Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia -- reperfusion-induced endothelial cell apoptosis Activation of cytoskeleton regulator Rho-kinase during ischemia -- reperfusion -LRB- I/R -RRB- plays a major role in I/R injury and apoptosis . Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase -LRB- PI3-kinase -RRB- / Akt pathway , we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement . Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632 , PI3-kinase inhibitor wortmannin , F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide . Intracellular ATP levels decreased following I/R . Y-27632 treatment reduced I/R-induced apoptosis by 31 % -LRB- P < 0.01 -RRB- and maintained Akt activity . Both effects were blocked by co-treatment with wortmannin . Y-27632 treatment prevented the formation of F-actin bundles during I/R . Similar results were observed with cytochalasinD treatment . In contrast , latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis . Apoptosis and Akt activity were inversely correlated -LRB- R2 = 0.68 , P < 0.05 -RRB- . In conclusion , prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity . Introduction Experimental ischemia and reperfusion -LRB- I/R -RRB- induces endothelial cell apoptosis in several organs -LSB- 1 , 2 -RSB- . We and others have previously shown that activation of RhoA and downstream effector Rho-kinase during experimental I/R plays a major role in I/R injury and associated apoptosis -LSB- 3 -- 7 -RSB- . Indeed , Rho-kinase inhibition has beneficial effects on I/R injury in several organs -LSB- 4 , 6 -- 12 -RSB- . Endothelial cells may be essential for mediating the beneficial effects of Rho-kinase inhibition on I/R injury , because , for instance inhibition of endothelial nitric oxide synthase blocked the beneficial effects of Rho-kinase inhibition on rat myocardial infarct size -LSB- 12 -RSB- . Rho-kinase activation during the execution of apoptosis results in increased myosin activity , bundling of F-actin by activated myosins , actin -- myosin contractile force generation , cell contraction and ultimately membrane blebbing -LSB- 13 -- 15 -RSB- . Recent in vivo and in vitro studies suggest that Rho-kinase and the effects of Rho-kinase on the actin -- myosin cytoskeleton may also be involved in the initiation of apoptosis -LSB- 4 , 12 , 16 -- 20 -RSB- . Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase -LRB- PI3-kinase -RRB- / Akt pathway and it upregulates mitochondrial death pathway protein Bax -LSB- 4 , 12 , 16 , 19 -RSB- . The effects of Rho-kinase on the cytoskeleton may therefore be important in linking Rho-kinase activation and PI3-kinase/Akt inactivation . We hypothesized that -LRB- 1 -RRB- inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and -LRB- 2 -RRB- prevention of F-actin rearrangement facilitates the protective effects of Rho-kinase inhibition -LRB- Fig. 1 -RRB- . To test this hypothesis , we assessed whether specific inhibition of Rho-kinase with Y-27632 -LSB- 21 -RSB- attenuates apoptosis via phosphorylation of Akt in an in vitro model of simulated I/R . The involvement of the F-actin cytoskeleton was explored by the use of F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide -LSB- 22 -RSB- . Fig. 1Proposed mechanism for modulation of I/R-induced apoptosis involving RhoA/Rho-kinase and PI3-kinase/Akt . Simulated I/R activates RhoA/Rho-kinase -LSB- 3 -- 7 -RSB- , which leads to F-actin rearrangement . F-actin rearrangement facilitates reduction of PI3-kinase/Akt activity and induction of apoptosis . This can be prevented by inhibition of Rho-kinase with Y-27632 . The protective effects of Y-27632 can be blocked with PI3-kinase inhibitor wortmannin . Several other pathways for I/R-induced apoptosis are represented by the dashed arrow -LSB- 2 , 19 -RSB- Materials and methods Cell culture Human Umbilical Vein Endothelial Cells -LRB- HUVECs -RRB- were isolated , cultured and characterized as previously described -LSB- 23 -RSB- . Briefly , HUVECs were cultured on gelatin-coated wells/glass cover slips until confluent at 37 °C under 5 % CO2/95 % air atmosphere . Culture medium was renewed every other day -LRB- medium 199 supplemented with 10 % heat-inactivated human serum , 10 % heat-inactivated new born calf serum , 150 μg/ml crude endothelial cell growth factor , 2 mM l-glutamine , 5 U/ml heparin , 100 IU/ml penicillin , and 100 μg/ml streptomycin -RRB- . Simulation of I/R We used an established in vitro endothelial cell model for simulation of ischemia and reperfusion -LSB- 24 , 25 -RSB- . Figure 2 shows a schematic representation of the experimental protocol . Cells were washed twice with washing buffer -LRB- 1.2 mM MgSO4 · 7H2O , 116 mM NaCl , 5.3 mM KCl , 1.13 mM NaH2PO4 · H2O , 1.8 mM CaCl2 · 2H2O , 20 mM HEPES -RRB- and treated for 1 h with washing buffer with or without 10 μM Y-27632 -LRB- Tocris Cookson Ltd. , Bristol , United Kingdom -LRB- UK -RRB- -RRB- , 250 nM cytochalasinD -LRB- Sigma-Aldrich , Saint Louis , Missouri , United States of America -LRB- USA -RRB- -RRB- , 150 nM latrunculinA -LRB- Calbiochem , Darmstadt , Germany -RRB- , 20 nM jasplakinolide -LRB- Calbiochem -RRB- and 50 nM wortmannin -LRB- Sigma-Aldrich -RRB- . This was followed by 1 h of simulated ischemia by covering cells with 1 ml mineral oil -LRB- nitrogen bubbled -RRB- and subsequently simulated reperfusion by replacement of culture medium for different time points : no reperfusion for adenosine tri-phosphate -LRB- ATP -RRB- measurement and F-actin staining , 1 h for ATP measurement , F-actin staining and western blotting to analyze phospho Akt -LRB- pAkt -RRB- levels and 24 h for quantification of apoptosis . Drugs were also present during simulated reperfusion . As controls , we used cells treated for 1 h with washing buffer followed by treatment with medium -LRB- control -RRB- or medium supplemented with 10 % mineral oil -LRB- control oil -RRB- to assess any injurious effects of products within the oil . Fig. 2Schematic representation of the experimental protocol . Cells were pre-treated for 1 h with Y-27632 , cytochalasinD , latrunculinA , jasplakinolide or wortmannin . One hour of simulated ischemia was followed by 1 -- 24 h of simulated reperfusion . No reperfusion for ATP measurement and F-actin staining , 1 h for ATP measurement , F-actin staining and analysis of Akt activity by western blotting and 24 h for quantification of apoptosis . The drugs were also present during the reperfusion phase ATP measurement We measured ATP after ischemia and I/R -LRB- 1 h of reperfusion -RRB- in order to confirm ischemic substrate deprivation . Cells were grown in a gelatin-coated 12-well chamber and treated according to the simulated I/R protocol . ATP was measured using the ENLITEN ATP Assay System Bioluminiscence Detection Kit for ATP -LRB- Promega Corporation , Madison , Wisconsin , USA -RRB- . Proteins in lysate were measured with BCA Protein Assay Kit -LRB- Pierce , Rockford , Illinois , USA -RRB- in order to calculate nmol ATP/μg protein . Quantification of apoptosis The cells were grown in a 24-well chamber on gelatin-coated glass cover slips and treated according to the simulated I/R protocol -LRB- 24 h of reperfusion -RRB- . Cells were fixed in 4 % formaldehyde and permeabilized with 0.2 % triton X-100 -LRB- Sigma-Aldrich -RRB- . The nucleus was stained with 4 ′ ,6-diamidino-2-phenylindole -LRB- DAPI -RRB- in Vectashield ® Mounting medium -LRB- Vector Laboratories , Inc. , Burlingame , California , USA -RRB- . Apoptosis on each glass cover slip was quantified using a DeadEndTM Fluorometric TdT-mediated dUTP Nick End Labeling -LRB- TUNEL -RRB- System -LRB- Promega Corporation -RRB- with fluorescence microscopy using a MarianasTM digital imaging microscope and Slidebook 4.2 software -LRB- Intelligent Imaging Innovations , Inc. , Denver , Colorado , USA -RRB- . The number of apoptotic nuclei -LRB- TUNEL-positive -RRB- and the total number of nuclei -LRB- DAPI-positive -RRB- were counted in three non-overlapping microscope fields/glass cover slip using a 10 × air lens -LRB- Carl Zeiss B.V. , Sliedrecht , The Netherlands -RRB- and averaged . The number of apoptotic nuclei was expressed as percentage of the total number of nuclei . F-actin cytoskeleton staining The cells were grown in a 24-well chamber on gelatin-coated glass cover slips and treated according to the simulated I/R protocol -LRB- 1 h of reperfusion -RRB- . Cells were fixed in 4 % formaldehyde and permeabilized with 0.2 % triton X-100 . F-actin was stained with rhodamin-phalloidin -LRB- 1:100 -RRB- -LRB- Molecular Probes , Inc. , Eugene , Oregon , USA -RRB- and the nucleus with DAPI in Vectashield ® Mounting medium . Cells were visualized with fluorescence microscopy and Slidebook software . Analysis of Akt phosphorylation Cells were grown in a gelatin coated 6-well chamber , treated according to the simulated I/R protocol -LRB- 1 h of reperfusion -RRB- and lysated with lysis buffer -LRB- 20 mM Tris/HCl pH 8.0 , 150 mM NaCl , 90 mM KCl , 2 mM EDTA/NaOH pH 8.0 , igepal -LRB- 1:200 -RRB- , triton X-100 -LRB- 1:200 -RRB- , 1 mM Na3VO4 , 10 mM NaF , protease inhibitors -LRB- 1:100 -RRB- , phosphatase inhibitors -LRB- 1:100 -RRB- -RRB- . Protein concentration was measured -LRB- BCA method -RRB- and equal amounts of protein from each sample were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane -LRB- Bio-Rad Laboratories , Hercules , California , USA -RRB- . Membranes were incubated with polyclonal antibodies against pAkt -LRB- serine-473 , rabbit , 1:1,000 -RRB- and total Akt -LRB- tAkt , rabbit , 1:1,000 -RRB- -LRB- Cell Signaling Technology , Inc. , Danvers , Massachusetts , USA -RRB- . tAkt was used as loading control . Goat anti-rabbit immunoglobulins horseradish peroxidase -LRB- HRP -RRB- from DakoCytomation -LRB- Glostrup , Denmark -RRB- was used for the detection of the primary antibodies at 1:1,000 . Detection of the HRP reaction was performed with ECL plus Western Blotting Detection System -LRB- Amersham Biosciences , Little Chalfont Buckinghamshire , UK -RRB- . Imaging and analysis were performed with LAS-3000 -LRB- Fuji Photo Film Co. , Ltd. , Tokyo , Japan -RRB- and AIDA Image Analyzer -LRB- Raytest GmbH , Straubenhardt , Germany -RRB- . Data were expressed as pAkt/tAkt as percentage of control . Statistical analysis A one-way analysis of variance -LRB- ANOVA -RRB- was conducted to explore the effects of I/R , Y-27632 , cytochalasinD or wortmannin treatment , and their interaction on apoptosis . The t-test was used to further analyse the differences between the groups when ANOVA indicated statistical significance . We adjusted for multiple testing with help of the Bonferroni Holm method . The paired t-test was used for paired ATP data and logarithmic transformed absolute pAkt/tAkt data . Absolute pAkt/tAkt data were logarithmic transformed to obtain a Gaussian distribution . We adjusted for multiple testing with help of the Bonferroni Holm method . The Pearson coefficient of determination -LRB- R2 -RRB- was used to express the relation between the percentage of apoptotic nuclei and the logarithmic transformed absolute pAkt/tAkt data . Differences were considered significant at the P < 0.05 level . Data are presented as means ± standard error of the mean -LRB- SEM -RRB- . Results ATP levels and apoptosis following simulated ischemia and I/R In order to confirm ischemic substrate deprivation in simulated I/R , we measured intracellular ATP levels . Ischemia was simulated by covering the monolayer of cells with nitrogen bubbled mineral oil . Reperfusion was simulated by replacement of culture medium -LRB- Fig. 2 -RRB- . Intracellular ATP levels decreased following simulated ischemia compared to the levels in cells treated with medium supplemented with 10 % mineral oil -LRB- control oil -RRB- -LRB- 0.15 ± 0.04 nmol ATP/μg protein versus 0.22 ± 0.04 nmol ATP/μg protein , P < 0.05 , n = 7 -RRB- . Intracellular ATP decreased even further during the reperfusion phase -LRB- 0.10 ± 0.04 nmol ATP/μg protein , P < 0.05 versus control oil , n = 7 -RRB- . ATP levels of control oil cells were comparable to those of control cells -LRB- 0.20 ± 0.04 nmol ATP/μg protein , n = 7 -RRB- . Simulated I/R induced a significant percentage of apoptotic nuclei -LRB- ANOVA for the effect of I/R on apoptosis , P < 0.0001 -RRB- , while hardly any apoptosis was observed in control and control oil cells -LRB- Fig. 3 -RRB- . Fig. 3Rho-kinase inhibition reduces the degree of apoptosis during I/R via PI3-kinase . Non-treated control -LRB- C , n = 11 -RRB- , control oil -LRB- C O , n = 2 -RRB- , Y-27632-treated control -LRB- C Y , n = 11 -RRB- and wortmannin-treated control -LRB- C W , n = 5 -RRB- showed hardly any apoptosis . Simulated I/R induced a significant percentage of apoptotic nuclei -LRB- minimum P < 0.005 versus the controls , n = 12 -RRB- . Y-27632 treatment reduced the percentage of apoptotic nuclei during simulated I/R -LRB- I/R Y , * P < 0.01 , n = 12 -RRB- . This protective effect was completely blocked by wortmannin -LRB- I/R YW , # P < 0.05 , n = 5 -RRB- . The number of apoptotic nuclei is expressed as percentage of total number of nuclei . Data are presented as means ± SEM The effect of Rho-kinase inhibition on apoptosis To investigate the role of Rho-kinase during I/R-induced apoptosis , HUVECs were treated with inhibitor Y-27632 during simulated I/R -LRB- Fig. 3 -RRB- . Y-27632 had no effect on basal apoptosis , but reduced the degree of apoptosis during I/R -LRB- ANOVA for the effects of Y-27632 and the interaction between I/R and Y-27632 on apoptosis , both P < 0.01 -RRB- . To determine whether the protective effects of Rho-kinase inhibition were mediated by PI3-kinase , cells were co-treated with PI3-kinase inhibitor wortmannin . Wortmannin had no effect on basal apoptosis , but blocked the protective effect of Rho-kinase inhibition during I/R -LRB- ANOVA for the effect of wortmannin on apoptosis , P < 0.05 -RRB- . Akt activity in Y-27632-mediated protection against apoptosis To investigate the effect of Y-27632 treatment during I/R on Akt activity , Akt phosphorylation was analyzed by western blotting -LRB- Fig. 4 -RRB- . Simulated I/R reduced Akt activity compared to non-treated or Y-27632-treated control cells . Y-27632 treatment maintained Akt activity in I/R cells . To determine whether this effect was mediated by PI3-kinase , cells were co-treated with wortmannin . Wortmannin blocked the Y-27632-induced maintenance of Akt activity . Akt activity in Y-27632 - -LRB- Fig. 4 -RRB- or wortmannin-treated control cells -LRB- preliminary data -RRB- was comparable to the activity in non-treated control cells . Fig. 4Rho-kinase inhibition maintains Akt activity during I/R . A representative western blot of pAkt and tAkt is shown in the upper panel and a graph of pAkt/tAkt as percentage of control in the lower panel . Simulated I/R -LRB- I/R , n = 15 -RRB- induced a decrease in pAkt compared to the control and the Y-27632-treated control -LRB- C Y , * P < 0.05 , n = 15 -RRB- . Y-27632 treatment during simulated I/R maintained pAkt levels -LRB- I/R Y , ** P < 0.05 , n = 14 -RRB- . Wortmannin blocked the Y-27632-induced maintenance of Akt activity -LRB- I/R YW , # P < 0.05 , n = 12 -RRB- . Data are presented as means ± SEM . Absolute pAkt/tAkt data in arbitrary units -LRB- a.u. -RRB- : control : 0.75 a.u. ; C Y : 0.64 a.u. ; I/R : 0.32 a.u. ; I/R Y : 0.36 a.u. ; I/R YW : 0.04 a.u The F-actin cytoskeleton in Y-27632-mediated protection against apoptosis To investigate whether Y-27632-mediated protection against apoptosis paralleled the absence of Rho-kinase-mediated F-actin rearrangement , the F-actin cytoskeleton was visualized with rhodamin-phalloidin in non-treated or Y-27632-treated control , simulated ischemia and simulated I/R cells -LRB- n = 9 , representative pictures are shown in Fig. 5 -RRB- . Y-27632-treated control cells showed a slight decrease in peripheral F-actin bundles compared non-treated control cells , indicating that in vitro inhibition of Rho-kinase was able to reduce F-actin bundle formation . After simulated ischemia in non-treated cells , some F-actin bundles appeared in the cytoplasm . Y-27632-treated ischemic cells showed only F-actin clumps . After simulated reperfusion , even more F-actin bundles appeared in the periphery and the cytoplasm of the non-treated cells . In contrast , Y-27632-treated I/R cells , showed no F-actin bundles . Fig. 5Y-27632 and cytochalasinD prevent F-actin bundle formation during I/R . Shown is fluorescent imaging of rhodamin-phalloidin stained F-actin structures and DAPI stained nuclei in non-treated , Y-27632 - , cytochalasinD - , latrunculinA - and jasplakinolide-treated control , simulated ischemia and simulated I/R cells . The arrow and * indicate F-actin bundles and F-actin clumps , respectively . Control cells : In non-treated cells , most F-actin bundles were seen in the periphery of the cell . Y-27632-treated cells showed a slight decrease in peripheral F-actin bundles . CytochalasinD-treated cells showed only F-actin clumps . Cells treated with latrunculinA or jasplakinolide showed a slight decrease and increase in F-actin bundles , respectively . Ischemic cells : Non-treated cells showed F-actin bundles throughout the whole cell . Cells treated with Y-27632 , cytochalasinD or latrunculinA showed only F-actin clumps . Jasplakinolide-treated cells showed F-actin bundles . I/R cells : Non-treated cells showed F-actin bundles throughout the whole cell . In Y-27632-treated cells , no F-actin bundles were seen . CytochalasinD-treated cells showed also no F-actin bundles , only F-actin clumps were visible . LatrunculinA-treated cells showed some F-actin bundles . Jasplakinolide-treated cells showed as many F-actin bundles as non-treated I/R cells To further investigate the role of the F-actin cytoskeleton in I/R-induced apoptosis , we used F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide -LRB- Fig. 5 -RRB- . CytochalasinD-treated control cells showed no F-actin bundles . LatrunculinA-treated control cells showed a slight decrease and jasplakinolide-treated control cells showed a slight increase in F-actin bundles compared to non-treated control cells . CytochalasinD - and latrunculinA-treated ischemic cells showed no F-actin bundles , only F-actin clumps were visible . Jasplakinolide-treated ischemic cells showed F-actin bundles throughout the whole cell . In cytochalasinD-treated I/R cells , no F-actin bundles were visible , only F-actin clumps could be seen . In contrast , latrunculinA-treated I/R cells still showed some F-actin bundles and jasplakinolide-treated I/R cells showed as many F-actin bundles as non-treated I/R cells . Since cytochalasinD treatment prevented F-actin rearrangement similarly to Y-27632 treatment , we investigated whether cytochalasinD treatment could also prevent apoptosis during simulated I/R -LRB- Fig. 6 -RRB- . CytochalasinD had no effect on basal apoptosis , but reduced the degree of apoptosis during I/R -LRB- ANOVA for the effects of cytochalasinD and the interaction between I/R and cytochalasinD on apoptosis , both P < 0.05 -RRB- . Wortmannin blocked the protective effect of cytochalasinD in I/R -LRB- ANOVA for the effect of wortmannin on apoptosis , P < 0.0001 -RRB- . In contrast to the protective effect of Y-27632 and cytochalasinD on apoptosis , latrunculinA and jasplakinolide did not affect I/R-induced apoptosis , also not in co-treatment with wortmannin -LRB- data not shown -RRB- . LatrunculinA and jasplakinolide had no effect on basal apoptosis -LRB- data not shown -RRB- . Fig. 6CytochalasinD reduces the degree of apoptosis during I/R via PI3-kinase . Non-treated control -LRB- C , n = 6 -RRB- and cytochalasinD-treated control -LRB- C CytoD , n = 6 -RRB- showed hardly any apoptosis . Simulated I/R induced a significant percentage of apoptotic nuclei -LRB- P < 0.0001 versus the controls , n = 6 -RRB- . Treatment with cytochalasinD reduced the percentage of apoptotic nuclei in simulated I/R -LRB- I/R CytoD , * P < 0.05 , n = 6 -RRB- . This protective effect was completely blocked by wortmannin -LRB- I/R CytoDW , # P < 0.005 , n = 6 -RRB- . The number of apoptotic nuclei is expressed as percentage of total number of nuclei . Data are presented as means ± SEM Furthermore , cytochalasinD maintained Akt activity during simulated I/R -LRB- Fig. 7 -RRB- . Co-treatment with wortmannin completely blocked the cytochalasinD-induced maintenance of Akt activity . Akt activity in cytochalasinD-treated control cells was comparable to the activity in non-treated control cells . Fig. 7CytochalasinD treatment maintains Akt activity during I/R . Simulated I/R -LRB- I/R , n = 11 -RRB- induced a decrease in pAkt compared to the control and the cytochalasinD-treated control -LRB- C CytoD , * P < 0.01 , n = 11 -RRB- . CytochalasinD treatment during simulated I/R maintained pAkt levels -LRB- I/R CytoD , ** P < 0.05 , n = 11 -RRB- . Wortmannin blocked the cytochalasinD-induced maintenance of Akt activity -LRB- I/R CytoDW , # P < 0.01 , n = 7 -RRB- . Data are presented as means ± SEM . Absolute pAkt/tAkt data in arbitrary units -LRB- a.u. -RRB- : control : 0.61 a.u. ; C CytoD : 0.84 a.u. ; I/R : 0.36 a.u. ; I/R CytoD : 0.54 a.u. ; I/R CytoDW : 0.05 a.u Interestingly , the percentage of apoptotic nuclei inversely correlated to Akt activity -LRB- Fig. 8 -RRB- , indicating that a decrease in Akt activity correlated to an increase in apoptosis . Fig. 8The percentage of apoptotic nuclei inversely correlates to Akt activity -LRB- R2 = 0.68 , P < 0.05 -RRB- . Mean percentage of apoptotic nuclei and mean logarithmic transformed absolute pAkt/tAkt levels in non-treated -LRB- C -RRB- , Y-27632 -LRB- C Y -RRB- - and cytochalasinD -LRB- C CytoD -RRB- - treated control cells and in non-treated -LRB- I/R -RRB- , Y-27632 -LRB- I/R Y -RRB- - , cytochalasinD -LRB- I/R CytoD -RRB- - and wortmannin -LRB- I/R YW and I/R CytoDW -RRB- - treated I/R cells are shown as * Discussion This study shows that -LRB- 1 -RRB- inhibition of Rho-kinase attenuates I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and -LRB- 2 -RRB- the effects of Rho-kinase inhibition on PI3-kinase/Akt and apoptosis are facilitated by prevention of F-actin rearrangement . Our data point to a novel mechanistic link between activation of Rho-kinase during I/R and negative regulation of PI3-kinase/Akt , namely the effect of Rho-kinase on F-actin rearrangement -LRB- Fig. 1 -RRB- . Prevention of F-actin rearrangement by Rho-kinase inhibition or by treatment with actin depolymerizer cytochalasinD , independent of Rho-kinase , both attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity . Indeed , it has been reported previously that cytochalasins inhibited in vivo and in vitro apoptosis induced by a variety of stimuli , including I/R -LSB- 5 , 13 , 26 -RSB- . This study is therefore an extension of and a link between reports on Rho-kinase as negative regulator of the PI3-kinase/Akt pathway and reports on the involvement of Rho-kinase-dependent cytoskeletal rearrangement in the initiation of apoptosis -LSB- 4 , 12 , 16 -- 20 -RSB- . Actin depolymerizer latrunculinA did not affect I/R-induced endothelial cell apoptosis . This may be explained by the nature and degree of F-actin rearrangement during I/R , since F-actin bundles were completely absent in Y-27632 - and cytochalasinD-treated I/R cells , while latrunculinA-treated cells still showed some bundles . Indeed , cytochalasinD and latrunculinA depolymerize F-actin by different mechanisms and induce different patterns in vitro -LSB- 22 , 27 -RSB- . CytochalasinD caps the barbed end of actin filaments , severs actin filaments , sequesters actin monomers or dimers , promotes nucleation and stimulates the ATPase activity of G-actin . In vitro in hamster fibroblasts , it caused a complete collapse of the stress fibers at concentrations comparable to ours -LSB- 22 , 27 -RSB- . LatrunculinA on the other hand binds to monomeric G-actin to form a non-polymerizable complex and caused concentration-dependent shortening and thickening of stress fibers in the fibroblasts at concentrations comparable to ours -LSB- 22 , 27 -RSB- . Jasplakinolide , which promotes actin polymerization and stabilizes actin filaments by binding to F-actin -LSB- 22 -RSB- , induced as many F-actin bundles in I/R as non-treated I/R cells and consequently did not affect I/R-induced endothelial apoptosis . However , in vivo it has been demonstrated that jasplakinolide prevented I/R-induced apoptosis in the rat kidney , while latrunculinB increased I/R-induced apoptosis in rat kidney -LSB- 28 , 29 -RSB- . Furthermore , we observed hardly any apoptosis in our Y-27632 - , cytochalasinD - , latrunculinA - and jasplakinolide-treated control cells , while some reports describe the induction of apoptosis in control cells by latrunculinA or jasplakinolide at much higher concentrations than ours -LSB- 30 , 31 -RSB- , by cytochalasinD in airway epithelial cells , but not in canine kidney epithelial cells -LSB- 32 -RSB- or by Y-27632 in airway epithelial cells -LSB- 32 -RSB- . Therefore , these discrepancies may be explained by the use of different concentrations , cell types or the use of an in vivo/in vitro model . Akt activation after 1 h of reperfusion inversely correlated to the percentage of apoptosis after 24 h of reperfusion . Therefore , early reduced Akt activity may have a predictive value for the development of late I/R-induced apoptosis . The preventive effect of Y-27632 and cytochalasinD treatment probably takes place early in the process of apoptosis , because the Y-27632-induced increase in Akt activity is rapid and transient with a maximum after 30 min -LSB- 12 , 16 -RSB- . Indeed , we could neither detect increased Akt activity in our Y-27632-treated control cells after 3 h nor in our Y-27632-treated I/R cells after 24 h of reperfusion -LRB- pilot study , data not shown -RRB- , possibly because this was to late to detect the peak in Akt activity . I/R-induced apoptosis was maximal as late as after 24 -- 48 h of reperfusion -LSB- 25 -RSB- . Therefore , apoptosis was quantified after 24 h of reperfusion . Indeed , we did not see any TUNEL-positive nuclei after 1 h of reperfusion -LRB- pilot study , data not shown -RRB- . Our percentage of apoptotic nuclei was comparable to the percentage reported by the developers of the simulated I/R model -LSB- 25 -RSB- . However , less endothelial cell apoptosis was observed in a model of hypoxia/reoxygenation -LSB- 33 -RSB- , possibly because this induces less severe metabolic deprivation . In addition , PI3-kinase inhibition did not decrease basal Akt activity nor increased basal apoptosis , while it blocked the effects of Rho-kinase inhibition on I/R-induced apoptosis and Akt activity . This suggests that the basal situation is independent of PI3-kinase , while the effects Rho-kinase inhibition during I/R are dependent on PI3-kinase . Although it is unclear by which mechanism F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis , several mechanisms can be proposed . First , RhoA and Rho-kinase may regulate pAkt levels by regulating phosphatase and tensin homologue -LRB- PTEN -RRB- activity . PTEN is in inactive state in the cytosol and translocates to the membrane upon RhoA or Rho-kinase-induced activation , where it can inhibit the phosphorylation of Akt via PI3-kinase -LSB- 34 -RSB- . Disruption of the F-actin bundles by Rho-kinase inhibition or cytochalasinD treatment or the direct effects of Rho-kinase inhibition on PTEN may prevent the translocation and activation of PTEN , thereby preventing its inhibitory effect on Akt phosphorylation . Furthermore , there may be some PI3-kinase independent mechanisms involved in the Y-27632-induced protection against apoptosis . It is reported that F-actin polymerization and actin -- myosin contractility are necessary for membrane blebbing and chromatin condensation during execution of apoptosis induced by tumor necrosis factor-α or overexpression of Rho-kinase -LSB- 13 , 26 -RSB- . Moreover , the actin -- myosin cytoskeleton may be involved in facilitating the cellular localization of specific components of the apoptotic machinery and be required for caspase-3 activation -LSB- 17 , 18 -RSB- . Our study carries some limitations . First , we can not conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis . Second , Rho-kinase inhibition and F-actin depolymerization could establish only a partial protection against apoptosis , in agreement with the literature -LSB- 4 , 5 , 8 , 12 -RSB- , possibly because other mechanisms are also involved and the I/R-induced injury is too extensive . Third , we may have underestimated the number of apoptotic cells , because endothelial cells that detached from the basement membrane during simulated I/R and did not reattach later were not counted in the quantification of apoptosis . Indeed , Prahalad et al. -LSB- 35 -RSB- describe this reversible cell detachment during ATP depletion of Madin-Darby canine kidney cells . Conclusion Prevention of F-actin rearrangement or F-actin bundle formation by Rho-kinase inhibition or by treatment with actin depolymerizator cytochalasinD , independent of Rho-kinase , attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity . The present finding suggests that the changes in the F-actin cytoskeleton play a pivotal role in the negative regulation of PI3-kinase and Akt by Rho-kinase . Further studies are required to conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis .

O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cytoskeleton cytochalasind latrunculina jasplakinolide


On-line

optimization

of

glutamate

production

based

on

balanced

metabolic

control

by

RQ

In

glutamate

fermentations

by

Corynebacterium

glutamicum

higher

glutamate

concentration

could

be

achieved

by

constantly

controlling

dissolved

oxygen

concentration

-LRB-

DO

-RRB-

at

a

lower

level

however

by-product

lactate

also

severely

accumulated

The

results

of

analyzing

activities

changes

of

the

two

key

enzymes

glutamate

and

lactate

dehydrogenases

involved

with

the

fermentation

and

the

entire

metabolic

network

flux

analysis

showed

that

the

lactate

overproduction

was

because

the

metabolic

flux

in

TCA

cycle

was

too

low

to

balance

the

glucose

glycolysis

rate

As

a

result

the

respiratory

quotient

-LRB-

RQ

-RRB-

adaptive

control

based

balanced

metabolic

control

-LRB-

BMC

-RRB-

strategy

was

proposed

and

used

to

regulate

the

TCA

metabolic

flux

rate

at

an

appropriate

level

to

achieve

the

metabolic

balance

among

glycolysis

glutamate

synthesis

and

TCA

metabolic

flux

Compared

with

the

best

results

of

various

DO

constant

controls

the

BMC

strategy

increased

the

maximal

glutamate

concentration

by

about

and

almost

completely

repressed

the

lactate

accumulation

with

competitively

high

glutamate

productivity

Introduction

For

more

than

a

few

decades

the

Corynebacterium

spp.

bacteria

have

been

used

for

amino

acids

productions

including

the

commercially

important

products

of

glutamate

glutamine

and

lysine

Among

the

amino

acids

mentioned

earlier

L-glutamate

is

the

largest

fermentative

product

which

occupies

about

of

the

world

's

amino

acids

market

-LSB-

-RSB-

It

is

also

particularly

important

in

food

industries

and

widely

used

as

an

important

starting

substance

for

the

synthesis

of

various

and

useful

pharmaceutical

and

healthy

products

The

previous

research

works

-LSB-

-RSB-

have

revealed

that

primary

by-products

such

as

lactate

accumulated

during

L-glutamate

fermentation

if

the

operating

condition

was

improperly

controlled

which

in

turn

deteriorated

the

fermentation

performance

in

terms

of

both

glutamate

productivity

and

yield

To

cope

with

the

problem

the

metabolic

reaction

network

-LRB-

flux

-RRB-

model

-LRB-

MR

model

-RRB-

or

technique

as

its

appearance

in

the

early

has

been

recognized

as

an

useful

system

analysis

tool

and

thus

widely

used

in

those

areas

such

as

metabolic

flux

distribution

analysis

-LSB-

-RSB-

determination

of

the

bottleneck

controlling

the

targeted

metabolic

product

formation

-LSB-

-RSB-

recognition

of

fermentation

phases

-LSB-

-RSB-

and

calculation

of

theoretical

or

maximum

yields

-LSB-

-RSB-

etc.

However

the

research

reports

of

using

MR

model

for

on-line

physiological

state

prediction

or

process

control

are

very

limited

-LSB-

-RSB-

and

the

study

stayed

on

on-line

recognition

of

different

fermentation

phases

or

physiological

states

so

as

to

provide

information

for

the

subsequent

process

control

such

as

whether

substrate

should

be

added

or

whether

the

fermentation

should

be

terminated

-LSB-

-RSB-

determination

of

glucose

feeding

rate

to

avoid

the

acetate

or

ethanol

overproduction

in

Escherichia

coli

or

Saccharomyces

cerevisiae

fed-batch

cultivation

-LSB-

-RSB-

etc.

Optimization

of

fermentation

processes

by

MR

model

was

mostly

limited

on

using

the

feed-forward

type

's

off-line

control

strategy

with

the

MR

model

as

the

constraint

conditions

-LSB-

-RSB-

On

the

other

hand

analysis

of

key

enzymes

in

metabolic

network

of

cells

was

also

very

important

as

those

key

enzyme

generally

play

important

roles

in

regulating

and

overproducing

the

targeted

metabolic

products

As

a

result

extensive

research

works

with

regard

to

the

enzymes

activities

under

different

operating

conditions

have

been

carried

out

Among

them

the

enzymes

activities

in

fermentation

by

Corynebacterium

glutamicum

such

as

the

influence

of

concentration

on

C.

glutamicum

growth

and

glutamate

dehydrogenase

-LRB-

GDH

-RRB-

-LSB-

-RSB-

activity

of

phosphoenolpyruvate

carboxylase

responsible

for

the

anaplerotic

reactions

in

lysine

continuous

production

by

C.

glutamicum

under

different

dilution

rates

-LSB-

-RSB-

as

well

as

lactate

dehydrogenase

-LRB-

LDH

-RRB-

for

pyruvate

overproduction

under

different

dilution

rates

with

lactate

as

the

sole

carbon

source

-LSB-

-RSB-

were

investigated

The

metabolic

network

models

were

also

built

for

C.

glutamicum

under

different

operating

conditions

aiming

at

achieving

metabolic

flux

distribution

information

concerning

the

overproduction

of

targeted

metabolites

or

substrate

utilization

-LSB-

-RSB-

as

well

as

on-line

prediction

of

the

targeted

products

formation

and

analysis

of

the

entire

metabolic

fluxes

-LSB-

-RSB-

In

our

previous

study

-LSB-

-RSB-

we

found

that

dissolved

oxygen

-LRB-

DO

-RRB-

largely

affected

the

metabolic

flux

distribution

and

the

glutamate

fermentation

performance

The

comprehensive

analysis

or

evaluation

of

a

fermentation

process

by

the

MR

model-based

metabolic

flux

analysis

integrated

with

the

intracellular

enzymes

activities

monitoring

might

gain

a

deeper

insight

into

the

entire

fermentation

process

and

the

metabolic

regulation

mechanisms

so

as

to

supply

a

more

comprehensive

and

accurate

information

base

for

the

subsequent

process

control

and

optimization

of

the

fermentation

process

In

this

study

combining

our

previous

study

on

on-line

metabolic

flux

analysis

of

the

glutamate

fermentation

by

C.

glutamicum

the

two

major

enzymes

-LRB-

GDH

and

LDH

-RRB-

which

possibly

dominated

the

overall

glutamate

metabolism

were

carefully

investigated

Then

based

on

the

comprehensive

evaluation

results

of

both

the

metabolic

flux

analysis

and

intracellular

enzymes

activities

monitoring

a

new

optimization

strategy

of

balanced

metabolic

control

-LRB-

BMC

-RRB-

was

proposed

and

verified

experimentally

aiming

to

increase

the

glutamate

production

yield

while

completely

repressing

the

by-product

overproduction

simultaneously

Materials

and

methods

Microorganism

and

fermentation

conditions

Corynebacterium

glutamicum

kept

by

the

laboratory

was

used

throughout

this

study

The

same

medium

and

seed

culture

conditions

described

in

our

previous

study

-LSB-

-RSB-

were

used

Corynebacterium

glutamicum

was

cultured

for

glutamate

production

at

°C

in

a

L

fermentor

-LRB-

Baoxing

Co.

China

-RRB-

containing

about

L

medium

Concentrated

glucose

was

fed

based

on

requirement

to

ensure

the

substrate

concentration

above

a

suitable

level

-LRB-

g/L

-RRB-

throughout

the

fermentation

period

pH

was

controlled

at

by

automatic

addition

of

-LRB-

w/w

-RRB-

ammonia

water

which

also

supplied

the

nitrogen

source

required

for

glutamate

synthesis

DO

was

controlled

at

various

levels

by

automatically

or

manually

controlling

the

agitation

speed

based

on

particular

requirements

The

air

aeration

rate

and

fermentor

pressure

were

kept

constantly

at

vvm

and

MPa

respectively

Analytical

methods

The

concentrations

of

cells

glucose

glutamate

and

lactate

were

measured

with

the

same

methods

as

reported

in

our

previous

study

-LSB-

-RSB-

The

and

concentrations

-LRB-

partial

pressure

-RRB-

in

the

inlet

and

exhaust

gas

were

on-line

measured

by

a

gas

analyzer

-LRB-

Lokas

Co.

Ltd

Korea

-RRB-

The

collected

on-line

data

were

smoothly

filtered

and

then

oxygen

uptake

rate

-LRB-

OUR

-RRB-

and

evolution

rate

-LRB-

CER

-RRB-

were

on-line

calculated

based

on

the

literature

reported

method

-LSB-

-RSB-

Respiratory

quotient

-LRB-

RQ

-RRB-

was

determined

by

its

definition

-LRB-

RQ

CER/OUR

-RRB-

using

the

on-line

measured

OUR

and

CER

data

Extraction

and

assay

of

the

GDH

and

LDH

The

two

key

enzymes

dominating

glutamate

fermentation

and

metabolism

the

NADPH

and

NADH

dependent

GDH

and

LDH

were

detected

by

spectrophotometric

method

and

the

relevant

stoichiometric

reactions

could

be

written

as

follows

The

enzymes

in

the

reverse

direction

of

glutamate

and

lactate

consumption

were

not

considered

and

assayed

in

this

case

Cells

were

collected

by

centrifugation

and

then

suspended

in

mmol/L

Tris

HCl

buffer

pH

The

suspension

was

sonicated

by

a

sonifier

-LRB-

Scientz

Biotechnology

Co.

China

-RRB-

at

°C

for

a

total

period

of

min

-LRB-

pulse

on

s

pulse

off

s

-RRB-

Cell

debris

was

removed

by

centrifugation

at

g

°C

for

min

the

supernatant

was

then

used

as

the

cell-free

crude

enzyme

GDH

was

assayed

with

a

spectrophotometer

-LRB-

Unico

Shanghai

China

-RRB-

at

nm

by

measuring

the

optical

variation

within

min

with

mL

supernatant

and

ml

GDH

reaction

mixture

consisting

of

mmol/L

α-ketoglutaric

acid

mmol/L

and

mmol/L

NADPH

-LRB-

Sigma

Chemical

Co.

St

Louis

MO

USA

-RRB-

in

phosphate

buffer

-LRB-

pH

-RRB-

at

°C

LDH

was

assayed

in

the

same

condition

but

with

the

LDH

reaction

mixture

containing

mmol/L

pyruvic

acid

and

mmol/L

NADH

-LRB-

Sigma

Chemical

Co.

St

Louis

MO

USA

-RRB-

Protein

was

measured

by

Bradford

method

with

bovine

serum

albumin

as

the

standard

Enzyme-specific

activity

-LRB-

GDH

or

LDH

-RRB-

was

expressed

as

units/mg-protein

where

U

was

defined

as

the

quantity

of

enzyme

that

converted

μmol

of

NAD

-LRB-

P

-RRB-

H

per

minute

On-line

control

system

The

on-line

measured

RQ

data

were

sent

to

a

PC

in

which

a

control

program

written

with

Visual-Basic

-LRB-

Microsoft

Inc.

USA

-RRB-

was

embedded

Based

on

the

RQ

set-points

and

the

measured

RQ

the

PC

on-line

regulated

the

agitation

rate

of

the

fermentor

-LRB-

AGT

-RRB-

with

a

discrete

PI

control

manner

described

by

Eq

which

renewed

the

agitation

rate

at

an

interval

of

min

In

Eq

k

represented

the

current

control

instant

RQset

was

the

RQ

set-point

which

might

be

subject

to

changes

during

the

control

KC

and

τI

were

proportional

and

integral

constants

of

the

feedback

controller

respectively

KC

and

τI

were

determined

by

Coon

Cohen

method

by

observing

the

RQ

response

to

a

step

change

in

the

input

-LRB-

agitation

rate

-RRB-

during

a

certain

period

of

the

glutamate

production

phase

Results

and

discussion

The

changing

patterns

of

glutamate

and

lactate

production

as

well

as

the

GDH

and

LDH

activities

at

different

DO

control

levels

Constant

control

of

DO

is

generally

considered

as

the

simplest

optimization

control

method

for

the

aerobic

fermentations

with

aeration

Fig.

c

d

showed

the

time

courses

of

glutamate

lactate

cells

and

residual

glucose

concentrations

when

DO

was

constantly

controlled

at

and

-LRB-

saturation

level

-RRB-

respectively

The

results

showed

that

glutamate

and

lactate

formation

pattern

strongly

depended

on

the

DO

control

level

A

higher

glutamate

production

rate

could

be

achieved

when

the

DO

was

controlled

at

a

lower

level

of

and

the

final

glutamate

concentration

reached

about

g/L

at

h

while

the

final

glutamate

concentration

stopped

at

a

lower

level

of

g/L

-LRB-

h

-RRB-

when

controlling

DO

at

On

the

other

hand

lactate

severely

accumulated

up

to

g/L

when

DO

was

controlled

at

a

lower

level

of

while

almost

no

lactate

accumulation

occurred

when

controlling

DO

at

These

results

suggested

that

the

enzymatic

activities

of

GDH

and

LDH

under

lower

and

higher

DO

level

might

be

quite

different.Fig

courses

of

glutamate

lactate

cells

and

glucose

concentrations

as

well

as

the

key

enzymes

activities

at

different

DO

control

levels

a

Glutamate

concentration

-LRB-

filled

circle

-RRB-

DO

-LRB-

filled

triangle

-RRB-

DO

lactate

concentration

-LRB-

open

circle

-RRB-

DO

-LRB-

open

triangle

-RRB-

DO

b

GDH

activity

-LRB-

filled

circle

-RRB-

DO

-LRB-

open

circle

-RRB-

DO

LDH

activity

-LRB-

filled

triangle

-RRB-

DO

-LRB-

open

triangle

-RRB-

DO

c

Cells

concentration

-LRB-

filled

circle

-RRB-

DO

-LRB-

filled

triangle

-RRB-

DO

d

Glucose

concentration

-LRB-

filled

circle

-RRB-

DO

-LRB-

filled

triangle

-RRB-

DO

Figure

indicated

the

changing

pattern

of

the

GDH

and

LDH

activities

under

the

same

DO

control

levels

The

GDH

activity

for

the

lower

DO

case

-LRB-

-RRB-

was

much

higher

and

declined

slower

than

that

of

the

higher

DO

case

-LRB-

-RRB-

after

experiencing

the

enzymatic

activity

peak

of

about

U/mg-protein

around

h

This

result

could

at

least

explain

or

account

for

the

fact

that

higher

glutamate

production

rate

and

accumulation

occurred

at

lower

DO

control

level

in

the

initial

and

middle

production

phases

of

h

The

slow

down

of

glutamate

production

rate

in

the

late

production

stage

-LRB-

h

-RRB-

and

the

final

stoppage

of

glutamate

production

were

due

to

the

reduction

of

the

co-enzyme

NADPH

regeneration

rate

which

will

be

further

discussed

in

the

following

section

As

for

the

LDH

activities

from

the

result

of

severe

lactate

's

accumulation

at

lower

DO

level

it

was

speculated

that

the

LDH

activity

under

lower

DO

level

-LRB-

-RRB-

should

also

be

much

higher

than

that

of

the

higher

DO

case

However

the

result

did

not

agree

with

the

expectation

Although

the

LDH

activity

under

lower

DO

level

was

slightly

higher

than

that

of

the

higher

DO

for

the

majority

of

the

fermentation

period

this

result

could

not

stand

for

the

large

difference

in

the

lactate

accumulation

under

different

DO

levels

It

should

be

noted

that

the

cell

concentration

could

reach

a

constant

and

stabilized

level

of

about

g/L

prior

to

production

phase

-LRB-

Fig.

-RRB-

for

all

of

the

constant

DO

control

cases

therefore

using

the

unit

of

U/mg-protein

could

actually

reflect

the

total

enzyme

activity

The

changing

patterns

of

glutamate

and

lactate

production

as

well

as

the

GDH

and

LDH

activities

at

anaerobic

fermentation

condition

Generally

it

is

considered

that

the

anaerobic

condition

is

extremely

harmful

to

glutamate

production

To

verify

the

above

speculation

an

experiment

under

extremely

low

DO

level

was

conducted

In

the

fermentation

DO

was

initially

controlled

at

and

the

agitation

rate

was

manually

reduced

to

bring

DO

down

to

instantly

at

h.

Then

the

same

agitation

rate

was

kept

for

the

next

h

During

this

period

the

fermentation

could

be

considered

as

implemented

under

anaerobic

condition

glutamate

production

stopped

and

lactate

overflowed

as

shown

in

Fig.

At

h

the

automatic

control

of

DO

was

resumed

to

quickly

bring

DO

back

to

a

partial

recovery

of

glutamate

production

was

observed

However

the

final

glutamate

concentration

ended

at

a

very

low

level

of

about

g/L

As

shown

in

Fig.

during

the

anaerobic

period

GDH

activity

sharply

dropped

to

a

level

of

U/mg

from

the

original

U/mg

and

could

not

be

recovered

again

even

when

DO

was

resumed

back

to

level

The

results

indicated

that

the

occurrence

of

anaerobic

condition

even

for

a

short

period

would

be

both

fatal

and

irretrievable

to

glutamate

fermentation.Fig

changing

pattern

of

the

products

formation

and

the

key

enzymes

activities

when

DO

control

level

was

subject

to

a

sudden

change

during

h

and

maintained

at

extremely

low

level

during

the

period

a

The

changing

patterns

of

glutamate

and

lactate

concentrations

as

well

as

the

DO

control

level

filled

circle

glutamate

concentration

open

triangle

lactate

concentration

solid

line

DO

b

The

changing

patterns

of

the

key

enzyme

activities

filled

circle

GDH

activity

open

triangle

LDH

activity

The

glucose

consumption

rates

-LRB-

glycolysis

rates

-RRB-

at

different

DO

control

levels

Figure

showed

the

average

glucose

consumption

rates

under

different

DO

control

levels

during

the

production

phase

Apparently

under

regular

-LRB-

aerobic

-RRB-

fermentation

the

glycolysis

rate

did

not

depend

on

the

DO

control

level

and

the

changing

patterns

of

glycolysis

rate

at

different

DO

levels

were

basically

the

same.Fig

time

courses

of

glucose

consumption

rate

at

different

DO

levels

Open

circle

DO

batch

no.

open

triangle

DO

batch

no.

The

mechanism

analysis

of

lactate

overflow

and

the

control

strategy

of

BMC

For

the

purpose

of

easily

interpreting

the

experimental

results

the

metabolic

flow

chart

of

glutamate

fermentation

by

C.

glutamicum

as

shown

in

our

previous

study

-LSB-

-RSB-

was

simplified

and

re-depicted

in

Fig.

The

metabolic

fluxes

changes

at

and

h

of

the

fermentations

with

the

DO

constant

controls

-LRB-

-RRB-

and

BMC

were

also

depicted

As

shown

in

the

map

glucose

was

converted

into

pyruvate

through

EMP

pathway

and

supplemented

with

PP

pathway

to

produce

NADPH

for

structuring

glutamate

synthesis

precursors

by

cells

The

pyruvate

formed

either

entered

TCA

cycle

via

pyruvate

dehydrogenase

generating

the

energy

as

well

as

co-enzyme

substances

such

as

ATP

NADH

etc.

for

the

entire

metabolic

network

operations

or

was

converted

into

lactate

with

LDH

as

the

enzymatic

catalyst

-LRB-

Eq

-RRB-

At

α-KG

-LRB-

α-ketoglutaric

acid

-RRB-

node

of

TCA

cycle

the

glutamate

synthesis

reaction

-LRB-

-RRB-

with

GDH

as

the

enzymatic

catalyst

-LRB-

Eq

-RRB-

occurred

As

shown

in

Fig.

with

the

constant

DO

control

strategy

the

decrease

of

the

DO

control

level

enhanced

the

metabolic

fluxes

of

both

glutamate

and

lactate

formation

while

the

metabolic

flux

of

TCA

after

the

α-KG

node

-LRB-

-RRB-

sharply

decreased

and

the

TCA

cycle

was

almost

completely

shut

down

when

controlling

DO

at

level

-LRB-

also

refer

to

Fig.

-RRB-

Fig.

metabolic

flow

chart

and

the

relevant

metabolic

fluxes

changes

during

the

fermentations

Broken

line

very

low

metabolic

flux

or

channels

shut

down

solid

line

moderate

metabolic

flux

bold

solid

line

enhanced

metabolic

flux

a

at

h

b

at

h

c

at

hFig

of

RQ

and

calculated

TCA

metabolic

flux

rate

at

different

DO

control

levels

A

higher

enzyme

activity

in

vivo

reflects

a

higher

potential

in

reaction

rate

catalyzed

by

the

enzyme

However

it

never

means

that

the

relevant

reaction

rate

must

be

high

as

the

reaction

rate

also

depends

on

other

factors

such

as

the

coenzyme

-LRB-

NADPH

NADH

-RRB-

activity

as

well

as

the

reactant

concentration

As

shown

in

Fig.

b

at

the

end

of

the

fermentation

the

glutamate

synthesis

completely

stopped

even

though

GDH

activity

still

remained

at

a

higher

level

The

calculation

results

in

our

previous

report

-LSB-

-RSB-

showed

that

at

the

end

of

the

fermentation

the

coenzyme

NADPH

regeneration

rate

had

decreased

to

of

its

peak

level

-LRB-

figure

not

presented

-RRB-

Therefore

it

could

be

concluded

that

the

glutamate

synthesis

stoppage

was

due

to

the

significant

reduction

of

NADPH

regeneration

rate

instead

of

the

reduction

in

GDH

activity

In

general

the

enzymes

catalyzing

interaction

assures

an

intracellular

carbon

fluxes

balance

This

means

that

in

glutamate

fermentation

glycolysis

rate

-LRB-

-RRB-

should

balance

with

glutamate

synthesis

-LRB-

-RRB-

lactate

formation

-LRB-

-RRB-

as

well

as

the

TCA

metabolic

flux

-LRB-

-RRB-

that

was

involved

with

release

As

a

result

from

Figs.

and

we

could

speculate

that

the

severe

lactate

accumulation

at

lower

DO

control

-LRB-

DO

-RRB-

was

due

to

the

carbon

metabolic

balance

rather

than

the

higher

LDH

activity

by

the

following

facts

First

the

changing

patterns

of

glycolysis

rate

-LRB-

-RRB-

at

different

DO

levels

were

almost

the

same

second

no

significant

differences

in

LDH

activities

were

shown

under

different

DO

level

Third

the

metabolic

flux

of

TCA

significantly

decreased

with

the

decrease

in

DO

control

level

Under

the

lower

DO

level

even

though

GDH

activity

was

higher

the

higher

glutamate

synthesis

rate

-LRB-

-RRB-

still

could

not

completely

balance

with

the

glycolysis

rate

-LRB-

-RRB-

as

TCA

cycle

was

almost

closed

completely

and

the

TCA

metabolic

flux

-LRB-

-RRB-

was

very

low

Under

this

circumstance

lactate

had

to

be

overflowed

or

excreted

-LRB-

-RRB-

into

the

broth

to

achieve

the

entire

intracellular

carbon

balance

On

the

other

hand

under

the

higher

DO

level

GDH

activity

was

relatively

low

but

the

TCA

cycle

was

nearly

open

for

a

complete

oxidation

Under

this

condition

lactate

was

not

necessarily

overflowed

as

the

glutamate

synthesis

rate

-LRB-

-RRB-

plus

the

higher

TCA

metabolic

flux

-LRB-

-RRB-

were

big

enough

to

balance

with

the

glycolysis

rate

-LRB-

-RRB-

even

though

LDH

exhibited

almost

equivalent

activity

as

compared

with

that

of

the

lower

DO

case

Is

there

an

effective

way

to

solve

the

problem

of

simultaneous

accumulation

of

the

main

product

-LRB-

glutamate

-RRB-

and

by-product

-LRB-

lactate

-RRB-

Two

ways

of

using

genetic

engineering

technique

could

be

considered

as

the

solution

-LRB-

-RRB-

to

use

genetic

engineering

technique

to

knock-out

the

genes

coding

for

LDH

-LRB-

-RRB-

to

use

certain

LDH

inhibitors

to

eliminate

the

LDH

action

without

affecting

GDH

and

entire

fermentation

activities

at

the

same

time

These

are

direct

and

straightforward

solutions

but

might

be

very

complicated

and

difficult

to

realize

Based

on

the

metabolic

analysis

mentioned

earlier

an

alternative

solution

the

BMC

strategy

as

shown

in

Fig.

which

is

adaptively

regulating

of

the

TCA

metabolic

fluxes

at

an

appropriate

level

to

achieve

the

metabolic

balance

among

glycolysis

glutamate

synthesis

and

TCA

metabolic

flux

for

enhancement

of

the

glutamate

synthesis

and

repression

of

lactate

overflow

seemed

to

be

easier

to

implement

and

more

realistic

Possibility

of

using

and

realizing

the

BMC

strategy

The

necessary

conditions

for

realization

of

BMC

existed

in

the

following

two

aspects

-LRB-

-RRB-

the

rates

of

the

glutamate

synthesis

-LRB-

-RRB-

plus

the

TCA

metabolic

flux

-LRB-

-RRB-

should

just

balance

with

the

glycolysis

rate

-LRB-

-RRB-

-LRB-

-RRB-

DO

should

be

controlled

at

a

reasonably

lower

level

to

assure

a

higher

GDH

activity

To

achieve

the

target

an

on-line

measurable

or

predictable

state

variable

that

could

actually

reflect

the

TCA

metabolic

flux

changes

must

be

available

Based

on

our

experimental

results

and

the

on-line

metabolic

flux

calculation

in

the

previous

study

we

found

RQ

are

related

with

the

TCA

metabolic

flux

-LRB-

-RRB-

closely

and

could

be

considered

as

the

on-line

feedback

index

for

realization

of

BMC

Figure

depicted

the

time

course

of

RQ

and

the

TCA

metabolic

flux

-LRB-

-RRB-

for

the

cases

of

constantly

controlling

DO

at

and

respectively

For

both

cases

RQ

almost

followed

the

same

changing

patterns

during

the

growth

phase

However

during

the

glutamate

production

phase

the

changing

patterns

of

RQ

and

TCA

metabolic

flux

under

different

DO

levels

were

apparently

different

RQ

almost

stayed

constantly

around

in

the

case

of

controlling

DO

at

while

RQ

gradually

declined

into

the

range

of

for

the

case

of

controlling

DO

at

Theoretically

RQ

means

that

TCA

cycle

is

fully

opened

and

glucose

is

completely

converted

into

and

via

TCA

cycle

In

glutamate

fermentation

a

lower

RQ

-LRB-

for

example

RQ

-RRB-

reflected

the

fact

that

TCA

cycle

was

almost

completely

shut

down

in

order

to

direct

the

carbon

flux

to

the

glutamate

synthesis

at

α-KG

node

On

the

other

hand

a

higher

RQ

meant

that

the

TCA

metabolic

flux

was

too

high

that

a

large

portion

of

carbon

was

oxidased

into

and

accompanyied

with

large

quantities

of

NADH

and

ATP

formation

which

actually

caused

the

inefficient

carbon

cycle

Based

on

Fig.

If

we

could

carefully

and

adaptively

control

RQ

at

a

suitable

level

maintaining

the

TCA

metabolic

flux

at

a

moderate

level

in

between

the

fluxes

of

lower

and

higher

DO

levels

while

still

directing

major

carbon

flux

to

the

glutamate

synthesis

then

the

BMC

might

be

realized

and

lactate

overflow

be

relieved

The

performance

and

experimental

result

of

the

BMC

strategy

Figure

showed

one

complete

data

set

of

the

BMC

control

results

The

BMC

was

activated

at

h

after

the

fermentation

entered

into

production

phase

where

the

two

controller

parameters

KC

and

τI

were

set

at

and

respectively

As

shown

in

Fig.

the

set-point

of

RQ

was

changed

three

times

during

the

fermentation

-LRB-

RQset

h

initial

stage

of

production

phase

h

middle

stage

h

final

stage

-RRB-

The

principle

of

changing

RQset

was

to

assure

that

DO

does

not

subject

to

either

a

higher

-LRB-

-RRB-

or

a

very

low

level

-LRB-

-RRB-

for

a

relatively

longer

time

-LRB-

h

-RRB-

in

order

to

keep

a

higher

GDH

activity

if

DO

stayed

at

higher

level

for

relatively

long

time

then

it

initiatively

reduced

the

RQset

down

if

DO

stayed

at

very

low

level

for

relatively

long

time

then

it

initiatively

brings

the

RQset

up

By

using

DO

as

the

complementary

indicator

in

this

way

RQ

could

be

controlled

around

its

set-point

with

the

agitation

rate

stably

staying

in

the

range

of

rpm

without

reaching

its

upper

limit

-LRB-

rpm

-RRB-

or

low

limit

-LRB-

rpm

-RRB-

even

for

a

short

time

With

this

control

strategy

DO

fluctuated

at

a

lower

range

of

so

that

a

higher

GDH

activity

could

be

maintained

The

BMC

control

led

to

an

excellent

fermentation

performance

as

compared

with

those

of

the

DO

constant

controls

the

maximal

glutamate

concentration

reached

g/L

at

h

which

was

about

an

increase

over

the

best

result

of

the

DO

constant

controls

-LRB-

g/L

DO

-RRB-

the

lactate

concentration

at

the

corresponding

time

was

only

g/L

which

was

only

of

the

best

result

of

the

DO

constant

controls

-LRB-

g/L

DO

-RRB-

To

verify

the

repeatability

of

BMC

control

's

performance

the

same

experiment

was

repeated

once

Again

a

maximal

glutamate

concentration

of

g/L

was

obtained

at

h

with

the

lactate

accumulation

of

only

g/L

at

the

same

time.Fig

experimental

results

and

control

performance

using

the

BMC

optimization

strategy

Filled

circle

glutamate

concentration

open

triangle

lactate

concentration

open

circle

cells

concentration

filled

square

glucose

concentration

It

should

be

noted

that

constant

RQ

control

strategy

could

not

lead

to

a

good

fermentation

performance

We

did

three

constant

RQ

control

experiments

setting

RQ

at

and

respectively

The

glutamate

production

stopped

at

very

low

level

-LRB-

g/L

-RRB-

for

all

of

the

three

cases

In

the

first

two

cases

constantly

controlling

RQ

at

and

caused

DO

to

drop

to

zero

level

or

the

anaerobic

environmental

condition

in

other

word

in

the

initial

production

stage

The

cells

metabolic

and

GDH

activities

were

largely

damaged

as

suggested

by

Fig.

and

relevant

discussion

In

the

third

case

the

high

RQ

set-point

-LRB-

RQ

-RRB-

caused

a

continuous

rise-up

and

high

level

of

DO

-LRB-

from

to

-RRB-

which

reduced

the

GDH

activity

and

deteriorated

glutamate

production

in

turn

As

a

result

the

adaptive

RQ

control

-LRB-

step-wise

changing

RQ

set-points

-RRB-

in

simultaneous

consideration

of

DO

change

has

to

be

adopted

to

satisfy

the

two

conditions

of

obtaining

an

appropriate

flux

and

a

reasonable

lower

DO

level

for

realizing

the

BMC

as

described

in

the

previous

section

Figure

showed

the

changing

patterns

of

the

TCA

metabolic

flux

-LRB-

-RRB-

when

using

the

BMC

control

and

the

DO

constant

controls

Apparently

the

BMC

strategy

controlled

the

TCA

metabolic

flux

at

an

appropriate

level

just

in

between

those

of

higher

and

lower

DO

levels

This

realized

the

target

of

balanced

metabolism

which

led

to

a

higher

glutamate

production

and

almost

completely

repressed

the

lactate

accumulation.Fig

calculated

TCA

metabolic

fluxes

of

the

BMC

strategy

and

the

DO

constant

controls

Table

indicated

that

yield

-LRB-

total

released

quantity

versus

total

glucose

consumed

quantity

-RRB-

of

the

BMC

was

also

just

in

between

those

of

higher

and

lower

DO

constant

control

cases

which

supported

the

BMC

idea

from

the

other

side

However

it

should

be

noted

that

besides

channel

the

release

also

occurred

at

the

other

routes

in

the

entire

metabolic

network

the

shutdown

of

TCA

cycle

never

meant

the

total

production

would

cease

The

effectiveness

of

the

BMC

strategy

was

also

supported

by

the

metabolic

fluxes

changes

results

shown

in

Fig.

The

moderate

TCA

metabolic

flux

-LRB-

-RRB-

simultaneously

ensured

a

higher

glutamate

synthetic

flux

and

an

almost

lactate

formation

flux.Table

materials

and

carbon

balances

of

the

balanced

metabolic

control

-LRB-

BMC

-RRB-

and

dissolved

oxygen

-LRB-

DO

-RRB-

constant

controlsBatch

no.Glucose

consumed

-LRB-

g

-RRB-

Glutamate

produced

-LRB-

g

-RRB-

Lactate

produced

-LRB-

g

-RRB-

Cellsa

-LRB-

g

-RRB-

released

-LRB-

g

-RRB-

Carbon

balanceb

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

DO

839.2384.0102.090.8352.9C-content335.6156

DO

aThe

dry

cell

weight

using

the

composition

of

strain

C.

glutamicum

-LRB-

N

Ref

-RRB-

as

the

calculation

referencebRatio

of

the

total

production

-LRB-

glutamate

lactate

cell

and

-RRB-

to

total

consumption

-LRB-

glucose

-RRB-

Table

summarized

the

results

and

performance

of

the

BMC

and

the

DO

constant

controls

In

consideration

of

the

repeatability

of

the

different

control

strategies

the

experiments

of

constantly

controlling

DO

at

and

as

well

as

BMC

were

repeated

at

least

twice

and

then

the

averaged

values

were

calculated

for

performance

evaluation

The

table

evaluated

the

two

different

control

strategies

in

terms

of

the

four

major

fermentation

performance

index

maximal

glutamate

concentration

lactate

accumulation

quantity

glutamate

productivity

and

conversion

rate

from

glucose

to

glutamate

The

BMC

showed

an

equivalent

performance

in

the

terms

of

productivity

and

conversion

rate

as

compared

with

the

best

results

of

the

DO

constant

controls

However

BMC

strategy

greatly

improved

the

other

two

performance

index

maximal

glutamate

concentration

was

increased

by

about

-LRB-

with

respect

to

DO

case

-RRB-

and

lactate

accumulation

was

decreased

by

about

-LRB-

with

respect

to

DO

case

-RRB-

Table

summarized

results

of

the

BMC

and

DO

constant

controlBatch

no.Conversion

rate

-LRB-

-RRB-

Glutamate

concentration

-LRB-

g/L

-RRB-

Lactate

concentration

-LRB-

g/L

-RRB-

Productivity

-LRB-

g/L/h

-RRB-

Constant

DO

controls

-LRB-

DO

-RRB-

-LRB-

DO

-RRB-

DO

-LRB-

DO

-RRB-

-LRB-

DO

-RRB-

DO

-LRB-

DO

-RRB-

Balanced

metabolic

control

by

RQ

-LRB-

RQ-BMC

-RRB-

-LRB-

RQ-BMC

-RRB-

-LRB-

RQ-BMC

-RRB-

RQ-BMC

Summary

A

novel

fermentation

optimization

method

the

balanced

metabolic

control

-LRB-

BMC

-RRB-

strategy

was

proposed

and

successfully

used

by

feedback

controlling

RQ

to

regulate

the

TCA

metabolic

flux

rate

at

an

appropriate

level

to

achieve

the

metabolic

balance

among

glycolysis

glutamate

synthesis

and

TCA

metabolic

flux

for

glutamate

fermentation

The

proposed

BMC

strategy

greatly

improved

the

fermentation

performance

in

the

two

terms

of

maximal

glutamate

concentration

and

the

lactate

overflow

repression

The

maximal

glutamate

concentration

increased

by

about

compared

with

the

best

results

of

the

DO

constant

controls

and

furthermore

the

lactate

overproduction

occurred

in

the

DO

constant

control

cases

could

be

completely

relieved

when

using

the

BMC

strategy

As

a

result

the

proposed

BMC

strategy

is

expected

to

be

applicable

for

optimization

of

other

aerobic

amino

acids

fermentations

in

potential

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I I O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

glutamate fermentation
intracellular enzymes activities
metabolic flux analysis

metabolic balancing
on-line optimization