Image_4_Regulatory Diversity and Functional Analysis of Two-Component Systems in Cyanobacterium Synechocystis sp. PCC 6803 by GC-MS Based Metabolomics.JPEG

Two-component signal transduction systems are still poorly functionally characterized in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, a GC-MS based comparative metabolomic analysis was conducted on a library of 44 knockout mutants for the response regulators (RRs) in Synechocystis. The metabolomic profiling analysis showed that 7 RRs mutants, namely Δslr1909, Δsll1291, Δslr6040, Δsll1330, Δslr2024, Δslr1584, and Δslr1693, were significantly different at metabolomic level, although their growth patterns are similar to the wild type under the normal autotrophic growth condition, suggesting regulatory diversity of RRs at metabolite level in Synechocystis. Additionally, a detailed metabolomic analysis coupled with RT-PCR verification led to useful clues for possible function of these 7 RRs, which were found involved in regulation of multiple aspects of cellular metabolisms in Synechocystis. Moreover, an integrative metabolomic and evolutionary analysis of all RR showed that four groups of RR genes clustered together in both metabolomic and evolutionary trees, suggesting of possible functional conservation of these RRs during the evolutionary process. Meanwhile, six groups of RRs with close evolutionary origin were found with different metabolomic profiles, suggesting possible functional changes during evolution. In contrast, more than 10 groups of RR genes with different clustering patterns in the evolutionary tree were found clustered together in metabolomics-based tree, suggesting possible functional convergences during the evolution. This study provided a metabolomic view of RR function, and the most needed functional clues for further characterization of these regulatory proteins in Synechocystis.</p

Image_2_Regulatory Diversity and Functional Analysis of Two-Component Systems in Cyanobacterium Synechocystis sp. PCC 6803 by GC-MS Based Metabolomics.JPEG

Two-component signal transduction systems are still poorly functionally characterized in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, a GC-MS based comparative metabolomic analysis was conducted on a library of 44 knockout mutants for the response regulators (RRs) in Synechocystis. The metabolomic profiling analysis showed that 7 RRs mutants, namely Δslr1909, Δsll1291, Δslr6040, Δsll1330, Δslr2024, Δslr1584, and Δslr1693, were significantly different at metabolomic level, although their growth patterns are similar to the wild type under the normal autotrophic growth condition, suggesting regulatory diversity of RRs at metabolite level in Synechocystis. Additionally, a detailed metabolomic analysis coupled with RT-PCR verification led to useful clues for possible function of these 7 RRs, which were found involved in regulation of multiple aspects of cellular metabolisms in Synechocystis. Moreover, an integrative metabolomic and evolutionary analysis of all RR showed that four groups of RR genes clustered together in both metabolomic and evolutionary trees, suggesting of possible functional conservation of these RRs during the evolutionary process. Meanwhile, six groups of RRs with close evolutionary origin were found with different metabolomic profiles, suggesting possible functional changes during evolution. In contrast, more than 10 groups of RR genes with different clustering patterns in the evolutionary tree were found clustered together in metabolomics-based tree, suggesting possible functional convergences during the evolution. This study provided a metabolomic view of RR function, and the most needed functional clues for further characterization of these regulatory proteins in Synechocystis.</p

Image_1_Regulatory Diversity and Functional Analysis of Two-Component Systems in Cyanobacterium Synechocystis sp. PCC 6803 by GC-MS Based Metabolomics.JPEG

Two-component signal transduction systems are still poorly functionally characterized in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, a GC-MS based comparative metabolomic analysis was conducted on a library of 44 knockout mutants for the response regulators (RRs) in Synechocystis. The metabolomic profiling analysis showed that 7 RRs mutants, namely Δslr1909, Δsll1291, Δslr6040, Δsll1330, Δslr2024, Δslr1584, and Δslr1693, were significantly different at metabolomic level, although their growth patterns are similar to the wild type under the normal autotrophic growth condition, suggesting regulatory diversity of RRs at metabolite level in Synechocystis. Additionally, a detailed metabolomic analysis coupled with RT-PCR verification led to useful clues for possible function of these 7 RRs, which were found involved in regulation of multiple aspects of cellular metabolisms in Synechocystis. Moreover, an integrative metabolomic and evolutionary analysis of all RR showed that four groups of RR genes clustered together in both metabolomic and evolutionary trees, suggesting of possible functional conservation of these RRs during the evolutionary process. Meanwhile, six groups of RRs with close evolutionary origin were found with different metabolomic profiles, suggesting possible functional changes during evolution. In contrast, more than 10 groups of RR genes with different clustering patterns in the evolutionary tree were found clustered together in metabolomics-based tree, suggesting possible functional convergences during the evolution. This study provided a metabolomic view of RR function, and the most needed functional clues for further characterization of these regulatory proteins in Synechocystis.</p

Data_Sheet_1_Regulatory Diversity and Functional Analysis of Two-Component Systems in Cyanobacterium Synechocystis sp. PCC 6803 by GC-MS Based Metabolomics.xlsx

Two-component signal transduction systems are still poorly functionally characterized in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, a GC-MS based comparative metabolomic analysis was conducted on a library of 44 knockout mutants for the response regulators (RRs) in Synechocystis. The metabolomic profiling analysis showed that 7 RRs mutants, namely Δslr1909, Δsll1291, Δslr6040, Δsll1330, Δslr2024, Δslr1584, and Δslr1693, were significantly different at metabolomic level, although their growth patterns are similar to the wild type under the normal autotrophic growth condition, suggesting regulatory diversity of RRs at metabolite level in Synechocystis. Additionally, a detailed metabolomic analysis coupled with RT-PCR verification led to useful clues for possible function of these 7 RRs, which were found involved in regulation of multiple aspects of cellular metabolisms in Synechocystis. Moreover, an integrative metabolomic and evolutionary analysis of all RR showed that four groups of RR genes clustered together in both metabolomic and evolutionary trees, suggesting of possible functional conservation of these RRs during the evolutionary process. Meanwhile, six groups of RRs with close evolutionary origin were found with different metabolomic profiles, suggesting possible functional changes during evolution. In contrast, more than 10 groups of RR genes with different clustering patterns in the evolutionary tree were found clustered together in metabolomics-based tree, suggesting possible functional convergences during the evolution. This study provided a metabolomic view of RR function, and the most needed functional clues for further characterization of these regulatory proteins in Synechocystis.</p

Image_3_Regulatory Diversity and Functional Analysis of Two-Component Systems in Cyanobacterium Synechocystis sp. PCC 6803 by GC-MS Based Metabolomics.JPEG

Two-component signal transduction systems are still poorly functionally characterized in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, a GC-MS based comparative metabolomic analysis was conducted on a library of 44 knockout mutants for the response regulators (RRs) in Synechocystis. The metabolomic profiling analysis showed that 7 RRs mutants, namely Δslr1909, Δsll1291, Δslr6040, Δsll1330, Δslr2024, Δslr1584, and Δslr1693, were significantly different at metabolomic level, although their growth patterns are similar to the wild type under the normal autotrophic growth condition, suggesting regulatory diversity of RRs at metabolite level in Synechocystis. Additionally, a detailed metabolomic analysis coupled with RT-PCR verification led to useful clues for possible function of these 7 RRs, which were found involved in regulation of multiple aspects of cellular metabolisms in Synechocystis. Moreover, an integrative metabolomic and evolutionary analysis of all RR showed that four groups of RR genes clustered together in both metabolomic and evolutionary trees, suggesting of possible functional conservation of these RRs during the evolutionary process. Meanwhile, six groups of RRs with close evolutionary origin were found with different metabolomic profiles, suggesting possible functional changes during evolution. In contrast, more than 10 groups of RR genes with different clustering patterns in the evolutionary tree were found clustered together in metabolomics-based tree, suggesting possible functional convergences during the evolution. This study provided a metabolomic view of RR function, and the most needed functional clues for further characterization of these regulatory proteins in Synechocystis.</p

Global Metabolomic and Network analysis of <i>Escherichia coli</i> Responses to Exogenous Biofuels

Although synthetic biology progress has made it possible to produce various biofuels in more user-friendly hosts, such as <i>Escherichia coli</i>, the large-scale biofuel production in these non-native systems is still challenging, mostly due to the very low tolerance of these non-native hosts to the biofuel toxicity. To address the issues, in this study we determined the metabolic responses of <i>E. coli</i> induced by three major biofuel products, ethanol, butanol, and isobutanol, using a gas chromatography–mass spectrometry (GC–MS) approach. A metabolomic data set of 65 metabolites identified in all samples was then subjected to principal component analysis (PCA) to compare their effects and a weighted correlation network analysis (WGCNA) to identify the metabolic modules specifically responsive to each of the biofuel stresses, respectively. The PCA analysis showed that cellular responses caused by the biofuel stress were in general similar to aging cells at stationary phase, inconsistent with early studies showing a high degree of dissimilarity between metabolite responses during growth cessation as induced through stationary phases or through various environmental stress applications. The WGCNA analysis allowed identification of 2, 4, and 2 metabolic modules specifically associated with ethanol, butanol, and isobutanol treatments, respectively. The biofuel-associated modules included amino acids and osmoprotectants, such as isoleucine, valine, glycine, glutamate, and trehalose, suggesting amino acid metabolism and osmoregulation are among the key protection mechanisms against biofuel stresses in <i>E. coli</i>. Interestingly, no module was found associated with all three biofuel products, suggesting differential effects of each biofuel on <i>E. coli</i>. The findings enhanced our understanding of <i>E. coli</i> responses to exogenous biofuels and also demonstrated the effectiveness of the metabolomic and network analysis in identifying key targets for biofuel tolerance

Global Metabolomic and Network analysis of <i>Escherichia coli</i> Responses to Exogenous Biofuels

Although synthetic biology progress has made it possible to produce various biofuels in more user-friendly hosts, such as <i>Escherichia coli</i>, the large-scale biofuel production in these non-native systems is still challenging, mostly due to the very low tolerance of these non-native hosts to the biofuel toxicity. To address the issues, in this study we determined the metabolic responses of <i>E. coli</i> induced by three major biofuel products, ethanol, butanol, and isobutanol, using a gas chromatography–mass spectrometry (GC–MS) approach. A metabolomic data set of 65 metabolites identified in all samples was then subjected to principal component analysis (PCA) to compare their effects and a weighted correlation network analysis (WGCNA) to identify the metabolic modules specifically responsive to each of the biofuel stresses, respectively. The PCA analysis showed that cellular responses caused by the biofuel stress were in general similar to aging cells at stationary phase, inconsistent with early studies showing a high degree of dissimilarity between metabolite responses during growth cessation as induced through stationary phases or through various environmental stress applications. The WGCNA analysis allowed identification of 2, 4, and 2 metabolic modules specifically associated with ethanol, butanol, and isobutanol treatments, respectively. The biofuel-associated modules included amino acids and osmoprotectants, such as isoleucine, valine, glycine, glutamate, and trehalose, suggesting amino acid metabolism and osmoregulation are among the key protection mechanisms against biofuel stresses in <i>E. coli</i>. Interestingly, no module was found associated with all three biofuel products, suggesting differential effects of each biofuel on <i>E. coli</i>. The findings enhanced our understanding of <i>E. coli</i> responses to exogenous biofuels and also demonstrated the effectiveness of the metabolomic and network analysis in identifying key targets for biofuel tolerance

Metabolomic Analysis Reveals Mechanism of Antioxidant Butylated Hydroxyanisole on Lipid Accumulation in <i>Crypthecodinium cohnii</i>

The heterotrophic dinoflagellate alga <i>Crypthecodinium cohnii</i> is known to accumulate lipids with a high fraction of docosahexaenoic acid (DHA). In this study, we first evaluated two antioxidant compounds, butylated hydroxyanisole (BHA) and propyl gallate (PG), for their effects on lipid accumulation in <i>C. cohnii</i>. The results showed that antioxidant BHA could increase lipid accumulation in <i>C. cohnii</i> by 8.80% at a final concentration of 30 μM, while PG had no obvious effect on lipid accumulation at the tested concentrations. To decipher the molecular mechanism responsible for the increased lipid accumulation by BHA, we employed an integrated GC-MS and LC-MS metabolomic approach to determine the time-series metabolic profiles with or without BHA, and then subjected the metabolomic data to a principal component analysis (PCA) and a weighted gene coexpression network analysis (WGCNA) network analyses to identify the key metabolic modules and metabolites possibly relevant to the increased lipid accumulation. LC-MS analysis showed that several metabolites, including NADPH, could be important for the stimulation role of BHA on lipid accumulation. Meanwhile GC-MS and network analyses allowed identification of eight metabolic modules and nine hub metabolites possibly relevant to the stimulation role of BHA in <i>C. cohnii</i>. The study provided a metabolomics view of the BHA mode of action on lipid accumulation in <i>C. cohnii</i>, and the information could be valuable for a better understanding of antioxidant effects on lipid accumulation in other microalgae as well

Table_1_13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii.XLSX

<p>The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using ¹³C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP⁺-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.</p

Table_2_13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii.XLSX

<p>The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using ¹³C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP⁺-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.</p