exogenous fatty acids and their 
 derivatives influence a wide variety of cellular processes including fatty acids 
 and phospholipids synthesis , organelle inheritance , vesicle fusion , protein 
 export and modification , enzyme activation or deactivation , cell signaling , 
 membrane permeability , bacterial pathogenesis , and transcriptional control [ 1 , 2 ] . the process governing the transport of fatty acids from environmental 
 conditions across the membrane is distinct from the transport of hydrophilic 
 substrates such as sugars and amino acids . in a number of cell types , the 
 process of fatty acid transport is inducible and commensurate with the 
 expression of specific sets of proteins . in wild - type escherichia coli , growth on fatty acids requires specific transport 
 system ( fadl ) , acyl - coa dehydrogenase ( fadd ) , enzymes of the -oxidation cycle ( fada , 
 fadb , fade , fadf , fadg , and fadh ) , and glyoxylate shunt ( acea , aceb , and acek ) , 
 and these genes are negatively regulated by a transcriptional factor , fadr . supply 
 of long - chain fatty acids that contain 12 or more carbons results in the derepression of the genes negatively controlled by fadr but leads to the decreased expression 
 of the genes ( e.g. , faba and fabb ) activated by fadr , indicating that 
 long - chain acyl - coa esters are the effector molecules that regulate fatty acids 
 metabolism and thereby mediate inductions . therefore , e. coli cells can grow on minimal 
 medium containing long - chain fatty acids but it can not grow on short- and medium - chain 
 fatty acids due to no induction of the enzymes associated with fatty acids 
 metabolism . so far , genes involved in fatty acids metabolism ( i.e. , fad regulon ) of e. coli have been reported at the transcriptional level by 
 biochemical and genetic analyses [ 36 ] . therefore , in this study , we looked at the 
 effects of long - chain fatty acids at the translational level of e. coli . previously , one or more proteins were chosen as models for 
 understanding local physiological phenomena . nowadays , proteomic studies allow 
 researchers to identify large members of stimulons , a set of proteins whose 
 amount or synthesis rate changes in response to a certain stimulus , and to 
 obtain information that indicates which specific proteins should be studied 
 further . comparative proteome profiling under various environmental conditions also 
 reveal new regulatory circuits and the relative abundances of protein sets at 
 the system - wide level . such analyses of every protein induced or repressed by the 
 stimulus may provide the necessary information to understand a response in the 
 cell . furthermore , proteome profiles can prove 
 invaluable when used in conjunction with various molecular biological tools 
 including recombinant dna technology . for example , conditional 
 promoters activated by the specific stimulus , such as stationary phase , ph , 
 temperature , and nutrient limitation have been used for efficient production of 
 heterologous proteins in bacteria . in this study , proteomic studies 
 that compared global translational differences between e. coli w3110 cells in the presence of glucose and oleic acid ( c18 ) 
 were conducted . the present study has three goals : ( i ) to identify the stimulon 
 of the oleic acid ; ( ii ) to select target proteins from the stimulon to utilize 
 them as the oleic acid - inducible promoter ; and ( iii ) further to apply it for 
 the production of recombinant proteins or other biotechnological systems . the bacterial strains and plasmids used in this 
 study are shown in table 1 . e. coli w3110 was used as a 
 host strain for proteomic studies and the production of a recombinant protein . primers for the amplification 
 of the promoter regions of alda and udp genes were designed based on the genome sequence of e. coli k-12 w3110 ( ac_000091 ) . the promoter region of alda gene was 
 amplified by pcr using primers 1 and 2 , and was cloned into the ecorv 
 and ecori sites of ptac99a to make pad99a 
 ( table 1 ) . uppsala , sweden ) , which was constructed by replacing the trc promoter of ptrc99a with the tac promoter from pkk223 - 3 ( pharmacia biotech ) digested by pvuii and ecori . also , the promoter region of udp gene was amplified by pcr using primers 3 and 4 , and was cloned 
 into the ecorv and ecori sites of the high - copy - number plasmid 
 ptac99a to make pup99a ( table 1 ) . both promoters were constructed with the ribosome 
 binding sites consisting of the agga sequence having an optimal distance length 
 of 8 bases from a start codon . pcr was performed in the pcr thermal cycler mp ( takara shuzo 
 co. , ltd . , shiga , japan ) using the expand high fidelity pcr system ( roche 
 molecular biochemicals , mannheim , germany ) . dna sequencing was carried out 
 using the bigdye terminator cycle sequencing kit ( perkin - elmer co. , boston , mass , 
 usa ) , taq polymerase and the abi prism 377 dna sequencer ( perkin - elmer co. , 
 mass , usa ) . cells were cultivated at 
 37c and 250 rpm in 100 ml of luria - bertani ( lb ) medium ( 10 g / l of tryptone , 
 5 g / l of yeast extract , and 5 g / l of nacl ) , or r/2 medium plus 10 g / l glucose 
 or 5 g / l oleic acid ( daejung chemicals & metals 
 co. , gyeonggi - do , korea ) as a carbon source . the r/2 medium ( ph 6.8 ) contains per liter : 2 g of ( nh4)2hpo4 , 
 6.75 g of kh2po4 , 0.85 g of citric acid , 0.7 g 
 of mgso47h2o , and 5 ml of a trace metal solution . the 
 trace metal solution contains per liter of 5 m hcl : 10 g of feso47h2o , 
 2.25 g of znso47h2o , 1 g of cuso45h2o , 0.5 g of mnso45h2o , 0.23 g of na2b4o710h2o , 
 2 g of cacl22h2o , and 0.1 g of ( nh4)6mo7o24 . 
 for the cultivation of 
 recombinant e. coli strains , ampicillin ( ap , 50 g / ml ) cell growth was 
 monitored by measuring the absorbance at 600 nm ( od600 ; du series 
 600 spectrophotometer , beckman , fullerton , calif , usa ) . at an od600 of 0.7 or 1.2 , isopropyl--d - thiogalactopyranoside 
 ( iptg , sigma chemical co. , st . louis , mo , usa ) was added at a final 
 concentration of 1 mm . for induction by oleic acid , the 
 defined medium supplemented with 10 g / l glucose was changed into the medium plus 
 5 g / l oleic acid after cells were collected by centrifugation at the same od600 of 0.7 or 1.2 . then , cells were further cultivated for 5 , 10 , and 20 hours , 
 and harvested by centrifugation at 3,500  g for 5 minutes at 4c . protein samples were analyzed by 
 electrophoresis on 12% ( w / v ) sodium dodecyl sulfate - polyacrylamide gel 
 electrophoresis ( sds - page ) as described by laemmli . the gels were stained 
 with coomassie brilliant blue r250 ( bio - rad , hercules , calif , usa ) , and the 
 protein bands were quantified by a gs-710 calibrated imaging densitometer 
 ( bio - rad ) . proteome analysis was performed by 2de using 
 the ipgphor ief system ( ge healthcare , chalfont st . giles , 
 uk ) and protean ii xi cell 
 ( bio - rad ) as described previously . in brief , e. coli w3110 cells grown in the presence of glucose and oleic acid 
 were 
 harvested at the exponential and stationary phases , respectively , by 
 centrifugation for 5 minutes at 
 3,500  g and 4c , and washed four 
 times with low - salt washing buffer . the pellet was then resuspended in 600 l 
 of a buffer containing 10 mm tris - hcl ( ph 8.0 ) , 1.5 mm mgcl2 , 10 mm 
 kcl , 0.5 mm dtt , 0.1% ( w / v ) sds , and 1% ( v / v ) cocktail protease inhibitor ( complete mini edta - free ; roche diagnostics gmbh , 
 germany ) . one l of this sample was mixed with 60 l of a solution consisting 
 of 8 m urea , 4% ( w / v ) chaps , 40 mm tris , 65 mm dtt , and a trace of bromophenol 
 blue . proteins ( 200 g ) quantified by bradford 
 assay   were resuspended in 350 l of ief denaturation buffer 
 composed of 8 m urea , 2% ( w / v ) chaps , 20 mm dtt , and 0.8% ( v / v ) ipg buffer ( ph 
 310 nl ; ge healthcare ) . the samples were carefully loaded on the ipg 
 strips ( 18 cm , ph 310 nl ; ge healthcare ) . the loaded ipg strips were rehydrated for 12 hours and focused at 20c for 15 
 minutes at 250 v , followed by 8,000 v until a total of 60,000 vh was reached . the 
 strips were equilibrated in two equilibration buffers as described previously 
 and then placed on 12% 
 ( w / v ) sds - page gels prepared by the standard protocol . protein spots were visualized using a 
 silver staining kit ( ge healthcare ) , and the stained gels were scanned by a gs-710 
 calibrated imaging densitometer ( bio - rad ) . imagemaster 2d platinum software ( version 
 5.0 ; ge healthcare ) was used to identify spots , to match gels , 
 and to quantify spot densities on a volume basis ( i.e. , integration of spot 
 optical intensity over the spot area ) . culture broth ( 3 ml ) was centrifuged at 3,500  g for 5 minutes at 4c , and the pellet was washed with 1 ml of 10 mm na2hpo4 buffer ( ph 7.2 ) , followed by centrifugation at 3,500  g for 5 minutes at 4c . the cell pellet was resuspended in 0.5 ml of 10 mm na2hpo4 buffer ( ph 7.2 ) . crude extracts of e. coli cells were prepared by five cycles 
 of sonication ( each for 15 seconds at 20% of maximum output ; high - intensity 
 ultrasonic liquid processors ; sonics & material inc . , partially disrupted cells were first removed by centrifugation of sonicated 
 samples at 12,000  g for 2 minutes at room temperature . membrane 
 proteins and lipid layers were isolated by centrifugation at 12,000  g for 
 30 minutes at 4c , followed by resuspension in 0.5 ml of 0.5% ( w / v ) sarcosyl in 
 10 mm na2hpo4 buffer ( ph 7.2 ) . after incubation at 37c 
 for 30 minutes , the insoluble pellet containing membrane proteins was obtained 
 by centrifugation at 12,000  g for 30 minutes at 4c . membrane proteins 
 were obtained by washing the insoluble pellet with 10 mm na2hpo4 buffer ( ph 7.2 ) , followed by resuspending in 50 l of tris - edta buffer ( ph 
 8.0 ) . briefly , 
 protein spots were excised and destained by incubating in 30 mm potassium 
 ferricyanide and 65 mm sodium thiosulfate for 10 minutes . gel pieces were 
 washed in milli - q water until they became colorless and transparent , and then vacuum - dried . these 
 pieces were proteolysed with 0.02 g/l of modified trypsin ( promega , madison , wis , 
 usa ) in 40 mm ammonium bicarbonate for overnight at 37c . tryptic peptides ( 10 l 
 aliquots ) were analyzed by a nano - lc / ms system consisting of an ultimate 
 hplc system ( lc packings , amsterdam , netherlands ) and a quadrupole - time - of - flight ( q - tof ) ms ( micromass , manchester , uk ) equipped 
 with a nano - esi source as 
 described previously . the mascot search server ( version 1.8 ; 
 http://www.matrixscience.com ) was used 
 for the identification of protein spots by querying sequence of the tryptic 
 peptide fragments . reference databases used for the identification of target 
 proteins were uniprot knowledgebase ( swiss - prot and trembl ; 

 http://kr.expasy.org ) 

  and ncbi ( http://www.ncbi.nlm.nih.gov ) . for 
 fluorescence imaging , cells were harvested by centrifugation for 5 minutes at 
 3,500  g and 4c , 
 washed with and resuspended in phosphate - buffered saline ( pbs ) solution . the 
 samples were mounted on microscopic slide glasses and examined by confocal 
 microscopy ( carl zeiss , jena , 
  germany ) . samples were 
 excited by a 364-nm argon laser , and images were filtered by a longpass 505-nm 
 filter . three - dimensional images were constructed from 510 serial images ( each 
 1-am thick ) made by automatic optical sectioning . fluorescence 
 intensities were measured at 395 nm ( excitation ) and 509 nm ( emission ) using 
 the spectramax m2 multi - detection system ( molecular devices , sunnyvale , calif , 
 usa ) , and a 96-well black and clear flat - bottom plate ( coastar , los angeles , calif , 
 usa ) . to understand physiological changes triggered by the long - chain fatty 
 acid , we analyzed the proteome profiles of e. coli k-12 w3110 grown in the presence of glucose and oleic acid , 
 respectively . the final concentration of cells cultured in oleic 
 acid as a carbon source was 4-fold higher than that of cells grown in glucose , 
 although the former took a longer lag - period for induction of the fad regulon ( see figure 1 ) . samples of proteome were taken at the exponential 
 and stationary phases in two different media ( see figure 1 ) : when the od600 of e. coli reached 0.57 and 
 1.25 in the presence of glucose , named g1 and g2 , respectively ; and when the od600 reached 0.56 and 5 in the presence of oleic acid , named o1 and o2 , respectively . the proteome profiles of the four samples , g1 , g2 , o1 , and o2 , were analyzed by 
 2d page using a strip of 310 pi range and 12% polyacrylamide gel for subsequent comparisons ( see figure 2 ) . the 
 overall profiles of whole cellular proteins were reproducible . from over 2,000 
 spots on each 2d gel shown in figure 2 , we identified 92 proteins by comparing 
 with our in - house e. coli proteome database or by conducting lc - ms / ms 
 analysis . the outer membrane proteins were enriched by fractionation , and 
 separated on 12% sds - page ( see figure 3 ) . membrane proteins are typically difficult 
 to be resolved in the ief denaturation buffer used commonly for 2d gels because 
 of their hydrophobic property . the highly abundant porin , ompf whose expression 
 level was regulated by osmolarity , was observed at the exponential phase 
 of e. coli in the presence of glucose . as expected , the long - chain fatty acid transporter protein , fadl , was newly 
 synthesized in the presence of oleic acid . this result proves that e. coli requires the specific transport 
 system ( fadl ) on the growth of fatty 
 acids . to examine the 
 influence of oleic acid on the proteome profile variation , we compared the 
 proteomes obtained from the exponential phase ( o1 versus g1 ) and the stationary 
 phase ( o2 versus g2 ) , as shown in table 3 . at the exponential growth phase , the 
 levels of 41 identified 
 proteins were altered in the presence of oleic acid . among them , 9 proteins including alda , cdd , fada , fadb , fadl , male , rbsb , udp , and yccu 
 were newly synthesized in response to oleic acid , while gapa ( the fragment ) , hypothetical 
 protein yfdx , and two unidentified proteins were not detectable . as expected , 
 the levels of proteins involved in fatty acid degradation ( fada and fadb ) , long - chain 
 fatty acid transport system ( fadl ) , glyoxylate shunt ( acea ) . and tca cycle ( mdh , 
 sdha , succ , and sucd ) were significantly increased to replenish the 
 dicarboxylic acid intermediates consumed in amino acid biosynthesis . particularly , 
 isocitrate lyase ( acea ) in the acebak operon was significantly synthesized by more than five folds in the presence of 
 oleic acid , making it the most abundant protein . concurrently , there were decreased 
 levels of proteins involved in the biosynthesis of fatty acids ( fabd and fabe ) 
 and amino acids ( arog , leuc , and serc ) . these results showed that the variation 
 patterns of most proteins identified as a fad regulon were in agreement with their corresponding transcriptional levels previously 
 reported [ 26 ] . furthermore , the growth of e. coli on oleic acid involves a significant contribution of the 
 pyrimidine salvage pathway ( cdd and udp ) and specific binding - protein - dependent 
 transport system ( male and rbsb ) because the levels of these proteins highly increased by oleic acid . the salvage pathway of e. coli functions to reutilize free bases and nucleosides produced 
 intracellularly from nucleotide turnover . also , the pyrimidine salvage 
 pathway has been reported to recycle the pentose moieties of exogenous 
 nucleosides to use them as carbon and energy sources and the amino groups of 
 cytosine compounds as a nitrogen source . the d - ribose - binding periplasmic 
 protein , rbsb in the rbsacbk operon , 
 mediates the entry of d - ribose across the cell membrane in the form of d - ribose 
 5-phosphate , which is an intermediate of the pentose phosphate cycle . therefore , 
 long - chain fatty acids seem to influence the status of the pyrimidine salvage 
 pathway and its associated transport system . interestingly , aldehyde dehydrogenase ( alda ) , which oxidize diverse 
 aldehydes throughout the cellular metabolism , received a special attention in 
 this study because of its possible applications in gene expression system with 
 oleic acid as an inducer . it has been reported that the expression of alda gene was induced on growth on 
 fucose , rhamnose , arabinose , glutamate , or 2-oxoglutarate during aerobic 
 condition , while that is repressed by glucose . our observation found in 
 this study demonstrated that oleic acid is another inducer of the alda gene . its application as an 
 inducible promoter is demonstrated in the next two sections . at the stationary phase , the levels of 45 identified proteins 
 were altered in response to oleic acid ( table 3 ) . sixteen proteins including acea , 
 acnb , alda , atpa , cdd , fada , fadb , fadl , hns , icd , male , rbsb , succ , sucd , udp , 
 and yccu were significantly increased or newly synthesized , while 29 proteins 
 were reduced or disappeared on 2d gels . although the variations of proteins at 
 the stationary phase were more complex , most proteins with altered levels on 2d 
 gels at this phase showed patterns similar to those at the exponential growth 
 phase . among proteins highly inducible by oleic acid , we 
 selected two target proteins , alda and udp according to the following two 
 criteria for their utilization as promoters : ( i ) they are only induced in the 
 presence of oleic acid to be strictly controlled ; ( ii ) they are strongly and 
 highly expressed for the enhanced bioproducts production . alda and udp were synthesized 
 in response to oleic acid with relatively high abundance from the exponential 
 to stationary phases , and were not synthesized in the presence of glucose , 
 suggesting that the native promoters of these proteins could be used as an oleic 
 acid - inducible promoter in e. coli w3110 . for the construction of the expression 
 systems controlled by alda or udp promoter , the representative 
 schematic plasmid map under the control of alda promoter is illustrated in figure 4 . this is a high - copy - number plasmid with replication 
 origin of pbr322 ( atcc 37017 ) . various cultivation strategies employing different 
 host strains and expression systems have been employed for the production of 
 recombinant proteins [ 10 , 21 ] . one of the most popular approaches is the use of 
 different promoters to regulate expression levels . in e. coli , many inducible promoters have been developed , which can be 
 induced by various mechanisms such as temperature upshifting , ph fluctuation , 
 nutrient starvation , and addition of chemical inducers . among these inducible 
 systems , t7 or lac - based promoters ( tac , trc , lac , lacuv5-t7 hybrid , etc . ) , which can be effectively induced by the 
 addition of iptg , are the most frequently used ones . in order to evaluate the effectiveness of oleic acid - inducible promoters discovered 
 in this study , we chose the iptg - inducible tac promoter as a control . green fluorescent protein ( gfp ) from the jellyfish aequorea 
 victoria was employed as a model recombinant protein to examine its expression 
 under the control of alda or udp promoter . for the induction of gfp 
 by oleic acid , the defined medium supplemented with glucose was transferred 
 into oleic acid medium at the od600 of 0.7 or 1.2 . for the control , cells 
 harboring the plasmid containing tac promoter were added with 1 mm iptg at the same values of od600 in 
 the defined medium supplemented with glucose . after induction by iptg or oleic 
 acid , cells were further cultured , harvested at each time , and analyzed by 12% 
 sds - page ( see figure 5 ) . when gfp is induced at the od600 of 1.2 in 
 recombinant e. coli w3110 harboring ptac99gfp , pad99gfp , and pup99gfp , its contents were approximately 27% , 42% , and 25% of the total proteins at 10 hours , 
 and 28% , 50% , and 25% at 20 hours , respectively . the gfp content induced by alda promoter was about 2-fold higher 
 than that obtained by iptg - inducible tac or udp promoter . under the 
 iptg - inducible promoter , the expression of gfp was low even in lb medium 
 compared to the oleic acid - inducible alda promoter ( data not shown ) . additionally , the final cell concentration of 
 recombinant e. coli w3110 cells in 
 the presence of oleic acid was 4-fold higher than that of recombinant e. coli cultured in glucose as a carbon 
 source . this result was further confirmed by fluorescence intensity 
 measurements and confocal microscopy ( see figure 6 ) . strong fluorescence was uniformly 
 detected in recombinant e. coli cells 
 under the control of alda promoter . the fluorescence intensity of gfp obtained from w3110 harboring pad99gfp was 
 more than 30-fold higher than that obtained from w3110 harboring ptac99gfp , indicating 
 that the alda promoter efficiently enhances 
 recombinant protein production compared to the tac promoter . since the alda promoter was not activated by glucose , gfp was not produced in the presence of 
 glucose in accordance with the proteome profiles . gfp was only produced under 
 the alda promoter along with the 
 supply of exogenous oleic acid . however , the tac promoter was not tightly controlled , leaking the recombinant protein 
 even without iptg induction . these results manifest that the alda promoter is very efficient for the production 
 of recombinant proteins in e. coli as 
 an inducible promoter . the maximum productivity can be achieved when the growth 
 and production phases are separated as conducted in this study . separation of 
 the two phases is often achieved by delaying induction time until the cell 
 density reaches a suitable value . until 
 now , the iptg - inducible promoters tac or trc have been widely used for basic 
 research . however , the use of iptg for the large - scale production of human 
 therapeutic proteins is undesirable because of its toxicity and relatively high 
 cost ; the high concentration of iptg can inhibit cell growth and recombinant 
 protein production [ 22 , 23 ] . therefore , determination of optimal induction time 
 point as well as the inducer concentration is crucial to increase the overall 
 productivity of recombinant protein . in this regard , there have been 
 significant efforts to overcome such problems by using different inducible or even 
 constitutive expression systems . thus , the alda promoter found in this study can be effectively used for the 
 enhanced production of recombinant proteins with the aforementioned problems 
 largely resolved . in summary , first , it is tightly controllable 
 with an appropriate inducer , oleic acid in this case . tight regulation of the 
 promoter is essential for the synthesis of proteins which may be detrimental to 
 the host cell . for example , the toxic rotavirus vp7 protein effectively kills 
 cells , and must be produced under tightly regulated conditions . second , its 
 expression is strong and long lasting , resulting in the accumulation of the 
 target protein constituting up to 50% of the total cellular proteins . the third 
 important characteristic of alda promoter is its inducibility in a cost - effective manner by using exogenous 
 oleic acid as an inducer . proteome analysis of the cells with focus on proteins 
 induced or repressed by the stimulus provides clues to the understanding of 
 cellular responses . this study revealed that 52 proteins showed significantly 
 altered levels in e. coli grown with oleic 
 acid compared to the glucose . based on the resulting proteome profiles , the 
 promoter of alda gene was found to be 
 strongly activated by oleic acid and subsequently to be demonstrated useful as 
 an inducible promoter for the enhanced production of desirable targets . thus , 
 this study demonstrates that e. coli proteome profiles not only provide invaluable information for physiological 
 status of the organism under specific conditions but also propose its biotechnological 
 applications .
in escherichia coli , a 
 long - chain acyl - coa is a regulatory 
 signal that modulates gene expression 
 through its binding to a transcription 
 factor fadr . in this study , 
 comparative proteomic analysis of 
 e. coli in the presence 
 of glucose and oleic acid 
 was 
 performed to understand cell 
 physiology in response to oleic acid . 
 among total of 52 proteins showing 
 altered expression levels with oleic 
 acid presence , 9 proteins including 
 alda , cdd , fada , fadb , fadl , male , 
 rbsb , udp , and yccu were newly 
 synthesized . among the genes 
 that were 
 induced by oleic acid , the promoter of 
 the alda gene was used 
 for the production of a green 
 fluorescent protein ( gfp ) . 
 analysis of 
 fluorescence intensities and confocal 
 microscopic images revealed that 
 soluble gfp was highly expressed under 
 the control of the alda 
 promoter . 
 these results suggest that 
 proteomics is playing an important 
 role not only in biological research 
 but also in various biotechnological 
 applications .