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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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Volcano plot result from JMP Genomics analysis showing significantly differentially expressed genes at 3 h (A) and 26 h (B) post-inoculation at 30°C. Green dots indicate oxygen up-regulated genes and red dots indicate the oxygen down-regulated genes. Grey colored dots were not considered significantly differentially expressed. The X axis shows the difference values between aerobic and anaerobic fermentations based on a log2 scale. The Y axis shows statistical significance values for expression values, based on a -log10 p-value. The red dashed line shows the statistical significance cut-off used in this study.
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Figure 3: Volcano plot result from JMP Genomics analysis showing significantly differentially expressed genes at 3 h (A) and 26 h (B) post-inoculation at 30°C. Green dots indicate oxygen up-regulated genes and red dots indicate the oxygen down-regulated genes. Grey colored dots were not considered significantly differentially expressed. The X axis shows the difference values between aerobic and anaerobic fermentations based on a log2 scale. The Y axis shows statistical significance values for expression values, based on a -log10 p-value. The red dashed line shows the statistical significance cut-off used in this study.

Mentions: Gene expression during the early exponential phase was surprisingly similar between anaerobic and aerobic conditions used in the present study (Fig. 3A). Eight genes were discovered that were considered differentially expressed at a significant level utilizing the ANOVA model using the False Discovery Rate (data not shown). Of these, only one gene (ZMO1752, encoding a hypothetical protein) showed a greater than a two-fold difference in relative expression levels. To confirm the microarray results, seventeen genes involving in ED and pyruvate pathways and different cellular functions were chosen for the qPCR analysis (see Additional file 6, 7). The data showed that qPCR was more sensitive and showed greater fold change differences compared to the microarray analysis, which was in keeping with previous reports [26]. We describe here only a rigorous analysis of stationary phase genes in this study and allow interested parties to conduct their own analyses on the entire dataset, which is available publicly through the GEO database. In the stationary phase 166 genes were significantly differentially expressed between anaerobic and aerobic conditions (Fig. 3B, 4, see Additional file 4, 5). This time point also showed the largest differences in extracellular metabolite profiles (Fig. 2). Fifty-five genes were up-regulated at 26 h post-inoculation under aerobic conditions and 111 genes were down-regulated (see Additional file 4, 5). Approximately two thirds of the genes down-regulated in the presence of oxygen for this time point were related to metabolism (see Additional file 4). In the presence of oxygen, genes related to regulation, cell processes, transport, and unknown function showed greater expression as compared to anaerobic conditions. Nearly half of the genes showing greater expression aerobically remain uncharacterized (see Additional file 5).


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)

Volcano plot result from JMP Genomics analysis showing significantly differentially expressed genes at 3 h (A) and 26 h (B) post-inoculation at 30°C. Green dots indicate oxygen up-regulated genes and red dots indicate the oxygen down-regulated genes. Grey colored dots were not considered significantly differentially expressed. The X axis shows the difference values between aerobic and anaerobic fermentations based on a log2 scale. The Y axis shows statistical significance values for expression values, based on a -log10 p-value. The red dashed line shows the statistical significance cut-off used in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Volcano plot result from JMP Genomics analysis showing significantly differentially expressed genes at 3 h (A) and 26 h (B) post-inoculation at 30°C. Green dots indicate oxygen up-regulated genes and red dots indicate the oxygen down-regulated genes. Grey colored dots were not considered significantly differentially expressed. The X axis shows the difference values between aerobic and anaerobic fermentations based on a log2 scale. The Y axis shows statistical significance values for expression values, based on a -log10 p-value. The red dashed line shows the statistical significance cut-off used in this study.
Mentions: Gene expression during the early exponential phase was surprisingly similar between anaerobic and aerobic conditions used in the present study (Fig. 3A). Eight genes were discovered that were considered differentially expressed at a significant level utilizing the ANOVA model using the False Discovery Rate (data not shown). Of these, only one gene (ZMO1752, encoding a hypothetical protein) showed a greater than a two-fold difference in relative expression levels. To confirm the microarray results, seventeen genes involving in ED and pyruvate pathways and different cellular functions were chosen for the qPCR analysis (see Additional file 6, 7). The data showed that qPCR was more sensitive and showed greater fold change differences compared to the microarray analysis, which was in keeping with previous reports [26]. We describe here only a rigorous analysis of stationary phase genes in this study and allow interested parties to conduct their own analyses on the entire dataset, which is available publicly through the GEO database. In the stationary phase 166 genes were significantly differentially expressed between anaerobic and aerobic conditions (Fig. 3B, 4, see Additional file 4, 5). This time point also showed the largest differences in extracellular metabolite profiles (Fig. 2). Fifty-five genes were up-regulated at 26 h post-inoculation under aerobic conditions and 111 genes were down-regulated (see Additional file 4, 5). Approximately two thirds of the genes down-regulated in the presence of oxygen for this time point were related to metabolism (see Additional file 4). In the presence of oxygen, genes related to regulation, cell processes, transport, and unknown function showed greater expression as compared to anaerobic conditions. Nearly half of the genes showing greater expression aerobically remain uncharacterized (see Additional file 5).

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