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Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations.

Yang S, Tschaplinski TJ, Engle NL, Carroll SL, Martin SL, Davison BH, Palumbo AV, Rodriguez M, Brown SD - BMC Genomics (2009)

Bottom Line: HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold.We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. yangs1@ornl.gov

ABSTRACT

Background: Zymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly.

Results: In this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point. Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.

Conclusion: High oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.

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Z. mobilis extracellular fermentation product analysis. The mean value for each metabolite identified by GC analysis from three independent fermentors for each condition is presented ± standard deviation (bars).
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Figure 2: Z. mobilis extracellular fermentation product analysis. The mean value for each metabolite identified by GC analysis from three independent fermentors for each condition is presented ± standard deviation (bars).

Mentions: Previous reports have indicated that ethanol production was decreased and other end-products such as acetaldehyde, acetate and acetoin were increased under aerobic conditions [12,18-20,25]. GC and HPLC were used to quantify and compare the kinetics of ethanol, acetate, acetaldehyde, lactate and acetoin production during aerobic and anaerobic fermentation processes and extracellular metabolites were often measured by more than one approach, which confirmed the observed trends. The more rapid production of ethanol under anaerobic conditions also corresponded with increased glucose uptake and growth under these conditions (Fig. 1). The ethanol concentration remained relatively stable post-peak production in anaerobic fermentations. In contrast, at 13 h the ethanol concentration dropped sharply from 52.7 mM to 3.2 mM at the end of the 26 h fermentation during aerobic fermentation. The decrease in ethanol concentration during this time was matched in nearly stoichiometric increases of acetate production, which went from 8.4 mM at 13 h to 72.1 mM at 26 h (Fig. 2). GC data also showed several other minor unidentified metabolites were being produced during aerobic fermentations relative to anaerobic conditions (data not shown). The concentration of acetaldehyde increased during the exponential growth and dropped appreciably during stationary phase, while acetoin was detected during stationary phase (Fig. 2). Lactate trended similarly to ethanol and acetoin profiles for respective conditions. The anaerobic ethanol yield at 13 h was 0.497 g/g of glucose or 97% theoretical and less than 1% of the yield went to solvent products other than ethanol or CO2 (Fig. 1). In contrast, under aerobic conditions at 13 h 0.14 g/g of glucose or 27% theoretical yield was obtained for ethanol and by 26 h around 3.2 mM ethanol remained (Fig. 1). The total measured solvents produced aerobically was 101 g/L, which was approximately 50% of theoretical total solvent yield at 13 h and at 26 h these figures had dropped to 76 g/L or approximately 37% of theoretical total solvent yield. As cell biomass (as measured by optical density) was approximately equivalent under the two conditions more carbon went to maintenance energy under aerobic conditions.


Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations.

Yang S, Tschaplinski TJ, Engle NL, Carroll SL, Martin SL, Davison BH, Palumbo AV, Rodriguez M, Brown SD - BMC Genomics (2009)

Z. mobilis extracellular fermentation product analysis. The mean value for each metabolite identified by GC analysis from three independent fermentors for each condition is presented ± standard deviation (bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651186&req=5

Figure 2: Z. mobilis extracellular fermentation product analysis. The mean value for each metabolite identified by GC analysis from three independent fermentors for each condition is presented ± standard deviation (bars).
Mentions: Previous reports have indicated that ethanol production was decreased and other end-products such as acetaldehyde, acetate and acetoin were increased under aerobic conditions [12,18-20,25]. GC and HPLC were used to quantify and compare the kinetics of ethanol, acetate, acetaldehyde, lactate and acetoin production during aerobic and anaerobic fermentation processes and extracellular metabolites were often measured by more than one approach, which confirmed the observed trends. The more rapid production of ethanol under anaerobic conditions also corresponded with increased glucose uptake and growth under these conditions (Fig. 1). The ethanol concentration remained relatively stable post-peak production in anaerobic fermentations. In contrast, at 13 h the ethanol concentration dropped sharply from 52.7 mM to 3.2 mM at the end of the 26 h fermentation during aerobic fermentation. The decrease in ethanol concentration during this time was matched in nearly stoichiometric increases of acetate production, which went from 8.4 mM at 13 h to 72.1 mM at 26 h (Fig. 2). GC data also showed several other minor unidentified metabolites were being produced during aerobic fermentations relative to anaerobic conditions (data not shown). The concentration of acetaldehyde increased during the exponential growth and dropped appreciably during stationary phase, while acetoin was detected during stationary phase (Fig. 2). Lactate trended similarly to ethanol and acetoin profiles for respective conditions. The anaerobic ethanol yield at 13 h was 0.497 g/g of glucose or 97% theoretical and less than 1% of the yield went to solvent products other than ethanol or CO2 (Fig. 1). In contrast, under aerobic conditions at 13 h 0.14 g/g of glucose or 27% theoretical yield was obtained for ethanol and by 26 h around 3.2 mM ethanol remained (Fig. 1). The total measured solvents produced aerobically was 101 g/L, which was approximately 50% of theoretical total solvent yield at 13 h and at 26 h these figures had dropped to 76 g/L or approximately 37% of theoretical total solvent yield. As cell biomass (as measured by optical density) was approximately equivalent under the two conditions more carbon went to maintenance energy under aerobic conditions.

Bottom Line: HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold.We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. yangs1@ornl.gov

ABSTRACT

Background: Zymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly.

Results: In this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point. Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.

Conclusion: High oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.

Show MeSH
Related in: MedlinePlus