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\f10 \fs13 \cf1 \f10 \fs13 \cf1 Biotechnol. J. 2011, 6, 343-356 \par}{\phpg\posx5055\pvpg\posy444\absw2061\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 DOI 10.1002/biot.201000316 \par}{\phpg\posx8787\pvpg\posy444\absw2250\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 www.biotechnology-journal.com \par}{\phpg\posx1131\pvpg\posy1559\absw10558\absh2087 \f10 \fs21 \cf1 \f10 \fs21 \cf1 Research Article \par}{\phpg\posx1131\pvpg\posy1559\absw10558\absh2087 \sl-343 \par\f10 \fs21 \cf1 {\b \fs32 Heterologous}{\b \fs32 production}{\b \fs32 of}{\b \fs32 polyunsaturated}{\b \fs32 fatty}{\b \fs32 acids }\par}{\phpg\posx1131\pvpg\posy1559\absw10558\absh2087 \sl-460 \f10 \fs21 \cf1 {\b \fs32 in}{\b\i \fs32 Saccharomyces}{\b\i \fs32 cerevisiae}{\b \fs32 causes}{\b \fs32 a}{\b \fs32 global}{\b \fs32 transcriptional }\par}{\phpg\posx1131\pvpg\posy1559\absw10558\absh2087 \sl-460 \f10 \fs21 \cf1 {\b \fs32 response}{\b \fs32 resulting}{\b \fs32 in}{\b \fs32 reduced}{\b \fs32 proteasomal}{\b \fs32 activity}{\b \fs32 and }\par}{\phpg\posx1131\pvpg\posy1559\absw10558\absh2087 \sl-459 \f10 \fs21 \cf1 {\b \fs32 increased}{\b \fs32 oxidative}{\b \fs32 stress }\par}{\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 Supapon 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 Technology,}{\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 Technology,}{\fs16 King}{\fs16 Mongkut's}{\fs16 University}{\fs16 of}{\fs16 Technology}{\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 Training}{\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 \fi90 {\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 National}{\fs16 Center}{\fs16 for}{\fs16 Genetic}{\fs16 Engineering}{\fs16 and }\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-230 \f10 \fs11 \cf1 \fi90 {\fs16 Biotechnology,}{\fs16 National}{\fs16 Sciences}{\fs16 and}{\fs16 Technology}{\fs16 Development}{\fs16 Agency}{\fs16 at}{\fs16 King}{\fs16 Mongkut's}{\fs16 University}{\fs16 of}{\fs16 Technology}{\fs16 Thonburi, }\par}{\phpg\posx1131\pvpg\posy5078\absw10394\absh1415 \sl-230 \f10 \fs11 \cf1 \fi90 {\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 processes for efficient \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 production 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 that accumu- \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 lated linoleic acid and{\f40 {\field{\*\fldinst SYMBOL 103 \\f Symbol }}}-linolenic acid in the different lipid species. Additionally, the strain contained \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 higher levels of phospholipids and lower levels of ergosterol than the reference strain. Integrated \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 analysis of the transcriptome revealed decreased expression of genes involved in ergosterol biosyn- \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 thesis, but more unexpectedly it also pointed towards attenuated activity of the ubiquitin-protea- \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 some 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 reduced levels of all three proteasomal activities and also increased levels of reactive oxidative \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 species in the PUFA-producing strain. Overall our results clearly show that PUFAs in yeast can be \par}{\phpg\posx1131\pvpg\posy7122\absw8264\absh2431 \sl-230 \f10 \fs16 \cf1 detrimental for several key cellular pathways, such as the oxidative stress response 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 these processes. \par}{\phpg\posx8784\pvpg\posy7211\absw2130\absh488 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Received 21 September 2010 \par}{\phpg\posx8784\pvpg\posy7211\absw2130\absh488 \sl-199 \f10 \fs13 \cf1 Revised 2 November 2010 \par}{\phpg\posx8784\pvpg\posy7211\absw2130\absh488 \sl-199 \f10 \fs13 \cf1 Accepted 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 used 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 \fs13 \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}{\b0 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 }}}{\b0 -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;} PUFAs{\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 fuels, chemicals, food ingredients and pharmaceu- \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 ticals [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 its high ethanol tolerance allows for efficient pro- \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 duction, reaching very high titers.Also a number of \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 recombinant proteins (i.e.,hormones and vaccines) \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 are being commercially produced by{\i S.}{\i cerevisiae}, \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 and here efficient protein production, folding and \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 secretion are of major importance [5].There is also \par}{\phpg\posx6148\pvpg\posy11021\absw4620\absh2230 \sl-229 \f20 \fs17 \cf1 much 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 \fi112 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\pgwsxn11905\pghsxn15817{\phpg\posx0\pvpg\posy0\absw11900\absh15820 \overlay{\*\shppict {\pict\jpegblip\picw595 \pich791\picwgoal11900 \pichgoal15820\picscalex135 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\b \f10 \fs16 \cf1 \b \f10 \fs16 \cf1 Biotechnology \par}{\phpg\posx1118\pvpg\posy315\absw1500\absh357 \sl-181 \b \f10 \fs16 \cf1 \fi801 Journal \par}{\phpg\posx8876\pvpg\posy467\absw2276\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 Biotechnol. 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 rated fatty acids (PUFAs), as this can lead to scala- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ble production of pure and defined oils compared \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 with fish oils. \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \fi283 Lipid metabolism has a central role in the phys- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 iology of the cell, e.g., the plasma membrane com- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 position,which influences tolerance to high ethanol \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 concentrations and other types of stress [6]. Lipid \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 metabolism also influences the membrane compo- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 sition of the endoplasmic reticulum (ER), which is \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 central to protein processing pathways [7]. The \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 processes of lipid metabolism are highly conserved \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 between{\i S.}{\i cerevisiae} and mammals, such as mouse \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 and human [8]. For example, several key regulatory \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 elements involved in transcriptional regulation of \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 genes involved in sterol biosynthesis and fatty acid \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 biosynthesis are found to be similar in yeast and \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 mammals. The yeast Snf1p, like its mammalian or- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tholog AMP-activated protein kinase (AMPK), reg- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ulates 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 back regulation of unsaturated fatty acid synthesis \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 through proteolytic activation of transcription fac- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tors like Mga2p and Spt23p resembles in function \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 the mammalian sterol regulatory element binding \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 proteins (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 mammalian cells,{\i S.cerevisiae} does,however,have a \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 quite different lipid composition as it contains no \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 PUFAs in its membranes.The presence of PUFAs in \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 the membranes of yeast was previously shown to \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 play important roles in cellular stress response to \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 various stimuli, e.g., resulting in increased resist- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ance to alkaline pH [11], oxidative stress [12], and \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 freezing and salt stress [13]. \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \fi283 Lipid metabolism is also of great interest in nu- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 tritional studies and it has been shown that fish oils, \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 which are rich in PUFAs such as eicosapentaenoic \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 acid (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 acid (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 itive effects on health, including reduced risk of \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 cardiovascular disease, cancer, neurodegeneration \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 and various other diseases [14-18]. In a recent \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 study by our group [19] it was found that mice fed a \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 herring-based diet that is rich in PUFAs resulted \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 not only in reduced expression of genes associated \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 with cholesterol biosynthesis, but also reduced ex- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 pression of genes involved in oxidative stress re- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 sponse, protein folding and protein degradation in \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 different tissues. It would be interesting to deter- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 mine whether some of these responses to the pres- \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 ence of PUFAs are conserved in eukaryotic cells. \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 \fi283 As it is both of interest to produce PUFAs in \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 yeast and this serves as a good model organism for \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 eukaryotes, we wanted to study the effect of PUFA \par}{\phpg\posx1131\pvpg\posy1591\absw4620\absh11545 \sl-229 \f20 \fs17 \cf1 production in yeast. Therefore, to obtain a yeast \par}{\phpg\posx6148\pvpg\posy1591\absw4620\absh160 \f20 \fs17 \cf1 \f20 \fs17 \cf1 strain that contains PUFAs, especially linoleic \par}{\phpg\posx6148\pvpg\posy1780\absw1868\absh429 \f20 \fs17 \cf1 \f20 \fs17 \cf1 acid (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}- desaturases 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 transcriptome}{\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 the}{\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 transcriptional }\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}{\i0 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 viability}{\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 proteotoxic }\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}{\i0 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 \cf1 {\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 \cf1 \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 rized in Table 1 and Fig. S1 (see Supplementary \par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 Information). \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 sole carbon source was used [20]. The cultures \par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 were 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 When 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 tivations. Batch cultivations were carried out in \par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 bioreactors (BIOSTAT {\up006 \fs12 }B, B. Braun Biotech, Ger- \par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-230 \f20 \fs17 \cf1 many) with a working volume of 1 L of a defined \par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 medium [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 constantly kept at 30C, and the pH was automati- \par}{\phpg\posx6148\pvpg\posy9641\absw4620\absh4300 \sl-229 \f20 \fs17 \cf1 cally controlled at 5.0 by addition of 2 N KOH. The \par}{\phpg\posx1131\pvpg\posy15150\absw308\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 344 \par}{\phpg\posx7217\pvpg\posy15150\absw3953\absh128 \f10 \fs13 \cf1 \f10 \fs13 \cf1 2011 Wiley-VCH Verlag GmbH & Co. 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