Effect of acetic acid on ethanol production by Zymomonas mobilis mutant strains through continuous adaptation

Abstract Background Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. Results In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. Conclusion The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation

The STAT3 phosphorylation and glutathionylation levels are dependent on the ROS levels in CO-treated ECs.

<p><b>A.</b> ECs subjected to IL-6 (10ng/ml) treatment for 10 min were pretreated with or without 0.5 or 1 mM GSSG for 30 minutes. Cell lysates were then subjected to western blotting with antibodies against pTyr-STAT3 or STAT3. <b>B.</b> ECs were pretreated with catalase at the indicated units for 30 min and then cultured without further treatment or exposed to TCDC or MC for 12 hours. S-glutathionylated STAT3 was then detected as above. The quantification of the band intensities was normalized to the control values. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100677#s3" target="_blank">Results</a> are presented as the mean ± SEM (n = 3). *<i>P</i><0.05 compared with untreated ECs. ^#<i>P</i><0.05 compared with TCDC or MC alone. <b>C.</b> ECs subjected to IL-6 treatment for 10 min were pretreated with 500 U catalase for 30 min and then cultured without further treatment or exposed to TCDC or MC for 12 hours. Cell lysates were then subjected to western blotting.</p

Carbon Monoxide Induces Heme Oxygenase-1 to Modulate STAT3 Activation in Endothelial Cells via S-Glutathionylation

<div><p>IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs.</p></div

CO increases oxidative stress in ECs.

<p><b>A.</b> and <b>B.</b> ECs were exposed to 25 µM TCDC or 5mM MC for 1, 3, 6 and 12 hours with or without pretreated 500U catalase for 30 min and the intracellular ROS levels were then measured. The results shown are the mean ± SEM. *<i>P</i><0.05 compared with untreated ECs. <b>C. and D.</b> ECs were subjected to similar treatments prior to the measurement of intracellular ROS levels. The folds of lucigenin-amplified chemiluminescence are shown as the mean ± SEM. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100677#s3" target="_blank">Results</a> are presented as the mean ± SEM (n = 3). *<i>P</i><0.05 compared with untreated ECs.</p

The effects of CO on STAT3 glutathionylation.

<p><b>A.</b> and <b>B.</b> ECs were treated with 100 µM BioGEE for one hour and loaded with TCDC or MC for the indicated times to induce protein glutathionylation. <b>C.</b> and <b>D.</b> ECs were exposed to TCDC or MC for the indicated periods. S-glutathionylated STAT3 was immunoprecipitated and detected using an antibody against protein-SSG. The quantification of the band intensities was normalized to the control values. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100677#s3" target="_blank">Results</a> are presented as the mean ± SEM (n = 3). *<i>P</i><0.05 compared with untreated ECs.</p

The effects of CO are dependent on the intracellular GSH level.

<p><b>A.</b> ECs were pretreated with 0.5-glutathionylated STAT3 was then detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100677#pone-0100677-g005" target="_blank">Figure 5</a>. *<i>P</i><0.05 compared with untreated ECs. ^#<i>P</i><0.05 compared with TCDC or MC alone. <b>B.</b> ECs subjected to IL-6 treatment for 10 min were pretreated with 0.5 mM BSO for 30 min and then cultured without further treatment or exposed to TCDC or MC for 12 hours. Cell lysates were then subjected to western blotting.</p

CO increases the GSSG level in ECs.

<p><b>A.</b> and <b>B.</b> ECs were exposed to TCDC or MC for 1, 3, 6 and 12 hours and the intracellular SGGS levels were then measured. The results shown are the mean ± SEM. *<i>P</i><0.05 compared with untreated ECs. <b>C. and D.</b> The intracellular GSH levels of ECs incubated with TCDC or MC for 1, 3, 6 and 12 hours. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100677#s3" target="_blank">Results</a> are presented as the mean ± SEM (n = 3). *<i>P</i><0.05 compared with untreated ECs.</p

Proposed model of the inhibitory effects of CO upon STAT3 activation.

<p>NO and CO have long been known to increase ROS via the inhibition of cytochrome <i>c</i> oxidase function by competing with oxygen binding. The increased intracellular low levels oxidative stress induces protein S-glutathionylation to prevent STAT3 nuclear translocation under inflammatory cytokine treatment. See the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100677#s4" target="_blank">Discussion</a>" section for details.</p

CO induces HO-1 expression upon STAT3 phosphorylation.

<p><b>A.</b> and <b>B.</b> EC cultures were incubated with 25 µM TCDC or 5mM MC for the indicated periods. Western blotting analysis was then performed with antibodies against HO-1. <b>C.</b> ECs were transfected with control or HO-1 siRNA vectors for 36 hours and exposed to TCDC or MC for 12 hours. Cell lysates were subjected to western blot analysis with antibodies against pTyr-STAT3 or STAT3. <b>D.</b> ECs were transiently transfected with pcDNA vector or HO-1 plasmid. ECs transfected with pcDNA vector were exposed to IL-6 as a positive control. Cell lysates were subjected to western blot analysis with antibodies against pTyr-STAT3 or STAT3 as indicated. The quantification of the band intensities from three independent experiments was normalized to the control values (if the control was at near background, the background was set to 1). The resulting data are the mean ± SEM. *<i>P</i><0.05 compared with untreated ECs.</p