula\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 ti
on, resulting in a shortened life-span.
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 \f
i283 When exposed to additional exogenous oxida\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 ti
ve stress by incubation of exponentially growing
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-146 \par\f20 \fs17 \cf
1 cells in 20 mM H {\dn006 \fs11 2}O {\dn006 \fs11 2} for 3 h prior to spottin
g,the LM
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-170 \f20 \fs17 \cf1 00
3-D126 strain again displayed augmented sensi\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 ti
vity (Fig. 3C). Although no difference in ROS lev\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 el
s under standard conditions was observed in ex\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 po
nentially growing cells on day 1 (Fig. 3A), the
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-229 \f20 \fs17 \cf1 pr
esence of PUFAs obviously decreased viability
\par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh3137 \sl-146 \par\f20 \fs17 \cf
1 upon addition of H {\dn006 \fs11 2}O {\dn006 \fs11 2}. Similar effects of PU
FAs on \par
}
{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
50 \par
}
{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
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0 and}{\b0 6}{\b0 are}{\b0 shown;}{\b0 white}{\b0 bar:}{\b0 10}{\b0 {\fi
eld{\*\fldinst SYMBOL 109 \\f Symbol }}M.}{\b0 (B)}{\b0 Spot}{\b0 tests}{\b0
used}{\b0 to}{\b0 investigate}{\b0 long-term}{\b0 survival}{\b0 under}{\b0
standard}{\b0 condi- }
\par}{\phpg\posx1131\pvpg\posy8560\absw10691\absh1600 \sl-199 \b \f10 \fs13 \cf1
{\b0 tions}{\b0 in}{\b0 the}{\b0 presence}{\b0 of}{\b0 PUFAs.}{\b0 The}{\
b0 strains}{\b0 were}{\b0 spotted}{\b0 in}{\b0 tenfold}{\b0 serial}{\b0 d
ilutions}{\b0 after}{\b0 incubation}{\b0 for}{\b0 1}{\b0 and}{\b0 6}{\b0
days.}{\b0 Representative}{\b0 images}{\b0 are}{\b0 shown. }
\par}{\phpg\posx1131\pvpg\posy8560\absw10691\absh1600 \sl-199 \b \f10 \fs13 \cf1
{\b0 (C)}{\b0 Spot}{\b0 tests}{\b0 used}{\b0 to}{\b0 examine}{\b0 the}{\b
0 effect}{\b0 of}{\b0 exogenous}{\b0 oxidizing}{\b0 agents}{\b0 on}{\b0 s
urvival}{\b0 of}{\b0 the}{\b0 reference}{\b0 and}{\b0 the}{\b0 PUFA-produc
ing}{\b0 strain.}{\b0 Exponential}{\b0 cells}{\b0 were }
\par}{\phpg\posx1131\pvpg\posy8560\absw10691\absh1600 \sl-124 \par\b \f10 \fs13
\cf1 {\b0 incubated}{\b0 for}{\b0 3}{\b0 h}{\b0 in}{\b0 the}{\b0 presence}
{\b0 of}{\b0 20}{\b0 mM}{\b0 H}{\b0\dn006 \fs9 2}{\b0 O }{\b0\dn006 \fs9 2
}{\b0 and}{\b0 then}{\b0 spotted}{\b0 in}{\b0 tenfold}{\b0 serial}{\b0 di
lutions.}{\b0 The}{\b0 control}{\b0 cultures}{\b0 were}{\b0 left}{\b0 untr

eated.}{\b0 Representative }
\par}{\phpg\posx1131\pvpg\posy8560\absw10691\absh1600 \sl-150 \b \f10 \fs13 \cf1
{\b0 images}{\b0 are}{\b0 shown. }\par
}
{\phpg\posx1131\pvpg\posy10791\absw4620\absh367 \f20 \fs17 \cf1 \f20 \fs17 \cf1
oxidative stress sensitivity in{\i
S.}{\i cerevisiae} have
\par}{\phpg\posx1131\pvpg\posy10791\absw4620\absh367 \sl-229 \f20 \fs17 \cf1 bee
n described previously [12, 34, 35]. \par
}
{\phpg\posx1131\pvpg\posy11470\absw342\absh177 \b \f10 \fs18 \cf1 \b \f10 \fs18
\cf1 3.5 \par
}
{\phpg\posx1603\pvpg\posy11470\absw4942\absh591 \b \f10 \fs18 \cf1 \b \f10 \fs18
\cf1 Presence of PUFAs decreases proteasomal
\par}{\phpg\posx1603\pvpg\posy11470\absw4942\absh591 \sl-229 \b \f10 \fs18 \cf1
activity and increases sensitivity to proteotoxic
\par}{\phpg\posx1603\pvpg\posy11470\absw4942\absh591 \sl-229 \b \f10 \fs18 \cf1
stress \par
}
{\phpg\posx1131\pvpg\posy13070\absw4620\absh1214 \f20 \fs11 \cf1 \fi1366 \f20 \f
s11 \cf1 6
12
\par}{\phpg\posx1131\pvpg\posy13070\absw4620\absh1214 \sl-292 \f20 \fs11 \cf1 {\
fs17 producing}{\fs17 strain,}{\fs17 but}{\fs17 it}{\fs17 also}{\fs17
showed}{\fs17 a}{\fs17 lower}{\fs17 ex- }
\par}{\phpg\posx1131\pvpg\posy13070\absw4620\absh1214 \sl-229 \f20 \fs11 \cf1 {\
fs17 pression}{\fs17 of}{\fs17 several}{\fs17 chaperones}{\fs17 and}{\fs17
even}{\fs17 of}{\i \fs17 BLM10 }
\par}{\phpg\posx1131\pvpg\posy13070\absw4620\absh1214 \sl-229 \f20 \fs11 \cf1 {\
fs17 (Supporting}{\fs17 information}{\fs17 S2),}{\fs17 which}{\fs17 is}{
\fs17 a}{\fs17 protea- }
\par}{\phpg\posx1131\pvpg\posy13070\absw4620\absh1214 \sl-229 \f20 \fs11 \cf1 {\
fs17 some}{\fs17 activator}{\fs17 subunit}{\fs17 presumed}{\fs17 to}{\fs17
be}{\fs17 involved}{\fs17 in }
\par}{\phpg\posx1131\pvpg\posy13070\absw4620\absh1214 \sl-229 \f20 \fs11 \cf1 {\
fs17 the}{\fs17 protection}{\fs17 against}{\fs17 oxidative}{\fs17 damage
}{\fs17 [36,}{\fs17 37]. }\par
}
{\phpg\posx1131\pvpg\posy12401\absw4620\absh806 \f20 \fs17 \cf1 \f20 \fs17 \cf1
As already mentioned, the analyses of the global
\par}{\phpg\posx1131\pvpg\posy12401\absw4620\absh806 \sl-229 \f20 \fs17 \cf1 tra
nscriptional response showed transcriptional
\par}{\phpg\posx1131\pvpg\posy12401\absw4620\absh806 \sl-229 \f20 \fs17 \cf1 dow
n-regulation of the oxidative stress response
\par}{\phpg\posx1131\pvpg\posy12401\absw4620\absh806 \sl-229 \f20 \fs17 \cf1 gen
es in the{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\f40 {\field{\*
\fldinst SYMBOL 68 \\f Symbol }}}
-desaturase-expressing, PUFA- \par
}
{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \f20 \fs17 \cf1 \f20 \fs17 \cf1
The latter effect could lead to down-regulation of
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 th
e proteasomal system,which in turn could lead to
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 an
accumulation of misfolded proteins in the
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 PU
FA-producing strain. That scenario would also
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 le
ad to blocking or occupying the available protea\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 so
mes with intrinsic cellular proteins that need to
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 be

degraded.We therefore measured all three pro\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 te
olytic activities of the 20S proteasome (the chy\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 mo
trypsin-,caspase- and trypsin-like proteases) in
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 ex
ponentially growing cells, under standard condi\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 ti
ons for both the reference and the LM 003-D126
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 st
rain (Fig. 4A).All three proteolytic activities were
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 in
deed lower in the LM 003-D126 strain.The addi\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 ti
on of AZE caused almost complete inhibition of
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 th
e proteasomal proteases in both strains (Fig. 4A).
\par}{\phpg\posx6148\pvpg\posy10791\absw4620\absh3265 \sl-229 \f20 \fs17 \cf1 Th
is suggests that an excess of misfolded proteins \par
}
{\phpg\posx1131\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
{\phpg\posx10545\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
351 \par
}
\sect\sectd\pard\plain
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28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
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28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
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28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
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28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a
28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a00fffd9
}}\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \b \f10 \fs16 \cf1 \b \f10
\fs16 \cf1 Biotechnology
\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \f
i801 Journal \par
}
{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Bi
otechnol. J. 2011, 6, 343-356 \par

}
{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \b \f10 \fs13 \cf1 \b \f10 \fs13
\cf1 Figure 4.{\b0 The}{\b0 presence}{\b0 of}{\b0 PUFAs}{\b0 lowers}{\b0
proteasomal}{\b0 activity}{\b0 and}{\b0 thus}{\b0 diminishes}{\b0 survival
}
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 under}{\b0 standard}{\b0 conditions}{\b0 as}{\b0 well}{\b0 as}{\b0 in
}{\b0 the}{\b0 presence}{\b0 of}{\b0 misfolded}{\b0 proteins.}{\b0 (A)}{\b
0 Lower}{\b0 ac- }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 tivity}{\b0 of}{\b0 the}{\b0 three}{\b0 proteasomal}{\b0 activities}{\
b0 in}{\b0 the}{\b0 PUFA-producing}{\b0 strain}{\b0 LM}{\b0 003-D126.}{\b0
Lumi- }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 nescence}{\b0 activity}{\b0 measurements}{\b0 of}{\b0 the}{\b0 chymotr
ypsin-,}{\b0 caspase-}{\b0 and}{\b0 trypsin-like}{\b0 proteolytic }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 activities}{\b0 of}{\b0 the}{\b0 20S}{\b0 proteasome}{\b0 are}{\b0 sh
own.}{\b0 The}{\b0 luminescent}{\b0 signal}{\b0 is}{\b0 proportional}{\b0
to}{\b0 the }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 amount}{\b0 of}{\b0 proteasome}{\b0 activity}{\b0 in}{\b0 cells.}{\b0
Different}{\b0 numbers}{\b0 of}{\b0 cells}{\b0 were}{\b0 monitored}{\b0 w
ith }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 and}{\b0 without}{\b0 3-h}{\b0 preincubation}{\b0 with}{\b0 30}{\b0 m
M}{\b0 of}{\b0 the}{\b0 protein}{\b0 misfolding-inducing}{\b0 agent}{\b0 A
ZE. }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 Luminescence}{\b0 was}{\b0 recorded}{\b0 as}{\b0 relative}{\b0 lumines
cence}{\b0 units}{\b0 (RLU).}{\b0 The}{\b0 data}{\b0 represent}{\b0 an }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 average}{\b0 of}{\b0 three}{\b0 independent}{\b0 experiments}{\b0 }{\b0
SEM.}{\b0 (B)}{\b0 Spot}{\b0 tests}{\b0 to}{\b0 analyze}{\b0 the}{\b0 e
ffect}{\b0 of }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 protein}{\b0 misfolding}{\b0 on}{\b0 survival}{\b0 in}{\b0 the}{\b0 r
eference}{\b0 and}{\b0 the}{\b0 LM}{\b0 003-D126.}{\b0 Exponential}{\b0 ce
lls }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 were}{\b0 incubated}{\b0 for}{\b0 3}{\b0 h}{\b0 in}{\b0 the}{\b0 pre
sence}{\b0 of}{\b0 30}{\b0 mM}{\b0 AZE}{\b0 and}{\b0 then}{\b0 spotted}{\
b0 in}{\b0 tenfold}{\b0 serial }
\par}{\phpg\posx5139\pvpg\posy4873\absw6239\absh2108 \sl-199 \b \f10 \fs13 \cf1
{\b0 dilutions.}{\b0 The}{\b0 control}{\b0 cultures}{\b0 were}{\b0 left}{\b
0 untreated.}{\b0 Representative}{\b0 images}{\b0 are}{\b0 shown. }\par
}
{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \f20 \fs17 \cf1 \f20 \fs17 \cf1
impedes access of further substrates to the prote\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 oly
tic sites.When the cells were spotted after 3 h of
\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 AZE
treatment, cell survival of the LM 003-D126
\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 str
ain was decreased (Fig. 4B).This strain was cer\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 tai
nly more sensitive to protein misfolding induced
\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 by
AZE, probably due to both the lower proteaso\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 mal

activity and saturation of the existing protea\par}{\phpg\posx1131\pvpg\posy7801\absw4620\absh1609 \sl-229 \f20 \fs17 \cf1 som
es with oxidized and misfolded proteins. \par
}
{\phpg\posx1131\pvpg\posy10069\absw165\absh203 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 4 \par
}
{\phpg\posx1491\pvpg\posy10069\absw1290\absh203 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 Discussion \par
}
{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \f20 \fs17 \cf1 \f20 \fs17 \cf1
Our study produced two main unexpected findings:
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 th
e first is linked to the lipid metabolism and its
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 re
gulation in yeast, and the second to the physio\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 lo
gical effect of the PUFAs on cell mechanisms in\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 vo
lved in oxidative stress and proteostasis. We
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 fo
und it surprising that the PUFA-producing LM
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 00
3-D126 strain expressing the{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }
}}{\up006 \fs11 12}- and{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\u
p006 \fs11 6}-desat\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-230 \f20 \fs17 \cf1 ur
ases had increased level of phospholipids (per
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 un
it of dry weight biomass), compared to the refer\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 en
ce strain and irrespective of the cultivation con\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 di
tions (carbon limiting or carbon excess). This
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 se
ems to be associated with our observations that
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 th
e PUFA-containing cells are smaller (Fig. 3A,
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 lo
wer panel), which means that the requirement
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 fo
r plasma membrane per gram of biomass in\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 cr
eases. We speculate that the altered composition
\par}{\phpg\posx1131\pvpg\posy10561\absw4620\absh3472 \sl-229 \f20 \fs17 \cf1 of
the phospholipids, due to the incorporation of \par
}
{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \f20 \fs17 \cf1 \f20 \fs17 \cf1
PUFAs is the cause of this effect. As we grew the
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 str
ains in chemostat cultures, where the specific
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 gro
wth rate was kept constant, the increased phos\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 pho
lipid level in the LM 003-D126 strain was asso\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 cia
ted with a higher flux through the fatty acid
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 bio
synthesis as shown in Fig. 1. From our mode
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 (Fi

g. 2), it is interesting to observe that the gene en\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 cod
ing Acc1p, which is generally considered a flux
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 con
trolling step in fatty acid biosynthesis [38], was
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 dow
n-regulated, whereas no other genes involved
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 in
fatty acid biosynthesis were significantly affect\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 ed.
However, genes involved in{\f40 {\field{\*\fldinst SYMBOL 98 \\f Symbol }
}}-oxidation were
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 als
o found to be down-regulated.We therefore con\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 clu
de that the increased flux towards fatty acid pro\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 duc
tion is mainly explained by reduced feedback
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 inh
ibition by oleic acid, which was at a 3-fold low\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 er
level in the PUFA-producing strain (Table 2),
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 and
this reduced inhibition can even compensate
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 for
a possibly reduced enzyme level due to a de\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 cre
ased transcription of{\i ACC1}. A reduced{\f40 {\field{\*\fldinst SYMBOL 98 \\
f Symbol }}}-oxida\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 tio
n activity may, however, also play a role in in\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 cre
asing the observed net flux towards fatty acids.
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 We
also analyzed a strain only expressing{\f40 {\field{\*\fldinst SYMBOL 68
\\f Symbol }}}{\up006 \fs11 12}-de\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-230 \f20 \fs17 \cf1 sat
urase, and this strain had also increased phos\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 pho
lipid levels, an increased flux into fatty acids
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 and
reduced levels of oleic acid (data not shown).
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 In
this strain there was no down-regulation of
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 gen
es involved in{\f40 {\field{\*\fldinst SYMBOL 98 \\f Symbol }}}-oxidation, so w
e conclude that
\par}{\phpg\posx6148\pvpg\posy7801\absw4620\absh5956 \sl-229 \f20 \fs17 \cf1 the
main regulation of flux into the pathway is by \par
}
{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
52 \par
}
{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
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w.biotechnology-journal.com \par
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feedback inhibition of Acc1p by oleic acid, and
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en the pathway is extended from oleic acid, the
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ee pool of this fatty acid is reduced,resulting in an
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 al
leviation of the feedback inhibition and an in\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 cr
eased flux through the pathway.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \f
i283 Interestingly,
genes
associated
with
lipid
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 bi
osynthesis were found to be transcriptionally
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 do
wn-regulated
in
the
PUFA-producing
strain
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (T
able 3),but,as seen in Fig.2,it is mainly genes as\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 so
ciated with ergosterol biosynthesis that are af\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 fe
cted. As mentioned above, it is particularly genes
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
at are regulated by the transcription factor Upc2p
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
at are down-regulated. This transcription factor

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
similar to SREBP-2 in mammals, and is found
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
th in yeast and mammalian cells in the ER mem\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ane. Upc2p is activated by proteolytic cleavage,
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ich releases the fragment that acts as a tran\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ription factor from the membrane spanning do\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
in. The changed composition of the ER mem\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ane might influence the activation mechanism of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
c2p, thus leading to the observed down-regula\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
on of the Upc2p-dependent gene transcription
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ch as the genes in the ergosterol pathway.The re\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ced expression of genes in the ergosterol path\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
y resulted in slightly lower ergosterol levels in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
e strain producing PUFAs (Fig. 2), which is par\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
cularly
surprising
considering
the
increased
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ospholipid level. Upc2p and Ecm22p are gener\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ly believed to be activated in response to low er\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
sterol levels in the ER, in analogy with SREBP-2
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
mammals [39-41], and it is interesting that the
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
esence of PUFAs seems to partly inactivate this
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
gulatory system. It is interesting that our obser\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
tion on transcriptional down-regulation in yeast
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
oducing LA and GLA is apparently completely
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
alogous to our recent study in mice fed a PUFA\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ch diet [19]. In that study we found a reduced ex\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ession of genes involved in cholesterol biosyn\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
esis (analogous to the ergosterol pathway in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
ast) in the liver, as well as reduced expression of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
nes associated with proteasomal activity, and a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
duction of oxidative stress response genes in dif\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229
rent tissues.In this respect,our study provides an

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\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ex


planation for the beneficial effects of PUFAs in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 th
e diet on cholesterol levels, and this finding can
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 be
used in the studies of development of cardio\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 va
scular diseases.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \f
i283 The transcriptome analysis also showed down\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 re
gulation of key genes involved in the defense
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ag
ainst oxidative stress, and down-regulation of
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ge
nes coding for proteins involved in proteome
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ma
intenance and proteostasis (e.g., chaperones),
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 su
ch as HSP104; STI1 (HSP90 chaperone), YDJ1
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 (c
haperone regulating HSP90 and HSP70 func\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ti
ons) andYOR289W (induced by the unfolded pro\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 te
in response) (Table 3 and Table S6). Accordingly, \par
}
{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \i \f20 \fs17 \cf1 \i \f20 \fs1
7 \cf1 YMR074c{\i0 ,}{\i0 an} S. cerevisiae{\i0 homologue}{\i0 of}{\i0 the}{
\i0 human }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
hPDCD5{\i0 ,}{\i0 which}{\i0 is}{\i0 a}{\i0 programmed}{\i0 cell}{\i0
death-pro- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 moting}{\i0 protein,}{\i0 was}{\i0 up-regulated}{\i0 in}{\i0 the
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{\i0 strain.}{\i0 Overexpression}{\i0 of} YMR074c{\i0 was}{\i0 recentl
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-146 \par\i \f20 \fs17
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-172 \i \f20 \fs17 \cf1
{\i0 [42],}{\i0
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transcriptional}{\i0
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of }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
YMR074c{\i0
in}{\i0
our}{\i0
PUFA-producing}{\i0
strain}{\i0
mig
ht }
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{\i0 point}{\i0 out}{\i0 that}{\i0 this}{\i0 strain}{\i0 is}{\i0 less}{\i
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 tive}{\i0 stress.}{\i0 Whether}{\i0 there}{\i0 is}{\i0 a}{\i0 s
imilar}{\i0 negative}{\i0 ef- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 fect}{\i0 of}{\i0 PUFAs}{\i0 on}{\i0 mammalian}{\i0 cells}{\i0
remains}{\i0 to}{\i0 be }

\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1


{\i0 seen. }
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\fi283 {\i0 It}{\i0 has}{\i0 been}{\i0 shown}{\i0 previously}{\i0 in}
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{\i0 that}{\i0 the}{\i0 membrane}{\i0 lipid}{\i0 composition}{\i0 is}
{\i0 important }
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{\i0 in}{\i0 conferring}{\i0 resistance}{\i0 to}{\i0 oxidative}{\i0 s
tress:}{\i0 cells }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 with}{\i0 membranes}{\i0 with}{\i0 a}{\i0 higher}{\i0 level}{\i0
of}{\i0 saturated }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 fatty}{\i0 acids}{\i0 are}{\i0 more}{\i0 resistant}{\i0 than}{\i
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 higher}{\i0 level}{\i0 of}{\i0 PUFAs}{\i0 [35].}{\i0 This}{\i0 is}{\
i0 probably}{\i0 due}{\i0 to }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 peroxidation}{\i0 of}{\i0 PUFAs}{\i0 esterified}{\i0 in}{\i0 mem
branes, }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 resulting}{\i0 in}{\i0 the}{\i0 production}{\i0 of}{\i0 reactive
}{\i0 lipid}{\i0 hy- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 droperoxides}{\i0
and}{\i0
lipid}{\i0
radicals}{\i0
[43-45].}{
\i0
Those }
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{\i0 ROS}{\i0 species}{\i0 in}{\i0 Fig.}{\i0 3A.}{\i0 Breakdown}{\i0
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{\i0 droperoxides}{\i0 leads}{\i0 to}{\i0 the}{\i0 generation}{\i0 of}{\i0
a}{\i0 great}{\i0 di- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 versity}{\i0 of}{\i0 highly}{\i0 reactive}{\i0 aldehydes}{\i0 [4
6].}{\i0 Interest- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 ingly,}{\i0 it}{\i0 has}{\i0 been}{\i0 found}{\i0 that}{\i0 there}{\
i0 is}{\i0 down-regula- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 tion}{\i0 of} GTT2{\i0 ,}{\i0 which}{\i0 encodes}{\i0 glutathione}
{\i0 S-trans- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 ferase}{\i0
forming}{\i0
part}{\i0
of}{\i0
the}{\i0
antioxi
dant-defense }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 systems,}{\i0 and}{\i0 thought}{\i0 to}{\i0 be}{\i0 important}{\i0 i
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 against}{\i0 lipid}{\i0 peroxidation}{\i0 [47].}{\i0 We}{\i0 also
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
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}
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{\i0 strain,}{\i0 suggesting}{\i0 that}{\i0 the}{\i0 cells}{\i0 have}{\i0


difficulties}{\i0 in }
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{\i0 coping}{\i0 with}{\i0 the}{\i0 accumulated}{\i0 toxic}{\i0 react
ive}{\i0 lipid }
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{\i0 hydroperoxides}{\i0 and}{\i0 lipid}{\i0 radicals,}{\i0 contributin
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 droxy-2-nonenal}{\i0 (HNE)}{\i0 [43].}{\i0 Besides}{\i0 deleteriou
s }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 effects}{\i0 like}{\i0 inhibition}{\i0 of}{\i0 DNA}{\i0 and}{\i0 pro
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 sis,}{\i0 HNE}{\i0 causes}{\i0 apoptosis}{\i0 in}{\i0 human}{\i0
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
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agreement }
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{\i0 with}{\i0 previous}{\i0 publications}{\i0 [12,}{\i0 34,}{\i0 35],}{\i
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{\i0 an}{\i0 increase}{\i0 in}{\i0 ROS}{\i0 and}{\i0 in}{\i0 the}{\i0 se
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
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{\i0 most}{\i0 likely}{\i0 result}{\i0 from}{\i0 lipid}{\i0 peroxidat
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 down-regulation}{\i0 of}{\i0 the}{\i0 oxidative}{\i0 stress}{\i0 defe
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 gen}{\i0
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on}{\i0
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 stress}{\i0 responses,}{\i0 but}{\i0 ultimately}{\i0 promoting}{\i0 p
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\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 and}{\i0 DNA}{\i0 damage}{\i0 and}{\i0 hence}{\i0 inducing}{\i0 prog

rammed }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 cell}{\i0 death. }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
\fi283 {\i0 The}{\i0 proteasome}{\i0 has}{\i0 been}{\i0 shown}{\i0 to}{\i0
serve}{\i0 an}{\i0 im- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 portant}{\i0 role}{\i0 in} S. cerevisiae{\i0 as}{\i0 a}{\i0 "secondar
y}{\i0 antiox- }
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh11545 \sl-229 \i \f20 \fs17 \cf1
{\i0 idant"}{\i0 by}{\i0 clearing}{\i0 the}{\i0 cell}{\i0 of}{\i0 oxidize
d}{\i0 proteins}{\i0 [51]. }\par
}
{\phpg\posx1131\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
{\phpg\posx10545\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
353 \par
}
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451401fffd9
}}\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \b \f10 \fs16 \cf1 \b \f10
\fs16 \cf1 Biotechnology
\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \f
i801 Journal \par
}
{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Bi
otechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \f20 \fs17 \cf1 \f20 \fs17 \cf1
The degradation of mildly oxidized proteins is an
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 im
portant function of the proteasomal system.The
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ma
jor recognition motifs of the substrates are hy\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 dr
ophobic surface patches that are recognized by
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 th
e 20S core proteasome. Such hydrophobic sur\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 fa
ce patches are formed by partial unfolding and
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ex
posure of hydrophobic amino acid residues dur\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 in
g oxidation. Oxidized proteins appear to be rela\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ti
vely poor substrates for ubiquitylation, and the
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ub
iquitin system does not seem to be involved in
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 th
e recognition or targeting of oxidized proteins
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 [5
2-55]. Heavily oxidized proteins however, appear
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 to
first aggregate and then to form covalent cross\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 li
nks that make them highly resistant to proteoly\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 si
s [44, 53].The inability to degrade extensively ox\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 id
ized proteins may contribute to the accumulation
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 of
protein aggregates during diseases and the age\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 in
g process [44, 53]. Heavily oxidized proteins can
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 al
so
act
as
proteasomal
inhibitors, as
clearly
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 de
monstrated for proteins cross-linked after mod\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 if
ication by the lipid peroxidation product HNE [56,
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 57
]. In addition, protein damage can result from
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pr
otein adduct formation with lipid peroxidation
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pr
oducts such as HNR [58]. HNE adducts were de\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 te
cted in{\i S.}{\i cerevisiae} expressing a{\f40 {\field{\*\fldinst SYMBO
L 68 \\f Symbol }}}{\up006 \fs12 12}-desaturase

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \f20 \fs17 \cf1 ge


ne from{\i Hevea}{\i brasiliensis} [34]. These strains
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 pr
oduced C18:2{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 9,12}
and C16:2{\f40 {\field{\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs12 9,12} P
UFAs and also
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-230 \f20 \fs17 \cf1 ex
hibited an increased sensitivity to oxidative chal\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 le
nges [34] similar to our LA- and GLA-producing
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 st
rain (Fig. 3C). Conjugation of several{\f40 {\field{\*\fldinst SYMBOL 97 \\f Sy
mbol }}} and{\f40 {\field{\*\fldinst SYMBOL 98 \\f Symbol }}} pro\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 te
asomal subunits with HNE (including active site\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 co
ntaining{\f40 {\field{\*\fldinst SYMBOL 98 \\f Symbol }}} subunits) was found t
o coincide with a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 de
crease in chymotrypsin-like activity of protea\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 so
mes in lymphocytes from healthy 50-63-year-old
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 hu
man donors when compared with 20-35-year\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ol
d donors [59], so the accumulation of lipid perox\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 id
ation products may be associated with ageing-re\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 la
ted diseases.
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 \f
i283 In conclusion, we have found that the presence
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 of
PUFAs in yeast{\i S.}{\i cerevisiae} results in a large
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 nu
mber of molecular effects that are detrimental to
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 th
e cell, such as increased sensitivity to oxidative
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 st
ress and reduced proteasomal activity.This clear\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ly
shows that the fatty acid composition of cellular
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 me
mbranes plays a very important role for correct
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 fu
nctions of many cellular processes and it may be
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 th
at{\i S.}{\i cerevisiae} has evolved towards not contain\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 in
g any PUFAs in its membranes, as opposed to
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ma
ny other closely associated yeasts, such as{\i Sac- }
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 {\
i charomyces}{\i kluyveri}, that do contain PUFAs. This
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ad
aptation has contributed to the development of a
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 mo
re resistant yeast that can cope better with
\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 ha
rsh and stressful conditions, such as high sugar

\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 an


d ethanol concentrations, and gives selective ad\par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11338 \sl-229 \f20 \fs17 \cf1 va
ntage in the nature. \par
}
{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \i \f20 \fs17 \cf1 \i \f20 \fs17
\cf1 Thailand Graduate Institute of Science and Technol\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
ogy is acknowledged for a grant to Rawisara Ruen\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
wai. Chalmers Foundation and Knut and Alice Wal\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
lenberg Foundation are acknowledged for financial
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
support to this project. We thank Lars Hellgren,
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
Anne-Marie Nepper and Mohammadali Asadollahi
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
for assistance with lipid and ergosterol analysis,
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
Goutham Vemuri for assistance with setting up the
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
chemostat
cultivation.
Jos
Manuel
Otero
and
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
Songsak Wattanachaisaereekul are acknowledged
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
for assistance with analyzing the fermentation data
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
and Maya Petrova Piddocke for assistance with the
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-229 \i \f20 \fs17 \cf1
DNA microarray experiment.
\par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh3057 \sl-230 \par\i \f20 \fs17 \
cf1 The authors have declared no conflict of interest. \par
}
{\phpg\posx6148\pvpg\posy5469\absw165\absh203 \b \f10 \fs21 \cf1 \b \f10 \fs21 \
cf1 5 \par
}
{\phpg\posx6508\pvpg\posy5469\absw1320\absh203 \b \f10 \fs21 \cf1 \b \f10 \fs21
\cf1 References \par
}
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368 110-116. \par
}
{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
54 \par
}
{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a

0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
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0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a
0028a28a0028a28a0028a28a0028a28a0028a28a0028a28a00ffd9
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i368 apoptosis
and
differentiation
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human
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i368 oxidation: 4-hydroxy-2-nonenal is a potential inducer of in- \par
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140

05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
05145140051451400514514005145140051451400514514005145140051451400514514005145140
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05145140051451401fffd9
}}\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \b \f10 \fs16 \cf1 \b \f10
\fs16 \cf1 Biotechnology
\par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \f
i801 Journal \par
}
{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Bi
otechnol. J. 2011, 6, 343-356 \par
}
{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \f20 \fs13 \cf1 \fi368 \f20 \fs1
3 \cf1 tracellular peroxide production{\i J.}{\i Biol.}{\i Chem.} 1999,
{\i 274},
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 2234-2242.
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 [51
] Chen, Q., Thorpe, J., Dohmen, J. R., Li, F., Keller, J. N., Ump1
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 extends yeast lifespan and enhances viability during oxida\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 tive stress:Central role for the proteasome?{\i Free}{\i Radic.Biol. }
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 {\i Med.} 2006,{\i 40}, 120-126.
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] Davies, K. J. A., Degradation of oxidized proteins by the 20S
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 proteasome.{\i Biochimie} 2001,{\i 83}, 301-310.
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 [53
] Grune, T., Merker, K., Sandig, G., Davies, K. J. A., Selective
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 degradation of oxidatively modified protein substrates by
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 the proteasome.{\i Biochem.}{\i Biophys.}{\i Res.}{\i Commun.} 2003,{\i
305},
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi

368 709-718.
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 [54
] Inai, Y., Nishikimi, M., Increased degradation of oxidized
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 proteins in yeast defective in 26S proteasome assembly.
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 {\i Arch.}{\i Biochem.}{\i Biophys.} 2002,{\i 404}, 279-284.
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 [55
] Shringarpure, R., Grune, T., Mehlhase, J., Davies, K. J. A.,
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 Ubiquitin conjugation is not required for the degradation of
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 oxidized proteins by proteasome.{\i J.}{\i Biol.}{\i Chem.} 2003,{\i
278},
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 311-318.
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 [56
] Friguet, B., Stadtman, E. R., Szweda, L. I., Modification of
\par}{\phpg\posx1131\pvpg\posy1596\absw4620\absh3726 \sl-199 \f20 \fs13 \cf1 \fi
368 glucose-6-phosphate
dehydrogenase
by
4-hydroxy-2- \par
}
{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \f20 \fs13 \cf1 \fi368 \f20 \fs1
3 \cf1 nonenal. Formation of cross-linked protein that inhibits the
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 multicatalytic protease.{\i J.}{\i Biol.}{\i Chem.} 1994,{\i 269},
21639\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 21643.
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 [57
] Friguet,B.,Szweda,L.I.,Inhibition of the multicatalytic pro\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 teinase (proteasome) by 4-hydroxy-2-nonenal cross-linked
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 protein.{\i FEBS}{\i Lett.} 1997,{\i 405}, 21-25.
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 [58
] Berlett,B.S.,Stadtman,E.R.,Protein oxidation in aging,dis\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 ease, and oxidative stress.{\i J.}{\i Biol.}{\i Chem.} 1997,{\i 272}, 20
313\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 20316.
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 [59
] Carrard, G., Dieu, M., Raes, M.,Toussaint, O., Friguet, B., Im\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 pact of ageing on proteasome structure and function in hu\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 man lymphocytes.{\i Int.J.Biochem.Cell}{\i Biol.} 2003,{\i 35},728-739.
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 [60
] Myers, A. M., Tzagoloff, A., Kinney, D. M., Lusty, C. J., Yeast
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 shuttle and integrative vectors with multiple cloning sites
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 suitable for construction of lacZ fusions.{\i Gene} 1986,{\i 45},
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 299-310.
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 [61
] Laoteng,
K.,
Cheevadhanarak,
S., Tanticharoen,
M.,
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 Maresca, B., Promoter analysis of{\i Mucor}{\i rouxii}{\f40 {\field{
\*\fldinst SYMBOL 68 \\f Symbol }}}{\up006 \fs9 9}-desat-

\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-200 \f20 \fs13 \cf1 \fi


368 urase: Its implication for transcriptional regulation in{\i Sac- }
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 {\i charomyces}{\i
cerevisiae}.{\i Biochem.}{\i Biophys.}{\i Res.}{
\i Commun. }
\par}{\phpg\posx6148\pvpg\posy1595\absw4620\absh3727 \sl-199 \f20 \fs13 \cf1 \fi
368 2005,{\i 335}, 400-405. \par
}
{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 3
56 \par
}
{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim \par
}
\sect\sectd\pard\plain
}

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